Constitutively active chimeric cytokine receptors

Abstract

The present invention provides a constitutively active chimeric cytokine receptor (CACCR). When present on immune cells carrying chimeric antigen receptors (CARs), such CACCRs can increase the activation, proliferation, persistence, and / or efficacy of immune cells. Also provided are methods for producing and using the CACCRs described herein.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of 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 entireties herein.

[0002] Sequence Listing This application contains a Sequence Listing, which has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The above ASCII copy, created on February 21, 2020, is named AT-023_03WO_SL.txt and is 251,652 bytes in size. [Background technology]

[0003] Adoptive transfer of immune cells (e.g., T cells) genetically modified to recognize malignant tumor-associated antigens has shown promise as a new approach to cancer treatment. T cells can be genetically modified to express chimeric antigen receptors (CARs), which are fusion proteins composed of an antigen recognition moiety and a T cell activation domain.

[0004] T cell proliferation, cytotoxicity potency, and persistence are driven by signaling pathways. Traditional CAR designs provide two signals: CD3zeta activation (signal 1) and costimulation (signal 2, e.g., 4-1BB, OX40, and / or CD28 expression). In some situations, a third signal (signal 3), cytokine-induced cytokine receptor signaling (e.g., cytokine support for immune enhancement), may be desirable. However, approaches that provide signal 3 have significant limitations.

[0005] One approach to provide cytokine support involves combining CAR-T cell therapy with systemic infusion of recombinant cytokine(s) / cytokine mimics and engineering CAR-T cells to secrete / express cytokines extracellularly. Because cytokines have pleiotropic effects and may 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 may cause systemic toxicity in humans, and (ii) in the context of allogeneic CAR-T cell therapy, these approaches may trigger bystander host immune activation that may accelerate rejection of allogeneic CAR-T cells and thereby impair therapeutic efficacy. Another approach to provide cytokine support is based on the introduction of a constitutively activated dimerized cytokine receptor, IL-7Ra - which limits the nature (IL-7 signaling only) and magnitude of the signaling output. Yet another approach to provide cytokine support involved incorporating signal 3 directly into the CAR molecule (Nat Med.2018 Mar;24(3):352-359). A 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 delivered.

[0006] A solution is needed to circumvent these drawbacks by specifically targeting cytokine signals to CAR-T cells in a tunable manner, improving the safety profile and therapeutic efficacy. Provided herein are compositions and methods that address this need. Summary of the Invention

[0007] The present disclosure provides a constitutively active chimeric cytokine receptor (CACCR). When present on immune cells carrying chimeric antigen receptors (CAR) (CAR-I cells, e.g., CAR-T cells), such CACCR allows for increased activation, proliferation, persistence, and / or efficacy of immune cells. Also provided are methods for making and using the CACCR described herein.

[0008] Thus, in one aspect, provided herein is a CACCR composed 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.

[0009] In some embodiments, the transmembrane domain and / or JAK binding domain are derived from a TPOR / MPLR receptor. In some embodiments, the transmembrane domain and / or JAK binding domain are 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 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 IL12Rb1, e.g., IL12Rb1(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).

[0010] Related aspects provided herein are polynucleotides encoding any one of the CACCRs of the present disclosure, and 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 claudin 18 isoform 2), DLL3 (delta-like protein 3, Drosophila delta homolog 3, Delta3), Muc17 (mucin 17, Muc3, Muc3), FAP alpha (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).

[0011] 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 may be selected from the group consisting of T cells, dendritic cells, killer dendritic cells, mast cells, NK cells, macrophages, monocytes, B cells, and immune cells derived from stem cells. In a related aspect, provided herein is a pharmaceutical composition comprising any of the engineered immune cells of the present disclosure, and a kit comprising such a pharmaceutical composition.

[0012] In another aspect, provided herein is a method of treating cancer in a subject comprising administering to the subject a therapeutically effective amount of any of the engineered immune cells described herein. [Brief description of the drawings]

[0013] [Figure 1]FIG. 1 shows a schematic diagram of an engineered CACCR of the present disclosure. [Diagram 2] Schematic diagram of the vector of the present disclosure that can be used to co-express the CACCR and CAR of the present disclosure. One or more cytotails can be linked in tandem to mimic signaling from one or more cytokines. Also shown is a schematic diagram of the vector expressing the control BFP (blue fluorescent protein) CAR. [Figure 3A-3B] We present the identification of a TpoR transmembrane (TM) mutant that constitutively activates cytokine receptor signaling. [Figure 4A-4C] 1 shows the results of expansion of CAR-T cells co-expressing constitutively active chimeric cytokine receptors. [Diagram 5] 1 shows the distribution of differentiation and memory T cell subsets in CAR-T cell products under different IL-2 conditions. [Figure 6A-6B] The degree of constitutive cytokine signaling mediated by each TpoR TM variant is shown. [Figure 7A-7D] We demonstrate the cytotoxic activity of TpoR TM mutants, demonstrating that constitutive cytokine receptor signaling enhances CAR-T cell potency. [Figure 8A-8B] Cytotoxic activity and durability of TpoR TM mutants. [Figure 9] Shows enrichment of CAR-T cells over time in a growth factor-independent assay. [Figure 10A-10B] Shows fold expansion of CAR-T cells over time in a growth factor-independent assay. [Figure 11] 1 shows the distribution of memory T cell subsets among CAR+ T cells over time in a growth factor-independent assay. [Figure 12] Activation of the STAT signaling pathway by CAR-T cells co-expressing the indicated CACCRs is shown. [Figure 13A-13B] Optimization of CACCR signaling strength as demonstrated by reporter assay in HEK293 T cells expressing full-length or truncated cytotails. [Figure 14A-14B]Optimization of CACCR signaling strength as demonstrated by reporter assay in HEK293 T cells expressing full-length or truncated cytotails. [Figure 15] Optimization of CACCR signaling strength demonstrated in primary CAR-T cells co-expressing full-length or truncated cytotails. [Figures 16A-16C] We show that CACCR CAR-T cells carrying a truncated IL2Rb cytotail more closely mimic IL-15, but not IL-2, signaling. [Figures 17A-17D] 1 shows the combinatorial signal output of different tandemly fused cytotails. [Figure 18A-18B] 1 shows the effect of CACCR on memory differentiation of CAR-T cells. [Figures 19A-19D] 1 shows the effect of CACCR on CAR-T cell survival and memory differentiation under growth factor-independent conditions. [Figure 20A-20B] Cytotoxic activity of CAR-T cells co-expressing various CACCRs. [Figure 21] 13 shows that CACCR improved the cytotoxic activity of CAR-T cells directed against the liquid tumor target BCMA. [Figures 22A-22C] We show that CACCR improved the in vivo antitumor activity and persistence of BCMA CAR-T cells against orthotopic multiple myeloma. [Figure 23] We show that CACCR improved the antitumor activity of CAR-T cells against established solid tumors. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] The present disclosure provides a constitutively active chimeric cytokine receptor (CACCR). The presence of a constitutively active and tunable chimeric cytokine receptor allows signal 3 immune enhancement to meet the need for immune enhancement. Thus, when present on immune cells carrying chimeric antigen receptors (CAR) (CAR-I cells, e.g., CAR-T cells), such CACCR allows for increased activation, proliferation, persistence, and / or potency of immune cells. Methods of making and using the CACCR described herein are also provided herein.

[0015] The CACCR of the present disclosure is tunable and has flexible cytokine signaling output for enhancing CAR-T cell activity, persistence, etc. The components, methods of making, and methods of use are described in turn below.

[0016] I. Constitutively active chimeric cytokine receptor (CACCR) The CACCR of the present disclosure is composed of two monomers, each of which contains (a) a transmembrane domain, (b) a Janus kinase (JAK) binding domain, and (c) a recruitment domain, and the monomers are constitutively dimerized. In some embodiments, the CACCR of the present disclosure does not contain an extracellular ligand-binding domain.

[0017] In some embodiments, the monomers are identical, resulting in constitutively active homodimers. In such embodiments, the number of proteins that need to be expressed in the vector is reduced. In some embodiments, the monomers are not identical, resulting in constitutively active heterodimers, which may be desirable under certain circumstances.

[0018] The monomer of CACCR of the present disclosure can spontaneously dimerize and can activate signal transduction in the absence of exogenous stimulation or ligand (ligand-independent dimerization).The level of activity can be controlled by the mutation introduced into the transmembrane domain of CACCR.Those skilled in the art will understand that the monomer of CACCR is not dimerized 100% of the time and can exist as a monomer.

[0019] A. Transmembrane Domains The CACCR of the present disclosure comprises a transmembrane domain.The transmembrane domain of the present disclosure comprises a sequence that allows them to constitutively dimerize with a monomer pair, thus allowing constitutive JAK activation in the intracellular portion, and constitutive recruitment and phosphorylation of STAT, for example, in the cytoplasmic region of the receptor.

[0020] The transmembrane domain is at the N-terminus and is linked to the C-terminus intracellular / cytoplasmic domain, in some embodiments, optionally via a linker.

[0021] As used herein, a transmembrane domain is capable of inserting into the membrane of a cell in which it is expressed. In some embodiments, a transmembrane domain of the present disclosure spans the cell membrane and includes an extracellular portion, and / or an intracellular portion.

[0022] In some embodiments, the transmembrane domains of the present disclosure are engineered (synthetic) and do not resemble any naturally occurring transmembrane domain, e.g., they are not naturally occurring.

[0023] In other embodiments, the transmembrane domain of the present disclosure is derived from a naturally occurring receptor.

[0024] In some embodiments, the transmembrane domain and / or JAK activation domain of the present disclosure are derived, for example, from 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, may not necessarily be necessary to achieve constitutive activation and constitutive JAK binding / activation in the intracellular portion. Thus, fragments of naturally occurring receptors can be utilized. Additionally, certain mutations may be introduced into the transmembrane domain derived from the naturally occurring receptor to further modulate downstream signaling.

[0025] In some embodiments, the transmembrane domain and / or the JAK activation domain of the present disclosure are derived from a naturally occurring EpoR receptor.

[0026] In some embodiments, the transmembrane domain and / or the JAK activation domain of the present disclosure are derived from a naturally occurring GP130 receptor.

[0027] In some embodiments, the transmembrane domain and / or the JAK activation domain of the present disclosure are derived from a naturally occurring PrlR receptor.

[0028] In some embodiments, the transmembrane domain and / or the JAK activation domain of the present disclosure are derived from a naturally occurring GHR receptor.

[0029] In some embodiments, the transmembrane domain and / or the JAK activation domain of the present disclosure are derived from a naturally occurring GCSF receptor.

[0030] In some embodiments, the transmembrane domain and / or JAK activation domain of the present disclosure is derived from a naturally occurring TPOR receptor.

[0031] Table 1a presents exemplary full-length sequences of naturally occurring receptors provided in the present disclosure from which transmembrane proteins are derived. The sequences provided in Table 1a are reference sequences for which later mutations are expressed, for example, in Tables 1b and 1c. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6]

[0032] In some embodiments, the transmembrane domains of the present disclosure are derived from truncated versions of the naturally occurring TPOR / MPLR (myeloproliferative leukemia protein) receptors shown in Table 1a.

[0033] In some embodiments, the transmembrane domain of CACCR comprises amino acids 478-582 of the TPOR receptor of Table 1a.

[0034] Table 1b provides exemplary transmembrane domain amino acid sequences of the present disclosure, where the transmembrane domain is derived from a naturally occurring TPOR receptor. [Table 2]

[0035] In some embodiments, the transmembrane domain of CACCR comprises amino acids 478-582 of the TPOR receptor and at least the amino acid substitution at H499. In some embodiments, the transmembrane domain of CACCR comprises amino acids 478-582 of the TPOR receptor and the amino acid substitution H499L.

[0036] In some embodiments, the transmembrane domain of 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 CACCR comprises amino acids 478-582 of the TPOR receptor and an amino acid substitution S505N.

[0037] In some embodiments, the transmembrane domain of 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 CACCR comprises amino acids 478-582 of the TPOR receptor and an amino acid substitution G509N.

[0038] In some embodiments, the transmembrane domain of 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 CACCR comprises amino acids 478-582 of the TPOR receptor and an amino acid substitution W515K.

[0039] In some embodiments, the transmembrane domain of CACCR comprises amino acids 478-582 of the TPOR receptor, as well as amino acid substitutions at H499 and S505 (sequences provided in Table 1b).

[0040] In some embodiments, the transmembrane domain of CACCR comprises amino acids 478-582 of the TPOR receptor, as well as amino acid substitutions at H499 and W515 (sequences provided in Table 1b).

[0041] In some embodiments, the transmembrane domain of CACCR comprises amino acids 478-582 of the TPOR receptor, as well as amino acid substitutions at H499, S505, and W515 (sequences provided in Table 1b).

[0042] In some embodiments, the transmembrane domain of CACCR comprises amino acids 478-582 of the TPOR receptor, as well as amino acid substitutions at S505 and W515 (sequences provided in Table 1b).

[0043] In some embodiments, the transmembrane domain of CACCR comprises amino acids 478-582 of the TPOR receptor, as well as amino acid substitutions at H499 and G509 (sequences provided in Table 1b).

[0044] In some embodiments, the transmembrane domain of CACCR comprises amino acids 478-582 of the TPOR receptor, as well as the amino acid substitutions H499L and S505N (sequences provided in Table 1b).

[0045] In some embodiments, the transmembrane domain of CACCR comprises amino acids 478-582 of the TPOR receptor, as well as the amino acid substitutions H499L and W515K (sequences provided in Table 1b).

[0046] In some embodiments, the transmembrane domain of CACCR comprises amino acids 478-582 of the TPOR receptor, as well as the amino acid substitutions H499L and G509N (sequences provided in Table 1b).

[0047] In some embodiments, the transmembrane domain of CACCR comprises amino acids 478-582 of the TPOR receptor, as well as the amino acid substitutions S505N and W515K (sequences provided in Table 1b).

[0048] In some embodiments, the transmembrane domain of CACCR comprises amino acids 478-582 of the TPOR receptor, as well as the amino acid substitutions H499L, S505N, and W515K (sequences provided in Table 1b).

[0049] In some embodiments, the transmembrane domain of CACCR comprises amino acids 478-582 of the TPOR receptor, as well as amino acid substitutions at H499 and S505 (sequences provided in Table 1b).

[0050] The CACCR of the present disclosure is required in CAR-bearing immune cells (e.g., CAR-T cells) and is tunable to achieve the level of signal 3 / immune enhancement desired in a particular situation or condition.

[0051] In some embodiments, low levels of STAT5 activation are desirable in CAR-bearing immune cells (e.g., CAR-T cells). By way of example, in such embodiments, the transmembrane domain of CACCR containing amino acids 478-582 of the TPOR receptor and the amino acid substitutions S505N, W515K, or H499L / G509N can be introduced.

[0052] In some embodiments, an increased level of STAT5 activation is desired in CAR-bearing immune cells (e.g., CAR-T cells). As an example, in such embodiments, a transmembrane domain of CACCR containing amino acids 478-582 of the TPOR receptor and amino acid substitutions H499L, S505N, and W515K can be introduced. As another example, in such embodiments, a transmembrane domain of CACCR containing amino acids 478-582 of the TPOR receptor and amino acid substitutions S505N and W515K can be introduced.

[0053] In some embodiments, increased differentiation into memory T cells is desired in CAR-bearing immune cells (e.g., CAR-T cells). By way of example, in such embodiments, the transmembrane domain of CACCR containing amino acids 478-582 of the TPOR receptor and the amino acid substitutions W515K, or H499L / G509N, can be introduced.

[0054] In some embodiments, increased differentiation into memory T cells is desired in CAR-bearing immune cells (e.g., CAR-T cells). By way of example, in such embodiments, the transmembrane domain of CACCR containing amino acids 478-582 of the TPOR receptor, as well as amino acid substitutions S505N / W515K and H499L / S505N / W515K, can be introduced.

[0055] Also provided herein are substitutions to increase cytotoxic potency, durability of response, and increased durability, for example, S505N / W515K and H499L / S505N / W515K substitutions. [Table 3-1] [Table 3-2] [Table 3-3]

[0056] B. Janus kinase (JAK) binding domain The CACCR of the present disclosure comprises an intracellular JAK binding domain. The JAK binding domain is linked to the C-terminus of the transmembrane domain directly or via a linker. The JAK binding domain is linked to the transmembrane domain on the cellular side of the chimeric cytokine receptor.

[0057] 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.

[0058] In some embodiments, the JAK-binding domain of a CACCR of the present disclosure is naturally occurring and is derived from a naturally occurring receptor.

[0059] In some embodiments, the JAK-binding domain of a CACCR of the present disclosure is synthetic.

[0060] Tables 1b and 1c provide exemplary amino acid sequences of transmembrane and JAK2-binding domains of the present disclosure. In some embodiments, the CACCR of the present disclosure comprises a transmembrane and JAK2-binding domain comprising an amino acid sequence selected from the sequences of Tables 1b and 1c. In some embodiments, the CACCR of the present disclosure comprises a transmembrane and JAK2-binding domain comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to any one of the sequences of Tables 1b and 1c.

[0061] C. Recruitment Domain The CACCR of the present disclosure comprises a cytoplasmic recruitment domain. The recruitment domain can be a STAT recruitment domain, an AP1 recruitment domain, a Myc / Max recruitment domain, or an NFkB recruitment domain. In some embodiments, the recruitment domain is a signal transducer and activator of transcription (STAT) recruitment (Stat activation) domain, e.g., from the receptor tail (cytotail) or cytokine receptor tail. These intracellular recruitment domains of the CACCR of the present disclosure allow for the propagation of signal 3 in immune cells comprising a CAR and a chimeric cytokine receptor (e.g., a CAR-T cell having a chimeric cytokine receptor of the present disclosure). The cytokine signaling propagated via the Stat recruitment domain allows for cytokine-based immune enhancement of cells. In some embodiments, the immune enhancement is homeostatic, e.g., the signaling causes an increase in immune cells bearing a CAR. In some embodiments, the immune enhancement is inflammatory, e.g., the signaling causes an increase in the efficacy of immune cells bearing a CAR. In some embodiments, the immune enhancement prevents exhaustion, e.g., the signaling maintains long-term function of immune cells bearing a CAR.

[0062] In some embodiments, the recruitment domains of the present disclosure are synthetic and do not resemble naturally occurring receptor fragments. In some embodiments, immune enhancement prevents exhaustion, e.g., signaling maintains long-term function of immune cells bearing the CAR.

[0063] In some embodiments, the Stat recruitment domains of the present disclosure are synthetic and do not resemble naturally occurring receptor fragments.

[0064] In other embodiments, the Stat recruitment domains of the present disclosure are derived from the cytoplasmic tails of naturally occurring receptors, e.g., from naturally occurring cytokine receptors. These cytoplasmic tails of naturally occurring receptors can be regions downstream of the JAK activation domain of the transmembrane domain of the receptor. The Stat recruitment domain of the chimeric cytokine receptor comprises at least one STAT recruitment domain from at least one receptor. In some embodiments, the Stat recruitment domain comprises at least one STAT1 recruitment domain. In some embodiments, the Stat recruitment domain comprises at least one STAT2 recruitment domain. In some embodiments, the Stat recruitment domain comprises at least one STAT3 recruitment domain. In some embodiments, the Stat recruitment domain comprises at least one STAT4 recruitment domain. In some embodiments, the Stat recruitment domain comprises at least one STAT5 recruitment domain. In some embodiments, the Stat recruitment domain comprises at least one STAT6 recruitment domain. In some embodiments, the Stat recruitment domain comprises at least one STAT7 recruitment domain.

[0065] In some embodiments, the naturally occurring receptor from which the Stat recruitment domain is derived is not a cytokine receptor.

[0066] In some embodiments, the naturally occurring receptor that Stat recruitment domain is derived from is a cytokine receptor.The exemplary cytokine receptor that T cell immune enhancing cytokine signals includes, but is not limited to, IL-2 receptor, IL-7 receptor, IL-15 receptor, and IL-21 receptor.In alternative embodiments, the receptor that Stat recruitment domain is derived from is not a cytokine receptor.By selecting the Stat recruitment domain of CACCR, the receptor can be redirected to the selected signal transduction.

[0067] In some embodiments, the CACCR of the present disclosure comprises a recruitment domain connected 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.

[0068] Table 2a presents exemplary receptors from which the recruitment domains of the CACCRs of the present disclosure are derived. Table 2b presents exemplary amino acid sequences of the recruitment domains of the present disclosure. In some embodiments, the CACCRs of the present disclosure comprise a recruitment domain comprising an amino acid sequence selected from one or more of the receptor sequences of Table 2b. In some embodiments, the CACCRs of the present disclosure comprise a recruitment domain comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to any one of the sequences of Table 2b. [Table 4] [Table 5-1] [Table 5-2] [Table 5-3] [Table 5-4] [Table 5-5]

[0069] In some embodiments, the Stat recruitment domain of a CACCR of the present disclosure comprises a STAT recruitment domain from one receptor.

[0070] To generate multiple outputs, two or more STAT recruitment domains can be linked in tandem to mimic signaling from one or more cytokines.

[0071] In some embodiments, two or more STAT recruitment domains may be linked in tandem, with or without a linker. In some embodiments, the linker comprises one or more amino acid residues.

[0072] In some embodiments, STAT recruitment domain comprises more than one receptor portion, for example, comprises more than one STAT recruitment domain.In such an embodiment, a tandem cytokine signaling domain is provided, enabling enhanced signaling.Thus, in some embodiments, the STAT recruitment domain of the monomer of CACCR of the present disclosure comprises more than one receptor-derived STAT recruitment domain, for example, comprises 2, 3, 4, 5, or even 6 receptor-derived STAT recruitment domains. For example, in some embodiments, STAT recruitment domains may be linked in tandem to stimulate multiple pathways (e.g., IL7R(316-459)-IL12Rb2(775-825) fragment fusions for sustained enhanced STAT5 and pro-inflammatory STAT4, IL7R(316-459)-IL2Rbsmall(393-433, 518-551) for sustained enhanced, IL7R(316-459)-EGFR(1122-1165) for sustained enhanced and anti-exhaustion, IL2Rbsmall(393-433, 518-551)-EGFR(1122-1165) for sustained enhanced and anti-exhaustion).

[0073] In generating multiple outputs, the proximity of individual STAT recruitment domains to the cell membrane can affect the strength of their respective signaling outputs. Table 2c shows examples of CACCRs with dual outputs, each of which can be located either proximal or distal to the cell membrane. In some embodiments, the CACCR of the present disclosure comprises a recruitment domain with dual outputs selected from Table 2c. [Table 6-1] [Table 6-2] [Table 6-3]

[0074] Without being bound by theory or mechanism, in some embodiments, JAK protein (JAK1, JAK2, JAK3 or TYK2) is bound to the dimerized CACCR of the present disclosure. The two bound JAK proteins are activated and can phosphorylate the tyrosine residue on the recruitment domain of CACCR. The phosphorylated recruitment domain can then bind to the recruited protein (e.g., phosphorylated STAT recruitment domain binds to STAT protein), which in turn leads to transcriptional events in the nucleus.

[0075] D. Exemplary CACCR Table 3 shows exemplary CACCR sequences of the present disclosure. The receptor can be expressed with a signal sequence, for example, the CD8SS of the sequence MALPVTALLLPLALLLHAARP (SEQ ID NO: 89).

[0076] In some embodiments, the CACCR of the present disclosure comprises any one 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 one of the amino acid sequences in SEQ ID NOs: 90-98 and 107-139. In some embodiments, the TPOR / MPLR receptor comprises any one of the amino acid sequences in SEQ ID NOs: 90-98 and 107-139.

[0077] In some embodiments, the CACCR comprises a transmembrane domain and / or a JAK binding domain derived from a TPOR / MPLR receptor. In some embodiments, the CACCR of the disclosure comprises amino acids 478-582 of the naturally occurring TPOR / MPLR receptor of SEQ ID NO:6. In some embodiments, the CACCR of the 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, the CACCR of the disclosure comprises the amino acid sequence of SEQ ID NO:17. In some embodiments, the CACCR further comprises a recruitment domain comprising one or more amino acid sequences of the receptor sequences shown in Table 2b. In some embodiments, the CACCR further comprises one or more recruitment domains selected from the group consisting of STAT recruitment domains derived from IL7Ra, IL2Rb, IL12Rb1, IL12Rb2, and IL21R. In some embodiments, the recruitment domain comprises a STAT recruitment domain derived 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 an 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 recruitment domain from IL12Rb1 or IL12Rb2 comprises an 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 recruitment 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 recruitment domain from IL21R comprises the amino acid sequence of SEQ ID NO: 54. In some embodiments, the CACCR comprises one or more recruitment domains shown in Table 2c. In some embodiments, the recruitment domain comprises a STAT recruitment domain from IL7Ra and IL2Rb. In some embodiments, the recruitment domain comprises a STAT recruitment domain from IL7Ra and IL12Rb1. In some embodiments, the recruitment domain comprises a STAT recruitment 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: 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.

[0078] 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 an H499L substitution. In some embodiments, the TPOR / MPLR receptor comprises an 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 one or more amino acid sequences of the receptor sequences shown 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 an 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 recruitment domain from IL12Rb1 or IL12Rb2 comprises an 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 recruitment 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 recruitment domain from IL21R comprises the amino acid sequence of SEQ ID NO: 54. In some embodiments, the CACCR comprises one or more recruitment domains shown in Table 2c. In some embodiments, the recruitment domain comprises a STAT recruitment domain from IL7Ra and IL2Rb. In some embodiments, the recruitment domain comprises a STAT recruitment domain from IL7Ra and IL12Rb1. In some embodiments, the recruitment domain comprises a STAT recruitment 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: 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.

[0079] 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 an H499L and S505N substitution. In some embodiments, the CACCR further comprises a recruitment domain comprising one or more amino acid sequences of the receptor sequences shown 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 recruitment domain from IL12Rb1 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 recruitment 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 recruitment domain from IL21R comprises the amino acid sequence of SEQ ID NO: 54. In some embodiments, CACCR comprises one or more recruitment domains shown in Table 2c. In some embodiments, the recruitment domain comprises a STAT recruitment domain from IL7Ra and IL2Rb. In some embodiments, the recruitment domain comprises a STAT recruitment domain from IL7Ra and IL12Rb1. In some embodiments, the recruitment domain comprises a STAT recruitment domain from IL7Ra and IL12Rb2. In some embodiments, CACCR comprises an amino acid sequence, with or without a signal 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. In some embodiments, CACCR comprises the amino acid sequence of SEQ ID NO: 91, 98, 120, or 127, with or without a signal sequence.

[0080] 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 an H499L and W515K substitution or an H499L and G509N substitution. In some embodiments, the CACCR further comprises a recruitment domain comprising one or more amino acid sequences of the receptor sequences shown 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 recruitment domain from IL12Rb1 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 recruitment 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 recruitment domain from IL21R comprises the amino acid sequence of SEQ ID NO: 54. In some embodiments, CACCR comprises one or more recruitment domains shown in Table 2c. In some embodiments, the recruitment domain comprises a STAT recruitment domain from IL7Ra and IL2Rb. In some embodiments, the recruitment domain comprises a STAT recruitment domain from IL7Ra and IL12Rb1. In some embodiments, the recruitment domain comprises a STAT recruitment domain from IL7Ra and IL12Rb2. In some embodiments, CACCR comprises an amino acid sequence, with or without a signal 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. In some embodiments, CACCR comprises the amino acid sequence of SEQ ID NO: 97 or 126, with or without a signal sequence.

[0081] 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 an S505N and W515K substitution. In some embodiments, the CACCR further comprises a recruitment domain comprising one or more amino acid sequences of the receptor sequences shown 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 recruitment domain from IL12Rb1 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 recruitment 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 recruitment domain from IL21R comprises the amino acid sequence of SEQ ID NO: 54. In some embodiments, CACCR comprises one or more recruitment domains shown in Table 2c. In some embodiments, the recruitment domain comprises a STAT recruitment domain from IL7Ra and IL2Rb. In some embodiments, the recruitment domain comprises a STAT recruitment domain from IL7Ra and IL12Rb1. In some embodiments, the recruitment domain comprises a STAT recruitment domain from IL7Ra and IL12Rb2. In some embodiments, 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, 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.

[0082] 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. In some embodiments, the CACCR further comprises a recruitment domain comprising one or more amino acid sequences of the receptor sequences shown 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 recruitment domain from IL12Rb1 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 recruitment 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 recruitment domain from IL21R comprises the amino acid sequence of SEQ ID NO: 54. In some embodiments, CACCR comprises one or more recruitment domains shown in Table 2c. In some embodiments, the recruitment domain comprises a STAT recruitment domain from IL7Ra and IL2Rb. In some embodiments, the recruitment domain comprises a STAT recruitment domain from IL7Ra and IL12Rb1. In some embodiments, the recruitment domain comprises a STAT recruitment domain from IL7Ra and IL12Rb2. In some embodiments, CACCR comprises an amino acid sequence, with or without a signal 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. In some embodiments, CACCR comprises the amino acid sequence of SEQ ID NO: 93, with or without a signal sequence.

[0083] 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 one or more amino acid sequences of the receptor sequences shown 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 recruitment domain from IL12Rb1 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 recruitment 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 recruitment domain from IL21R comprises the amino acid sequence of SEQ ID NO: 54. In some embodiments, CACCR comprises one or more recruitment domains shown in Table 2c. In some embodiments, the recruitment domain comprises a STAT recruitment domain from IL7Ra and IL2Rb. In some embodiments, the recruitment domain comprises a STAT recruitment domain from IL7Ra and IL12Rb1. In some embodiments, the recruitment domain comprises a STAT recruitment domain from IL7Ra and IL12Rb2. In some embodiments, 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, 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. [Table 7-1] [Table 7-2] [Table 7-3] [Table 7-4] [Table 7-5] [Table 7-6] [Table 7-7] [Table 7-8] [Table 7-9] [Table 7-10]

[0084] E. Expression of CACCR Provided herein is a polynucleotide that encodes any one of the CACCRs provided herein.Also provided herein is an expression vector that comprises such a polynucleotide.In some embodiments, the vector is a viral vector.In some embodiments, the vector is not a viral vector.

[0085] In some embodiments, the expression vector comprises a polynucleotide expressing CACCR and a chimeric antigen receptor (CAR).

[0086] In some embodiments, the expression of CACCR and CAR is expressed as a single polypeptide chain separated by a linker. Figure 2 shows a schematic diagram of a vector that can be used to co-express CACCR and CAR of the present disclosure. One or more recruitment domains can be linked in tandem to mimic signaling from one or more cytokines.

[0087] II. CAR-bearing immune cells Provided herein is an engineered immune cell comprising a polynucleotide encoding the chimeric antigen receptor (CAR) and CACCR of the present disclosure. Provided herein is an engineered immune cell expressing the chimeric antigen receptor (CAR-I cell) and CACCR of the present disclosure. Examples of immune cells include T cells, such as 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, bone marrow-derived phagocytes, dendritic cells, killer dendritic cells, macrophages, and monocytes. Immune cells also refer to cells derived from stem cells, for example, but not limited to. Stem cells can be adult stem cells, non-human embryonic stem cells, more specifically non-human stem cells, umbilical cord blood stem cells, progenitor cells, bone marrow stem cells, induced pluripotent stem cells, totipotent stem cells, or hematopoietic stem cells.

[0088] Thus, in some embodiments, provided herein is a CAR-T cell comprising a CACCR of the present disclosure.

[0089] In some embodiments, the CAR may 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., a single chain. Multichain CARs and polypeptides are also provided herein. In some embodiments, the multichain 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, where the polypeptides assemble together to form a multichain CAR.

[0090] The extracellular ligand-binding domain of the CAR specifically binds to a target of interest. The target of interest may 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 claudin 18 isoform 2), DLL3 (delta-like protein 3, Drosophila delta homolog 3, Delta3), Muc17 (mucin 17, Muc3, Muc3), FAP alpha (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).

[0091] 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 linked by a flexible linker. Single-chain variable region fragments are generated by linking the variable regions of the light chain and / or heavy chain by using a short linking peptide (e.g., a glycine-serine-containing linker) (Bird et al., Science 242:423-426, 1988). In general, the linker is a short, flexible polypeptide, generally composed of about 20 or less amino acid residues. The linker can then be modified for additional functions, such as attachment of drugs or attachment to a solid support. Single-chain variants can be produced either recombinantly or synthetically. For synthetic production of scFvs, 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, either eukaryotic, such as yeast, plant, insect or mammalian cells, or prokaryotic, such as E. coli. The polynucleotide encoding the desired scFv can be produced by routine manipulation, such as polynucleotide ligation. The resulting scFv can be isolated using standard protein purification techniques known in the art.

[0092] The intracellular signaling domain of the CAR according to the present invention is responsible for intracellular signaling following the binding of the extracellular ligand binding domain to a target, resulting in the activation of immune cells and immune responses (signal 1 and / or 2). The intracellular signaling domain has the ability to activate at least one of the normal effector functions of the immune cell in which the CAR is expressed. For example, the effector function of a T cell can be a cytolytic activity or a helper activity, including the secretion of cytokines.

[0093] In some embodiments, the intracellular signaling domain for use in the CAR can be, for example, without limitation, the cytoplasmic sequences of T cell receptors and co-receptors that act in concert to initiate signal transduction following antigen receptor engagement, as well as any analogs or variants of these sequences, and any synthetic sequences with the same functional capabilities. Intracellular signaling domains include two different classes of cytoplasmic signaling sequences, those that initiate antigen-dependent primary activation, and those that act in an antigen-independent manner to provide secondary or costimulatory signals. Primary cytoplasmic signaling sequences can include signaling motifs known as ITAM immunoreceptor tyrosine-based activation motifs. ITAMs are well-defined signaling motifs found in the intracytoplasmic tails of various receptors that function as binding sites for the syk / zap70 class of tyrosine kinases. Examples of ITAMs used in the present invention can include, by way of non-limiting example, those derived from TCRzeta, FcRgamma, FcRbeta, FcRepsilon, CD3gamma, CD3delta, CD3epsilon, CD5, CD22, CD79a, CD79b, and CD66d. In some embodiments, the intracellular signaling domain of the CAR can comprise a CD3ζ signaling domain. In some embodiments, the intracellular signaling domain of the CAR of the invention comprises a domain of a costimulatory molecule.

[0094] In some embodiments, the intracellular signaling domain of the CAR of the present invention comprises a portion of a costimulatory molecule selected from the group consisting of fragments of 41BB (GenBank: AAA53133.) and CD28 (NP_006130.1).

[0095] CAR is expressed on the surface membrane of a cell. Thus, CAR may include a transmembrane domain. A suitable transmembrane domain for the CAR disclosed herein has (a) the ability to be expressed on the surface of a cell, preferably an immune cell, such as, for example, but not limited to, a lymphocyte cell or a natural killer (NK) cell, and (b) the ability to interact with a ligand binding domain and an intracellular signaling domain to direct the cellular response of the immune cell to a predefined target cell. The transmembrane domain may be derived from either natural or synthetic sources. The transmembrane domain may be derived from any membrane-bound or transmembrane protein. As a non-limiting example, the transmembrane polypeptide may be a subunit of a T cell receptor, such as the α, β, γ or δ polypeptides that make up the CD3 complex, the IL-2 receptor p55 (chain), p75 (β chain) or γ chain, the subunit chain of the Fc receptor, particularly the Fcγ receptor III or CD protein. Alternatively, the transmembrane domain may be synthetic and may include primarily hydrophobic residues, such as leucine and valine. In some embodiments, the transmembrane domain is derived from the human CD8 α chain (e.g., NP_001139345.1). The transmembrane domain may further comprise a stalk domain between the extracellular ligand binding domain and the transmembrane domain. The stalk domain may comprise up to 300 amino acids, preferably 10-100 amino acids, most preferably 25-50 amino acids. The stalk region may be derived from all or a portion of a naturally occurring molecule, such as from all or a portion of the extracellular region of CD8, CD4, or CD28, or from all or a portion of an antibody constant region. Alternatively, the stalk domain may be a synthetic sequence that corresponds to a naturally occurring stalk sequence, or may be a completely synthetic stalk sequence. In some embodiments, the stalk domain is a portion of the human CD8 α chain (e.g., NP_001139345.1). In another particular embodiment, the transmembrane domain and hinge domain comprise a portion of the human CD8 α chain. In some embodiments, the intracellular signaling domain comprises a CD3 ζ signaling domain. In some embodiments, the intracellular signaling domain comprises a CD3 zeta signaling domain and further comprises a second signaling domain.In some embodiments, the intracellular signaling domain comprises a CD3ζ signaling domain and a 4-1BB signaling domain. In some embodiments, the CAR disclosed herein may comprise an extracellular ligand binding domain that specifically binds to BCMA or EGFRvIII, a CD8α human hinge and transmembrane domain, a CD3ζ signaling domain, and a 4-1BB signaling domain. In some embodiments, the EGFRvIII-specific CAR comprises the amino acid sequence of SEQ ID NO: 140. 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.

[0096] In some aspects, the CAR immune cell is a BCMA CAR-T cell comprising the CACCR of the present disclosure. In some embodiments, the CACCR of the BCMA CAR-T cell comprises a transmembrane / JAK binding domain of amino acids 478-582 of the naturally occurring TPOR / MPLR receptor 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.

[0097] In some embodiments, the CAR can be introduced into immune cells as a transgene via a plasmid vector. In some embodiments, the plasmid vector can also include a selection marker, for example, that provides for identification and / or selection of cells that have received the vector.

[0098] Table 4 provides exemplary sequences of CAR components that may be used in the CARs disclosed herein, as well as the antibody and / or CAR sequences exemplified herein. [Table 8-1] [Table 8-2]

[0099] In some embodiments, the CAR immune cells (e.g., CAR-T cells) of the present disclosure comprise a polynucleotide encoding a suicide polypeptide, such as, for example, RQR8. See, for example, WO2013153391A, which is incorporated herein by reference in its entirety. In some embodiments, the suicide polypeptide is expressed on the surface of the cell. In some embodiments, the suicide polypeptide is included in a CAR construct. In some embodiments, the suicide polypeptide is not part of a CAR construct.

[0100] In some embodiments, the extracellular domain of any one of the CARs disclosed herein may contain 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. WO2016 / 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). CARs that contain mAb-specific epitopes can be single-chain or multi-chain.

[0101] The inclusion of an epitope specific for a monoclonal antibody in the extracellular domain of a CAR described herein allows for the selection and depletion of engineered immune cells expressing the CAR. In some embodiments, allowing depletion provides a safety switch, for example in the event of adverse effects upon administration to a subject.

[0102] Also provided herein is a method for preparing engineered immune cells for use in immunotherapy. In some embodiments, the method includes introducing CACCR and CAR into immune cells and expanding the cells. In some embodiments, the present invention relates to a method for engineering immune cells, comprising providing a cell, expressing CACCR, and expressing at least one CAR on the surface of the cell. In some embodiments, the method includes transfecting the cell with at least one polynucleotide encoding CACCR and at least one polynucleotide encoding CAR, and expressing the polynucleotide in the cell. In some embodiments, the method includes transfecting the cell with at least one polynucleotide encoding CACCR and at least one polynucleotide encoding CAR, and expressing the polynucleotide in the cell.

[0103] In some embodiments, the polynucleotides encoding CACCR and CAR are present in one or more expression vectors for stable expression in cells.In some embodiments, the polynucleotides are present in a viral vector for stable expression in cells.In some embodiments, the viral vector can be, for example, a lentiviral vector or an adenoviral vector.

[0104] In some embodiments, the polynucleotide encoding the polypeptide according to the present invention can be, for example, mRNA that is directly introduced into cells by electroporation. In some embodiments, CytoPulse electroporation technology, such as PulseAgile, can be used to transiently permeabilize live cells for delivery of materials into cells (e.g., US 6,078,490, PCT / US2011 / 000827, and PCT / US2004 / 005237). Parameters can be altered to determine conditions for high transfection efficiency with minimal mortality.

[0105] Also provided herein are methods of transfecting immune cells, e.g., T cells. In some embodiments, the methods include contacting a T cell with RNA and applying an agile pulse sequence to the T cell. In some embodiments, the methods of transfecting immune cells (e.g., T cells) include contacting an immune cell with RNA and applying an agile pulse sequence to the cell.

[0106] In some embodiments, the method may further comprise genetically modifying the cell, for example, by inactivating at least one gene expressing a component of TCR, a target of an immunosuppressant, an HLA gene, and / or an immune checkpoint protein, such as, but not limited to, PDCD1 or CTLA-4. By inactivating a gene, it is intended that the gene of interest is not expressed in a functional protein form. In some embodiments, the inactivated gene is selected from the group consisting of, for example, but not limited to, TCRα, TCRβ, CD52, GR, deoxycytidine kinase (DCK), PD-1, and CTLA-4. In some embodiments, the method comprises inactivating one or more genes by introducing a rare-cutting endonuclease into the cell, which can selectively inactivate the gene by selective DNA cleavage. In some embodiments, the rare-cutting endonuclease may be, for example, a transcription activator-like effector nuclease (TALE nuclease), or a CRISPR-based endonuclease (e.g., Cas-9 or Cas12a).

[0107] In another aspect, the step of genetically modifying the cells may include modifying the immune cells by inactivating at least one gene that expresses a target of an immunosuppressant drug, and optionally expanding the cells in the presence of the immunosuppressant drug.

[0108] In some embodiments, the engineered immune cells (e.g., T cells) provided herein exhibit improved cytotoxicity, increased expansion, and / or increased levels of memory phenotypic markers compared to engineered immune cells that do not express CACCR.

[0109] In some embodiments, the engineered immune cells (e.g., T cells) provided herein exhibit (i) increased in vivo persistence, (ii) increased STAT activation, (iii) increased cytotoxicity, (iv) increased levels of memory phenotype markers, (v) increased expansion (proliferation), or a combination of these functional characteristics, compared to engineered immune cells that do not express CACCR. 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 STATs activated by the engineered immune cells comprising one or more of the disclosed CACCRs are STAT1, STAT2, STAT3, STAT4, STAT5, STAT6, or a combination thereof. In some embodiments, the memory phenotype markers increased or maintained by the immune cells comprising CACCR include stem cell memory (Tscm) markers and central memory (Tcm) markers.

[0110] In some embodiments, the improvement in one or more functional characteristics exhibited by immune cells comprising CACCR provided herein is at least about 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 125-fold, 150-fold, 200-fold, 250-fold, 300-fold, 350-fold, 400-fold, 450-fold, or even up to about 10,500-fold, including values ​​and ranges therebetween, as compared to immune cells that do not express CACCR.

[0111] In some embodiments, the improvement in one or more functional characteristics exhibited by immune cells comprising CACCR provided herein 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 up to about 500%, including values ​​and ranges therebetween, compared to immune cells that do not express CACCR.

[0112] III. Treatment Pharmaceutical compositions comprising cells bearing the CACCR and CAR of the present disclosure are provided herein.

[0113] The engineered CACCR-bearing and CAR-bearing immune cells (e.g., T cells) obtained by the above-mentioned method, or cell lines derived from such engineered immune cells, can be used as medicines. In some embodiments, such medicines can be used to treat disorders such as viral diseases, bacterial diseases, cancer, inflammatory diseases, immune diseases, or age-related diseases. 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 gastric cancer, sarcoma, lymphoma, leukemia, head and neck cancer, thymic 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).

[0114] In some embodiments, the engineered immune cells, or cell lines derived from the engineered immune cells, may be used in the manufacture of a medicament for the treatment of a disorder in a subject in need thereof, in some embodiments, the disorder may be, for example, cancer, an autoimmune disorder, or an infectious disease.

[0115] Also provided herein are methods of treating a subject in need of such treatment.

[0116] 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 ape and monkey species), livestock (e.g., cows, sheep, pigs, goats, and horses), domestic mammals (e.g., dogs and cats), laboratory animals (e.g., rodents such as rabbits, mice, rats, and guinea pigs), and birds (e.g., domestic, wild, and game birds such as chickens, turkeys, and other poultry birds, ducks, geese, and the like). In some embodiments, the subject is a mammal. In an exemplary embodiment, the subject is a human.

[0117] In some embodiments, the method comprises providing an immune cell of the present disclosure carrying a CACCR and a CAR as described herein to a subject in need thereof.

[0118] In some embodiments, T cells bearing the CACCR and CAR of the present invention can undergo robust in vivo cell expansion and remain for long periods of time.

[0119] The therapeutic methods of the invention may be ameliorative, curative or prophylactic. The methods of the invention may be either part of an autoimmunotherapy or part of an alloimmunotherapy treatment.

[0120] In another aspect, the present invention provides a method of inhibiting tumor growth or progression in a subject having a tumor, comprising administering to the subject an effective amount of an immune cell expressing CACCR and expressing CAR as described herein.In another aspect, the present invention provides a method of inhibiting or preventing metastasis of cancer cells in a subject, comprising administering to a subject in need thereof an effective amount of an engineered immune cell as described herein.In another aspect, the present invention provides a method of inducing tumor regression in a subject having a tumor, comprising administering to the subject an effective amount of an engineered immune cell as described herein.

[0121] In some embodiments, the engineered T cells herein may be administered parenterally in a subject.

[0122] Further provided herein is the use of any of the engineered T cells provided herein in the manufacture of a medicament for treating cancer or inhibiting tumor growth or progression in a subject in need thereof.

[0123] In some embodiments, the treatment may be administered in a subject undergoing immunosuppressive treatment. Indeed, the present invention preferably relies on a cell or a population of cells that has been rendered resistant to at least one immunosuppressive agent due to inactivation of a gene encoding a receptor for such an immunosuppressive agent. In this aspect, the immunosuppressive treatment should aid in the selection and expansion of immune cells according to the present invention in the subject. The administration of a cell or a population of cells according to the present invention may be performed in any convenient manner, including by aerosol inhalation, injection, ingestion, transfusion, implantation or transplantation. The compositions described herein may be administered to a subject subcutaneously, intradermally, intratumorally, intranodal, intramedullary, intramuscularly, intravenously or intralymphatic injection, or intraperitoneally. The cells bearing CACCR and CAR of the present disclosure or pharmaceutical compositions thereof may be administered via 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, via the ear, or intranasally.

[0124] In some embodiments, administration of cells or cell populations (bearing a CACCR and CAR of the present disclosure) includes all integer values ​​of cell numbers within these ranges, for example, about 10 per kg of body weight. 4 ~about 10 9 In some embodiments, the administration of cells or cell populations is at least about 10 per kg of body weight. 4 ~10 5 Cells, 10 per kg body weight 5 ~10 6 Cells, 10 per kg body weight 6 ~107 Cells, 10 per kg body weight 7 ~10 8 cells or 10 per kg of body weight 8 ~10 9 The administration of cells may include administration of cells. The cells or population of cells may be administered in one or more doses. In some embodiments, the effective amount of cells may be administered as a single dose. In some embodiments, the effective amount of cells may be administered as two or more doses over a period of time. The timing of administration is within the discretion of the attending physician and depends on the clinical condition of the subject. The cells or population of cells may be obtained from any source, such as a blood bank or a donor. While individual needs vary, determination of the optimal range of effective amounts of a given cell type for a particular disease or condition is within the skill of the art. An effective amount means an amount that provides a therapeutic or prophylactic benefit. The dosage administered will depend on the age, health, and weight of the recipient, type of concurrent treatment, frequency of treatment, if any, and the nature of the desired effect. In some embodiments, an effective amount of the cells or a composition comprising the cells is administered parenterally. In some embodiments, administration may be intravenous. In some embodiments, administration may be by injection directly into the tumor.

[0125] The method may further include administering one or more therapeutic 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, a method of lymphodepleting a subject in need of such therapy may include administering to the subject a specific effective dose of cyclophosphamide (200 mg / m 2 / day~2000mg / m 2 / day, about 100mg / m 2 / day~about 2000mg / m 2 / day, e.g., about 100 mg / m 2 / day, about 200mg / m 2 / day, about 300mg / m 2 / day, about 400mg / m 2 / day, about 500mg / m 2 / day, about 600mg / m 2 / day, about 700mg / m 2 / day, about 800mg / m 2 / day, approximately 900mg / m 2 / day, about 1000mg / m 2 / day, about 1500mg / m 2 / day or approximately 2000 mg / m 2 / day) and a specific dose of fludarabine (20 mg / m 2 / day~900mg / m 2 / day, about 10mg / m 2 / day~about 900mg / m 2 / day, e.g., about 10 mg / m 2 / day, about 20mg / m 2 / day, about 30mg / m 2 / day, about 40mg / m 2 / day, about 40mg / m 2 / day, about 50mg / m 2 / day, about 60mg / m 2 / day, about 70mg / m 2 / day, about 80mg / m 2 / day, approximately 90mg / m 2 / day, about 100mg / m 2 / day, about 500mg / m 2 / day or approximately 900 mg / m 2 An exemplary dosing regimen includes administering about 30 mg / m2 for three days prior to administration of a therapeutically effective amount of engineered immune cells to a patient. 2 in combination with, before, or after administration of fludarabine at a dose of about 300 mg / m 2 The method includes administering daily to the patient 100 mg / day of cyclophosphamide.

[0126] 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 at a dose of about 1-20 mg / day IV, e.g., about 13 mg / day IV, for 1, 2, 3 or more days. The antibody may be administered in combination with, prior to, or following administration of other components of the lymphodepletion regimen (e.g., cyclophosphamide and / or fludarabine).

[0127] In certain embodiments, the compositions comprising the CACCR- and CAR-expressing immune effector cells disclosed herein may be administered in combination with any number of chemotherapeutic agents.

[0128] IV. KITS AND PRODUCTS The present disclosure provides kits comprising any one or more of the cells carrying CACCR and CAR described herein, and pharmaceutical compositions thereof. The present disclosure also provides articles of manufacture comprising any one or more of the CAR-1 cells carrying CACCR and CAR described herein, pharmaceutical compositions thereof, and kits described herein.

[0129] The following examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention in any way.

[0130] All patent and non-patent literature referenced throughout this disclosure is hereby incorporated by reference in its entirety for all purposes. EXAMPLES

[0131] Example 1: Identification of TpoR TM mutants that constitutively activate cytokine signaling A prototypic constitutively active chimeric cytokine receptor (CACCR) was designed using the sequence of the thrombopoietin receptor (TpoR). TpoR can activate the JAK-Stat signaling pathway and transmits signals as a homodimeric receptor. Single point mutations (amino acid substitutions) in the TpoR receptor have been shown to modulate receptor activity (Proc Natl Acad Sci US 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, a constitutively active chimeric cytokine receptor was engineered from the naturally occurring TpoR receptor. The extracellular domain of the native TpoR receptor was removed, thus losing its ligand binding ability. One to three mutations were introduced into its transmembrane domain. The TpoR cytotail was replaced with that of the desired / described cytokine receptor. FIG. 1 shows a schematic diagram of an engineered constitutively active chimeric cytokine receptor.

[0132] To demonstrate the utility of a 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 the CAR, both genes were linked via a P2A peptide. To facilitate detection of transduced cells, a v5 epitope tag (KPIPNPLLGLDST (SEQ ID NO: 144)) was inserted between the scFv and the CD8 hinge domain.

[0133] FIG. 2 shows a schematic diagram of the lentiviral vector used to co-express a constitutively active chimeric cytokine receptor and a CAR.

[0134] Table 4 shows the sequences associated with the constructs used.

[0135] A HEK293T cell reporter assay was used to screen for TpoR TM variants capable of constitutive cytokine signaling. Briefly, 20,000 HEK293T cells were seeded into each well of a 96-well flat-bottom plate coated with poly-L-lysine and allowed to adhere overnight. Cytokine receptor-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 a final volume of 5 uL in Opti-MEM (Gibco) ("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 (erythropoietin receptor in place of the cytokine receptor-CAR construct) to allow induction of Stat5 signaling by addition of exogenous recombinant human Epo. 0.3uL Lipofectamine 2000 (Invitrogen) in 5uL Opti-MEM was incubated at room temperature for 5 minutes and then added to the DNA mixture. The mixture was incubated at room temperature for 20 minutes and a total volume of 10uL was added to each well containing HEK-293T. 48 hours after transfection, Stat5 reporter activity was assessed using the Dual-Glo Luciferase Assay System (Promega). The fold induction of Stat5 reporter activity was normalized to that of HEK293T cells transfected with all vectors except for the cytokine receptors and left untreated.

[0136] Figures 3a and 3b show the identification of TpoR TM mutants that constitutively activate cytokine receptor signaling. Figure 3a shows a schematic of the lentiviral vector used. It carries the IL7R(316-459) cytotail, which mimics IL7 signaling in CAR-T cells. Figure 3b shows Stat5 reporter activity as determined by Dual-Glo luciferase assay. Cytokine receptors carrying the wild-type TpoR TM domain (TpoR(478-582)) did not spontaneously activate Stat5. TpoR(478-582, S505N), TpoR(478-582, W515K), and TpoR(478-582, H499L, G509N) mutants resulted in weak Stat5 activation. TpoR(478–582, H499L, S505N, W515K) allowed moderate Stat5 activity, and TpoR(478–582, S505N, W515K) generated the strongest Stat5 signal.

[0137] Example 2: Generation of CAR-T cells expressing constitutively active chimeric cytokine receptors We next tested whether these cytokine receptors signal in the context of primary human CAR-T cells. To generate lentiviruses encoding cytokine receptor CARs, HEK293T cells were seeded at 450,000 cells per mL in 2 mL of DMEM (Gibco) supplemented with 10% FBS (Hyclone) per well of a 6-well plate the day before transfection. On the day of transfection, lentiviruses were prepared by mixing together lentiviral packaging vectors 1.5 ug psPAX2, 0.5 ug pMD2G, and 0.5 ug of the appropriate CAR vector in 250 uL of Opti-MEM (Gibco) per well of a 6-well plate ("DNA mixture"). 10 uL of Lipofectamine 2000 (Invitrogen) was incubated at room temperature for 5 minutes in 250 uL of Opti-MEM and then added to the DNA mixture. The mixture 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. One day after transfection, the medium from each well of HEK293T cells in the 6-well plate was replaced with 2 mL per well of T cell transduction medium, i.e., X-Vivo-15 supplemented with 10% FBS. Two days after transfection, the lentiviral supernatant from the HEK293T cells was harvested, passed through a 0.45 micron filter (EMD Millipore) to remove cell debris, concentrated 25-fold using a Lenti-X concentrator (Takara Bio) according to the manufacturer's instructions, and flash-frozen in aliquots. Lentiviral titers were determined by thawing an aliquot of frozen lentivirus, performing 4-fold serial dilutions, and performing limiting dilution titrations on Jurkat cells (clone E6-1; ATCC).On day 0, purified T cells were activated with 10% FBS (Hyclone) and human T TransAct (Miltenyi Biotec, catalog number 130-111-160, 1:100 dilution) in X-Vivo-15 medium (Lonza) supplemented with 100 IU / mL human IL-2 (Miltenyi Biotec) in Grex-24 plates (Wilson Wolf, catalog number 80192M). On day 2, T cells were resuspended at 500,000 cells / mL in T cell transduction medium and transduced with the respective lentiviral stocks at MOI=5 with 100 IU / mL human IL-2 in Grex-24 plates. On day 5, when transduction was completed, cells were harvested and washed to remove residual IL-2. They were then resuspended in T cell expansion medium, i.e., X-Vivo-15 supplemented with 5% human AB serum (Gemini Bio), and each sample was divided equally into two parts, one part 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 in larger G-Rex vessels (Wilson Wolf) as required, using T cell expansion medium and the respective concentration of human IL-2. On days 5, 9, and 14, the absolute number of T cells in each sample was counted, and transduction efficiency was determined by detecting the percentage of T cells bound to a FITC-conjugated v5 tag monoclonal antibody (Thermo Fisher) using flow cytometry. On days 14 or 15, the CAR-T cell products were cryopreserved and thawed as required for further assays.

[0138] Figures 4a-4c show the results of the generation of CAR-T cells co-expressing constitutively active chimeric cytokine receptors. Compared to CAR-T cell cultures with wild-type TpoR TM cytokine receptor (TpoR(478-582)), CAR-T cell cultures carrying TpoR TM mutants underwent a more robust expansion in terms of both total T cell numbers (Figure 4a) and CAR-T cell numbers (Figure 4b). Figures 4a-4c show that the transduction efficiency of the TpoR TM mutants is comparable or even higher than their wild-type TpoR(478-582) counterparts. The TpoR TM mutants allow for a higher yield of CAR-T cell product. Furthermore, expanding the TpoR TM mutants at lower IL-2 concentrations did not affect the expansion or yield of CAR-T cells (Figure 4c).

[0139] On the 14th day of CAR-T cell generation, the memory phenotype of CAR-T cells was determined. Briefly, samples were washed with PBS, blocked with Fc, and then stained with the following antibody cocktails 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 with PBS and cell pellets were resuspended in 130 uL of PBS+1% BSA for FACS analysis.

[0140] Figure 5 shows the distribution of memory T cell subsets in CAR-T cell products. Compared to their wild-type TpoR(478-582) counterparts, TpoR(478-582, W515K) and TpoR(478-582, H499L, G509N) mutants showed greater differentiation when expanded in standard 100 IU / mL IL-2 conditions. Growth in low IL-2 conditions improved differentiation. In concert with standard concentrations of IL-2, stronger Stat5 signaling induced by TpoR(478-582, W515K) and TpoR(478-582, H499L, G509N) mutants may result in accelerated CAR-T cell differentiation, and expansion in low IL-2 conditions may be more favorable in the context of CAR-T cells expressing constitutive cytokine receptors.

[0141] Example 3: TpoR TM mutants constitutively activate cytokine signaling in human CAR-T cells To determine the strength of cytokine signaling mediated by TpoR TM variants, CAR-T cells carrying TpoR TM cytokine receptor variants were serum-starved in 100uL of serum-free RPMI (Corning) for 4 hours in a humidified incubator at 37°C with 5% CO2. As a positive control, exogenous recombinant human IL-7 (10ng / mL; Miltenyi) was added for the last 30 minutes of the 4-hour serum starvation. After 4 hours, an antibody cocktail containing BUV395-conjugated anti-human CD3 (Biolegend) and FITC-conjugated v5 tag monoclonal antibody (Thermo Fisher) was added to the cells and incubated for the final 20 minutes. Cells were then fixed by adding 35uL of 16% paraformaldehyde to each 100uL sample and incubated at 37°C for 15 minutes. Cells were then washed three times with PBS and permeabilized with 100% cold methanol at -20% for one or two nights. On the day of FACS analysis, cells were washed 3 times with PBS, blocked with Fc, and stained with AlexaFluor647-conjugated anti-mouse / human Stat5(pY694) (BD Biosciences) diluted in PBS+1% BSA. After 1 hour incubation at room temperature in the dark, cells were washed 3 times before FACS analysis.

[0142] Figures 6a-6b show the degree 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). TpoR TM single mutants (TpoR(478-582, S505N) and TpoR(478-582, W515K)) did not induce appreciable Stat5 activation, whereas TpoR TM double mutant (TpoR(478-582, S505N, W515K) and triple mutant (TpoR(478-582, H499L, S505N, W515K)) induced relatively strong constitutive Stat5 activation. CAR-T cells expanded in low and standard IL-2 concentrations generated comparable Stat activation profiles. Stat5 was activated only in CAR-bearing (CAR+) and non-CAR-bearing (CAR-) T cells in the same cultures, indicating that cytokine signaling is CAR-T cell specific.

[0143] Example 4: Constitutive cytokine receptors enhanced CAR-T cell cytotoxic potency and long-term persistence of responses 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 kind gift from Cellectis SA (Paris, France). U87KO-EGFRvIII was derived from the parent cell line U87MG (ATCC) by first knocking out endogenous wild-type EGFR using transcription activator-like effector nucleases (TALENs) and then stably overexpressing full-length human EGFRvIII via lentiviral transduction. To facilitate target cell imaging via the IncuCyte live cell analysis imaging system, U87KO-EGFRvIII-nucGFP target cells were derived from U87KO-EGFRvIII by a second lentiviral transduction with IncuCyte NucLight Green lentiviral reagent (Sartorius). 5,000 U87KO-EGFRvIII-nucGFP target cells were seeded and allowed to adhere in 96-well plates with black walls and flat, clear bottom in 50 uL of RPMI containing 10% FBS (Hyclone), non-essential amino acids, sodium pyruvate, and 20-25 mM HEPES. EGFRvIII CAR (2173 scFv) T cells carrying the TpoR TM variant cytokine receptor 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 floating cells from the original plate to a new plate of target cells. Duplicate wells were set up for each condition. Cytotoxicity was determined by enumerating the number of live target cells at each time point using an IncuCyte live cell analysis imaging system.

[0144] Figures 7a-7d show the cytotoxic activity of TpoR TM mutants at an E:T ratio of 1:8. Consistent with their inability to effectively activate Stat5 (Figures 6a-6b), the single TpoR TM mutants TpoR(478-582, S505N) (Figure 7a) and TpoR(478-582, W515KN) (Figure 7b) showed no functional enhancement compared to their counterparts carrying the wild-type TpoR(478-582) control. Figure 7c shows that TpoR double mutant CAR-T cells expanded in standard IL-2 concentrations were not enhanced, whereas TpoR double mutant CAR-T cells expanded in low IL-2 conditions were more potent in lysis of target cells. Figure 7d shows that TpoR triple mutant CAR-T cells were more potent in target cell lysis, regardless of IL-2 concentration during CAR-T cell generation. This indicates that constitutive cytokine receptor signaling enhances the potency of CAR-T cells.

[0145] Figures 8a-8b show the cytotoxic activity of the TpoR TM double (Figure 8a) and triple mutants (Figure 8b) at an E:T ratio of 1:2. During the primary response, CAR-T cells eliminated targeted cells equally effectively, regardless of cytokine receptor activity. However, when re-challenged with new targets, only CAR-T cells expressing constitutively active chimeric cytokine receptors maintained function, indicating that constitutive cytokine receptor signaling enhances the persistence of CAR-T cell responses.

[0146] Example 5: Constitutive cytokine receptors enhanced CAR-T cell persistence and promoted expansion of CAR+Tscm. To confirm the enhancing effect of constitutive cytokine receptor signaling on CAR-T cell persistence in the absence of target or exogenous cytokines, a growth factor-independent assay was performed. Briefly, the percentage of CAR-T cells across all samples was normalized to the sample with the lowest transduction efficiency (35.7%) by the addition of non-transduced (NTD) T cells. 0.25 × 10 CAR-T cells were cultured in 4 mL of RPMI containing 10% FBS (Hyclone), non-essential amino acids, sodium pyruvate, and 20–25 mM HEPES. 6 100 μg / mL of CAR-bearing T cells. Cells were then seeded into 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)). Duplicate samples of 200 uL were collected from each condition and stained using the Zombie NIR Fixable Viability Kit (Biolegend). Samples were washed with PBS, blocked with Fc, and then stained with the following antibody cocktails 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 123count eBeads (Thermo Fisher) (10 uL counting beads in 120 uL PBS+1% BSA) prior to FACS analysis.

[0147] Figure 9 shows enrichment of CAR-T cells over time in a growth factor-independent assay. CAR-T cells carrying wild-type TpoR TM (TpoR(478-582) and TpoR TM single mutants (TpoR(478-582, S505N) and TpoR(478-582, W515KN)) were not enriched, whereas CAR-T cells carrying TpoR TM double and triple mutants were enriched over time, indicating that CAR-bearing T cells that received constitutive cytokine receptor signaling preferentially survived.

[0148] Figures 10a-10b show the fold expansion of CAR-T cells over time in a growth factor-independent assay. The fold expansion was determined by normalizing the absolute number of CAR-T cells at each time point to the number of CAR-T cells on day 0 of the assay. Figure 10a shows that the TpoR TM single mutant, which failed to productively activate cytokine receptor signaling, declined at the same rate as CAR-T cells carrying wild-type TpoR TM (TpoR(478-582)). In contrast, Figure 10b shows that CAR-T cells carrying the TpoR TM double mutant or triple mutant had extended survival in the absence of target and exogenous cytokines. TpoR TM double mutant CAR-T cells expanded in low IL-2 conditions showed increased persistence compared to their counterparts grown in standard IL-2 conditions. TpoR TM triple mutant CAR-T cells produced in low and standard IL-2 conditions showed comparable intermediate enhancements in persistence. Notably, TpoR double- and triple-mutant CAR-T cells persisted longer but eventually declined, indicating that constitutive cytokine receptor signaling is unlikely to result in the immortalization or transformation of CAR-T cells.

[0149] Figure 11 shows the distribution of memory T cell subsets over time among CAR+ T cells in a growth factor-independent assay. Compared to wild-type TpoR TM (TpoR(478-582)), CAR-T cells carrying a constitutively active cytokine receptor, in this case the TpoR TM triple mutant expanded in low IL-2 conditions, show an expansion in the absolute number of stem cell memory T cells (Tscm), a subset that mediates long-lived anti-tumor immunity. Notably, constitutive signaling from the TpoR TM triple mutant was more effective than exogenous human IL-7 supplementation in expanding Tscm CAR-T cells.

[0150] Example 6: Constitutive Cytotails Can Be Tuned to Activate Signaling Pathways of Interest The ability of cytokines to regulate CAR-T cell fate and function stems from their ability to induce various downstream signaling pathways. For example, IL-7 / IL-2 / IL-15-mediated activation of STAT5 promotes T cell survival and expansion, whereas IL-12-mediated activation of STAT4 promotes terminal differentiation into short-lived effectors. Engineering recruitment domains (i.e., cytotails) that can mimic a broader range of cytokine signals provides flexibility for user-programmable signaling outcomes, thereby resulting in control of CAR-T cell fate and function.

[0151] To investigate whether CACCR can transduce signals mediated through additional cytokine receptors, the IL7Ra(316-459) cytotail in Figure 3 was replaced with an alternative cytotail derived from the intracellular signaling domain 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 in each well of a poly-L-lysine coated 96-well flat-bottom plate and allowed to adhere overnight. The cytokine receptor-CAR construct (2.5 ng), a Stat response element driving firefly luciferase (100 ng, Promega), and a Renilla luciferase control reporter vector (1 ng, Promega) were mixed in a final volume of 5 uL in Opti-MEM (Gibco) ("DNA mixture"). As a negative control, cells were transfected with either a BFP-CAR construct lacking all cytokine signaling domains, or a TpoR(478–582).IL7Ra(316–459) construct lacking transmembrane mutations and therefore unable to signal constitutively. 0.3 uL of Lipofectamine 2000 (Invitrogen) in 5 uL of Opti-MEM was incubated at room temperature for 5 min and then added to the DNA mixture. The mixture was incubated at room temperature for 20 min and a total volume of 10 uL was added to each well containing HEK-293T. 48 h after transfection, the activity of the respective Stat 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.

[0152] Figure 12 shows that constructs carrying different signaling domains preferentially activate different STAT pathways. Specifically, IL2Rb(333-551) and IL7Ra(316-459) activate STAT5, whereas IL12Rb2(714-862) activates STAT4, mirroring the signaling expected for their respective parent receptors. This indicates that CACCR can be programmed to activate desired signaling pathways by fusing it with a signaling domain of interest. Furthermore, consistent with Figure 3, CACCRs carrying the TpoR(478-582, S505N, W515K) dimerization domain resulted in stronger signaling than their TpoR(478-582, H499L, S505N, W515K) counterparts. Thus, the strength of CACCR signaling output can be further tuned by fusion with either of these dimerization domains.

[0153] Example 7: The CACCR signaling domain can be optimized to modulate signal strength while reducing vector cargo size Currently, virus-based gene delivery methods (e.g., lentivirus- and retrovirus-mediated gene transfer) are routinely used to manufacture CAR-T cells. As cargo size increases, transduction efficiency and CAR-T cell yield decrease. Therefore, reducing the size of the cargo is beneficial to ensure successful manufacturing. Optimization of the recruitment / signaling domain of CACCR provides a means to achieve this goal for two reasons. First, as in the case of IL2Rb (333-551), signaling domains derived from cytokine receptors can reach over 200 amino acids in length and exceed 650 base pairs in transfer vectors. Second, although tyrosine residues within the signaling domain are important for initiating and propagating downstream signaling, some of them can also participate in negative feedback loops that limit the duration and strength of signaling. Thus, trimming the cytotail signaling domain not only allows the size of the vector cargo to be reduced, but also provides an opportunity to modulate cytotail 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 cytotail signaling.

[0154] Full-length IL12Rb2 (714-862) contains two phosphorylatable tyrosine residues, Y767 and Y800, which may be involved in downstream signaling. A truncated IL12Rb2 (775-825) tail containing only Y800 was generated and assessed for its ability to activate STAT4 using a HEK293T cell reporter assay. Briefly, 20,000 HEK293T cells were seeded in each well of a poly-L-lysine-coated 96-well flat-bottom plate and allowed to adhere overnight. CACCR-CAR construct (2.5 ng), a Stat response element driving firefly luciferase (100 ng, Promega), and a Renilla luciferase control reporter vector (1 ng, Promega) were mixed in a final volume of 5 uL in Opti-MEM (Gibco) ("DNA mixture"). 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 min and then added to the DNA mixture. The mixture was incubated at room temperature for 20 min and a total volume of 10 uL was added to each well containing HEK-293T. 48 h after transfection, the activity of the Stat4 reporter 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.

[0155] Figures 13A-B show the identification of a truncated IL12Rb2(775-825) cytotail capable of STAT4 activation comparable to the full-length IL12Rb2(714-862) cytotail. Figure 13A shows a schematic of the full-length IL12Rb(714-862) tail and the truncated IL12Rb(775-825) cytotail. The positions of the tyrosine residues (Y) contained in each tail are as indicated. Figure 13B shows that when fused to the more potent TpoR(478-582, S505N, W515K) dimerization domain, the truncated IL12Rb2(775-825) cytotail fully recapitulated the STAT4 signaling strength of the full-length IL12Rb2(714-862) cytotail. When fused to the weaker TpoR(478–582, H499L, S505N, W515K) dimerization domain, the truncated IL12Rb2(775–825) cytotail partially recapitulated the STAT4 signaling strength of the full-length IL12Rb2(714–862) cytotail.

[0156] The full-length IL2Rb (333–551) cytotail contains six tyrosine residues that may be involved in 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, and to promote cytoskeletal rearrangements to induce receptor internalization. Thus, Y364 is capable of promoting T cell effector functions and may also limit the strength and duration of IL2Rb signaling. We generated truncated IL2Rb cytotails containing three out of the six tyrosine residues (Y364, Y418, and Y436) or two out 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 in each well of a poly-L-lysine-coated 96-well flat-bottom plate and allowed to adhere overnight. CACCR-CAR construct (2.5ng), Stat response element driving firefly luciferase (100ng, Promega), and Renilla luciferase control reporter vector (1ng, Promega) were mixed in Opti-MEM (Gibco) in a final volume of 5uL ("DNA mixture"). As a negative control, cells were transfected with a BFP CAR construct lacking all cytokine signaling domains. 0.3uL of Lipofectamine 2000 (Invitrogen) in 5uL of Opti-MEM was incubated at room temperature for 5 minutes and then added to the DNA mixture. The mixture was incubated at room temperature for 20 minutes and a total volume of 10uL was added to each well containing HEK-293T. 48 hours after transfection, the activity of the Stat5 reporter was evaluated using the Dual-Glo Luciferase Assay System (Promega). Fold induction of Stat5 reporter activity was normalized to the fold induction of HEK293T cells transfected with a control BFP-CAR construct.

[0157] Figures 14A-B show the identification of truncated IL2Rb tails capable of equal or greater STAT5 activation compared to the full-length IL2Rb(333-551) tail. Figure 14A shows a schematic diagram of the full-length IL2Rb(333-551) tail and two truncated IL2Rb tails. The positions of the tyrosine residues (Y) contained in each tail are as indicated. The dotted lines represent the interconnection region of the full-length IL2Rb(333-551) tail that was deleted from the truncated tail. Figure 14B shows the results of HEK293T cell reporter assays, in which STAT5 signaling of full-length IL2Rb(333-551) is fully recapitulated by TpoR(478-582, H499L, S505N, W515K) IL2Rb(339-379, 393-433, 518-551) cytotail containing Y364, Y418, and Y536. Further removal of Y364, which mediates receptor internalization, in TpoR(478-582, S505N, W515K).IL2Rb(393-433, 518-551) cytotail dramatically enhanced STAT5 signaling strength.

[0158] The HEK293T cell assay is a short-term assay with readouts measured within 48 hours from cytotail transfection. Although it provides an efficient screening platform for cytotail activity, the limited duration of this assay does not reflect the complexity of the negative feedback loops triggered following prolonged constitutive cytokine and cytotail signaling. To more accurately evaluate the long-term signaling activity of reduced IL2Rb tail variants, we generated CACCR CAR-T cells using a 2-week production process and assessed activation of STAT5 by intracellular flow cytometry. To this end, CACCR CAR-T cells were serum-starved in 100 uL of serum-free RPMI (Corning) for 4 h in a humidified incubator at 37 °C with 5% CO2. As a positive control, exogenous recombinant human IL-2 (10 ng / mL; Miltenyi) was added during the last 30 min of the 4 h serum starvation. After 4 hours, an antibody cocktail containing BUV395-conjugated anti-human CD3 (Biolegend) and FITC-conjugated v5 tag monoclonal antibody (Thermo Fisher) was added to the cells and incubated for the final 20 minutes. Then, 35uL of 16% paraformaldehyde was added to each 100uL sample and incubated at 37°C for 15 minutes to fix the cells. The cells were then washed three times with PBS and permeabilized with 100% cold methanol at -20% for one or two nights. On the day of FACS analysis, the cells were washed three times with PBS, blocked with Fc, 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, the cells were washed three times before FACS analysis.

[0159] Figure 15 shows STAT5 activation in primary CACCR CAR-T cells carrying full-length or truncated IL2Rb cytotails. Maximal STAT5 activation was elicited by CACCR CAR-T cells carrying a truncated IL2Rb(393-433, 518-551) cytotail lacking the Y364 internalization motif. Intermediate STAT5 activation was observed in CACCR CAR-T cells carrying a truncated IL2Rb(339-379, 393-433, 518-551) cytotail. No STAT5 activation was observed in the CAR-negative population within the same culture, indicating the CAR-T cell-specific nature of cytotail signaling. Notably, little to no STAT5 activity was detected in CACCR CAR-T cells carrying the full-length IL2Rb(333-551) cytotail. This may be due to the other three tyrosine residues present in the full-length IL2Rb (333-551) cytotail, which may induce long-term negative regulation. Therefore, optimization of the cytotail signaling domain to eliminate such negative regulatory motifs would be beneficial to ensure that constitutive and productive signaling is maintained long-term. Because a strong cytotail may induce a strong negative feedback response, a weak cytotail may be preferable for long-term stimulation.

[0160] Example 7: Optimized IL2Rb-derived cytotails more closely mimic IL-15 but not IL-2 signaling IL-2 and IL-15 are two cytokines that naturally signal through a heterodimeric cytokine receptor composed of a common gamma chain and IL2Rb. Despite sharing the same natural receptor, IL-2 and IL-15 have different effects on T cell differentiation and persistence. IL-2 induces short-lived effector differentiation, whereas IL-15 promotes the generation of long-lived memory T cells. Furthermore, elevated serum concentrations of IL-15 have been shown to positively correlate with patient response to CAR-T cell therapy. Thus, a cytotail that mimics the signaling and effects of IL-15 rather than IL-2 would be preferable. We sought to determine whether a reduced IL2Rb cytotail more closely mimics IL-2 or IL-15 signaling.

[0161] To this end, we utilized CAR-T cells containing an exemplary CAR carrying the P5A2 scFv directed against BCMA, coupled to the rituximab mimotope, 4-1BB and CD3z signaling domain (see US10,294,304, 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 generate lentiviruses encoding CACCR and CAR, HEK293T cells were seeded at 450,000 cells per mL in 2 mL of 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 together lentiviral packaging vectors 1.5ug psPAX2, 0.5ug pMD2G, 0.5ug of the appropriate CAR vector in 250uL Opti-MEM (Gibco) per well of a 6-well plate ("DNA mixture"). 10uL Lipofectamine 2000 (Invitrogen) in 250uL Opti-MEM was incubated at room temperature for 5 minutes and then added to the DNA mixture. The mixture was incubated at room temperature for 20 minutes and a total of 500uL was slowly added to the side of the well containing HEK293T. One day after transfection, the medium from each well of HEK293T cells in the 6-well plate was replaced with 2mL per well of T cell transduction medium, i.e., X-Vivo-15 supplemented with 10% FBS. Two days after transfection, lentiviral supernatant was harvested from HEK293T cells and passed through a 0.45 micron filter (EMD Millipore) to remove cellular debris, and crude lentiviral supernatant was used directly for T cell transduction.On day 0, purified T cells were activated in Grex-24 plates (Wilson Wolf, catalog no. 80192M) 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 no. 130-111-160, 1:100 dilution) in X-Vivo-15 medium (Lonza) supplemented with 100 IU / mL human IL-2 (Miltenyi Biotec). On day 2, T cells were resuspended at 500,000 cells / mL in T cell transduction medium and transduced with an equal volume of crude lentiviral supernatant along with 100 IU / mL human IL-2 in Grex-24 plates. On day 5, cytotail-expressing CAR-T cells were cultured 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 cytotail were expanded with either 100 U / mL human IL-2 alone or 100 U / mL human IL-2 and 10 ng / mL human IL-15 (Miltenyi Biotec). Cells were expanded in larger G-Rex vessels (Wilson Wolf) as required using T cell expansion medium and respective concentrations of recombinant cytokines. On day 13, cells were stained with Zombie NIR Fixable Viability Kit (Biolegend), labeled with BUV395-conjugated CD3 antibody (Biolegend) and anti-idiotypic antibody specific for P5A2 scFv, and then sorted by FACS 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, with CACCR CAR+ T cells left in the absence of exogenous cytokines and sorted control CAR+ T cells left either in the absence of exogenous cytokines, treated with 100 U / mL human IL-2, or treated with 10 ng / mL human IL-15.On day 15, viable CAR+ T cells were enriched using the Easy Sep Dead Cell Removal Kit (StemCell Technologies), and cell pellets were flash frozen for subsequent RNA extraction and NanoString gene expression analysis (Human CAR-T Panel; NanoString Technologies).

[0162] The data show that CACCR CAR-T cells carrying a truncated IL2Rb tail more closely mimic the signaling of IL-15 rather than IL-2. As examples, we tested cytotailed 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 shows the gene expression profile of CACCR CAR-T cells compared to that of control CAR-T cells treated with IL-2 from days 13-15. Figure 16C shows the gene expression profile of CACCR CAR-T cells compared to that of control CAR-T cells treated with IL-15 from days 13-15. The Log2 fold change (FC) of each sample was calculated by normalization to control CAR-T cells left untreated from days 13-15. R determined by linear regression analysis 2 Values ​​and best-fit lines (solid lines) are shown on each graph. Data shown are representative of two donors. Gene expression profiles of CACCR CAR-T cells showed no correlation with IL-2 treated samples (Figure 16B), but positive correlation with IL-15 treated samples (Figure 16C). These suggest that cytotail carrying a truncated IL2Rb tail more closely mimics downstream signaling and transcriptional responses of IL-15 rather than IL-2.

[0163] Example 9: Constitutive Cytotails Can Be Programmed for Combinatorial Signaling Output As shown in Figure 12, CACCR, which carries signaling domains derived from various cytokine receptors, can activate signaling reminiscent of the parent receptor. We hypothesized that cytotails could be designed to mimic simultaneous signaling from multiple parent receptors by fusing more than one cytotail in tandem, achieving combinatorial signaling outcomes. To test combinatorial signaling output from tandem cytotails, we generated a constitutive 7.12tail by fusing the IL7Ra(316-459) cytotail with a truncated IL12Rb2(775-825) cytotail and assessed signaling using a HEK293T cell reporter assay.

[0164] Figures 17A-D show the design and signaling capabilities of constitutive tandem cytotails, as exemplified by 7.12tail. Figure 17A shows a schematic of constitutive 7.12tail. Figure 17B shows a schematic of lentiviral vectors used to co-express 7.12tail variants and CAR, differing only in their TpoR(478-582) dimerization / JAK binding domains. Figures 17C-D show the STAT reporter activity of constructs carrying TpoR(478-582;S505N;W515K) and TpoR(478-582;H499L;S505NW515K) dimerization / JAK binding domains, respectively, fused to the indicated cytotails. The IL7Ra(316-459) cytotail potently activated STAT5, whereas the IL12Rb2(775-825) cytotail potently activated STAT4. However, as observed with IL7Ra(316-459), the IL12Rb2(775-825) cytotail, which fuses both cytotails in tandem, simultaneously combinatorially activated both STAT5 and STAT4. This indicates that multiple signaling pathways that normally require two or more distinct negative cytokine receptors can be achieved with a single cytotail.

[0165] Example 10: Constitutive cytotails can be tuned with single or multiple outputs to direct CAR-T cell phenotype and function We next determined whether constitutive cytotails carrying distinct signaling outputs could differentially impact the phenotype and function of primary human CAR-T cells. Unlike IL-7, which promotes T cell survival and memory maintenance, IL12 is a proinflammatory cytokine that promotes T cell differentiation. Therefore, we sought to examine whether signaling through IL7Ra- or IL12Rb-derived cytotails could differentially direct these distinct phenotypes and to assess the net combinatorial effect of fusing both tails in tandem.

[0166] To this end, human primary CAR-T cells were generated that co-express either the 7-tail (i.e., IL7Ra(316-459)) or the 12-tail variants (i.e., IL12Rb2(775-825) or IL12Rb2(714-862)). See Figure 13A. To generate lentivirus encoding CACCR and CAR, HEK293T cells were seeded at 450,000 cells per mL in 2 mL of 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 together lentiviral packaging vectors 1.5ug psPAX2, 0.5ug pMD2G, 0.5ug of the appropriate CAR vector in 250uL of Opti-MEM (Gibco) per well of a 6-well plate ("DNA mix"). 10uL of Lipofectamine 2000 (Invitrogen) in 250uL of Opti-MEM was incubated at room temperature for 5 minutes and then added to the DNA mixture. The mixture was incubated at room temperature for 20 minutes and a total volume of 500uL was slowly added to the side of the well containing HEK293T. One day after transfection, the medium from each well of HEK293T cells in the 6-well plate was replaced with 2mL per well of T cell transduction medium, i.e., X-Vivo-15 supplemented with 10% FBS. Two days after transfection, the lentiviral supernatant was collected from the HEK293T cells and passed through a 0.45 micron filter (EMD Millipore) to remove cell debris, and the crude lentiviral supernatant was used directly for T cell transduction. On day 0, purified T cells were activated with human T TransAct (Miltenyi Biotec, catalog no. 130-111-160, 1:100 dilution) in X-Vivo-15 medium (Lonza) supplemented with 100 IU / mL human IL-2 (Miltenyi Biotec) in Grex-24 plates (Wilson Wolf, catalog no. 80192M) in X-Vivo-15 medium (Lonza) supplemented with 100 IU / mL human IL-2 (Miltenyi Biotec).On day 2, T cells were resuspended at 500,000 cells / mL in T cell transduction medium and transduced with an equal volume of crude lentiviral supernatant along with 100 IU / mL human IL-2 in Grex-24 plates. On day 5, cells were cultured 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. Cells were expanded in larger G-Rex vessels (Wilson Wolf) as required using T cell expansion medium and the respective concentrations of human IL-2. On day 14, memory phenotype analysis of the CAR-T cell product was performed by flow cytometry by detecting CAR-transduced cells using a FITC-conjugated v5 tag monoclonal antibody (Thermo Fisher) along with co-staining with PE / Cy7-conjugated CD62L antibody (Biolegend) and BV785-conjugated CD45RO antibody (Biolegend). As a negative control lacking CACCR signaling, we generated CAR-T cells in parallel co-expressing wild-type TpoR(478–582) transmembrane domain linked to BFP or 7tail.

[0167] Figure 18 shows the effect of cytotails on memory differentiation of CAR-T cell products. The memory phenotype of day 14 CACCR CAR-T cell products generated from two healthy donors is shown. CAR-T cells co-expressing IL7Ra(316-459) cytotails retained the stem cell memory (Tscm) population, whereas CAR-T cells carrying IL12Rb2-derived cytotails showed a marked decrease in the Tscm population accompanied by an increase in the central memory (Tcm) population. These suggested that in the absence of CAR engagement, constitutive IL12-like signaling through IL12Rb2-derived cytotails promotes progressive differentiation of Tscm to Tcm. Furthermore, CAR-T cells co-expressing tandem IL7Ra(316-459); IL12Rb2(775-825) cytotails showed restoration of the Tscm population, mimicking the phenotype of CAR-T cells carrying a single IL7Ra(316-459) cytotail. This indicates that the negative effects of a single cytotail can be alleviated by combinatorial signaling of tandem cytotails.

[0168] We further investigated whether CACCR signaling could direct CAR-T cell functional outcomes, including survival and cytotoxicity. Compared to IL-7 signaling, which promotes long-term T cell survival, IL-12 signaling instead promotes differentiation into short-lived terminally differentiated effectors. In support of this, the Tscm population, which is capable of long-term survival and is thought to mediate long-term CAR-T cell persistence, was largely absent in CAR-T cells carrying IL12Rb2-derived tails. To investigate whether signaling through different cytotails could program CAR-T cell survival and differentiation, we performed a growth factor-independent assay in which CAR-T cells co-expressing CACCR were cultured in the absence of target cells or exogenously supplemented cytokines. Under these conditions, CAR-T cell numbers and memory differentiation were monitored over time.

[0169] Briefly, cryopreserved CAR-T cells were thawed and counted, and the percentage of CAR-T cells across all samples was normalized to the sample with the lowest transduction efficiency by the addition of non-transduced (NTD) T cells. As a control, CAR-T cells co-expressing BFP instead of cytotail (BFP CAR) were used. 0.25 × 10 cells were cultured in 1.5 mL of RPMI containing 10% FBS (Hyclone), non-essential amino acids, sodium pyruvate, and 20–25 mM HEPES con. 6 CAR+ T cells / mL were seeded into 24-well tissue culture plates. On the days indicated, 100uL replicate samples were taken from each condition and stained using the Zombie NIR Fixable Viability Kit (Biolegend). Samples were washed with PBS, blocked with Fc, and then stained with the following antibody cocktails 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 123count eBeads (Thermo Fisher) (10 uL counting beads in 120 uL PBS+1% BSA) prior to FACS analysis.

[0170] Figures 19A-B show representative data for cells from two donors. Figures 19A and 19B show fold expansion of CAR-T cells compared 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 cytotails, respectively. Compared to control BFP CAR-T cells, CAR-T cells carrying the IL7Ra(316-459) cytotail declined at a slower rate, indicating that constitutive signaling through the IL7Ra(316-459) cytotail improved CAR-T cell survival. In contrast, CAR-T cells carrying IL12Rb2-derived cytotails declined at a rate comparable to BFP CAR-T cells, suggesting a lack of survival benefit. In particular, CAR-T cells carrying the tandem IL7Ra(316-459). IL12Rb2(775-825) cytotail conferred a survival benefit comparable to that of the IL7Ra(316-459) cytotail. Figure 19C-D show CAR-T cell differentiation at day 7 of a growth factor-independent assay of constructs carrying TpoR(478-582, S505N, W515K) and TpoR(478-582, H499L, S505N, W515K) dimerization / JAK-binding domains, respectively, fused to the indicated cytotails. Compared to control BFP CAR-T cells and CAR-T cells carrying IL12Rb2-derived cytotails, CAR-T cells carrying IL7Ra(316-459) cytotails were not only more abundant but also more enriched in the Tscm population. See Figure 19C-D. CAR-T cells carrying tandem IL7Ra(316-459)IL12Rb2(775-825) cytotails improved Tscm enrichment. Without being limited to a specific mechanism, the results suggest that fusion with IL7Ra(316-459) cytotails abrogated the phenotype of a single IL12Rb2(775-825) cytotail. Collectively, these data recapitulate the combinatorial functional effects of tandem cytotail signaling and demonstrate the tunability of different cytotail constructs.

[0171] CAR-T cell products enriched for Tscm populations are associated with improved expansion, persistence, and activity. However, terminally differentiated CAR-T cells are short-lived and have limited proliferative potential, resulting in reduced efficacy. Given that these properties were differentially affected by IL7Ra(316-459)- and IL12Rb2-derived cytotails, we assessed the ability of these cytotails to affect CAR-T cell cytotoxicity.

[0172] For this purpose, 5,000 U87KO-EGFRvIII-nucGFP target cells were seeded and allowed to adhere in 96-well plates with black walls and flat, clear bottom in 50 uL of RPMI containing 10% FBS (Hyclone), non-essential amino acids, sodium pyruvate, and 20–25 mM HEPES. EGFRvIII CAR (2173 scFv) T cells carrying cytotails derived from IL7Ra (316–459) or IL12Rb were thawed and added to the seeded target cells at an E:T ratio of 1:3. As the target cells outnumber the CAR-T cells, repeated or sequential killing of CAR-T cells is necessary to control the proliferation of target cells. As a control, CAR-T cells co-expressing the BFP CAR instead of the cytotail were used. The number of live target cells over time was monitored via an IncuCyte live cell analysis imaging system.

[0173] Figures 20A-20B show the cytotoxic activity of CAR-T cells co-expressing various CACCRs. Figures 20A-B show target cell clearance by CACCR CAR-T cells carrying TpoR(478-582, S505N, W515K) and TpoR(478-582, H499L, S505N, W515K) dimerization / JAK binding domains, respectively, fused to the indicated cytotails. Compared to BFP CAR-T cells, CAR-T cells carrying IL12Rb2-derived cytotails showed some or no improvement in serial killing activity in vitro, likely due to their limited proliferation capacity and short life span. In contrast, CAR-T cells carrying IL7Ra(316-459) cytotails 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) cytotail exhibited sequential killing activity equal to or greater than that of the IL7Ra(316-459) cytotail, suggesting that the combination of the persistence-promoting IL7Ra(316-459) signaling domain and the effector-promoting IL12Rb2(775-825) signaling domain within a single cytotail can simultaneously enhance CAR-T cell longevity, expansion, and immediate effector function.

[0174] Example 11: Constitutive cytokine receptors enhance in vitro cytotoxicity of CARs directed against liquid tumor targets We demonstrated that CACCR can enhance the activity of CARs directed against EGFRvIII, a target for solid tumors such as glioblastoma. To determine whether CACCR is broadly applicable across different scFvs for hematological tumor targets, we further cloned CACCR into a CAR construct directed against a marker for hematological malignancies (i.e., BCMA) and assessed long-term cytotoxicity against BCMA-positive target cell lines.

[0175] Target cells stably expressing firefly luciferase and a GFP reporter were generated by lentiviral transduction. 10,000 Luc-GFP-labeled target cells were seeded at 100uL per well in white flat-bottom 96-well tissue culture plates. Cryopreserved CAR-T cells were thawed and counted, and the percentage of CAR-T cells across all samples was normalized to the sample with the lowest transduction efficiency by the addition of non-transduced (NTD) T cells. A volume of 100uL of CAR-T cells was then added to each well of target cells in triplicate at the indicated effector:target (E:T) ratios. As a "target only" negative control, 100μL of media was added to the target cells instead of T cells. After 2–3 days, wells were mixed by gentle pipetting and 100uL of each T cell-containing well was transferred to a new white flat-bottom 96-well tissue culture plate containing 10,000 freshly plated 100µL of Luc-GFP-labeled target cells. "Target only" wells received fresh medium instead of T cells. The new plates were incubated at 37°C and the number of viable 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 viable target cells was calculated by normalizing the luciferase signal to the signal of the "target only" wells and the percentage of cytotoxicity was calculated as 100%-% viable target cells. Serial transfers to fresh target cells and luciferase readouts were performed every 2 or 3 days until all cytotoxic activity ceased.

[0176] Figure 21 shows that CACCR improved the cytotoxic activity of CAR-T cells directed against the liquid tumor target BCMA. Figure 21 shows the cytotoxicity of BCMA CAR (P5A2 scFv) against the MM1.S multiple myeloma cell line at E:T=10:1, demonstrating that co-expression of CACCR increased the long-term cytotoxicity of CAR-T cells.

[0177] Example 12: CACCR enhances the in vivo activity of CAR-T cells CAR-T cell therapies, such as those targeting CD19 and BCMA, have achieved unprecedented clinical success in the treatment of hematological malignancies. Although a high rate of complete responses has been achieved, this is temporary as most patients eventually relapse. Furthermore, CAR-T cells have had more limited success in the treatment of solid tumors. Among the reasons for relapse and lack of response include insufficient CAR-T cell expansion and persistence, as well as functional inhibition of CAR-T cells by an immunosuppressive microenvironment. As in vitro characterization of CACCR CAR-T cells revealed improved target-driven proliferation, persistence, potency, and exhaustion profiles, we next investigated whether these functional enhancements would translate into improved antitumor activity in vivo.

[0178] To investigate the in vivo activity of CACCR CAR-T cells in the context of hematological malignancies, we utilized CAR-T cells carrying the BCMA-specific P5A2 scFv linked to 4-1BB and CD3ζ signaling domains in an orthotopic xenograft model of multiple myeloma. T cell receptor (TCR)-deficient BCMA CAR-T cells were generated by transcription activator-like effector nuclease (TALEN)-mediated knockout to avoid potential confounds from TCR-driven xenoreactivity. Eight- to ten-week-old female NSG mice were pre-incubated with 5 × 10 6 Mice were irradiated with 1 Gy the day before intravenous inoculation of MM1.S-Luc-GFP. Fourteen days after tumor implantation, mice were randomized based on tumor burden and inoculated with 1 × 10 of the indicated CAR-T cells. 6個 , or 3 × 10 6 Each group was administered either 3×10 T cells or 1×10 T cells intravenously (n=10 per group). Tumor progression was monitored by bioluminescence imaging. Thirty days after T cell administration, 3×10 T cells were administered either 3×10 T cells or 1×10 T cells. 6Mice administered CAR-T cells were bled for enumeration of BCMA CAR-T cells in the periphery. Specifically, 50 uL of whole blood from each mouse was subjected to red blood cell lysis using ACK Lysing Buffer (Gibco), blocked with Fc, 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 anti-idiotypic antibody specific for P5A2 scFv. Finally, samples were washed in PBS and cell pellets were resuspended in 130 uL of PBS+1% BSA containing 123count eBeads (Thermo Fisher) (10 uL of counting beads in 120 uL of PBS+1% BSA) prior to FACS analysis.

[0179] Figures 22A-C show that CACCR improved the in vivo antitumor activity and persistence of BCMA CAR-T cells against orthotopic multiple myeloma. Figures 24A-B show that CACCR improved the in vivo antitumor activity and persistence of BCMA CAR-T cells against orthotopic multiple myeloma after administration of 1 x 10 6 pcs or 3×10 6 Figure 24 shows tumor progression in response to treatment with either BCMA CAR-T cells or CAR-T cells co-expressing CACCR. Although control BCMA CAR-T cells were able to mediate initial tumor regression, this response was short-lived as tumors recurred 22 days after cell injection. However, CAR-T cells co-expressing CACCR significantly delayed tumor recurrence and improved the durability of the response. Statistics in Figures 24A-B are based on repeated measures one-way ANOVA with Tukey's multiple comparisons from days 6-34 in Figure 22A and days 6-44 in Figure 22B. ** p<0.01 and *** p<0.001. Figure 22C shows that 3×10 6Figure 24C shows 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 periphery. In contrast, CACCR BCMA CAR-T cells, which were superior in preventing tumor recurrence, were also significantly more abundant in vivo. Statistics in Figure 24C are based on ordinary one-way ANOVA with Tukey's multiple comparisons. * p<0.05 and **** p<0.0001, suggesting that improved CACCR CAR-T cell persistence mediated, in part, enhanced long-term tumor control and prolonged durability of responses.

[0180] Furthermore, we assessed the impact of CACCR on CAR-T cell activity in the context of solid tumors known to be resistant to CAR-T cell therapy, such as glioblastoma. For this purpose, we utilized an EGFRvIII-specific CAR carrying 2173 scFv bound to 4-1BB and CD3ζ signaling domains, and the LN229 human glioblastoma cell line stably overexpressing EGFRvIII (LN229-EGFRvIII). 8–10 week old female NSG mice were injected with 3 × 10 6 LN229-EGFRvIII were implanted subcutaneously. 25 days after tumor establishment, mice were randomized based on tumor burden and administered 1.5 × 10 of the indicated CAR-T cells. 6 pcs, or 3×10 6 Each patient was administered one of the following drugs intravenously (n = 8–10 per group). Tumor progression was monitored twice weekly by caliper measurement.

[0181] Figure 23 shows that CACCR improved the antitumor activity of CAR-T cells against established solid tumors. Treatment with control EGFRvIII CAR-T cells was able to slow the growth of LN229-EGFRvIII tumors, but the response was transient and suboptimal as the tumors eventually progressed. In contrast, treatment with CACCR CAR-T cells led to complete tumor regression. Notably, 1.5 × 106 Even a low dose of 3 × 10 CACCR CAR-T cells was sufficient for tumor elimination. Statistics are based on repeated measures one-way ANOVA with Tukey's multiple comparisons from days 3 to 38. 6 Compared to treatment with control CAR-T cells ** p<0.01 These results recapitulate the ability of CACCR to non-redundantly synergize with the signaling domains of CAR-T cells to confer improved activity.

Claims

1. A chimeric cytokine receptor (CCR) composed of two monomers, each monomer comprising: a) a transmembrane domain; b) a Janus kinase (JAK) binding domain; and c) a STAT recruitment domain; the transmembrane domain and the JAK binding domain have 95% or more sequence identity with the amino acid sequence of 478 to 582 of SEQ ID NO:6, and have the amino acid substitutions S505N and W515K of SEQ ID NO:6; A chimeric cytokine receptor (CCR), wherein the CCR does not contain an extracellular ligand binding domain.

2. The CCR of claim 1, wherein the JAK binding domain comprises a JAK1 binding domain.

3. The CCR of claim 1, wherein the JAK binding domain comprises a JAK2 binding domain.

4. The CCR of claim 1, wherein the JAK binding domain comprises a JAK3 binding domain.

5. The CCR of claim 1, wherein the JAK binding domain comprises a TYK2 binding domain.

6. 6. The CCR of any one of claims 1 to 5, wherein the STAT recruitment domain comprises a STAT recruitment domain selected from STAT-1, STAT-2, STAT-3, STAT-4, STAT-5, STAT-6, or STAT-7 recruitment domains from at least one receptor.

7. The CCR described in claim 1, wherein the amino acid sequence of 478 to 582 of SEQ ID NO: 6 further contains an amino acid substitution of H499L.

8. The CCR described in claim 1, wherein the amino acid sequence of 478 to 582 of SEQ ID NO: 6 further contains an amino acid substitution of G509N.

9. The CCR described in claim 1, wherein the amino acid sequence of 478 to 582 of SEQ ID NO: 6 further contains amino acid substitutions of H499L and G509N.

10. A CCR described in any one of claims 1 to 9, wherein the two monomers can spontaneously dimerize in the absence of an extracellular ligand.

11. The CCR according to any one of claims 1 to 10, wherein the two monomers are identical.

12. The CCR according to any one of claims 1 to 10, wherein the two monomers are different.

13. 13. The CCR of any one of claims 1 to 12, wherein the STAT recruitment domain comprises a STAT recruitment domain from a receptor selected from the group consisting of BLNK, IL2RG, EGFR, EpoR, GHR, IFNAR1, IFNAR2, IFNAR1 / 2, IFNLR1, IL10R1, IL12Rb1, IL12Rb2, IL2Rb, IL2small, IL7R, IL7Ra, IL9R, IL15R, and IL21R.

14. The CCR of any one of claims 1 to 12, wherein the STAT recruitment domain comprises one or more amino acid sequences of receptor sequences selected from the group consisting of SEQ ID NOs: 46-88, 106 and 143.

15. The CCR of any one of claims 1 to 14, wherein the STAT recruitment domain comprises a STAT recruitment domain from IL7Ra.

16. The CCR of claim 15, wherein the STAT recruitment domain derived from IL7Ra comprises the amino acid sequence of SEQ ID NO:

46.

17. The CCR of any one of claims 1 to 14, wherein the STAT recruitment domain comprises a STAT recruitment domain from IL2Rb.

18. 18. The CCR of claim 17, wherein the STAT recruitment domain comprises a STAT recruitment domain from IL7Ra.

19. The CCR of any one of claims 1 to 14, wherein the STAT recruitment domain comprises a STAT recruitment domain from IL12Rb2.

20. The CCR of claim 19, wherein the STAT recruitment domain derived from IL12Rb2 comprises the amino acid sequence of SEQ ID NO: 86 or 67.

21. The CCR of any one of claims 1 to 20, wherein the STAT recruitment domain comprises STAT recruitment domains from two receptors.

22. The CCR of any one of claims 1 to 21, wherein the STAT recruitment domain comprises STAT recruitment domains from two cytokine receptors.

23. 23. The CCR of claim 22, wherein the two cytokine receptors are selected from the group consisting of IL7Ra, IL2Rb, and IL12Rb2.

24. A polynucleotide encoding any one of the CCRs according to any one of claims 1 to 23.

25. 25. An expression vector comprising the polynucleotide of claim 24.

26. The expression vector according to claim 25, comprising the polynucleotide of claim 24 and a polynucleotide that expresses a chimeric antigen receptor (CAR).

27. The CAR is selected from the group consisting of 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 claudin 18 isoform 2), DLL3 (delta-like protein 3, Drosophila delta homolog 3, Delta-like protein 3, a3), Mucl7 (mucin 17, Muc3, Muc3), FAP alpha (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).

28. The expression vector according to any one of claims 25 to 27, wherein the vector is a lentiviral vector.

29. An engineered immune cell comprising an expression vector according to any one of claims 25 to 28.

30. 30. The engineered immune cell of claim 29, wherein the immune cell is a T cell.

31. 24. An engineered immune cell comprising a chimeric antigen receptor (CAR) and at least one CCR according to any one of claims 1 to 23.

32. 32. The engineered immune cell of claim 31 , wherein the CAR and the CCR are expressed in stoichiometrically equivalent amounts.

33. 33. The engineered immune cell of any one of claims 29 to 32, wherein the immune cell is a T cell.

34. The CAR is selected from the group consisting of 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 claudin 18 isoform 2), DLL3 (delta-like protein 3, Drosophila delta homolog 3, Delta3), 33. The engineered immune cell of claim 31 or 32, which binds to Mucl7 (mucin 17, Muc3, Muc3), FAP alpha (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).

35. 35. The engineered immune cell of any one of claims 29 to 34, wherein the cell is an allogeneic immune cell.

36. The engineered immune cell of any one of claims 29 to 34, wherein the cell is an autoimmune cell.

37. 37. The engineered immune cell of any one of claims 29 to 36, wherein the immune cell is selected from the group consisting of T cells, dendritic cells, killer dendritic cells, mast cells, NK cells, macrophages, monocytes, B cells, and immune cells derived from stem cells.

38. A method for preparing an engineered immune cell, comprising introducing the polynucleotide according to claim 24 or the expression vector according to any one of claims 25 to 28 into an immune cell.

39. 39. The method of claim 38, wherein the immune cells are selected from the group consisting of T cells, dendritic cells, killer dendritic cells, mast cells, NK cells, macrophages, monocytes, B cells, and immune cells derived from stem cells.

40. A pharmaceutical composition comprising an engineered immune cell according to any one of claims 29 to 37.

41. A kit comprising an engineered immune cell according to any one of claims 29 to 37 or a pharmaceutical composition according to claim 40.

42. 41. The pharmaceutical composition of claim 40 for treating cancer in a subject.

43. 43. The pharmaceutical composition of claim 42, wherein the cancer comprises a solid tumor.

44. 43. The pharmaceutical composition of claim 42, wherein the cancer comprises a liquid tumor.

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