RU2824672C2 - Constitutively active chimeric cytokine receptors - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: disclosed is a constitutively active chimeric cytokine receptor (CACCR) for increasing the cytotoxicity of an engineered immune cell, wherein the CACCR consists of two monomers, where each monomer contains: a thrombopoietin receptor (TpoR) transmembrane domain containing amino acids 478–582 of SEQ ID NO: 6 with amino acid substitutions S505N and W515K according to SEQ ID NO: 6, a domain which binds Janus kinase (JAK) and an intracellular STAT recruiting domain. Also disclosed are: a polynucleotide coding CACCR, an expression vector, engineered T-cells having increased cytotoxicity and a method for production thereof, a pharmaceutical composition and a kit for treating cancer in a subject. Also disclosed is the use of a therapeutically effective amount of engineered T-cells or a pharmaceutical composition for treating cancer in a subject.

EFFECT: invention provides increased activation, proliferation, resistance and/or activity of immune cells and is used for treating cancer in a subject, in particular for treating a solid tumor or haemablastosis.

45 cl, 23 dwg, 8 tbl, 12 ex

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 62/812,911, filed March 1, 2019; and U.S. Provisional Application No. 62/980,823, filed February 24, 2020, the entire contents of both of which are incorporated herein by reference.

SEQUENCE LIST

[0002] This application includes a sequence listing that was filed electronically in ASCII format, the entire contents of which are incorporated by reference. Said ASCII copy, created on February 21, 2020, is designated AT-023_03WO_SL.txt and is 251,652 bytes in size.

LEVEL OF TECHNOLOGY

[0003] Adoptive transfer of immune cells (e.g., T cells) genetically modified to recognize antigens associated with malignancies is a promising new approach to cancer treatment. For example, T cells can be genetically modified to express chimeric antigen receptors (CARs), which are fusion proteins consisting of an antigen-recognition moiety and T cell activation domains.

[0004] T cell proliferation, cytotoxic activity, and persistence are controlled by signaling pathways. Conventional CAR constructs provide two signals, CD3zeta activation (Signal 1) and costimulation (Signal 2, e.g., through expression of 4-1BB, OX40, and/or CD28). In some cases, a third signal (Signal 3), cytokine-induced cytokine receptor signaling (e.g., cytokine support for immune stimulation), may be desirable. However, approaches to providing Signal 3 have significant limitations.

[0005] One approach to providing cytokine support involves combining CAR-T cell therapy with systemic infusions of recombinant cytokines/cytokine mimetics and modifying CAR-T cells to secrete/express cytokines extracellularly. Because cytokines have pleiotropic effects and can also influence the function of other cell types, systemic administration or production of immune-stimulatory cytokines by CAR-T cells has at least two major drawbacks: (i) these approaches can cause systemic toxicity in humans, and (ii) in the case of allogeneic CAR-T cell therapy, these approaches can cause bystander activation of the host immune system, which can accelerate rejection of the allogeneic CAR-T cells, thereby compromising therapeutic efficacy. Another approach to providing cytokine support has been to introduce a constitutively activated dimerized cytokine receptor, IL-7Ra, which limits the nature (IL-7 signaling only) and magnitude of the signal output. Another approach to providing cytokine support has been to incorporate 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 a target (and CAR activation), Signal 3 will not be transmitted.

[0006] Solutions are needed to overcome these shortcomings by targeting cytokine signals specifically to CAR-T cells in a controlled manner, which will improve the safety profile and therapeutic efficacy. The present invention provides compositions and methods that meet this need.

ESSENCE OF THE INVENTION

[0007] The present disclosure provides constitutively active chimeric cytokine receptors (CACCRs). When present on immune cells bearing a chimeric antigen receptor (CAR) (CAR-1 cells, such as CAR-T cells), CACCRs provide enhanced activation, proliferation, resistance, and/or activity of immune cells. Methods for producing and using the CACCRs described herein are also provided.

[0008] Accordingly, according to one aspect of the present invention, there is provided a CACCR consisting of two monomers, wherein each monomer comprises: (a) a trans membrane domain; (b) a Janus kinase (JAK) binding domain; and (c) a recruitment domain, wherein said monomers are constitutively dimerized. In some embodiments, the CACCR does not comprise a ligand binding domain of the extracellular domain.

[0009] In some embodiments, the transmembrane domain and/or the JAK binding domain is derived from a TPOR/MPLR receptor. In some embodiments, the transmembrane domain and/or the JAK binding domain is derived from amino acids 478-582 of the naturally occurring TPOR/MPLR receptor of SEQ ID NO:6. In some embodiments, the TPOR/MPLR receptor comprises one or more 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, such as; IL7Ra(316-459). In some embodiments, the recruitment domain comprises the STAT recruitment domain of IL2Rb, such as 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 the STAT recruitment domain of IL12Rbl, such as IL12Rb 1(622-662). In some embodiments, the recruitment domain comprises the STAT recruitment domain of IL12Rb2, such as IL12Rb2(714-862) or IL12Rb2(775-825). In some embodiments, the recruitment domain comprises the STAT recruitment domain of IL21R, such as IL21R(322-538).

[0010] According to a related aspect of the present invention, there is provided a polynucleotide encoding any of the CACCRs of the present disclosure and an expression vector comprising such a polynucleotide. In some embodiments, the polynucleotide further encodes a chimeric antigen receptor (CAR), wherein the CAR binds to BCMA, EGFRvIII, Flt-3, WT-1, CD20, CD23, CD30, CD38, CD70, CD33, CD133, LeY, NKG2D, CS1, CD44v6, ROR1, CD19, claudin-18.2 (claudin-18A2 or claudin18 isoform 2), DLL3 (Drosophila delta-like protein 3, delta homolog 3, Delta3), Muc17 (mucin17, Muc3, Muc3), FAP alpha (fibroblast activation protein alpha), Ly6G6D (lymphocyte antigen complex 6 G6d locus protein, c6orf23, G6D, MEGT1, NG25) and/or RNF43 (RNF43 ubiquitin ligase (E3), RING finger protein 43).

[0011] According to another aspect of the present invention, there are engineered immune cells comprising at least one chimeric antigen receptor (CAR) and at least one CACCR according to the present disclosure. According to some embodiments, the immune cell is a T cell. According to some embodiments, the immune cell is an allogeneic immune cell. According to other embodiments, the immune cell is an autologous immune cell. The immune cell can be selected from the group consisting of: a T cell, a dendritic cell; a dendritic killer cell, a mast cell, an NK cell, a macrophage, a monocyte, a B cell, and a stem cell-derived immune cell. According to a related aspect, the present invention provides a pharmaceutical composition comprising any of the engineered immune cells according to the present disclosure, and a kit comprising such a pharmaceutical composition.

[0012] According to another aspect of the present invention, there is provided a method of treating cancer in a subject, comprising administering to said subject a therapeutically effective amount of any of the engineered immune cells described herein.

BRIEF DESCRIPTION OF DRAWINGS

[0013] FIG. 1 schematically shows a designed CACCR according to the present disclosure.

[0014] FIG. 2 is a schematic representation of a vector according to the present disclosure that can be used to co-express a CACCR and a CAR according to the present disclosure. One or more cytoplasmic tails can be joined in tandem to mimic signaling from one or more cytokines. A schematic representation of a vector expressing a control BFP (blue fluorescent protein) CAR is also shown.

[0015] FIGS. 3A-3B show the identification of TpoR transmembrane domain (TM) mutants that constitutively activate cytokine receptor signaling.

[0016] FIGS. 4A-4C show results regarding the expansion of CAR-T cells co-expressing a constitutively active chimeric cytokine receptor.

[0017] FIG. 5 shows the differentiation and distribution of a memory T cell subset in a CAR T cell product under conditions differing in IL-2.

[0018] FIGS. 6A-6B show the extent of constitutive cytokine signaling mediated by each TpoR TM variant.

[0019] FIGS. 7A-7D show the cytotoxic activity of TpoR mutants, indicating that constitutive cytokine receptor signaling enhances CAR-T cell activity.

[0020] FIGS. 8A-8B show the cytotoxic activity and resistance of TpoR TM mutants.

[0021] FIG. 9 shows enrichment of CAR-T cells over time in a growth factor independent assay.

[0022] FIGS. 10A-10B show the fold expansion of CAR-T cells over time in a growth factor independent assay.

[0023] FIG. 11 shows the distribution of the memory T cell subpopulation among CAR ⁺ T cells over time in a growth factor independent assay.

[0024] FIG. 12 shows activation of STAT signaling pathways by CAR-T cells co-expressing the indicated CACCR.

[0025] FIGS. 13A-13B and FIGS. 14A-14B show optimization of CACCR signaling strength by T cells expressing full-length or truncated cytoplasmic tails as demonstrated in a reporter assay in HEK293 cells.

[0026] FIG. 15 shows optimization of CACCR signaling strength demonstrated in primary CAR-T cells co-expressing full-length or truncated cytoplasmic tails.

[0027] FIGS. 16A-16C show that CACCR CAR-T cells bearing truncated IL2Rb cytoplasmic tails more closely mimic IL-15 signaling, but not IL-2 signaling.

[0028] FIGS. 17A-17D show the combinatorial output signals of different cytoplasmic tails fused in tandem.

[0029] FIGS. 18A-18B depict the effects of CACCR on the differentiation of CAR-T cells into memory cells.

[0030] FIGS. 19A-19D depict the effect of CACCR on CAR-T cell survival and differentiation into memory cells under growth factor-independent conditions.

[0031] FIGS. 20A-20B depict the cytotoxic activity of CAR-T cells co-expressing various CACCRs.

[0032] FIG. 21 shows that CACCRs improved the cytotoxic activity of CAR-T cells directed against a hematologic malignancy targeting BCMA.

[0033] FIGS. 22A-22C show that CACCRs improved in vivo antitumor activity and persistence of BCMA CAR-T cells against orthotopic multiple myeloma.

[0034] FIG. 23 shows that CACCRs improved the antitumor activity of CAR T cells against established solid tumors.

DETAILED DESCRIPTION

[0035] The present disclosure provides constitutively active chimeric cytokine receptors (CACCRs). The presence of a constitutively active, regulated chimeric cytokine receptor provides immune stimulation of Signal 3 to meet the need for immune stimulation. Accordingly, when such CACCRs are present on immune cells bearing a chimeric antigen receptor (CAR) (CAR-1 cells, such as CAR-T cells), they provide increased activation, proliferation, resistance, and/or activity of immune cells. The present invention also provides methods for making and using the CACCRs described herein.

[0036] The CACCRs of the present disclosure are regulable and have readily modifiable cytokine output signals to enhance the activity, stability, etc. of CAR-T cells. The components, methods of preparation, and uses are described sequentially below.

I. Constitutively active chimeric cytokine receptors (CACCR)

[0037] The CACCRs of the present disclosure are comprised of two monomers, each monomer comprising: (a) a transmembrane domain; (b) a JAK binding domain; and (c) a recruitment domain, wherein the monomers are constitutively dimerized. In some embodiments, the CACCRs of the present disclosure do not comprise an extracellular ligand binding domain.

[0038] In some embodiments, the monomers are identical, resulting in the formation of a constitutively active homodimer. 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 the formation of a constitutively active heterodimer, which may be desirable under certain circumstances.

[0039] The CACCR monomers of the present disclosure are capable of spontaneous dimerization and can activate signaling in the absence of any exogenous stimulation or ligand (ligand-independent dimerization). The level of activity can be controlled by mutations introduced into the transmembrane domain of CACCR. The skilled artisan will appreciate that CACCR monomers are not 100% dimerized and can exist as a monomer.

A. Transmembrane domains

[0040] The CACCRs of the present disclosure comprise transmembrane domains. The transmembrane domains of the present disclosure comprise sequences that provide for constitutive dimerization of a pair of monomers, thereby providing for constitutive activation of JAK in the intracellular portion and constitutive recruitment and phosphorylation of, for example, STAT in the cytoplasmic region of the receptor.

[0041] The transmembrane domains are at the N-terminus and are linked to the intracellular/cytoplasmic domains at the C-terminus. In some embodiments, linkage is achieved optionally through a linker.

[0042] Herein, transmembrane domains are capable of inserting into the membrane of a cell in which they are expressed. In some embodiments, transmembrane domains of the present disclosure span the cell membrane and comprise an extracellular portion and/or an intracellular portion.

[0043] In some embodiments, the transmembrane domains of the present disclosure are engineered (synthetic) and are not similar to any naturally occurring transmembrane domain, e.g., they are not found in nature.

[0044] In other embodiments, the transmembrane domains of the present disclosure are derived from naturally occurring receptors.

[0045] In some embodiments, the trans membrane domains and/or JAK activating domains of the present disclosure are derived from, for example, one or more of the following receptors: erythropoietin receptor (EpoR), interleukin 6 signal transducer (GP130 or IL6ST), prolactin receptor (PrlR), growth hormone receptor (GHR), granulocyte colony stimulating factor receptor (GCSFR), and thrombopoietin receptor/myeloproliferative leukemia protein receptor (TPOR/MPLR). When derived from naturally occurring receptors, the entire receptor or the entire transmembrane sequence of the receptor may not be required to achieve constitutive activation and constitutive binding/activation of JAK in the intracellular portion. Accordingly, fragments of naturally occurring receptors can be used. Additionally, specific mutations can be introduced into transmembrane domains derived from naturally occurring receptors to further regulate downstream signaling.

[0046] In some embodiments, the transmembrane domain and/or JAK activating domain of the present disclosure is derived from a naturally occurring EpoR receptor.

[0047] In some embodiments, the transmembrane domain and/or JAK activating domain of the present disclosure is derived from a naturally occurring GP130 receptor.

[0048] In some embodiments, the transmembrane domain and/or JAK activating domain of the present disclosure is derived from a naturally occurring PrlR receptor.

[0049] In some embodiments, the transmembrane domain and/or JAK activating domain of the present disclosure is derived from a naturally occurring GHR receptor.

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

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

[0052] Table 1a provides exemplary full-length sequences of naturally occurring receptors provided in the present disclosure from which the transmembrane proteins are derived. The sequences provided in Table 1a are reference sequences against which later mutations are reflected, such as in Tables 1b and 1c.

[0053] In some embodiments, the trans membrane domain of the present disclosure is derived from a truncated version of the naturally occurring TPOR/MPLR (myeloproliferative leukemia protein) receptor shown in Table 1a.

[0054] In some embodiments, the trans membrane domain of CACCR comprises amino acids 478-582 of the TPOR receptor in Table 1a.

[0055] Table 1b provides exemplary amino acid sequences of a trans membrane domain according to the present disclosure, wherein said trans membrane domain is derived from a naturally occurring TPOR receptor.

[0056] In some embodiments, the transmembrane domain of CACCR comprises amino acids 478-582 of the TPOR receptor and an amino acid substitution of at least H499. In some embodiments, the transmembrane domain of CACCR comprises amino acids 478-582 of the TPOR receptor and an amino acid substitution of H499L.

[0057] 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.

[0058] In some embodiments, the transmembrane domain of CACCR comprises amino acids 478-582 of the TPOR receptor and an amino acid substitution of at least G509. In some embodiments, the transmembrane domain of CACCR comprises amino acids 478-582 of the TPOR receptor and an amino acid substitution of G509N.

[0059] 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 of W515K.

[0060] In some embodiments, the trans membrane domain of CACCR comprises amino acids 478-582 of the TPOR receptor and an amino acid substitution at H499 and S505 (sequence is presented in Table 1b).

[0061] In some embodiments, the trans membrane domain of CACCR comprises amino acids 478-582 of the TPOR receptor and an amino acid substitution at H499 and W515 (sequence is presented in Table 1b).

[0062] In some embodiments, the trans membrane domain of CACCR comprises amino acids 478-582 of the TPOR receptor and an amino acid substitution at H499, S505, and W515 (sequence is presented in Table 1b).

[0063] In some embodiments, the trans membrane domain of CACCR comprises amino acids 478-582 of the TPOR receptor and an amino acid substitution at S505 and W515 (sequence is presented in Table 1b).

[0064] In some embodiments, the trans membrane domain of CACCR comprises amino acids 478-582 of the TPOR receptor and an amino acid substitution at H499 and G509 (sequence is presented in Table 1b).

[0065] In some embodiments, the trans membrane domain of CACCR comprises amino acids 478-582 of the TPOR receptor and amino acid substitutions H499L and S505N (sequence presented in Table 1b).

[0066] In some embodiments, the trans membrane domain of CACCR comprises amino acids 478-582 of the TPOR receptor and amino acid substitutions H499L and W515K (sequence presented in Table 1b).

[0067] In some embodiments, the trans membrane domain of CACCR comprises amino acids 478-582 of the TPOR receptor and amino acid substitutions H499L and G509N (sequence presented in Table 1b).

[0068] In some embodiments, the trans membrane domain of CACCR comprises amino acids 478-582 of the TPOR receptor and amino acid substitutions S505N and W515K (sequence presented in Table 1b).

[0069] In some embodiments, the trans membrane domain of CACCR comprises amino acids 478-582 of the TPOR receptor and amino acid substitutions H499L, S505N, and W515K (sequence presented in Table 1b).

[0070] In some embodiments, the trans membrane domain of CACCR comprises amino acids 478-582 of the TPOR receptor and an amino acid substitution at H499 and S505 (sequence is presented in Table 1b).

[0071] The CACCR of the present disclosure can be adjusted to achieve the level of Signal 3/immune stimulation required in the CAR-bearing immune cell (e.g., CAR-T cell) and desired in a particular case or condition.

[0072] In some embodiments, a low level of STAT5 activation is desirable in an immune cell carrying a CAR (e.g., a CAR-T cell). As an example, in such embodiments, a CACCR trans membrane domain comprising amino acids 478-582 of the TPOR receptor and an amino acid substitution of S505N, W515K, or H499L/G509N may be introduced.

[0073] In some embodiments, an increased level of STAT5 activation is desirable in an immune cell carrying a CAR (e.g., a CAR-T cell). As an example, in such embodiments, a CACCR transmembrane domain comprising amino acids 478-582 of the TPOR receptor and the amino acid substitutions H499L, S505N, and W515K may be introduced. As another example, in such embodiments, a CACCR transmembrane domain comprising amino acids 478-582 of the TPOR receptor and the amino acid substitutions S505N and W515K may be introduced.

[0074] In some embodiments, it is desirable to enhance the differentiation of CAR-bearing immune cells (e.g., CAR-T cells) into memory T cells. As an example, in such embodiments, a CACCR trans membrane domain comprising amino acids 478-582 of the TPOR receptor and the amino acid substitutions W515K or H499L/G509N may be introduced.

[0075] In some embodiments, it is desirable to enhance the differentiation of CAR-bearing immune cells (e.g., CAR-T cells) into memory T cells. As an example, in such embodiments, a CACCR trans membrane domain comprising amino acids 478-582 of the TPOR receptor and the amino acid substitutions S505N/W515K and H499L/S505N/W515K may be introduced.

[0076] The present invention also provides substitutions for enhancing cytotoxic activity, robustness of response and increased resistance, such as S505N/W515K and H499L/S505N/W515K substitutions.

B. Janus kinase (JAK) binding domains

[0077] The CACCRs of the present disclosure comprise intracellular JAK binding domains. The JAK binding domain is linked to the C-terminus of the trans membrane domain either directly or through a linker. The JAK binding domain is linked to the trans membrane domain on the intracellular side of the chimeric cytokine receptor.

[0078] 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.

[0079] In some embodiments, the JAK binding domains of the CACCRs of the present disclosure are naturally occurring and are derived from a naturally occurring receptor.

[0080] In some embodiments, the JAK binding domains of the CACCRs of the present disclosure are synthetic.

[0081] Table 1b and Table 1c provide exemplary amino acid sequences of transmembrane and JAK2 binding domains of the present disclosure. In some embodiments, a CACCR of the present disclosure comprises a trans membrane and JAK2 binding domain comprising an amino acid sequence selected from the sequences in Tables 1b and 1c. In some embodiments, a CACCR of the present disclosure comprises a trans membrane and JAK2 binding domain comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to any of the sequences in Tables 1b and 1c.

C. Attraction domains

[0082] The CACCRs of the present disclosure comprise cytoplasmic recruitment domains. The recruitment domain may 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 signal transducer and activator of transcription (STAT) recruitment domains (STAT-activating), such as from receptor tails (cytoplasmic tails) or from cytokine receptor tails. These intracellular recruitment domains of the CACCRs of the present disclosure mediate the propagation of Signal 3 in an immune cell comprising a CAR and a chimeric cytokine receptor (e.g., a chimeric cytokine receptor CAR-T cell of the present disclosure). The cytokine signaling propagated by the Stat recruitment domain mediates cytokine-based immune stimulation of the cell. In some embodiments, immune stimulation is homeostatic, such as signaling that increases the number of CAR-bearing immune cells. In some embodiments, immune stimulation is inflammatory, such as signaling that increases the activity of CAR-bearing immune cells. In some embodiments, immune stimulation prevents exhaustion, such as signaling that maintains the long-term functionality of CAR-bearing immune cells.

[0083] In some embodiments, the engagement domains of the present disclosure are synthetic and do not resemble any portion of a naturally occurring receptor. In some embodiments, immune stimulation prevents exhaustion, such as signaling that maintains long-term functionality of immune cells bearing a CAR.

[0084] In some embodiments, the Stat engagement domains of the present disclosure are synthetic and do not resemble any fragment of a naturally occurring receptor.

[0085] In other embodiments, the Stat recruitment domains of the present disclosure are derived from the cytoplasmic tails of naturally occurring receptors, such as derived from naturally occurring cytokine receptors. These cytoplasmic tails of naturally occurring receptors can be regions located downstream of the JAK activating domains of the trans membrane domain of the receptor. The Stat recruitment domains of the chimeric cytokine receptors comprise 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.

[0086] In some embodiments, the naturally occurring receptor from which the Stat recruitment domain is derived is not a cytokine receptor.

[0087] In some embodiments, the naturally occurring receptor from which the Stat recruitment domain is derived is a cytokine receptor. Exemplary cytokine receptors that transmit cytokine signals that enhance immune stimulation of T cells include, but are not limited to, the IL-2 receptor, the IL-7 receptor, the IL-15 receptor, and the IL-21 receptor. In alternative embodiments, the receptor from which the Stat recruitment domain is derived is not a cytokine receptor. The receptor can be redirected to selected signaling by selecting the Stat recruitment domain of the CACCR.

[0088] In some embodiments, a CACCR of the present disclosure comprises an attraction domain connected to the C-terminus of a transmembrane/JAK2 binding domain, with or without a linker. In some embodiments, the linker comprises one or more amino acid residues.

[0089] Table 2a provides exemplary receptors from which the CACCR engagement domains of the present disclosure are derived. Table 2b provides exemplary amino acid sequences of engagement domains of the present disclosure. In some embodiments, a CACCR of the present disclosure comprises an engagement domain comprising an amino acid sequence selected from one or more receptor sequences in Table 2b. In some embodiments, a CACCR of the present disclosure comprises an engagement domain comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to any of the sequences in Table 2b.

[0090] In some embodiments, the Stat recruitment domain of a CACCR of the present disclosure comprises a STAT recruitment domain from a single receptor.

[0091] To produce multiple signal outputs, two or more STAT recruitment domains can be linked in tandem to mimic signaling from one or more cytokines.

[0092] In some embodiments, two or more STAT recruitment domains may be connected in tandem with or without a linker. In some embodiments, the linker comprises one or more amino acid residues.

[0093] In some embodiments, the STAT recruitment domain comprises portions of more than one receptor, such as comprising more than one STAT recruitment domain. In such embodiments, a tandem cytokine signaling domain is provided that provides enhanced signaling. Accordingly, in some embodiments, the STAT recruitment domain of a CACCR monomer of the present disclosure comprises STAT recruitment domains from more than one receptor, such as comprising STAT recruitment domains from two, three, four, five, or even six receptors. For example, in some embodiments, STAT recruitment domains can be linked in tandem to stimulate multiple signaling pathways (e.g., IL7R(316-459)-IL12Rb2(775-825) fusion to promote STAT5 signaling and STAT4 proinflammatory signaling; IL7R(316-459)-IL2Rbsmall(393-433,518-551) to promote resistance; IL7R(316-459)-EGFR(1122-1165) to promote resistance and prevent exhaustion; IL2Rbsmall(393-433,518-551)-EGFR(1122-1165) to promote resistance and prevent exhaustion).

[0094] When generating multiple output signals, the proximity of individual STAT recruitment domains to the cell membrane can influence the strength of their respective output signals. Table 2c provides examples of dual output CACCRs, where each output signal can be positioned closer to or further away from the cell membrane. In some embodiments, CACCRs of the present disclosure comprise a dual output recruitment domain selected from Table 2c.

[0095] Without being limited by any theory or mechanism, in some embodiments, a JAK protein (JAK1, JAK2, JAK3, or TYK2) is associated with a dimerized CACCR according to the present disclosure. Two associated JAK proteins are activated and are capable of phosphorylating tyrosine residues on the recruitment domain of CACCR. The phosphorylated recruitment domains are then capable of binding recruited proteins (e.g., a phosphorylated STAT recruitment domain binds a STAT protein), which in turn mediate transcriptional events in the nucleus.

D. Approximate CACCR

[0096] Table 3 shows exemplary CACCR sequences according to the present disclosure. The receptors can be expressed with a signal sequence, for example, a CD8SS having the sequence MALPVTALLLPLALLLHAARP (SEQ ID NO: 89).

[0097] In some embodiments, a CACCR of the present disclosure comprises any of the sequences in Table 3. In some embodiments, a CACCR comprises an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% identical to any of the amino acid sequences of SEQ ID NOS: 90-98 and 107-139. In some embodiments, a TPOR/MPLR receptor comprises any of the amino acid sequences of SEQ ID NOS: 90-98 and 107-139.

[0098] 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 present 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 present disclosure comprises an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 17. In some embodiments, the CACCR of the present disclosure comprises the amino acid sequence of SEQ ID NO: 17. In some embodiments, the CACCR further comprises an attraction domain comprising the amino acid sequence of one or more 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 of 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 of IL7Ra comprises the amino acid sequence of SEQ ID NO: 46, 68, 69, 70, 71, or 72. In some embodiments, the recruitment domain comprises the STAT recruitment domain of IL2Rb. In some embodiments, the STAT recruitment domain of 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 of 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 the STAT recruitment domain of IL12Rb1 or IL12Rb2. In some embodiments, the STAT recruitment domain of 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 of IL12Rb1 or IL12Rb2 comprises the amino acid sequence of SEQ ID NO: 67, 86, or 87. In some embodiments, the recruitment domain comprises the STAT recruitment domain of IL21R. In some embodiments, the STAT recruitment domain of 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 of 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 domains comprise the STAT recruitment domains of IL7Ra and IL2Rb. In some embodiments, the recruitment domain comprises the STAT recruitment domains of IL7Ra and IL12Rb1. In some embodiments, the recruitment domain comprises the STAT recruitment domains of 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.

[0099] In some embodiments, a CACCR of the present disclosure comprises a transmembrane domain and/or a JAK binding domain from a TPOR/MPLR receptor that comprises 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 an recruitment domain comprising an amino acid sequence of one or more of the receptor sequences set forth 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 of 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 of IL7Ra comprises the amino acid sequence of SEQ ID NO: 46, 68, 69, 70, 71, or 72. In some embodiments, the recruitment domain comprises the STAT recruitment domain of IL2Rb. In some embodiments, the STAT recruitment domain of 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 of 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 the STAT recruitment domain of IL12Rb1 or IL12Rb2. In some embodiments, the STAT recruitment domain of 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 of IL12Rb1 or IL12Rb2 comprises the amino acid sequence of SEQ ID NO: 67, 86, or 87. In some embodiments, the recruitment domain comprises the STAT recruitment domain of IL21R. In some embodiments, the STAT recruitment domain of 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 of 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 domains comprise the STAT recruitment domains of IL7Ra and IL2Rb. In some embodiments, the recruitment domain comprises the STAT recruitment domains of IL7Ra and IL12Rb1. In some embodiments, the recruitment domain comprises the STAT recruitment domains of 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.

[00100] In some embodiments, a CACCR of the present disclosure comprises a transmembrane domain and/or a JAK binding domain from a TPOR/MPER receptor that comprises the H499L and S505N substitutions. In some embodiments, the CACCR further comprises an attraction domain comprising an amino acid sequence of one or more of the receptor sequences set forth in Table 2b. In some embodiments, the CACCR further comprises one or more attraction domains selected from the group consisting of STAT attraction domains from IL7Ra, IL2Rb, IL12Rb1, IL12Rb2, and IL21R. In some embodiments, the attraction domain comprises a STAT attraction domain from IL7Ra. In some embodiments, the STAT recruitment domain of 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 of IL7Ra comprises the amino acid sequence of SEQ ID NO: 46, 68, 69, 70, 71, or 72. In some embodiments, the recruitment domain comprises the STAT recruitment domain of IL2Rb. In some embodiments, the STAT recruitment domain of 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 of 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 the STAT recruitment domain of IL12Rb1 or IL12Rb2. In some embodiments, the STAT recruitment domain of 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 of IL12Rb1 or IL12Rb2 comprises the amino acid sequence of SEQ ID NO: 67, 86, or 87. In some embodiments, the recruitment domain comprises the STAT recruitment domain of IL21R. In some embodiments, the STAT recruitment domain of 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 of 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 domains comprise the STAT recruitment domains of IL7Ra and IL2Rb. In some embodiments, the recruitment domain comprises the STAT recruitment domains of IL7Ra and IL12Rb1. In some embodiments, the recruitment domain comprises the STAT recruitment domains of IL7Ra and IL12Rb2. In some embodiments, the CACCR comprises an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 91, 98, 120, or 127, with or without a signal sequence. In some embodiments, the CACCR comprises the amino acid sequence of SEQ ID NO: 91, 98, 120, or 127, with or without a signal sequence.

[00101] In some embodiments, a CACCR of the present disclosure comprises a transmembrane domain and/or a JAK binding domain from a TPOR/MPLR receptor that comprises the H499L and W515K substitutions or the H499L and G509N substitutions. In some embodiments, the CACCR further comprises an attraction domain comprising an amino acid sequence of one or more of the receptor sequences set forth in Table 2b. In some embodiments, the CACCR further comprises one or more attraction domains selected from the group consisting of STAT attraction domains from IL7Ra, IL2Rb, IL12Rb1, IL12Rb2, and IL21R. In some embodiments, the attraction domain comprises a STAT attraction domain from IL7Ra. In some embodiments, the STAT recruitment domain of 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 of IL7Ra comprises the amino acid sequence of SEQ ID NO: 46, 68, 69, 70, 71, or 72. In some embodiments, the recruitment domain comprises the STAT recruitment domain of IL2Rb. In some embodiments, the STAT recruitment domain of 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 of 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 the STAT recruitment domain of IL12Rb1 or IL12Rb2. In some embodiments, the STAT recruitment domain of 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 of IL12Rb1 or IL12Rb2 comprises the amino acid sequence of SEQ ID NO: 67, 86, or 87. In some embodiments, the recruitment domain comprises the STAT recruitment domain of IL21R. In some embodiments, the STAT recruitment domain of 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 of 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 domains comprise the STAT recruitment domains of IL7Ra and IL2Rb. In some embodiments, the recruitment domain comprises the STAT recruitment domains of IL7Ra and IL12Rb1. In some embodiments, the recruitment domain comprises the STAT recruitment domains of IL7Ra and IL12Rb2. In some embodiments, the CACCR comprises an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 97 or 126, with or without a signal sequence. In some embodiments, the CACCR comprises the amino acid sequence of SEQ ID NO: 97 or 126, with or without a signal sequence.

[00102] In some embodiments, a CACCR of the present disclosure comprises a transmembrane domain and/or a JAK binding domain from a TPOR/MPLR receptor that comprises the S505N and W515K substitutions. In some embodiments, the CACCR further comprises an attraction domain comprising an amino acid sequence of one or more of the receptor sequences set forth in Table 2b. In some embodiments, the CACCR further comprises one or more attraction domains selected from the group consisting of STAT attraction domains from IL7Ra, IL2Rb, IL12Rb1, IL12Rb2, and IL21R. In some embodiments, the attraction domain comprises a STAT attraction domain from IL7Ra. In some embodiments, the STAT recruitment domain of 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 of IL7Ra comprises the amino acid sequence of SEQ ID NO: 46, 68, 69, 70, 71, or 72. In some embodiments, the recruitment domain comprises the STAT recruitment domain of IL2Rb. In some embodiments, the STAT recruitment domain of 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 of 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 the STAT recruitment domain of IL12Rb1 or IL12Rb2. In some embodiments, the STAT recruitment domain of 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 of IL12RM or IL12Rb2 comprises the amino acid sequence of SEQ ID NO: 67, 86, or 87. In some embodiments, the recruitment domain comprises the STAT recruitment domain of IL21R. In some embodiments, the STAT recruitment domain of 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 of 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 domains comprise the STAT recruitment domains of IL7Ra and IL2Rb. In some embodiments, the recruitment domain comprises the STAT recruitment domains of IL7Ra and IL12Rb1. In some embodiments, the recruitment domain comprises the STAT recruitment domains of IL7Ra and IL12Rb2. In some embodiments, the CACCR comprises an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 96, 107, 109, 111, 113, 115, 117, 125, 128, 129, 132, 134, 136, or 138, with or without a signal sequence. In some embodiments, the CACCR comprises the amino acid sequence of SEQ ID NO: 96, 107, 109, 111, 113, 115, 117, 125, 128, 129, 132, 134, 136, or 138, with or without a signal sequence.

[00103] In some embodiments, a CACCR of the present disclosure comprises a transmembrane domain and/or a JAK binding domain from a TPOR/MPLR receptor that comprises the H499L and W515K substitutions. In some embodiments, the CACCR further comprises an attraction domain comprising an amino acid sequence of one or more of the receptor sequences set forth in Table 2b. In some embodiments, the CACCR further comprises one or more attraction domains selected from the group consisting of STAT attraction domains from IL7Ra, IL2Rb, IL12Rb1, IL12Rb2, and IL21R. In some embodiments, the attraction domain comprises a STAT attraction domain from IL7Ra. In some embodiments, the STAT recruitment domain of 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 of IL7Ra comprises the amino acid sequence of SEQ ID NO: 46, 68, 69, 70, 71, or 72. In some embodiments, the recruitment domain comprises the STAT recruitment domain of IL2Rb. In some embodiments, the STAT recruitment domain of 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 of 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 the STAT recruitment domain of IL12Rb1 or IL12Rb2. In some embodiments, the STAT recruitment domain of 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 of IL12Rb1 or IL12Rb2 comprises the amino acid sequence of SEQ ID NO: 67, 86, or 87. In some embodiments, the recruitment domain comprises the STAT recruitment domain of IL21R. In some embodiments, the STAT recruitment domain of 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 of 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 domains comprise the STAT recruitment domains of IL7Ra and IL2Rb. In some embodiments, the recruitment domain comprises the STAT recruitment domains of IL7Ra and IL12Rb1. In some embodiments, the recruitment domain comprises the STAT recruitment domains of IL7Ra and IL12Rb2. In some embodiments, the CACCR comprises an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 93, with or without a signal sequence. In some embodiments, the CACCR comprises the amino acid sequence of SEQ ID NO: 93, with or without a signal sequence.

[00104] In some embodiments, a CACCR of the present disclosure comprises a transmembrane domain and/or a JAK binding domain from a TPOR/MPLR receptor that comprises the substitutions H499L, S505N, and W515K. In some embodiments, the CACCR further comprises an attraction domain comprising an amino acid sequence of one or more of the receptor sequences set forth in Table 2b. In some embodiments, the CACCR further comprises one or more attraction domains selected from the group consisting of STAT attraction domains from IL7Ra, IL2Rb, IL12Rb1, IL12Rb2, and IL21R. In some embodiments, the attraction domain comprises a STAT attraction domain from IL7Ra. In some embodiments, the STAT recruitment domain of 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 of IL7Ra comprises the amino acid sequence of SEQ ID NO: 46, 68, 69, 70, 71, or 72. In some embodiments, the recruitment domain comprises the STAT recruitment domain of IL2Rb. In some embodiments, the STAT recruitment domain of 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 of 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 the STAT recruitment domain of IL12Rb1 or IL12Rb2. In some embodiments, the STAT recruitment domain of 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 of IL12RM or IL12Rb2 comprises the amino acid sequence of SEQ ID NO: 67, 86, or 87. In some embodiments, the recruitment domain comprises the STAT recruitment domain of IL21R. In some embodiments, the STAT recruitment domain of 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 of 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 domains comprise the STAT recruitment domains of IL7Ra and IL2Rb. In some embodiments, the recruitment domain comprises the STAT recruitment domains of IL7Ra and IL12Rb1. In some embodiments, the recruitment domain comprises the STAT recruitment domains of IL7Ra and IL12Rb2. In some embodiments, the CACCR comprises an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 95, 108, 110, 112, 114, 116, 118, 124, 130, 131, 133, 135, 137, or 139, with or without a signal sequence. In some embodiments, the CACCR comprises the amino acid sequence of SEQ ID NO: 95, 108, 110, 112, 114, 116, 118, 124, 130, 131, 133, 135, 137, or 139, with or without a signal sequence.

E. CACCR expression

[00105] The present invention provides polynucleotides encoding any of the CACCRs provided herein. Likewise, the present invention provides expression vectors comprising such polynucleotides. In some embodiments, the vector is a viral vector. In some embodiments, the vector is not a viral vector.

[00106] In some embodiments, the expression vector comprises a CACCR and a polynucleotide expressing a chimeric antigen receptor (CAR).

[00107] In some embodiments, the CACCR and CAR are expressed as a single polypeptide chain separated by a linker. FIG. 2 schematically shows a vector that can be used to co-express the 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.

II. CAR-bearing immune cells

[00108] The present invention provides engineered immune cells comprising a polynucleotide encoding a chimeric antigen receptor (CAR) and a CACCR of the present disclosure; the present invention also provides engineered immune cells expressing a chimeric antigen receptor (CAR-I cell) and a 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 cells (NK cells), natural killer T cells (NKT cells), invariant NKT cells, mast cells, myeloid-derived phagocytes, dendritic cells, dendritic killer cells, macrophages, and monocytes. Immune cells also include cells derived from, for example, but not limited to, a stem cell. Stem cells may be adult stem cells, non-human embryonic stem cells, more specifically non-human stem cells, cord blood stem cells, progenitor cells, bone marrow stem cells, induced pluripotent stem cells, totipotent stem cells, or hematopoietic stem cells.

[00109] Accordingly, some embodiments of the present invention provide CAR-T cells comprising a CACCR according to the present disclosure.

[00110] In some embodiments, CARs may comprise an extracellular ligand binding domain (e.g., a single chain variable fragment (scFv)), a trans membrane domain, and an intracellular signaling domain. In some embodiments, the extracellular ligand binding domain, the trans membrane domain, and the intracellular signaling domain are in a single polypeptide, i.e., a single chain. The present invention also provides multi-chain CARs and polypeptides. In some embodiments, multi-chain CARs comprise: a first polypeptide comprising a trans membrane domain and at least one extracellular ligand binding domain, and a second polypeptide comprising a trans membrane domain and at least one intracellular signaling domain, wherein the polypeptides are assembled together to form a multi-chain CAR.

[00111] The extracellular ligand binding domain of the CAR specifically binds to a target of interest. The target of interest can be any molecule of interest, including, for example, but not limited to, BCMA, EGFRvIII, Flt-3, WT-1, CD20, CD23, CD30, CD38, CD70, CD33, CD133, LeY, NKG2D, CS1, CD44v6, ROR1, CD 19, claudin-18.2 (claudin-18A2 or claudin 18 isoform 2), DLL3 (Drosophila delta-like protein 3, delta homolog 3, Delta3), Muc17 (mucin17, Muc3, Muc3), FAP alpha (fibroblast activation protein alpha), Ly6G6D (Ly6G6D locus protein G6d lymphocyte antigen complex 6, c6orf23, G6D, MEGT1, NG25) and/or RNF43 (RNF43 ubiquitin ligase (E3), RING finger protein 43).

[00112] In some embodiments, the extracellular ligand binding domain of a CAR comprises an scFv comprising a light chain variable region (VL) and a heavy chain variable region (VH) of a monoclonal antibody (mAb) specific for a target antigen, connected by a flexible linker. The single-chain variable region fragments are prepared by linking the light and/or heavy chain variable regions using a short linking peptide (Bird et al., Science 242:423-426, 1988) (e.g., glycine-serine linkers). In general, linkers can be short flexible polypeptides and typically consist of about 20 or fewer amino acid residues. Linkers, in turn, can be modified to perform additional functions, such as attachment of drugs or attachment to solid supports. Single chain variants can be produced either recombinantly or synthetically. An automatic synthesizer can be used to produce scFv synthetically. For recombinant production of scFv, a suitable plasmid containing a polynucleotide that encodes said 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. Polynucleotides encoding the scFv of interest can be obtained by routine manipulations, such as ligation of polynucleotides. The resulting scFv can be isolated using standard protein purification techniques known in the art.

[00113] The intracellular signaling domain of the CAR of the present invention is responsible for intracellular signaling upon binding of the extracellular ligand binding domain to a target, resulting in activation of an immune cell and an immune response (Signals 1 and/or 2). The intracellular signaling domain is capable of activating at least one of the normal effector functions of an immune cell in which the CAR is expressed. For example, the effector function of a T cell can be cytolytic activity or helper activity, including cytokine secretion.

[00114] In some embodiments, an intracellular signaling domain for use in a CAR may be cytoplasmic sequences, such as, but not limited to, a T cell receptor and coreceptors that act together to initiate signaling upon engagement with an antigen receptor, as well as any derivative or variant of these sequences, and any synthetic sequence having the same functional capability. Intracellular signaling domains comprise two distinct classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation and those that act in an antigen-independent manner to provide a secondary or costimulatory signal. Primary cytoplasmic signaling sequences may comprise signaling motifs that are known as immunoreceptor tyrosine-based activation motifs, ITAMs. ITAMs are well-defined signaling motifs found in the intracytoplasmic tail of various receptors that serve as binding sites for syk/zap70 class tyrosine kinases. Examples of ITAMs used in the present invention may include, but are not limited to, those derived from TCRζ, FcRγ, FcRβ, FcRε, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, and CD66d. In some embodiments, the intracellular signaling domain of a CAR may comprise a CD3ζ signaling domain.

In some embodiments, the intracellular signaling domain of a CAR of the present invention comprises a costimulatory molecule domain.

[00115] According to some embodiments, the intracellular signaling domain of a CAR of the present invention comprises a portion of a costimulatory molecule selected from the group consisting of fragment 41BB (GenBank: AAA53133.) and CD28 (NP 006130.1).

[00116] CARs are expressed on the cell surface membrane. Thus, a CAR comprises a transmembrane domain. Suitable transmembrane domains for a CAR disclosed herein have the ability to (a) be expressed on the surface of a cell, preferably an immune cell such as, for example but not limited to, lymphocytes or natural killer cells (NK cells), and (b) interact with a ligand binding domain and an intracellular signaling domain to direct the cellular response of the immune cell to a predetermined target cell. The transmembrane domain may be from either a natural or synthetic source. The transmembrane domain may be from any membrane-bound or trans membrane protein. As non-limiting examples, the trans membrane polypeptide can be a T cell receptor subunit such as α, β, γ or δ, a polypeptide comprising the CD3 complex, p55 (a chain), p75 (β chain) or γ chain of the IL-2 receptor, a chain of a subunit of Fc receptors, in particular the FcγIII receptor, or CD proteins. Alternatively, the trans membrane domain can be synthetic and can comprise predominantly hydrophobic residues such as leucine and valine. In some embodiments, said trans membrane domain is derived from the human CD8a chain (e.g., NP 001139345.1). The trans membrane domain can further comprise a stalk domain between the extracellular ligand binding domain and said trans membrane domain. The stalk domain may comprise up to 300 amino acids, preferably 10 to 100 amino acids, and most preferably 25 to 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 domain sequence, or may be a completely synthetic stalk domain sequence. In some embodiments, the stalk domain is a portion of a human CD8α chain (e.g., NP 001139345.1). In another specific embodiment, the trans membrane and hinge domains comprise a portion of a human CD8α chain. In some embodiments, the intracellular signaling domain comprises a CD3ζ signaling domain. In some embodiments, the intracellular signaling domain comprises a CD3ζ signaling domain, and optionally 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 CARs disclosed herein may comprise an extracellular ligand binding domain that specifically binds BCMA or EGFRvIII, the hinge and transmembrane domains of human CD8α, a CD3ζ signaling domain, and a 4-1BB signaling domain. In some embodiments, an EGFRvIII-specific CAR comprises the amino acid sequence of SEQ ID NO: 140. In some embodiments, a BCMA-specific CAR comprises the amino acid sequence of SEQ ID NO: 141 or 142, with or without a signal sequence.

[00117] In some aspects, the CAR immune cell is a BCMA CAR-T cell comprising a CACCR of the present disclosure. In some embodiments, the CACCR of the BCMA CAR-T cell comprises a transmembrane/JAK binding domain comprising amino acids 478-582 of the naturally occurring TPOR/MPLR receptor of SEQ ID NO: 6, with three substitutions H499L, S505N, and W515K, or two substitutions S505N and W515K (e.g., SEQ ID NO: 12 or 13). In some embodiments, the CACCR further comprises an engagement domain from IL2Rb. In some embodiments, the CACCR of the BCMA CAR T cell further comprises an engagement 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 cells comprise a CACCR that comprises the amino acid sequence of SEQ ID NO: 113, 114, or 116, with or without a signal sequence.

[00118] In some embodiments, a CAR may be introduced into an immune cell as a transgene using a plasmid vector. In some embodiments, the plasmid vector may also contain, for example, a selectable marker that allows for identification and/or selection of cells that have received the vector.

[00119] Table 4 provides exemplary sequences of CAR components that can be used in the CARs disclosed herein, as well as sequences of antibodies and/or CARs exemplified herein.

[00120] In some embodiments, a CAR immune cell (e.g., a CAR T cell) of the present disclosure comprises a polynucleotide encoding a "self-destruct" polypeptide, such as, for example, RQR8. See, for example, WO2013153391A, the entire contents of which are incorporated herein by reference. In some embodiments, the "self-destruct" polypeptide is expressed on the surface of the cell. In some embodiments, the "self-destruct" polypeptide is included in the CAR construct. In some embodiments, the "self-destruct" polypeptide is not part of the CAR construct.

[00121] In some embodiments, the extracellular domain of any of the CARs disclosed herein may comprise one or more epitopes specific to (specifically recognized by) a monoclonal antibody (mAb). These epitopes are also referred to herein as mAb-specific epitopes. Exemplary mAb-specific epitopes are disclosed in International Patent Publication No. WO 2016/120216, which is incorporated herein in its entirety. In these embodiments, the extracellular domain of the CAR comprises antigen-binding domains that specifically bind to a target of interest and one or more epitopes that bind to one or more monoclonal antibodies (mAbs). CARs comprising mAb-specific epitopes may be single-chain or multi-chain.

[00122] Incorporation of monoclonal antibody-specific epitopes into the extracellular domain of the CARs described herein allows for the sorting and depletion of engineered immune cells expressing the CAR. In some embodiments, the depletion capability provides a "safety switch" in the event of deleterious effects, such as when administered to a subject.

[00123] The present invention also provides methods for preparing engineered immune cells for use in immunotherapy. In some embodiments, the methods comprise introducing a CACCR and a CAR into immune cells and expanding the cells. In some embodiments, the present invention relates to a method of modifying an immune cell, comprising: providing a cell with and expressing a CACCR, and expressing at least one CAR on the surface of the cell. In some embodiments, the method comprises: transfecting a cell with at least one polynucleotide encoding a CACCR and at least one polynucleotide encoding a CAR, and expressing the polynucleotides in the cell. In some embodiments, the method comprises: transfecting a cell with at least one polynucleotide encoding a CACCR, at least one polynucleotide encoding a CAR, and expressing the polynucleotides in the cell.

[00124] In some embodiments, 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 viral vectors for stable expression in cells. In some embodiments, the viral vectors can be, for example, lentiviral vectors or adenoviral vectors.

[0100] In some embodiments, polynucleotides encoding polypeptides according to the present disclosure can be mRNA that is introduced directly into cells, such as by electroporation. In some embodiments, CytoPulse electroporation technology, such as PulseAgile (e.g., US 6,078,490; PCT/US 2011/000827; and PCT/US 2004/005237), can be used to transiently permeabilize living cells to deliver genetic material into the cells. Parameters can be altered to determine conditions for high transfection efficiency with minimal mortality.

[0101] The present invention also provides methods for transfecting an immune cell, such as a T cell. In some embodiments, the method comprises: contacting the T cell with RNA and applying a dynamic pulse sequence to the T cell. In some embodiments, the method for transfecting an immune cell (e.g., a T cell) comprises contacting the immune cell with RNA and applying a dynamic pulse sequence to the cell.

[0102] In some embodiments, the method may further comprise the step of genetically modifying the cell by inactivating at least one gene expressing, for example, but not limited to, a TCR component, a target for an immunosuppressive agent, an HLA gene, and/or an immune checkpoint protein such as, for example, PDCD1 or CTLA-4. By inactivating a gene, it is meant that the gene of interest is not expressed in a functional protein form. In some embodiments, the gene to be inactivated 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 into the cells a rare-cutting endonuclease capable of selectively inactivating the gene by selectively cleaving DNA. In some embodiments, the rare-cutting endonuclease may be, for example, a transcription activator-like effector (TALE)-based nuclease or a CRISPR-based endonuclease (e.g., Cas-9 or Cas12a).

[0103] In another aspect, the step of genetically modifying cells may comprise: modifying immune cells (e.g., T cells) by inactivating at least one gene expressing a target for an immunosuppressive agent, and; expanding the cells, optionally in the presence of an immunosuppressive agent.

[0104] In some embodiments, engineered immune cells (e.g., T cells) provided herein exhibit improved cytotoxicity, increased proliferation, and/or increased levels of memory phenotype markers compared to engineered immune cells that do not express CACCR.

[0105] In some embodiments, engineered immune cells (e.g., T cells) provided herein constitutively exhibit (i) increased in vivo resistance, (ii) increased STAT activation, (iii) increased cytotoxicity, (iv) increased levels of memory phenotype markers, (v) increased proliferation, or a combination of these functional features, compared to engineered immune cells that do not express CACCRs. In some embodiments, the improvement in one or more functional features described herein is adjustable, depending on the mutations/modifications introduced into the CACCRs. In some embodiments, the STATs activated by an engineered immune cell comprising one or more of the disclosed CACCRs are STAT1, STAT2, STAT3, STAT4, STAT5, STAT6, or combinations thereof. In one embodiment, the memory phenotype markers that are elevated or maintained by an immune cell containing CACCR include a memory T stem cell marker (Tscm) and a central memory T cell marker (Tcm).

[0106] According to some embodiments, one or more functional features exhibited by an engineered immune cell comprising a CACCR provided herein are improved by 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 about 10,500-fold, including values and ranges therebetween, compared to an immune cell that does not express a CACCR.

[0107] According to some embodiments, one or more functional features exhibited by an engineered immune cell comprising a CACCR provided herein are improved by at least about 10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 75%, 80%, 90%, 100%, 125%, 150%, 200%, 250%, 300%, 350%, 400%, or even about 80%500%, including values and ranges therebetween, compared to an immune cell that does not express a CACCR.

III. Treatment methods

[0108] The present invention provides pharmaceutical compositions comprising cells bearing a CACCR and a CAR according to the present disclosure.

[0109] Engineered immune cells that carry a CACCR and carry a CAR (e.g., T cells) obtained using the methods described above, or cell lines derived from such engineered immune cells, can be used as a medicine. In some embodiments, such a medicine can be used to treat a disorder such as, for example, a viral disease, a bacterial disease, a cancer, an inflammatory disease, an immune disease, or a disease associated with aging. In some embodiments, the cancer is a solid cancer. In some embodiments, the cancer is a hematological malignancy. The cancer may be selected from the group consisting of gastric cancer, sarcoma, lymphoma, leukemia, head and neck cancer, thymus cancer, epithelial cancer, salivary gland cancer, liver cancer, gastric 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 carcinoma of the head and neck (SCHNC), ovarian carcinoma, sarcoma, or relapsed or refractory classical Hodgkin lymphoma (cHL).

[0110] In some embodiments, engineered immune cells or a cell line derived from engineered immune cells can be used in the manufacture of a medicament for treating a disorder in a subject in need thereof. In some embodiments, the disorder can be, for example, cancer, an autoimmune disorder, or an infection.

[0111] The present invention also provides methods for treating subjects in need of such treatment.

[0112] As used herein, the term "subject" refers to any vertebrate, including, but not limited to, humans and other primates (e.g., chimpanzees, cynomolgus macaques, and various species of great apes and other monkeys), farm animals (e.g., cattle, sheep, pigs, goats, and horses), domestic mammals (e.g., dogs and cats), laboratory animals (e.g., rabbits, rodents such as mice, rats, and guinea pigs), and birds (e.g., domestic, wild, and game birds such as chickens, turkeys, and other chickens, ducks, geese, etc.). In some embodiments, the subject is a mammal. In exemplary embodiments, the subject is a human.

[0113] According to some embodiments, the method comprises providing immune cells of the present disclosure carrying the CACCR and CAR described herein to a subject in need thereof.

[0114] In some embodiments, T cells bearing a CACCR or CAR according to the present invention can undergo robust T cell expansion in vivo and can be maintained for an extended period of time.

[0115] The methods of treatment according to the present invention may be ameliorative, curative, or prophylactic. The method according to the present invention may be either part of an autologous immunotherapy or part of an allogeneic immunotherapy treatment.

[0116] According to another aspect of the present invention, there is provided a method of inhibiting the growth or progression of a tumor in a subject having a tumor, comprising administering to said subject an effective amount of CACCR-expressing and CAR-expressing immune cells as described herein. According to another aspect of the present invention, there is provided 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 engineered immune cells as described herein. According to another aspect of the present invention, there is provided a method of inducing regression of a tumor in a subject having a tumor, comprising administering to the subject an effective amount of engineered immune cells as described herein.

[0117] In some embodiments, engineered T cells of the present invention can be administered to a subject parenterally.

[0118] Also provided is the use of any engineered T cells provided herein in the manufacture of a medicament for the treatment of cancer or for inhibiting the growth or progression of a tumor in a subject in need thereof.

[0119] In some embodiments, the treatment can be performed in subjects undergoing immunosuppressive treatment. Indeed, the present invention preferably relies on cells or a population of cells that have been rendered resistant to at least one immunosuppressive agent by inactivating a gene encoding a receptor for such an immunosuppressive agent. In this aspect, the immunosuppressive treatment should facilitate the selection and expansion of T cells in accordance with the present invention in the subject. Administration of the cells or a population of cells in accordance with the present invention can be performed by any convenient route, including aerosol inhalation, injection, ingestion, transfusion, implantation, or transplantation. The compositions described herein can be administered to the subject subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, by intravenous or intralymphatic injection, or intraperitoneally. The cells bearing the CACCR and CAR of the present disclosure, or pharmaceutical compositions thereof, may be administered via one or more of the following routes of administration: intravenous, intraocular, intravitreal, intramuscular, subcutaneous, topical, oral, transdermal, intraperitoneal, intraorbital, by implantation, by inhalation, intrathecal, intraventricular, otic, or intranasal.

[0120] In some embodiments, administering the cells or population of cells (bearing a CACCR or CAR of the present disclosure) may comprise administering, for example, from about 10 ⁴ to about 10 ⁹ cells per kg body weight, including all integer cell numbers within these ranges. In some embodiments, administering the cells or population of cells may comprise administering from about 10 ⁴ to 10 ⁵ cells per kg body weight, from 10 ⁵ to 10 ⁶ cells per kg body weight, from 10 ⁶ to 10 ⁷ cells per kg body weight, from 10 ⁷ to 10 ⁸ cells per kg body weight, or from 10 ⁸ to 10 ⁹ cells per kg body weight. The cells or population of cells may be administered in one or more doses. In some embodiments, said effective amount of cells may be administered in a single dose. In some embodiments, said effective amount of cells may be administered in more than one dose over a period of time. The time of administration is determined by the attending physician and depends on the clinical condition of the subject. The cells or cell population can be obtained from any source, such as a blood bank or donor. Although individual needs vary, it is within the skill of the art to determine optimal ranges of effective amounts of a particular cell type for a particular disease or condition. 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, the type of concomitant treatment, if any, the frequency of treatment and the nature of the desired effect. In some embodiments, an effective amount of cells or a composition containing these cells is administered parenterally. In some embodiments, the administration can be intravenous administration. In some embodiments, the administration can be carried out directly by injection into the tumor.

[0121] The methods may further comprise administering one or more agents to the subject prior to administering the engineered immune cells bearing the CAR and CACCR provided herein. In certain embodiments, the agent is a lymphodepletion (preconditioning) regimen. For example, methods for lymphodepletion of a subject in need of such therapy comprise administering to said subject the indicated useful doses of cyclophosphamide (from 200 mg/ ^m2 /day to 2000 mg/ ^m2 /day, about 100 mg/ ^m2 /day and about 2000 mg/ ^m2 /day; e.g., about 100 mg/ ^m2 /day, about 200 mg/ ^m2 /day, about 300 mg/ ^m2 /day, about 400 mg/ ^m2 /day, about 500 mg/ ^m2 /day, about 600 mg/ ^m2 /day, about 700 mg/ ^m2 /day, about 800 mg/ ^m2 /day, about 900 mg/ ^m2 /day, about 1000 mg/ ^m2 /day, about 1500 mg/ ^m2 /day or about 2000 mg/ ^m2 /day) and the indicated doses of fludarabine (from 20 mg/ ^m2 /day to 900 mg/ ^m2 /day, from about 10 mg/ ^m2 /day to about 900 mg/ ^m2 /day; for example, about 10 mg/ ^m2 /day, about 20 mg/ ^m2 /day, about 30 mg/ ^m2 /day, about 40 mg/ ^m2 /day, about 40 mg/ ^m2 /day, about 50 mg/ ^m2 /day, about 60 mg/ ^m2 /day, about 70 mg/ ^m2 /day, about 80 mg/ ^m2 /day, about 90 mg/ ^m2 /day, about 100 mg/ ^m2 /day, about 500 mg/ ^m2 /day or about 900 mg/ ^m2 /day). An exemplary dosing regimen includes treating a subject comprising daily administration to the patient of about 300 mg/ ^m2 /day of cyclophosphamide in combination with or before or after administration of about 30 mg/ ^m2 /day of fludarabine for three days prior to administration of a therapeutically effective amount of engineered immune cells to the patient.

[0122] In some embodiments, particularly where the engineered cells provided herein have been gene edited to eliminate or minimize surface expression of CD52, lymphodepletion further comprises administering an anti-CD52 antibody, such as alemtuzumab. In some embodiments, the anti-CD52 antibody is administered at a dose of about 1-20 mg/day intravenously, such as about 13 mg/day intravenously for 1, 2, 3, or more days. The antibody may be administered in combination with, prior to, or following administration of other elements of the lymphodepletion regimen (e.g., cyclophosphamide and/or fludarabine).

[0123] According to certain embodiments, compositions comprising CACCR- and CAR-expressing immune effector cells disclosed herein can be administered in combination with any number of chemotherapeutic agents.

IV. Kits and finished products

[0124] The present disclosure provides kits comprising any one or more of the CACCR and CAR-bearing CAR-I cells described herein, and pharmaceutical compositions thereof. The present disclosure also provides articles of manufacture comprising any one or more of the CACCR and CAR-bearing CAR-I cells described herein, and pharmaceutical compositions thereof, and the kits described herein.

[0125] The following examples are included for illustrative purposes only and are not intended to limit the scope of the present disclosure.

[0126] All patent and non-patent documents referenced in this disclosure are herein incorporated by reference in their entirety for all purposes.

EXAMPLES

Example 1: Identification of TpoR mutants that constitutively activate cytokine signaling

[0127] A prototypical constitutively active chimeric cytokine receptor (CACCR) was constructed using sequences from the thrombopoietin receptor (TpoR). TpoR is capable of activating the JAK-Stat signaling pathway and signaling as a homodimeric receptor. With regard to TpoR activity, single point mutations (amino acid substitutions) have been shown to modulate receptor activity (Proc Natl Acad Sci USA. 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 constructed from a naturally occurring TpoR receptor: the extracellular domain of the naturally occurring TpoR receptor was removed so that it no longer has ligand binding capacity; 1-3 mutations were introduced into its trans membrane domain; and the cytoplasmic tail of TpoR was replaced with the cytoplasmic tail of a target/characterized cytokine receptor. FIG. 1 schematically shows the constructed constitutively active chimeric cytokine receptor.

[0128] To demonstrate the utility of the constitutively active chimeric cytokine receptor in CAR-T cells, each TpoR transmembrane (TM) domain 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 ensure stoichiometric co-expression of the cytokine receptor and CAR, the two genes were linked via the 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.

[0129] FIG. 2 schematically shows a lentiviral vector used to co-express a constitutively active chimeric cytokine receptor and CAR.

[0130] Table 4 shows the sequences associated with the constructs used.

[0131] A HEK293T cell reporter gene 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 poly-L-lysine-coated flat-bottom plate and allowed to attach overnight. The cytokine receptor CAR construct (2.5 ng), the Stat response element that drives firefly luciferase (100 ng; Promega), and the Renilla luciferase control reporter vector (1 ng; Promega) were mixed in a final volume of 5 μl in Opti-MEM (Gibco) (“DNA mix”). As a negative control, cells were transfected with the BFP CAR construct, which lacks all cytokine signaling domains. As a positive control, cells were transfected with a vector encoding full-length human EpoR (erythropoietin receptor instead of the cytokine receptor-CAR construct) so that StatS signaling could be induced by the addition of exogenous recombinant human Epo. 0.3 μl Lipofectamine 2000 (Invitrogen) in 5 μl 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 μl was added to each well containing HEK-293T. Stat5 reporter activity was assessed 48 h post-transfection using the Dual-Glo Luciferase Assay System (Promega). The fold induction of StatS reporter activity was normalized to that of HEK293T cells transfected with all vectors except the cytokine receptor and not treated.

[0132] FIG. 3a and 3b show the identification of TpoR TM mutants that constitutively activate cytokine receptor signaling. FIG. 3a is a schematic representation of the lentiviral vector used. The vector carries the cytoplasmic tail of IL7R(316-459) to mimic IL7 signaling in CAR-T cells. FIG. 3b shows Stat5 reporter activity determined by the Dual-Glo luciferase assay. The cytokine receptor carrying the wild-type TpoR TM domain (TpoR(478-582)) did not spontaneously activate StatS. Mutants TpoR(478-582;S505N), TpoR(478-582;W515K), and TpoR(478-582;H499L,G509N) resulted in weak Stat5 activation. TpoR(478-582;H499L,S505N,W515K) resulted in moderate Stat5 activity, while TpoR(478-582; S505N,W515K) elicited the strongest Stat5 signal.

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

[0133] We next tested whether these cytokine receptors signaled in primary human CAR-T cells. To generate a lentivirus encoding a cytokine receptor-CAR, HEK293T cells were seeded at 0.45 million cells/mL in 2 mL of DMEM (Gibco) supplemented with 10% fetal bovine serum (FBS) (Hyclone) per well of a 6-well plate the day before transfection. On the day of transfection, lentivirus was generated by mixing 1.5 μg of psPAX2 lentiviral packaging vectors, 0.5 μg of pMD2G, and 0.5 μg of the appropriate CAR transfer vector in 250 μL of Opti-MEM (Gibco) per well of a 6-well plate (“DNA mix”). 10 μl of Lipofectamine 2000 (Invitrogen) in 250 μl of Opti-MEM were 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 500 μl was slowly added to the sides of the wells containing HEK293T. One day after transfection, the media from each well containing HEK293T cells in a 6-well plate was replaced with 2 ml per well of T cell transduction media, i.e. X-Vivo-15 supplemented with 10% FBS. Two days after transfection, lentiviral supernatants from HEK293T cells were collected and passed through a 0.45-μm filter (EMD Millipore) to remove cell debris, concentrated 25-fold using a Lenti-X concentrator (Takara Bio) according to the manufacturer's instructions, and snap-frozen as 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 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, Cat. #130-111-160, 1:100 dilution) in a Grex-24 plate (Wilson Wolf, Cat. #80192M). On Day 2, T cells were resuspended at 0.5 million cells/ml in T cell transduction media, transduced with the appropriate lentivirus concentrates at MOI=5 with 100 IU/ml human IL-2 in a Grex-24 plate. On Day 5, when transduction was complete, cells were harvested and washed to remove residual IL-2. Cells were then resuspended in T cell expansion media, i.e., X-Vivo-15 supplemented with 5% human AB serum (Gemini Bio), and each sample was split into 2 equal parts, one part receiving 100 IU/ml human IL-2 according to the standard protocol and the other part receiving a lower concentration of 25 IU/ml human IL-2. When necessary, cells were expanded in larger G-Rex flasks (Wilson Wolf) using T cell expansion media and appropriate concentrations 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 that bound FITC-conjugated anti-v5 monoclonal antibody (Thermo Fisher) using flow cytometry. On Day 14 or 15, CAR-T cell products were cryopreserved and thawed as needed for further analysis.

[0134] FIGS. 4a-4c show results regarding the generation of CAR-T cells co-expressing a constitutively active chimeric cytokine receptor. Compared to CAR-T cell cultures bearing a cytokine receptor with the wild-type TpoR TM (TpoR(478-582)), CAR-T cell cultures bearing the TpoR TM mutants underwent more robust expansion in terms of both total T cell numbers (FIG. 4a) and CAR-T cell numbers (FIG. 4b). FIGS. 4a-4c show that the transduction efficiency of the TpoR TM mutants was equal to or greater than that of the wild-type TpoR(478-582) counterparts. The TpoR TM mutants yield higher yields of CAR-T cell product. Furthermore, expansion of TpoR mutants at lower IL-2 concentrations did not affect CAR-T cell expansion or yield (FIG. 4c).

[0135] On Day 14 of CAR-T cell production, the memory phenotype of CAR-T cells was determined. Briefly, samples were washed with phosphate-buffered saline (PBS), blocked with Fc, then stained with the following antibody mixture diluted in PBS + 1% BSA: BUV395-conjugated anti-human CD3, BV510-conjugated anti-human CD8, BV605-conjugated anti-human CD4, and FITC-conjugated anti-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 µl PBS+1% BSA for FACS analysis.

[0136] FIG. 5 shows the distribution of the memory T cell subpopulation in a CAR T cell product. Compared to their wild-type TpoR(478-582) counterpart, the TpoR(478-582;W515K) and TpoR(478-582;H499L,G509N) mutants showed greater differentiation when expanded under standard 100 IU/mL IL-2 conditions. Expansion under low IL-2 conditions reduced differentiation. When combined with standard IL-2 concentrations, 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 under low IL-2 conditions may be more favorable for CAR-T cells expressing constitutive cytokine receptors.

Example 3: TpoR TM mutants constitutively activate cytokine signaling in human CAR-T cells

[0137] To determine the potency of cytokine signaling mediated by TpoR TM mutants, CAR-T cells bearing cytokine receptors with TpoR TM variants were serum-starved in 100 μl 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-7 (10 ng/ml; Miltenyi) was added for the last 30 min of a 4-h serum-starved incubation. After 4 h, an antibody cocktail containing BUV395-conjugated anti-human CD3 antibody (Biolegend) and FITC-conjugated anti-v5 tag monoclonal antibody (Thermo Fisher) was added to the cells and incubation was continued for the last 20 min. Cells were then fixed by adding 35 μl 16% paraformaldehyde to 100 μl of each sample and incubation was continued for 15 min at 37°C. Cells were then washed three times with PBS and permeabilized in 100% cold methanol for 1 or 2 nights at -20°C. On the day of FACS analysis, cells were washed three times with PBS, blocked with Fc, and stained with A1exaFluor647-conjugated anti-mouse/human Stat5 (pY694) (BD Biosciences) diluted in PBS+1% BSA. After 1 h of incubation at room temperature in the dark, cells were washed three times before FACS analysis.

[0138] FIGS. 6a-6b show the extent of constitutive cytokine signaling mediated by each TpoR TM variant, represented as the percentage of pStat5 ⁺ cells (FIG. 6a) and the geometric mean fluorescence intensity (gMFI) of pStat5 staining (FIG. 6b). While the single point mutants of TM TpoR (TpoR(478-582;S505N) and TpoR(478-582;W515K)) did not induce detectable Stat5 activation, the double mutant of TM TpoR (TpoR(478-582;S505N,W515K) and the triple mutant (TpoR(478-582;H499L,S505N,W515K)) induced comparatively strong constitutive Stat5 activation. CAR T cells expanded in low and standard IL-2 concentrations yielded comparable Stat activation profiles. Since Stat5 was activated only by CAR-bearing T cells (CAR ⁺ ), but not by CAR-non-bearing T cells (CAR′), in the same culture, this suggests that cytokine signaling was specific to CAR-T cells.

Example 4: Constitutive cytokine receptor enhanced CAR-T cell cytotoxic activity and prolonged response durability

[0139] To investigate 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 generated from the parent cell line, U87MG (ATCC), by first knocking down endogenous wild-type EGFR using transcription activator-like effector nucleases (TALENs) and then stably overexpressing full-length human EGFRvIII by lentiviral transduction. To facilitate visualization of target T cells using the IncuCyte Live Cell Imaging System, U87KO-EGFRvIII-nucGFP target cells were generated from U87KO-EGFRvIII by a second lentiviral transduction using IncuCyte NucLight Green Lentivirus Reagent (Sartorius). 5,000 U87KO-EGFRvIII-nucGFP target cells were seeded and allowed to adhere in 96-well black-walled, flat-bottomed, clear-bottomed plates in 50 μl RPMI containing 10% FBS (Hyclone), nonessential amino acids, sodium pyruvate, and 20-25 mM HEPES. EGFRvIII CAR (2173 scFv) T cells bearing cytokine receptors with TM variants of TpoR were thawed and added to plated target cells at effector:target (E:T) ratios of 1:8 and 1:2. For comparison, CAR T cells bearing wild-type TpoR(478-582) were included in the analysis with or without exogenous recombinant human IL-7. Where applicable, CAR T cells were restimulated at the indicated time points by transferring the cell suspension from the original plate to a fresh plate with target cells. Duplicate wells were used for each condition. Cytotoxicity was determined by enumerating the number of live target cells at each time point using the mcuCyte Live Cell Imaging Assay System.

[0140] FIGS. 7a-7d show the cytotoxic activity of TpoR TM mutants at a 1:8 E:M ratio. The single point mutants TpoR(478-582;S505N) (FIG. 7a) and TpoR(478-582;W515KN) (FIG. 7b) showed no functional improvement over their wild-type TpoR(478-582) control counterparts, consistent with their inability to efficiently activate Stat5 (FIG. 6a-6b). FIG. 7c shows that TpoR double mutant CAR-T cells expanded with standard concentrations of IL-2 failed to exhibit enhancement; while TpoR double mutant CAR-T cells expanded under low IL-2 conditions were more active in target cell lysis. FIG. 7d shows that TpoR triple mutant CAR-T cells were more active in target cell lysis, regardless of the IL-2 concentration during CAR-T cell production. This indicates that constitutive cytokine receptor signaling enhances CAR-T cell activity.

[0141] FIGS. 8a-8b show the cytotoxic activity of double mutants (FIG. 8a) and triple mutants (FIG. 8b) of TpoR TM at a 1:2 E:M ratio. During the primary response, CAR-T cells eliminated target cells with equal efficiency, regardless of cytokine receptor activity. However, upon restimulation with fresh targets, only CAR-T cells expressing constitutively active chimeric cytokine receptors remained functional, indicating that constitutive cytokine receptor signaling enhances the durability of CAR-T cell responses.

Example 5: Constitutive cytokine receptor enhanced CAR-T cell resistance and stimulated CAR ⁺ Tscm expansion

[0142] To observe the enhancing effects of constitutive cytokine receptor signaling on CAR-T cell persistence in the absence of targets or exogenous cytokines, a growth factor-independent assay was performed. Briefly, the percentage of CAR-T cells in all samples was normalized to the sample with the lowest transduction efficiency (35.7%) by adding 0.25 x 10 ⁶ CAR-bearing T cells/mL in 4 mL RPMI containing 10% FBS (Hyclone), nonessential amino acids, sodium pyruvate, and 20-25 mM HEPES. Cells were then seeded in T25 tissue culture flasks. As positive controls, exogenous human IL-7 (10 ng/mL; Miltenyi) was added to CAR-T cells lacking constitutive cytokine receptor signaling (wild-type TpoR(478-582)). On the indicated days for each condition, duplicate 200 μL samples were collected and stained using the Zombie NIR Fixable Viability Kit (Biolegend). Samples were washed with PBS, blocked with Fc, then stained with the following antibody mixture diluted in PBS + 1% BSA: BUV395-conjugated anti-human CD3, BV510-conjugated anti-human CD8, BV605-conjugated anti-human CD4, and FITC-conjugated anti-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 µl PBS+1% BSA containing 123count eBeads (Thermo Fisher) (10 µl counting beads in 120 µl PBS+1% BSA) prior to FACS analysis.

[0143] FIG. 9 shows enrichment of CAR-T cells over time in a growth factor independent assay. While CAR-T cells bearing wild-type TpoR TM (TpoR(478-582) and single point mutants of TpoR TM (TpoR(478-582;S505N) and TpoR(478-582;W515KN)) were not enriched, CAR-T cells bearing double mutant and triple mutants of TpoR TM were enriched over time, indicating that predominantly CAR-bearing T cells that had received constitutive cytokine receptor signaling survived.

[0144] FIGS. 10a-10b show the fold expansion of CAR-T cells over time in a growth factor independent assay. 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. FIG. 10a shows that single point mutants of TpoR that were unable to efficiently activate cytokine receptor signaling declined in number at the same rate as CAR-T cells bearing wild-type TpoR (TpoR(478-582)). In contrast, FIG. 10b shows that CAR-T cells bearing the double mutant or triple mutant of TpoR had prolonged survival in the absence of targets and exogenous cytokines. TpoR double mutant CAR-T cells expanded under low IL-2 conditions showed enhanced resistance compared to their counterparts expanded under standard IL-2 conditions. TpoR triple mutant CAR-T cells made under low and standard IL-2 conditions showed comparable intermediate increases in resistance. Notably, although TpoR double and triple mutant CAR-T cells persisted longer, their numbers eventually declined, indicating that constitutive cytokine receptor signaling was unlikely to result in CAR-T cell immortalization or transformation.

[0145] FIG. 11 shows the distribution of the memory T cell subset among CAR ⁺ T cells over time in a growth factor-independent assay. Compared to wild-type TpoR (TpoR(478-582)) CAR-T cells bearing a constitutively active cytokine receptor (here shown is the TpoR triple mutant expanded under low IL-2 conditions) showed an expansion in absolute numbers of stem cell memory (Tscm) T cells, which are a subset that mediates long-lasting anti-tumor immunity. Notably, constitutive signaling from the TpoR triple mutant was more efficient in expanding Tscm CAR-T cells than the addition of exogenous human IL-7.

Example 6: Constitutive cytoplasmic tails can be adapted to activate signaling pathways of interest

[0146] The ability of cytokines to regulate CAR-T cell fate and function is due to their ability to induce activation of various downstream signaling pathways. For example, IL-7/IL-2/IL-15-mediated STAT5 activation enhances T cell survival and expansion, while IL-12-mediated STAT4 activation drives terminal differentiation into short-lived effectors. Engineering recruitment domains (i.e., the cytoplasmic tail) that can mimic a broader range of cytokine signals can provide flexibility for user-programmable signaling outcomes, enabling control of CAR-T cell fate and function.

[0147] To analyze whether CACCRs can transmit signals mediated by additional cytokine receptors, the IL7Ra(316-459) cytoplasmic tail of FIG. 3 was replaced with alternative cytoplasmic tails derived from the intracellular signaling domains of IL2Rb or IL12Rb2. A HEK293T cell reporter gene assay was then used to assess the signaling ability of these chimeras. In general, 20,000 HEK293T cells were seeded into each well of a 96-well poly-L-lysine-coated flat-bottom plate and allowed to attach overnight. The cytokine receptor-CAR construct (2.5 ng), the Stat response element that drives firefly luciferase (100 ng; Promega), and the Renilla luciferase control reporter vector (1 ng; Promega) were mixed in a final volume of 5 μl in Opti-MEM (Gibco) (“DNA mix”). As a negative control, cells were transfected with either the BFP-CAR construct, which lacks all cytokine signaling domains, or the TpoR(478-582).IL7Ra(316-459) construct, which lacks transmembrane mutations and is therefore incapable of constitutive signaling. 0.3 μl Lipofectamine 2000 (hwitrogen) in 5 μl Opti-MEM was incubated at room temperature for 5 min and then added to the DNA mix. The mixture was incubated at room temperature for 20 min and a total volume of 10 μl was added to each well containing HEK-293T. At 48 h post-transfection, the activity of the respective Stat5 reporters 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 the control BFP-CAR construct.

[0148] FIG. 12 shows that constructs bearing different signaling domains preferentially activate different STAT pathways. Specifically, IL2Rb(333-551) and IL7Ra(316-459) activated STAT5, while IL12Rb2(714-862) activated STAT4, reflecting the signaling expected from the respective parent receptors. This demonstrates that CACCRs can be programmed to activate target signaling pathways by fusing to the signaling domain of interest. Furthermore, consistent with FIG. 3, CACCRs bearing the TpoR(478-582;S505N,W515K) dimerization domain elicited stronger signaling than their TpoR(478-582;H499L,S505N,W515K) counterpart. The strength of CACCR output signals can therefore be further regulated by fusion with any of these dimerization domains.

Example 7: The CACCR signaling domain can be optimized to modulate signal strength while reducing the size of the target vector cargo

[0149] Currently, viral-based gene delivery methods (e.g., lentivirus- and retrovirus-mediated gene transfers) are commonly used to manufacture CAR-T cells. As the size of the target cargo increases, the transduction efficiency and yield of CAR-T cells decrease. Thus, reducing the size of the target cargo would be beneficial to ensure successful manufacturing. The CACCR recruitment/signaling optimization domain provides a means to achieve this goal for two reasons. First, as with IL2Rb(333-551), cytokine receptor-derived signaling domains can be over 200 amino acids in length and can represent over 650 base pairs in the transfer vector. Second, although tyrosine residues in the signaling domains are important for the initiation and propagation of downstream signaling, some of them may also participate in negative feedback loops that limit the duration and strength of signaling. Thus, truncation of the cytoplasmic tail signaling domain not only allows for a reduction in the size of the vector's target cargo, but also provides the ability to modulate cytoplasmic tail signaling. To this end, we identified tyrosine residues in the full-length tails of IL12Rb2(714-862) and IL2Rb(331-551) and generated variants to identify truncated constructs capable of mediating cytoplasmic tail signaling.

[0150] Full-length IL12Rb2(714-862) contains two phosphorylatable tyrosine residues, Y767 and Y800, which may be involved in downstream signaling. We generated a truncated tail of IL12Rb2(775-825) containing only Y800 and assessed its ability to activate STAT4 using a HEK293T cell reporter gene assay. In general, 20,000 HEK293T cells were seeded in each well of a 96-well poly-L-lysine-coated flat-bottom plate and allowed to attach overnight. The CACCR-CAR construct (2.5 ng), the Stat response element that drives firefly luciferase (100 ng; Promega), and the Renilla luciferase control reporter vector (1 ng; Promega) were mixed in a final volume of 5 μl in Opti-MEM (Gibco) (“DNA mix”). As a negative control, cells were transfected with the BFP-CAR construct, which lacks all cytokine signaling domains. 0.3 μl of Lipofectamine 2000 (Invitrogen) in 5 μl of Opti-MEM was incubated at room temperature for 5 min and then added to the DNA mix. The mix was incubated at room temperature for 20 min and a total volume of 10 μl was added to each well containing HEK-293T. At 48 h post-transfection, Stat4 reporter activity was assessed using the Dual-Glo Luciferase Assay System (Promega). The fold induction of Stat4 reporter activity was normalized to that of HEK293T cells transfected with the control BFP-CAR construct.

[0151] FIG. 13A-B show the identification of the truncated cytoplasmic tail of IL12Rb2(775-825) capable of STAT4 activation comparable to the full-length cytoplasmic tail of IL12Rb2(714-862). FIG. 13A shows a schematic drawing of the full-length tail of IL12Rb(714-862) and the truncated cytoplasmic tail of IL12Rb(775-825). The positions of the tyrosine (Y) residues included in each tail correspond to those indicated. FIG. 13B shows that when fused to the stronger dimerization domain of TpoR(478-582;S505N,W515K), the truncated cytoplasmic tail of IL12Rb2(775-825) fully recapitulated the STAT4 signaling potency of the full-length cytoplasmic tail of IL12Rb2(714-862). When fused to the weaker dimerization domain of TpoR(478-582;H499L,S505N,W515K), the truncated cytoplasmic tail of IL12Rb2(775-825) partially recapitulated the STAT4 signaling potency of the full-length cytoplasmic tail of IL12Rb2(714-862).

[0152] The full-length cytoplasmic tail of IL2Rb(333-551) contains six tyrosine residues that may be involved in downstream signaling. Of these residues, Y364 (the tyrosine residue closest to the trans membrane domain of the receptor) has been reported to activate PI3K to stimulate T cell differentiation and proliferation, as well as cytoskeletal reorganization to induce receptor internalization; thus, although Y364 may stimulate T cell effector functions, it may also limit the strength and duration of IL2Rb signaling. We generated truncated IL2Rb cytoplasmic tails containing three (Y364, Y418, and Y436) of the six tyrosine residues or two (Y418 and Y436) of the six tyrosine residues and assessed their ability to activate STAT5 using a HEK293T cell reporter gene assay. Briefly, 20,000 HEK293T cells were seeded in each well of a 96-well poly-L-lysine-coated flat-bottom plate and allowed to attach overnight. The CACCR-CAR construct (2.5 ng), the Stat response element that drives firefly luciferase (100 ng; Promega), and the Renilla luciferase control reporter vector (1 ng; Promega) were mixed in a final volume of 5 μl in Opti-MEM (Gibco) (“DNA mix”). As a negative control, cells were transfected with a BFP-CAR construct lacking all cytokine signaling domains. 0.3 μl Lipofectamine 2000 (Invitrogen) in 5 μl 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 μl was added to each well containing HEK-293T. Stat5 reporter activity was assessed 48 h post-transfection using the Dual-Glo Luciferase Assay System (Promega). The fold induction of Stat5 reporter activity was normalized to that of HEK293T cells transfected with the BFP-CAR control construct.

[0153] FIG. 14A-B show the identification of truncated IL2Rb tails capable of equal or better STAT5 activation compared to the full-length IL2Rb(333-551) tail. FIG. 14A is a schematic diagram of the full-length IL2Rb(333-551) tail and two truncated IL2Rb tails. The positions of the tyrosine (Y) residues included in each tail are as indicated. The dotted lines represent the connecting regions in the full-length IL2Rb(333-551) tail that were removed from the truncated tails. FIG. 14B shows the results of a reporter gene assay in HEK293T cells in which STAT5 signaling of full-length IL2Rb(333-551) was fully recapitulated by the cytoplasmic tail of TpoR(478-582;H499L,S505N,W515K)IL2Rb(339-379,393-433,518-551), which contained Y364, Y418, and Y536. In the cytoplasmic tail of TpoR(478-582;S505N,W515K).IL2Rb(393-433,518-551), additional deletion of Y364, which mediates receptor internalization, resulted in a highly significant improvement in the strength of STAT5 signaling.

[0154] The HEK293T cell-based assay is a rapid assay that measures STAT5 activity within 48 hours of cytoplasmic tail transfection. Although it provides an efficient platform for screening cytoplasmic tail activity, the limited duration of this assay does not reflect the complexity of the negative feedback loops that are triggered by long-term constitutive cytokine and cytoplasmic tail signaling. To more accurately assess the long-term signaling activity of the IL2Rb reduced tail variants, we produced CACCR CAR-T cells using a 2-week manufacturing process and assessed STAT5 activation using intracellular flow cytometry. To this end, CACCR CAR-T cells were serum starved in 100 μL 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-2 (10 ng/ml; Miltenyi) was added for the last 30 min of a 4-h incubation under serum starvation. After 4 h, an antibody cocktail containing BUV395-conjugated anti-human CD3 antibody (Biolegend) and FITC-conjugated anti-v5 tag monoclonal antibody (Thermo Fisher) was added to the cells and incubation was continued for the last 20 min. Cells were then fixed by adding 35 μl of 16% paraformaldehyde to 100 μl of each sample and incubation was continued for 15 min at 37°C. Cells were then washed three times with PBS and permeabilized in 100% cold methanol for 1 or 2 nights at -20°C. On the day of FACS analysis, cells were washed three times with PBS, blocked with Fc, and stained with A1exaFluor647-conjugated mouse/human Stat5 (pY694) (BD Biosciences) diluted in PBS+1% BSA. After 1 h of incubation at room temperature in the dark, cells were washed three times before FACS analysis.

[0155] FIG. 15 shows STAT5 activation in primary CACCR CAR-T cells bearing full-length or truncated IL2Rb cytoplasmic tails. The greatest STAT5 activation was caused by CACCR CAR-T cells bearing the truncated IL2Rb(393-433,518-551) cytoplasmic tail, which lacked the Y364 internalization motif. Intermediate STAT5 activation was observed in CACCR CAR-T cells bearing the truncated IL2Rb(339-379,393-433,518-551) cytoplasmic tail. STAT5 activation was not observed in CAR-negative populations in the same culture, demonstrating the CAR-T cell-specific nature of cytoplasmic tail signaling. Notably, in CACCR CAR-T cells harboring the full-length IL2Rb(333-551) cytoplasmic tail, STAT5 activity was negligible or undetectable; this may be due to three other tyrosine residues present in the full-length IL2Rb(333-551) cytoplasmic tail that can induce long-term downregulation. Thus, optimization of the cytoplasmic tail signaling domains to eliminate such downregulation motifs is beneficial to ensure long-term maintenance of constitutive and efficient signaling. Since strong cytoplasmic tails can induce strong negative feedback, a weak cytoplasmic tail may be preferable for long-term stimulation.

Example 7: Optimized IL2Rb-derived cytoplasmic tails more closely mimic IL-15 but not IL-2 signaling

[0156] IL-2 and IL-15 are two cytokines that in vivo signal through a heterodimeric cytokine receptor consisting of a common gamma chain and IL2Rb. Despite using the same native receptors, IL-2 and IL-15 have distinct effects on T cell differentiation and persistence. While IL-2 induces the differentiation of short-lived effectors, IL-15 stimulates the generation of long-lived memory T cells. Furthermore, elevated serum IL-15 concentrations have been shown to positively correlate with patient response to CAR T cell therapy. Thus, cytoplasmic tails that mimic IL-15 signaling and effects but not IL-2 are preferred. The present inventors sought to determine whether reduced IL2Rb cytoplasmic tails could more closely mimic IL-2 or IL-15 signaling.

[0157] To this end, the present inventors used CAR-T cells comprising an exemplary CAR carrying a P5A2 scFv directed to BCMA linked to rituximab mimotopes, 4-1BB signaling domains, and CD3z (see US 10294304, incorporated herein by reference). BCMA-specific CAR-T cells co-expressing IL2Rb truncated tails were generated and their gene expression profiles were compared to control CAR-T cells that were exposed to exogenous recombinant human IL-2 or IL-15. To generate lentivirus encoding CACCR and CAR, HEK293T cells were seeded at 0.45 million cells/ml in 2 ml DMEM (Gibco) supplemented with 10% FBS (Hyclone) per well of a 6-well plate the day before transfection. On the day of transfection, lentivirus was generated by mixing 1.5 μg psPAX2 lentiviral packaging vectors, 0.5 μg pMD2G, and 0.5 μg of the appropriate CAR transfer vector in 250 μl Opti-MEM (Gibco) per well of a 6-well plate (“DNA mix”). 10 μl Lipofectamine 2000 (hwitrogen) in 250 μl Opti-MEM was incubated at room temperature for 5 min and then added to the DNA mix. The mixture was incubated at room temperature for 20 min and a total volume of 500 μl was slowly added to the sides of the wells containing HEK293T. One day after transfection, the medium from each well of HEK293T cells in a 6-well plate was replaced with 2 ml per well of T cell transduction media, i.e., X-Vivo-15 supplemented with 10% FBS. Two days after transfection, lentiviral supernatants from HEK293T cells were collected and passed through a 0.45 micron filter (EMD Millipore) to remove cellular debris, and the crude lentiviral supernatants were used directly for T cell transduction. On Day 0, purified T cells were activated in X-Vivo-15 medium (Lonza) supplemented with 100 IU/ml human IL-2 (Miltenyi Biotec), 10% FBS (Hyclone), and human T Trans Act (Miltenyi Biotec, Cat. #130-111-160, 1:100 dilution) in a Grex-24 plate (Wilson Wolf, Cat. #80192M). On Day 2, T cells were resuspended at 0.5 million cells/ml in T cell transduction media and transduced with an equal volume of crude lentiviral supernatant supplemented with 100 IU/ml human IL-2 in a Grex-24 plate. On Day 5, CAR-T cells expressing a cytoplasmic tail were nutrient-replenished by replacing spent media with T cell expansion media, i.e., X-Vivo-15 supplemented with 5% human AB serum (Gemini Bio) with 100 IU/mL human IL-2. At this time, control CAR-T cells lacking cytoplasmic tails were expanded in the presence of either 100 U/mL human IL-2 alone or 100 U/mL human IL-2 and 10 ng/mL human IL-15 (Miltenyi Biotec). When required, cells were expanded in larger G-Rex vessels (Wilson Wolf) using T cell expansion media and appropriate concentrations of recombinant cytokines. On Day 13, cells were stained with the Zombie NIR Fixable Viability Kit (Biolegend), labeled with BUV395-conjugated anti-CD3 antibody (Biolegend) and an anti-idiotype antibody specific for P5A2 scFv, then sorted by FACS to enrich for CAR ⁺ T cells. Sorted CAR ⁺ T cells were then cultured on Grex-24 plates for a further 2 days in T cell expansion media, with CACCR CAR ⁺ T cells starved of exogenous cytokines and sorted control CAR ⁺ T cells starved of exogenous cytokines, treated with 100 U/ml human IL-2, or treated with 10 ng/ml human IL-15. On Day 15, live CAR ⁺ T cells were enriched using the Easy Sep™ Dead Cell Removal Kit (StemCell Technologies) and cell pellets were snap-frozen for subsequent RNA extraction and gene expression analysis by NanoString (human CAR T cell panel; NanoString Technologies).

[0158] The data show that CACCR CAR-T cells bearing truncated IL2Rb tails more closely mimic IL-15 signaling, but not IL-2 signaling. As an example, we tested the cytoplasmic tails of TpoR(478-582;S505N,W515K).IL2Rb(393-433,518-551) and TpoR(478-582;H499L,S505N,W515K).IL2Rb(339-379,393-433,518-551). FIG. 16A is a schematic drawing of the experimental design and workflow for sample preparation. FIG. 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. FIG. 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) for each sample was calculated by normalizing to control CAR-T cells that were left untreated from Days 13-15. ^R2 values and the line of best fit (solid line) determined by linear regression analysis are shown in each graph. The data shown are from one representative of two donors. Although the gene expression profiles of CACCR CAR-T cells showed no correlation with IL-2-treated samples (FIG. 16B), they were positively correlated with IL-15-treated samples (FIG. 16C), suggesting that cytoplasmic tails bearing truncated IL2Rb tails more closely mimic the downstream signaling and transcriptional responses of IL-15, but not IL-2.

Example 9: Constitutive cytoplasmic tails can be programmed for combinatorial outputs

[0159] As shown in FIG. 12, CACCRs bearing signaling domains derived from different cytokine receptors can activate signaling similar to that of the parent receptor. The present inventors hypothesized that cytoplasmic tails could be engineered to mimic simultaneous signaling from multiple parent receptors by fusing more than one cytoplasmic tail in tandem, thereby achieving combinatorial signaling outputs. To test combinatorial signaling outputs from tandem cytoplasmic tails, the inventors generated a constitutive 7.12 tail by fusing the IL7Ra(316-459) cytoplasmic tail with the truncated IL12Rb2(775-825) cytoplasmic tail and assessed signaling using a HEK293T cell reporter gene assay.

[0160] FIGS. 17A-D show the design and signaling capacity of constitutive tandem cytoplasmic tails exemplified by the 7.12-tail. FIG. 17A is a schematic diagram of a constitutive 7.12-tail. FIG. 17B is a schematic diagram of lentiviral vectors differing only in their TpoR(478-582) dimerization/JAK binding domains used to coexpress 7.12-tail and CAR variants. FIG. 17C-D show STAT reporter activity for constructs bearing TpoR(478-582;S505N;W515K) and TpoR(478-582;H499L;S505N;W515K) dimerization JAK-binding domains, respectively, fused to the indicated cytoplasmic tails. While the IL7Ra(316-459) cytoplasmic tail potently activated STAT5, the IL12Rb2(775-825) cytoplasmic tail potently activated STAT4. However, consistent with the observations for the IL7Ra(316-459).IL12Rb2(775-825) cytoplasmic tail, fusion of both cytoplasmic tails in tandem resulted in simultaneous and combinatorial activation of both STAT5 and STAT4. This demonstrates that multiple signaling pathways that would normally require two or more different native cytokine receptors can be achieved via a single cytoplasmic tail.

Example 10: Constitutive cytoplasmic tails can be tailored with one or more output signals to control CAR-T cell phenotype and function

[0161] The present inventors then determined whether constitutive cytoplasmic tails carrying different output signals could differentially influence the phenotype and function of primary human CAR-T cells. Unlike IL-7, which drives T cell survival and memory cell maintenance, IL-12 is a proinflammatory cytokine that stimulates T cell differentiation. Therefore, the present inventors sought to investigate whether signaling through IL7Ra- or IL12Rb-derived cytoplasmic tails could differentially drive these divergent phenotypes and to assess the overall combinatorial effect of fusing both tails in tandem.

[0162] To this end, we generated primary human CAR-T cells that co-expressed either the 7-tail (i.e., IL7Ra(316-459)) or 12-tail variants (i.e., IL12Rb2(775-825) or IL12Rb2(714-862)). See FIG. 13A. To generate lentivirus encoding CACCR and CAR, HEK293T cells were seeded at 0.45 million cells/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 1.5 μg psPAX2 lentiviral packaging vectors, 0.5 μg pMD2G, and 0.5 μg of the appropriate CAR transfer vector in 250 μl Opti-MEM (Gibco) per well of a 6-well plate (“DNA mix”). 10 μl Lipofectamine 2000 (Invitrogen) in 250 μl Opti-MEM was incubated at room temperature for 5 min and then added to the DNA mix. The mix was incubated at room temperature for 20 min and a total volume of 500 μl was slowly added to the sides of the wells containing HEK293T. One day after transfection, the media from each well of HEK293T cells in a 6-well plate was replaced with 2 ml per well of T cell transduction media, i.e., X-Vivo-15 supplemented with 10% FBS. Two days after transfection, lentiviral supernatants from HEK293T cells were collected and passed through a 0.45 μm filter (EMD Millipore) to remove cell debris, and the crude lentiviral supernatants were used directly for T cell transduction. On Day 0, purified T cells were activated in X-Vivo-15 medium (Lonza) supplemented with 100 IU/ml human IL-2 (Miltenyi Biotec), 10% FBS (Hyclone), and human T Trans Act (Miltenyi Biotec, Cat. #130-111-160, 1:100 dilution) in a Grex-24 plate (Wilson Wolf, Cat. #80192M). On Day 2, T cells were resuspended at 0.5 million cells/ml in T cell transduction media and transduced with an equal volume of crude lentiviral supernatant supplemented with 100 IU/ml human IL-2 in a Grex-24 plate. On Day 5, cells were nutrient-replenished by replacing spent media with T cell expansion media, i.e., X-Vivo-15 supplemented with 5% human AB serum (Gemini Bio) with 100 IU/ml human IL-2. When necessary, cells were expanded in larger G-Rex flasks (Wilson Wolf) using T cell expansion media and appropriate concentrations of human IL-2. On Day 14, memory phenotyping of CAR T cell products was performed by detecting CAR-transduced cells using FITC-conjugated monoclonal antibody to the v5 tag (Thermo Fisher) co-staining with PE/Cy7-conjugated anti-CD62L (Biolegend) and BV785-conjugated anti-CD45RO (Biolegend) by flow cytometry. As negative controls lacking CACCR signaling, CAR-T cells coexpressing BFP or the wild-type TpoR(478-582) transmembrane domain linked to the 7-tail were simultaneously generated.

[0163] FIG. 18 depicts the effect of cytoplasmic tails on the differentiation into memory cells of CAR-T cell products. The memory cell phenotype of CACCR CAR-T cell products obtained from 2 healthy donors is shown at Day 14. While CAR-T cells co-expressing the IL7Ra(316-459) cytoplasmic tail maintained the memory stem cell (Tscm) population, CAR-T cells bearing IL12Rb2-derived cytoplasmic tails showed a marked decrease in the Tscm population accompanied by an increase in the central memory (Tcm) population. These results suggest that in the absence of CAR engagement, constitutive IL12-like signaling via IL12Rb2-derived cytoplasmic tails drives progressive differentiation from Tscm to Tcm. Furthermore, CAR-T cells co-expressing the tandem cytoplasmic tail of IL7Ra(316-459).IL12Rb2(775-825) showed reconstitution of the Tscm population and mimicked the phenotype of CAR-T cells bearing the single cytoplasmic tail of IL7Ra(316-459), suggesting that the negative effects of single cytoplasmic tails can be ameliorated by combinatorial signaling of tandem cytoplasmic tails.

[0164] The present inventors next examined whether CACCR signaling could drive functional endpoints of CAR-T cells, including survival and cytotoxicity. In contrast, IL-7 signaling, which drives long-term survival of T cells, drives differentiation into short-lived end effectors. In support of this, the Tscm population, which is capable of long-term survival and is thought to mediate long-term persistence of CAR-T cells, was deficient in CAR-T cells bearing IL12Rb2-derived tails. To analyze whether signaling through different cytoplasmic tails can program CAR-T cell survival and differentiation, we performed a growth factor-independent assay in which CACCR-coexpressing CAR-T cells were cultured in the absence of target cells or exogenously supplemented cytokines. The number and differentiation of CAR-T cells into memory cells under these conditions was monitored over time.

[0165] In general, cryopreserved CAR-T cells were thawed, counted, and the percentage of CAR-T cells in all samples was normalized to the sample with the lowest transduction efficiency by adding non-transduced (NTD) T cells. CAR-T cells co-expressing BFP (BFP-CAR) in place of the cytoplasmic tail were used as a control. 0.25× ¹⁰⁶ CAR ⁺ T cells/mL were added to 1.5 mL of RPMI containing 10% FBS (Hyclone), nonessential amino acids, sodium pyruvate, and 20-25 mM HEPES con, then seeded into 24-well tissue culture plates. On the indicated days for each condition, duplicate 100 μl samples were collected and stained using the Zombie NIR Fixable Viability Kit (Biolegend). Samples were washed with PBS, blocked with Fc, then stained with the following antibody mixture diluted in PBS + 1% BSA: BUV395-conjugated anti-human CD3, BV510-conjugated anti-human CD8, BV605-conjugated anti-human CD4, and FITC-conjugated anti-v5 (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 µl PBS + 1% BSA containing 123count eBeads (Thermo Fisher) (10 µl counting beads in 120 µl PBS + 1% BSA) prior to FACS analysis.

[0166] FIGS. 19A-B show representative data for cells from 2 donors. FIGS. 19A and 19B show the fold expansion of CAR-T cells relative to the input amount at the start of the assay (Day 0) for constructs bearing the TpoR(478-582;S505NiW515K) and TpoR(478-582;H499b;8505^W515K) dimerization/JAK binding domains, respectively, fused to the indicated cytoplasmic tails. Compared with control BFP CAR-T cells, the number of CAR-T cells bearing the IL7Ra(316-459) cytoplasmic tail declined more slowly, suggesting that constitutive signaling through the IL7Ra(316-459) cytoplasmic tail improved CAR-T cell survival. In contrast, the number of CAR-T cells bearing IL12Rb2-derived cytoplasmic tails declined at a rate more comparable to that of BFP CAR-T cells, suggesting no survival advantage. Notably, CAR-T cells bearing the tandem IL7Ra(316-459).IL12Rb2(775-825) cytoplasmic tail conferred a survival advantage more comparable to that of the IL7Ra(316-459) cytoplasmic tail. FIGS. 19C-D show CAR-T cell differentiation at Day 7 in a growth factor-independent assay for constructs bearing the TpoR(478-582;S505N;W515K) and TpoR(478-582;H499L;S505N;W515K) dimerization/JAK binding domains, respectively, fused to the indicated cytoplasmic tails. Compared with control BFP CAR-T cells and CAR-T cells bearing IL12Rb2-derived cytoplasmic tails, CAR-T cells bearing the IL7Ra(316-459) cytoplasmic tail were not only more numerous but also more enriched in the Tscm population. See FIG. 19C-D. CAR-T cells bearing the tandem IL7Ra(316-459) IL12Rb2(775-825) cytoplasmic tail had improved Tscm enrichment. Without being bound by any specific mechanisms, the results suggest that fusion with the IL7Ra(316-459) cytoplasmic tail occluded the phenotype of the single IL12Rb2(775-825) cytoplasmic tail. Taken together, these data support combinatorial functional effects of tandem cytoplasmic tail signaling and demonstrate the tunability of different cytoplasmic tail constructs.

[0167] CAR-T cell products enriched in the Tscm population are associated with improved expansion, persistence, and potency. However, terminally differentiated CAR-T cells are short-lived and have limited proliferative potential, resulting in decreased efficacy. Given that the cytoplasmic tails derived from IL7Ra(316-459) and IL12Rb2 differentially impacted these characteristics, we assessed the ability of these cytoplasmic tails to impact CAR-T cell cytotoxicity.

[0168] For this purpose, 5000 U87KO-EGFRvIII-nucGFP target cells were seeded and allowed to adhere in 96-well black-walled, flat-bottomed plates in 50 µl RPMI containing 10% FBS (Hyclone), nonessential amino acids, sodium pyruvate, and 20-25 mM HEPES. EGFRvIII CAR (2173 scFv) T cells bearing IL7Ra(316-459)- or IL12Rb-derived cytoplasmic tails were thawed and added to the seeded target cells at an effector:target (E:T) ratio of 1:3. Because target cells outnumber CAR-T cells, control of target cell expansion may require repeated or serial killing of CAR-T cells. CAR-T cells coexpressing the BFP CAR in place of the cytoplasmic tail were used as controls. Live target cell numbers were monitored over time using the IncuCyte Live Cell Imaging Assay.

[0169] FIGS. 20A-B depict the cytotoxic activity of CAR-T cells co-expressing various CACCRs. FIGS. 20A-B show the killing of CACCR target cells by CAR-T cells bearing the TpoR(478-582;S505N;W515K) and TpoR(478-582;H499L;S505N;W515K) dimerization/JAK binding domains, respectively, fused to the indicated cytoplasmic tails. Compared to BFP CAR-T cells, CAR-T cells bearing IL12Rb2-derived cytoplasmic tails exhibited little or no improvement in serial cytolytic activity in vitro, likely due to their limited proliferative potential and short lifespan. In contrast, CAR-T cells bearing the IL7Ra(316-459) cytoplasmic tail exhibited enhanced serial cytolytic activity, likely due to enhanced proliferation and persistence. Notably, CAR-T cells bearing the tandem IL7Ra(316-459) cytoplasmic tail. IL12Rb2(775-825) cytoplasmic tail exhibited equal or better serial cytolytic activity than the IL7Ra(316-459) cytoplasmic tail, suggesting that combining the persistence-enhancing IL7Ra(316-459) signaling domain and the effector differentiation-enhancing IL12Rb2(775-825) signaling domain in a single cytoplasmic tail may simultaneously enhance CAR-T cell survival, proliferation, and immediate effector functions.

Example 11: Constitutive cytokine receptors enhance the in vitro cytotoxicity of CARs targeting hematologic malignancies

[0170] We have demonstrated that CACCRs can enhance the activity of CARs directed to EGFRvIII, a solid tumor target such as glioblastoma. To determine whether CACCRs can be broadly applied across different scFvs for a hematological malignancy target, we further cloned CACCRs into a CAR construct directed to a hematological malignancy marker (i.e., BCMA) and assessed long-term cytotoxicity against a BCMA-positive target cell line.

[0171] Target cells stably expressing firefly luciferase and GFP reporters were generated by lentiviral transduction. 10,000 Luc-GFP-labeled target cells were seeded in 100 μl per well in a white flat-bottomed 96-well tissue culture plate. Cryopreserved CAR-T cells were thawed, counted, and the percentage of CAR-T cells in all samples was normalized to the sample with the lowest transduction efficiency by adding non-transduced (NTD) T cells. CAR-T cells were then added to each well of target cells in a volume of 100 μl at the indicated effector:target (E:T) ratios in triplicate. As a negative control, “target only” wells were supplemented with 100 μl of media instead of T cells. After two or three days, the well contents were mixed by gentle pipetting and 100 μl from each well containing T cells was transferred to a new white flat-bottomed 96-well tissue culture plate containing 10,000 freshly seeded Luc-GFP-labeled target cells in 100 μl. Fresh media were added to the “target only” wells instead of T cells. The new plate was incubated at 37°C while the number of live target cells remaining in the old 96-well plate was determined using the ONE-Glo Luciferase Assay System (Promega) according to the manufacturer’s instructions. The percentage of viable target cells was calculated by normalizing the luciferase signal to that from the "target only" wells, and the percentage of cytotoxicity was calculated as 100% - % viable target cells. Serial transfers to fresh target cells and luciferase readings were performed every two or three days until cytotoxic activity ceased.

[0172] FIG. 21 shows that CACCRs improved the cytotoxic activity of CAR-T cells directed against BCMA, a hematologic malignancy target. FIG. 21 shows the cytotoxicity of BCMA CAR (P5A2 scFv) against the multiple myeloma cell line MM1.S at an E:M ratio of 10:1, indicating that co-expression of CACCRs increased the long-term cytotoxicity of CAR-T cells.

Example 12: CACCRs enhance CAR-T cell activity in vivo

[0173] CAR-T cell-based therapeutics, such as those targeting CD 19 and BCMA, have achieved unprecedented clinical success in the treatment of hematological malignancies. Although high complete response rates have been achieved, these are short-lived, as most patients eventually relapse. Additionally, CAR-T cells have achieved more limited success in the treatment of solid tumors. 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 immunosuppressive microenvironments. Because our in vitro characterization of CACCR CAR-T cells revealed improvements in target-driven proliferation, persistence, activity, and exhaustion profiles, we next examined whether these functional improvements translate into improved antitumor activity in vivo.

[0174] To examine the in vivo activity of CACCR CAR-T cells in hematologic malignancies, we used CAR-T cells expressing a BCMA-specific P5A2 scFv linked to the 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 using transcription activator-like effector nuclease (TALEN)-mediated gene silencing to avoid potential interference from TCR-driven xenoreactivity. Female 8-10 week-old NSG mice were irradiated at 1 Gy one day prior to intravenous inoculation with 5×10 ⁶ MM1.S-Luc-GFP. Fourteen days after tumor implantation, mice were randomized based on tumor burden and injected intravenously with 1× ¹⁰⁶ or 3× ¹⁰⁶ of the indicated CAR-T cells (n=10 per group). Tumor progression was monitored by bioluminescence imaging. On day 30 after T cell dosing, mice receiving 3× ¹⁰⁶ CAR-T cells had blood drawn to enumerate peripheral BCMA CAR-T cells. Specifically, 50 μl of whole blood from each mouse was subjected to red blood cell lysis using ASC Lysis Buffer (Gibco), Fc blocked, and stained with the following antibody mixture diluted in PBS + 1% BSA: FITC-conjugated anti-mouse CD45 (Biolegend), BV21-conjugated anti-human CD45 (Biolegend), and an anti-idiotype antibody specific for P5A2 scFv. Finally, samples were washed in PBS and cell pellets were resuspended in 130 μl PBS + 1% BSA containing 123count eBeads (Thermo Fisher) (10 μl counting beads in 120 μl PBS + 1% BSA) prior to FACS analysis.

[0175] FIGS. 22A-C show that CACCR improved the antitumor activity and persistence of BCMA CAR-T cells against orthotopic multiple myeloma in vivo. FIGS. 24A-B show tumor progression in response to treatment with 1×10 ⁶ or 3×10 ⁶ of the indicated CAR-T cells, respectively. Although control BCMA CAR-T cells were able to mediate initial tumor regression, this response was short-lived as tumors relapsed 22 days after cell infusion. However, CACCR-coexpressing CAR-T cells significantly delayed tumor relapse and improved the persistence of the response. Statistics in FIG. 24A-B represent **p<0.01 and ***p<0.001 based on one-way repeated measures ANOVA with Tukey's test for multiple comparisons for Days 6-34 for FIG. 22A and Days 6-44 for FIG. 22B. FIG. 22C shows the number of BCMA CAR-T cells present in the peripheral blood of mice that received 3× ¹⁰⁶ CAR-T cells 30 days after T cell infusion. Concomitant with the tumor recurrence observed in mice that received 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 better at preventing tumor recurrence, were also more numerous in vivo. Statistics in FIG. 24C represents *p<0.05 and ****p<0.0001 based on ordinary one-way ANOVA with Tukey's test for multiple comparisons, suggesting that improved CACCR CAR-T cell persistence partially mediated improved long-term tumor control and prolonged response durability.

[0176] We further assessed the impact of CACCR on CAR-T cell activity in solid tumors known to be resistant to CAR-T cell therapy, such as glioblastoma. To this end, we used an EGFRvIII-specific CAR carrying 2173 scFv linked to the 4-1BB and CD3ζ signaling domains and the human glioblastoma cell line LN229 stably overexpressing EGFRvIII (LN229-EGFRvIII). Female NSG mice aged 8-10 weeks were implanted subcutaneously with 3×10 ⁶ LN229-EGFRvIII. After 25 days, when tumors had developed, mice were randomized based on tumor burden and injected intravenously with 1.5× ¹⁰⁶ or 3× ¹⁰⁶ of the indicated CAR-T cells (n=8-10 per group). Tumor progression was monitored twice weekly using caliper measurements.

[0177] FIG. 23 shows that CACCR improved the antitumor activity of CAR-T cells against established solid tumors. Although treatment with control EGFRvIII CAR-T cells was able to slow the growth of LN229-EGFRvIII tumors, the response was short-lived and suboptimal as the tumors eventually progressed. In contrast, treatment with CACCR CAR-T cells resulted in complete tumor regression. Notably, even a low dose of 1.5 x 10 ⁶ CACCR CAR-T cells was sufficient to eliminate the tumor. Statistics represent **p < 0.01 compared to treatment with 3 x 10 ⁶ control CAR-T cells based on a one-way repeated measures ANOVA with Tukey's test for multiple comparisons for Days 3-38. These results confirm the ability of CACCR to act synergistically without excessive effects with signaling domains in CAR-T cells to provide enhanced activity.

Claims (49)

1. A constitutively active chimeric cytokine receptor (CACCR) for enhancing the cytotoxicity of an engineered immune cell, wherein the CACCR is composed of two monomers, each monomer containing: a. the transmembrane domain of the thrombopoietin receptor (TpoR) comprising amino acids 478-582 of SEQ ID NO: 6 with the amino acid substitutions S505N and W515K according to SEQ ID NO: 6; b. Janus kinase (JAK) binding domain; and c. intracellular STAT recruitment domain, in this case, the indicated monomers are constitutively dimerized. 2. CACCR according to claim 1, characterized in that said JAK-binding domain comprises a JAK1-binding domain. 3. CACCR according to claim 1, characterized in that said JAK-binding domain comprises a JAK2-binding domain. 4. CACCR according to claim 1, characterized in that said JAK-binding domain comprises a JAK3-binding domain. 5. CACCR according to claim 1, characterized in that said JAK-binding domain comprises a TYK2-binding domain. 6. CACCR according to any one of claims 1-5, characterized in that said JAK-binding domain comprises one of the transmembrane amino acid sequences selected from SEQ ID NO: 12 and 13. 7. A CACCR according to any one of claims 1 to 6, wherein said STAT recruitment domain comprises a STAT recruitment domain selected from the recruitment domain of STAT-1, STAT-2, STAT-3, STAT-4, STAT-5, STAT-6 or STAT-7 of at least one receptor. 8. A CACCR according to any one of claims 1 to 7, wherein the STAT recruitment domain comprises more than one of the STAT-1, STAT-2, STAT-3, STAT-4, STAT-5, STAT-6 or STAT-7 recruitment domains from at least one receptor. 9. CACCR according to any one of claims 1-7, characterized in that said transmembrane domain and JAK-binding domain are derived from the TPOR/MPLR receptor. 10. The CACCR of claim 1, wherein the monomers are capable of (i) spontaneous dimerization or (ii) can activate signaling in the absence of any exogenous stimulation or ligand. 11. CACCR according to claim 1, characterized in that said TPOR/MPLR transmembrane domain additionally contains the amino acid substitution H499L. 12. CACCR according to claim 1, characterized in that said TPOR/MPLR transmembrane domain additionally contains the amino acid substitution G509N. 13. CACCR according to claim 1, characterized in that said TPOR/MPLR transmembrane domain additionally contains the amino acid substitutions H499L and G509N. 14. CACCR according to any one of paragraphs 11-13, characterized in that the said monomers are identical. 15. CACCR according to any one of paragraphs 11-14, characterized in that said monomers are different from each other. 16. A CACCR according to any one of claims 1-15, wherein said STAT recruitment domain comprises a STAT recruitment domain from a cytokine receptor. 17. The CACCR of any one of claims 1 to 15, wherein said 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, IL21R, IL2Rb, IL2small, IL7R, IL7Ra, IL9R, IL15R and IL21R. 18. The CACCR of any one of claims 1-15, wherein said STAT recruitment domain comprises an amino acid sequence of one or more receptor sequences selected from the group consisting of SEQ ID NOs: 46-88, 106 and 143. 19. The CACCR according to any one of claims 1-18, wherein said STAT recruitment domain comprises the STAT recruitment domain of IL7Ra. 20. CACCR according to claim 19, characterized in that said STAT recruitment domain from IL7Ra comprises SEQ ID NO: 69. 21. CACCR according to any one of claims 1-18, characterized in that said STAT recruitment domain comprises a STAT recruitment domain from IL2Rb. 22. CACCR according to claim 21, characterized in that said IL2Rb contains a STAT recruitment domain containing the STAT recruitment domain of IL7Ra. 23. The CACCR according to any one of claims 1-18, wherein said STAT recruitment domain comprises the STAT recruitment domain of IL12Rb2. 24. CACCR according to claim 23, characterized in that said STAT recruitment domain from IL12Rb2 comprises SEQ ID NO: 86 or 67. 25. The CACCR according to any one of claims 1-24, characterized in that said STAT recruitment domain comprises STAT recruitment domains from two receptors. 26. The CACCR according to any one of claims 1-25, characterized in that said STAT recruitment domain comprises STAT recruitment domains from two cytokine receptors. 27. The CACCR of claim 26, wherein said two cytokine receptors are selected from the group consisting of IL7Ra, IL2Rb and IL12Rb2. 28. A polynucleotide encoding a CACCR according to any of paragraphs 6 or 18. 29. An expression vector containing a polynucleotide according to claim 28. 30. An expression vector according to claim 29, comprising a polynucleotide according to claim 28 and a polynucleotide expressing a chimeric antigen receptor (CAR). 31. The expression vector of claim 30, wherein said 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 (Drosophila delta-like protein 3, delta homolog 3, Delta3), Muc17 (mucin17, Muc3, Muc3), FAP alpha (fibroblast activation protein alpha), Ly6G6D (lymphocyte antigen complex 6 G6d locus protein, c6orf23, G6D, MEGT1, NG25) and/or RNF43 (RNF43 ubiquitin ligase (E3), RING finger protein 43). 32. An expression vector according to any one of paragraphs 29-31, characterized in that said vector is a lentiviral vector. 33. An engineered T cell having enhanced cytotoxicity, wherein the cell comprises an expression vector according to any one of paragraphs 29-32. 34. An engineered T cell having enhanced cytotoxicity, wherein the cell comprises a chimeric antigen receptor (CAR) and at least one CACCR according to any one of claims 1-27. 35. The engineered T cell of claim 34, wherein said CAR and CACCR are expressed in stoichiometrically equal amounts. 36. An engineered T cell according to any one of claims 33-35, characterized in that the said 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 (Drosophila delta-like protein 3, delta homolog 3, Delta3), Muc17 (mucin17, Muc3, Muc3), FAP alpha (fibroblast activation protein alpha), Ly6G6D (lymphocyte antigen complex 6 G6d locus protein, c6orf23, G6D, MEGT1, NG25) and/or RNF43 (RNF43 ubiquitin ligase (E3), RING finger protein 43). 37. An engineered T cell according to any one of paragraphs 33-36, characterized in that said T cell is an allogeneic T cell. 38. An engineered T cell according to any one of paragraphs 33-36, characterized in that said T cell is an autologous T cell. 39. A method for producing an engineered T cell, characterized in that said method includes introducing a polynucleotide according to claim 28 or an expression vector according to any of claims 29-32 into the T cell. 40. The method according to claim 39, characterized in that said T-cell is a T-cell derived from a stem cell. 41. A pharmaceutical composition for treating cancer in a subject comprising engineered T cells according to any one of claims 33-38. 42. A kit for treating cancer in a subject comprising engineered T cells according to any one of claims 33-38 or a pharmaceutical composition according to claim 41. 43. Use of a therapeutically effective amount of engineered T cells according to any one of claims 33-38 or a pharmaceutical composition according to claim 41 for treating cancer in a subject. 44. The use according to paragraph 43, characterized in that said cancer includes a solid tumor. 45. The use according to paragraph 43, characterized in that said cancer includes hemoblastosis.