TGFBeta signal convertor

Abstract

The present disclosure provides improved compositions for adoptive T cell therapies for treating, preventing, or ameliorating at least one symptom of a cancer, infectious disease, autoimmune disease, inflammatory disease, and immunodeficiency, or condition associated therewith. 20 24 20 36 04 29 M ay 2 02 4 A B S T R A C T 2 0 2 4 2 0 3 6 0 4 2 9 M a y 2 0 2 4

Description

2024203604   29 May 2024 comprising an extracellular TGF01 -binding domain of TGF0R1, a transmembrane domain, and an IL-2Ry intracellular signaling domain; a polypeptide cleavage signal; and a second polypeptide comprising an extracellular TGF01 -binding domain of TGF0R2, a transmembrane domain, and an IL-2R0 intracellular signaling domain. 5         In particular embodiments, the CTBR15 signal convertor is a complex of polypeptides comprising a first polypeptide comprising a polypeptide comprising an extracellular TGF^l-binding domain of TGF0R1, a transmembrane domain, and an IL-2R0 intracellular signaling domain; and a polypeptide comprising an extracellular TGF01-binding domain of TGF0R2, a transmembrane domain, and an IL-2Ry intracellular 10 signaling domain. In particular embodiments, the CTBR15 signal convertor is a complex of polypeptides comprising a first polypeptide comprising a polypeptide comprising an extracellular TGF01 -binding domain of TGF0R1, a transmembrane domain, and an IL-2Ry intracellular signaling domain; and a polypeptide comprising an extracellular TGF01-binding domain of TGF0R2, a transmembrane domain, and an IL-2R0 intracellular 15 signaling domain. In certain embodiments, a polypeptide comprises a transmembrane domain of TGF0R1 or TGF^R2. In certain embodiments, a polypeptide comprises a transmembrane domain of IL-2R0 or IL-2Ry. In one embodiment, a polypeptide comprises an extracellular TGFJG -binding domain of TGF0R1 and an IL-2R0 transmembrane domain and 20 intracellular signaling domain. In one embodiment, a polypeptide comprises an extracellular TGF01-binding domain of TGF0R2 and an IL-2Ry transmembrane domain and intracellular signaling domain. In one embodiment, a polypeptide comprises an extracellular TGF01-binding domain of TGF0R1 and an IL-2Ry transmembrane domain and intracellular signaling domain. In one embodiment, a polypeptide comprises an 25 extracellular TGF01-binding domain of TGF0R2 and an IL-2R0 transmembrane domain and intracellular signaling domain. In particular embodiments, the polypeptide cleavage signal is a viral self-cleaving polypeptide; more preferably, a viral self-cleaving 2A polypeptide; and more preferably a viral self-cleaving polypeptide selected from the group consisting of: a foot-and-mouth 2024203604   29 May 2024 disease virus (FMDV) (F2A) peptide, an equine rhinitis A virus (ERAV) (E2A) peptide, a Thosea asigna virus (TaV) (T2A) peptide, a porcine teschovirus-1 (PTV-1) (P2A) peptide, a Theilovirus 2A peptide, and an encephalomyocarditis virus 2A peptide. In one embodiment, the polypeptide cleavage signal is a P2A or T2A viral self-cleaving 5 polypeptide. 4.    CTBR21 Signal Convertor Interleukin-21 (IL-21) is a cytokine that promotes T cell function and activity by, in part, improving T cell precursor survival and proliferation. IL-21 binds to interleukin 21 receptor (IL-21R, also known as CD360) and IL-2Ry (also known as CD132 and yc). IL- 10  21 signaling activates the JAK / STAT, PI-3K, and Src kinase pathways and results in transcription of anti-apoptotic genes and genes that promote proliferation of T cell precursors. In various embodiments, one or more immune effector cells, including immune effector cells expressing an engineered antigen receptor, are modified by introducing one 15 or more polynucleotides or vectors encoding a CTBR21 signal convertor. In various embodiments, one or more immune effector cells are modified by introducing one or more polynucleotides or vectors encoding a CTBR21 signal convertor and an engineered antigen receptor. In particular embodiments, the TGF0 signal convertor converts an 20 immunosuppressive TGF0 signal to an IL-21-mediated immunostimulatory signal. In particular embodiments, a CTBR21 signal convertor contemplated herein comprises: an extracellular TGF^l-binding domain of TGF0R1, a transmembrane domain, and an IL-21R intracellular signaling domain; a polypeptide cleavage signal; and an extracellular TGF01-binding domain of TGF^R2, a transmembrane domain, and an IL-2Ry intracellular 25 signaling domain. In particular embodiments, a CTBR21 signal convertor contemplated herein comprises: an extracellular TGF01 -binding domain of TGF^Rl, a transmembrane domain, and an IL-2Ry intracellular signaling domain; a polypeptide cleavage signal; and 2024203604   29 May 2024 an extracellular TGF01 -binding domain of TGF0R2, a transmembrane domain, and an IL-21R intracellular signaling domain. In particular embodiments a CTBR21 signal convertor contemplated herein comprises a fusion polypeptide comprising: a first polypeptide comprising an extracellular 5 TGF^l-binding domain of TGF0R1, a transmembrane domain, and an IL-21R intracellular signaling domain; a polypeptide cleavage signal; and a second polypeptide comprising an extracellular TGF01 -binding domain of TGF^R2, a transmembrane domain, and an IL-2Ry intracellular signaling domain. In particular embodiments, a CTBR21 signal convertor contemplated herein comprises a fusion polypeptide comprising: a first polypeptide 10 comprising an extracellular TGF01 -binding domain of TGF^Rl, a transmembrane domain, and an IL-2Ry intracellular signaling domain; a polypeptide cleavage signal; and a second polypeptide comprising an extracellular TGFJG -binding domain of TGF^R2, a transmembrane domain, and an IL-21R intracellular signaling domain. In particular embodiments, the CTBR21 signal convertor is a complex of 15 polypeptides comprising a first polypeptide comprising a polypeptide comprising an extracellular TGF^l-binding domain of TGF0R1, a transmembrane domain, and an IL-21R intracellular signaling domain; and a polypeptide comprising an extracellular TGFJG -binding domain of TGF0R2, a transmembrane domain, and an IL-2Ry intracellular signaling domain. In particular embodiments, the CTBR21 signal convertor is a complex 20 of polypeptides comprising a first polypeptide comprising a polypeptide comprising an extracellular TGF01 -binding domain of TGF0R1, a transmembrane domain, and an IL-2Ry intracellular signaling domain; and a polypeptide comprising an extracellular TGFJG -binding domain of TGF0R2, a transmembrane domain, and an IL-21R intracellular signaling domain. 25         In certain embodiments, a polypeptide comprises a transmembrane domain of TGF0R1 or TGF0R2. In certain embodiments, a polypeptide comprises a transmembrane domain of IL-21R or IL-2Ry. In one embodiment, a polypeptide comprises an extracellular TGF01 -binding domain of TGF^R! and an IL-21R transmembrane domain and intracellular signaling domain. In one embodiment, a polypeptide comprises an 2024203604   29 May 2024 extracellular TGF01-binding domain of TGF0R2 and an IL-2Ry transmembrane domain and intracellular signaling domain. In one embodiment, a polypeptide comprises an extracellular TGF01-binding domain of TGF0R1 and an IL-2Ry transmembrane domain and intracellular signaling domain. In one embodiment, a polypeptide comprises an 5 extracellular TGF^l-binding domain of TGF0R2 and an IL-21R transmembrane domain and intracellular signaling domain. In particular embodiments, the polypeptide cleavage signal is a viral self-cleaving polypeptide; more preferably, a viral self-cleaving 2A polypeptide; and more preferably a viral self-cleaving polypeptide selected from the group consisting of: a foot-and-mouth 10 disease virus (FMDV) (F2A) peptide, an equine rhinitis A virus (ERAV) (E2A) peptide, a Thosea asigna virus (TaV) (T2A) peptide, a porcine teschovirus-1 (PTV-1) (P2A) peptide, a Theilovirus 2A peptide, and an encephalomyocarditis virus 2A peptide. In one embodiment, the polypeptide cleavage signal is a P2A or T2A viral self-cleaving polypeptide. 15        5.    CTBR18 SIGNAL CONVERTOR Interleukin-18 (IL-18) is a cytokine that promotes T cell function and activity by, in part, increasing IFNy expression, increasing T cell proliferation, and protecting against activation induced cell death (AICD). IL-18 binds interleukin 18 receptor 1, (IL-18R1, also known as CD218a) and interleukin 18 receptor accessory protein (IL-18RAP, 20  CD218b). IL-18 signaling through IL-18R1 and IL-18RAP results in activation through the MyD88 adaptor protein and IRAK4 phosphorylation. Phosphorylation of IRAK4 and subsequent phosphorylation of IRAK1 / 2 ultimately leads to activation of NF-kappa B and AP-1 transcription factors to increase IFNy expression and increase sensitivity to IL-12. 25 The transcriptional program induced by IL-18 also increases T cell proliferation and protects against AICD. In various embodiments, one or more immune effector cells, including immune effector cells expressing an engineered antigen receptor, are modified by introducing one 2024203604   29 May 2024 or more polynucleotides or vectors encoding a CTBR18 signal convertor. In various embodiments, one or more immune effector cells are modified by introducing one or more polynucleotides or vectors encoding a CTBR18 signal convertor and an engineered antigen receptor. 5          In particular embodiments, the TGF0 signal convertor converts an immunosuppressive TGF0 signal to an IL-18-mediated immunostimulatory signal. In particular embodiments, a CTBR18 signal convertor contemplated herein comprises: an extracellular TGF01 -binding domain of TGF0R1, a transmembrane domain, and an IL-18RAP intracellular signaling domain; a polypeptide cleavage signal; and an extracellular 10 TGF01 -binding domain of TGF0R2, a transmembrane domain, and an IL- 18R1 intracellular signaling domain. In particular embodiments, a CTBR18 signal convertor contemplated herein comprises: an extracellular TGF01 -binding domain of TGF0R1, a transmembrane domain, and an IL-18R1 intracellular signaling domain; a polypeptide cleavage signal; and an extracellular TGF01 -binding domain of TGF0R2, a transmembrane 15 domain, and an IL-18RAP intracellular signaling domain. In particular embodiments, a CTBR18 signal convertor contemplated herein comprises a fusion polypeptide comprising: a first polypeptide comprising an extracellular TGF01 -binding domain of TGF0R1, a transmembrane domain, and an IL-18R1 intracellular signaling domain; a polypeptide cleavage signal; and a second polypeptide 20 comprising an extracellular TGF01-binding domain of TGF0R2, a transmembrane domain, and an IL-18RAP intracellular signaling domain. In particular embodiments, a CTBR18 signal convertor contemplated herein comprises a fusion polypeptide comprising: a first polypeptide comprising an extracellular TGF^l-binding domain of TGF0R1, a transmembrane domain, and an IL-18RAP intracellular signaling domain; a polypeptide 25 cleavage signal; and a second polypeptide comprising an extracellular TGF01-binding domain of TGF0R2, a transmembrane domain, and an IL-18R1 intracellular signaling domain. In particular embodiments, the CTBR18 signal convertor is a complex of polypeptides comprising a first polypeptide comprising a polypeptide comprising an 2024203604   29 May 2024 extracellular TGF01 -binding domain of TGF0R1, a transmembrane domain, and an IL-18RAP intracellular signaling domain; and a polypeptide comprising an extracellular TGF01 -binding domain of TGF0R2, a transmembrane domain, and an IL-18R1 intracellular signaling domain. In particular embodiments, the CTBR18 signal convertor is 5 a complex of polypeptides comprising a first polypeptide comprising a polypeptide comprising an extracellular TGF01 -binding domain of TGF0R1, a transmembrane domain, and an IL-18R1 intracellular signaling domain; and a polypeptide comprising an extracellular TGF01 -binding domain of TGF^R2, a transmembrane domain, and an IL-18RAP intracellular signaling domain. 10         In certain embodiments, a polypeptide comprises a transmembrane domain of TGF0R1 or TGF0R2. In certain embodiments, a polypeptide comprises a transmembrane domain of IL-18R1 or IL-18RAP. In one embodiment, a polypeptide comprises an extracellular TGF01 -binding domain of TGF0R1 and an IL-18RAP transmembrane domain and intracellular signaling domain. In one embodiment, a polypeptide comprises an 15 extracellular TGF01 -binding domain of TGF0R2 and an IL-18R1 transmembrane domain and intracellular signaling domain. In one embodiment, a polypeptide comprises an extracellular TGF01 -binding domain of TGF0R1 and an IL-18R1 transmembrane domain and intracellular signaling domain. In one embodiment, a polypeptide comprises an extracellular TGF01 -binding domain of TGF0R2 and an IL-18RAP transmembrane domain 20 and intracellular signaling domain. In particular embodiments, the polypeptide cleavage signal is a viral self-cleaving polypeptide; more preferably, a viral self-cleaving 2A polypeptide; and more preferably a viral self-cleaving polypeptide selected from the group consisting of: a foot-and-mouth disease virus (FMDV) (F2A) peptide, an equine rhinitis A virus (ERAV) (E2A) peptide, a 25 Thosea asigna virus (TaV) (T2A) peptide, a porcine teschovirus-1 (PTV-1) (P2A) peptide, a Theilovirus 2A peptide, and an encephalomyocarditis virus 2A peptide. In one embodiment, the polypeptide cleavage signal is a P2A or T2A viral self-cleaving polypeptide. 2024203604   29 May 2024 6.    CTBR1 Signal Convertor Interleukin-1 (IL-1) is a cytokine that promotes T cell function and activity by, in part, increasing IFNy expression, increasing T cell proliferation, and potentiating protecting against activation induced cell death (AICD). IL-1 binds interleukin 1 receptor 1, (IL-1R1, 5 also known as CD121a) and interleukin 1 receptor accessory protein (IL-1RAP). IL-1 signaling through IL-1R1 and IL-1RAP results in activation through the MyD88 adaptor protein and IRAK4 phosphorylation. Phosphorylation of IRAK4 and subsequent phosphorylation of IRAK1 / 2 ultimately leads to activation of NF-kappa B and AP-1 transcription factors to increase IFNy expression and increase sensitivity to IL-12. 10 The transcriptional program induced by IL-1 also increases T cell proliferation and protects against AICD. In various embodiments, one or more immune effector cells, including immune effector cells expressing an engineered antigen receptor, are modified by introducing one or more polynucleotides or vectors encoding a CTBR1 signal convertor. In various 15 embodiments, one or more immune effector cells are modified by introducing one or more polynucleotides or vectors encoding a CTBR1 signal convertor and an engineered antigen receptor. In particular embodiments, the TGF0 signal convertor converts an immunosuppressive TGF0 signal to an IL-1-mediated immunostimulatory signal. In 20 particular embodiments, a CTBR1 signal convertor contemplated herein comprises: an extracellular TGF01 -binding domain of TGF0R1, a transmembrane domain, and an IL-1RAP intracellular signaling domain; a polypeptide cleavage signal; and an extracellular TGFpi-binding domain of TGF0R2, a transmembrane domain, and an IL-1R1 intracellular signaling domain. In particular embodiments, a CTBR1 signal convertor contemplated 25 herein comprises: an extracellular TGF01 -binding domain of TGF0R1, a transmembrane domain, and an IL-1R1 intracellular signaling domain; a polypeptide cleavage signal; and an extracellular TGF01 -binding domain of TGF0R2, a transmembrane domain, and an IL-1RAP intracellular signaling domain. 2024203604   29 May 2024 In particular embodiments, a CTBR1 signal convertor contemplated herein comprises a fusion polypeptide comprising: a first polypeptide comprising an extracellular TGFpi-bmdmg domain of TGF0R1, a transmembrane domain, and an IL-1R1 intracellular signaling domain; a polypeptide cleavage signal; and a second polypeptide comprising an 5 extracellular TGF01 -binding domain of TGF0R2, a transmembrane domain, and an IL-1RAP intracellular signaling domain. In particular embodiments, a CTBR1 signal convertor contemplated herein comprises a fusion polypeptide comprising: a first polypeptide comprising an extracellular TGF01 -binding domain of TGF0R1, a transmembrane domain, and an IL-1RAP intracellular signaling domain; a polypeptide 10 cleavage signal; and a second polypeptide comprising an extracellular TGF01 -binding domain of TGF0R2, a transmembrane domain, and an IL-1R1 intracellular signaling domain. In particular embodiments, the CTBR1 signal convertor is a complex of polypeptides comprising a first polypeptide comprising a polypeptide comprising an 15 extracellular TGF01 -binding domain of TGF0R1, a transmembrane domain, and an IL-1RAP intracellular signaling domain; and a polypeptide comprising an extracellular TGFpi-binding domain of TGF0R2, a transmembrane domain, and an IL-1R1 intracellular signaling domain. In particular embodiments, the CTBR1 signal convertor is a complex of polypeptides comprising a first polypeptide comprising a polypeptide comprising an 20 extracellular TGF^l-binding domain of TGF0R1, a transmembrane domain, and an IL-1R1 intracellular signaling domain; and a polypeptide comprising an extracellular TGF01-binding domain of TGF0R2, a transmembrane domain, and an IL-1RAP intracellular signaling domain. In certain embodiments, a polypeptide comprises a transmembrane domain of 25 TGF0R1 or TGF0R2. In certain embodiments, a polypeptide comprises a transmembrane domain of IL-1R1 or IL-1RAP. In one embodiment, a polypeptide comprises an extracellular TGF^l-binding domain of TGF0R1 and an IL-1RAP transmembrane domain and intracellular signaling domain. In one embodiment, a polypeptide comprises an extracellular TGF^l-binding domain of TGF^R2 and an IL-1R1 transmembrane domain 2024203604   29 May 2024 and intracellular signaling domain. In one embodiment, a polypeptide comprises an extracellular TGFpi-bmdmg domain of TGF0R1 and an IL-1R1 transmembrane domain and intracellular signaling domain. In one embodiment, a polypeptide comprises an extracellular TGFpi-bmdmg domain of TGF0R2 and an IL-1RAP transmembrane domain 5 and intracellular signaling domain. In particular embodiments, the polypeptide cleavage signal is a viral self-cleaving polypeptide; more preferably, a viral self-cleaving 2A polypeptide; and more preferably a viral self-cleaving polypeptide selected from the group consisting of: a foot-and-mouth disease virus (FMDV) (F2A) peptide, an equine rhinitis A virus (ERAV) (E2A) peptide, a 10 Thosea asigna virus (TaV) (T2A) peptide, a porcine teschovirus-1 (PTV-1) (P2A) peptide, a Theilovirus 2A peptide, and an encephalomyocarditis virus 2A peptide. In one embodiment, the polypeptide cleavage signal is a P2A or T2A viral self-cleaving polypeptide. 7.    CTBR.TLR Signal Convertor 15          Toll like receptors (TLR1 through TLR10) are pattern recognition receptors that detect invading pathogens and activate the innate and adaptive immune responses. Activation of TLRs by various ligands leads to induction of a pro-inflammatory transcriptional program and expression of multiple inflammatory cytokines. TLR signaling occurs via homodimerization of TLR signaling domains leading to 20 activation through the MyD88 adaptor protein and IRAK4 phosphorylation. Phosphorylation of IRAK4 and subsequent phosphorylation of IRAK1 / 2 ultimately leads to activation of NF-kappa B and AP-1 transcription factors to increase inflammatory cytokine production and induce proliferation. TLR activation can also lead to the activation of IRF3 and IRF7 transcription factors. 25         In various embodiments, one or more immune effector cells, including immune effector cells expressing an engineered antigen receptor, are modified by introducing one or more polynucleotides or vectors encoding a CTBR.TLR signal convertor. In various embodiments, one or more immune effector cells are modified by introducing one or more 2024203604   29 May 2024 polynucleotides or vectors encoding a CTBR.TLR signal convertor and an engineered antigen receptor. In particular embodiments, the TGF0 signal convertor converts an immunosuppressive TGF0 signal to a TLR-mediated immunostimulatory signal. In 5 particular embodiments, a CTBR.TLR signal convertor contemplated herein comprises: an extracellular TGFJG -binding domain of TGF0R1, a transmembrane domain, and a TLR intracellular signaling domain; a polypeptide cleavage signal; and an extracellular TGF01-binding domain of TGF0R2, a transmembrane domain, and an identical TLR signaling domain. 10         In particular embodiments, a CTBR.TLR signal convertor contemplated herein comprises a fusion polypeptide comprising: a first polypeptide comprising an extracellular TGFJG -binding domain of TGF0R1, a transmembrane domain, and a TLR intracellular signaling domain; a polypeptide cleavage signal; and a second polypeptide comprising an extracellular TGFJG -binding domain of TGF^R2, a transmembrane domain, and an 15 identical TLR signaling domain. In particular embodiments, the CTBR.TLR signal convertor is a complex of polypeptides comprising a first polypeptide comprising a polypeptide comprising an extracellular TGFJG -binding domain of TGF0R1, a transmembrane domain, and a TLR intracellular signaling domain; and a polypeptide comprising an extracellular TGF01- 20 binding domain of TGF0R2, a transmembrane domain, and an identical TLR intracellular signaling domain. In certain embodiments, a polypeptide comprises a transmembrane domain of TGF0R1 or TGF0R2. In certain embodiments, a polypeptide comprises a transmembrane domain of a TLR. In one embodiment, a polypeptide comprises an extracellular TGFJG -25 binding domain of TGF0R1 and a TLR transmembrane domain and intracellular signaling domain. In one embodiment, a polypeptide comprises an extracellular TGF^l-binding domain of TGF0R2 and a TLR transmembrane domain and intracellular signaling domain. In particular embodiments, the polypeptide cleavage signal is a viral self-cleaving polypeptide; more preferably, a viral self-cleaving 2A polypeptide; and more preferably a 2024203604   29 May 2024 viral self-cleaving polypeptide selected from the group consisting of: a foot-and-mouth disease virus (FMDV) (F2A) peptide, an equine rhinitis A virus (ERAV) (E2A) peptide, a Thosea asigna virus (TaV) (T2A) peptide, a porcine teschovirus-1 (PTV-1) (P2A) peptide, a Theilovirus 2A peptide, and an encephalomyocarditis virus 2A peptide. In one 5 embodiment, the polypeptide cleavage signal is a P2A or T2A viral self-cleaving polypeptide. D.   Engineered Antigen Receptors In particular embodiments, a polypeptide comprises an engineered antigen receptor, a polypeptide cleavage signal and a CTBR. In other particular embodiments, a 10 polynucleotide or vector encoding a CTBR is introduced into an immune effector cell that comprises an engineered antigen receptor. Without wishing to be bound by any particular theory, it is contemplated in particular embodiments, that any mechanism known in the art may be used to introduce and co-express an engineered antigen receptor and a CTBR in the same immune effector cell or population of cells to increase the resistance of the immune 15 effector cells to the TME and potentiate and increase the efficiency, potency, and durability of the immune effector cell response. In particular embodiments, immune effector cells contemplated herein comprise an engineered antigen receptor and a CTBR. In particular embodiments, the engineered antigen receptor is an engineered T cell receptor (TCR), a chimeric antigen receptor 20 (CAR), a DARIC receptor or components thereof, or a zetakine. 1.    Engineered TCRs In particular embodiments, immune effector cells contemplated herein comprise an engineered TCR and a CTBR signal convertor. In one embodiment, T cells are engineered by introducing a polynucleotide or vector encoding an engineered TCR and a CTBR signal 25 convertor separated by one or more polypeptide cleavage signals. In one embodiment, T cells are engineered by introducing a polynucleotide or vector encoding an engineered TCR and a polynucleotide or vector encoding a CTBR signal convertor. In one embodiment, T 2024203604   29 May 2024 cells are engineered to express an engineered TCR are further engineered by introducing a polynucleotide or vector encoding a CTBR signal convertor. Naturally occurring T cell receptors comprise two subunits, an alpha chain and a beta chain subunit, each of which is a unique protein produced by recombination event in 5 each T cell’s genome. Libraries of TCRs may be screened for their selectivity to particular target antigens. In this manner, natural TCRs, which have a high-avidity and reactivity toward target antigens may be selected, cloned, and subsequently introduced into a population of T cells used for adoptive immunotherapy. In one embodiment, T cells are modified by introducing a TCR subunit has the 10 ability to form TCRs that confer specificity to T cells for tumor cells expressing a target antigen. In particular embodiments, the subunits have one or more amino acid substitutions, deletions, insertions, or modifications compared to the naturally occurring subunit, so long as the subunits retain the ability to form TCRs and confer upon transfected T cells the ability to home to target cells, and participate in immunologically-relevant 15 cytokine signaling. The engineered TCRs preferably also bind target cells displaying the relevant tumor-associated peptide with high avidity, and optionally mediate efficient killing of target cells presenting the relevant peptide in vivo. The nucleic acids encoding engineered TCRs are preferably isolated from their natural context in a (naturally-occurring) chromosome of a T cell, and can be incorporated 20 into suitable vectors as described elsewhere herein. Both the nucleic acids and the vectors comprising them can be transferred into a cell, preferably a T cell in particular embodiments. The modified T cells are then able to express one or more chains of a TCR encoded by the transduced nucleic acid or nucleic acids. In preferred embodiments, the engineered TCR is an exogenous TCR because it is introduced into T cells that do not 25 normally express the particular TCR. The essential aspect of the engineered TCRs is that it has high avidity for a tumor antigen presented by a major histocompatibility complex (MHC) or similar immunological component. In contrast to engineered TCRs, CARs are engineered to bind target antigens in an MHC independent manner. 2024203604   29 May 2024 The TCR can be expressed with additional polypeptides attached to the aminoterminal or carboxyl-terminal portion of the alpha chain or beta chain of a TCR so long as the attached additional polypeptide does not interfere with the ability of the alpha chain or beta chain to form a functional T cell receptor and the MHC dependent antigen recognition. 5         Antigens that are recognized by the engineered TCRs contemplated in particular embodiments include, but are not limited to cancer antigens, including antigens on both hematological cancers and solid tumors. Illustrative antigens include, but are not limited to alpha folate receptor, alpha folate receptor, 5T4, av06 integrin, BCMA, B7-H3, B7-H6, CAIX, CD19, CD20, CD22, CD30, CD33, CD44, CD44v6, CD44v7 / 8, CD70, CD79a, 10 CD79b, CD123, CD138, CD171, CEA, CSPG4, EGFR, EGFR family including ErbB2 (HER2), EGFRvIII, EGP2, EGP40, EPCAM, EphA2, EpCAM, FAP, fetal AchR, FRa, GD2, GD3, Glypican-3 (GPC3), HLA-A1+MAGE1, HLA-A2+MAGE1, HLA-A3+MAGE1, HLA-A1+NY-ESO-1, HLA-A2+NY-ESO-1, HLA-A3+NY-ESO-1, IL-11Ra, IL-13Ra2, Lambda, Lewis-Y, Kappa, Mesothelin, Muc1, Muc16, NCAM, NKG2D 15 Ligands, NY-ESO-1, PRAME, PSCA, PSMA, ROR1, SSX, Survivin, TAG72, TEMs, VEGFR2, and WT-1. 2.    Chimeric Antigen Receptors In various embodiments, immune effector cells express CARs that redirect cytotoxicity toward tumor cells. CARs are molecules that combine antibody-based 20 specificity for a target antigen (e.g., tumor antigen) with a T cell receptor-activating intracellular domain to generate a chimeric protein that exhibits a specific anti-tumor cellular immune activity. As used herein, the term, “chimeric,” describes being composed of parts of different proteins or DNAs from different origins. In particular embodiments, immune effector cells contemplated herein comprise 25 CAR and a CTBR signal convertor. In one embodiment, T cells are engineered by introducing a polynucleotide or vector encoding a CAR and a CTBR signal convertor separated by one or more polypeptide cleavage signals. In one embodiment, T cells are engineered by introducing a polynucleotide or vector encoding a CAR and a polynucleotide 2024203604   29 May 2024 or vector encoding a CTBR signal convertor. In one embodiment, T cells are engineered to express a CAR are further engineered by introducing a polynucleotide or vector encoding a CTBR signal convertor. In various embodiments, a CAR comprises an extracellular domain that binds to a 5 specific target antigen (also referred to as a binding domain or antigen-specific binding domain), a transmembrane domain and an intracellular signaling domain. The main characteristic of CARs is their ability to redirect immune effector cell specificity, thereby triggering proliferation, cytokine production, phagocytosis or production of molecules that can mediate cell death of the target antigen expressing cell in a major histocompatibility 10 (MHC) independent manner, exploiting the cell specific targeting abilities of monoclonal antibodies, soluble ligands or cell specific coreceptors. In particular embodiments, CARs comprise an extracellular binding domain that specifically binds to a target polypeptide, e.g., target antigen, expressed on tumor cell. As used herein, the terms, “binding domain,” “extracellular domain,” “extracellular binding 15 domain,” “antigen binding domain,” “antigen-specific binding domain,” and “extracellular antigen specific binding domain,” are used interchangeably and provide a chimeric receptor, e.g., a CAR or DARIC, with the ability to specifically bind to the target antigen of interest. A binding domain may comprise any protein, polypeptide, oligopeptide, or peptide that possesses the ability to specifically recognize and bind to a biological molecule 20   (e.g., a cell surface receptor or tumor protein, lipid, polysaccharide, or other cell surface target molecule, or component thereof). A binding domain includes any naturally occurring, synthetic, semi-synthetic, or recombinantly produced binding partner for a biological molecule of interest. In particular embodiments, the extracellular binding domain comprises an antibody 25 or antigen binding fragment thereof. An “antibody” refers to a binding agent that is a polypeptide comprising at least a light chain or heavy chain immunoglobulin variable region which specifically recognizes and binds an epitope of a target antigen, such as a peptide, lipid, polysaccharide, or nucleic acid containing an antigenic determinant, such as those recognized by an immune cell. 2024203604   29 May 2024 Antibodies include antigen binding fragments, e.g., Camel Ig (a camelid antibody or VHH fragment thereof), Ig NAR, Fab fragments, Fab' fragments, F(ab)'2 fragments, F(ab)'3 fragments, Fv, single chain Fv antibody (“scFv”), bis-scFv, (scFv)2, minibody, diabody, triabody, tetrabody, disulfide stabilized Fv protein (“dsFv”), and single-domain antibody 5 (sdAb, Nanobody) or other antibody fragments thereof. The term also includes genetically engineered forms such as chimeric antibodies (for example, humanized murine antibodies), heteroconjugate antibodies (such as, bispecific antibodies) and antigen binding fragments thereof. See also, Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, IL); Kuby, J., Immunology, 3rd Ed., W. H. Freeman & Co., New York, 1997. 10         In one preferred embodiment, the binding domain is an scFv. In another preferred embodiment, the binding domain is a camelid antibody. In particular embodiments, the CAR comprises an extracellular domain that binds an antigen selected from the group consisting of: alpha folate receptor, 5T4, av06 integrin, BCMA, B7-H3, B7-H6, CAIX, CD16, CD19, CD20, CD22, CD30, CD33, CD44, CD44v6, 15  CD44v7 / 8, CD70, CD79a, CD79b, CD123, CD138, CD171, CEA, CSPG4, EGFR, EGFR family including ErbB2 (HER2), EGFRvIII, EGP2, EGP40, EPCAM, EphA2, EpCAM, FAP, fetal AchR, FRa, GD2, GD3, Glypican-3 (GPC3), HLA-A1+MAGE1, HLA-A2+MAGE1, HLA-A3+MAGE1, HLA-A1+NY-ESO-1, HLA-A2+NY-ESO-1, HLA-A3+NY-ESO-1, IL-11Ra, IL-13Ra2, Lambda, Lewis-Y, Kappa, Mesothelin, Muc1, 20 Muc16, NCAM, NKG2D Ligands, NY-ESO-1, PRAME, PSCA, PSMA, ROR1, SSX, Survivin, TAG72, TEMs, VEGFR2, and WT-1. In particular embodiments, the CARs comprise an extracellular binding domain, e.g., antibody or antigen binding fragment thereof that binds an antigen, wherein the antigen is an MHC-peptide complex, such as a class I MHC-peptide complex or a class II 25 MHC-peptide complex. In certain embodiments, the CARs comprise linker residues between the various domains. A “variable region linking sequence,” is an amino acid sequence that connects a heavy chain variable region to a light chain variable region and provides a spacer function compatible with interaction of the two sub-binding domains so that the resulting 2024203604   29 May 2024 polypeptide retains a specific binding affinity to the same target molecule as an antibody that comprises the same light and heavy chain variable regions. In particular embodiments, CARs comprise one, two, three, four, or five or more linkers. In particular embodiments, the length of a linker is about 1 to about 25 amino acids, about 5 to about 20 amino acids, 5 or about 10 to about 20 amino acids, or any intervening length of amino acids. In some embodiments, the linker is 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, or more amino acids long. In particular embodiments, the binding domain of the CAR is followed by one or more “spacer domains,” which refers to the region that moves the antigen binding domain 10 away from the effector cell surface to enable proper cell / cell contact, antigen binding and activation (Patel et al., Gene Therapy, 1999; 6: 412-419). The spacer domain may be derived either from a natural, synthetic, semi-synthetic, or recombinant source. In certain embodiments, a spacer domain is a portion of an immunoglobulin, including, but not limited to, one or more heavy chain constant regions, e.g., CH2 and CH3. The spacer 15 domain can include the amino acid sequence of a naturally occurring immunoglobulin hinge region or an altered immunoglobulin hinge region. In one embodiment, the spacer domain comprises the CH2 and CH3 of IgG1, IgG4, or IgD. In one embodiment, the binding domain of the CAR is linked to one or more “hinge 20 domains,” which plays a role in positioning the antigen binding domain away from the effector cell surface to enable proper cell / cell contact, antigen binding and activation. A CAR generally comprises one or more hinge domains between the binding domain and the transmembrane domain (TM). The hinge domain may be derived either from a natural, synthetic, semi-synthetic, or recombinant source. The hinge domain can include the amino 25 acid sequence of a naturally occurring immunoglobulin hinge region or an altered immunoglobulin hinge region. Illustrative hinge domains suitable for use in the CARs described herein include the hinge region derived from the extracellular regions of type 1 membrane proteins such as 2024203604   29 May 2024 CD8a, and CD4, which may be wild-type hinge regions from these molecules or may be altered. In another embodiment, the hinge domain comprises a CD8a hinge region. In one embodiment, the hinge is a PD-1 hinge or CD152 hinge. The “transmembrane domain” is the portion of the CAR that fuses the extracellular 5 binding portion and intracellular signaling domain and anchors the CAR to the plasma membrane of the immune effector cell. The TM domain may be derived either from a natural, synthetic, semi-synthetic, or recombinant source. Illustrative TM domains may be derived from (i.e., comprise at least the transmembrane region(s) of the alpha or beta chain of the T-cell receptor, CD38, CD3s, 10 CD3y, CD3Z, CD4, CD5, CD8a, CD9, CD 16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD 134, CD137, CD152, CD154, AMN, and PD-1. In one embodiment, a CAR comprises a TM domain derived from CD8a. In another embodiment, a CAR contemplated herein comprises a TM domain derived from CD8a and a short oligo- or polypeptide linker, preferably between 1, 2, 3, 4, 5, 6, 7, 8, 9, or 15   10 amino acids in length that links the TM domain and the intracellular signaling domain of the CAR. A glycine-serine linker provides a particularly suitable linker. In particular embodiments, a CAR comprises an intracellular signaling domain. An “intracellular signaling domain,” refers to the part of a CAR that participates in transducing the message of effective CAR binding to a target antigen into the interior of the immune 20 effector cell to elicit effector cell function, e.g., activation, cytokine production, proliferation and cytotoxic activity, including the release of cytotoxic factors to the CARbound target cell, or other cellular responses elicited with antigen binding to the extracellular CAR domain. The term “effector function” refers to a specialized function of the cell. Effector 25   function of the T cell, for example, may be cytolytic activity or help or activity including the secretion of a cytokine. Thus, the term “intracellular signaling domain” refers to the portion of a protein which transduces the effector function signal and that directs the cell to perform a specialized function. While usually the entire intracellular signaling domain can be employed, in many cases it is not necessary to use the entire domain. To the extent that 2024203604   29 May 2024 a truncated portion of an intracellular signaling domain is used, such truncated portion may be used in place of the entire domain as long as it transduces the effector function signal. The term intracellular signaling domain is meant to include any truncated portion of the intracellular signaling domain sufficient to transducing effector function signal. 5          It is known that signals generated through the TCR alone are insufficient for full activation of the T cell and that a secondary or costimulatory signal is also required. Thus, T cell activation can be said to be mediated by two distinct classes of intracellular signaling domains: primary signaling domains that initiate antigen-dependent primary activation through the TCR (e.g., a TCR / CD3 complex) and costimulatory signaling domains that act 10 in an antigen-independent manner to provide a secondary or costimulatory signal. In preferred embodiments, a CAR comprises an intracellular signaling domain that comprises one or more “costimulatory signaling domains” and a “primary signaling domain.” Primary signaling domains regulate primary activation of the TCR complex either in a stimulatory way, or in an inhibitory way. Primary signaling domains that act in a 15 stimulatory manner may contain signaling motifs which are known as immunoreceptor tyrosine-based activation motifs or ITAMs. Illustrative examples of ITAM containing primary signaling domains suitable for use in CARs contemplated in particular embodiments include those derived from FcRy, FcRp, CD3y, CD38, CD3s, CD3Z, CD22, CD79a, CD79b, and CD66d. In particular 20 preferred embodiments, a CAR comprises a CD3Z primary signaling domain and one or more costimulatory signaling domains. The intracellular primary signaling and costimulatory signaling domains may be linked in any order in tandem to the carboxyl terminus of the transmembrane domain. In particular embodiments, a CAR comprises one or more costimulatory signaling 25 domains to enhance the efficacy and expansion of T cells expressing CAR receptors. As used herein, the term, “costimulatory signaling domain,” or “costimulatory domain”, refers to an intracellular signaling domain of a costimulatory molecule. Illustrative examples of such costimulatory molecules suitable for use in CARs contemplated in particular embodiments include TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, 2024203604   29 May 2024 TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD278 (ICOS), DAP10, LAT, NKD2C, SLP76, TRIM, and ZAP70. In one embodiment, a CAR comprises one or more costimulatory signaling domains selected from the group consisting of CD28, CD137, and 5  CD134, and a CD3Z primary signaling domain. In various embodiments, the CAR comprises: an extracellular domain that binds an antigen selected from the group consisting of: BCMA, CD19, CSPG4, PSCA, ROR1, and TAG72; a transmembrane domain isolated from a polypeptide selected from the group consisting of: CD4, CD8a, CD154, and PD-1; one or more intracellular costimulatory 10 signaling domains isolated from a polypeptide selected from the group consisting of: CD28, CD134, and CD137; and a signaling domain isolated from a polypeptide selected from the group consisting of: FcRy, FcR0, CD3y, CD38, CD3s, CD3Z, CD22, CD79a, CD79b, and CD66d. 3.    DARIC 15         In particular embodiments, immune effector cells comprise one or more chains of a DARIC receptor. As used herein, the term “DARIC receptor” refers to a multi-chain engineered antigen receptor. In particular embodiments, immune effector cells contemplated herein comprise one or more chains of a DARIC receptor and a CTBR signal convertor. In one 20 embodiment, T cells are engineered by introducing a polynucleotide or vector encoding one or more chains of a DARIC receptor and a CTBR signal convertor separated by one or more polypeptide cleavage signals. In one embodiment, T cells are engineered by introducing a polynucleotide or vector encoding one or more chains of a DARIC receptor and a polynucleotide or vector encoding a CTBR signal convertor. In one embodiment, T 25 cells are engineered to express one or more chains of a DARIC receptor are further engineered by introducing a polynucleotide or vector encoding a CTBR signal convertor. 2024203604   29 May 2024 Illustrative examples of DARIC architectures and components are disclosed in PCT Publication No. WO2015 / 017214 and U.S. Patent Publication No. 20150266973, each of which is incorporated here by reference in its entirety. In one embodiment, a donor repair template comprises the following DARIC 5 components: a signaling polypeptide comprising a first multimerization domain, a first transmembrane domain, and one or more intracellular co-stimulatory signaling domains and / or primary signaling domains; and a binding polypeptide comprising a binding domain, a second multimerization domain, and optionally a second transmembrane domain. A functional DARIC comprises a bridging factor that promotes the formation of a DARIC 10 receptor complex on the cell surface with the bridging factor associated with and disposed between the multimerization domains of the signaling polypeptide and the binding polypeptide. In particular embodiments, the first and second multimerization domains associate with a bridging factor selected from the group consisting of: rapamycin or a rapalog 15 thereof, coumermycin or a derivative thereof, gibberellin or a derivative thereof, abscisic acid (ABA) or a derivative thereof, methotrexate or a derivative thereof, cyclosporin A or a derivative thereof, FKCsA or a derivative thereof, trimethoprim (Tmp)-synthetic ligand for FKBP (SLF) or a derivative thereof, and any combination thereof. Illustrative examples of rapamycin analogs (rapalogs) include those disclosed in 20 U.S. Pat. No. 6,649,595, which rapalog structures are incorporated herein by reference in their entirety. In certain embodiments, a bridging factor is a rapalog with substantially reduced immunosuppressive effect as compared to rapamycin. A “substantially reduced immunosuppressive effect” refers to a rapalog having at least less than 0.1 to 0.005 times the immunosuppressive effect observed or expected for an equimolar amount of rapamycin, 25 as measured either clinically or in an appropriate in vitro (e.g., inhibition of T cell proliferation) or in vivo surrogate of human immunosuppressive activity. In one embodiment, “substantially reduced immunosuppressive effect” refers to a rapalog having an EC50 value in such an in vitro assay that is at least 10 to 250 times larger than the EC50 value observed for rapamycin in the same assay. 2024203604   29 May 2024 Other illustrative examples of rapalogs include, but are not limited to everolimus, novolimus, pimecrolimus, ridaforolimus, tacrolimus, temsirolimus, umirolimus, and zotarolimus. In certain embodiments, multimerization domains will associate with a bridging 5 factor being a rapamycin or rapalog thereof. For example, the first and second multimerization domains are a pair selected from FKBP and FRB. FRB domains are polypeptide regions (protein “domains”) that are capable of forming a tripartite complex with an FKBP protein and rapamycin or rapalog thereof. FRB domains are present in a number of naturally occurring proteins, including mTOR proteins (also referred to in the 10 literature as FRAP, RAPT1, or RAFT) from human and other species; yeast proteins including Tor1 and Tor2; and a Candida FRAP homolog. Information concerning the nucleotide sequences, cloning, and other aspects of these proteins is already known in the art. For example, a protein sequence accession number for a human mTOR is GenBank Accession No. L34075.1 (Brown et al., Nature 369:756, 1994). 15         FRB domains suitable for use in particular embodiments contemplated herein generally contain at least about 85 to about 100 amino acid residues. In certain embodiments, an FRB amino acid sequence for use in fusion proteins of this disclosure will comprise a 93 amino acid sequence Ile-2021 through Lys-2113 and a mutation of T2098L, based the amino acid sequence of GenBank Accession No. L34075.1. An FRB domain for 20 use in DARICs contemplated in particular embodiments will be capable of binding to a complex of an FKBP protein bound to rapamycin or a rapalog thereof. In certain embodiments, a peptide sequence of an FRB domain comprises (a) a naturally occurring peptide sequence spanning at least the indicated 93 amino acid region of human mTOR or corresponding regions of homologous proteins; (b) a variant of a naturally occurring FRB 25 in which up to about ten amino acids, or about 1 to about 5 amino acids or about 1 to about 3 amino acids, or in some embodiments just one amino acid, of the naturally-occurring peptide have been deleted, inserted, or substituted; or (c) a peptide encoded by a nucleic acid molecule capable of selectively hybridizing to a DNA molecule encoding a naturally occurring FRB domain or by a DNA sequence which would be capable, but for the 2024203604   29 May 2024 degeneracy of the genetic code, of selectively hybridizing to a DNA molecule encoding a naturally occurring FRB domain. FKBPs (FK506 binding proteins) are the cytosolic receptors for macrolides, such as FK506, FK520 and rapamycin, and are highly conserved across species lines. FKBPs are 5 proteins or protein domains that are capable of binding to rapamycin or to a rapalog thereof and further forming a tripartite complex with an FRB-containing protein or fusion protein. An FKBP domain may also be referred to as a “rapamycin binding domain.” Information concerning the nucleotide sequences, cloning, and other aspects of various FKBP species is known in the art (see, e.g., Staendart et al., Nature 346:671, 1990 (human FKBP12); Kay, 10 Biochem. J. 314:361, 1996). Homologous FKBP proteins in other mammalian species, in yeast, and in other organisms are also known in the art and may be used in the fusion proteins disclosed herein. An FKBP domain contemplated in particular embodiments will be capable of binding to rapamycin or a rapalog thereof and participating in a tripartite complex with an FRB-containing protein (as may be determined by any means, direct or 15 indirect, for detecting such binding). Illustrative examples of FKBP domains suitable for use in a DARIC contemplated in particular embodiments include, but are not limited to: a naturally occurring FKBP peptide sequence, preferably isolated from the human FKBP12 protein (GenBank Accession No. AAA58476.1) or a peptide sequence isolated therefrom, from another 20 human FKBP, from a murine or other mammalian FKBP, or from some other animal, yeast or fungal FKBP; a variant of a naturally occurring FKBP sequence in which up to about ten amino acids, or about 1 to about 5 amino acids or about 1 to about 3 amino acids, or in some embodiments just one amino acid, of the naturally-occurring peptide have been deleted, inserted, or substituted; or a peptide sequence encoded by a nucleic acid molecule 25 capable of selectively hybridizing to a DNA molecule encoding a naturally occurring FKBP or by a DNA sequence which would be capable, but for the degeneracy of the genetic code, of selectively hybridizing to a DNA molecule encoding a naturally occurring FKBP. 2024203604   29 May 2024 Other illustrative examples of multimerization domain pairs suitable for use in a DARIC contemplated in particular embodiments include, but are not limited to include from FKBP and FRB, FKBP and calcineurin, FKBP and cyclophilin, FKBP and bacterial DHFR, calcineurin and cyclophilin, PYL1 and ABI1, or GIB1 and GAI, or variants thereof. 5          In yet other embodiments, an anti-bridging factor blocks the association of a signaling polypeptide and a binding polypeptide with the bridging factor. For example, cyclosporin or FK506 could be used as anti-bridging factors to titrate out rapamycin and, therefore, stop signaling since only one multimerization domain is bound. In certain embodiments, an anti-bridging factor (e.g., cyclosporine, FK506) is an immunosuppressive 10 agent. For example, an immunosuppressive anti-bridging factor may be used to block or minimize the function of the DARIC components contemplated in particular embodiments and at the same time inhibit or block an unwanted or pathological inflammatory response in a clinical setting. In one embodiment, the first multimerization domain comprises FRB T2098L, the 15 second multimerization domain comprises FKBP12, and the bridging factor is rapalog AP21967. In another embodiment, the first multimerization domain comprises FRB, the second multimerization domain comprises FKBP12, and the bridging factor is Rapamycin, temsirolimus or everolimus. 20          In particular embodiments, a signaling polypeptide a first transmembrane domain and a binding polypeptide comprises a second transmembrane domain or GPI anchor. Illustrative examples of the first and second transmembrane domains are isolated from a polypeptide independently selected from the group consisting of: CD38, CD3s, CD3y, CD3Z, CD4, CD5, CD8a, CD9, CD 16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, 25  CD80, CD86, CD 134, CD137, CD152, CD154, AMN, and PD-1. In one embodiment, a signaling polypeptide comprises one or more intracellular costimulatory signaling domains and / or primary signaling domains. Illustrative examples of primary signaling domains suitable for use in DARIC signaling components contemplated in particular embodiments include those derived from 2024203604   29 May 2024 FcRY, FcRp, CD3y, CD38, CD3s, CD3Z, CD22, CD79a, CD79b, and CD66d. In particular preferred embodiments, a DARIC signaling component comprises a CD3Z primary signaling domain and one or more costimulatory signaling domains. The intracellular primary signaling and costimulatory signaling domains may be linked in any order in 5 tandem to the carboxyl terminus of the transmembrane domain. Illustrative examples of such costimulatory molecules suitable for use in DARIC signaling components contemplated in particular embodiments include TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD278 10 (ICOS), DAP10, LAT, NKD2C, SLP76, TRIM, and ZAP70. In one embodiment, a DARIC signaling component comprises one or more costimulatory signaling domains selected from the group consisting of CD28, CD137, and CD134, and a CD3Z primary signaling domain. In particular embodiments, a DARIC binding component comprises a binding 15 domain. In one embodiment, the binding domain is an antibody or antigen binding fragment thereof. The antibody or antigen binding fragment thereof comprises at least a light chain or heavy chain immunoglobulin variable region which specifically recognizes and binds an epitope of a target antigen, such as a peptide, lipid, polysaccharide, or nucleic acid 20 containing an antigenic determinant, such as those recognized by an immune cell. Antibodies include antigen binding fragments, e.g., Camel Ig (a camelid antibody or VHH fragment thereof), Ig NAR, Fab fragments, Fab' fragments, F(ab)'2 fragments, F(ab)'3 fragments, Fv, single chain Fv antibody (“scFv”), bis-scFv, (scFv)2, minibody, diabody, triabody, tetrabody, disulfide stabilized Fv protein (“dsFv”), and single-domain antibody 25 (sdAb, Nanobody) or other antibody fragments thereof. The term also includes genetically engineered forms such as chimeric antibodies (for example, humanized murine antibodies), heteroconjugate antibodies (such as, bispecific antibodies) and antigen binding fragments thereof. See also, Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, IL); Kuby, J., Immunology, 3rd Ed., W. H. Freeman & Co., New York, 1997. 2024203604   29 May 2024 In one preferred embodiment, the binding domain is an scFv. In another preferred embodiment, the binding domain is a camelid antibody. In particular embodiments, the DARIC binding component comprises an extracellular domain that binds an antigen selected from the group consisting of: alpha 5 folate receptor, 5T4, av06 integrin, BCMA, B7-H3, B7-H6, CAIX, CD16, CD19, CD20, CD22, CD30, CD33, CD44, CD44v6, CD44v7 / 8, CD70, CD79a, CD79b, CD123, CD138, CD171, CEA, CSPG4, EGFR, EGFR family including ErbB2 (HER2), EGFRvIII, EGP2, EGP40, EPCAM, EphA2, EpCAM, FAP, fetal AchR, FRa, GD2, GD3, Glypican-3 (GPC3), HLA-A1+MAGE1, HLA-A2+MAGE1, HLA-A3+MAGE1, HLA-A1+NY-ESO- 10  1, HLA-A2+NY-ESO-1, HLA-A3+NY-ESO-1, IL-11Ra, IL-13Ra2, Lambda, Lewis-Y, Kappa, Mesothelin, Muc1, Muc16, NCAM, NKG2D Ligands, NY-ESO-1, PRAME, PSCA, PSMA, ROR1, SSX, Survivin, TAG72, TEMs, VEGFR2, and WT-1. In one embodiment, the DARIC binding component comprises an extracellular domain, e.g., antibody or antigen binding fragment thereof that binds an MHC-peptide 15 complex, such as a class I MHC-peptide complex or class II MHC-peptide complex. In particular embodiments, the DARIC components contemplated herein comprise a linker or spacer that connects two proteins, polypeptides, peptides, domains, regions, or motifs. In certain embodiments, a linker comprises about two to about 35 amino acids, or about four to about 20 amino acids or about eight to about 15 amino acids or about 15 to 20 about 25 amino acids. In other embodiments, a spacer may have a particular structure, such as an antibody CH2CH3 domain, hinge domain or the like. In one embodiment, a spacer comprises the CH2 and CH3 domains of IgG1, IgG4, or IgD. In particular embodiments, the DARIC components contemplated herein comprise one or more “hinge domains,” which plays a role in positioning the domains to enable 25 proper cell / cell contact, antigen binding and activation. A DARIC may comprise one or more hinge domains between the binding domain and the multimerization domain and / or the transmembrane domain (TM) or between the multimerization domain and the transmembrane domain. The hinge domain may be derived either from a natural, synthetic, semi-synthetic, or recombinant source. The hinge domain can include the amino acid 2024203604   29 May 2024 sequence of a naturally occurring immunoglobulin hinge region or an altered immunoglobulin hinge region. In particular embodiment, the hinge is a CD8a hinge or a CD4 hinge. In one embodiment, a DARIC comprises a signaling polypeptide comprises a first 5 multimerization domain of FRB T2098L, a CD8 transmembrane domain, a 4-1BB costimulatory domain, and a CD3Z primary signaling domain; the binding polypeptide comprises an scFv that binds CD19, a second multimerization domain of FKBP12 and a CD4 transmembrane domain; and the bridging factor is rapalog AP21967. In one embodiment, a DARIC comprises a signaling polypeptide comprises a first 10 multimerization domain of FRB, a CD8 transmembrane domain, a 4-1BB costimulatory domain, and a CD3Z primary signaling domain; the binding polypeptide comprises an scFv that binds CD19, a second multimerization domain of FKBP12 and a CD4 transmembrane domain; and the bridging factor is Rapamycin, temsirolimus or everolimus. 4.    Zetakines 15         In various embodiments, immune effector cells comprise chimeric cytokine receptor that redirect cytotoxicity toward tumor cells. Zetakines are chimeric transmembrane immunoreceptors that comprise an extracellular domain comprising a soluble receptor ligand linked to a support region capable of tethering the extracellular domain to a cell surface, a transmembrane region and an intracellular signaling domain. 20 Zetakines, when expressed on the surface of T lymphocytes, direct T cell activity to those cells expressing a receptor for which the soluble receptor ligand is specific. Zetakine chimeric immunoreceptors redirect the antigen specificity of T cells, with application to treatment of a variety of cancers, particularly via the autocrine / paracrine cytokine systems utilized by human malignancy. 25         In particular embodiments, immune effector cells contemplated herein comprise one or more chains of a zetakine receptor and a CTBR signal convertor. In one embodiment, T cells are engineered by introducing a polynucleotide or vector encoding one or more chains of a zetakine receptor and a CTBR signal convertor separated by one or 2024203604   29 May 2024 more polypeptide cleavage signals. In one embodiment, T cells are engineered by introducing a polynucleotide or vector encoding one or more chains of a zetakine receptor and a polynucleotide or vector encoding a CTBR signal convertor. In one embodiment, T cells are engineered to express one or more chains of a zetakine receptor are further 5 engineered by introducing a polynucleotide or vector encoding a CTBR signal convertor. In particular embodiments, the zetakine comprises an immunosuppressive cytokine or cytokine receptor binding variant thereof, a linker, a transmembrane domain, and an intracellular signaling domain. In particular embodiments, the cytokine or cytokine receptor binding variant thereof 10 is selected from the group consisting of: interleukin-4 (IL-4), interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-10 (IL-10), and interleukin-13 (IL-13). In certain embodiments, the linker comprises a CH2CH3 domain, hinge domain, or the like. In one embodiment, a linker comprises the CH2 and CH3 domains of IgG1, IgG4, or IgD. In one embodiment, a linker comprises a CD8a or CD4 hinge domain. 15         In particular embodiments, the transmembrane domain is selected from the group consisting of: the alpha or beta chain of the T-cell receptor, CD38, CD3s, CD3y, CD3Z, CD4, CD5, CD8a, CD9, CD 16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD 134, CD137, CD152, CD154, AMN, and PD-1. In particular embodiments, the intracellular signaling domain is selected from the 20 group consisting of: an ITAM containing primary signaling domain and / or a costimulatory domain. In particular embodiments, the intracellular signaling domain is selected from the group consisting of: FcRy, FcR0, CD3y, CD38, CD3s, CD3Z, CD22, CD79a, CD79b, and CD66d. 25          In particular embodiments, the intracellular signaling domain is selected from the group consisting of: TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD278 (ICOS), DAP10, LAT, NKD2C, SLP76, TRIM, and ZAP70. 2024203604   29 May 2024 In one embodiment, a chimeric cytokine receptor comprises one or more costimulatory signaling domains selected from the group consisting of CD28, CD137, and CD134, and a CD3Z primary signaling domain. E.    Polypeptides 5          Various polypeptides are contemplated herein, including, but not limited to, TGF0 signal convertor polypeptides, CTBRs, engineered TCRs, CARs, DARICs, zetakines, fusion proteins comprising the foregoing polypeptides and fragments thereof. In preferred embodiments, a polypeptide comprises an amino acid sequence set forth in any one of SEQ ID NOs: 1-71. “Polypeptide,” “peptide” and “protein” are used interchangeably, unless 10 specified to the contrary, and according to conventional meaning, i.e., as a sequence of amino acids. In one embodiment, a “polypeptide” includes fusion polypeptides and other variants. Polypeptides can be prepared using any of a variety of well-known recombinant and / or synthetic techniques. Polypeptides are not limited to a specific length, e.g., they may comprise a full-length protein sequence, a fragment of a full length protein, or a fusion 15 protein, and may include post-translational modifications of the polypeptide, for example, glycosylations, acetylations, phosphorylations and the like, as well as other modifications known in the art, both naturally occurring and non-naturally occurring. An “isolated peptide” or an “isolated polypeptide” and the like, as used herein, refer to in vitro isolation and / or purification of a peptide or polypeptide molecule from a cellular 20 environment, and from association with other components of the cell, i.e., it is not significantly associated with in vivo substances. Polypeptides include “polypeptide variants.” Polypeptide variants may differ from a naturally occurring polypeptide in one or more substitutions, deletions, additions and / or insertions. Such variants may be naturally occurring or may be synthetically generated, for 25 example, by modifying one or more of the above polypeptide sequences. For example, in particular embodiments, it may be desirable to improve the binding affinity and / or other biological properties of a polypeptide by introducing one or more substitutions, deletions, additions and / or insertions the polypeptide. In particular embodiments, polypeptides include polypeptides having at least about 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 2024203604   29 May 2024 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 86%, 97%, 98%, or 99% amino acid identity to any of the reference sequences contemplated herein, typically where the variant maintains at least one biological activity of the reference sequence. 5           Polypeptides variants include biologically active “polypeptide fragments.” Illustrative examples of biologically active polypeptide fragments include DNA binding domains, nuclease domains, and the like. As used herein, the term “biologically active fragment” or “minimal biologically active fragment” refers to a polypeptide fragment that retains at least 100%, at least 90%, at least 80%, at least 70%, at least 60%, at least 50%, at least 40%, at least 10   30%, at least 20%, at least 10%, or at least 5% of the naturally occurring polypeptide activity. In certain embodiments, a polypeptide fragment can comprise an amino acid chain at least 5 to about 1700 amino acids long. It will be appreciated that in certain embodiments, fragments are at least 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, 55, 60, 65, 70, 15   75, 80, 85, 90, 95, 100, 110, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700 or more amino acids long. In particular embodiments, the polypeptides set forth herein may comprise one or more amino acids denoted as “X.” “X” if present in an amino acid SEQ ID NO, refers to any one or more amino acids. In particular embodiments, SEQ ID NOs denoting a fusion protein 20 comprise a sequence of continuous X residues that cumulatively represent any amino acid sequence. As noted above, polypeptides may be altered in various ways including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are generally known in the art. For example, amino acid sequence variants of a reference 25 polypeptide can be prepared by mutations in the DNA. Methods for mutagenesis and nucleotide sequence alterations are well known in the art. See, for example, Kunkel (1985, Proc. Natl. Acad. Sci. USA. 82: 488-492), Kunkel et al., (1987, Methods in Enzymol, 154: 367382), U.S. Pat. No. 4,873,192, Watson, J. D. et al., (Molecular Biology of the Gene, Fourth Edition, Benjamin / Cummings, Menlo Park, Calif., 1987) and the references cited therein. 30 Guidance as to appropriate amino acid substitutions that do not affect biological activity of the 2024203604   29 May 2024 protein of interest may be found in the model of Dayhoff et al., (1978) Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, D.C.). In certain embodiments, a polypeptide variant comprises one or more conservative substitutions. A “conservative substitution” is one in which an amino acid is substituted 5 for another amino acid that has similar properties, such that one skilled in the art of peptide chemistry would expect the secondary structure and hydropathic nature of the polypeptide to be substantially unchanged. Modifications may be made in the structure of the polynucleotides and polypeptides contemplated in particular embodiments and still obtain a functional molecule that encodes a variant or derivative polypeptide with desirable 10 characteristics. When it is desired to alter the amino acid sequence of a polypeptide to create an equivalent, or even an improved, variant polypeptide, one skilled in the art, for example, can change one or more of the codons of the encoding DNA sequence, e.g., according to Table 1. TABLE 1- Amino Acid Codons Amino Acids One letter code Three letter code Codons Alanine A Ala GCA GCC GCG GCU Cysteine C Cys UGC UGU Aspartic acid D Asp GAC GAU Glutamic acid E Glu GAA GAG Phenylalanine F Phe UUC UUU Glycine G Gly GGA GGC GGG GGU Histidine H His CAC CAU Isoleucine I Iso AUA AUC AUU Lysine K Lys AAA AAG Leucine L Leu UUA UUG CUA CUC CUG CUU Methionine M Met AUG Asparagine N Asn AAC AAU Proline P Pro CCA CCC CCG CCU Glutamine Q Gln CAA CAG 2024203604   29 May 2024 Arginine R Arg AGA AGG CGA CGC CGG CGU Serine S Ser AGC AGU UCA UCC UCG UCU Threonine T Thr ACA ACC ACG ACU Valine V Val GUA GUC GUG GUU Tryptophan W Trp UGG Tyrosine Y Tyr UAC UAU Guidance in determining which amino acid residues can be substituted, inserted, or deleted without abolishing biological activity can be found using computer programs well known in the art, such as DNASTAR, DNA Strider, Geneious, Mac Vector, or Vector NTI software. Preferably, amino acid changes in the protein variants disclosed herein are 5 conservative amino acid changes, i.e., substitutions of similarly charged or uncharged amino acids. A conservative amino acid change involves substitution of one of a family of amino acids which are related in their side chains. Naturally occurring amino acids are generally divided into four families: acidic (aspartate, glutamate), basic (lysine, arginine, histidine), non-polar (alanine, valine, leucine, isoleucine, proline, phenylalanine, 10 methionine, tryptophan), and uncharged polar (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine) amino acids. Phenylalanine, tryptophan, and tyrosine are sometimes classified jointly as aromatic amino acids. In a peptide or protein, suitable conservative substitutions of amino acids are known to those of skill in this art and generally can be made without altering a biological activity of a resulting molecule. Those 15 of skill in this art recognize that, in general, single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, e.g., Watson et al. Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin / Cummings Pub. Co., p.224). In one embodiment, where expression of two or more polypeptides is desired, the 20 polynucleotide sequences encoding them can be separated by an IRES sequence as disclosed elsewhere herein. Polypeptides contemplated in particular embodiments include fusion polypeptides. In particular embodiments, fusion polypeptides and polynucleotides encoding fusion polypeptides are provided. Fusion polypeptides and fusion proteins refer to a polypeptide 25 having at least two, three, four, five, six, seven, eight, nine, or ten polypeptide segments. 2024203604   29 May 2024 In another embodiment, two or more polypeptides can be expressed as a fusion protein that comprises one or more self-cleaving polypeptide sequences as disclosed elsewhere herein. Fusion polypeptides can comprise one or more polypeptide domains or segments 5 including, but are not limited to signal peptides, cell permeable peptide domains (CPP), DNA binding domains, nuclease domains, etc., epitope tags (e.g., maltose binding protein (“MBP”), glutathione S transferase (GST), HIS6, MYC, FLAG, V5, VSV-G, and HA), polypeptide linkers, and polypeptide cleavage signals. Fusion polypeptides are typically linked C-terminus to N-terminus, although they can also be linked C-terminus to C-terminus, N-terminus to N- 10 terminus, or N-terminus to C-terminus. In particular embodiments, the polypeptides of the fusion protein can be in any order. Fusion polypeptides or fusion proteins can also include conservatively modified variants, polymorphic variants, alleles, mutants, subsequences, and interspecies homologs, so long as the desired activity of the fusion polypeptide is preserved. Fusion polypeptides may be produced by chemical synthetic methods or by chemical linkage 15 between the two moieties or may generally be prepared using other standard techniques. Ligated DNA sequences comprising the fusion polypeptide are operably linked to suitable transcriptional or translational control elements as disclosed elsewhere herein. Fusion polypeptides may optionally comprise a linker that can be used to link the one or more polypeptides or domains within a polypeptide. A peptide linker sequence may be 20 employed to separate any two or more polypeptide components by a distance sufficient to ensure that each polypeptide folds into its appropriate secondary and tertiary structures so as to allow the polypeptide domains to exert their desired functions. Such a peptide linker sequence is incorporated into the fusion polypeptide using standard techniques in the art. Suitable peptide linker sequences may be chosen based on the following factors: (1) their ability to 25 adopt a flexible extended conformation; (2) their inability to adopt a secondary structure that could interact with functional epitopes on the first and second polypeptides; and (3) the lack of hydrophobic or charged residues that might react with the polypeptide functional epitopes. Preferred peptide linker sequences contain Gly, Asn and Ser residues. Other near neutral amino acids, such as Thr and Ala may also be used in the linker sequence. Amino acid 30 sequences which may be usefully employed as linkers include those disclosed in Maratea et al., 2024203604   29 May 2024 Gene 40:39-46, 1985; Murphy et al., Proc. Natl. Acad. Sci. USA 83:8258-8262, 1986; U.S. Patent No. 4,935,233 and U.S. Patent No. 4,751,180. Linker sequences are not required when a particular fusion polypeptide segment contains non-essential N-terminal amino acid regions that can be used to separate the functional domains and prevent steric interference. Preferred 5 linkers are typically flexible amino acid subsequences which are synthesized as part of a recombinant fusion protein. Linker polypeptides can be between 1 and 200 amino acids in length, between 1 and 100 amino acids in length, or between 1 and 50 amino acids in length, including all integer values in between. Exemplary polypeptide cleavage signals include polypeptide cleavage recognition sites 10 such as protease cleavage sites, nuclease cleavage sites (e.g., rare restriction enzyme recognition sites, self-cleaving ribozyme recognition sites), and self-cleaving viral oligopeptides (see deFelipe and Ryan, 2004. Traffic, 5(8); 616-26). Suitable protease cleavages sites and self-cleaving peptides are known to the skilled person (see, e.g., in Ryan et al., 1997. J. Gener. Virol. 78, 699-722; Scymczak et al. (2004) 15 Nature Biotech. 5, 589-594). Exemplary protease cleavage sites include, but are not limited to the cleavage sites of potyvirus NIa proteases (e.g., tobacco etch virus protease), potyvirus HC proteases, potyvirus P1 (P35) proteases, byovirus NIa proteases, byovirus RNA-2-encoded proteases, aphthovirus L proteases, enterovirus 2A proteases, rhinovirus 2A proteases, picorna 3C proteases, comovirus 24K proteases, nepovirus 24K proteases, RTSV (rice tungro spherical 20 virus) 3C-like protease, PYVF (parsnip yellow fleck virus) 3C-like protease, heparin, thrombin, factor Xa and enterokinase. Due to its high cleavage stringency, TEV (tobacco etch virus) protease cleavage sites are preferred in one embodiment, e.g., EXXYXQ(G / S) (SEQ ID NO: 47), for example, ENLYFQG (SEQ ID NO: 48) and ENLYFQS (SEQ ID NO: 49), wherein X represents any amino acid (cleavage by TEV occurs between Q and G or Q and S). 25          In certain embodiments, the self-cleaving polypeptide site comprises a 2A or 2A-like site, sequence or domain (Donnelly et al., 2001. J. Gen. Virol. 82:1027-1041). In a particular embodiment, the viral 2A peptide is an aphthovirus 2A peptide, a potyvirus 2A peptide, or a cardiovirus 2A peptide. In one embodiment, the viral 2A peptide is selected from the group consisting of: a 30 foot-and-mouth disease virus (FMDV) (F2A) peptide, an equine rhinitis A virus (ERAV) 2024203604   29 May 2024 (E2A) peptide, a Thosea asigna virus (TaV) (T2A) peptide, a porcine teschovirus-1 (PTV-1) (P2A) peptide, a Theilovirus 2A peptide, and an encephalomyocarditis virus 2A peptide. Illustrative examples of 2A sites are provided in Table 2. TABLE 2: SEQ ID NO: 50 GSGATNFSLLKQAGDVEENPGP SEQ ID NO: 51 ATNFSLLKQAGDVEENPGP SEQ ID NO: 52 LLKQAGDVEENPGP SEQ ID NO: 53 GSGEGRGSLLTCGDVEENPGP SEQ ID NO: 54 EGRGSLLTCGDVEENPGP SEQ ID NO: 55 LLTCGDVEENPGP SEQ ID NO: 56 GSGQCTNYALLKLAGDVESNPGP SEQ ID NO: 57 QCTNYALLKLAGDVESNPGP SEQ ID NO: 58 LLKLAGDVESNPGP SEQ ID NO: 59 GSGVKQTLNFDLLKLAGDVESNPGP SEQ ID NO: 60 VKQTLNFDLLKLAGDVESNPGP SEQ ID NO: 61 LLKLAGDVESNPGP SEQ ID NO: 62 LLNFDLLKLAGDVESNPGP SEQ ID NO: 63 TLNFDLLKLAGDVESNPGP SEQ ID NO: 64 LLKLAGDVESNPGP SEQ ID NO: 65 NFDLLKLAGDVESNPGP SEQ ID NO: 66 QLLNFDLLKLAGDVESNPGP SEQ ID NO: 67 APVKQTLNFDLLKLAGDVESNPGP SEQ ID NO: 68 VTELLYRMKRAETYCPRPLLAIHPTEARHKQKIVAPVKQT SEQ ID NO: 69 LNFDLLKLAGDVESNPGP SEQ ID NO: 70 LLAIHPTEARHKQKIVAPVKQTLNFDLLKLAGDVESNPGP SEQ ID NO: 71 EARHKQKIVAPVKQTLNFDLLKLAGDVESNPGP 5         In preferred embodiments, a polypeptide comprises a CTBR signal convertor polypeptide. 2024203604   29 May 2024 F.    Polynucleotides In particular embodiments, polynucleotides encoding TGF0 signal convertor polypeptides, CTBRs, engineered TCRs, CARs, DARICs, zetakines, fusion proteins comprising the foregoing polypeptides and fragments thereof are provided. As used herein, 5 the terms “polynucleotide” or “nucleic acid” refer to deoxyribonucleic acid (DNA), ribonucleic acid (RNA) and DNA / RNA hybrids. Polynucleotides may be single-stranded or doublestranded and either recombinant, synthetic, or isolated. Polynucleotides include, but are not limited to: pre-messenger RNA (pre-mRNA), messenger RNA (mRNA), RNA, short interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), ribozymes, 10 genomic RNA (gRNA), plus strand RNA (RNA(+)), minus strand RNA (RNA(-)), tracrRNA, crRNA, single guide RNA (sgRNA), synthetic RNA, synthetic mRNA, genomic DNA (gDNA), PCR amplified DNA, complementary DNA (cDNA), synthetic DNA, or recombinant DNA. Polynucleotides refer to a polymeric form of nucleotides of at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 100, at least 200, at least 15   300, at least 400, at least 500, at least 1000, at least 5000, at least 10000, or at least 15000 or more nucleotides in length, either ribonucleotides or deoxyribonucleotides or a modified form of either type of nucleotide, as well as all intermediate lengths. It will be readily understood that “intermediate lengths,” in this context, means any length between the quoted values, such as 6, 7, 8, 9, etc., 101, 102, 103, etc.; 151, 152, 153, etc.; 201, 202, 203, etc. In particular 20 embodiments, polynucleotides or variants have at least or about 50%, 55%, 60%, 65%, 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% sequence identity to a reference sequence. In particular embodiments, polynucleotides may be codon-optimized. As used herein, 25 the term “codon-optimized” refers to substituting codons in a polynucleotide encoding a polypeptide in order to increase the expression, stability and / or activity of the polypeptide. Factors that influence codon optimization include, but are not limited to one or more of: (i) variation of codon biases between two or more organisms or genes or synthetically constructed bias tables, (ii) variation in the degree of codon bias within an organism, gene, or set of genes, 30 (iii) systematic variation of codons including context, (iv) variation of codons according to 2024203604   29 May 2024 their decoding tRNAs, (v) variation of codons according to GC %, either overall or in one position of the triplet, (vi) variation in degree of similarity to a reference sequence for example a naturally occurring sequence, (vii) variation in the codon frequency cutoff, (viii) structural properties of mRNAs transcribed from the DNA sequence, (ix) prior knowledge about the 5 function of the DNA sequences upon which design of the codon substitution set is to be based, (x) systematic variation of codon sets for each amino acid, and / or (xi) isolated removal of spurious translation initiation sites. As used herein the term “nucleotide” refers to a heterocyclic nitrogenous base in N-glycosidic linkage with a phosphorylated sugar. Nucleotides are understood to include natural 10 bases, and a wide variety of art-recognized modified bases. Such bases are generally located at the 1 ' position of a nucleotide sugar moiety. Nucleotides generally comprise a base, sugar and a phosphate group. In ribonucleic acid (RNA), the sugar is a ribose, and in deoxyribonucleic acid (DNA) the sugar is a deoxyribose, i.e., a sugar lacking a hydroxyl group that is present in ribose. Exemplary natural nitrogenous bases include the purines, adenosine (A) and guanidine 15   (G), and the pyrimidines, cytidine (C) and thymidine (T) (or in the context of RNA, uracil (U)). The C-1 atom of deoxyribose is bonded to N-1 of a pyrimidine or N-9 of a purine. Nucleotides are usually mono, di- or triphosphates. The nucleotides can be unmodified or modified at the sugar, phosphate and / or base moiety, (also referred to interchangeably as nucleotide analogs, nucleotide derivatives, modified nucleotides, non-natural nucleotides, and non-standard 20 nucleotides; see for example, WO 92 / 07065 and WO 93 / 15187). Examples of modified nucleic acid bases are summarized by Limbach et al., (1994, Nucleic Acids Res. 22, 21832196). A nucleotide may also be regarded as a phosphate ester of a nucleoside, with esterification occurring on the hydroxyl group attached to C-5 of the sugar. As used herein, the 25 term “nucleoside” refers to a heterocyclic nitrogenous base in N-glycosidic linkage with a sugar. Nucleosides are recognized in the art to include natural bases, and also to include well known modified bases. Such bases are generally located at the 1 ' position of a nucleoside sugar moiety. Nucleosides generally comprise a base and sugar group. The nucleosides can be unmodified or modified at the sugar, and / or base moiety, (also referred to interchangeably as 30 nucleoside analogs, nucleoside derivatives, modified nucleosides, non-natural nucleosides, or 2024203604   29 May 2024 non-standard nucleosides). As also noted above, examples of modified nucleic acid bases are summarized by Limbach et al., (1994, Nucleic Acids Res. 22, 2183-2196). Illustrative examples of polynucleotides include, but are not limited to polynucleotides encoding SEQ ID NOs: 1-71. 5          In various illustrative embodiments, polynucleotides contemplated herein include, but are not limited to polynucleotides encoding TGF0 signal convertors, CTBR signal convertors, engineered antigen receptors, fusion polypeptides, and expression vectors, viral vectors, and transfer plasmids comprising polynucleotides contemplated herein. As used herein, the terms “polynucleotide variant” and “variant” and the like refer 10 to polynucleotides displaying substantial sequence identity with a reference polynucleotide sequence or polynucleotides that hybridize with a reference sequence under stringent conditions that are defined hereinafter. These terms also encompass polynucleotides that are distinguished from a reference polynucleotide by the addition, deletion, substitution, or modification of at least one nucleotide. Accordingly, the terms “polynucleotide variant” 15 and “variant” include polynucleotides in which one or more nucleotides have been added or deleted, or modified, or replaced with different nucleotides. In this regard, it is well understood in the art that certain alterations inclusive of mutations, additions, deletions and substitutions can be made to a reference polynucleotide whereby the altered polynucleotide retains the biological function or activity of the reference polynucleotide. 20         In one embodiment, a polynucleotide comprises a nucleotide sequence that hybridizes to a target nucleic acid sequence under stringent conditions. To hybridize under “stringent conditions” describes hybridization protocols in which nucleotide sequences at least 60% identical to each other remain hybridized. Generally, stringent conditions are selected to be about 5°C lower than the thermal melting point (Tm) for the specific 25 sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to the target sequence hybridize to the target sequence at equilibrium. Since the target sequences are generally present at excess, at Tm, 50% of the probes are occupied at equilibrium. 2024203604   29 May 2024 The recitations “sequence identity” or, for example, comprising a “sequence 50% identical to,” as used herein, refer to the extent that sequences are identical on a nucleotide-by-nucleotide basis or an amino acid-by-amino acid basis over a window of comparison. Thus, a “percentage of sequence identity” may be calculated by comparing two optimally 5 aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, I) or the identical amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window 10 of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. Included are nucleotides and polypeptides having at least about 50%, 55%, 60%, 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%, 86%, 97%, 98%, or 99% sequence identity to any of the 15 reference sequences described herein, typically where the polypeptide variant maintains at least one biological activity of the reference polypeptide. Terms used to describe sequence relationships between two or more polynucleotides or polypeptides include “reference sequence,” “comparison window,” “sequence identity,” “percentage of sequence identity,” and “substantial identity”. A 20 “reference sequence” is at least 12 but frequently 15 to 18 and often at least 25 monomer units, inclusive of nucleotides and amino acid residues, in length. Because two polynucleotides may each comprise (1) a sequence (i.e., only a portion of the complete polynucleotide sequence) that is similar between the two polynucleotides, and (2) a sequence that is divergent between the two polynucleotides, sequence comparisons 25 between two (or more) polynucleotides are typically performed by comparing sequences of the two polynucleotides over a “comparison window” to identify and compare local regions of sequence similarity. A “comparison window” refers to a conceptual segment of at least 6 contiguous positions, usually about 50 to about 100, more usually about 100 to about 150 in which a sequence is compared to a reference sequence of the same number of contiguous 30 positions after the two sequences are optimally aligned. The comparison window may 2024203604   29 May 2024 comprise additions or deletions (i.e., gaps) of about 20% or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. Optimal alignment of sequences for aligning a comparison window may be conducted by computerized implementations of algorithms (GAP, BESTFIT, 5 FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, WI, USA) or by inspection and the best alignment (i.e., resulting in the highest percentage homology over the comparison window) generated by any of the various methods selected. Reference also may be made to the BLAST family of programs as for example disclosed by Altschul et al., 1997, Nucl. Acids 10  Res. 25:3389. A detailed discussion of sequence analysis can be found in Unit 19.3 of Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons Inc, 19941998, Chapter 15. As used herein, “isolated polynucleotide” refers to a polynucleotide that has been purified from the sequences which flank it in a naturally-occurring state, e.g., a DNA 15 fragment that has been removed from the sequences that are normally adjacent to the fragment. An “isolated polynucleotide” also refers to a complementary DNA (cDNA), a recombinant DNA, or other polynucleotide that does not exist in nature and that has been made by the hand of man. In various embodiments, a polynucleotide comprises an mRNA encoding a 20 polypeptide contemplated herein. In certain embodiments, the mRNA comprises a cap, one or more nucleotides, and a poly(A) tail. Terms that describe the orientation of polynucleotides include: 5' (normally the end of the polynucleotide having a free phosphate group) and 3' (normally the end of the polynucleotide having a free hydroxyl (OH) group). Polynucleotide sequences can be 25 annotated in the 5' to 3' orientation or the 3' to 5' orientation. For DNA and mRNA, the 5' to 3' strand is designated the “sense,” “plus,” or “coding” strand because its sequence is identical to the sequence of the premessenger (premRNA) [except for uracil (U) in RNA, instead of thymine (T) in DNA]. For DNA and mRNA, the complementary 3' to 5' strand which is the strand transcribed by the RNA polymerase is designated as “template,” 30 “antisense,” “minus,” or “non-coding” strand. As used herein, the term “reverse 2024203604   29 May 2024 orientation” refers to a 5' to 3' sequence written in the 3' to 5' orientation or a 3' to 5' sequence written in the 5' to 3' orientation. The terms “complementary” and “complementarity” refer to polynucleotides (i.e., a sequence of nucleotides) related by the base-pairing rules. For example, the 5 complementary strand of the DNA sequence 5' A G T C A T G 3' is 3' T C A G T A C 5'. The latter sequence is often written as the reverse complement with the 5' end on the left and the 3' end on the right, 5' C A T G A C T 3'. A sequence that is equal to its reverse complement is said to be a palindromic sequence. Complementarity can be “partial,” in which only some of the nucleic acids’ bases are matched according to the base pairing 10 rules. Or, there can be “complete” or “total” complementarity between the nucleic acids. Moreover, it will be appreciated by those of ordinary skill in the art that, as a result of the degeneracy of the genetic code, there are many nucleotide sequences that encode a polypeptide, or fragment of variant thereof, as described herein. Some of these polynucleotides bear minimal homology to the nucleotide sequence of any native gene. 15 Nonetheless, polynucleotides that vary due to differences in codon usage are specifically contemplated in particular embodiments, for example polynucleotides that are optimized for human and / or primate codon selection. In particular embodiments, the polynucleotides are codon optimized for expression and / or stability. Further, alleles of the genes comprising the polynucleotide sequences provided herein may also be used. Alleles are 20 endogenous genes that are altered as a result of one or more mutations, such as deletions, additions and / or substitutions of nucleotides. The term “nucleic acid cassette” or “expression cassette” as used herein refers to genetic sequences within the vector which can express an RNA, and subsequently a polypeptide. In one embodiment, the nucleic acid cassette contains a gene(s)-of-interest, e.g., a 25   polynucleotide(s)-of-interest. In another embodiment, the nucleic acid cassette contains one or more expression control sequences, e.g., a promoter, enhancer, poly(A) sequence, and a gene(s)-of-interest, e.g., a polynucleotide(s)-of-interest. Vectors may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more nucleic acid cassettes. The nucleic acid cassette is positionally and sequentially oriented within the vector such that the nucleic acid in the cassette can be 30 transcribed into RNA, and when necessary, translated into a protein or a polypeptide, undergo 2024203604   29 May 2024 appropriate post-translational modifications required for activity in the transformed cell, and be translocated to the appropriate compartment for biological activity by targeting to appropriate intracellular compartments or secretion into extracellular compartments. Preferably, the cassette has its 3 ' and 5 ' ends adapted for ready insertion into a vector, e.g., it has restriction 5 endonuclease sites at each end. In a preferred embodiment, the nucleic acid cassette contains the sequence of a therapeutic gene used to treat, prevent, or ameliorate a genetic disorder. The cassette can be removed and inserted into a plasmid or viral vector as a single unit. Polynucleotides include polynucleotide(s)-of-interest. As used herein, the term “polynucleotide-of-interest” refers to a polynucleotide encoding a polypeptide or fusion 10 polypeptide or a polynucleotide that serves as a template for the transcription of an inhibitory polynucleotide, as contemplated herein. The polynucleotides contemplated herein, regardless of the length of the coding sequence itself, may be combined with other DNA sequences, such as promoters and / or enhancers, untranslated regions (UTRs), signal sequences, Kozak sequences, 15 polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, internal ribosomal entry sites (IRES), recombinase recognition sites (e.g., LoxP, FRT, and Att sites), termination codons, transcriptional termination signals, and polynucleotides encoding self-cleaving polypeptides, epitope tags, as disclosed elsewhere herein or as known in the art, such that their overall length may vary considerably. It is therefore 20 contemplated that a polynucleotide 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 DNA protocol. Polynucleotides can be prepared, manipulated, expressed and / or delivered using any of a variety of well-established techniques known and available in the art. In order to express a 25 desired polypeptide, a nucleotide sequence encoding the polypeptide, can be inserted into appropriate vector. Illustrative examples of vectors include, but are not limited to plasmid, autonomously replicating sequences, and transposable elements, e.g., Sleeping Beauty, PiggyBac. Additional Illustrative examples of vectors include, without limitation, plasmids, 30 phagemids, cosmids, artificial chromosomes such as yeast artificial chromosome (YAC), 2024203604   29 May 2024 bacterial artificial chromosome (BAC), or P1-derived artificial chromosome (PAC), bacteriophages such as lambda phage or M13 phage, and animal viruses. Illustrative examples of viruses useful as vectors include, without limitation, retrovirus (including lentivirus), adenovirus, adeno-associated virus, herpesvirus (e.g., herpes simplex 5 virus), poxvirus, baculovirus, papillomavirus, and papovavirus (e.g., SV40). Illustrative examples of expression vectors include, but are not limited to pClneo vectors (Promega) for expression in mammalian cells; pLenti4 / V5-DEST™, pLenti6 / V5-DEST™, and pLenti6.2 / V5-GW / lacZ (Invitrogen) for lentivirus-mediated gene transfer and expression in mammalian cells. In particular embodiments, coding sequences of polypeptides 10 disclosed herein can be ligated into such expression vectors for the expression of the polypeptides in mammalian cells. In particular embodiments, the vector is an episomal vector or a vector that is maintained extrachromosomally. As used herein, the term “episomal” refers to a vector that is able to replicate without integration into host’s chromosomal DNA and without gradual loss 15 from a dividing host cell also meaning that said vector replicates extrachromosomally or episomally. “Expression control sequences,” “control elements,” or “regulatory sequences” present in an expression vector are those non-translated regions of the vector—origin of replication, selection cassettes, promoters, enhancers, translation initiation signals (Shine Dalgarno 20 sequence or Kozak sequence) introns, a polyadenylation sequence, 5' and 3' untranslated regions—which interact with host cellular proteins to carry out transcription and translation. Such elements may vary in their strength and specificity. Depending on the vector system and host utilized, any number of suitable transcription and translation elements, including ubiquitous promoters and inducible promoters may be used. 25          In particular embodiments, a polynucleotide comprises a vector, including but not limited to expression vectors and viral vectors. A vector may comprise one or more exogenous, endogenous, or heterologous control sequences such as promoters and / or enhancers. An “endogenous control sequence” is one which is naturally linked with a given gene in the genome. An “exogenous control sequence” is one which is placed in juxtaposition 30 to a gene by means of genetic manipulation (i.e., molecular biological techniques) such that 2024203604   29 May 2024 transcription of that gene is directed by the linked enhancer / promoter. A “heterologous control sequence” is an exogenous sequence that is from a different species than the cell being genetically manipulated. A “synthetic” control sequence may comprise elements of one more endogenous and / or exogenous sequences, and / or sequences determined in vitro or in silico that 5 provide optimal promoter and / or enhancer activity for the particular therapy. The term “promoter” as used herein refers to a recognition site of a polynucleotide (DNA or RNA) to which an RNA polymerase binds. An RNA polymerase initiates and transcribes polynucleotides operably linked to the promoter. In particular embodiments, promoters operative in mammalian cells comprise an AT-rich region located approximately 10  25 to 30 bases upstream from the site where transcription is initiated and / or another sequence found 70 to 80 bases upstream from the start of transcription, a CNCAAT region where N may be any nucleotide. The term “enhancer” refers to a segment of DNA which contains sequences capable of providing enhanced transcription and in some instances can function independent of 15 their orientation relative to another control sequence. An enhancer can function cooperatively or additively with promoters and / or other enhancer elements. The term “promoter / enhancer” refers to a segment of DNA which contains sequences capable of providing both promoter and enhancer functions. The term “operably linked”, refers to a juxtaposition wherein the components 20 described are in a relationship permitting them to function in their intended manner. In one embodiment, the term refers to a functional linkage between a nucleic acid expression control sequence (such as a promoter, and / or enhancer) and a second polynucleotide sequence, e.g., a polynucleotide-of-interest, wherein the expression control sequence directs transcription of the nucleic acid corresponding to the second sequence. 25          As used herein, the term “constitutive expression control sequence” refers to a promoter, enhancer, or promoter / enhancer that continually or continuously allows for transcription of an operably linked sequence. A constitutive expression control sequence may be a “ubiquitous” promoter, enhancer, or promoter / enhancer that allows expression in a wide variety of cell and tissue types or a “cell specific,” “cell type specific,” “cell lineage 2024203604   29 May 2024 specific,” or “tissue specific” promoter, enhancer, or promoter / enhancer that allows expression in a restricted variety of cell and tissue types, respectively. Illustrative ubiquitous expression control sequences suitable for use in particular embodiments include, but are not limited to, a cytomegalovirus (CMV) immediate early 5 promoter, a viral simian virus 40 (SV40) (e.g., early or late), a Moloney murine leukemia virus (MoMLV) LTR promoter, a Rous sarcoma virus (RSV) LTR, a herpes simplex virus (HSV) (thymidine kinase) promoter, H5, P7.5, and P11 promoters from vaccinia virus, an elongation factor 1-alpha (EF1a) promoter, early growth response 1 (EGR1), ferritin H (FerH), ferritin L (FerL), Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), 10 eukaryotic translation initiation factor 4A1 (EIF4A1), heat shock 70kDa protein 5 (HSPA5), heat shock protein 90kDa beta, member 1 (HSP90B1), heat shock protein 70kDa (HSP70), p-kinesm (P-KIN), the human ROSA 26 locus (Irions et al., Nature Biotechnology 25, 1477 - 1482 (2007)), a Ubiquitin C promoter (UBC), a phosphoglycerate kinase-1 (PGK) promoter, a cytomegalovirus enhancer / chicken p-actin (CAG) promoter, a 15 p-actin promoter and a myeloproliferative sarcoma virus enhancer, negative control region deleted, dl587rev primer-binding site substituted (MND) promoter (Challita et al., J Virol. 69(2):748-55 (1995)). In one embodiment, a vector comprises an MND promoter. In one embodiment, a vector comprises an EF1a promoter comprising the first 20 intron of the human EF1a gene. In one embodiment, a vector comprises an EF1a promoter that lacks the first intron of the human EF1a gene. In a particular embodiment, it may be desirable to use a cell, cell type, cell lineage or tissue specific expression control sequence to achieve cell type specific, lineage specific, 25 or tissue specific expression of a desired polynucleotide sequence (e.g., to express a particular nucleic acid encoding a polypeptide in only a subset of cell types, cell lineages, or tissues or during specific stages of development). In a particular embodiment, it may be desirable to express a polynucleotide a T cell specific promoter. 2024203604   29 May 2024 As used herein, “conditional expression” may refer to any type of conditional expression including, but not limited to, inducible expression; repressible expression; expression in cells or tissues having a particular physiological, biological, or disease state, etc. This definition is not intended to exclude cell type or tissue specific expression. 5 Certain embodiments provide conditional expression of a polynucleotide-of-interest, e.g., expression is controlled by subjecting a cell, tissue, organism, etc., to a treatment or condition that causes the polynucleotide to be expressed or that causes an increase or decrease in expression of the polynucleotide encoded by the polynucleotide-of-interest. Illustrative examples of inducible promoters / systems include, but are not limited to, 10 steroid-inducible promoters such as promoters for genes encoding glucocorticoid or estrogen receptors (inducible by treatment with the corresponding hormone), metallothionine promoter (inducible by treatment with various heavy metals), MX-1 promoter (inducible by interferon), the “GeneSwitch” mifepristone-regulatable system (Sirin et al., 2003, Gene, 323:67), the cumate inducible gene switch (WO 2002 / 088346), 15 tetracycline-dependent regulatory systems, etc. Inducer agents include, but are not limited to glucocorticoids, estrogens, mifepristone (RU486), metals, interferons, small molecules, cumate, tetracycline, doxycycline, and variants thereof. Conditional expression can also be achieved by using a site specific DNA recombinase. According to certain embodiments the vector comprises at least one 20 (typically two) site(s) for recombination mediated by a site specific recombinase. As used herein, the terms “recombinase” or “site specific recombinase” include excisive or integrative proteins, enzymes, co-factors or associated proteins that are involved in recombination reactions involving one or more recombination sites (e.g., two, three, four, five, seven, ten, twelve, fifteen, twenty, thirty, fifty, etc.), which may be wild-type proteins 25   (see Landy, Current Opinion in Biotechnology 3:699-707 (1993)), or mutants, derivatives (e.g., fusion proteins containing the recombination protein sequences or fragments thereof), fragments, and variants thereof. Illustrative examples of recombinases suitable for use in particular embodiments include, but are not limited to: Cre, Int, IHF, Xis, Flp, Fis, Hin, Gin, OC31, Cin, Tn3 resolvase, TndX, XerC, XerD, TnpX, Hjc, Gin, SpCCEl, and ParA. 2024203604   29 May 2024 The polynucleotides may comprise one or more recombination sites for any of a wide variety of site specific recombinases. It is to be understood that the target site for a site specific recombinase is in addition to any site(s) required for integration of a vector, e.g., a retroviral vector or lentiviral vector. As used herein, the terms “recombination 5 sequence,” “recombination site,” or “site specific recombination site” refer to a particular nucleic acid sequence to which a recombinase recognizes and binds. For example, one recombination site for Cre recombinase is loxP which is a 34 base pair sequence comprising two 13 base pair inverted repeats (serving as the recombinase binding sites) flanking an 8 base pair core sequence (see FIG. 1 of Sauer, B., Current 10 Opinion in Biotechnology 5:521-527 (1994)). Other exemplary loxP sites include, but are not limited to: lox511 (Hoess et al., 1996; Bethke and Sauer, 1997), lox5171 (Lee and Saito, 1998), lox2272 (Lee and Saito, 1998), m2 (Langer et al., 2002), lox71 (Albert et al., 1995), and lox66 (Albert et al., 1995). Suitable recognition sites for the FLP recombinase include, but are not limited to: 15 FRT (McLeod, et al., 1996), F1, F2, F3 (Schlake and Bode, 1994), F4, F5 (Schlake and Bode, 1994), FRT(LE) (Senecoff et al., 1988), FRT(RE) (Senecoff et al., 1988). Other examples of recognition sequences are the attB, attP, attL, and attR sequences, which are recognized by the recombinase enzyme X Integrase, e.g., phi-c31. The y C31 SSR mediates recombination only between the heterotypic sites attB (34 bp in 20 length) and attP (39 bp in length) (Groth et al., 2000). attB and attP, named for the attachment sites for the phage integrase on the bacterial and phage genomes, respectively, both contain imperfect inverted repeats that are likely bound by y C31 homodimers (Groth et al., 2000). The product sites, attL and attR, are effectively inert to further yC31-mediated recombination (Belteki et al., 2003), making the reaction irreversible. For 25 catalyzing insertions, it has been found that attB-bearing DNA inserts into a genomic attP site more readily than an attP site into a genomic attB site (Thyagarajan et al., 2001; Belteki et al., 2003). Thus, typical strategies position by homologous recombination an attP-bearing “docking site” into a defined locus, which is then partnered with an attB-bearing incoming sequence for insertion. 2024203604   29 May 2024 As used herein, an “internal ribosome entry site” or “IRES” refers to an element that promotes direct internal ribosome entry to the initiation codon, such as ATG, of a cistron (a protein encoding region), thereby leading to the cap-independent translation of the gene. See, e.g., Jackson et al., 1990. Trends Biochem Sci 15(12):477-83) and Jackson and Kaminski. 5   1995. RNA 1(10):985-1000. Examples of IRES generally employed by those of skill in the art include those described in U.S. Pat. No. 6,692,736. Further examples of “IRES” known in the art include, but are not limited to IRES obtainable from picornavirus (Jackson et al., 1990) and IRES obtainable from viral or cellular mRNA sources, such as for example, immunoglobulin heavy-chain binding protein (BiP), the vascular endothelial growth factor (VEGF) (Huez et al. 10   1998. Mol. Cell. Biol. 18(11):6178-6190), the fibroblast growth factor 2 (FGF-2), and insulin like growth factor (IGFII), the translational initiation factor eIF4G and yeast transcription factors TFIID and HAP4, the encephelomycarditis virus (EMCV) which is commercially available from Novagen (Duke et al., 1992. J. Virol 66(3):1602-9) and the VEGF IRES (Huez et al., 1998. Mol Cell Biol 18(11):6178-90). IRES have also been reported in viral genomes of 15 Picornaviridae, Dicistroviridae and Flaviviridae species and in HCV, Friend murine leukemia virus (FrMLV) and Moloney murine leukemia virus (MoMLV). In one embodiment, the IRES used in polynucleotides contemplated herein is an EMCV IRES. In particular embodiments, the polynucleotides comprise polynucleotides that have a 20 consensus Kozak sequence and that encode a desired polypeptide. As used herein, the term “Kozak sequence” refers to a short nucleotide sequence that greatly facilitates the initial binding of mRNA to the small subunit of the ribosome and increases translation. The consensus Kozak sequence is (GCC)RCCATGG (SEQ ID NO:72), where R is a purine (A or G) (Kozak, 1986. Cell. 44(2):283-92, and Kozak, 1987. Nucleic Acids Res. 15(20):8125-48). 25          Elements directing the efficient termination and polyadenylation of the heterologous nucleic acid transcripts increases heterologous gene expression. Transcription termination signals are generally found downstream of the polyadenylation signal. In particular embodiments, vectors comprise a polyadenylation sequence 3’ of a polynucleotide encoding a polypeptide to be expressed. The term “polyA site” or “polyA sequence” as used herein 30 denotes a DNA sequence which directs both the termination and polyadenylation of the nascent 2024203604   29 May 2024 RNA transcript by RNA polymerase II. Polyadenylation sequences can promote mRNA stability by addition of a polyA tail to the 3’ end of the coding sequence and thus, contribute to increased translational efficiency. Cleavage and polyadenylation is directed by a poly(A) sequence in the RNA. The core poly(A) sequence for mammalian pre-mRNAs has two 5 recognition elements flanking a cleavage-polyadenylation site. Typically, an almost invariant AAUAAA hexamer lies 20-50 nucleotides upstream of a more variable element rich in U or GU residues. Cleavage of the nascent transcript occurs between these two elements and is coupled to the addition of up to 250 adenosines to the 5' cleavage product. In particular embodiments, the core poly(A) sequence is an ideal polyA sequence (e.g., AATAAA, 10 ATTAAA, AGTAAA). In particular embodiments, the poly(A) sequence is an SV40 polyA sequence, a bovine growth hormone polyA sequence (BGHpA), a rabbit p-globin polyA sequence (r^gpA), or another suitable heterologous or endogenous polyA sequence known in the art. In some embodiments, a polynucleotide or cell harboring the polynucleotide utilizes a 15 suicide gene, including an inducible suicide gene to reduce the risk of direct toxicity and / or uncontrolled proliferation. In specific embodiments, the suicide gene is not immunogenic to the host harboring the polynucleotide or cell. A certain example of a suicide gene that may be used is caspase-9 or caspase-8 or cytosine deaminase. Caspase-9 can be activated using a specific chemical inducer of dimerization (CID). 20         In certain embodiments, polynucleotides comprise gene segments that cause the immune effector cells, e.g., T cells, to be susceptible to negative selection in vivo. By “negative selection” is meant that the infused cell can be eliminated as a result of a change in the in vivo condition of the individual. The negative selectable phenotype may result from the insertion of a gene that confers sensitivity to an administered agent, for example, a 25 compound. Negative selectable genes are known in the art, and include, inter alia the following: the Herpes simplex virus type I thymidine kinase (HSV-I TK) gene (Wigler et al., Cell 11:223, 1977) which confers ganciclovir sensitivity; the cellular hypoxanthine phosphribosyltransferase (HPRT) gene, the cellular adenine phosphoribosyltransferase (APRT) gene, and bacterial cytosine deaminase, (Mullen et al., Proc. Natl. Acad. Sci. USA. 30   89:33 (1992)). 2024203604   29 May 2024 In some embodiments, genetically modified immune effector cells, such as T cells, comprise a polynucleotide further comprising a positive marker that enables the selection of cells of the negative selectable phenotype in vitro. The positive selectable marker may be a gene which, upon being introduced into the host cell expresses a dominant phenotype 5 permitting positive selection of cells carrying the gene. Genes of this type are known in the art, and include, inter alia, hygromycin-B phosphotransferase gene (hph) which confers resistance to hygromycin B, the amino glycoside phosphotransferase gene (neo or aph) from Tn5 which codes for resistance to the antibiotic G418, the dihydrofolate reductase (DHFR) gene, the adenosine deaminase gene (ADA), and the multi-drug resistance (MDR) 10 gene. In one embodiment, the positive selectable marker and the negative selectable element are linked such that loss of the negative selectable element necessarily also is accompanied by loss of the positive selectable marker. In a particular embodiment, the positive and negative selectable markers are fused so that loss of one obligatorily leads to loss of the other. An 15 example of a fused polynucleotide that yields as an expression product a polypeptide that confers both the desired positive and negative selection features described above is a hygromycin phosphotransferase thymidine kinase fusion gene (HyTK). Expression of this gene yields a polypeptide that confers hygromycin B resistance for positive selection in vitro, and ganciclovir sensitivity for negative selection in vivo. See also the publications of PCT 20  US91 / 08442 and PCT / US94 / 05601, by S. D. Lupton, describing the use of bifunctional selectable fusion genes derived from fusing a dominant positive selectable markers with negative selectable markers. Preferred positive selectable markers are derived from genes selected from the group consisting of hph, nco, and gpt, and preferred negative selectable markers are derived from 25 genes selected from the group consisting of cytosine deaminase, HSV-I TK, VZV TK, HPRT, APRT and gpt. Exemplary bifunctional selectable fusion genes contemplated in particular embodiments include, but are not limited to genes wherein the positive selectable marker is derived from hph or neo, and the negative selectable marker is derived from cytosine deaminase or a TK gene or selectable marker. 2024203604   29 May 2024 In particular embodiments, polynucleotides encoding one or more polypeptides, or fusion polypeptides may be introduced into immune effector cells, e.g., T cells, by both non-viral and viral methods. In particular embodiments, delivery of one or more polynucleotides may be provided by the same method or by different methods, and / or by 5 the same vector or by different vectors. The term “vector” is used herein to refer to a nucleic acid molecule capable transferring or transporting another nucleic acid molecule. The transferred nucleic acid is generally linked to, e.g., inserted into, the vector nucleic acid molecule. A vector may include sequences that direct autonomous replication in a cell, or may include sequences sufficient to allow integration 10 into host cell DNA. In particular embodiments, non-viral vectors are used to deliver one or more polynucleotides contemplated herein to a T cell. Illustrative examples of non-viral vectors include, but are not limited to plasmids (e.g., DNA plasmids or RNA plasmids), transposons, cosmids, and bacterial artificial chromosomes. 15          Illustrative methods of non-viral delivery of polynucleotides contemplated in particular embodiments include, but are not limited to: electroporation, sonoporation, lipofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, nanoparticles, polycation or lipid:nucleic acid conjugates, naked DNA, artificial virions, DEAE-dextran-mediated transfer, gene gun, and heat-shock. 20          Illustrative examples of polynucleotide delivery systems suitable for use in particular embodiments contemplated in particular embodiments include, but are not limited to those provided by Amaxa Biosystems, Maxcyte, Inc., BTX Molecular Delivery Systems, and Copernicus Therapeutics Inc. Lipofection reagents are sold commercially (e.g., Transfectam™ and Lipofectin™). Cationic and neutral lipids that are suitable for efficient 25 receptor-recognition lipofection of polynucleotides have been described in the literature. See e.g., Liu et al. (2003) Gene Therapy. 10:180-187; and Balazs et al. (2011) Journal of Drug Delivery. 2011:1-12. Antibody-targeted, bacterially derived, non-living nanocell-based delivery is also contemplated in particular embodiments. Viral vectors comprising polynucleotides contemplated in particular embodiments 30 can be delivered in vivo by administration to an individual patient, typically by systemic 2024203604   29 May 2024 administration (e.g., intravenous, intraperitoneal, intramuscular, subdermal, or intracranial infusion) or topical application, as described below. Alternatively, vectors can be delivered to cells ex vivo, such as cells explanted from an individual patient (e.g., mobilized peripheral blood, lymphocytes, bone marrow aspirates, tissue biopsy, etc.) or universal donor 5 hematopoietic stem cells, followed by reimplantation of the cells into a patient. In one embodiment, viral vectors comprising nuclease variants and / or donor repair templates are administered directly to an organism for transduction of cells in vivo. Alternatively, naked DNA can be administered. Administration is by any of the routes normally used for introducing a molecule into ultimate contact with blood or tissue cells 10 including, but not limited to, injection, infusion, topical application and electroporation. Suitable methods of administering such nucleic acids are available and well known to those of skill in the art, and, although more than one route can be used to administer a particular composition, a particular route can often provide a more immediate and more effective reaction than another route. 15          Illustrative examples of viral vector systems suitable for use in particular embodiments contemplated in particular embodiments include, but are not limited to adeno-associated virus (AAV), retrovirus, herpes simplex virus, adenovirus, and vaccinia virus vectors. In various embodiments, one or more polynucleotides are introduced into an immune 20 effector cell, e.g., T cell, by transducing the cell with a recombinant adeno-associated virus (rAAV), comprising the one or more polynucleotides. AAV is a small (~26 nm) replication-defective, primarily episomal, non-enveloped virus. AAV can infect both dividing and non-dividing cells and may incorporate its genome into that of the host cell. Recombinant AAV (rAAV) are typically composed of, at a 25 minimum, a transgene and its regulatory sequences, and 5' and 3' AAV inverted terminal repeats (ITRs). The ITR sequences are about 145 bp in length. In particular embodiments, the rAAV comprises ITRs and capsid sequences isolated from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10. In some embodiments, a chimeric rAAV is used the ITR sequences are isolated from 30 one AAV serotype and the capsid sequences are isolated from a different AAV serotype. For 2024203604   29 May 2024 example, a rAAV with ITR sequences derived from AAV2 and capsid sequences derived from AAV6 is referred to as AAV2 / AAV6. In particular embodiments, the rAAV vector may comprise ITRs from AAV2, and capsid proteins from any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10. In a preferred 5 embodiment, the rAAV comprises ITR sequences derived from AAV2 and capsid sequences derived from AAV6. In a preferred embodiment, the rAAV comprises ITR sequences derived from AAV2 and capsid sequences derived from AAV2. In some embodiments, engineering and selection methods can be applied to AAV capsids to make them more likely to transduce cells of interest. 10          Construction of rAAV vectors, production, and purification thereof have been disclosed, e.g., in U.S. Patent Nos. 9,169,494; 9,169,492; 9,012,224; 8,889,641; 8,809,058; and 8,784,799, each of which is incorporated by reference herein, in its entirety. In various embodiments, one or more polynucleotides are introduced into an immune effector cell, e.g., T cell, by transducing the cell with a retrovirus, e.g., lentivirus, comprising 15 the one or more polynucleotides. As used herein, the term “retrovirus” refers to an RNA virus that reverse transcribes its genomic RNA into a linear double-stranded DNA copy and subsequently covalently integrates its genomic DNA into a host genome. Illustrative retroviruses suitable for use in particular embodiments, include, but are not limited to: Moloney murine leukemia virus (M- 20 MuLV), Moloney murine sarcoma virus (MoMSV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), gibbon ape leukemia virus (GaLV), feline leukemia virus (FLV), spumavirus, Friend murine leukemia virus, Murine Stem Cell Virus (MSCV) and Rous Sarcoma Virus (RSV)) and lentivirus. As used herein, the term “lentivirus” refers to a group (or genus) of complex 25 retroviruses. Illustrative lentiviruses include, but are not limited to: HIV (human immunodeficiency virus; including HIV type 1, and HIV type 2); visna-maedi virus (VMV) virus; the caprine arthritis-encephalitis virus (CAEV); equine infectious anemia virus (EIAV); feline immunodeficiency virus (FIV); bovine immune deficiency virus (BIV); and simian immunodeficiency virus (SIV). In one embodiment, HIV based vector backbones 30   (i.e., HIV cis-acting sequence elements) are preferred. 2024203604   29 May 2024 In various embodiments, a lentiviral vector contemplated herein comprises one or more LTRs, and one or more, or all, of the following accessory elements: a cPPT / FLAP, a Psi (T) packaging signal, an export element, poly (A) sequences, and may optionally comprise a WPRE or HPRE, an insulator element, a selectable marker, and a cell suicide gene, as 5 discussed elsewhere herein. In particular embodiments, lentiviral vectors contemplated herein may be integrative or non-integrating or integration defective lentivirus. As used herein, the term “integration defective lentivirus” or “IDLV” refers to a lentivirus having an integrase that lacks the capacity to integrate the viral genome into the genome of the host cells. Integration-incompetent viral 10 vectors have been described in patent application WO 2006 / 010834, which is herein incorporated by reference in its entirety. Illustrative mutations in the HIV-1 pol gene suitable to reduce integrase activity include, but are not limited to: H12N, H12C, H16C, H16V, S81 R, D41A, K42A, H51A, Q53C, D55V, D64E, D64V, E69A, K71A, E85A, E87A, D116N, D1161, D116A, N120G, 15  N1201, N120E, E152G, E152A, D35E, K156E, K156A, E157A, K159E, K159A, K160A, R166A, D167A, E170A, H171A, K173A, K186Q, K186T, K188T, E198A, R199c, R199T, R199A, D202A, K211A, Q214L, Q216L, Q221 L, W235F, W235E, K236S, K236A, K246A, G247W, D253A, R262A, R263A and K264H. The term “long terminal repeat (LTR)” refers to domains of base pairs located at the 20 ends of retroviral DNAs which, in their natural sequence context, are direct repeats and contain U3, R and U5 regions. As used herein, the term “FLAP element” or “cPPT / FLAP” refers to a nucleic acid whose sequence includes the central polypurine tract and central termination sequences (cPPT and CTS) of a retrovirus, e.g., HIV-1 or HIV-2. Suitable FLAP elements are described in U.S. 25 Pat. No. 6,682,907 and in Zennou, et al., 2000, Cell, 101:173. As used herein, the term “packaging signal” or “packaging sequence” refers to psi [T] sequences located within the retroviral genome which are required for insertion of the viral RNA into the viral capsid or particle, see e.g., Clever et al., 1995. J. of Virology, Vol. 69, No. 4; pp. 2101-2109. 2024203604   29 May 2024 The term “export element” refers to a cis-acting post-transcriptional regulatory element which regulates the transport of an RNA transcript from the nucleus to the cytoplasm of a cell. Examples of RNA export elements include, but are not limited to, the human immunodeficiency virus (HIV) rev response element (RRE) (see e.g., Cullen et al., 1991. J. 5 Virol. 65: 1053; and Cullen et al., 1991. Cell 58: 423), and the hepatitis B virus post-transcriptional regulatory element (HPRE). In particular embodiments, expression of heterologous sequences in viral vectors is increased by incorporating posttranscriptional regulatory elements, efficient polyadenylation sites, and optionally, transcription termination signals into the vectors. A variety of 10 posttranscriptional regulatory elements can increase expression of a heterologous nucleic acid at the protein, e.g., woodchuck hepatitis virus posttranscriptional regulatory element (WPRE; Zufferey et al., 1999, J. Virol., 73:2886); the posttranscriptional regulatory element present in hepatitis B virus (HPRE) (Huang et al., Mol. Cell. Biol., 5:3864); and the like (Liu et al., 1995, Genes Dev., 9:1766). 15          Lentiviral vectors preferably contain several safety enhancements as a result of modifying the LTRs. “Self-inactivating” (SIN) vectors refers to replication-defective vectors, e.g., retroviral or lentiviral vectors, in which the right (3') LTR enhancer-promoter region, known as the U3 region, has been modified (e.g., by deletion or substitution) to prevent viral transcription beyond the first round of viral replication. Self-inactivation is 20 preferably achieved through in the introduction of a deletion in the U3 region of the 3' LTR of the vector DNA, i.e., the DNA used to produce the vector RNA. Thus, during reverse transcription, this deletion is transferred to the 5' LTR of the proviral DNA. In particular embodiments, it is desirable to eliminate enough of the U3 sequence to greatly diminish or abolish altogether the transcriptional activity of the LTR, thereby greatly diminishing or 25 abolishing the production of full-length vector RNA in transduced cells. In the case of HIV based lentivectors, it has been discovered that such vectors tolerate significant U3 deletions, including the removal of the LTR TATA box (e.g., deletions from -418 to -18), without significant reductions in vector titers. An additional safety enhancement is provided by replacing the U3 region of the 5' LTR 30 with a heterologous promoter to drive transcription of the viral genome during production of 2024203604   29 May 2024 viral particles. Examples of heterologous promoters which can be used include, for example, viral simian virus 40 (SV40) (e.g., early or late), cytomegalovirus (CMV) (e.g., immediate early), Moloney murine leukemia virus (MoMLV), Rous sarcoma virus (RSV), and herpes simplex virus (HSV) (thymidine kinase) promoters. 5          The terms “pseudotype” or “pseudotyping” as used herein, refer to a virus whose viral envelope proteins have been substituted with those of another virus possessing preferable characteristics. For example, HIV can be pseudotyped with vesicular stomatitis virus G-protein (VSV-G) envelope proteins, which allows HIV to infect a wider range of cells because HIV envelope proteins (encoded by the env gene) normally target the virus to 10   CD4+ presenting cells. In certain embodiments, lentiviral vectors are produced according to known methods. See e.g., Kutner et al., BMC Biotechnol. 2009;9:10. doi: 10.1186 / 1472-6750-9-10; Kutner et al. Nat. Protoc. 2009;4(4):495-505. doi: 10.1038 / nprot.2009.22. According to certain specific embodiments contemplated herein, most or all of the 15 viral vector backbone sequences are derived from a lentivirus, e.g., HIV-1. However, it is to be understood that many different sources of retroviral and / or lentiviral sequences can be used, or combined and numerous substitutions and alterations in certain of the lentiviral sequences may be accommodated without impairing the ability of a transfer vector to perform the functions described herein. Moreover, a variety of lentiviral vectors are known 20 in the art, see Naldini et al., (1996a, 1996b, and 1998); Zufferey et al., (1997); Dull et al., 1998, U.S. Pat. Nos. 6,013,516; and 5,994,136, many of which may be adapted to produce a viral vector or transfer plasmid contemplated herein. In various embodiments, one or more polynucleotides are introduced into an immune effector cell, by transducing the cell with an adenovirus comprising the one or more 25 polynucleotides. Adenoviral based vectors are capable of very high transduction efficiency in many cell types and do not require cell division. With such vectors, high titer and high levels of expression have been obtained. This vector can be produced in large quantities in a relatively simple system. Most adenovirus vectors are engineered such that a transgene replaces the Ad 30 E1a, E1b, and / or E3 genes; subsequently the replication defective vector is propagated in 2024203604   29 May 2024 human 293 cells that supply deleted gene function in trans. Ad vectors can transduce multiple types of tissues in vivo, including non-dividing, differentiated cells such as those found in liver, kidney and muscle. Conventional Ad vectors have a large carrying capacity. Generation and propagation of the current adenovirus vectors, which are replication 5 deficient, may utilize a unique helper cell line, designated 293, which was transformed from human embryonic kidney cells by Ad5 DNA fragments and constitutively expresses E1 proteins (Graham et al., 1977). Since the E3 region is dispensable from the adenovirus genome (Jones & Shenk, 1978), the current adenovirus vectors, with the help of 293 cells, carry foreign DNA in either the E1, the D3 or both regions (Graham & Prevec, 1991). 10 Adenovirus vectors have been used in eukaryotic gene expression (Levrero et al., 1991; Gomez-Foix et al., 1992) and vaccine development (Grunhaus & Horwitz, 1992; Graham & Prevec, 1992). Studies in administering recombinant adenovirus to different tissues include trachea instillation (Rosenfeld et al., 1991; Rosenfeld et al., 1992), muscle injection (Ragot et al., 1993), peripheral intravenous injections (Herz & Gerard, 1993) and stereotactic 15 inoculation into the brain (Le Gal La Salle et al., 1993). An example of the use of an Ad vector in a clinical trial involved polynucleotide therapy for antitumor immunization with intramuscular injection (Sterman et al., Hum. Gene Ther. 7:1083-9 (1998)). In various embodiments, one or more polynucleotides are introduced into an immune effector cell by transducing the cell with a herpes simplex virus, e.g., HSV-1, HSV-2, 20 comprising the one or more polynucleotides. The mature HSV virion consists of an enveloped icosahedral capsid with a viral genome consisting of a linear double-stranded DNA molecule that is 152 kb. In one embodiment, the HSV based viral vector is deficient in one or more essential or non-essential HSV genes. In one embodiment, the HSV based viral vector is replication deficient. Most 25 replication deficient HSV vectors contain a deletion to remove one or more intermediate-early, early, or late HSV genes to prevent replication. For example, the HSV vector may be deficient in an immediate early gene selected from the group consisting of: ICP4, ICP22, ICP27, ICP47, and a combination thereof. Advantages of the HSV vector are its ability to enter a latent stage that can result in long-term DNA expression and its large viral DNA genome that can 30 accommodate exogenous DNA inserts of up to 25 kb. HSV-based vectors are described in, for 2024203604   29 May 2024 example, U.S. Pat. Nos. 5,837,532, 5,846,782, and 5,804,413, and International Patent Applications WO 91 / 02788, WO 96 / 04394, WO 98 / 15637, and WO 99 / 06583, each of which are incorporated by reference herein in its entirety. G.   Genetically Modified Cells 5          In various embodiments, cells are modified to express TGF0 signal convertor polypeptides, CTBRs, engineered TCRs, CARs, DARICs, zetakines, and fusion proteins contemplated herein, for use in the treatment of cancer. Cells may be non-genetically modified to express the polypeptides contemplated herein, or in particular preferred embodiments, cells may be genetically modified to express the polypeptides contemplated 10 herein. As used herein, the term “genetically engineered” or “genetically modified” refers to the addition of extra genetic material in the form of DNA or RNA into the total genetic material in a cell. The terms, “genetically modified cells,” “modified cells,” and “redirected cells,” are used interchangeably in particular embodiments. In particular embodiments, the CTBR signal convertor polypeptides contemplated 15 herein are introduced and expressed in immune effector cells to improve the resistance of the cells to the immunosuppressive signals in the TME mediated by TGF0. In particular embodiments, CTBR signal convertor polypeptides are introduced and expressed in immune effector cells that have been redirected to a target cell by virtue of co-expressing an engineered antigen receptor in the cell. 20         An “immune effector cell,” is any cell of the immune system that has one or more effector functions (e.g., cytotoxic cell killing activity, secretion of cytokines, induction of ADCC and / or CDC). The illustrative immune effector cells contemplated herein are T lymphocytes, in particular cytotoxic T cells (CTLs; CD8+ T cells), TILs, and helper T cells (HTLs; CD4+ T cells. In one embodiment, immune effector cells include natural killer (NK) 25 cells. In one embodiment, immune effector cells include natural killer T (NKT) cells. Immune effector cells can be autologous / autogeneic (“self”) or non-autologous (“non-self,” e.g., allogeneic, syngeneic or xenogeneic). “Autologous,” as used herein, refers to cells from the same subject. “Allogeneic,” as used herein, refers to cells of the same species that differ genetically to the cell in 2024203604   29 May 2024 comparison. “Syngeneic,” as used herein, refers to cells of a different subject that are genetically identical to the cell in comparison. “Xenogeneic,” as used herein, refers to cells of a different species to the cell in comparison. In preferred embodiments, the cells are autologous. 5          Illustrative immune effector cells suitable for introducing the CTBR signal convertor polypeptides contemplated herein include T lymphocytes. The terms “T cell” or “T lymphocyte” are art-recognized and are intended to include thymocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, or activated T lymphocytes. A T cell can be a T helper (Th) cell, for example a T helper 1 (Th1) or a T helper 2 (Th2) 10 cell. The T cell can be a helper T cell (HTL; CD4+ T cell) CD4+ T cell, a cytotoxic T cell (CTL; CD8+ T cell), CD4+CD8+ T cell, CD4-CD8- T cell, or any other subset of T cells. Other illustrative populations of T cells suitable for use in particular embodiments include naive T cells and memory T cells. As would be understood by the skilled person, other cells may also be used as 15 immune effector cells with CTBR signal convertor polypeptides contemplated herein. In particular, immune effector cells also include NK cells, NKT cells, neutrophils, and macrophages. Immune effector cells also include progenitors of effector cells wherein such progenitor cells can be induced to differentiate into an immune effector cells in vivo or in vitro. Thus, in particular embodiments, immune effector cell includes progenitors of 20 immune effectors cells such as hematopoietic stem cells (HSCs) contained within the CD34+ population of cells derived from cord blood, bone marrow or mobilized peripheral blood which upon administration in a subject differentiate into mature immune effector cells, or which can be induced in vitro to differentiate into mature immune effector cells. As used herein, immune effector cells genetically engineered to contain a specific 25 chimeric receptor may be referred to as, “antigen specific redirected immune effector cells.” The term, “CD34+ cell,” as used herein refers to a cell expressing the CD34 protein on its cell surface. “CD34,” as used herein refers to a cell surface glycoprotein (e.g., sialomucin protein) that often acts as a cell-cell adhesion factor and is involved in T cell 30 entrance into lymph nodes. The CD34+ cell population contains hematopoietic stem cells 2024203604   29 May 2024 (HSC), which upon administration to a patient differentiate and contribute to all hematopoietic lineages, including T cells, NK cells, NKT cells, neutrophils and cells of the monocyte / macrophage lineage. Methods for making the immune effector cells which express a TGF0 signal 5 convertor polypeptide contemplated herein are provided in particular embodiments. In one embodiment, the method comprises transfecting or transducing immune effector cells isolated from an individual such that the immune effector cells express one or more TGF0 signal convertor polypeptides as contemplated herein. In one embodiment, the method comprises transfecting or transducing immune effector cells isolated from an individual 10 such that the immune effector cells express one or more TGF0 signal convertor polypeptides and engineered antigen receptors contemplated herein. In certain embodiments, the immune effector cells are isolated from an individual and genetically modified without further manipulation in vitro. Such cells can then be directly readministered into the individual. In further embodiments, the immune effector cells are 15 first activated and stimulated to proliferate in vitro prior to being genetically modified. In this regard, the immune effector cells may be cultured before and / or after being genetically modified. In particular embodiments, prior to in vitro manipulation or genetic modification of the immune effector cells described herein, the source of cells is obtained from a subject. 20 In particular embodiments, the modified immune effector cells comprise T cells. T cells can be obtained from a number of sources including, but not limited to, peripheral blood mononuclear cells, bone marrow, lymph nodes tissue, cord blood, thymus issue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. In certain embodiments, T cells can be obtained from a unit of blood collected from a subject 25 using any number of techniques known to the skilled person, such as sedimentation, e.g., FICOLLTM separation. In other embodiments, an isolated or purified population of T cells is used. In some embodiments, after isolation of PBMC, both cytotoxic and helper T lymphocytes can be sorted into naive, memory, and effector T cell subpopulations either before or after activation, 30 expansion, and / or genetic modification. 2024203604   29 May 2024 In one embodiment, an isolated or purified population of T cells expresses one or more of the markers including, but not limited to a CD3+, CD4+, CD8+, or a combination thereof In certain embodiments, the T cells are isolated from an individual and first activated and stimulated to proliferate in vitro prior to being modified to express a TGF0 signal 5 convertor polypeptide. In order to achieve sufficient therapeutic doses of T cell compositions, T cells are often subjected to one or more rounds of stimulation, activation and / or expansion. T cells can be activated and expanded generally using methods as described, for example, in U.S. Patents 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 10   7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; and 6,867,041, each of which is incorporated herein by reference in its entirety. In particular embodiments, T cells are activated and expanded for about 6 hours, about 12 hours, about 18 hours or about 24 hours prior to introduction of vectors or polynucleotides encoding the TGF0 signal convertor polypeptides. Optionally in combination with an engineered antigen receptor contemplated 15 herein. In one embodiment, T cells are activated at the same time that they are modified. In various embodiments, a method of generating an immune effector cell comprises activating a population of cells comprising T cells and expanding the population of T cells. T cell activation can be accomplished by providing a primary stimulation signal through the T 20 cell TCR / CD3 complex and by providing a secondary costimulation signal through an accessory molecule, e.g., CD28. The TCR / CD3 complex may be stimulated by contacting the T cell with a suitable CD3 binding agent, e.g., a CD3 ligand or an anti-CD3 monoclonal antibody. Illustrative examples of CD3 antibodies include, but are not limited to, OKT3, G19-4, BC3, and 64.1. 25          In addition to the primary stimulation signal provided through the TCR / CD3 complex, induction of T cell responses requires a second, costimulatory signal. In particular embodiments, a CD28 binding agent can be used to provide a costimulatory signal. Illustrative examples of CD28 binding agents include but are not limited to: natural CD 28 ligands, e.g., a natural ligand for CD28 (e.g., a member of the B7 family of proteins, such as B7-1(CD80) and 30 B7-2 (CD86); and anti-CD28 monoclonal antibody or fragment thereof capable of crosslinking 2024203604   29 May 2024 the CD28 molecule, e.g., monoclonal antibodies 9.3, B-T3, XR-CD28, KOLT-2, 15E8, 248.23.2, and EX5.3D10. In one embodiment, the molecule providing the primary stimulation signal, for example a molecule which provides stimulation through the TCR / CD3 complex and the costimulatory 5 molecule are coupled to the same surface. In certain embodiments, binding agents that provide stimulatory and costimulatory signals are localized on the surface of a cell. This can be accomplished by transfecting or transducing a cell with a nucleic acid encoding the binding agent in a form suitable for its expression on the cell surface or alternatively by coupling a binding agent to the cell surface. 10          In another embodiment, the molecule providing the primary stimulation signal, for example a molecule which provides stimulation through the TCR / CD3 complex and the costimulatory molecule are displayed on antigen presenting cells. In one embodiment, the molecule providing the primary stimulation signal, for example a molecule which provides stimulation through the TCR / CD3 complex and the costimulatory 15 molecule are provided on separate surfaces. In a certain embodiment, one of the binding agents that provides stimulatory and costimulatory signals is soluble (provided in solution) and the other agent(s) is provided on one or more surfaces. In a particular embodiment, the binding agents that provide stimulatory and 20 costimulatory signals are both provided in a soluble form (provided in solution). In various embodiments, the methods for making T cells contemplated herein comprise activating T cells with anti-CD3 and anti-CD28 antibodies. In one embodiment, expanding T cells activated by the methods contemplated herein further comprises culturing a population of cells comprising T cells for several hours (about 3 25 hours) to about 7 days to about 28 days or any hourly integer value in between. In another embodiment, the T cell composition may be cultured for 14 days. In a particular embodiment, T cells are cultured for about 21 days. In another embodiment, the T cell compositions are cultured for about 2-3 days. Several cycles of stimulation / activation / expansion may also be desired such that culture time of T cells can be 60 days or more. 2024203604   29 May 2024 In particular embodiments, conditions appropriate for T cell culture include an appropriate media (e.g., Minimal Essential Media or RPMI Media 1640 or, X-vivo 15, (Lonza)) and one or more factors necessary for proliferation and viability including, but not limited to serum (e.g., fetal bovine or human serum), interleukin-2 (IL-2), insulin, IFN-y, IL-4, 5  IL-7, IL-21, GM-CSF, IL-10, IL-12, IL-15, TGF0, and TNF-a or any other additives suitable for the growth of cells known to the skilled artisan. Further illustrative examples of cell culture media include, but are not limited to RPMI 1640, Clicks, AIM-V, DMEM, MEM, a-MEM, F-12, X-Vivo 15, and X-Vivo 20, Optimizer, with added amino acids, sodium pyruvate, and vitamins, either serum-free or supplemented 10 with an appropriate amount of serum (or plasma) or a defined set of hormones, and / or an amount of cytokine(s) sufficient for the growth and expansion of T cells. Antibiotics, e.g., penicillin and streptomycin, are included only in experimental cultures, not in cultures of cells that are to be infused into a subject. The target cells are maintained under conditions necessary to support growth, for example, an appropriate 15 temperature (e.g., 37° C) and atmosphere (e.g., air plus 5% C02). In particular embodiments, PBMCs or isolated T cells are contacted with a stimulatory agent and costimulatory agent, such as anti-CD3 and anti-CD28 antibodies, generally attached to a bead or other surface, in a culture medium with appropriate cytokines, such as IL-2, IL-7, and / or IL-15. 20         In other embodiments, artificial APC (aAPC) made by engineering K562, U937, 721.221, T2, and C1R cells to direct the stable expression and secretion, of a variety of costimulatory molecules and cytokines. In a particular embodiment K32 or U32 aAPCs are used to direct the display of one or more antibody-based stimulatory molecules on the AAPC cell surface. Populations of T cells can be expanded by aAPCs expressing a variety of 25 costimulatory molecules including, but not limited to, CD137L (4-1BBL), CD134L (OX40L), and / or CD80 or CD86. Finally, the aAPCs provide an efficient platform to expand genetically modified T cells and to maintain CD28 expression on CD8 T cells. aAPCs provided in WO 03 / 057171 and US2003 / 0147869 are hereby incorporated by reference in their entirety. In a particular embodiment, polynucleotide encoding a TGF0 signal convertor and an 30 engineered antigen receptor are introduced into the population of T cells. In a particular 2024203604   29 May 2024 embodiment, polynucleotide encoding a TGF0 signal convertor is introduced into a population of T cells that express an engineered antigen receptor. The polynucleotides may be introduced into the T cells by microinjection, transfection, lipofection, heat-shock, electroporation, transduction, gene gun, microinjection, DEAE-dextran-mediated transfer, and 5 the like. In a preferred embodiment, polynucleotides are introduced into a T cell by viral transduction. Illustrative examples of viral vector systems suitable for introducing a polynucleotide into an immune effector cell or CD34+ cell include, but are not limited to adeno-associated 10 virus (AAV), retrovirus, herpes simplex virus, adenovirus, vaccinia virus vectors for gene transfer. In one embodiment, polynucleotides are introduced into a T cell by AAV transduction. In one embodiment, polynucleotides are introduced into a T cell by retroviral transduction. 15          In one embodiment, polynucleotides are introduced into a T cell by lentiviral transduction. In one embodiment, polynucleotides are introduced into a T cell by adenovirus transduction. In one embodiment, polynucleotides are introduced into a T cell by herpes simplex 20   virus transduction. In one embodiment, polynucleotides are introduced into a T cell by vaccinia virus transduction. H.   Compositions and Formulations The compositions contemplated herein may comprise one or more polypeptides, 25 polynucleotides, vectors comprising same, genetically modified immune effector cells, etc. Compositions include, but are not limited to pharmaceutical compositions. A “pharmaceutical composition” refers to a composition formulated in pharmaceutically-acceptable or physiologically-acceptable solutions for administration to a cell or an animal, either alone, or in combination with one or more other modalities of therapy. It will also be 2024203604   29 May 2024 understood that, if desired, the compositions may be administered in combination with other agents as well, such as, e.g., cytokines, growth factors, hormones, small molecules, chemotherapeutics, pro-drugs, drugs, antibodies, or other various pharmaceutically-active agents. There is virtually no limit to other components that may also be included in the 5 compositions, provided that the additional agents do not adversely affect the ability of the composition to deliver the intended therapy. The phrase “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and 10 animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio. The term “pharmaceutically acceptable carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the polypeptides, polynucleotides, vectors comprising same, or genetically modified immune effector cells are administered. Illustrative examples of 15 pharmaceutical carriers can be sterile liquids, such as cell culture media, water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients in particular embodiments, include starch, glucose, lactose, sucrose, 20 gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions. 25         In one embodiment, a composition comprising a pharmaceutically acceptable carrier is suitable for administration to a subject. In particular embodiments, a composition comprising a carrier is suitable for parenteral administration, e.g., intravascular (intravenous or intraarterial), intraperitoneal or intramuscular administration. In particular embodiments, a composition comprising a pharmaceutically acceptable 30 carrier is suitable for intraventricular, intraspinal, or intrathecal administration. 2024203604   29 May 2024 Pharmaceutically acceptable carriers include sterile aqueous solutions, cell culture media, or dispersions. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the polypeptides, polynucleotides, vectors comprising same, or 5 genetically modified immune effector cells, use thereof in the pharmaceutical compositions is contemplated. In particular embodiments, compositions contemplated herein comprise genetically modified T cells and a pharmaceutically acceptable carrier. A composition comprising a cell-based composition contemplated herein can be administered separately by enteral or 10 parenteral administration methods or in combination with other suitable compounds to effect the desired treatment goals. The pharmaceutically acceptable carrier must be of sufficiently high purity and of sufficiently low toxicity to render it suitable for administration to the human subject being treated. It further should maintain or increase the stability of the composition. The 15 pharmaceutically acceptable carrier can be liquid or solid and is selected, with the planned manner of administration in mind, to provide for the desired bulk, consistency, etc., when combined with other components of the composition. For example, the pharmaceutically acceptable carrier can be, without limitation, a binding agent (e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.), a filler (e.g., lactose 20 and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates, calcium hydrogen phosphate, etc.), a lubricant (e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycols, sodium benzoate, sodium acetate, etc.), a disintegrant (e.g., starch, sodium starch glycolate, etc.), or a wetting agent (e.g., sodium 25 lauryl sulfate, etc.). Other suitable pharmaceutically acceptable carriers for the compositions contemplated herein include, but are not limited to, water, salt solutions, alcohols, polyethylene glycols, gelatins, amyloses, magnesium stearates, talcs, silicic acids, viscous paraffins, hydroxymethylcelluloses, polyvinylpyrrolidones and the like. Such carrier solutions also can contain buffers, diluents and other suitable 30 additives. The term “buffer” as used herein refers to a solution or liquid whose chemical 2024203604   29 May 2024 makeup neutralizes acids or bases without a significant change in pH. Examples of buffers contemplated herein include, but are not limited to, Dulbecco's phosphate buffered saline (PBS), Ringer's solution, 5% dextrose in water (D5W), normal / physiologic saline (0.9% NaCl). 5          The pharmaceutically acceptable carriers may be present in amounts sufficient to maintain a pH of the composition of about 7. Alternatively, the composition has a pH in a range from about 6.8 to about 7.4, e.g., 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, and 7.4. In still another embodiment, the composition has a pH of about 7.4. Compositions contemplated herein may comprise a nontoxic pharmaceutically 10 acceptable medium. The compositions may be a suspension. The term “suspension” as used herein refers to non-adherent conditions in which cells are not attached to a solid support. For example, cells maintained as a suspension may be stirred or agitated and are not adhered to a support, such as a culture dish. In particular embodiments, compositions contemplated herein are formulated in a 15 suspension, where the modified T cells are dispersed within an acceptable liquid medium or solution, e.g., saline or serum-free medium, in an intravenous (IV) bag or the like. Acceptable diluents include, but are not limited to water, PlasmaLyte, Ringer's solution, isotonic sodium chloride (saline) solution, serum-free cell culture medium, and medium suitable for cryogenic storage, e.g., Cryostor® medium. 20          In certain embodiments, a pharmaceutically acceptable carrier is substantially free of natural proteins of human or animal origin, and suitable for storing a composition comprising a population of modifed T cells. The therapeutic composition is intended to be administered into a human patient, and thus is substantially free of cell culture components such as bovine serum albumin, horse serum, and fetal bovine serum. 25         In some embodiments, compositions are formulated in a pharmaceutically acceptable cell culture medium. Such compositions are suitable for administration to human subjects. In particular embodiments, the pharmaceutically acceptable cell culture medium is a serum free medium. Serum-free medium has several advantages over serum containing medium, 30 including a simplified and better defined composition, a reduced degree of contaminants, 2024203604   29 May 2024 elimination of a potential source of infectious agents, and lower cost. In various embodiments, the serum-free medium is animal-free, and may optionally be protein-free. Optionally, the medium may contain biopharmaceutically acceptable recombinant proteins. “Animal-free” medium refers to medium wherein the components are derived 5 from non-animal sources. Recombinant proteins replace native animal proteins in animal-free medium and the nutrients are obtained from synthetic, plant or microbial sources. “Protein-free” medium, in contrast, is defined as substantially free of protein. Illustrative examples of serum-free media used in particular compositions includes, but is not limited to QBSF-60 (Quality Biological, Inc.), StemPro-34 (Life Technologies), 10 and X-VIVO 10. In a preferred embodiment, the compositions comprising modifed T cells are formulated in PlasmaLyte. In various embodiments, compositions comprising modified T cells are formulated in a cryopreservation medium. For example, cryopreservation media with 15 cryopreservation agents may be used to maintain a high cell viability outcome post-thaw. Illustrative examples of cryopreservation media used in particular compositions includes, but is not limited to, CryoStor CS10, CryoStor CS5, and CryoStor CS2. In one embodiment, the compositions are formulated in a solution comprising 50:50 PlasmaLyte A to CryoStor CS10. 20          In particular embodiments, the composition is substantially free of mycoplasma, endotoxin, and microbial contamination. By “substantially free” with respect to endotoxin is meant that there is less endotoxin per dose of cells than is allowed by the FDA for a biologic, which is a total endotoxin of 5 EU / kg body weight per day, which for an average 70 kg person is 350 EU per total dose of cells. In particular embodiments, compositions 25 comprising hematopoietic stem or progenitor cells transduced with a retroviral vector contemplated herein contain about 0.5 EU / mL to about 5.0 EU / mL, or about 0.5 EU / mL, 1.0 EU / mL, 1.5 EU / mL, 2.0 EU / mL, 2.5 EU / mL, 3.0 EU / mL, 3.5 EU / mL, 4.0 EU / mL, 4.5 EU / mL, or 5.0 EU / mL. In particular embodiments, formulation of pharmaceutically-acceptable carrier 30 solutions is well-known to those of skill in the art, as is the development of suitable dosing 2024203604   29 May 2024 and treatment regimens for using the particular compositions described herein in a variety of treatment regimens, including e.g., enteral and parenteral, e.g., intravascular, intravenous, intrarterial, intraosseously, intraventricular, intracerebral, intracranial, intraspinal, intrathecal, and intramedullary administration and formulation. It would be 5 understood by the skilled artisan that particular embodiments contemplated herein may comprise other formulations, such as those that are well known in the pharmaceutical art, and are described, for example, in Remington: The Science and Practice of Pharmacy, volume I and volume II. 22nd Edition. Edited by Loyd V. Allen Jr. Philadelphia, PA: Pharmaceutical Press; 2012, which is incorporated by reference herein, in its entirety. 10         In particular embodiments, compositions comprise an amount of immune effector cells, including CAR T cells, that express a CTBR signal convertor contemplated herein. As used herein, the term “amount” refers to “an amount effective” or “an effective amount” of cells comprising a CTBR signal convertor contemplated herein, etc., to achieve a beneficial or desired prophylactic or therapeutic result, including clinical results. 15          A “prophylactically effective amount” refers to an amount of cells comprising a CTBR signal convertor contemplated herein, etc., effective to achieve the desired prophylactic result. Typically but not necessarily, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount is less than the therapeutically effective amount. 20          A “therapeutically effective amount” refers to an amount of cells comprising a CTBR signal convertor contemplated herein that is effective to “treat” a subject (e.g., a patient). When a therapeutic amount is indicated, the precise amount of the compositions to be administered can be determined by a physician with consideration of individual differences in age, weight, tumor size, extent of infection or metastasis, and condition of 25 the patient (subject). It can generally be stated that a pharmaceutical composition comprising the immune effector cells described herein may be administered at a dosage of 102 to 1010 cells / kg body weight, preferably 105 to 106 cells / kg body weight, including all integer values within those ranges. The number of cells will depend upon the ultimate use for which the composition is intended as will the type of cells included therein. For uses 30 provided herein, the cells are generally in a volume of a liter or less, can be 500 mLs or 2024203604   29 May 2024 less, even 250 mLs or 100 mLs or less. Hence the density of the desired cells is typically greater than 106 cells / ml and generally is greater than 107 cells / ml, generally 108 cells / ml or greater. The clinically relevant number of immune cells can be apportioned into multiple infusions that cumulatively equal or exceed 105, 106, 107, 108, 109, 1010, 1011, or 1012 cells. 5 In some embodiments, particularly since all the infused cells will be redirected to a particular target antigen, lower numbers of cells, in the range of 106 / kilogram (106-1011 per patient) may be administered. If desired, the treatment may also include administration of mitogens (e.g., PHA) or lymphokines, cytokines, and / or chemokines (e.g., IFN-y, IL-2, IL-12, TNF-alpha, IL-18, and TNF-beta, GM-CSF, IL-4, IL-13, Flt3-L, RANTES, MIPla, 10 etc.) as described herein to enhance induction of the immune response. Generally, compositions comprising the cells activated and expanded as described herein may be utilized in the treatment and prevention of diseases that arise in individuals who are immunocompromised. In particular, compositions contemplated herein are used in the treatment of cancer. In particular embodiments, the immune effector cells may be 15 administered either alone, or as a pharmaceutical compositions in combination with carriers, diluents, excipients, and / or with other components such as IL-2 or other cytokines or cell populations. In particular embodiments, pharmaceutical compositions comprise an amount of genetically modified T cells, in combination with one or more pharmaceutically or 20 physiologically acceptable carriers, diluents or excipients. In a particular embodiment, compositions comprise an effective amount of immune effector cells comprising a CTBR signal convertor contemplated herein, alone or in combination with one or more therapeutic agents, such as radiation therapy, chemotherapy, transplantation, immunotherapy, hormone therapy, photodynamic therapy, etc. The 25 compositions may also be administered in combination with antibiotics. Such therapeutic agents may be accepted in the art as a standard treatment for a particular disease state as described herein, such as a particular cancer. Exemplary therapeutic agents contemplated include cytokines, growth factors, steroids, NSAIDs, DMARDs, anti-inflammatories, chemotherapeutics, radiotherapeutics, therapeutic antibodies, or other active and ancillary 30 agents. 2024203604   29 May 2024 In certain embodiments, compositions comprising immune effector cells comprising a CTBR signal convertor contemplated herein may be administered in conjunction with any number of chemotherapeutic agents. Illustrative examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide 5 (CYTOXAN™); alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethylenethiophosphaoramide and trimethylolomelamine resume; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, 10 mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, calicheamicin, carabicin, carminomycin, carzinophilin, chromomycins, dactinomycin, 15 daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, 20 methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; 25 folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; podophyllinic acid; 2-ethylhydrazide; procarbazine; 30 PSK®; razoxane; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2, 2',2”- 2024203604   29 May 2024 trichlorotriethylamine; urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, e.g. paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, N.J.) and doxetaxel (TAXOTERE®., Rhne-Poulenc Rorer, Antony, France); chlorambucil; 5 gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; difluoromethylomithine (DMFO); retinoic acid derivatives such as Targretin™ 10 (bexarotene), Panretin™ (alitretinoin) ; ONTAK™ (denileukin diftitox) ; esperamicins; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above. Also included in this definition are anti-hormonal agents that act to regulate or inhibit hormone action on cancers such as anti-estrogens including for example tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, 15 keoxifene, LY117018, onapristone, and toremifene (Fareston); and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the above. A variety of other therapeutic agents may be used in conjunction with the compositions described herein. In one embodiment, the composition comprising immune 20 effector cells comprising a CTBR signal convertor contemplated herein is administered with an anti-inflammatory agent. Anti-inflammatory agents or drugs include, but are not limited to, steroids and glucocorticoids (including betamethasone, budesonide, dexamethasone, hydrocortisone acetate, hydrocortisone, hydrocortisone, methylprednisolone, prednisolone, prednisone, triamcinolone), nonsteroidal anti- 25 inflammatory drugs (NSAIDS) including aspirin, ibuprofen, naproxen, methotrexate, sulfasalazine, leflunomide, anti-TNF medications, cyclophosphamide and mycophenolate. Other exemplary NSAIDs are chosen from the group consisting of ibuprofen, naproxen, naproxen sodium, Cox-2 inhibitors such as VIOXX® (rofecoxib) and CELEBREX® (celecoxib), and sialylates. Exemplary analgesics are chosen from the 30 group consisting of acetaminophen, oxycodone, tramadol of proporxyphene hydrochloride. 2024203604   29 May 2024 Exemplary glucocorticoids are chosen from the group consisting of cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisolone, or prednisone. Exemplary biological response modifiers include molecules directed against cell surface markers (e.g., CD4, CD5, etc.), cytokine inhibitors, such as the TNF antagonists (e.g., 5 etanercept (ENBREL®), adalimumab (HUMIRA®) and infliximab (REMICADE®), chemokine inhibitors and adhesion molecule inhibitors. The biological response modifiers include monoclonal antibodies as well as recombinant forms of molecules. Exemplary DMARDs include azathioprine, cyclophosphamide, cyclosporine, methotrexate, penicillamine, leflunomide, sulfasalazine, hydroxychloroquine, Gold (oral (auranofin) and 10 intramuscular) and minocycline. Illustrative examples of therapeutic antibodies suitable for combination with the modified T cells comprising a CTBR signal convertor contemplated herein, include but are not limited to, bavituximab, bevacizumab (avastin), bivatuzumab, blinatumomab, conatumumab, daratumumab, duligotumab, dacetuzumab, dalotuzumab, elotuzumab 15 (HuLuc63), gemtuzumab, ibritumomab, indatuximab, inotuzumab, lorvotuzumab, lucatumumab, milatuzumab, moxetumomab, ocaratuzumab, ofatumumab, rituximab, siltuximab, teprotumumab, and ublituximab. In certain embodiments, the compositions described herein are administered in conjunction with a cytokine. By “cytokine” as used herein is meant a generic term for 20 proteins released by one cell population that act on another cell as intercellular mediators. Examples of such cytokines are lymphokines, monokines, and traditional polypeptide hormones. Included among the cytokines are growth hormones such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as 25 follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH); hepatic growth factor; fibroblast growth factor; prolactin; placental lactogen; tumor necrosis factor-alpha and -beta; mullerian-inhibiting substance; mouse gonadotropin-associated peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factors such as NGF-beta; platelet-growth 30 factor; transforming growth factors (TGFs) such as TGF-alpha and TGF-beta; insulin-like 2024203604   29 May 2024 growth factor-I and -II; erythropoietin (EPO); osteoinductive factors; interferons such as interferon-alpha, beta, and -gamma; colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF); interleukins (ILs) such as IL-1, IL-1alpha, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, 5  IL-8, IL-9, IL-10, IL-11, IL-12; IL-15, a tumor necrosis factor such as TNF-alpha or TNF- beta; and other polypeptide factors including LIF and kit ligand (KL). As used herein, the term cytokine includes proteins from natural sources or from recombinant cell culture, and biologically active equivalents of the native sequence cytokines. I.    Therapeutic Methods 10         The immune effector cells, including CAR T cells, comprising a CTBR contemplated herein provide improved methods of adoptive immunotherapy for use in the prevention, treatment, and amelioration cancers, or for preventing, treating, or ameliorating at least one symptom associated with a cancer. The immune effector cells that comprise an engineered receptor and a CTBR 15 contemplated herein provide improved drug products for use in the prevention, treatment, or amelioration of at least one symptom of a cancer, GVHD, an infectious disease, an autoimmune disease, an inflammatory disease, or an immunodeficiency. As used herein, the term “drug product” refers to modified cells produced using the compositions and methods contemplated herein. In particular embodiments, the drug product comprises genetically 20 modified immune effector cells, T cells comprising an engineered receptor, or CAR T cells further modified to express a CTBR signal convertor. Moreover, the modified T cells contemplated in particular embodiments provide safer and more efficacious adoptive cell therapies because they are resistant to T cell exhaustion and display increased durability and persistence in the tumor microenvironment that can lead to sustained therapy. 25          In particular embodiments, an effective amount of modified immune effector cells or T cells comprising an engineered receptor and a CTBR signal convertor are administered to a subject to prevent, treat, or ameliorate at least one symptom of a cancer, GVHD, an infectious disease, an autoimmune disease, an inflammatory disease, or an immunodeficiency. 2024203604   29 May 2024 In particular embodiments, a method of preventing, treating, or ameliorating at least one symptom of a cancer comprises administering the subject an effective amount of modified immune effector cells or T cells comprising a CTBR signal convertor and an engineered TCR, CAR, or Daric, or other therapeutic transgene to redirect the cells to a tumor or cancer. 5 The genetically modified cells are a more durable and persistent drug product because the cells are more resistant to immunosuppressive signals from the tumor microenvironment by virtue of converting an immunosuppressive TGF0 signal to an immunostimulatory signal. In particular embodiments, the modified immune effector cells contemplated herein are used in the treatment of solid tumors or cancers. 10          In particular embodiments, the modified immune effector cells contemplated herein are used in the treatment of solid tumors or cancers including, but not limited to: adrenal cancer, adrenocortical carcinoma, anal cancer, appendix cancer, astrocytoma, atypical teratoid / rhabdoid tumor, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brain / CNS cancer, breast cancer, bronchial tumors, cardiac tumors, cervical cancer, 15 cholangiocarcinoma, chondrosarcoma, chordoma, colon cancer, colorectal cancer, craniopharyngioma, ductal carcinoma in situ (DCIS) endometrial cancer, ependymoma, esophageal cancer, esthesioneuroblastoma, Ewing’s sarcoma, extracranial germ cell tumor, extragonadal germ cell tumor, eye cancer, fallopian tube cancer, fibrous histiosarcoma, fibrosarcoma, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumors, 20 gastrointestinal stromal tumor (GIST), germ cell tumors, glioma, glioblastoma, head and neck cancer, hemangioblastoma, hepatocellular cancer, hypopharyngeal cancer, intraocular melanoma, kaposi sarcoma, kidney cancer, laryngeal cancer, leiomyosarcoma, lip cancer, liposarcoma, liver cancer, lung cancer, non-small cell lung cancer, lung carcinoid tumor, malignant mesothelioma, medullary carcinoma, medulloblastoma, menangioma, melanoma, 25 Merkel cell carcinoma, midline tract carcinoma, mouth cancer, myxosarcoma, myelodysplastic syndrome, myeloproliferative neoplasms, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, oligodendroglioma, oral cancer, oral cavity cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, pancreatic islet cell tumors, papillary carcinoma, paraganglioma, parathyroid cancer, penile cancer, pharyngeal 30 cancer, pheochromocytoma, pinealoma, pituitary tumor, pleuropulmonary blastoma, primary 2024203604   29 May 2024 peritoneal cancer, prostate cancer, rectal cancer, retinoblastoma, renal cell carcinoma, renal pelvis and ureter cancer, rhabdomyosarcoma, salivary gland cancer, sebaceous gland carcinoma, skin cancer, soft tissue sarcoma, squamous cell carcinoma, small cell lung cancer, small intestine cancer, stomach cancer, sweat gland carcinoma, synovioma, testicular cancer, 5 throat cancer, thymus cancer, thyroid cancer, urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer, vascular cancer, vulvar cancer, and Wilms Tumor. In particular embodiments, the modified immune effector cells contemplated herein are used in the treatment of solid tumors or cancers including, without limitation, liver cancer, pancreatic cancer, lung cancer, breast cancer, bladder cancer, brain cancer, bone cancer, thyroid 10 cancer, kidney cancer, or skin cancer. In particular embodiments, the modified immune effector cells contemplated herein are used in the treatment of various cancers including but not limited to pancreatic, bladder, and lung. In particular embodiments, the modified immune effector cells contemplated herein are 15 used in the treatment of liquid cancers or hematological cancers. In particular embodiments, the modified immune effector cells contemplated herein are used in the treatment of B-cell malignancies, including but not limited to: leukemias, lymphomas, and multiple myeloma. In particular embodiments, the modified immune effector cells contemplated herein are 20 used in the treatment of liquid cancers including, but not limited to leukemias, lymphomas, and multiple myelomas: acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), myeloblastic, promyelocytic, myelomonocytic, monocytic, erythroleukemia, hairy cell leukemia (HCL), chronic lymphocytic leukemia (CLL), and chronic myeloid leukemia (CML), chronic myelomonocytic leukemia (CMML) and polycythemia vera, Hodgkin lymphoma, 25 nodular lymphocyte-predominant Hodgkin lymphoma, Burkitt lymphoma, small lymphocytic lymphoma (SLL), diffuse large B-cell lymphoma, follicular lymphoma, immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, mantle cell lymphoma, marginal zone lymphoma, mycosis fungoides, anaplastic large cell lymphoma, Sezary syndrome, precursor T-lymphoblastic lymphoma, multiple myeloma, overt multiple myeloma, smoldering multiple 2024203604   29 May 2024 myeloma, plasma cell leukemia, non-secretory myeloma, IgD myeloma, osteosclerotic myeloma, solitary plasmacytoma of bone, and extramedullary plasmacytoma. Preferred cells for use in the methods contemplated herein include autologous / autogeneic (“self”) cells, preferably hematopoietic cells, more preferably T 5 cells, and more preferably immune effector cells. In particular embodiments, methods comprising administering a therapeutically effective amount of modified immune effector cells contemplated herein or a composition comprising the same, to a patient in need thereof, alone or in combination with one or more therapeutic agents, are provided. In certain embodiments, the cells are used in the treatment of 10 patients at risk for developing a cancer, GVHD, an infectious disease, an autoimmune disease, an inflammatory disease, or an immunodeficiency. Thus, particular embodiments comprise the treatment or prevention or amelioration of at least one symptom of a cancer, an infectious disease, an autoimmune disease, an inflammatory disease, or an immunodeficiency comprising administering to a subject in need thereof, a therapeutically effective amount of the genome 15 edited cells contemplated herein. In one embodiment, a method of treating a cancer, GVHD, an infectious disease, an autoimmune disease, an inflammatory disease, or an immunodeficiency in a subject in need thereof comprises administering an effective amount, e.g., therapeutically effective amount of a composition comprising modified immune effector cells contemplated herein. The quantity 20 and frequency of administration will be determined by such factors as the condition of the patient, and the type and severity of the patient's disease, although appropriate dosages may be determined by clinical trials. In one illustrative embodiment, the effective amount of modified immune effector cells provided to a subject is at least 2 x 106 cells / kg, at least 3 x 106 cells / kg, at least 4 x 25   106 cells / kg, at least 5 x 106 cells / kg, at least 6 x 106 cells / kg, at least 7 x 106 cells / kg, at least 8 x 106 cells / kg, at least 9 x 106 cells / kg, or at least 10 x 106 cells / kg, or more cells / kg, including all intervening doses of cells. In another illustrative embodiment, the effective amount of modified immune effector cells provided to a subject is about 2 x 106 cells / kg, about 3 x 106 cells / kg, about 4 30 x 106 cells / kg, about 5 x 106 cells / kg, about 6 x 106 cells / kg, about 7 x 106 cells / kg, about 8 2024203604   29 May 2024 x 106 cells / kg, about 9 x 106 cells / kg, or about 10 x 106 cells / kg, or more cells / kg, including all intervening doses of cells. In another illustrative embodiment, the effective amount of modified immune effector cells provided to a subject is from about 2 x 106 cells / kg to about 10 x 106 cells / kg, 5 about 3 x 106 cells / kg to about 10 x 106 cells / kg, about 4 x 106 cells / kg to about 10 x 106 cells / kg, about 5 x 106 cells / kg to about 10 x 106 cells / kg, 2 x 106 cells / kg to about 6 x 106 cells / kg, 2 x 106 cells / kg to about 7 x 106 cells / kg, 2 x 106 cells / kg to about 8 x 106 cells / kg, 3 x 106 cells / kg to about 6 x 106 cells / kg, 3 x 106 cells / kg to about 7 x 106 cells / kg, 3 x 106 cells / kg to about 8 x 106 cells / kg, 4 x 106 cells / kg to about 6 x 106 cells / kg, 4 x 106 cells / kg 10 to about 7 x 106 cells / kg, 4 x 106 cells / kg to about 8 x 106 cells / kg, 5 x 106 cells / kg to about 6 x 106 cells / kg, 5 x 106 cells / kg to about 7 x 106 cells / kg, 5 x 106 cells / kg to about 8 x 106 cells / kg, or 6 x 106 cells / kg to about 8 x 106 cells / kg, including all intervening doses of cells. One of ordinary skill in the art would recognize that multiple administrations of the 15 compositions contemplated in particular embodiments may be required to effect the desired therapy. For example, a composition may be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more times over a span of 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, 2 years, 5, years, 10 years, or more. In certain embodiments, it may be desirable to administer activated T cells to a subject 20 and then subsequently redraw blood (or have an apheresis performed), activate T cells therefrom, and reinfuse the patient with these activated and expanded T cells. This process can be carried out multiple times every few weeks. In certain embodiments, T cells can be activated from blood draws of from 10cc to 400cc. In certain embodiments, T cells are activated from blood draws of 20cc, 30cc, 40cc, 50cc, 60cc, 70cc, 80cc, 90cc, 100cc, 150cc, 25  200cc, 250cc, 300cc, 350cc, or 400cc or more. Not to be bound by theory, using this multiple blood draw / multiple reinfusion protocol may serve to select out certain populations of T cells. In one embodiment, a method of treating a subject diagnosed with a cancer, comprises removing immune effector cells from the subject, modifying the immune effector cells by introducing one or more vectors encoding an engineered antigen receptor 30 and a TGF0 signal convertor and producing a population of modified immune effector 2024203604   29 May 2024 cells, and administering the population of modified immune effector cells to the same subject. In a preferred embodiment, the immune effector cells comprise T cells. The methods for administering the cell compositions contemplated in particular embodiments include any method which is effective to result in reintroduction of ex vivo 5 modified immune effector cells or on reintroduction of the modified progenitors of immune effector cells that on introduction into a subject differentiate into mature immune effector cells. One method comprises modifying peripheral blood T cells ex vivo by introducing one or more vectors encoding an engineered antigen receptor and a TGF0 signal convertor and returning the transduced cells into the subject. 10 All publications, patent applications, and issued patents cited in this specification are herein incorporated by reference as if each individual publication, patent application, or issued patent were specifically and individually indicated to be incorporated by reference. 15         Although the foregoing embodiments have been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to one of ordinary skill in the art in light of the teachings contemplated herein that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims. The following examples are provided by way of illustration only and not by 20 way of limitation. Those of skill in the art will readily recognize a variety of noncritical parameters that could be changed or modified to yield essentially similar results. 2024203604   29 May 2024 EXAMPLES EXAMPLE 1 T Cells Expressing a TGF0 IL-12R Signal Convertor (CTBR12) and a Chimeric Antigen Receptor (CAR) 5         Illustrative TGF0 IL-12R based signal convertor constructs were designed as shown in Figure 1. optimal IL-12 receptor signaling is initiated by dimerization of the intracellular domains of the IL-12R01 and IL-12R02 subunits following IL-12 ligation. To convert a TGF0 signal to induce IL-12 receptor signaling after exposure to TGF0, the intracellular 10 domains of TGF0 receptor 1 (TGF0R1) and TGF0 receptor 2 (TGF0R2) were replaced with the IL-12R01 and IL-12R02 signaling domains, respectively. The IL-12R01 and IL-12R02 transmembrane and signaling domains were cloned into a lentiviral vector encoding a CAR and separated by 2A self-cleaving polypeptide sequences (CAR.CTBR12). Primary human T cells from healthy donor PBMCs were activated with soluble 15 anti-CD3 (1 gg / mL) and anti-CD28 (5 gg / mL) and transduced with vehicle or lentiviral vectors expressing (i) an anti-RoR1 CAR; (ii) an anti-RoR1 CAR and dominant negative TGF0 receptor (anti-ROR1.DNR); (iii) an anti-ROR1 CAR and a TGF0R2 subunit; or (iv) an anti-RoR1 CAR and CTBR12 (anti-RoR1.CTBR12). After 10 days of culture in IL-2 containing growth media, cell surface expression of the anti-ROR1 CAR and TGF0R2 was 20 determined by flow cytometry. A recombinant human RoR1 protein conjugated to R-phycoerythrin (R-PE) was used to specifically stain the anti-RoR1 CAR expressing T cells. A commercially available antibody to TGFBR2 was used to detect CTBR12. Representative expression data is shown in Figure 2. Fifty percent of T cells transduced with the lentiviral vector encoding the anti- 25 RoR1 CAR and CTBR12 co-expressed the anti-RoR1 CAR and CTBR12 (rightmost panel of Figure 2). In contrast, neither the anti-RoR1 CAR nor CTBR12 was detected in 2024203604   29 May 2024 untransduced T cells, indicating that the antibody to TGFBR2 did not detect endogenous TGFBR2. EXAMPLE 2 Immunosuppressive TGF0 Signaling Inhibited by CTBR12 5         TGF01 ligation to a tetrameric complex containing 2 units of TGF0R1 and 2 units of TGF0R2 induces SMAD2 and SMAD3 phosphorylation to propagate an immunosuppressive signal to the cell nucleus. Overexpression of a truncated TGF0R2 (dominant negative TGF0 receptor - DNR) renders T cells insensitive to TGF0 as shown by loss of SMAD2 / 3 phosphorylation in response to TGF0 treatment. Thus, phospho- 10 SMAD2 / 3 expression was used to interrogate TGF0 signaling pathway activation. primary human T cells from healthy donor pBMCs were activated with soluble anti-CD3 (1 ug mL) and anti-CD28 (5 ug / mL) and transduced with vehicle or lentiviral vectors expressing (i) an anti-ROR1 CAR; (ii) an anti-ROR1 CAR and dominant negative TGF0 receptor (anti-RORl.DNR); (iii) an anti-RORl CAR and a TGF0R2 subunit; or (iv) 15 an anti-ROR1 CAR and CTBR12 (anti-ROR1.CTBR12). After 10 days of culture in iL-2 containing growth media, cultures were treated with 10 ng / mL of recombinant human TGF01 for 20 minutes. SMAD2 / 3 phosphorylation was evaluated with antibodies specific to phosphorylated SMAD2 / 3. T cells expressing either CTBR12 or DNR were completely protected from phosphorylation of SMAD2 / 3 (Figure 3). These data demonstrated that 20 expression of CTBR12 rendered anti-RORl CAR T cells insensitive to TGF0 immunosuppressive signaling. EXAMPLE 3 CTBR12 Transduces IL-12R Signaling Upon Exposure to TGF01 The cellular response to iL-12 is initiated by receptor dimerization and 25 phosphorylation of STAT4 and STAT5. Thus, phospho-STAT4 and phospho-STAT5 expression was used to assess iL-12 receptor signaling pathway activation. 2024203604   29 May 2024 Primary human T cells from healthy donor PBMCs were activated with soluble anti-CD3 (1 ug mL) and anti-CD28 (5 ug / mL) and transduced with vehicle or lentiviral vectors expressing (i) an anti-ROR1 CAR; (ii) an anti-ROR1 CAR and dominant negative TGF0 receptor (anti-RORl.DNR); (iii) an anti-RORl CAR and a TGF0R2 subunit; or (iv) 5 an anti-ROR1 CAR and CTBR12 (anti-ROR1.CTBR12). After 10 days of culture in IL-2 containing growth media, cultures were treated with 50 ng / mL of recombinant human IL-12 or with 10 ng / mL of recombinant human TGF01 for 20 minutes. T cells expressing anti-ROR1 CARs cells exhibited increased levels of phosphorylated STAT4 (Figure 4, top row, compare rightmost 4 panels to untransduced 10 control (UTD)). Only CAR T cells expressing CTBR12 showed detectable levels of phospho-STAT4 expression when treated with recombinant human TGF01 (Figure 4, bottom row, compare rightmost panel to other panels). In contrast, CAR T cells expressing only the TGF0R2 portion of the signal converter did not phosphorylate STAT4 in response to TGF0 treatment (Figure 4, bottom row, fourth panel from the right). 15         CAR T cells expressing CTBR12 also exhibited detectable levels of phospho- STAT5 when treated with either IL-12 or TGF01, confirming that the converted TGF0 signal induces endogenous IL-12 receptor signaling (Figure 5). The gene expression of CAR T cells expressing the anti-ROR1.CTBR12 was measured in an antigen-driven serial expansion assay in the presence or absence of TGF01. 20 Briefly, GFP-labeled K562 target cells that express human ROR1 antigen were used to serially expand the CAR T cells in the presence or absence of recombinant human TGF01. CAR T cells were stimulated with target cells at a 1:1 ratio once every seven days in the presence or absence of 5 ng / mL recombinant human TGF01. T cells were harvested and mRNA for gene expression analysis was isolated on day 21 following the initial 25 stimulation. Gene expression analysis was performed using the Nanostring immune profiling panel. Significant gene expression changes driven by TGF01 treatment were identified (Figure 6, left panel) in the anti-ROR1.CTBR12 expressing cells, including upregulation of the known IL-12R-regulated transcripts IFNG, SELL, IL18RAP, IL18R1, and IL21R (Figure 6, right panel). 2024203604   29 May 2024 EXAMPLE 4 CAR T Cells Expressing CTBR12 Secrete Increased IFNy Upon Exposure to Antigen and TGF01 IL-12 receptor signaling in human T cells drives TH1 differentiation and increases 5 effector function. IL-12 receptor signaling can cooperate with TCR signals to increase the release of IFNy in response to antigen stimulation. The R2 / R1 signal converter amplified IFNy production when T cells were stimulated through either a TCR or CAR in the presence of recombinant human TGF01. primary human T cells from healthy donor pBMCs were activated with soluble anti-CD3 10  (1 gg / mL) and anti-CD28 (5 ug / mL) and transduced with vehicle or lentiviral vectors expressing (i) an anti-RORl CAR; (ii) an anti-RORl CAR and dominant negative TGF0 receptor (anti-RoR1.DNR); or (iii) an anti-RoR1 CAR and CTBR12 (anti-RoR1.CTBR12). After 10 days of culture in IL-2 containing growth media, the cells were plated on either plate-bound anti-CD3 antibody (1 ug / mL) or recombinant human RoR1 15 protein (100 ng / mL) in the presence or absence of 5 ng / mL recombinant human TGF01. Forty eight hours post-plating, supernatants were collected and analyzed via Luminex for soluble cytokine content. CTBR12 expressing cells produced significantly greater amounts of IFNy than the other cell types when stimulated through either TCR or CAR in the presence of 20 recombinant human TGF01 (Figure 7). EXAMPLE 5 CAR T Cells Expressing CTBR12 are Resistant to TGF01 Immunosuppressive Signals TGF0 signaling decreases T cell expansion in response to antigen stimulation. In 25 contrast, iL-12 signaling increases T cell proliferation and reduces T cell hypofunction resulting from chronic antigen exposure. 2024203604   29 May 2024 Primary human T cells from healthy donor PBMCs were activated with soluble anti-CD3 (1 ug mL) and anti-CD28 (5 ug / mL) and transduced with vehicle or lentiviral vectors expressing (i) an anti-ROR1 CAR; (ii) an anti-ROR1 CAR and dominant negative TGF0 receptor (anti-RORl.DNR); or (iii) an anti-RORl CAR and CTBR12 (anti- 5 ROR1.CTBR12). After 10 days of culture in IL-2 containing growth media, the cells were subject to an in vitro serial re-stimulation assay. Briefly, GFP-labeled K562 target cells that express human ROR1 antigen were used to serially expand the CAR T cells in the presence or absence of recombinant human TGF01. CART cells were stimulated with target cells at a 1:1 ratio once every seven days 10 in the presence or absence of 5 ng / mL recombinant human TGF01. Control anti-RORl CAR-T cells displayed minimal expansion in the presence of 5 ng / mL recombinant human TGF01 over the course of the assay. In contrast, anti-ROR1 CAR T cells co-expressing either the TGF0 DNR or CTBR12 were significantly protected from immunosuppressive TGF01 mediated signaling. Figure 8. These results correlated with both the DNR’s and 15 CTBR12’s ability to block SMAD phosphorylation (Figure 8). EXAMPLE 6 T Cells Expressing a TGF0-IL-7R Signal Convertor R2 / R1 (CTBR7) and a Chimeric Antigen Receptor (CAR) Illustrative TGF0 IL-7R based signal convertor (CTBR7) constructs were designed 20 as shown in Figure 1. Optimal iL-7 receptor signaling is initiated by dimerization of the intracellular domains of the IL-7Ra and the common gamma chain (yc; IL-2Ry) following IL-7 ligation. To convert a TGF0 signal to induce IL-7 receptor signaling after exposure to TGF0, the intracellular domains of TGF0 receptor 1 (TGF0R1) and TGF0 receptor 2 (TGF0R2) were 25 replaced with the IL-2Ry and IL-7Ra signaling domains, respectively to produce an IL-7 signaling chimeric TGF0 receptor (CTBR7). The IL-2Ry and IL-7Ra transmembrane and signaling domains were cloned into a lentiviral vector encoding a CAR and separated by 2A self-cleaving polypeptide sequences (CAR.CTBR7). 2024203604   29 May 2024 Primary human T cells from healthy donor PBMCs were activated with soluble anti-CD3 (1 ug mL) and anti-CD28 (5 ug / mL) and transduced with vehicle or lentiviral vectors expressing (i) an anti-ROR1 CAR; (ii) an anti-ROR1 CAR and dominant negative TGF0 receptor (anti-RORl.DNR); (iii) an anti-RORl CAR and CTBR7 (anti- 5 ROR1.CTBR7). After 10 days of culture in IL-2 containing growth media, cell surface expression of the anti-RORl CAR and TGF0R2 were determined by flow cytometry. A recombinant human RORl protein conjugated to R-phycoerythrin (R-PE) was used to specifically stain the anti-RORl CAR expressing T cells. A commercially available antibody to TGFBR2 was used to detect CTBR7. Representative expression data is shown l0 in Figure 9. Forty percent of T cells transduced with the lentiviral vector encoding the anti-RORl CAR and CTBR7 co-expressed the anti-RORl CAR and CTBR7 (rightmost panel of Figure 9). In contrast, neither the anti-RORl CAR nor CTBR7 was detected in untransduced T cells, indicating that the antibody to TGFBR2 did not detect endogenous l5 TGFBR2. EXAMPLE 7 Immunosuppressive TGF0 Signaling Inhibited by CTBR7 TGFpi ligation to a tetrameric complex containing 2 units of TGF0R1 and 2 units of TGF0R2 induces SMAD2 and SMAD3 phosphorylation to propagate an 20 immunosuppressive signal to the cell nucleus. Overexpression of a truncated TGF0R2 (dominant negative TGF0 receptor - DNR) renders T cells insensitive to TGF0 as shown by loss of SMAD2 / 3 phosphorylation in response to TGF0 treatment. Thus, phospho-SMAD2 / 3 expression was used to interrogate TGF0 signaling pathway activation. primary human T cells from healthy donor pBMCs were activated with soluble 25 anti-CD3 (1 ug / mL) and anti-CD28 (5 ug / mL) and transduced with vehicle or lentiviral vectors expressing (i) an anti-ROR1 CAR; (ii) an anti-ROR1 CAR and dominant negative TGF0 receptor (anti-RORl.DNR); (iii) an anti-RORl CAR and CTBR7 (anti- 2024203604   29 May 2024 ROR1.CTBR7). After 10 days of culture in IL-2 containing growth media, cultures were treated with 10 ng / mL of recombinant human TGF01 for 20 minutes. SMAD2 / 3 phosphorylation was evaluated with antibodies specific to phosphorylated SMAD2 / 3. T cells expressing either CTBR7 or DNR were protected from phosphorylation of SMAD2 / 3 5 (Figure 10). These data demonstrated that expression of CTBR7 rendered anti-ROR1 CART cells insensitive to TGF0 immunosuppressive signaling. EXAMPLE 8 CTBR7 Transduces IL-7R Signaling Upon Exposure to TGF01 The cellular response to IL-7 is initiated by receptor dimerization and 10 phosphorylation of sTAT5. Thus, phospho-sTAT5 expression was used to assess iL-7 receptor signaling pathway activation for T cells expressing cTBR7. primary human T cells from healthy donor pBMcs were activated with soluble anti-CD3 (1 ug mL) and anti-CD28 (5 ug / mL) and transduced with vehicle or lentiviral vectors expressing (i) an anti-RoR1 cAR; (ii) an anti-RoR1 cAR and dominant negative 15 TGF0 receptor (anti-RORl.DNR); (iii) an anti-RORl CAR and CTBR7 (anti-RoR1.CTBR7). After 10 days of culture in iL-2 containing growth media, cultures were treated with 10 ng / mL of recombinant human TGF01 for 20 minutes. Only CAR T cells expressing CTBR7 showed detectable levels of phospho-sTAT5 expression when treated with recombinant human TGF01 (Figure 11, compare rightmost panel to other panels). 20          To further interrogate the converted iL-7R signaling, the ability of CTBR7 expressing cells to upregulate Bcl-2 protein expression in response to continuous TGF01 exposure was determined. Control CAR T cells or CAR T cells co-expressing either the DNR (anti-RoR1.DNR) or CTBR7 (anti-RoR1.CTBR7) were subjected to an antigen-driven serial expansion assay in absence of exogenous cytokine support and either the 25 presence or absence of TGF01. Briefly, GFP-labeled K562 target cells that express human RoR1 antigen were used to serially expand the CAR T cells in the presence or absence of recombinant human TGF01. CAR T cells were stimulated with target cells at a 1:1 ratio 2024203604   29 May 2024 once every seven days in the presence or absence of 5 ng / mL recombinant human TGFpi. Six days following the second stimulation, anti-ROR1 CAR, anti-ROR1 CAR.DNR, or anti-ROR1 CAR.CTBR7 T cells were interrogated for Bcl-2 protein expression by flow cytometry. Only CAR T cells expressing CTBR7 demonstrated increased levels of Bcl-2 5 protein expression when expanded in the presence of TGF01 (Figure 12). EXAMPLE 9 CAR T Cells Co-Expressing CTBR7 Demonstrate Sustained Effector Activity in the Absence of Exogenous IL-2 and Presence of TGF01 TGF0 signaling decreases T cell expansion in response to antigen stimulation. In 10 contrast, IL-7 signaling can induce T cell proliferation and survival, an activity that is particularly apparent for memory T cell populations. To assess whether cTBR7 signaling could increase CAR T cell effector activity in the presence of TGF01, we compared cAR.cTBR7 expansion and anti-tumor activity against control cAR T cells and cAR.DNR T cells in a serial re-stimulation assay where exogenous IL-2 cytokine support 15 was not provided. Primary human T cells from healthy donor PBMcs were activated with soluble anti-CD3 (1 ug mL) and anti-CD28 (5 ug / mL) and transduced with vehicle or lentiviral vectors expressing (i) an anti-RoR1 cAR; (ii) an anti-RoR1 cAR and dominant negative TGF0 receptor (anti-RORl.DNR); or (iii) an anti-RORl CAR and CTBR7 (anti- 20 RoR1.cTBR7). After 10 days of culture in IL-2 containing growth media, the cells were subjected to an in vitro serial re-stimulation assay in the absence of exogenous IL-2 cytokine support. Briefly, GFP-labeled K562 target cells that express human RoR1 antigen were used to serially expand the cAR T cells in the presence or absence of recombinant human 25 TGF01. CAR T cells were stimulated with target cells at a 1:1 ratio once every seven days in the presence or absence of 5 ng / mL recombinant human TGF01. No exogenous IL-2 was used for support in this assay. Control anti-RoR1 CAR T cells displayed minimal expansion in the presence of 5 ng / mL recombinant human TGF01 over the course of the 2024203604   29 May 2024 assay. CAR T cells co-expressing the DNR also demonstrated reduced expansion when expanded in the presence of TGF01. In contrast, anti-RORl CAR T cells co-expressing CTBR7 demonstrated enhanced expansion compared to the same cells expanded in the absence of TGF01 (Figure 13). These data demonstrated that active CTBR7 signaling 5 increased T cell expansion compared to the CAR alone. CAR T cells co-expressing CTBR7 clear tumor cells from culture in the abovedescribed serial re-stimulation assay with no IL-2 support. After the second round of stimulation, only CAR T cells co-expressing CTBR7 and treated with TGF01 completely clear the tumor population (as monitored by the presence of GFP positive tumor cells 10 remaining in culture) (Figure 13). These data demonstrated that CTBR7 signaling was sufficient to support effector function in conditions where CAR signaling alone was not sufficient. EXAMPLE 10 T Cells Expressing a Chimeric Antigen Receptor (CAR) and a CTBR12 or CTBR7 15         Illustrative TGF0 IL-12R or TGF0 IL-7R signal convertor constructs were designed as shown in Figure 1. IL-12R01 and IL-12R02 transmembrane and signaling domains were cloned into a lentiviral vector encoding an anti-EGFR CAR and separated by 2A self-cleaving polypeptide sequences (anti-EGFR.CTBR12). 20         IL-2Ry and IL-7Ra transmembrane and signaling domains were cloned into a lentiviral vector encoding anti-EGFR CAR and separated by 2A self-cleaving polypeptide sequences (anti-EGFR.CTBR7). Primary human T cells from healthy donor PBMCs were activated with soluble anti-CD3 (1 ug mL) and anti-CD28 (5 ug / mL) and transduced with vehicle or lentiviral 25 vectors expressing (i) an anti-EGFR CAR; (ii) an anti-EGFR CAR and dominant negative TGF0 receptor (anti-EGFR.DNR); (iii) an anti-EGFR CAR and CTBR12 (anti- EFGR.CTBR12); and (iv) an anti-EGFR CAR and CTBR7 (anti-EFGR.CTBR7). After 10 2024203604   29 May 2024 days of culture in IL-2 containing growth media, cell surface expression of the anti-EGFR CAR and TGF0R2 was determined by flow cytometry. Representative expression data is shown in Figure 15 (top panel). EXAMPLE 11 5         Immunosuppressive TGF0 Signaling Inhibited by T Cells Expressing anti-EGFR CAR and CTBR12 or anti-EGFR CAR and CTBR7 primary human T cells from healthy donor pBMCs were activated with soluble anti-CD3 (1 ug mL) and anti-CD28 (5 ug / mL) and transduced with vehicle or lentiviral vectors expressing (i) an anti-EGFR CAR; (ii) an anti-EGFR CAR and dominant negative 10 TGF0 receptor (anti-EGFR.DNR); (iii) an anti-EGFR CAR and CTBR12 (anti-EFGR.CTBR12); and (iv) an anti-EGFR CAR and CTBR7 (anti-EFGR.CTBR7). After 10 days of culture in iL-2 containing growth media, cultures were treated with 10 ng / mL of recombinant human TGF01 for 20 minutes. SMAD2 / 3 phosphorylation was evaluated with antibodies specific to phosphorylated sMAD2 / 3. T cells expressing the DNR, 15 CTBR12 or CTBR7 were completely protected from phosphorylation of sMAD2 / 3 (Figure 15, bottom panel). These data demonstrated that expression of either CTBR12 or CTBR7 rendered anti-EGFR CAR T cells insensitive to TGF0 immunosuppressive signaling. EXAMPLE 12 CTBR Transduce IL-R Signaling Upon Exposure to TGF01 20          The cellular response to iL-12 is initiated by receptor dimerization and phosphorylation of sTAT4 and sTAT5. phospho-sTAT4 expression was used to assess iL-12 receptor signaling pathway activation for T cells expressing anti-EGFR.CTBR12. The cellular response to iL-7 is initiated by receptor dimerization and phosphorylation of sTAT5. Thus, phospho-sTAT5 expression was used to assess iL-7 25 receptor signaling pathway activation for T cells expressing anti-EGFR.CTBR7. 2024203604   29 May 2024 Primary human T cells from healthy donor PBMCs were activated with soluble anti-CD3 (1 ug mL) and anti-CD28 (5 ug / mL) and transduced with vehicle or lentiviral vectors expressing (i) an anti-EGFR CAR; (ii) an anti-EGFR CAR and CTBR12 (anti-EFGR.CTBR12); and (iii) an anti-EGFR CAR and CTBR7 (anti-EFGR.CTBR7). After 10 5 days of culture in IL-2 containing growth media, T cell cultures were treated with recombinant human IL-12 or recombinant human TGF01 for 20 minutes (Figure 16) or with recombinant human IL-7 or recombinant human TGF01 for 20 minutes (Figure 17). T cells expressing anti-EGFR CAR or anti-EFGR.CTBR12 shows increased levels of phosphorylated STAT4 in the presence of IL-12 (Figure 16, left panels), but only T cells 10 expressing anti-EFGR.CTBR12 show increased levels of phosphorylated STAT4 in the presence of TGF01 (Figure 16, lower right panel). T cells expressing anti-EGFR CAR or anti-EFGR.CTBR7 shows increased levels of phosphorylated STAT5 in the presence of IL-7 (Figure 17, left panels), but only T cells expressing anti-EFGR.CTBR7 show increased levels of phosphorylated STAT4 in the 15 presence of TGF01 (Figure 17, lower right panel). EXAMPLE 13 CAR T Cells Expressing CTBR12 Secrete Increased IFNy Upon Exposure to Antigen and TGF01 Primary human T cells from healthy donor PBMCs were activated with soluble 20 anti-CD3 (1 ug / mL) and anti-CD28 (5 ug / mL) and transduced with vehicle or lentiviral vectors expressing: (i) an anti-EGFR CAR; (ii) an anti-EGFR CAR and dominant negative TGF0 receptor (anti-EGFR.DNR); or (iii) an anti-EGFR CAR and CTBR12 (anti-EFGR.CTBR12). After 10 days of culture in IL-2 containing growth media, CAR and CTBR expressing T cells were cultured with Jurkat cells (EGFR(-)), A549 cells 25  (EGFR(+)), or HT1080 cells (EGFR(+)) for 48 hours either in the presence or absence of 5 ng / mL recombinant human TGF01. Supernatants were collected and analyzed via Luminex for soluble cytokine content. 2024203604   29 May 2024 CTBR12 expressing cells produced significantly greater amounts of IFNy when cultured with EGFR(+) cell lines compared to EGFR(-) cell lines in the presence of recombinant human TGF01 (Figure 18). EXAMPLE 14 5   anti-EGFR CAR T Cells Co-Expressing CTBR Demonstrate Sustained Effector Activity in the Absence of Exogenous IL-2 and Presence of TGF01 Primary human T cells from healthy donor PBMCs were activated with soluble anti-CD3 (1 ug mL) and anti-CD28 (5 ug / mL) and transduced with vehicle or lentiviral 10 vectors expressing (i) an anti-EGFR cAR; (ii) an anti-EGFR cAR and dominant negative TGF0 receptor (anti-EGFR.DNR); (iii) an anti-EGFR CAR and CTBR12 (anti-EFGR.cTBR12); and (iv) an anti-EGFR cAR and cTBR7 (anti-EFGR.cTBR7). After 10 days of culture in iL-2 containing growth media, the cells were subjected to an in vitro serial re-stimulation assay in the absence of exogenous iL-2 cytokine support. 15         Briefly, GFP-labeled target cells that express human EGFR antigen were used to serially expand the CAR T cells in the presence or absence of recombinant human TGF01. cAR T cells were stimulated with target cells at a 1:1 ratio once every seven days in the presence or absence of 5 ng / mL recombinant human TGF01. No exogenous IL-2 was used for support in this assay. control anti-EGFR cAR T cells displayed minimal expansion in 20 the presence of 5 ng / mL recombinant human TGF01 through the first stimulation and were not cultured futher. cAR T cells co-expressing the DNR also demonstrated reduced expansion when expanded in the presence of TGF01. In contrast, anti-EGFR CAR T cells co-expressing cTBR12 or cTBR7 demonstrated enhanced expansion compared to the same cells expanded in the absence of TGF01 (Figure 19). These data demonstrated that 25 active CTBR12 or CTBR7 signaling increased T cell expansion compared to the CAR alone. 2024203604   29 May 2024 EXAMPLE 15 NY-ESO1 TCR T Cells Co-Expressing CTBR Demonstrate Sustained Effector Activity in the Absence of Exogenous IL-2 and Presence of TGF01 5         Illustrative TCR-based TGF0 IL-12R and TGF0 IL-R signal convertor constructs were designed as shown in Figure 20. IL-12R01 and IL-12R02 transmembrane and signaling domains were cloned into a lentiviral vector encoding an anti-NY-Eso1 TcR and separated by 2A self-cleaving polypeptide sequences (NY-Eso1.cTBR12). 10         IL-2Ry and IL-7Ra transmembrane and signaling domains were cloned into a lentiviral vector encoding NY-Eso1 TcR and separated by 2A self-cleaving polypeptide sequences (NY-Eso1.cTBR7). Primary human T cells from healthy donor PBMcs were activated with soluble anti-CD3 (1 ug mL) and anti-CD28 (5 ug / mL) and transduced with vehicle or lentiviral 15 vectors expressing (i) an NY-Eso1 TcR; (ii) an NY-Eso1 TcR and dominant negative TGF0 receptor (NY-ESO1.DNR); (iii) an NY-ESO1 TCR and CTBR12 (NY-Eso1.CTBR12); and (iv) an NY-Eso1 TCR and CTBR7 (NY-Eso1.CTBR7). After 10 days of culture in iL-2 containing growth media, cell surface expression of the NY-Eso1 TCRR and TGF0R2 was determined by flow cytometry. All constructs were expressed. 20                            EXAMPLE 16 IMMUNOSUPPRESSIVE TGF0 SIGNALING INHIBITED BY T CELLS EXPRESSING NY-Eso TCR and CTBR12 or NY-Eso TCR and CTBR7 Primary human T cells from healthy donor PBMCs were activated with soluble anti-CD3 (1 ug / mL) and anti-CD28 (5 ug / mL) and transduced with vehicle or lentiviral 25 vectors expressing (i) an NY-ESo1 TCR; (ii) an NY-ESo1 TCR and dominant negative TGF0 receptor (NY-ESO1.DNR); (iii) an NY-ESO1 TCR and CTBR12 (NY-ESo1.CTBR12); and (iv) an NY-ESo1 TCR and CTBR7 (NY-ESo1.CTBR7). After 10 2024203604   29 May 2024 days of culture in IL-2 containing growth media, cultures were treated with 10 ng / mL of recombinant human TGF01 for 20 minutes. SMAD2 / 3 phosphorylation was evaluated with antibodies specific to phosphorylated SMAD2 / 3. T cells expressing the DNR, CTBR12 or CTBR7 were completely protected from phosphorylation of SMAD2 / 3 (Figure 5  21). These data demonstrated that expression of either CTBR12 or CTBR7 rendered NY- ESO1 TCR T cells insensitive to TGF0 immunosuppressive signaling. EXAMPLE 17 CTBR Transduce IL-R Signaling Upon Exposure to TGF01 The cellular response to IL-12 is initiated by receptor dimerization and 10 phosphorylation of sTAT4 and sTAT5. phospho-sTAT4 expression was used to assess iL-12 receptor signaling pathway activation for T cells expressing NY-Eso1.cTBR12. The cellular response to iL-7 is initiated by receptor dimerization and phosphorylation of sTAT5. Thus, phospho-sTAT5 expression was used to assess iL-7 receptor signaling pathway activation for T cells expressing NY-Eso1.cTBR7. 15         primary human T cells from healthy donor pBMcs were activated with soluble anti-CD3 (1 ug mL) and anti-CD28 (5 ug / mL) and transduced with vehicle or lentiviral vectors expressing (i) an NY-Eso1 TcR and cTBR12 (NY-Eso1.cTBR12); and (ii) an NY-Eso1 TCR and CTBR7 (NY-Eso1.CTBR7). After 10 days of culture in iL-2 containing growth media, T cell cultures were treated with recombinant human iL-7 or 20 recombinant human TGF01 for 20 minutes (Figure 22, top panel) or with recombinant human IL-12 or recombinant human TGF01 for 20 minutes (Figure 22, bottom panel). EXAMPLE 18 CAR T Cells Expressing CTBR12 Secrete Increased IFNy Upon Exposure to Antigen and TGF01 25         Primary human T cells from healthy donor PBMCs were activated with soluble anti-CD3 (1 ug / mL) and anti-CD28 (5 ug / mL) and transduced with vehicle or lentiviral 2024203604   29 May 2024 vectors expressing: (i) an NY-ESO1 TCR; (ii) an NY-ESO1 TCR and dominant negative TGF0 receptor (NY-ESO1.DNR); (iii) an NY-ESO1 TCR and CTBR12 (NY-ESO1.CTBR12); and (iv) an NY-ESO1 TCR and CTBR7 (NY-ESO1.CTBR7). After 10 days of culture in IL-2 containing growth media, CAR and CTBR expressing T cells were 5 cultured with SaOs2 cells (A2, NY-ESO1(+)) or A549.A2.NY-ESO1 cells (A2, NY- ESO1(+)) at a 5:1 ratio of T cells to target cells for 48 hours either in the presence or absence of 5 ng / mL recombinant human TGF01. Supernatants were collected and analyzed via Luminex for soluble cytokine content. CTBR12 expressing cells produced significantly greater amounts of IFNy when 10 cultured with A2 and NY-ESO1 (+) cell lines in the presence of recombinant human TGF01 compared to A2 and NY-ESO1 (+) cell lines (Figure 23). CTBR expressing cells demonstrates resistance to immunosuppressive TGF0 signaling. The reference in this specification to any prior publication (or information derived 15 from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates. 20          In general, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and the claims, but should be construed to include all possible embodiments along with the full scope of equivalents to which such claims are entitled. Accordingly, the claims are not limited by the disclosure.

Claims

1. A polypeptide complex comprising:(a) a TGF0R2 polypeptide comprising:(i) an extracellular TGF01 -binding domain of TGF0R2;(ii) an IL-12R02 transmembrane domain; and(iii) an IL-12R02 intracellular signaling domain; and(b) a TGF0R1 polypeptide comprising:(i) an extracellular TGF01 -binding domain of TGF^Rl;(ii) an IL-12R01 transmembrane domain; and(iii) an IL-12R01 intracellular signaling domain.

2. The polypeptide complex of claim 1, wherein the TGF0R1 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 26.

3. The polypeptide complex of claim 1 or claim 2, wherein the TGF0R2 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 27.

4. A polypeptide complex comprising:(a) a TGF0R2 polypeptide comprising:(i) an extracellular TGF01 -binding domain of TGF^R2;(ii) an IL-12R01 transmembrane domain; and(iii) an IL-12R01 intracellular signaling domain; and(b) a TGF0R1 polypeptide comprising:(i) an extracellular TGFJG -binding domain of TGF^Rl;(ii) an IL-12R02 transmembrane domain; and(iii) an IL-12R02 intracellular signaling domain.

5. One or more polynucleotides encoding the polypeptide complex of any one of claims 1-4.2024203604   15 Jun 20266. A cell comprising the polypeptide complex of any one of claims 1-4 or the one or more polynucleotides of claim 5.

7. The cell of claim 6, wherein the cell further comprises an engineered antigen receptor selected from the group consisting of: an engineered T cell receptor (TCR), a chimeric antigen receptor (CAR), a DARIC receptor or components thereof, and a chimeric cytokine receptor.

8. The cell of claim 7, wherein the engineered antigen receptor recognizes an antigen selected from the group consisting of: alpha folate receptor, 5T4, av06 integrin, BCMA, B7-H3, B7-H6, CAIX, CD16, CD19, CD20, CD22, CD30, CD33, CD44, CD44v6, CD44v7 / 8, CD70, CD79a, CD79b, CD123, CD138, CD171, CEA, CSPG4, EGFR, EGFR family including ErbB2 (HER2), EGFRvIII, EGP2, EGP40, EphA2, EpCAM, FAP, fetal AchR, FRa, GD2, GD3, Glypican-3 (GPC3), HLA-A1+MAGE1, HLA-A2+MAGE1, HLA-A3+MAGE1, HLA-A1+NY-ESO-1, HLA-A2+NY-ESO-1, HLA-A3+NY-ESO-1, IL-11Ra, IL-13Ra2, Lambda, Lewis-Y, Kappa, Mesothelin, Muc1, Muc16, NCAM, NKG2D Ligands, NY-ESO-1, PRAME, PSCA, PSMA, ROR1, SSX, Survivin, TAG72, TEMs, VEGFR2, and WT-1.

9. The cell of any one of claims 6-8, wherein the cell is an immune effector cell.

10. The cell of any one of claims 6-9, wherein the cell is:(a) a hematopoietic cell;(b) a T cell;(c) a CD3+, CD4+, and / or CD8+ cell;(d) a cytotoxic T lymphocyte (CTL), a tumor infiltrating lymphocyte (TIL), or a helper T cell; or(e) a natural killer (NK) cell or natural killer T (NKT) cell.

11. A composition comprising a pharmaceutically acceptable carrier and the cell of any one of claims 6-10.

12. A method of treating a solid cancer comprising administering to the subject an effective amount of the composition of claim 11.2024203604   15 Jun 202613. The method of claim 12, wherein the solid cancer comprises liver cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, prostate cancer, testicular cancer, bladder cancer, brain cancer, sarcoma, head and neck cancer, bone cancer, thyroid cancer, kidney cancer, esophageal cancer, synovioma, or skin cancer.

14. The method of claim 12 or claim 13, wherein the solid cancer is a pancreatic cancer, a lung cancer, or a breast cancer.

15. The method of claim 12 or claim 13, wherein the solid cancer is esophageal cancer.

16. The method of claim 12 or claim 13, wherein the solid cancer is synovioma cancer.

17. A method of treating a hematological malignancy comprising administering to a subject an effective amount of the composition of claim 11.

18. The method of claim 17, wherein the hematological malignancy is a leukemia, lymphoma, or a multiple myeloma.

19. Use of the composition of claim 11 in the manufacture of a medicament for treating a solid cancer.

20. The use of claim 19, wherein the solid cancer comprises liver cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, prostate cancer, testicular cancer, bladder cancer, brain cancer, sarcoma, head and neck cancer, bone cancer, thyroid cancer, kidney cancer, esophageal cancer, synovioma, or skin cancer.

21. The use of claim 19 or claim 20, wherein the solid cancer is a pancreatic cancer, a lung cancer, or a breast cancer.

22. The use of claim 19 or claim 20, wherein the solid cancer is esophageal cancer.

23. The use of claim 19 or claim 20, wherein the solid cancer is synovioma.

24. Use of the composition of claim 11 in the manufacture of a medicament for treating a hematological malignancy.2024203604   15 Jun 202625. The use of claim 24, wherein the hematological malignancy is a leukemia, lymphoma, or multiple myeloma.

Images