Constitutively active chimeric cytokine receptors

Abstract

Provided herein are constitutively active chimeric cytokine receptors (CACCRs). Such CACCRs allow for increased immune cell activation, proliferation, persistence, and / or potency when present on immune cells bearing chimeric antigen receptors (CARs). Also provided are methods of making and using the CACCRs described herein.

Description

[0001] Cross Reference to Related Applications

[0002] This application claims priority to U.S. Provisional Application No. 62 / 812,911, filed March 1, 2019, and U.S. Provisional Application No. 62 / 980,823, filed February 24, 2020, the contents of both of which are incorporated by reference in their entirety.

[0003] SEQUENCE LISTING

[0004] The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy, created on February 21, 2020, is named AT-023_03WO_SL.txt and is 251,652 bytes in size. BACKGROUND

[0005] Adoptive transfer of immune cells (e.g., T cells) genetically modified to recognize malignancy-associated antigens shows promise as a new approach to treating cancer. For example, T cells can be genetically modified to express a chimeric antigen receptor (CAR), which is a fusion protein composed of an antigen recognition moiety and a T cell activating domain.

[0006] T cell proliferation, cytotoxic potency, and persistence are driven by signaling pathways. Conventional CAR designs provide two signals - CD3 zeta activation (signal 1) and costimulation (signal 2, e.g., by expression of 4-1BB, OX40, and / or CD28). In some cases, a third signal (signal 3), cytokine-induced cytokine receptor signaling (e.g., cytokine support immune enhancement), can be needed. However, approaches to provide signal 3 have met with significant limitations.

[0007] One approach to provide cytokine support involves combining CAR-T cell therapy with systemic infusion of recombinant cytokine / cytokine mimetic, and engineering CAR-T cells to secrete / express cytokines extracellularly. Since cytokines are pleiotropic and can also affect the function of other cell types, systemic administration or production of immune-enhancing cytokines by CAR-T cells has at least two major drawbacks: (i) these approaches can lead to systemic toxicity in humans, and (ii) in the context of allogeneic CAR-T cell therapy, these approaches can lead to bystander host immune activation that accelerates rejection of allogeneic CAR-T cells, thereby compromising therapeutic efficacy. Another approach to provide cytokine support is based on introducing a constitutively activated dimerized cytokine receptor IL-7Ra - this limits the nature (only IL-7 signaling) and magnitude of the signaling output. Yet another approach to provide cytokine support involves directly incorporating signal 3 into the CAR molecule (Nat Med. 2018 Mar;24(3):352-359). One limitation of this approach is that the strength of signal 3 depends on the strength of CAR activation. In the absence of target (and CAR activation), signal 3 is not transduced.

[0008] There is a need for a solution that circumvents these drawbacks by specifically targeting cytokine signals to CAR-T cells in a tunable manner, thereby allowing for improved safety and therapeutic efficacy. Provided herein are compositions and methods that address this need. SUMMARY

[0009] The present disclosure provides constitutively active chimeric cytokine receptors (CACCRs). Such CACCRs allow for increased immune cell activation, proliferation, persistence, and / or potency when present on immune cells carrying chimeric antigen receptors (CAR-I cells, e.g., CAR-T cells). Methods of making and using the CACCRs described herein are also provided.

[0010] Accordingly, in one aspect, provided herein is a CACCR consisting of two monomers, each monomer comprising: (a) a transmembrane domain; (b) a Janus kinase (JAK) binding domain; and (c) a recruitment domain, wherein the monomers are constitutively dimerized. In some embodiments, the CACCR does not comprise an extracellular domain ligand binding domain.

[0011] In some embodiments, the transmembrane domain and / or the JAK-binding domain is derived from a TPOR / MPLR receptor. In some embodiments, the transmembrane domain and / or the JAK-binding domain is derived from amino acids 478-582 of a naturally occurring TPOR / MPLR receptor of SEQ ID NO: 6. In some embodiments, the TPOR / MPLR receptor comprises one or more of the amino acid substitutions selected from H499L, S505N, W515K, and G509N. In some embodiments, the TPOR / MPLR receptor comprises H499L, S505N, and W515K substitutions, or S505N and W515K substitutions. In some embodiments, the recruitment domain is a STAT-recruitment domain. In some embodiments, the recruitment domain comprises a STAT-recruitment domain from IL7Ra, e.g., IL7Ra(316-459). In some embodiments, the recruitment domain comprises a STAT-recruitment domain from IL2Rb, e.g., IL2Rb(333-551), IL2Rb(393-433, 518-551), IL2Rb(339-379, 393-433, 518-551), IL2Rb(333-551, Y381S, Y384S, Y387S), IL2Rb(333-551, Y364S, Y381S, Y384S, Y387S). In some embodiments, the recruitment domain comprises a STAT-recruitment domain from IL12Rbl, e.g., IL12Rbl(622-662). In some embodiments, the recruitment domain comprises a STAT-recruitment domain from IL12Rb2, e.g., IL12Rb2(714-862) or IL12Rb2(775-825). In some embodiments, the recruitment domain comprises a STAT-recruitment domain from IL21R, e.g., IL21R(322-538).

[0012] In related aspects, provided herein is a polynucleotide encoding any one of the CACCRs of the present disclosure, as well as expression vectors comprising such polynucleotides. In some embodiments, the polynucleotide further encodes a chimeric antigen receptor (CAR), wherein the CAR binds to BCMA, EGFRvIII, Flt-3, WT-1, CD20, CD23, CD30, CD38, CD70, CD33, CD133, LeY, NKG2D, CS1, CD44v6, ROR1, CD19, Claudin-18.2 (Claudin-18A2 or Claudin18 isoform 2), DLL3 (Delta-like protein 3, Drosophila Delta homolog 3, Delta3), Muc17 (Mucin 17, Muc3, Muc3), FAPa (fibroblast activation protein alpha), Ly6G6D (lymphocyte antigen 6 complex locus protein G6d, c6orf23, G6D, MEGT1, NG25), and / or RNF43 (E3 ubiquitin-protein ligase RNF43, RING finger protein 43).

[0013] In another aspect, provided herein is an engineered immune cell comprising at least one chimeric antigen receptor (CAR) and at least one CACCR of the present disclosure. In some embodiments, the immune cell is a T cell. In some embodiments, the immune cell is an allogeneic immune cell. In other embodiments, the immune cell is an autologous immune cell. The immune cell can be selected from the group consisting of a T cell, a dendritic cell, a killer dendritic cell, a mast cell, an NK cell, a macrophage, a monocyte, a B cell, and an immune cell derived from a stem cell. In related aspects, provided herein is a pharmaceutical composition comprising any of the engineered immune cells of the present disclosure, as well as a kit comprising such a pharmaceutical composition.

[0014] In another aspect, provided herein is a method of treating a cancer in a subject, the method comprising administering to the subject a therapeutically effective amount of any of the engineered immune cells described herein. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 A schematic diagram showing engineered CACCRs of the present disclosure.

[0016] Figure 2 A schematic diagram showing vectors of the present disclosure that can be used to co-express CACCRs and CARs of the present disclosure. One or more cytotails can be connected in tandem to mimic signaling from one or more cytokines. Also shown is a schematic diagram of a vector expressing a control BFP (blue fluorescent protein) CAR.

[0017] Figures 3A-3BIdentification of TpoR transmembrane (TM) mutants that constitutively activate cytokine receptor signaling is shown.

[0018] Figures 4A-4C Results of expanding CAR-T cells co-expressing constitutively active chimeric cytokine receptors are shown.

[0019] Figure 5 Distribution of differentiated and memory T cell subsets in CAR-T cell products under different IL-2 conditions is shown.

[0020] Figures 6A-6B Extent of constitutive cytokine signaling mediated by each TpoR TM variant is shown.

[0021] Figures 7A-7D Cytotoxic activity of TpoR TM mutants is shown, which indicates that constitutive cytokine receptor signaling enhances CAR-T cell potency.

[0022] Figures 8A-8B Cytotoxic activity and persistence of TpoR TM mutants is shown.

[0023] Figure 9 Enrichment of CAR-T cells over time in growth factor-independent assays is shown.

[0024] Figures 10A-10B Fold expansion of CAR-T cells over time in growth factor-independent assays is shown.

[0025] Figure 11 Distribution of memory T cell subsets over time between CAR+ T cells in growth factor-independent assays is shown.

[0026] Figure 12 Activation of STAT signaling pathway by CAR-T cells co-expressing indicated CACCR is shown.

[0027] Figures 13A-13B And Figures 14A-14B Optimization of CACCR signaling strength shown in reporter assays in HEK293 T cells expressing full-length or truncated cytoplasmic tails is shown.

[0028] Figure 15 Optimization of CACCR signaling strength shown in primary CAR-T cells co-expressing full-length or truncated cytoplasmic tails is shown.

[0029] Figures 16A-16C It is shown that CACCR CAR-T cells carrying truncated IL2Rb cytoplasmic tails more closely mimic IL-15 than IL-2 signaling.

[0030] Figures 17A-17D The combined signal output of different cell tails fused in series is shown.

[0031] Figures 18A-18B The effect of CACCR on memory differentiation of CAR-T cells is depicted.

[0032] Figures 19A-19D The effect of CACCR on CAR-T cell survival and memory differentiation under growth factor independent conditions is depicted.

[0033] Figures 20A-20B The cytotoxic activity of CAR-T cells co-expressing multiple CACCRs is depicted.

[0034] Figure 21 It is shown that CACCRs improve the cytotoxic activity of CAR-T cells against the liquid tumor target BCMA.

[0035] Figures 22A-22C It is shown that CACCRs improve the in vivo anti-tumor activity and persistence of BCMA CAR-T cells against orthotopic multiple myeloma.

[0036] Figure 23 It is shown that CACCRs improve the anti-tumor activity of CAR-T cells against established solid tumors. DETAILED DESCRIPTION

[0037] The present disclosure provides constitutively active chimeric cytokine receptors (CACCRs). The presence of constitutively active, tunable chimeric cytokine receptors allows for immune enhancement of signal 3 to meet the need for immune enhancement. Thus, when present on immune cells bearing chimeric antigen receptors (CARs) (CAR-I cells, e.g., CAR-T cells), such CACCRs allow for increased immune cell activation, proliferation, persistence, and / or potency. Methods of making and using the CACCRs described herein are also provided.

[0038] The CACCRs of the present disclosure are tunable and have flexible cytokine signaling outputs for enhancing CAR-T cell activity, persistence, etc. Their components, methods of making, and methods of use are presented in turn below.

[0039] I. Constitutively Active Chimeric Cytokine Receptors (CACCRs)

[0040] The CACCRs of the present disclosure are composed of two monomers, each monomer comprising: (a) a transmembrane domain; (b) a JAK-binding domain; and (c) a recruitment domain, wherein the monomers are constitutively dimerized. In some embodiments, the CACCRs of the present disclosure do not include an extracellular ligand-binding domain.

[0041] In some embodiments, the monomers are identical, resulting in a constitutively active homodimer. In such embodiments, the amount of protein that needs to be expressed in the vector is reduced. In some embodiments, the monomers are different, resulting in a constitutively active heterodimer, which may be desirable in certain situations.

[0042] The monomers of the CACCR disclosed herein can spontaneously dimerize and can activate signal transduction without any exogenous stimulation or ligands (ligand-independent dimerization). The activity level can be controlled by introducing mutations into the transmembrane domain of CACCR. Those skilled in the art will understand that the monomers of CACCR do not dimerize 100% of the time and can exist as monomers.

[0043] A. Transmembrane domain

[0044] The CACCR disclosed herein includes a transmembrane domain. The transmembrane domain contains sequences that allow constitutive dimerization with monomer pairs, thereby enabling constitutive JAK activation on the intracellular portion and constitutive recruitment and phosphorylation of receptor cytoplasmic regions, such as STAT.

[0045] The transmembrane domain is located at the N-terminus and coupled to the intracellular / cytoplasmic domain at the C-terminus. In some embodiments, the coupling is optionally achieved via a adapter.

[0046] As used herein, transmembrane domains are capable of inserting into the membrane of a cell, where they are expressed. In some embodiments, the transmembrane domains of this disclosure span the cell membrane and include extracellular and / or intracellular portions.

[0047] In some embodiments, the transmembrane domains of this disclosure are engineered (synthetic) and do not resemble any naturally occurring transmembrane domains, for example, the transmembrane domains of this disclosure are non-naturally occurring.

[0048] In other embodiments, the transmembrane domains of this disclosure are derived from naturally occurring receptors.

[0049] In some embodiments, the transmembrane domain and / or JAK-activating domain of the present disclosure is derived from, for example, one or more of the following receptors: erythropoietin receptor (EpoR), interleukin 6 signal transducer (GP130 or IL6ST), prolactin receptor (PrlR), growth hormone receptor (GHR), granulocyte colony-stimulating factor receptor (GCSFR), and thrombopoietin receptor / myeloproliferative leukemia protein receptor (TPOR / MPLR). When derived from a naturally occurring receptor, the entire receptor or the entire transmembrane sequence of the receptor can not be necessary to achieve constitutive activation on the intracellular portion and constitutive JAK binding / activation. Fragments of the naturally occurring receptor can thus be utilized. In addition, certain mutations can be introduced into the transmembrane domain derived from the natural receptor to further tune downstream signaling.

[0050] In some embodiments, the transmembrane domain and / or JAK-activating domain of the present disclosure is derived from a naturally occurring EpoR receptor.

[0051] In some embodiments, the transmembrane domain and / or JAK-activating domain of the present disclosure is derived from a naturally occurring GP130 receptor.

[0052] In some embodiments, the transmembrane domain and / or JAK-activating domain of the present disclosure is derived from a naturally occurring PrlR receptor.

[0053] In some embodiments, the transmembrane domain and / or JAK-activating domain of the present disclosure is derived from a naturally occurring GHR receptor.

[0054] In some embodiments, the transmembrane domain and / or JAK-activating domain of the present disclosure is derived from a naturally occurring GCSF receptor.

[0055] In some embodiments, the transmembrane domain and / or JAK-activating domain of the present disclosure is derived from a naturally occurring TPOR receptor.

[0056] Table la provides exemplary full-length sequences of naturally occurring receptors from which the transmembrane proteins provided in the present disclosure are derived. The sequences provided in Table la are reference sequences against which mutations are expressed, for example, in Tables lb and lc.

[0057] Table la: Exemplary naturally occurring receptors

[0058]

[0059]

[0060]

[0061]

[0062] In some embodiments, the transmembrane domain of the present disclosure is derived from a naturally occurring TPOR / MPLR (myelopro liferative leukemia protein) receptor shown in Table la.

[0063] In some embodiments, the transmembrane domain of the CACCR comprises amino acids 478-582 of the TPOR receptor of Table la.

[0064] Table lb provides exemplary transmembrane domain amino acid sequences of the present disclosure, wherein the transmembrane domain is derived from a naturally occurring TPOR receptor.

[0065] Table lb: Exemplary transmembrane domain amino acid sequences

[0066]

[0067] In some embodiments, the transmembrane domain of the CACCR comprises amino acids 478-582 of the TPOR receptor, and an amino acid substitution at least at H499. In some embodiments, the transmembrane domain of the CACCR comprises amino acids 478-582 of the TPOR receptor, and the amino acid substitution H499L.

[0068] In some embodiments, the transmembrane domain of the CACCR comprises amino acids 478-582 of the TPOR receptor, and an amino acid substitution at least at S505. In some embodiments, the transmembrane domain of the CACCR comprises amino acids 478-582 of the TPOR receptor, and the amino acid substitution S505N.

[0069] In some embodiments, the transmembrane domain of the CACCR comprises amino acids 478-582 of the TPOR receptor, and an amino acid substitution at least at G509. In some embodiments, the transmembrane domain of the CACCR comprises amino acids 478-582 of the TPOR receptor, and the amino acid substitution G509N.

[0070] In some embodiments, the transmembrane domain of the CACCR comprises amino acids 478-582 of the TPOR receptor, and an amino acid substitution at least at W515. In some embodiments, the transmembrane domain of the CACCR comprises amino acids 478-582 of the TPOR receptor, and the amino acid substitution W515K.

[0071] In some embodiments, the transmembrane domain of the CACCR comprises amino acids 478-582 of the TPOR receptor, and amino acid substitutions at H499 and S505 (sequences provided in Table lb).

[0072] In some embodiments, the transmembrane domain of CACCR comprises amino acids 478-582 of the TPOR receptor, and amino acid substitutions at H499 and W515 (sequence provided in Table lb).

[0073] In some embodiments, the transmembrane domain of CACCR comprises amino acids 478-582 of the TPOR receptor, and amino acid substitutions at H499, S505 and W515 (sequence provided in Table lb).

[0074] In some embodiments, the transmembrane domain of CACCR comprises amino acids 478-582 of the TPOR receptor, and amino acid substitutions at S505 and W515 (sequence provided in Table lb).

[0075] In some embodiments, the transmembrane domain of CACCR comprises amino acids 478-582 of the TPOR receptor, and amino acid substitutions at H499 and G509 (sequence provided in Table lb).

[0076] In some embodiments, the transmembrane domain of CACCR comprises amino acids 478-582 of the TPOR receptor, and amino acid substitutions H499L and S505N (sequence provided in Table lb).

[0077] In some embodiments, the transmembrane domain of CACCR comprises amino acids 478-582 of the TPOR receptor, and amino acid substitutions H499L and W515K (sequence provided in Table lb).

[0078] In some embodiments, the transmembrane domain of CACCR comprises amino acids 478-582 of the TPOR receptor, and amino acid substitutions H499L and G509N (sequence provided in Table lb).

[0079] In some embodiments, the transmembrane domain of CACCR comprises amino acids 478-582 of the TPOR receptor, and amino acid substitutions S505N and W515K (sequence provided in Table lb).

[0080] In some embodiments, the transmembrane domain of CACCR comprises amino acids 478-582 of the TPOR receptor, and amino acid substitutions H499L, S505N and W515K (sequence provided in Table lb).

[0081] In some embodiments, the transmembrane domain of CACCR comprises amino acids 478-582 of the TPOR receptor, and amino acid substitutions at H499 and S505 (sequence provided in Table lb).

[0082] The CACCR of the present disclosure is tunable to achieve the level of signal 3 / immune enhancement desired and under particular contexts or conditions in CAR-bearing immune cells, e.g., CAR-T cells.

[0083] In some embodiments, a low level of STAT5 activation is desired in CAR-bearing immune cells, e.g., CAR-T cells. For example, in such embodiments, the transmembrane domain of the CACCR can be introduced that includes amino acids 478-582 of the TPOR receptor and the amino acid substitutions S505N, W515K, or H499L / G509N.

[0084] In some embodiments, an increased level of STAT5 activation is desired in CAR-bearing immune cells, e.g., CAR-T cells. For example, in such embodiments, the transmembrane domain of the CACCR can be introduced that includes amino acids 478-582 of the TPOR receptor and the amino acid substitutions H499L, S505N, and W515K. For example, in such embodiments, the transmembrane domain of the CACCR can be introduced that includes amino acids 478-582 of the TPOR receptor and the amino acid substitutions S505N and W515K.

[0085] In some embodiments, increased differentiation to memory T cells is desired in CAR-bearing immune cells, e.g., CAR-T cells. For example, in such embodiments, the transmembrane domain of the CACCR can be introduced that includes amino acids 478-582 of the TPOR receptor and the amino acid substitution W515K or H499L / G509N.

[0086] In some embodiments, increased differentiation to memory T cells is desired in CAR-bearing immune cells, e.g., CAR-T cells. For example, in such embodiments, the transmembrane domain of the CACCR can be introduced that includes amino acids 478-582 of the TPOR receptor and the amino acid substitutions S505N / W515K and H499L / S505N / W515K.

[0087] Also provided herein are substitutions that increase cytotoxic potency, persistence of response, and increase persistence, e.g., S505N / W515K and H499L / S505N / W515K substitutions.

[0088] Table 1c: Exemplary transmembrane + JAK2 binding domain sequences

[0089]

[0090]

[0091]

[0092] B. JAK-binding domain

[0093] The CACCRs of the present disclosure include an intracellular JAK-binding domain. The JAK-binding domain is coupled directly or through a linker to the C-terminus of the transmembrane domain. The JAK-binding domain is coupled to the transmembrane domain on the intracellular side of the chimeric cytokine receptor.

[0094] In some embodiments, the JAK-binding domain is a JAK-1 binding domain, a JAK-2 binding domain, a JAK-3 binding domain, or a TYK2 binding domain.

[0095] In some embodiments, the JAK-binding domain of the CACCRs of the present disclosure is naturally occurring and is derived from a naturally occurring receptor.

[0096] In some embodiments, the JAK-binding domain of the CACCRs of the present disclosure is synthetic.

[0097] Table lb and Table lc provide exemplary amino acid sequences of transmembrane and JAK2 binding domains of the present disclosure. In some embodiments, the CACCRs of the present disclosure include a transmembrane and JAK2 binding domain that includes an amino acid sequence selected from the sequences in Table lb and lc. In some embodiments, the CACCRs of the present disclosure include a transmembrane and JAK2 binding domain that includes an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to any of the sequences in Table lb and lc.

[0098] C. Recruitment domain

[0099] The CACCRs of the present disclosure include a cytoplasmic recruitment domain. The recruitment domain can be a STAT-recruitment domain, an API -recruitment domain, a Myc / Max-recruitment domain; or a NFkB-recruitment domain. In some embodiments, the recruitment domain is a signal transducer and activator of transcription (STAT)-recruitment (STAT-activating) domain, for example from a receptor tail (cell tail) or from a cytokine receptor tail. These intracellular recruitment domains of the CACCRs of the present disclosure allow signal 3 to propagate in an immune cell comprising a CAR and a chimeric cytokine receptor (e.g. a CAR-T cell having a chimeric cytokine receptor of the present disclosure). Cytokine signaling propagated by the Stat-recruitment domain allows cytokine-based immune enhancement of the cell. In some embodiments, the immune enhancement is homeostatic, e.g. the signaling results in an increase in CAR-bearing immune cells. In some embodiments, the immune enhancement is inflammatory, e.g. the signaling results in an increase in potency of CAR-bearing immune cells. In some embodiments, the immune enhancement prevents exhaustion, e.g. the signaling maintains long-term function of CAR-bearing immune cells.

[0100] In some embodiments, the recruitment domain of the present disclosure is synthetic and does not resemble any naturally occurring receptor fragment. In some embodiments, the immune enhancement is homeostatic, e.g. the signaling maintains long-term function of CAR-bearing immune cells.

[0101] In some embodiments, the Stat-recruitment domain of the present disclosure is synthetic and does not resemble any naturally occurring receptor fragment.

[0102] In other embodiments, the Stat-recruitment domain of the present disclosure is derived from a cytoplasmic tail of a naturally occurring receptor, e.g. from a naturally occurring cytokine receptor. These cytoplasmic tails of naturally occurring receptors can be regions downstream of the JAK-activating domain of the receptor transmembrane domain. The Stat-recruitment domain of the chimeric cytokine receptor includes at least one STAT-recruitment domain from at least one receptor. In some embodiments, the Stat-recruitment domain includes at least one STAT1 -recruitment domain. In some embodiments, the Stat-recruitment domain includes at least one STAT2-recruitment domain. In some embodiments, the Stat-recruitment domain includes at least one STAT3-recruitment domain. In some embodiments, the Stat-recruitment domain includes at least one STAT4-recruitment domain. In some embodiments, the Stat-recruitment domain includes at least one STAT5-recruitment domain. In some embodiments, the Stat-recruitment domain includes at least one STAT6-recruitment domain. In some embodiments, the Stat-recruitment domain includes at least one STAT7-recruitment domain.

[0103] In some embodiments, the naturally occurring receptor from which the Stat-recruiting domain is derived is not a cytokine receptor.

[0104] In some embodiments, the naturally occurring receptor from which the Stat-recruiting domain is derived is a cytokine receptor. Exemplary cytokine receptors through which T cell immunity enhancing cytokines signal include, but are not limited to, IL-2 receptor, IL-7 receptor, IL-15 receptor, and IL-21 receptor. In alternative embodiments, the receptor from which the Stat-recruiting domain is derived is not a cytokine receptor. By selecting a Stat-recruiting domain of CACCR, the receptor can be redirected to a selected signaling.

[0105] In some embodiments, the CACCR of the present disclosure comprises a recruiting domain linked to the C-terminus of the transmembrane / JAK2 binding domain with or without a linker. In some embodiments, the linker comprises one or more amino acid residues.

[0106] Table 2a provides exemplary receptors from which the recruiting domain of the CACCR of the present disclosure is derived. Table 2b provides exemplary amino acid sequences of the recruiting domains of the present disclosure. In some embodiments, the CACCR of the present disclosure comprises a recruiting domain comprising an amino acid sequence selected from one or more of the receptor sequences in Table 2b. In some embodiments, the CACCR of the present disclosure comprises a recruiting domain comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to any of the sequences in Table 2b.

[0107] Table 2a

[0108] Sources of Recruitment Domains BLNK IL2RG EGFR EpoR GHR IFNAR1 IFNAR2 IFNAR1 / 2 IFNLR1 IL10R1 IL12Rb1 IL12Rb2 IL21R IL2Rb IL2small IL7R IL7Ra IL9R IL15R IL21R

[0109] Table 2b. Recruiting domain (cell tail) sequences

[0110]

[0111]

[0112]

[0113]

[0114]

[0115] In some embodiments, the Stat-recruiting domain of the CACCR of the present disclosure comprises a STAT-recruiting domain from one receptor.

[0116] To generate multiple outputs, two or more STAT-recruiting domains can be connected in tandem to mimic signaling from one or more cytokines.

[0117] In some embodiments, two or more STAT-recruiting domains can be connected in tandem with or without a linker. In some embodiments, the linker comprises one or more amino acid residues.

[0118] In some embodiments, a STAT-recruiting domain comprises a portion of more than one receptor, e.g., comprises more than one STAT-recruiting domain. In such embodiments, tandem cytokine signaling domains are provided, allowing for enhanced signaling. Thus, in some embodiments, a STAT-recruiting domain of a monomer of a CACCR of the present disclosure comprises STAT-recruiting domains from more than one receptor, e.g., comprises STAT-recruiting domains from two, three, four, five, or even six receptors. For example, in some embodiments, STAT-recruiting domains can be connected in tandem to stimulate multiple pathways (e.g., IL7R(316-459)-IL12Rb2(775-825) fragment fusion for pro-persistence STAT5 and pro-inflammatory STAT4; IL7R(316-459)-IL2Rbsmall(393-433, 518-551) for pro-persistence; IL7R(316-459)-EGFR(1122-1165) for pro-persistence and anti-fatigue; IL2Rbsmall(393-433, 518-551)-EGFR(1122-1165) for pro-persistence and anti-fatigue).

[0119] When multiple outputs are generated, the proximity of individual STAT-recruiting domains to the cell membrane can affect the strength of their respective signaling outputs. Table 2c shows examples of CACCRs with dual outputs, where each output can be placed proximal or distal to the cell membrane. In some embodiments, a CACCR of the present disclosure comprises a recruiting domain with dual outputs selected from Table 2c.

[0120] Table 2c: Examples of CACCRs with dual outputs

[0121]

[0122]

[0123]

[0124] Without being bound by theory or mechanism, in some embodiments, a JAK-protein (JAK1, JAK2, JAK3, or TYK2) binds to a dimerized CACCR of the present disclosure. Two bound JAK-proteins are activated, which can phosphorylate tyrosine residues on the CACCR recruitment domain. The phosphorylated recruitment domain can then bind to recruited proteins (e.g., the phosphorylated STAT-recruitment domain binds to STAT-proteins), which in turn enables transcriptional events in the nucleus.

[0125] D. Exemplary CACCRs

[0126] Table 3 shows exemplary CACCR sequences of the present disclosure. The receptor can be expressed with a signal sequence, such as CD8SS of sequence MALPVTALLLPLALLLHAARP (SEQ ID NO: 89).

[0127] In some embodiments, a CACCR of the present disclosure comprises any of the sequences in Table 3. In some embodiments, the CACCR comprises an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% identical to any of the amino acid sequences in SEQ ID NOs: 90-98 and 107-139. In some embodiments, the TPOR / MPLR receptor comprises any of the amino acid sequences in SEQ ID NOs: 90-98 and 107-139.

[0128] In some embodiments, the CACCR comprises a transmembrane domain and / or a JAK-binding domain derived from a TPOR / MPLR receptor. In some embodiments, a CACCR of the present disclosure comprises amino acids 478-582 of a naturally occurring TPOR / MPLR receptor of SEQ ID NO: 6. In some embodiments, a CACCR of the present disclosure comprises an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 17. In some embodiments, a CACCR of the present disclosure comprises the amino acid sequence of SEQ ID NO: 17. In some embodiments, the CACCR further comprises a recruitment domain comprising an amino acid sequence of one or more receptor sequences presented in Table 2b. In some embodiments, the CACCR further comprises one or more recruitment domains selected from the group consisting of: STAT-recruitment domains from IL7Ra, IL2Rb, IL12Rbl, IL12Rb2, and IL21R. In some embodiments, the recruitment domain comprises a STAT-recruitment domain from IL7Ra. In some embodiments, the STAT-recruitment domain from IL7Ra comprises an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 46, 68, 69, 70, 71, or 72. In some embodiments, the STAT-recruitment domain from IL7Ra comprises the amino acid sequence of SEQ ID NO: 46, 68, 69, 70, 71, or 72. In some embodiments, the recruitment domain comprises a STAT-recruitment domain from IL2Rb. In some embodiments, the STAT-recruitment domain from IL2Rb comprises an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 47, 73, 74, 75, 76, 77, 78, 106, or 143. In some embodiments, the STAT-recruitment domain from IL2Rb comprises the amino acid sequence of SEQ ID NO: 47, 73, 74, 75, 76, 77, 78, 106, or 143. In some embodiments, the recruitment domain comprises a STAT-recruitment domain from IL12Rbl or IL12Rb2. In some embodiments, the STAT-recruitment domain from IL12Rbl or IL12Rb2 comprises an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 67, 86, or 87.In some embodiments, the STAT-recruiting domain from IL12Rbl or IL12Rb2 comprises the amino acid sequence of SEQ ID NO: 67, 86, or 87. In some embodiments, the recruiting domain comprises a STAT-recruiting domain from IL21R. In some embodiments, the STAT-recruiting domain from IL21R comprises an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 54. In some embodiments, the STAT-recruiting domain from IL21R comprises the amino acid sequence of SEQ ID NO: 54. In some embodiments, the CACCR comprises one or more recruiting domains presented in Table 2c. In some embodiments, the recruiting domain comprises STAT-recruiting domains from IL7Ra and IL2Rb. In some embodiments, the recruiting domain comprises STAT-recruiting domains from IL7Ra and IL12Rbl. In some embodiments, the recruiting domain comprises STAT-recruiting domains from IL7Ra and IL12Rb2. In some embodiments, the CACCR comprises an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 90 or 119, with or without a signal sequence. In some embodiments, the CACCR comprises the amino acid sequence of SEQ ID NO: 90 or 119, with or without a signal sequence.

[0129] In some embodiments, the CACCR of the present disclosure comprises a transmembrane domain and / or a JAK-binding domain from a TPOR / MPLR receptor comprising one or more amino acid substitutions at H499, S505, G509, or W515. In some embodiments, the TPOR / MPLR receptor comprises a H499L substitution. In some embodiments, the TPOR / MPLR receptor comprises a S505N substitution. In some embodiments, the TPOR / MPLR receptor comprises a G509N substitution. In some embodiments, the TPOR / MPLR receptor comprises a W515K substitution. In some embodiments, the CACCR further comprises a recruitment domain comprising an amino acid sequence of one or more receptor sequences presented in Table 2b. In some embodiments, the CACCR further comprises one or more recruitment domains selected from the group consisting of: STAT-recruitment domains from IL7Ra, IL2Rb, IL12Rb1, IL12Rb2, and IL21R. In some embodiments, the recruitment domain comprises a STAT-recruitment domain from IL7Ra. In some embodiments, the STAT-recruitment domain from IL7Ra comprises an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 46, 68, 69, 70, 71, or 72. In some embodiments, the STAT-recruitment domain from IL7Ra comprises the amino acid sequence of SEQ ID NO: 46, 68, 69, 70, 71, or 72. In some embodiments, the recruitment domain comprises a STAT-recruitment domain from IL2Rb. In some embodiments, the STAT-recruitment domain from IL2Rb comprises an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 47, 73, 74, 75, 76, 77, 78, 106, or 143. In some embodiments, the STAT-recruitment domain from IL2Rb comprises the amino acid sequence of SEQ ID NO: 47, 73, 74, 75, 76, 77, 78, 106, or 143. In some embodiments, the recruitment domain comprises a STAT-recruitment domain from IL12Rb1 or IL12Rb2. In some embodiments, the STAT-recruitment domain from IL12Rb1 or IL12Rb2 comprises an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 67, 86, or 87.In some embodiments, the STAT-recruiting domain from IL12Rbl or IL12Rb2 comprises the amino acid sequence of SEQ ID NO: 67, 86, or 87. In some embodiments, the recruiting domain comprises a STAT-recruiting domain from IL21R. In some embodiments, the STAT-recruiting domain from IL21R comprises an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 54. In some embodiments, the STAT-recruiting domain from IL21R comprises the amino acid sequence of SEQ ID NO: 54. In some embodiments, the CACCR comprises one or more recruiting domains presented in Table 2c. In some embodiments, the recruiting domain comprises STAT-recruiting domains from IL7Ra and IL2Rb. In some embodiments, the recruiting domain comprises STAT-recruiting domains from IL7Ra and IL12Rbl. In some embodiments, the recruiting domain comprises STAT-recruiting domains from IL7Ra and IL12Rb2. In some embodiments, the CACCR comprises an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 92, 94, 121, or 123, with or without a signal sequence. In some embodiments, the CACCR comprises the amino acid sequence of SEQ ID NO: 92, 94, 121, or 123, with or without a signal sequence.

[0130] In some embodiments, the CACCR of the present disclosure comprises a transmembrane domain and / or a JAK-binding domain from a TPOR / MPLR receptor, including H499L and S505N substitutions. In some embodiments, the CACCR further comprises a recruitment domain comprising an amino acid sequence of one or more receptor sequences presented in Table 2b. In some embodiments, the CACCR further comprises one or more recruitment domains selected from the group consisting of: STAT-recruitment domains from IL7Ra, IL2Rb, IL12Rbl, IL12Rb2, and IL21R. In some embodiments, the recruitment domain comprises a STAT-recruitment domain from IL7Ra. In some embodiments, the STAT-recruitment domain from IL7Ra comprises an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 46, 68, 69, 70, 71, or 72. In some embodiments, the STAT-recruitment domain from IL7Ra comprises the amino acid sequence of SEQ ID NO: 46, 68, 69, 70, 71, or 72. In some embodiments, the recruitment domain comprises a STAT-recruitment domain from IL2Rb. In some embodiments, the STAT-recruitment domain from IL2Rb comprises an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 47, 73, 74, 75, 76, 77, 78, 106, or 143. In some embodiments, the STAT-recruitment domain from IL2Rb comprises the amino acid sequence of SEQ ID NO: 47, 73, 74, 75, 76, 77, 78, 106, or 143. In some embodiments, the recruitment domain comprises a STAT-recruitment domain from IL12Rbl or IL12Rb2. In some embodiments, the STAT-recruitment domain from IL12Rbl or IL12Rb2 comprises an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 67, 86, or 87. In some embodiments, the STAT-recruitment domain from IL12Rbl or IL12Rb2 comprises the amino acid sequence of SEQ ID NO: 67, 86, or 87. In some embodiments, the recruitment domain comprises a STAT-recruitment domain from IL21R.In some embodiments, the STAT-recruiting domain from IL21R comprises an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 54. In some embodiments, the STAT-recruiting domain from IL21R comprises the amino acid sequence of SEQ ID NO: 54. In some embodiments, the CACCR comprises one or more recruiting domains presented in Table 2c. In some embodiments, the recruiting domains comprise STAT-recruiting domains from IL7Ra and IL2Rb. In some embodiments, the recruiting domains comprise STAT-recruiting domains from IL7Ra and IL12Rb1. In some embodiments, the recruiting domains comprise STAT-recruiting domains from IL7Ra and IL12Rb2. In some embodiments, the CACCR comprises an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 91, 98, 120, or 127, with or without a signal sequence. In some embodiments, the CACCR comprises the amino acid sequence of SEQ ID NO: 91, 98, 120, or 127, with or without a signal sequence.

[0131] In some embodiments, the CACCR of the present disclosure comprises a transmembrane domain and / or a JAK-binding domain from a TPOR / MPLR receptor comprising H499L and W515K substitutions or H499L and G509N substitutions. In some embodiments, the CACCR further comprises a recruiting domain comprising an amino acid sequence of one or more receptor sequences presented in Table 2b. In some embodiments, the CACCR further comprises one or more recruiting domains selected from the group consisting of STAT-recruiting domains from IL7Ra, IL2Rb, IL12Rb1, IL12Rb2, and IL21R. In some embodiments, the recruiting domains comprise STAT-recruiting domains from IL7Ra. In some embodiments, the STAT-recruiting domain from IL7Ra comprises an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 46, 68, 69, 70, 71, or 72.

[0132] In some embodiments, the STAT-recruiting domain from IL7Ra comprises the amino acid sequence of SEQ ID NO: 46, 68, 69, 70, 71, or 72. In some embodiments, the recruiting domains comprise STAT-recruiting domains from IL2Rb.

[0133] In some embodiments, the STAT-recruiting domain from IL2Rb comprises an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 47, 73, 74, 75, 76, 77, 78, 106, or 143. In some embodiments, the STAT-recruiting domain from IL2Rb comprises the amino acid sequence of SEQ ID NO: 47, 73, 74, 75, 76, 77, 78, 106, or 143. In some embodiments, the recruiting domain comprises a STAT-recruiting domain from IL12Rbl or IL12Rb2. In some embodiments, the STAT-recruiting domain from IL12Rbl or IL12Rb2 comprises an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 67, 86, or 87. In some embodiments, the STAT-recruiting domain from IL12Rbl or IL12Rb2 comprises the amino acid sequence of SEQ ID NO: 67, 86, or 87. In some embodiments, the recruiting domain comprises a STAT-recruiting domain from IL21R. In some embodiments, the STAT-recruiting domain from IL21R comprises an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 54. In some embodiments, the STAT-recruiting domain from IL21R comprises the amino acid sequence of SEQ ID NO: 54. In some embodiments, the CACCR comprises one or more recruiting domains presented in Table 2c. In some embodiments, the recruiting domain comprises a STAT-recruiting domain from IL7Ra and IL2Rb. In some embodiments, the recruiting domain comprises a STAT-recruiting domain from IL7Ra and IL12Rbl. In some embodiments, the recruiting domain comprises a STAT-recruiting domain from IL7Ra and IL12Rb2. In some embodiments, the CACCR comprises an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 97 or 126, with or without a signal sequence. In some embodiments, the CACCR comprises the amino acid sequence of SEQ ID NO: 97 or 126, with or without a signal sequence.

[0134] In some embodiments, the CACCR of the present disclosure comprises a transmembrane domain and / or a JAK-binding domain from a TPOR / MPLR receptor comprising S505N and W515K substitutions. In some embodiments, the CACCR further comprises a recruitment domain comprising an amino acid sequence of one or more receptor sequences presented in Table 2b. In some embodiments, the CACCR further comprises one or more recruitment domains selected from the group consisting of: STAT-recruitment domains from IL7Ra, IL2Rb, IL12Rbl, IL12Rb2, and IL21R. In some embodiments, the recruitment domain comprises a STAT-recruitment domain from IL7Ra. In some embodiments, the STAT-recruitment domain from IL7Ra comprises an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 46, 68, 69, 70, 71, or 72. In some embodiments, the STAT-recruitment domain from IL7Ra comprises the amino acid sequence of SEQ ID NO: 46, 68, 69, 70, 71, or 72. In some embodiments, the recruitment domain comprises a STAT-recruitment domain from IL2Rb. In some embodiments, the STAT-recruitment domain from IL2Rb comprises an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 47, 73, 74, 75, 76, 77, 78, 106, or 143. In some embodiments, the STAT-recruitment domain from IL2Rb comprises the amino acid sequence of SEQ ID NO: 47, 73, 74, 75, 76, 77, 78, 106, or 143. In some embodiments, the recruitment domain comprises a STAT-recruitment domain from IL12Rbl or IL12Rb2. In some embodiments, the STAT-recruitment domain from IL12Rbl or IL12Rb2 comprises an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 67, 86, or 87. In some embodiments, the STAT-recruitment domain from IL12Rbl or IL12Rb2 comprises the amino acid sequence of SEQ ID NO: 67, 86, or 87. In some embodiments, the recruitment domain comprises a STAT-recruitment domain from IL21R.In some embodiments, the STAT-recruiting domain from IL21R comprises an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 54. In some embodiments, the STAT-recruiting domain from IL21R comprises the amino acid sequence of SEQ ID NO: 54. In some embodiments, the CACCR comprises one or more recruiting domains presented in Table 2c. In some embodiments, the recruiting domains comprise STAT-recruiting domains from IL7Ra and IL2Rb. In some embodiments, the recruiting domains comprise STAT-recruiting domains from IL7Ra and IL12Rb1. In some embodiments, the recruiting domains comprise STAT-recruiting domains from IL7Ra and IL12Rb2. In some embodiments, the CACCR comprises an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 96, 107, 109, 111, 113, 115, 117, 125, 128, 129, 132, 134, 136, or 138, with or without a signal sequence. In some embodiments, the CACCR comprises the amino acid sequence of SEQ ID NO: 96, 107, 109, 111, 113, 115, 117, 125, 128, 129, 132, 134, 136, or 138, with or without a signal sequence.

[0135] In some embodiments, the CACCR of the present disclosure comprises a transmembrane domain and / or a JAK-binding domain from a TPOR / MPLR receptor, including H499L and W515K substitutions. In some embodiments, the CACCR further comprises a recruitment domain comprising an amino acid sequence of one or more receptor sequences presented in Table 2b. In some embodiments, the CACCR further comprises one or more recruitment domains selected from the group consisting of: STAT-recruitment domains from IL7Ra, IL2Rb, IL12Rbl, IL12Rb2, and IL21R. In some embodiments, the recruitment domain comprises a STAT-recruitment domain from IL7Ra. In some embodiments, the STAT-recruitment domain from IL7Ra comprises an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 46, 68, 69, 70, 71, or 72. In some embodiments, the STAT-recruitment domain from IL7Ra comprises the amino acid sequence of SEQ ID NO: 46, 68, 69, 70, 71, or 72. In some embodiments, the recruitment domain comprises a STAT-recruitment domain from IL2Rb. In some embodiments, the STAT-recruitment domain from IL2Rb comprises an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 47, 73, 74, 75, 76, 77, 78, 106, or 143. In some embodiments, the STAT-recruitment domain from IL2Rb comprises the amino acid sequence of SEQ ID NO: 47, 73, 74, 75, 76, 77, 78, 106, or 143. In some embodiments, the recruitment domain comprises a STAT-recruitment domain from IL12Rbl or IL12Rb2. In some embodiments, the STAT-recruitment domain from IL12Rbl or IL12Rb2 comprises an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 67, 86, or 87. In some embodiments, the STAT-recruitment domain from IL12Rbl or IL12Rb2 comprises the amino acid sequence of SEQ ID NO: 67, 86, or 87. In some embodiments, the recruitment domain comprises a STAT-recruitment domain from IL21R.In some embodiments, the STAT-recruiting domain from IL21R comprises an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 54. In some embodiments, the STAT-recruiting domain from IL21R comprises the amino acid sequence of SEQ ID NO: 54. In some embodiments, the CACCR comprises one or more recruiting domains presented in Table 2c. In some embodiments, the recruiting domains comprise STAT-recruiting domains from IL7Ra and IL2Rb. In some embodiments, the recruiting domains comprise STAT-recruiting domains from IL7Ra and IL12Rb1. In some embodiments, the recruiting domains comprise STAT-recruiting domains from IL7Ra and IL12Rb2. In some embodiments, the CACCR comprises an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 93, with or without a signal sequence. In some embodiments, the CACCR comprises the amino acid sequence of SEQ ID NO: 93, with or without a signal sequence.

[0136] In some embodiments, the CACCR of the present disclosure comprises a transmembrane domain and / or a JAK-binding domain from a TPOR / MPLR receptor comprising H499L, S505N, and W515K substitutions. In some embodiments, the CACCR further comprises a recruitment domain comprising an amino acid sequence of one or more receptor sequences presented in Table 2b. In some embodiments, the CACCR further comprises one or more recruitment domains selected from the group consisting of: STAT-recruitment domains from IL7Ra, IL2Rb, IL12Rbl, IL12Rb2, and IL21R. In some embodiments, the recruitment domain comprises a STAT-recruitment domain from IL7Ra. In some embodiments, the STAT-recruitment domain from IL7Ra comprises an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 46, 68, 69, 70, 71, or 72. In some embodiments, the STAT-recruitment domain from IL7Ra comprises the amino acid sequence of SEQ ID NO: 46, 68, 69, 70, 71, or 72. In some embodiments, the recruitment domain comprises a STAT-recruitment domain from IL2Rb. In some embodiments, the STAT-recruitment domain from IL2Rb comprises an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 47, 73, 74, 75, 76, 77, 78, 106, or 143. In some embodiments, the STAT-recruitment domain from IL2Rb comprises the amino acid sequence of SEQ ID NO: 47, 73, 74, 75, 76, 77, 78, 106, or 143. In some embodiments, the recruitment domain comprises a STAT-recruitment domain from IL12Rbl or IL12Rb2. In some embodiments, the STAT-recruitment domain from IL12Rbl or IL12Rb2 comprises an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 67, 86, or 87. In some embodiments, the STAT-recruitment domain from IL12Rbl or IL12Rb2 comprises the amino acid sequence of SEQ ID NO: 67, 86, or 87. In some embodiments, the recruitment domain comprises a STAT-recruitment domain from IL21R.In some embodiments, the STAT-recruiting domain from IL21R comprises an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 54. In some embodiments, the STAT-recruiting domain from IL21R comprises the amino acid sequence of SEQ ID NO: 54. In some embodiments, the CACCR comprises one or more recruiting domains presented in Table 2c. In some embodiments, the recruiting domains comprise STAT-recruiting domains from IL7Ra and IL2Rb. In some embodiments, the recruiting domains comprise STAT-recruiting domains from IL7Ra and IL12Rb1. In some embodiments, the recruiting domains comprise STAT-recruiting domains from IL7Ra and IL12Rb2. In some embodiments, the CACCR comprises an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 95, 108, 110, 112, 114, 116, 118, 124, 130, 131, 133, 135, 137, or 139, with or without a signal sequence. In some embodiments, the CACCR comprises the amino acid sequence of SEQ ID NO: 95, 108, 110, 112, 114, 116, 118, 124, 130, 131, 133, 135, 137, or 139, with or without a signal sequence.

[0137] Table 3

[0138]

[0139]

[0140]

[0141]

[0142]

[0143]

[0144]

[0145]

[0146] The underlined LE and SR are exemplary optional linkers that can be inserted between two domains.

[0147] E. Expression of CACCR

[0148] Provided herein are polynucleotides encoding any one of the CACCRs provided herein. Also provided herein are expression vectors comprising such polynucleotides. In some embodiments, the vector is a viral vector. In some embodiments, the vector is not a viral vector.

[0149] In some embodiments, the expression vector comprises a CACCR and a polynucleotide expressing a chimeric antigen receptor (CAR).

[0150] In some embodiments, expression of the CACCR and the CAR are expressed as a single polypeptide chain, separated by a linker. Figure 2 A schematic of a vector that can be used to co-express a CACCR and a CAR of the disclosure is shown. One or more recruitment domains can be linked in tandem to mimic signaling from one or more cytokines.

[0151] II. CAR- bearing immune cells

[0152] Provided herein are engineered immune cells comprising polynucleotides encoding a chimeric antigen receptor (CAR) and a CACCR of the disclosure; and provided herein are engineered immune cells expressing a chimeric antigen receptor (CAR-I cell) and a CACCR of the disclosure. Examples of immune cells include T cells (e.g., alpha / beta T cells and gamma / delta T cells), B cells, natural killer (NK) cells, natural killer T (NKT) cells, invariant NKT cells, mast cells, myeloid phagocytes, dendritic cells, killer dendritic cells, macrophages, and monocytes. Immune cells also refer to cells derived from, for example, but not limited to, stem cells. Stem cells can be adult stem cells, non-human embryonic stem cells, more specifically, non-human stem cells, cord blood stem cells, progenitor cells, bone marrow stem cells, induced pluripotent stem cells, totipotent stem cells, or hematopoietic stem cells.

[0153] Accordingly, in some embodiments, provided herein are CAR-T cells comprising a CACCR of the disclosure.

[0154] In some embodiments, a CAR can comprise an extracellular ligand binding domain (e.g., a single chain variable fragment (scFv)), a transmembrane domain, and an intracellular signaling domain. In some embodiments, the extracellular ligand binding domain, the transmembrane domain, and the intracellular signaling domain are in one polypeptide, i.e., in a single chain. Also provided herein are multi-chain CARs and polypeptides. In some embodiments, a multi-chain CAR comprises a first polypeptide comprising a transmembrane domain and at least one extracellular ligand binding domain, and a second polypeptide comprising a transmembrane domain and at least one intracellular signaling domain, wherein the polypeptides assemble together to form a multi-chain CAR.

[0155] The extracellular ligand-binding domain of the CAR specifically binds to a target of interest. The target of interest can be any molecule of interest including, for example, but not limited to, BCMA, EGFRvIII, Flt-3, WT-1, CD20, CD23, CD30, CD38, CD70, CD33, CD133, LeY, NKG2D, CS1, CD44v6, ROR1, CD19, Claudin-18.2 (Claudin-18A2 or Claudin18 isoform 2), DLL3 (Delta-like protein 3, Drosophila Delta homolog 3, Delta3), Muc17 (Mucin 17, Muc3, Muc3), FAPa (fibroblast activation protein alpha), Ly6G6D (lymphocyte antigen 6 complex locus protein G6d, c6orf23, G6D, MEGT1, NG25), and / or RNF43 (E3 ubiquitin-protein ligase RNF43, RING finger protein 43).

[0156] In some embodiments, the extracellular ligand-binding domain of the CAR comprises an scFv comprising a light chain variable (VL) region and a heavy chain variable (VH) region of a target antigen-specific monoclonal antibody connected by a flexible linker. Single-chain variable region fragments are prepared by linking light and / or heavy chain variable regions using a short linking peptide (Bird et al., Science 242:423-426, 1988) (e.g., a glycine-serine containing linker). Typically, the linker can be a short, flexible polypeptide and generally consists of about 20 or fewer amino acid residues. The linker, in turn, can be modified to achieve additional functionality, such as attachment of a drug or attachment to a solid support. Single-chain variants can be produced recombinantly or synthetically. For synthetic production of scFv, an automated synthesizer can be used. For recombinant production of scFv, a suitable plasmid containing a polynucleotide encoding the scFv can be introduced into a suitable host cell (such as a eukaryotic cell, e.g., a yeast, plant, insect, or mammalian cell, or a prokaryotic cell, e.g., E. coli). The polynucleotide encoding the scFv of interest can be prepared by conventional procedures, such as ligation of polynucleotides. The resulting scFv can be isolated using standard protein purification techniques known in the art.

[0157] The intracellular signaling domain of the CAR according to the present application is responsible for generating intracellular signaling after binding of the extracellular ligand-binding domain to the target, leading to activation of the immune cell and an immune response (signal 1 and / or 2). The intracellular signaling domain is capable of activating at least one normal effector function of the immune cell expressing the CAR. For example, the effector function of a T cell can be cytolytic activity or helper activity (including secretion of cytokines).

[0158] In some embodiments, the intracellular signaling domain for a CAR can be, for example, but not limited to, the cytoplasmic sequences of the T cell receptor and co-receptors that act in concert upon antigen receptor engagement to initiate signal transduction, and any derivatives or variants of these sequences and any synthetic sequences with the same functional ability. Intracellular signaling domains include two different types of cytoplasmic signaling sequences: cytoplasmic signaling sequences that initiate antigen-dependent primary activation, and cytoplasmic signaling sequences that act in an antigen-independent manner to provide a secondary or costimulatory signal. Primary cytoplasmic signaling sequences can include signaling motifs known as immunoreceptor tyrosine-based activation motifs, ITAMs. ITAMs are well-defined signaling motifs found within the cytoplasmic tails of various receptors that serve as binding sites for syk / zap70 class tyrosine kinases. Examples of ITAMs used in the present application can include, by way of non-limiting example, those derived from TCR zeta, FcR gamma, FcR beta, FcR epsilon, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d. In some embodiments, the intracellular signaling domain of a CAR can include a CD3 zeta signaling domain. In some embodiments, the intracellular signaling domain of a CAR of the present application includes a domain of a costimulatory molecule.

[0159] In some embodiments, the intracellular signaling domain of a CAR of the present application includes a portion of a costimulatory molecule selected from the group consisting of a fragment of 41BB (GenBank: AAA53133.) and CD28 (NP_006130.1).

[0160] The CAR is expressed on the surface membrane of a cell. Thus, the CAR includes a transmembrane domain. A suitable transmembrane domain of the CARs disclosed herein has the ability to: (a) be expressed on the surface of a cell, preferably an immune cell, such as, for example and without limitation, a lymphocyte or a natural killer (NK) cell; and (b) interact with the ligand binding domain and the intracellular signaling domain to direct a cellular response of the immune cell to a predetermined target cell. The transmembrane domain can be derived from a natural or synthetic source. The transmembrane domain can be derived from any membrane-bound or transmembrane protein. As non-limiting examples, the transmembrane polypeptide can be a subunit of a T cell receptor (such as alpha, beta, gamma or delta, a polypeptide that constitutes the CD3 complex, IL-2 receptor p55 (a chain), p75 (beta chain), or gamma chain, a subunit chain of an Fc receptor, specifically Fcy receptor III, or a CD protein). Alternatively, the transmembrane domain can be synthetic and can include primarily hydrophobic residues, such as leucine and valine. In some embodiments, the transmembrane domain is derived from a human CD8 alpha chain (e.g., NP_001139345.1). The transmembrane domain can further include a stalk domain between the extracellular ligand binding domain and the transmembrane domain. The stalk domain can include up to 300 amino acids, preferably, 10 to 100 amino acids, and most preferably, 25 to 50 amino acids. The stalk region can be derived from all or a portion of a naturally occurring molecule, such as all or a portion of the extracellular region of CD8, CD4, or CD28, or all or a portion of an antibody constant region. Alternatively, the stalk domain can be a synthetic sequence corresponding to a naturally occurring stalk sequence, or can be a completely synthetic stalk sequence. In some embodiments, the stalk domain is part of a human CD8 alpha chain (e.g., NP_001139345.1). In another particular embodiment, the transmembrane and hinge domains include part of a human CD8 alpha chain. In some embodiments, the intracellular signaling domain includes a CD3 zeta signaling domain. In some embodiments, the intracellular signaling domain includes a CD3 zeta signaling domain and a second additional signaling domain. In some embodiments, the intracellular signaling domain includes a CD3 zeta signaling domain and a 4-1BB signaling domain. In some embodiments, the CARs disclosed herein can include an extracellular ligand binding domain that specifically binds to BCMA or EGFRvIII, a CD8 alpha human hinge and transmembrane domain, a CD3 zeta signaling domain, and a 4-1BB signaling domain. In some embodiments, the EGFRvIII-specific CAR includes the amino acid sequence of SEQ ID NO: 140. In some embodiments, the BCMA-specific CAR includes the amino acid sequence of SEQ ID NO: 141 or 142, with or without a signal sequence.

[0161] In some aspects, the CAR-immune cell is a BCMA CAR-T cell comprising a CACCR of the disclosure. In some embodiments, the CACCR of the BCMA CAR-T cell comprises the transmembrane / JAK-binding domain of the naturally occurring TPOR / MPLR receptor of amino acids 478-582 of SEQ ID NO: 6, with a triple substitution of H499L, S505N, and W515K, or a double substitution of S505N and W515K (e.g., SEQ ID NO: 12 or 13). In some embodiments, the CACCR further comprises a recruitment domain from IL2Rb. In some embodiments, the CACCR of the BCMA CAR-T cell further comprises a recruitment domain from IL2Rb (393-433, 518-551) or IL2Rb (339-379, 393-433, 518-551) (e.g., SEQ ID NO: 77 or 78). In some embodiments, the BCMA-specific CAR comprises the amino acid sequence of SEQ ID NO: 141 or 142, with or without a signal sequence. In some embodiments, the BCMA CAR-T cell comprises a CACCR comprising the amino acid sequence of SEQ ID NO: 113, 114, or 116, with or without a signal sequence.

[0162] In some embodiments, the CAR can be introduced into the immune cell as a transgene via a plasmid vector. In some embodiments, the plasmid vector can also contain, for example, a selection marker that provides for identification and / or selection of cells that have received the vector.

[0163] Table 4 provides exemplary sequences for CAR components that can be used in the CARs disclosed herein and the antibody and / or CAR sequences exemplified herein.

[0164] Table 4: Sequences Related to CARs

[0165]

[0166]

[0167] In some embodiments, the CAR-immune cell (e.g., CAR-T cell) of the disclosure comprises a polynucleotide encoding a suicide polypeptide, such as, for example, RQR8. See, e.g., WO2013153391A, which is incorporated by reference herein in its entirety. In some embodiments, the suicide polypeptide is expressed on the cell surface. In some embodiments, the suicide polypeptide is comprised in a CAR construct. In some embodiments, the suicide polypeptide is not part of a CAR construct.

[0168] In some embodiments, the extracellular domain of any of the CARs disclosed herein can comprise one or more epitopes specific for (specifically recognized by) a monoclonal antibody. These epitopes are also referred to herein as mAb-specific epitopes. Exemplary mAb-specific epitopes are disclosed in International Patent Publication No. WO 2016 / 120216, which is incorporated herein in its entirety. In these embodiments, the extracellular domain of the CAR comprises an antigen binding domain that specifically binds to a target of interest and one or more epitopes that bind to one or more monoclonal antibodies (mAbs). The CARs comprising mAb-specific epitopes can be single chain or multichain.

[0169] Inclusion of epitopes specific for a monoclonal antibody in the extracellular domain of the CARs described herein allows for the sorting and depletion of engineered immune cells expressing the CAR. In some embodiments, allowing for depletion provides a safety switch in the event of deleterious effects, e.g., upon administration to a subject.

[0170] Also provided herein are methods of making engineered immune cells for immunotherapy. In some embodiments, the methods comprise introducing a CACCR and a CAR into an immune cell, and expanding the cell. In some embodiments, the present disclosure relates to a method of engineering an immune cell, comprising: providing a cell and expressing a CACCR, and expressing at least one CAR on the surface of the cell. In some embodiments, the method comprises: transfecting a cell with at least one polynucleotide encoding a CACCR and at least one polynucleotide encoding a CAR, and expressing the polynucleotides in the cell. In some embodiments, the method comprises: transfecting a cell with at least one polynucleotide encoding a CACCR, at least one polynucleotide encoding a CAR, and expressing the polynucleotides in the cell.

[0171] In some embodiments, the polynucleotides encoding the CACCR and the CAR are present in one or more expression vectors for stable expression in the cell. In some embodiments, the polynucleotides are present in viral vectors for stable expression in the cell. In some embodiments, the viral vector can be, e.g., a lentiviral vector or an adenoviral vector.

[0172] In some embodiments, the polynucleotides encoding the polypeptides according to the present disclosure can be mRNA introduced directly into the cell, e.g., by electroporation. In some embodiments, the CytoPulse electroporation technology, such as PulseAgile, can be used to transiently permeabilize living cells to deliver materials into the cells (e.g., US 6,078,490; PCT / US2011 / 000827; and PCT / US2004 / 005237). Parameters can be modified to determine conditions of high transfection efficiency and minimal mortality.

[0173] Also provided herein are methods of transfecting immune cells (e.g., T cells). In some embodiments, the methods comprise contacting a T cell with RNA and applying a smart pulse sequence to the T cell. In some embodiments, the methods of transfecting immune cells (e.g., T cells) comprise contacting an immune cell with RNA and applying a smart pulse sequence to the cell.

[0174] In some embodiments, the methods can further comprise the step of genetically modifying the cell by inactivating at least one gene expressing a component of, for example but not limited to, a TCR, a target of an immunosuppressant, an HLA gene, and / or an immune checkpoint protein (such as, for example, PDCD1 or CTLA-4). Inactivating a gene aims to prevent the expression of the gene of interest in the form of a functional protein. In some embodiments, the gene to be inactivated is selected from the group consisting of, for example but not limited to, TCRa, TCRp, CD52, GR, deoxycytidine kinase (DCK), PD-1, and CTLA-4. In some embodiments, the methods comprise inactivating one or more genes by introducing into the cell a rare-cutting endonuclease capable of selectively inactivating a gene by selective DNA cleavage. In some embodiments, the rare-cutting endonuclease can be, for example, a transcription activator-like effector nuclease (TALE-nuclease) or a CRISPR-based endonuclease (such as Cas-9 or Casl2a).

[0175] In another aspect, the step of genetically modifying the cell can comprise modifying an immune cell (e.g., a T cell) by inactivating at least one gene expressing a target of an immunosuppressant, and; expanding the cell, optionally in the presence of the immunosuppressant.

[0176] In some embodiments, the engineered immune cells (e.g., T cells) provided herein exhibit increased cytotoxicity, increased expansion, and / or increased levels of memory phenotype markers relative to engineered immune cells that do not express CACCR.

[0177] In some embodiments, the engineered immune cells (e.g., T cells) provided herein, relative to engineered immune cells that do not express CACCR, structurally exhibit (i) increased in vivo persistence, (ii) increased STAT activation, (iii) increased cytotoxicity, (iv) increased levels of memory phenotypic markers, (v) increased expansion (proliferation), or a combination of these functional characteristics. In some embodiments, the improvement of one or more functional characteristics described herein is tunable, depending on the mutation / modification introduced into CACCR. In some embodiments, the STAT activated by engineered immune cells including one or more disclosed CACCRs is STAT1, STAT2, STAT3, STAT4, STAT5, STAT6, or a combination thereof. In one embodiment, the memory phenotypic markers increased or maintained by immune cells including CACCR include stem cell memory (Tscm) markers and central memory (Tcm) markers.

[0178] In some embodiments, the improvement in one or more functional characteristics exhibited by engineered immune cells including the CACCR provided herein, compared to immune cells that do not express CACCR, is at least about 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 200, 250, 300, 350, 400, 450, or even about 10,500 times, values ​​and ranges in between, is between.

[0179] In some embodiments, the improvement in one or more functional characteristics exhibited by engineered immune cells including the CACCR provided herein, compared to engineered immune cells that do not express CACCR, is at least about 10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 75%, 80%, 90%, 100%, 125%, 150%, 200%, 250%, 300%, 350%, 400%, or even about 80% to 500%, values ​​and ranges in between.

[0180] III. Treatment Methods

[0181] This document provides pharmaceutical compositions comprising cells carrying the CACCR and CAR disclosed herein.

[0182] The engineered CACCR and CAR-bearing immune cells (e.g., T cells) obtained by the above methods or cell lines derived from such engineered immune cells can be used as a medicament. In some embodiments, such medicaments can be used to treat a disorder, such as, for example, a viral disease, a bacterial disease, a cancer, an inflammatory disease, an immune disease, or an aging-related disease. In some embodiments, the cancer is a solid cancer. In some embodiments, the cancer is a liquid cancer. The cancer can be selected from the group consisting of stomach cancer, sarcoma, lymphoma, leukemia, head and neck cancer, thymus cancer, epithelial cancer, salivary cancer, liver cancer, stomach cancer, thyroid cancer, lung cancer, ovarian cancer, breast cancer, prostate cancer, esophageal cancer, pancreatic cancer, glioma, leukemia, multiple myeloma, renal cell carcinoma, bladder cancer, cervical cancer, choriocarcinoma, colon cancer, oral cancer, skin cancer, and melanoma. In some embodiments, the subject is a previously treated adult subject with locally advanced or metastatic melanoma, squamous cell head and neck cancer (SCHNC), ovarian cancer, sarcoma, or relapsed or refractory classical Hodgkin lymphoma (cHL).

[0183] In some embodiments, the engineered immune cells or cell lines derived from engineered immune cells can be used to manufacture a medicament for treating a disorder in a subject in need thereof. In some embodiments, the disorder can be, for example, a cancer, an autoimmune disorder, or an infection.

[0184] Also provided herein are methods for treating a subject in need of such treatment.

[0185] As used herein, the term "subject" refers to any vertebrate, including but not limited to, humans and other primates (e.g., chimpanzees, cynomolgus monkeys, and other apes and monkey species), farm animals (e.g., cows, sheep, pigs, goats, and horses), domestic mammals (e.g., dogs and cats), laboratory animals (e.g., rabbits, rodents such as mice, rats, and guinea pigs), and birds (e.g., poultry, game birds, and birds of prey, such as chickens, turkeys and other gallinaceous birds, ducks, geese, and the like). In some embodiments, the subject is a mammal. In exemplary embodiments, the subject is a human.

[0186] In some embodiments, the methods comprise providing to a subject in need thereof an immune cell of the disclosure bearing a CACCR and a CAR described herein.

[0187] In some embodiments, the CACCR and CAR-bearing T cells of the present invention can undergo robust in vivo T cell expansion and can persist for an extended amount of time.

[0188] The methods of treatment of the present invention can be ameliorative, curative, or prophylactic. The methods of the present invention can be part of an autologous immunotherapy or part of an allogeneic immunotherapy.

[0189] In another aspect, the present application provides a method of inhibiting tumor growth or progression in a subject having a tumor, the method comprising administering to the subject an effective amount of an immune cell expressing CACCR and a CAR as described herein. In another aspect, the present application provides a method of inhibiting or preventing metastasis of cancer cells in a subject, the method comprising administering to a subject in need thereof an effective amount of an engineered immune cell as described herein. In another aspect, the present application provides a method of inducing tumor regression in a subject having a tumor, the method comprising administering to the subject an effective amount of an engineered immune cell as described herein.

[0190] In some embodiments, the engineered T cells herein can be administered parenterally in a subject.

[0191] Also provided is the use of any one of the engineered T cells provided herein in the manufacture of a medicament for treating cancer or for inhibiting tumor growth or progression in a subject in need thereof.

[0192] In some embodiments, the treatment can be administered to a subject undergoing immunosuppressive therapy. Indeed, the present application preferably relies on cells or cell populations that are resistant to at least one immunosuppressive agent due to inactivation of the gene encoding the receptor for such immunosuppressive agent. In this respect, the immunosuppressive therapy should contribute to the selection and expansion of T cells according to the application in the subject. Administration of the cells or cell populations according to the application can be performed in any convenient manner, including nebulization inhalation, injection, ingestion, infusion, implantation or transplantation. The compositions described herein can be administered to a subject subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, by intravenous or intralymphatic injection or intraperitoneally. Cells carrying CACCR and CAR of the present disclosure or pharmaceutical compositions thereof can be administered by one or more of the following routes of administration: intravenously, intraocularly, intravitreally, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly, through the ear or intranasally.

[0193] In some embodiments, administration of the cells or cell populations (carrying CACCR and CAR of the present disclosure) can include administration of about 10 4 to about 10 9 cells per kg of body weight, including all integer values of cell numbers within these ranges. In some embodiments, administration of the cells or cell populations can include administration of about 10 4 to 10 5 cells / kg of body weight, 10 5 to 10 6 cells / kg of body weight, 10 6 to 10 7 cells / kg of body weight, 10 7to 10 8 cells / kg body weight or 10 8 to 10 9 cells / kg body weight. The cells or cell populations can be administered in one or more doses. In some embodiments, the effective amount of cells can be administered as a single dose. In some embodiments, the effective amount of cells can be administered as more than one dose over a period of time. The timing of administration is within the judgment of the attending physician and depends on the clinical condition of the subject. The cells or cell populations can be obtained from any source, such as a blood bank or a donor. While individual needs vary, determination of optimal ranges of effective amounts of a given cell type for treatment of a particular disease or condition is within the skill of the art. An effective amount means an amount which provides the therapeutic or prophylactic benefit. The dose administered will depend on the age, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired. In some embodiments, an effective amount of cells or a composition comprising those cells is administered parenterally. In some embodiments, the administration can be intravenous administration. In some embodiments, the administration can be done directly by injection into a tumor.

[0194] The methods can further comprise administering one or more agents to the subject prior to administering the engineered immune cells carrying the CAR and CACCR provided herein. In certain embodiments, the agent is a lymphodepleting (preconditioning) regimen. For example, methods of lymphodepleting a subject in need of such therapy comprise administering to the subject a specified beneficial dose of cyclophosphamide (between 200 mg / m 2 per day and 2000 mg / m 2 per day; e.g., about 100 mg / m 2 per day and about 2000 mg / m 2 per day; e.g., about 100 mg / m 2 per day, about 200 mg / m 2 per day, about 300 mg / m 2 per day, about 400 mg / m 2 per day, about 500 mg / m 2 per day, about 600 mg / m 2 per day, about 700 mg / m 2 per day, about 800 mg / m 2 per day, about 900 mg / m 2 per day, about 1000 mg / m 2 per day, about 1500 mg / m 2 per day, or about 2000 mg / m 2 per day) and a specified dose of fludarabine (between 20 mg / m 2 per day and 900 mg / m 2between about 10 mg / m 2 between about 10 mg / m 2 between about 10 mg / m 2 between about 10 mg / m 2 between about 10 mg / m 2 between about 10 mg / m 2 between about 10 mg / m 2 between about 10 mg / m 2 between about 10 mg / m 2 between about 10 mg / m 2 between about 10 mg / m 2 between about 10 mg / m 2 between about 10 mg / m 2 between about 10 mg / m 2 between about 10 mg / m 2 between about 10 mg / m 2 between about 10 mg / m 2 between about 10 mg / m

[0195] In some embodiments, particularly where the engineered cells provided herein have been genetically edited to eliminate or minimize surface expression of CD52, lymphodepletion further comprises administration of an anti-CD52 antibody, such as alemtuzumab. In some embodiments, the CD52 antibody is administered intravenously (IV) at a dose of about 1-20 mg / day (e.g., about 13 mg / day) for 1 day, 2 days, 3 days, or more. The antibody can be administered in conjunction with other components of the lymphodepletion regimen (e.g., cyclophosphamide and / or fludarabine), or prior to or after administration of the other components.

[0196] In certain embodiments, compositions comprising immune effector cells expressing CACCR and CAR disclosed herein can be administered in conjunction with any number of chemotherapeutic agents.

[0197] IV. Kits and Articles

[0198] The present disclosure provides kits comprising any one or more of the CACCR and CAR- bearing cells and pharmaceutical compositions thereof described herein. The present disclosure also provides articles comprising any one or more of the CACCR and CAR-bearing CAR-I cells, pharmaceutical compositions thereof, and kits described herein.

[0199] The following examples are included for illustrative purposes only and are not intended to limit the scope of the disclosure.

[0200] All patents and non-patent literature cited throughout this disclosure are incorporated herein by reference in their entirety for all purposes

[0201] Example

[0202] Example 1: Identification of TpoR TM mutants that constitutively activate cytokine signaling

[0203] A prototype constitutively active chimeric cytokine receptor (CACCR) was designed using sequences from the thrombopoietin receptor (TpoR). TpoR is capable of activating the JAK-Stat signaling pathway and signals as a homodimeric receptor. Single point mutations (amino acid substitutions) in TpoR activity have been shown to modulate receptor activity (Proc Natl Acad Sci U S A. 2013 Feb 12; 110(7):2540-5; FASEB J. 2011 Jul; 25(7):2234-44; J Biol Chem. 2016 Feb 5; 291(6):2974-87). In this example, the constitutively active chimeric cytokine receptor was engineered from the naturally occurring TpoR receptor: the extracellular domain of the native TpoR receptor was removed, thus it no longer has ligand binding ability; 1-3 mutations were introduced into its transmembrane domain; and the TpoR cell tail was replaced with the cell tail of the desired / described cytokine receptor. Figure 1 A schematic of the engineered constitutively active chimeric cytokine receptor is shown.

[0204] To demonstrate the utility of the constitutively active chimeric cytokine receptor in the context of CAR-T cells, each TpoR transmembrane (TM) variant was cloned into a lentiviral vector encoding a second generation EGFRvIII-specific CAR (2173scFv; described in Sci Transl Med. 2015 Feb 18; 7(275):275ra22.). To allow stoichiometric co-expression of the cytokine receptor and CAR, the two genes were linked by a P2A peptide. To facilitate detection of transduced cells, a v5 epitope tag (KPIPNPLLGLDST) SEQ ID NO: 144) was inserted between the scFv and CD8 hinge domains.

[0205] Figure 2 A schematic of the lentiviral vector used to co-express the constitutively active chimeric cytokine receptor and CAR is shown.

[0206] Table 4 shows the sequences associated with the constructs used.

[0207] HEK293T cell reporter assays were used to screen TpoR™ variants capable of constitutive cytokine signaling. Briefly, 20,000 HEK293T cells were seeded into each well of a poly-L-lysine coated 96-well flat bottom plate and allowed to adhere overnight. Cytokine receptor-CAR constructs (2.5 ng), Stat response elements driving firefly luciferase (100 ng; Promega), and Renilla luciferase control reporter vector (1 ng; Promega) were mixed in Opti-MEM (Gibco) in a final volume of 5 uL (“DNA mix”). As a negative control, cells were transfected with a BFP CAR construct lacking all cytokine signaling domains. As a positive control, cells were transfected with a vector encoding full-length human EpoR (in place of the erythropoietin receptor of the cytokine receptor-CAR constructs) to induce Stat5 signaling by the addition of exogenous recombinant human Epo. 0.3 uL Lipofectamine 2000 (Invitrogen) in 5 uL Opti-MEM was incubated at room temperature for 5 minutes before being added to the DNA mix. The mix was incubated at room temperature for 20 minutes and a total volume of 10 uL was added to each well containing HEK-293T. Forty-eight hours post-transfection, Stat5 reporter gene activity was assessed using the Dual-Glo luciferase assay system (Promega). The fold-induction of Stat5 reporter gene activity was normalized to the fold-induction of HEK293T cells transfected with all vectors except the chimeric cytokine receptor and untreated cells.

[0208] Figures 3a and 3b show the identification of TpoR™ mutants that constitutively activate cytokine receptor signaling. Figure 3a shows a schematic of the lentiviral vectors used. It carries an IL7R (316-459) cytoplasmic tail to mimic IL7 signaling in CAR-T cells. Figure 3b shows Stat5 reporter gene activity determined by Dual-Glo luciferase assay. Cytokine receptors carrying the wild-type TpoR™ domain (TpoR(478-582)) do not spontaneously activate Stat5. The TpoR(478-582; S505N), TpoR(478-582; W515K), and TpoR(478-582; H499L, G509N) mutants result in weak Stat5 activation. TpoR(478-582; H499L, S505N, W515K) allows for moderate Stat5 activity, while TpoR(478-582; S505N, W515K) generates the strongest Stat5 signal.

[0209] Example 2: Generation of CAR-T cells expressing constitutively active chimeric cytokine receptors

[0210] We next tested whether these cytokine receptors signal in the context of primary human CAR-T cells. To prepare lentivirus encoding cytokine receptor-CARs, HEK293T cells were seeded at 450,000 cells / mL in 6-well plates in 2 mL DMEM (Gibco) supplemented with 10% FBS (Hyclone) per well the day before transfection. On the day of transfection, lentivirus was prepared by mixing 1.5 ug psPAX2, 0.5 ug pMD2G, and 0.5 ug of the appropriate transfer CAR vector in 250 uL Opti-MEM (Gibco) per well of a 6-well plate (“DNA mix”). 10 uL Lipofectamine 2000 (Invitrogen) in 250 uL Opti-MEM was incubated at room temperature for 5 minutes before adding to the DNA mix. The mix was incubated at room temperature for 20 minutes and the total volume of 500 uL was added slowly to the side of the well containing HEK293T. One day after transfection, the media from the HEK293T cells in the 6-well plate was replaced with 2 mL of T cell transduction media, X-Vivo-15 supplemented with 10% FBS, per well. Two days after transfection, lentivirus supernatant from the HEK293T cells was collected and cell debris was removed by a 0.45 micron filter (EMD Millipore), concentrated 25-fold using a Lenti-X concentrator (Takara Bio) according to the manufacturer’s instructions, and then aliquoted and frozen. Lentivirus titers were determined by thawing an aliquot of frozen lentivirus, performing a 4-fold serial dilution, and performing a limiting dilution titration on Jurka T cells (clone E6-1; ATCC). On day 0, purified T cells were activated in X-Vivo-15 media (Lonza) supplemented with 100 IU / mL human IL-2 (Miltenyi Biotec), 10% FBS (Hyclone), and human T TransAct (Miltenyi Biotec, catalog number 130-111-160, 1:100 dilution) in Grex-24 plates (Wilson Wolf, catalog number 80192M). On day 2, T cells were resuspended at 5 million cells / mL in T cell transduction media, transduced with the appropriate lentivirus stock at an MOI = 5, and 100 IU / mL human IL-2 in Grex-24 plates. On day 5, when transduction was complete, cells were harvested and washed to remove residual IL-2. Cells were then resuspended in T cell expansion media, X-Vivo-15 supplemented with 5% human AB serum (Gemini Bio), and each sample was split into 2, with one receiving 100 IU / mL human IL-2 according to standard protocols and the other receiving a lower concentration of 25 IU / mL human IL-2.Cells were expanded into larger G-Rex flasks (Wilson Wolf) using T cell expansion media and corresponding concentrations of human IL-2 as needed. On days 5, 9, and 14, the absolute number of T cells in each sample was calculated and transduction efficiency was determined by using flow cytometry to detect the percentage of T cells binding a FITC-conjugated v5 tag monoclonal antibody (Thermo Fisher). On day 14 or 15, CAR-T cell products were cryopreserved and thawed as needed for further analysis.

[0211] Figures 4a-4c show results of generating CAR-T cells co-expressing a constitutively active chimeric cytokine receptor. CAR-T cell cultures carrying TpoR TM mutants experienced a more robust expansion in both total T cell number (Figure 4a) and CAR-T cell number (Figure 4b) compared to CAR-T cell cultures with wild-type TpoR TM cytokine receptor (TpoR(478-582)). Figures 4a-4c show that the transduction efficiency of TpoR TM mutants was equal to or better than their wild-type TpoR(478-582) counterparts. TpoR TM mutants allowed for higher yield of CAR-T cell products. Furthermore, expanding TpoR TM mutants at lower IL-2 concentrations did not affect the expansion or yield of CAR-T cells (Figure 4c).

[0212] On day 14 of CAR-T cell production, the memory phenotype of CAR-T cells was determined. Briefly, samples were washed with PBS, Fc-blocked, and then stained with the following antibody cocktail diluted in PBS + 1% BSA: BUV395-conjugated anti-human CD3, BV510-conjugated anti-human CD8, BV605-conjugated human CD4, and FITC-conjugated v5 tag (for CAR detection), PE / Cy7-conjugated anti-human CD62L (Biolegend), and BV785-conjugated anti-human CD45RO (Biolegend). Finally, samples were washed in PBS and cell pellets were resuspended in 130 uL PBS + 1% BSA for FACS analysis.

[0213] Figure 5Memory T cell subpopulation distribution in CAR-T cell products is shown. TpoR(478-582; W515K) and TpoR(478-582; H499L, G509N) mutants exhibited greater differentiation when expanded under standard 100 IU / mL IL-2 conditions compared to their wild-type TpoR(478-582) counterpart. Expansion under low IL-2 conditions improved differentiation. In line with the standard concentration of IL-2, the stronger Stat5 signaling induced by TpoR(478-582; W515K) and TpoR(478-582; H499L, G509N) mutants can lead to accelerated CAR-T cell differentiation, and expansion under low IL-2 conditions can be more advantageous in the context of CAR-T cells expressing constitutive cytokine receptors.

[0214] Example 3: TpoR TM mutant constitutively activates cytokine signaling in human CAR-T cells

[0215] To determine the strength of cytokine signaling mediated by TpoR Tm mutants, CAR-T cells bearing TpoR TM cytokine receptor variants were serum starved for 4 hours in 100 uL of serum-free RPMI (Corning) in a humidified incubator at 37 °C and 5% CO2. As a positive control, exogenous recombinant human IL-7 (10 ng / mL; Miltenyi) was added during the last 30 minutes of the 4-hour serum starvation. After 4 hours, an antibody cocktail including BUV395-conjugated anti-human CD3 (Biolegend) and FITC-conjugated v5-tag monoclonal antibody (Thermo Fisher) was added to the cells and allowed to incubate for the last 20 minutes. Cells were then fixed by adding 35 uL of 16% paraformaldehyde to each 100 uL sample and incubated at 37 °C for 15 minutes. Cells were then washed three times with PBS and permeabilized in 100% cold methanol at -20 °C for 1 night or 2 nights. On the day of FACS analysis, cells were washed three times with PBS, Fc-blocked, and stained with AlexaFluor647-conjugated anti-mouse / human Stat5 (pY694) (BD Biosciences) diluted in PBS + 1% BSA. After 1 hour of incubation at room temperature in the dark, cells were washed three times and then subjected to FACS analysis.

[0216] Figures 6a-6b show the extent of constitutive cytokine signaling mediated by each TpoR TM variant as reflected by the percentage of pStat5+ cells (Figure 6a) and the geometric mean fluorescence intensity (gMFI) of pStat5 staining (Figure 6b). While the TpoR TM single mutants (TpoR(478-582; S505N) and TpoR(478-582; W515K)) did not induce significant Stat5 activation, the TpoR TM double mutant (TpoR(478-582; S505N, W515K) and triple mutant (TpoR(478-582; H499L, S505N, W515K)) induced comparable strong constitutive Stat5 activation. CAR-T cells expanded at low IL-2 and standard IL-2 concentrations generated comparable Stat activation profiles. Since Stat5 was activated in both CAR-bearing (CAR+) and non-CAR-bearing (CAR-) T cells in the same culture, this indicates that cytokine signaling is CAR-T cell specific.

[0217] Example 4: Constitutive Cytokine Receptor Enhanced CAR-T Cell Cytotoxicity Potency and Prolonged Response Durability

[0218] To test whether constitutive cytokine receptor signaling enhances the cytotoxic activity of CAR-T cells, we used U87KO-EGFRvIII-nucGFP as target cells. U87KO-EGFRvIII was a gift from Cellectis SA (Paris, France). U87KO-EGFRvIII was derived from the parental cell line U87MG (ATCC) by first knocking out the endogenous wild-type EGFR using Transcription Activator-Like Effector Nucleases (TALENs) and then stably overexpressing full-length human EGFRvIII by lentiviral transduction. To facilitate target T cell imaging by the IncuCyte Live-Cell Analysis Imaging System, U87KO-EGFRvIII-nucGFP target cells were derived from U87KO-EGFRvIII by a second lentiviral transduction using IncuCyte NucLight Green lentivirus reagent (Sartorius). 5,000 U87KO-EGFRvIII-nucGFP target cells were seeded and allowed to adhere in 50 uL RPMI containing 10% FBS (Hyclone), non-essential amino acids, sodium pyruvate, and 20-25 mM HEPES in a 96-well plate with black-walled and flat transparent bottom. EGFRvIII CAR (2173scFv) T cells bearing TpoR TM variant cytokine receptors were thawed and added to the seeded target cells at effector:target (E:T) ratios of 1:8 and 1:2. For comparison, wild-type TpoR (478-582) CAR-T cells with and without the addition of exogenous recombinant human IL-7 were included in the assay. Where applicable, CAR-T cells were re-challenged at the indicated time points by transferring the suspended cells from the original plate to a new plate of target cells. Duplicate wells were established for each condition. Cytotoxicity was determined by calculating the number of live target cells at each time point using the IncuCyte Live-Cell Analysis Imaging System.

[0219] FIGs. 7a-7d show cytotoxic activity of TpoR TM mutants at an E:T ratio of 1 :8. Individual TpoR TM mutants TpoR(478-582; S505N) (FIG. 7a) and TpoR(478-582; W515KN) (FIG. 7b) did not show enhanced function compared to their counterparts carrying the wild-type TpoR(478-582) control, which is consistent with their inability to efficiently activate Stat5 (FIGs. 6a-6b). FIG. 7c shows that TpoR double mutant CAR-T cells expanded under standard IL-2 concentrations were not enhanced; whereas TpoR double mutant CAR-T cells expanded under low IL-2 conditions were more effective at target cell lysis. FIG. 7d shows that TpoR triple mutant CAR-T cells were more effective at target cell lysis regardless of IL-2 concentration during CAR-T cell production. This indicates that constitutive cytokine receptor signaling enhances CAR-T cell potency.

[0220] FIGs. 8a-8b show cytotoxic activity of TpoR TM double mutants (FIG. 8a) and triple mutants (FIG. 8b) at an E:T ratio of 1 :2. CAR-T cells were equally effective at eliminating target cells during the primary response regardless of cytokine receptor activity. However, when re-challenged with fresh targets, only CAR-T cells expressing constitutively active chimeric cytokine receptors retained function, which indicates that constitutive cytokine receptor signaling enhances CAR-T cell response durability.

[0221] Example 5: Constitutive cytokine receptor enhances CAR-T cell durability and promotes CAR+ Tscm expansion

[0222] To observe the enhancing effect of constitutive cytokine receptor signaling on CAR-T cell durability in the absence of targets or exogenous cytokines, a growth factor-independent assay was performed. Briefly, the percentage of CAR-T cells in all samples was normalized to the sample with the lowest transduction efficiency (35.7%) by adding non-transduced (NTD) T cells. CAR-T cells carrying 0.25x10 6T cells / mL in 4 mL of RPMI containing 10% FBS (Hyclone), non-essential amino acids, sodium pyruvate, and 20-25 mM HEPES. Cells were then seeded in T25 tissue culture flasks. As a positive control, exogenous human IL-7 (10 ng / mL; Miltenyi) was added to CAR-T cells lacking constitutive cytokine receptor signaling (wild-type TpoR(478-582)). On the indicated days, 200 uL of duplicate samples were harvested from each condition and stained using the Zombie NIR Viability kit (Biolegend). Samples were washed with PBS, Fc-blocked, and then stained with the following antibody cocktail diluted in PBS + 1% BSA: BUV395-conjugated anti-human CD3, BV510-conjugated anti-human CD8, BV605-conjugated human CD4, and FITC-conjugated v5 tag (for CAR detection), PE / Cy7-conjugated anti-human CD62L (Biolegend), and BV785-conjugated anti-human CD45RO (Biolegend). Finally, samples were washed in PBS and cell pellets were resuspended in 130 uL of PBS + 1% BSA containing 123 eBead Counting Beads (Thermo Fisher) (10 uL of counting beads in 120 uL of PBS + 1% BSA) prior to FACS analysis.

[0223] Figure 9 CAR-T cells were shown to enrich over time in a growth factor-independent assay. While CAR-T cells bearing the wild-type TpoR TM (TpoR(478-582) and TpoR TM single mutants (TpoR(478-582; S505N) and TpoR(478-582; W515KN)) did not enrich, CAR-T cells bearing the TpoR TM double and triple mutants enriched over time, indicating that CAR-bearing T cells receiving constitutive cytokine receptor signaling preferentially survive.

[0224] Figures 10a-10b show the expansion fold of CAR-T cells over time in a growth factor independent assay. The expansion fold was determined by normalizing the absolute number of CAR-T cells at each time point to the number of CAR-T cells on assay day 0. Figure 10a shows that TpoR TM single mutants, which are unable to efficiently activate cytokine receptor signaling, decline at the same rate as CAR-T cells bearing the wild-type TpoR TM (TpoR(478-582)). In contrast, Figure 10b shows that CAR-T cells bearing TpoR TM double or triple mutants survive longer in the absence of target and exogenous cytokine. TpoR TM double mutant CAR-T cells expanded under low IL-2 conditions show increased persistence compared to their counterparts expanded under standard IL-2 conditions. TpoR TM triple mutant CAR-T cells manufactured under low and standard IL-2 conditions show comparable moderate enhancement of persistence. Notably, although the TpoR double and triple mutant CAR-T cells persist longer, both eventually decline, suggesting that constitutive cytokine receptor signaling is unlikely to result in CAR-T cell immortalization or transformation.

[0225] Figure 11 Figure 10c shows the distribution of memory T cell subsets among CAR+ T cells over time in a growth factor independent assay. CAR-T cells bearing constitutively active cytokine receptors (shown in this case is TpoR TM triple mutant expanded under low IL-2 conditions) show an increase in the absolute number of stem cell memory T cells (Tscm), which are a subset that mediate long-lived anti-tumor immunity, compared to wild-type TpoR TM (TpoR(478-582)). Notably, constitutive signaling by TpoR TM triple mutant is more effective than exogenous human IL-7 supplementation in expanding Tscm CAR-T cells.

[0226] Example 6: Constitutive cytokine tails can be customized to activate signaling pathways of interest

[0227] The ability of cytokines to regulate CAR-T cell fate and function stems from their ability to elicit different downstream signaling pathways. For example, IL-7 / IL-2 / IL-15 mediated STAT5 activation enhances T cell survival and expansion, while IL-12 mediated STAT4 activation drives terminal differentiation into short-lived effectors. Designing recruitment domains (i.e., cytokine tails) that can mimic a broader range of cytokine signals will provide flexibility in user-programmable signaling outcomes, thereby controlling CAR-T cell fate and function.

[0228] To interrogate whether CACCRs can transmit signals through additional cytokine receptor-mediated signals, the IL7Ra (316-459) cell tail from Figure 3 was replaced with alternative cell tails derived from the intracellular signaling domains of IL2Rb or IL12Rb2. The signaling capacity of these chimeras was then assessed using a HEK293T cell reporter assay. Briefly, 20,000 HEK293T cells were seeded into each well of a poly-L-lysine coated 96-well flat bottom plate and allowed to adhere overnight. Cytokine receptor-CAR constructs (2.5 ng), a Stat-responsive element driving firefly luciferase (100 ng; Promega), and a Renilla luciferase control reporter vector (1 ng; Promega) were mixed in Opti-MEM (Gibco) in a final volume of 5 uL (“DNA mix”). As negative controls, cells were transfected with either a BFP-CAR construct lacking all cytokine signaling domains or a TpoR (478-582).IL7Ra (316-459) construct lacking a transmembrane mutation and thus unable to signal constitutively. 0.3 uL Lipofectamine 2000 (Invitrogen) in 5 uL Opti-MEM was incubated at room temperature for 5 minutes before being added to the DNA mix. The mix was incubated at room temperature for 20 minutes and a total volume of 10 uL was added to each well containing HEK-293T. Forty-eight hours post-transfection, the activity of the individual Stat reporter genes was assessed using the Dual-Glo luciferase assay system (Promega). The fold-induction of Stat5 reporter gene activity was normalized to the fold-induction of HEK293T cells transfected with the control BFP-CAR construct.

[0229] Figure 12 It was shown that constructs with different signaling domains preferentially activated different STAT pathways. Specifically, IL2Rb (333-551) and IL7Ra (316-459) activated STAT5, while IL12Rb2 (714-862) activated STAT4, which reflects the expected signaling of the individual parent receptors. This demonstrates that CACCRs can be programmed to activate desired signaling pathways by fusing to the signaling domain of interest. Furthermore, consistent with Figure 3, CACCRs bearing the TpoR (478-582; S505N, W515K) dimerization domain produced stronger signaling than their TpoR (478-582; H499L, S505N, W515K) counterparts. Thus, the strength of CACCR signaling output can be further tuned by fusing to any of these dimerization domains.

[0230] Example 7: CACCR signaling domains can be optimized to modulate signal strength while reducing vector cargo size

[0231] Currently, viral-based gene delivery methods (e.g., lentivirus and retrovirus- mediated gene transfer) are commonly used for CAR-T cell manufacturing. With increasing cargo size, transduction efficiency and CAR-T cell yield decrease. Therefore, reducing the size of the cargo would be beneficial to ensure manufacturing success. For two reasons, CACCR-optimized recruitment / signaling domains provide a means to do so. First, as is the case with IL2Rb (333-551), cytokine receptor-derived signaling domains can be up to 200 amino acids in length and represent over 650 base pairs in the transfer vector. Second, while tyrosine residues within the signaling domain are important for initiating and propagating downstream signal transduction, some of these residues can also participate in negative feedback loops that limit the duration and strength of signaling. Therefore, trimming the cell tail signaling domain can not only reduce the size of the vector cargo, but also provide an opportunity to modulate cell tail signaling. To this end, we identified tyrosine residues within the full-length IL12Rb2 (714-862) and IL2Rb (331-551) tails and generated variants to identify truncated constructs capable of mediating cell tail signaling.

[0232] Full length IL12Rb2 (714-862) contains two phosphorylatable tyrosine residues Y767 and Y800 that can be involved in downstream signaling. We generated a truncated IL12Rb2 (775-825) tail containing only Y800 and assessed its ability to activate STAT4 using a HEK293T cell reporter assay. Briefly, 20,000 HEK293T cells were seeded into each well of a poly-L-lysine coated 96 well flat bottom plate and allowed to adhere overnight. CACCR-CAR constructs (2.5 ng), Stat response element driving firefly luciferase (100 ng; Promega), and Renilla luciferase control reporter vector (1 ng; Promega) were mixed in Opti-MEM (Gibco) in a final volume of 5 uL (“DNA mix”). As a negative control, cells were transfected with a BFP-CAR construct lacking all cytokine signaling domains. 0.3 uL Lipofectamine 2000 (Invitrogen) in 5 uL Opti-MEM was incubated at room temperature for 5 minutes before being added to the DNA mix. The mix was incubated at room temperature for 20 minutes and a total volume of 10 uL was added to each well containing HEK-293T. Forty-eight hours post-transfection, Stat4 reporter activity was assessed using the Dual-Glo Luciferase Assay System (Promega). The fold-induction of Stat4 reporter activity was normalized to the fold-induction of HEK293T cells transfected with the control BFP-CAR construct.

[0233] Figure 13A -B shows the identification of a truncated IL12Rb2 (775-825) cell tail that can activate STAT4 comparable to the full length IL12Rb2 (714-862) cell tail. Figure 13A A schematic of the full length IL12Rb (714-862) tail and the truncated IL12Rb (775-825) cell tail is shown. The position of the tyrosine residues (Y) included in each tail is shown. Figure 13B It is shown that the truncated IL12Rb2 (775-825) cell tail fully recapitulates the STAT4 signaling strength of the full length IL12Rb2 (714-862) cell tail when fused to a stronger TpoR (478-582; S505N, W515K) dimerization domain. The truncated IL12Rb2 (775-825) cell tail partially recapitulates the STAT4 signaling strength of the full length IL12Rb2 (714-862) cell tail when fused to a weaker TpoR (478-582; H499L, S505N, W515K) dimerization domain.

[0234] The full-length IL2Rb (333-551) cytoplasmic tail contains six tyrosine residues that can potentially engage downstream signaling. Of these, Y364 (the tyrosine residue closest to the transmembrane domain of the receptor) has been reported to activate PI3K to promote T cell differentiation and proliferation, as well as cytoskeletal reorganization to induce receptor internalization; thus, while Y364 can promote T cell effector functions, it can also limit the strength and duration of IL2Rb signaling. We generated truncated IL2Rb cytoplasmic tails containing either three of the six tyrosine residues (Y364, Y418, and Y436) or two of the six tyrosine residues (Y418 and Y436) and assessed their ability to activate STAT5 using a HEK293T cell reporter assay. Briefly, 20,000 HEK293T cells were seeded into each well of a poly-L-lysine coated 96-well flat bottom plate and allowed to adhere overnight. CACCR-CAR constructs (2.5 ng), a Stat-responsive element driving firefly luciferase (100 ng; Promega), and a Renilla luciferase control reporter vector (1 ng; Promega) were mixed in Opti-MEM (Gibco) in a final volume of 5 uL (“DNA mix”). As a negative control, cells were transfected with a BFP-CAR construct lacking all cytokine signaling domains. 0.3 uL Lipofectamine 2000 (Invitrogen) in 5 uL Opti-MEM was incubated at room temperature for 5 minutes before being added to the DNA mix. The mix was incubated at room temperature for 20 minutes, and a total volume of 10 uL was added to each well containing HEK-293T. Forty-eight hours post-transfection, the activity of the Stat5 reporter was assessed using the Dual-Glo Luciferase Assay System (Promega). The fold-induction of Stat5 reporter activity was normalized to the fold-induction of HEK293T cells transfected with the control BFP-CAR construct.

[0235] Figure 14A -B shows the identification of truncated IL2Rb tails that are able to activate STAT5 equally or better than the full-length IL2Rb (333-551) tail. Figure 14A A schematic showing the full-length IL2Rb (333-551) tail and the two truncated IL2Rb tails is shown. The positions of the tyrosine residues (Y) included in each tail are shown. Dashed lines represent the interconnecting regions in the full-length IL2Rb (333-551) tail that have been removed from the truncated tails. Figure 14BResults from HEK293T cell reporter assays are shown, where STAT5 signaling of full-length IL2Rb (333-551) is recapitulated by a cell tail containing Y364, Y418, and Y536 of TpoR (478-582; H499L, S505N, W515K) IL2Rb (339-379, 393-433, 518-551). In the TpoR (478-582; S505N, W515K).IL2Rb (393-433, 518-551) cell tail, the additional removal of Y364, which mediates receptor internalization, results in a significant increase in STAT5 signaling intensity.

[0236] The HEK293T cell assay is a short-term assay with readout measured within 48 hours of cell tail transfection. While it provides an efficient screening platform for cell tail activity, the limited duration of this assay does not reflect the complexity of long-term constitutive cytokine and cell tail signaling- triggered negative feedback loops. To more accurately assess the long-term signaling activity of reduced IL2Rb tail variants, we generated CACCR CAR-T cells using a 2-week production process and assessed STAT5 activation by intracellular flow cytometry. To this end, CACCR CAR-T cells were serum starved for 4 hours in 100 uL of serum-free RPMI (Corning) in a humidified incubator at 37°C and 5% CO2. As a positive control, exogenous recombinant human IL-2 (10 ng / mL; Miltenyi) was added during the last 30 minutes of the 4-hour serum starvation. After 4 hours, an antibody cocktail including BUV395-conjugated anti-human CD3 (Biolegend) and FITC-conjugated v5-tag monoclonal antibody (Thermo Fisher) was added to the cells and allowed to incubate for the last 20 minutes. Cells were then fixed by adding 35 uL of 16% paraformaldehyde to each 100 uL sample and incubated at 37°C for 15 minutes. Cells were then washed three times with PBS and permeabilized in 100% cold methanol at -20°C for 1 night or 2 nights. On the day of FACS analysis, cells were washed three times with PBS, Fc-blocked, and stained with AlexaFluor647-conjugated anti-mouse / human Stat5 (pY694) (BD Biosciences) diluted in PBS + 1% BSA. After 1 hour of incubation at room temperature in the dark, cells were washed three times and then subjected to FACS analysis.

[0237] Figure 15STAT5 activation in primary CACCR CAR-T cells carrying full length or truncated IL2Rb cell tail is shown. The greatest STAT5 activation was elicited by CACCR CAR-T cells carrying a truncated IL2Rb (393-433, 518-551) cell tail that lacks the Y364 internalization motif. Intermediate STAT5 activation was observed in CACCR CAR-T cells carrying a truncated IL2Rb (339-379, 393-433, 518-551) cell tail. No STAT5 activation was observed in the CAR-negative population in the same culture, suggesting a CAR-T cell specific nature of the cell tail signaling. Notably, little STAT5 activity was detected in CACCR CAR-T cells carrying a full length IL2Rb (333-551) cell tail; this can be due to the presence of three additional tyrosine residues in the full length IL2Rb (333-551) cell tail that can induce long-term negative regulation. Thus, optimizing the cell tail signaling domain to eliminate such negative regulatory motifs is beneficial to ensure long-term maintenance of constitutive and productive signaling. Since a strong cell tail can elicit a strong negative feedback response, a weak cell tail can be preferred for long-term stimulation.

[0238] Example 7: Optimized IL2Rb-derived cell tail more closely mimics IL-15 than IL-2 signaling

[0239] IL-2 and IL-15 are two cytokines that signal naturally through a heterodimeric cytokine receptor composed of common gamma chain and IL2Rb. Despite sharing the same native receptor, IL-2 and IL-15 have different effects on T cell differentiation and persistence. IL-2 induces differentiation of short-lived effector cells, while IL-15 promotes the generation of long-lived memory T cells. Furthermore, increased IL-15 serum concentrations have been shown to positively correlate with patient response to CAR-T cell therapy. Thus, a cell tail that mimics IL-15 rather than IL-2 signaling and effects is preferred. We sought to determine whether a reduced IL2Rb cell tail more closely mimics IL-2 or IL-15 signaling.

[0240] To this end, we used CAR-T cells including an exemplary CAR carrying a P5A2 scFv against BCMA coupled to a rituximab mimotope, 4-1 BB and CD3z signaling domains (see US 10,294,304, incorporated herein by reference). BCMA-specific CAR-T cells co-expressing a truncated IL2Rb tail were generated and their gene expression profile was compared to control CAR-T cells exposed to exogenous recombinant human IL-2 or IL-15. To prepare lentivirus encoding CACCR and CAR, HEK293T cells were seeded at 450,000 cells / mL in 2 mL DMEM (Gibco) supplemented with 10% FBS (Hyclone) per well of a 6-well plate the day before transfection. On the day of transfection, lentivirus was prepared by mixing 1.5 ug psPAX2, 0.5 ug pMD2G and 0.5 ug of the appropriate transfer CAR vector in 250 uL Opti-MEM (Gibco) per well of a 6-well plate (“DNA mix”). 10 uL Lipofectamine 2000 (Invitrogen) in 250 uL Opti-MEM was incubated at room temperature for 5 minutes before being added to the DNA mix. The mix was incubated at room temperature for 20 minutes and a total volume of 500 uL was slowly added to the side of the well containing HEK293T. The day after transfection, the medium of the HEK293T cells in the 6-well plate was replaced by 2 mL of T cell transduction medium, i.e. X-Vivo-15 supplemented with 10% FBS, per well. Two days after transfection, lentivirus supernatant from the HEK293T cells was collected and cell debris was removed by a 0.45 micron filter (EMD Millipore) and the crude lentivirus supernatant was used directly for T cell transduction. On day 0, purified T cells were activated in X-Vivo-15 medium (Lonza) supplemented with 100 IU / mL human IL-2 (Miltenyi Biotec), 10% FBS (Hyclone) and human T TransAct (Miltenyi Biotec, catalog number 130-111-160, 1 : 100 dilution) in Grex-24 plates (Wilson Wolf, catalog number 80192M). On day 2, T cells were resuspended at 5 million cells / mL in T cell transduction medium, transduced with an equal volume of crude lentivirus supernatant, and 100 IU / mL human IL-2 in Grex-24 plates. On day 5, CAR-T cells expressing the cell tail were fed by replacing spent medium with T cell expansion medium, i.e. X-Vivo-15 supplemented with 5% human AB serum (Gemini Bio) and 100 IU / mL human IL-2.At this time, control CAR-T cells lacking a cell tail were expanded in 100 U / mL human IL-2 alone, or in 100 U / mL human IL-2 and 10 ng / mL human IL-15 (Miltenyi Biotec). Cells were expanded into larger G-Rex flasks (Wilson Wolf) as needed using T Cell Expansion Medium and the corresponding concentration of recombinant cytokine. At day 13, cells were stained with Zombie NIR Viability Kit (Biolegend), labeled with BUV395-conjugated CD3 antibody (Biolegend) and an anti-idiotype antibody specific for the P5A2 scFv, followed by FACS sorting to enrich for CAR+ T cells. Sorted CAR+ T cells were then cultured in Grex-24 plates in T Cell Expansion Medium for an additional 2 days, CACCR CAR+ T cells were left without exogenous cytokines, while sorted control CAR+ T cells were left without exogenous cytokines, treated with 100 U / mL human IL-2, or treated with 10 ng / mL human IL-15. At day 15, live CAR+ T cells were enriched using Easy Sep Dead Cell Removal Kit (StemCell Technologies), and cell pellets were then snap-frozen for subsequent RNA extraction and NanoString gene expression analysis (Human CAR-T Panel; NanoString Technologies).

[0241] The data show that CACCR CAR-T cells carrying the truncated IL2Rb tail more closely mimic IL-15 than IL-2 signaling. For example, we tested the cell tail TpoR (478-582; S505N, W515K).IL2Rb (393-433, 518-551) and TpoR (478-582; H499L, S505N, W515K).IL2Rb (339-379, 393-433, 518-551). Figure 16A is a schematic of the experimental design and workflow for sample preparation. Figure 16B Gene expression profiles of CACCR CAR-T cells compared to control CAR-T cells treated with IL-2 at days 13-15 are shown. Figure 16C Gene expression profiles of CACCR CAR-T cells compared to control CAR-T cells treated with IL-15 at days 13-15 are shown. Log2 fold change (FC) was calculated for each sample by normalizing to control CAR-T cells untreated at days 13-15. R2determined by linear regression analysis 2Values and best fit line (solid line) are shown on each plot. Data shown is one representative of two donors. While the gene expression profile of CACCR CAR-T cells did not correlate with IL-2 treated samples Figure 16B ), the gene expression profile did positively correlate with IL-15 treated samples Figure 16C . These indicate that the cell tail carrying the truncated IL2Rb tail more closely mimics the downstream signaling and transcriptional response of IL-15 than IL-2.

[0242] Example 9: Constitutive cell tails can be programmed for combinatorial signaling outputs

[0243] As shown in Figure 12 , CACCRs carrying signaling domains from various cytokine receptors can activate signaling reminiscent of the parent receptors. We hypothesized that cell tails could be designed to mimic simultaneous signaling from multiple parent receptors by fusing more than one cell tail in tandem, thereby achieving combinatorial signaling outcomes. To test combinatorial signaling outputs from tandem cell tails, we generated a constitutive 7.12 tail by fusing the IL7Ra (316-459) cell tail with the truncated IL12Rb2 (775-825) cell tail and assessed signaling using a HEK293T cell reporter assay.

[0244] Figure 17A - D shows the design and signaling capacity of constitutive tandem cell tails, as exemplified by the 7.12 tail. Figure 17A A schematic of the constitutive 7.12 tail is shown. Figure 17B A schematic of lentiviral vectors used to co-express 7.12 tail variants and CARs, differing only in their TpoR (478-582) dimerization / JAK-binding domains is shown. Figure 17C - D shows STAT reporter activity of constructs carrying TpoR (478-582; S505N; W515K) and TpoR (478-582; H499L; S505N; W515K) dimerization / JAK-binding domains fused to the indicated cell tails, respectively. While the IL7Ra (316-459) cell tail strongly activates STAT5, the IL12Rb2 (775-825) cell tail strongly activates STAT4. However, as observed in the IL7Ra (316-459) IL12Rb2 (775-825) cell tail, the tandem fusion of the two cell tails leads to simultaneous and combinatorial activation of STAT5 and STAT4. This indicates that multiple signaling pathways typically required from two or more different native cytokine receptors can be achieved with a single cell tail.

[0245] Example 10: Constitutive cell tails can be tailored by single or multiple outputs to direct CAR-T cell phenotype and function

[0246] We next determined whether constitutive cell tails with different signaling outputs could differentially affect the phenotype and function of primary human CAR-T cells. Unlike IL-7, which drives T cell survival and memory maintenance, IL12 is a proinflammatory cytokine that can promote T cell differentiation. Thus, we sought to ask whether signaling through a cell tail derived from IL7Ra or IL12Rb could differentially direct these different phenotypes and assess the net combinatorial effect of fusing the two tails in tandem.

[0247] To this end, we generated human primary CAR-T cells that co-express a 7-tail (i.e., IL7Ra (316-459)) or 12-tail variant (i.e., IL12Rb2 (775-825) or IL12Rb2 (714-862)). See Figure 13ATo prepare lentivirus encoding CACCR and CAR, HEK293T cells were seeded at 450,000 cells / mL in 2 mL DMEM (Gibco) supplemented with 10% FBS (Hyclone) per well of a 6-well plate the day before transfection. On the day of transfection, lentivirus was prepared by mixing 1.5 ug psPAX2, 0.5 ug pMD2G, and 0.5 ug of the appropriate transfer CAR vector in 250 uL Opti-MEM (Gibco) per well of a 6-well plate (“DNA mix”). 10 uL Lipofectamine 2000 (Invitrogen) in 250 uL Opti-MEM was incubated at room temperature for 5 minutes before adding to the DNA mix. The mix was incubated at room temperature for 20 minutes and a total volume of 500 uL was added slowly to the side of the well containing the HEK293T. One day post-transfection, the media from the HEK293T cells in the 6-well plate was replaced with 2 mL of T cell transduction media, X-Vivo-15 supplemented with 10% FBS, per well. Two days post-transfection, lentivirus supernatant from the HEK293T cells was collected and cell debris was removed by a 0.45 micron filter (EMD Millipore), and the crude lentivirus supernatant was used directly for T cell transduction. On day 0, purified T cells were activated in X-Vivo-15 media (Lonza) supplemented with 100 IU / mL human IL-2 (Miltenyi Biotec), 10% FBS (Hyclone), and human T TransAct (Miltenyi Biotec, catalog number 130-111-160, 1:100 dilution) in Grex-24 plates (Wilson Wolf, catalog number 80192M). On day 2, T cells were resuspended at 5 million cells / mL in T cell transduction media, transduced with an equal volume of crude lentivirus supernatant, and 100 IU / mL human IL-2 in Grex-24 plates. On day 5, cells were fed by replacing spent media with T cell expansion media, X-Vivo-15 supplemented with 5% human AB serum (Gemini Bio), and 100 IU / mL human IL-2. Cells were expanded into larger G-Rex vessels (Wilson Wolf) as needed using T cell expansion media and corresponding concentrations of human IL-2. On day 14, memory phenotype analysis of CAR-T cell product was performed by detecting CAR transduced cells using a FITC conjugated v5 tag monoclonal antibody (Thermo Fisher) and co-staining with a PE / Cy7 conjugated CD62L antibody (Biolegend) and a BV785 conjugated CD45RO antibody (Biolegend) by flow cytometry.As a negative control lacking CACCR signaling, CAR-T cells were generated in parallel co-expressing BFP or the wild-type TpoR (478-582) transmembrane domain coupled to a 7-tail.

[0248] Figure 18 depicts the influence of cell tails on memory differentiation of CAR-T cell products. Shown is the memory phenotype of day 14 CACCR CAR-T cell products generated from 2 healthy donors. While CAR-T cells co-expressing the IL7Ra (316-459) cell tail retained a stem cell memory (Tscm) population, CAR-T cells carrying the IL12Rb2 derived cell tail showed a significant reduction of the Tscm population, while the central memory (Tcm) population increased. These indicate that constitutive IL12-like signaling by the IL12Rb2 derived cell tail can drive a progressive differentiation from Tscm to Tcm in the absence of CAR engagement. Furthermore, CAR-T cells co-expressing the tandem IL7Ra (316-459) IL12Rb2 (775-825) cell tail showed a restoration of the Tscm population and mimicked the phenotype of CAR-T cells carrying the single IL7Ra (316-459) cell tail, indicating that the negative effects of a single cell tail can be mitigated by combinatorial signaling of tandem cell tails.

[0249] We further investigated whether CACCR signaling can direct CAR-T cell functional outcomes, including survival rate and cytotoxicity. In contrast to IL-7 signaling, which promotes long-lived T cells, IL-12 signaling instead drives differentiation into short-lived terminal effectors. To support this, the Tscm population, which is capable of long-term survival and is thought to mediate prolonged CAR-T cell persistence, was rare in CAR-T cells carrying the IL12Rb2 derived tail. To interrogate whether signaling by different cell tails can program CAR-T cell survival and differentiation, we performed a growth factor independent assay, in which CACCR co-expressing CAR-T cells were cultured in the absence of target cells or exogenous supplemental cytokines. Under these conditions, CAR-T cell numbers and memory differentiation were monitored over time.

[0250] Briefly, cryopreserved CAR-T cells were thawed, counted, and the percentage of CAR-T cells in all samples was normalized to the sample with the lowest transduction efficiency by adding non-transduced (NTD) T cells. As a control, CAR-T cells co-expressing BFP (BFP CAR) were used instead of cell tails. Then 0.25 x 105CAR-T cells were cultured in 1.5 mL RPMI containing 10% FBS (Hyclone), non-essential amino acids, sodium pyruvate, and 20-25 mM HEPES with 0.5 x 105NTD T cells in a 24-well plate. The number of CAR-T cells was determined by flow cytometry on day 7. 6CAR+ T cells / mL were seeded into 24-well tissue culture plates. On the indicated days, 100 uL of duplicate samples were harvested from each condition and stained using the Zombie NIR Viability Kit (Biolegend). Samples were washed with PBS, Fc-blocked, and then stained with the following antibody cocktail diluted in PBS + 1% BSA: BUV395-conjugated anti-human CD3, BV510-conjugated anti-human CD8, BV605-conjugated human CD4, and FITC-conjugated v5 tag (for CAR detection), PE / Cy7-conjugated anti-human CD62L (Biolegend), and BV785-conjugated anti-human CD45RO (Biolegend). Finally, samples were washed in PBS and cell pellets were resuspended in 130 uL PBS + 1% BSA containing 123 eBead Counting Beads (Thermo Fisher) (10 uL Counting Beads in 120 uL PBS + 1% BSA) prior to FACS analysis.

[0251] Figure 19A -B shows representative data from cells from 2 donors. Figure 19A and Figure 19B shows the fold expansion of CAR-T cells relative to input at the start of the assay (day 0) for constructs carrying TpoR (478-582; S505N; W515K) and TpoR (478-582; H499L; S505N; W515K) dimerization / JAK-binding domains fused to the indicated cell tails. CAR-T cells carrying the IL7Ra (316-459) cell tail declined at a slower rate compared to control BFP CAR-T cells, indicating that constitutive signaling through the IL7Ra (316-459) cell tail improved CAR-T cell survival. In contrast, CAR-T cells carrying the IL12Rb2-derived cell tail declined at a similar rate to BFP CAR-T cells, indicating a lack of survival benefit. Notably, CAR-T cells carrying the tandem IL7Ra (316-459) IL12Rb2 (775-825) cell tail conferred a survival benefit more similar to the IL7Ra (316-459) cell tail. Figure 19C- D shows the CAR-T cell differentiation at day 7 of the growth factor independent assay for constructs carrying TpoR (478-582; S505N; W515K) and TpoR (478-582; H499L; S505N; W515K) dimerization / JAK-binding domains fused to the indicated cell tails, respectively. CAR-T cells carrying the IL7Ra (316-459) cell tail were not only more abundant, but also enriched in the Tscm population compared to control BFP CAR-T cells and CAR-T cells carrying the IL12Rb2 derived cell tail. See Figure 19C - D. CAR-T cells carrying the IL7Ra (316-459) IL12Rb2 (775-825) cell tail in tandem improved Tscm enrichment. Without being limited by any particular mechanism, the results suggest that the fusion of the IL7Ra (316-459) cell tail overrides the phenotype of the single IL12Rb2 (775-825) cell tail. Altogether, these data reiterate the combinatorial functional effect of tandem cell tail signaling and demonstrate the tunability of different cell tail constructs.

[0252] CAR-T cell products enriched in the Tscm population are associated with improved expansion, persistence and activity. However, terminally differentiated CAR-T cells have a short life span and limited proliferative potential, resulting in reduced therapeutic efficacy. Given that these characteristics are differentially affected by the IL7Ra (316-459) and IL12Rb2 derived cell tails, we assessed the ability of these cell tails to influence CAR-T cell cytotoxicity.

[0253] To this end, 5,000 U87KO-EGFRvIII-nucGFP target cells were seeded and allowed to adhere in 50 uL RPMI containing 10% FBS (Hyclone), non-essential amino acids, sodium pyruvate and 20-25 mM HEPES in 96-well plates with black walls and flat transparent bottom. EGFRvIII CAR (2173scFv) T cells carrying the IL7Ra (316-459) or IL12Rb derived cell tail were thawed and added to the seeded target cells at a 1 :3 E:T ratio. Since the number of target cells exceeds the CAR-T cells, repeated or continuous killing by the CAR-T cells is required to control target cell growth. As a control, CAR-T cells co-expressing a BFP CAR were used instead of the cell tail. The number of live target cells over time was monitored by the IncuCyte live cell analysis imaging system.

[0254] Figure 20A - B depicts the cytotoxic activity of CAR-T cells co-expressing multiple CACCRs. Figure 20A-B shows the elimination of target cells by CACCR CAR-T cells carrying TpoR (478-582; S505N; W515K) and TpoR (478-582; H499L; S505N; W515K) dimerization / JAK-binding domains fused to the indicated cell tails, respectively. CAR-T cells carrying IL12Rb2- derived cell tails showed some improvement to no improvement in serial killing activity in vitro compared to BFP CAR-T cells, likely due to their limited proliferative potential and short lifespan. In contrast, CAR-T cells carrying IL7Ra (316-459) cell tails showed improved serial killing activity, likely due to enhanced proliferation and persistence. Notably, CAR-T cells carrying the tandem IL7Ra (316-459).IL12Rb2 (775-825) cell tail showed the same or better serial killing activity as the IL7Ra (316-459) cell tail, suggesting that combining the pro-persistence IL7Ra (316-459) signaling domain and the pro-effector IL12Rb2 (775-825) signaling domain in a single cell tail can simultaneously enhance the lifespan, expansion, and immediate effector functions of CAR-T cells.

[0255] Example 11: Constitutive Cytokine Receptor Enhances In Vitro Cytotoxicity of CARs Against Liquid Tumor Targets

[0256] We have demonstrated that CACCRs can enhance the activity of CARs against EGFRvIII, a target for solid tumors (e.g., glioblastoma). To determine whether CACCRs are broadly applicable to different scFvs against hematological tumor targets, we additionally cloned CACCRs into CAR constructs against a hematological malignancy marker, namely BCMA, and assessed long-term cytotoxicity against BCMA-positive target cell lines.

[0257] Luc-GFP labeled target cells were seeded at 10,000 cells per well in white flat-bottom 96-well tissue culture plates in 100 uL / well. Cryopreserved CAR-T cells were thawed, counted, and the percentage of CAR-T cells in all samples was normalized to the sample with the lowest transduction efficiency by adding non-transduced (NTD) T cells. Volumes of 100 uL of CAR-T cells were then added to each well of target cells at the indicated effector:target (E:T) ratios in triplicate. As a "target only" negative control, 100 uL of media (instead of T cells) was added to target cells. After two to three days, the wells were mixed by gentle pipetting, and 100 uL of each T cell-containing well was transferred to a new white flat-bottom 96-well tissue culture plate containing 10,000 freshly seeded Luc-GFP labeled target cells (100 uL). The "target only" wells received fresh media instead of T cells. The new plate was incubated at 37°C while the number of live target cells remaining in the old 96-well plate was determined using the ONE-Glo luciferase assay system (Promega) according to the manufacturer's instructions. The percentage of live target cells was calculated by normalizing the luciferase signal to that of the "target only" wells, and the percentage of cytotoxicity was calculated as 100% - % live target cells. The serial transfer to fresh target cells and luciferase readout was continued every two to three days until all cytotoxic activity ceased.

[0258] Figure 21 CACCR was shown to improve cytotoxic activity of CAR-T cells against BCMA, a liquid tumor target. Figure 21 BCMA CAR (P5A2 scFv) cytotoxicity against MM1.S multiple myeloma cell line at E:T = 10:1 is shown, which indicates that co-expression of CACCR increases long-term cytotoxicity of CAR-T cells.

[0259] Example 12: CACCR enhances in vivo activity of CAR-T cells

[0260] CAR-T cell therapies, such as those targeting CD19 and BCMA, have achieved unprecedented clinical success in treating hematological malignancies. While very high complete remission rates have been achieved, this is transient as most patients eventually relapse. In addition, CAR-T cells have had more limited success in treating solid tumors. Causes of relapse and lack of response include insufficient CAR-T cell expansion and persistence, as well as suppression of CAR-T cell function by immunosuppressive microenvironments. As our in vitro characterization of CACCR CAR-T cells revealed improvements in target-driven proliferation, persistence, potency, and exhaustion, we next investigated whether these functional enhancements would translate into improved anti-tumor activity in vivo.

[0261] To interrogate CACCR CAR-T cell activity in vivo in the context of hematological malignancies, we used CAR-T cells bearing a BCMA-specific P5A2 scFv coupled to 4-1BB and CD3 zeta signaling domains in a multiple myeloma orthotopic xenograft model. T cell receptor (TCR)-deficient BCMA CAR-T cells were generated by transcription activator-like effector nuclease (TALEN)-mediated knockout to avoid potential confounding from TCR-driven xenoreactivity. Eight- to 10-week-old female NSG mice were irradiated with 1 Gy the day before intravenous inoculation with 5 x 105MM1.S-Luc-GFP. Fourteen days after tumor implantation, mice were randomized according to tumor burden and intravenously injected with 1 x 105or 3 x 105designated CAR-T cells (n = 10 per group). Tumor progression was monitored by bioluminescence imaging. On day 30 after T cell administration, mice that received 3 x 105CAR-T cells were bled to enumerate peripheral BCMA CAR-T cells. Specifically, 50 uL whole blood from each mouse was subjected to red blood cell lysis using ACK lysis buffer (Gibco), Fc-blocked, and stained with the following antibody cocktail diluted in PBS + 1% BSA: FITC-conjugated anti-mouse CD45 (Biolegend), BV421-conjugated anti-human CD45 (Biolegend), and an anti-idiotype antibody specific for the P5A2 scFv. Finally, samples were washed in PBS and cell pellets were resuspended in 130 uL PBS + 1% BSA containing 123 eBeads Counting Beads (Thermo Fisher) (10 uL counting beads in 120 uL PBS + 1% BSA) prior to FACS analysis. 6 6 6 6

[0262] Figure 22A ​​​​-C shows that CACCR enhances the in vivo antitumor activity and durability of BCMA CAR-T cells against orthotopic multiple myeloma. Figures 24A-B show the response to 1×10 6 One or 3×10 6 Tumor progression after targeted CAR-T cell therapy. Although control BCMA CAR-T cells were able to mediate initial tumor regression, this response was transient, as the tumor relapsed 22 days after cell infusion. However, CAR-T cells co-expressing CACCR significantly delayed tumor relapse and improved the durability of the response. Statistical data in Figures 24A-B represent **p<0.01 and ***p<0.001, based on repeated measures one-way ANOVA and... Figure 22A Days 6-34 and Figure 22B Tukey multiple comparisons from day 6 to day 44. Figure 22C This demonstrates the use of 3×10⁻⁶ cells 30 days after T-cell infusion. 6 The number of BCMA CAR-T cells present in the peripheral blood of mice treated with CAR-T cells. Consistent with the tumor recurrence observed in mice treated with control BCMA CAR-T cells, control BCMA CAR-T cells were no longer detectable in the peripheral blood. In contrast, CACCR BCMA CAR-T cells, which have an advantage in preventing tumor recurrence, were also more abundant in vivo. The statistics in Figure 24C represent *p<0.05 and ****p<0.0001, based on ordinary one-way ANOVA versus Tukey multiple comparisons. These suggest that enhanced CACCR CAR-T cell persistence partially mediates enhanced long-term tumor control and prolonged response durability.

[0263] We also evaluated the effect of CACCR on CAR-T cell activity in solid tumors known to be resistant to CAR-T cell therapy, such as glioblastoma. To this end, we utilized an EGFRvIII-specific CAR carrying 2173scFv conjugated to the 4-1BB and CD3ζ signaling domains, and the LN229 human glioblastoma cell line stably overexpressing EGFRvIII (LN229-EGFRvIII). 3 × 10⁶ cells were subcutaneously implanted into 8-10 week old female NSG mice. 6 1 LN229-EGFRvIII. 25 days later, when tumors formed, mice were randomly assigned based on tumor burden and intravenously injected with 1.5 × 10⁻⁶ LN229-EGFRvIII. 6 One or 3×10 6 Selected CAR-T cells (n = 8-10 per group). Tumor progression was monitored twice weekly using calipers.

[0264] Figure 23The results showed that CACCR enhanced the antitumor activity of CAR-T cells against established solid tumors. Although treatment with control EGFRvIII CAR-T cells could delay the growth of LN229-EGFRvIII tumors, this response was transient and suboptimal as the tumor eventually progressed. In contrast, treatment with CACCR CAR-T cells led to complete tumor regression. Notably, even at low doses (1.5 × 10⁻⁶), the tumor regressed significantly. 6 One CACCR CAR-T cell is sufficient to eliminate the tumor. Compared to using 3 × 10⁻⁶ cells... 6 Compared with control CAR-T cell therapy, statistical data indicate **p<0.01, based on repeated measures one-way ANOVA and Tukey multiple comparisons from day 3 to 38. These results reaffirm the ability of CACCR to non-redundantly synergize with signal transduction domains in CAR-T cells to enhance activity.

Claims

1. A constitutively active chimeric cytokine receptor (CACCR) consisting of two monomers, each monomer comprising: a. a transmembrane domain; b. a Janus kinase (JAK)-binding domain; and c. a recruitment domain, wherein the monomers are constitutively dimerized; wherein the transmembrane domain and / or the JAK-binding domain is derived from amino acids 478-582 of a naturally occurring TPOR / MPLR receptor of SEQ ID NO: 6, and an amino acid substitution selected from one of the following groups is introduced into the transmembrane domain and / or the JAK-binding domain compared to the amino acid sequence 478-582 of SEQ ID NO: 6: i) W515K; ii) H499L, G509N; iii) H499L, S505N, W515K; or iv) S505N, W515K.

2. The CACCR of claim 1, wherein the TPOR / MPLR receptor comprises the amino acid substitution W515K.

3. The CACCR of claim 1, wherein the TPOR / MPLR receptor comprises the amino acid substitutions S505N and W515K.

4. The CACCR of claim 1, wherein the TPOR / MPLR receptor comprises the amino acid substitutions H499L and G509N.

5. The CACCR of claim 1, wherein the TPOR / MPLR receptor comprises the amino acid substitutions H499L, S505N, and W515K.

6. The CACCR of any one of claims 1-5, wherein the monomers are identical.

7. The CACCR of any one of claims 1-5, wherein the monomers are different.

8. The CACCR of any one of claims 1-5, wherein the recruitment domain comprises a STAT- recruitment domain from a cytokine receptor.

9. The CACCR of any one of claims 1-5, wherein the recruitment domain comprises a STAT- recruitment domain from a receptor selected from the receptors presented in the following table:

10. The CACCR of any one of claims 1-5, wherein the recruitment domain comprises an amino acid sequence selected from one or more of SEQ ID NOs: 46-88, 106, and 143.

11. The CACCR of any one of claims 1-5, wherein the recruitment domain comprises a STAT- recruitment domain from IL7Ra.

12. The CACCR of claim 11, wherein the IL7Ra is the IL7Ra set forth in SEQ ID NO:

46.

13. The CACCR of any one of claims 1-5, wherein the recruitment domain comprises a STAT- recruitment domain from IL2Rb.

14. The CACCR of claim 13, wherein the recruitment domain comprises a STAT- recruitment domain from IL7Ra.

15. The CACCR of any one of claims 1-5, wherein the recruitment domain comprises a STAT-recruitment domain from IL12Rb2.

16. The CACCR of claim 15, wherein the IL12Rb2 comprises IL12Rb2 set forth in SEQ ID NO: 86 or IL12Rb2 set forth in SEQ ID NO:

67.

17. The CACCR of any one of claims 1-5, wherein the recruitment domain comprises STAT- recruitment domains from two receptors.

18. The CACCR of any one of claims 1-5, wherein the recruitment domain comprises STAT- recruitment domains from two cytokine receptors.

19. The CACCR of claim 18, wherein the two cytokine receptors are selected from the group consisting of IL7Ra, IL2Rb, and IL12Rb2.

20. The CACCR of any one of claims 1-5 and 19, which does not comprise an extracellular ligand binding domain.

21. A polynucleotide encoding any one of the CACCRs of any one of claims 1-20.

22. An expression vector comprising the polynucleotide of claim 21.

23. The expression vector of claim 22, comprising the polynucleotide of claim 21 and a polynucleotide expressing a chimeric antigen receptor (CAR).

24. The expression vector of claim 23, wherein the CAR binds BCMA, EGFRvIII, Flt-3, WT-1, CD20, CD23, CD30, CD38, CD70, CD33, CD133, LeY, NKG2D, CS1, CD44v6, ROR1, CD19, Claudin-18.2, DLL3, Muc17, FAPa, Ly6G6D, and / or RNF43.

25. The expression vector of any one of claims 22-24, wherein the vector is a lentiviral vector.

26. An engineered immune cell comprising the expression vector of any one of claims 22-25.

27. The engineered immune cell of claim 26, wherein the immune cell is a T cell.

28. An engineered immune cell comprising a chimeric antigen receptor (CAR) and at least one CACCR of any one of claims 1-20.

29. The engineered immune cell of claim 28, wherein the CAR and the CACCR are expressed in stoichiometrically equivalent amounts.

30. The engineered immune cell of any one of claims 26-29, wherein the immune cell is a T cell.

31. The engineered immune cell of any one of claims 26-29, wherein the CAR binds BCMA, EGFRvIII, Flt-3, WT-1, CD20, CD23, CD30, CD38, CD70, CD33, CD133, LeY, NKG2D, CS1, CD44v6, ROR1, CD19, Claudin-18.2, DLL3, Muc17, FAPa, Ly6G6D, and / or RNF43.

32. The engineered immune cell of any one of claims 26-29, wherein the immune cell is an allogeneic immune cell.

33. The engineered immune cell of any one of claims 26-29, wherein the immune cell is an autologous immune cell.

34. The engineered immune cell of any one of claims 26-29, wherein the immune cell is selected from the group consisting of a T cell, a dendritic cell, a killer dendritic cell, a mast cell, an NK cell, a macrophage, a monocyte, a B cell, and an immune cell derived from a stem cell.

35. A method of making an engineered immune cell, the method comprising introducing the polynucleotide of claim 21 or the expression vector of any one of claims 22-25 into an immune cell.

36. The method of claim 35, wherein the immune cell is selected from the group consisting of a T cell, a dendritic cell, a killer dendritic cell, a mast cell, an NK cell, a macrophage, a monocyte, a B cell, and an immune cell derived from a stem cell.

37. A pharmaceutical composition comprising the engineered immune cell of any one of claims 26-34.

38. A kit comprising the engineered immune cell of any one of claims 26-34 or the pharmaceutical composition of claim 37.

39. Use of a therapeutically effective amount of the engineered immune cell of any one of claims 26-34 or the pharmaceutical composition of claim 37 in the manufacture of a medicament for treating cancer in a subject.

40. The use of claim 39, wherein the cancer comprises a solid tumor.

41. The use of claim 39, wherein the cancer comprises a liquid tumor.

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