Inducible cytokine transgenes to enhance immune cell function

Abstract

[0003] Artificial expression constructs are described that include a cytokine transgene under the control of an inducible promoter. The artificial expression constructs of the present disclosure can be used to enhance the function of immune cells (e.g., CAR-T cells) bearing the recombinant receptor. The cytokine transgenes disclosed herein enhance the efficacy of immune cells (e.g., CAR-T cells) bearing the recombinant receptor, enhancing killing, immune cell proliferation, and / or cytokine production.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 482,270, filed January 30, 2023, the contents of which are incorporated herein by reference in their entirety as if set forth herein.

[0002] Sequence Listing Reference The Sequence Listing accompanying this application is provided in XML format rather than in hard copy, and is incorporated herein by reference. The XML file containing this Sequence Listing is named S281-0048PCT.xml. This file is 182 KB in size, was created on January 29, 2024, and was submitted electronically via the Patent Center.

[0003] The present disclosure provides artificial expression constructs comprising a cytokine transgene under the control of an inducible promoter for enhancing the function of immune cells (e.g., CAR-T cells). The cytokine transgenes disclosed herein enhance immune cell function by enhancing (i) target cell killing by the immune cell, (ii) immune cell proliferation, and / or (iii) cytokine production within the immune cell's environment. [Background technology]

[0004] According to the World Health Organization, cancer is one of the leading causes of death worldwide, with cancer deaths reaching nearly 10 million in 2020.

[0005] For many years, the treatments of choice for cancer have been surgery, chemotherapy, and / or radiation therapy. Recently, more targeted therapies have been developed, primarily by identifying and exploiting specific molecular and / or immunophenotypic changes found in cancer cells. For example, many cancer cells selectively express certain markers on their cell surface, and these markers have been used as targets for antibody-based therapies.

[0006] Great advances have been made in genetically engineering immune system cells, enabling them to target and kill unwanted cells, such as cancer cells. Many of these immune cells are T cells engineered to express recombinant receptors, such as chimeric antigen receptors (CARs). CARs are proteins containing several distinctive components that enable engineered T cells to recognize and kill targeted cell types. These components include at least an extracellular portion and an intracellular portion, which are either expressed as a single protein or assembled into a functional unit. The extracellular portion contains a binding domain that specifically binds to a marker (e.g., an antigen) present selectively on the surface of unwanted cells. Binding of the binding domain to such a marker transmits a signal from the intracellular portion to the T cell, instructing it to destroy the bound cell. CARs can also contain a transmembrane domain that can link the extracellular portion to the intracellular portion.

[0007] Other components that can enhance the function of the CAR can also be used. For example, a spacer can often enhance the binding ability of the binding domain to the target cell marker by providing additional flexibility to the CAR's three-dimensional structure, thereby enhancing cytolytic activity. The appropriate length of the spacer for a particular CAR depends on many factors, including how close or far the target marker is located from the surface of the cell membrane of undesired cells.

[0008] Although CAR T cells have been highly successful in treating various cancers, challenges remain. For example, CAR T cells can exhibit suboptimal proliferation, functional unresponsiveness or exhaustion, impaired memory differentiation, and / or limited persistence, limiting their ability to induce or maintain remission in patients. Therefore, strategies to improve the efficacy of CAR T cells are needed, especially in situations involving prolonged antigen exposure, which can lead to functional unresponsiveness and exhaustion. Summary of the Invention [Means for solving the problem]

[0009] The present disclosure provides artificial expression constructs comprising a cytokine transgene under the control of an inducible promoter for enhancing the function of immune cells (e.g., T cells or chimeric antigen receptor (CAR)-T cells). The inducible cytokine transgenes disclosed herein enhance the efficacy of immune cells (e.g., T cells or CAR-T cells) to enhance killing, immune cell proliferation, and / or cytokine production. In certain embodiments, the cytokine transgene encodes a soluble cytokine. In certain embodiments, the cytokine transgene encodes transforming growth factor beta 2-7m (TGFβ2-7m), mini TGFβ2-7m*, interleukin 21 (IL21), interleukin 15 (IL15), single-chain interleukin 12 (scIL12), decoy-resistant interleukin 18 (DR-IL18), or interleukin 36γ (IL36γ).

[0010] In certain embodiments, inducible expression of TGFβ2-7m, mTGFβ2-7m*, IL21, IL15, scIL12, DR-IL18, or IL36γ is used to enhance immune cell-mediated cell killing.

[0011] In certain embodiments, inducible expression of TGFβ2-7m, mTGFβ2-7m*, IL21, IL15, scIL12, DR-IL18, or IL36γ is used to enhance T cell proliferation.

[0012] In certain embodiments, inducible expression of TGFβ2-7m, mTGFβ2-7m*, IL21, IL15, scIL12, DR-IL18, or IL36γ is used to enhance cytokine production, hi certain embodiments, cytokine production includes IFNγ production and / or TNFα production.

[0013] In certain embodiments, inducible expression of IL21 is used to enhance immune cell-mediated cell killing, hi certain embodiments, inducible expression of IL15 is used to enhance immune cell-mediated cell killing.

[0014] In certain embodiments, inducible expression of IL21 is used to enhance immune cell proliferation, hi certain embodiments, inducible expression of IL15 is used to enhance immune cell proliferation.

[0015] In certain embodiments, inducible expression of scIL12 is used to enhance cytokine production. In certain embodiments, inducible expression of DR-IL18 is used to enhance cytokine production. In certain embodiments, inducible expression of IL36γ is used to enhance cytokine production. In certain embodiments, inducible expression of TGFβ2-7m or mTGFβ2-7m* is used to enhance cytokine production.

[0016] In certain embodiments, inducible expression of scIL12 is used to enhance IFNγ production. In certain embodiments, inducible expression of DR-IL18 is used to enhance IFNγ production. In certain embodiments, inducible expression of IL36γ is used to enhance IFNγ production. In certain embodiments, inducible expression of TGFβ2-7m or mTGFβ2-7m* is used to enhance IFNγ production.

[0017] In certain embodiments, inducible expression of scIL12 is used to enhance TNFα production. In certain embodiments, inducible expression of DR-IL18 is used to enhance TNFα production. In certain embodiments, inducible expression of IL36γ is used to enhance TNFα production.

[0018] In certain embodiments, a combination of inducible expression of IL21 and inducible expression of IL15, scIL12, DR-IL18, IL36γ, TGFβ2-7m or mTGFβ2-7m* is utilized.

[0019] In certain embodiments, a combination of inducible expression of IL15 and inducible expression of IL21, scIL12, DR-IL18, IL36γ, TGFβ2-7m or mTGFβ2-7m* is utilized.

[0020] In certain embodiments, expression of the cytokine transgene is under the control of the iSynPro promoter. In certain embodiments, the iSynPro promoter comprises S1-61 (SEQ ID NO: 8). In certain embodiments, the iSynPro promoter comprises S1-61 (SEQ ID NO: 8) operably linked to the IL2 minimal promoter (SEQ ID NO: 138). In certain embodiments, the artificial expression construct comprising the cytokine transgene further comprises or encodes a skip sequence and a regulatory mechanism.

[0021] In certain embodiments, the immune cells comprise a T cell receptor. In certain embodiments, the immune cells can be engineered to express a recombinant receptor. In certain embodiments, the artificial expression construct expressing the recombinant receptor may be included in an artificial expression construct comprising a cytokine transgene, or may be included in a separate artificial expression construct from the artificial expression construct comprising the cytokine transgene. In certain embodiments, the artificial expression construct comprising the recombinant receptor comprises a promoter and the recombinant receptor, or encodes a promoter and the recombinant receptor. In certain embodiments, the artificial expression construct comprising the recombinant receptor comprises a promoter, the recombinant receptor, a skip sequence, and a control mechanism. In certain embodiments, the recombinant receptor comprises a CAR or a recombinant TCR (eTCR). In certain embodiments, the recombinant receptor comprises an extracellular portion that binds to a target antigen, a transmembrane domain, and an intracellular portion.

[0022] Thus, the present disclosure provides a system for enhancing immune cell killing, proliferation, and / or cytokine production. The immune cells of the present disclosure may include immune cells having recombinant receptors that can be engineered to target various target cells (e.g., cancer cells, viruses, bacteria, fungi) that express a target antigen by engineering the extracellular domain to include a binding domain that binds to the target antigen. [Brief explanation of the drawings]

[0023] Some of the drawings submitted in this application may be more easily understood in color, and applicants hereby include color versions of these drawings as part of the original application and reserve the right to submit color images of such drawings in subsequent proceedings.

[0024] [Figure 1] We present an example of an artificial expression construct used as a template to screen for putative potent enhancers for enhancing target cell killing, immune cell proliferation, and cytokine production by chimeric antigen receptor (CAR) T cells.

[0025] [Figure 2] This section shows the structure of constructs for facilitating T cell production and screening. To verify that T cells expressing a single soluble cytokine transgene, including transforming growth factor β2-7m (TGFβ2-7m), mini TGFβ2-7m* (mTGFβ2-7m*), interleukin (IL) 21, IL-15, single-chain IL12, decoy-resistant (DR)-IL18, and IL36γ, are potent enhancers of CAR T cells, the open reading frame (ORF) of each cytokine transgene was placed under the control of the inducible CAR promoter, iSynPro, and arranged in a dual-promoter construct format. The orientation of the ORFs for Her2tG and the soluble cytokine transgenes was reversed to ensure no trace of 2A was present in the soluble cytokine transgenes. Downstream of this cargo were the EF1a(L) promoter, which drives the expression of CAR (CD19CAR), double-mutated dihydrofolate reductase (DHFRdm), and truncated epidermal growth factor receptor (EGFRt). CAR and DHFRdm were separated by P2A, and DHFRdm and EGFRt were separated by T2A.

[0026] [Figure 3]A schematic diagram of the screening pipeline is shown. A schematic diagram of the screening for potent enhancers is shown. CD4 T cells or CD8 T cells were engineered with a transferable construct expressing CD19CAR or a transferable construct expressing CD19CAR with each of the putative potent inducible soluble enhancers (soluble cytokine transgenes). Each T cell was expanded using a method referred to herein as the TICLE method, and the CAR positivity rate was confirmed by expression of the EGFRt marker. A killing assay designed to screen for soluble cytokine transgenes was performed using a scoring system that tracks T cell killing and proliferation capacity. Briefly, the TICLE method involves obtaining CD8+, CD4+, and negative fractions (e.g., peripheral blood mononuclear cells) from a sample; introducing the above-described artificial expression construct into CD8+ or CD4+ cells using electroporation or viral transduction; co-culturing the resulting transduced cells with the CD4+ or CD8+ and negative fractions; and co-culturing in a gas-permeable container in medium supplemented with 7.5 U / mL IL2, 20 ng / mL IL4, 10 ng / mL IL7, and 20 ng / mL IL21.

[0027] [Figure 4] We demonstrate that all T cell lines generated maintained a high CD19CAR positivity rate, as determined by EGFRt detection. After cell production using the TICLE method described above, the CAR positivity rate of CD4 T cells was examined by staining for EGFRt and analyzing by flow cytometry. For each of the CD19CAR or CD19CAR + iSynPro soluble cytokine transgenes (a single soluble cytokine transgene for each generated cell line), CD4 T cells were enriched based on the presence or absence of the delivered construct, as determined by a high EGFRt positivity rate.

[0028] [Figure 5]CD4+ cells enriched for expression of either CD19CAR alone or CD19CAR + iSynPro soluble cytokine transgene (single soluble cytokine transgene line) were subjected to repeated killing assays with the neuroblastoma cell line Be2 + mCherry + CD19t. Effectors and targets were cocultured for 72 hours, followed by two rounds of repeated stimulation at 144 and 216 hours. Effectors and targets were set at various E:T ratios. In this experiment, the E:T ratio for the killing assay was 0.25:1, and the E:T ratio for the proliferation assay was 1:1. The killing score (the sum of the differences in Be2 cell killing between CD19CAR and CD19CAR + iSynPro soluble cytokine transgene) was used to determine the differences between the assessed groups. In CD4 cells, the soluble cytokine transgenes huTGFβ2-7m*, IL21, or IL15 enhanced killing compared to CD19CAR alone, suggesting that secretion of these cytokines can improve CAR T cell function. In the same assay, T cell proliferation was measured by quantifying the number of "dark red" objects, representing T cells, using live cell imaging software. T cell proliferation was assessed compared to CD19CAR alone using the same scoring method as above. In CD4 cells, IL21 or IL15 enhanced T cell proliferation compared to CD19CAR alone.

[0029] [Figure 6] In a separate donor, a repeat killing assay was performed using CD4 cells prepared similarly to those in Figure 5. All tested soluble cytokine transgenes enhanced killing in CD4 cells compared to CD19CAR alone, suggesting that secretion of these cytokines can enhance CAR T cell function. In the same assay, T cell proliferation was measured similarly to the method described in Figure 5. All tested soluble cytokine transgenes enhanced T cell proliferation compared to CD19CAR alone.

[0030] [Figure 7]Supernatants were collected from cultures containing CD4+ cells carrying CD19CAR or CD19CAR+iSynPro soluble cytokine transgenes and Be2 mCherry CD19t target cells. Cytokine concentrations of the effector cytokines IFNγ and TNFα were measured in the collected supernatants. IFNγ production was enhanced by the soluble cytokine transgenes mTGFβ2-7m*, scIL12, DR-IL18, and IL36γ. Furthermore, significant increases in TNFα production were observed with only scIL12, DR-IL18, or IL-36γ.

[0031] [Figure 8]Sequences supporting the present disclosure are set forth below: IL2 minimal promoter (SEQ ID NO: 138), huTGFβ2-7m* coding sequence (SEQ ID NO: 1), IL-21 coding sequence (SEQ ID NO: 2), IL-15 coding sequence (SEQ ID NO: 3), scIL12 coding sequence (SEQ ID NO: 4), drIL18(6-12) (SEQ ID NO: 6), IL36γ (SEQ ID NO: 7), S1-61-version 1 (SEQ ID NO: 8), S1-61-version 2 (SEQ ID NO: 9), S1-17 (SEQ ID NO: 10), S1-37 (SEQ ID NO: 11), S1-4 (SEQ ID NO: 12), S1-1 (SEQ ID NO: 13), S1-3 (SEQ ID NO: 14), S1-42 (SEQ ID NO: 15), S1-62 (SEQ ID NO: 16), S1-15 (SEQ ID NO: 17), S1-2 (SEQ ID NO: 18), S1-27 (SEQ ID NO: 19), S1-8 (SEQ ID NO: 20), S1-30 (SEQ ID NO: 21), S1-33 ​​(SEQ ID NO: 22), S1-41 (SEQ ID NO: 23), S1-59 (SEQ ID NO: 24), S1-66 (SEQ ID NO: 25), S1-71 (SEQ ID NO: 26), S1-56 (SEQ ID NO: 27), S1-6 (SEQ ID NO: 28), S1-60 (SEQ ID NO: 29), S1-86 (SEQ ID NO: 30), S1-32 (SEQ ID NO: 31), S1-10 (SEQ ID NO: 3 2), S1-18 (SEQ ID NO: 33), S1-14 (SEQ ID NO: 34), S1-16 (SEQ ID NO: 35), S1-19 (SEQ ID NO: 36), S1-26 (SEQ ID NO: 37), S1-65 (SEQ ID NO: 38), S2-n1 (SEQ ID NO: 39), S4-n1 (SEQ ID NO: 40), S6-n1 (SEQ ID NO: 41), S1-325 (SEQ ID NO: 42), S1-60 (SEQ ID NO: 43), S2-274 (SEQ ID NO: 44), S2-310 (SEQ ID NO: 45), S1-367 (SEQ ID NO: 46), S1-7 (SEQ ID NO: 47), huTGFβ2-7m (SEQ ID NO: 48), huTGFβ2-7m* (SEQ ID NO: 49), IL21 (SEQ ID NO: 50), IL-15 (SEQ ID NO: 51), scIL12 (SEQ ID NO: 52), DR-IL18 (SEQ ID NO: 54), EF1a promoter (long chain; Ef1a-HTLV hybrid) (SEQ ID NO: 55), core insulator-version 1 (SEQ ID NO: 56), core insulator-version 2 (SEQ ID NO: 57), IgG4 hinge-A (SEQ ID NO: 58), coding sequence for IgG4 hinge-A (SEQ ID NO: 59), coding sequence for IgG4 hinge-B (SEQ ID NO: 60), coding sequence for IgG4 hinge-C (SEQ ID NO: 61),Coding sequence of IgG4-CH2 domain (SEQ ID NO: 62), coding sequence of IgG4-CH3 (SEQ ID NO: 63), CD28 transmembrane domain-A (SEQ ID NO: 64), CD28 transmembrane domain-B (SEQ ID NO: 65), CD28 transmembrane domain-C (SEQ ID NO: 66), coding sequence of CD28 transmembrane domain-A (SEQ ID NO: 67), coding sequence of CD28 transmembrane domain-B (SEQ ID NO: 68), coding sequence of CD28 transmembrane domain-C (SEQ ID NO: 69), coding sequence of CD28 transmembrane domain-D (SEQ ID NO: 70), 4-1BB costimulatory domain-A (SEQ ID NO: 71), 4-1BB costimulatory domain-B (SEQ ID NO: 72), No. 72), 4-1BB costimulatory domain-C (SEQ ID NO: 73), 4-1BB signaling domain coding sequence-A (SEQ ID NO: 74), 4-1BB signaling domain coding sequence-B (SEQ ID NO: 75), 4-1BB signaling domain coding sequence-C (SEQ ID NO: 76), CD3ζ signaling domain-A (SEQ ID NO: 77), CD3ζ signaling domain-B (SEQ ID NO: 78), CD3ζ signaling domain-C (SEQ ID NO: 79), CD3ζ signaling domain coding sequence-A (SEQ ID NO: 80), CD3ζ signaling domain coding sequence-B (SEQ ID NO: 81), Thosea asigna virus 2A (T2A) peptide-v1 (SEQ ID NO: 82), Thosea asigna virus 2A (T2A) peptide-v2 (SEQ ID NO: 83), Thosea asigna virus 2A (T2A) peptide-v3 (SEQ ID NO: 84), Porcine teschovirus-1 2a (P2A) peptide (SEQ ID NO: 85), Equine rhinitis A virus (ERAV) 2A (E2A) peptide (SEQ ID NO: 86), Foot-and-mouth disease virus 2A (F2A) peptide (SEQ ID NO: 87), coding sequence for T2A-v1 (SEQ ID NO: 88), coding sequence for T2A-v2 (SEQ ID NO: 89), EGFRt-version 1 (SEQ ID NO: 90), EGFRt-version 2 (SEQ ID NO: 91), coding sequence for EGFRt (SEQ ID NO: 92), coding sequence for tCD19 (SEQ ID NO: 93), HER2tG (no start codon) (SEQ ID NO: 94), DHFRdm (SEQ ID NO: 95),Vector pj05999: s1-61-il2mp_her2tg-t2a-hutgfb2-7m_rbgpolya_2x3hsa_ef1al_hucd19scfvg01s-igg4hinge-cd28tm-41bb-zeta-p2a-dhfrdm-t2a-egfrt_bghpolya_pbnp (SEQ ID NO: 96), vector pj06000: s1-61-il2mp_her2tg-t2a-il21_rbgpolya _2x3hsa_ef1al_hucd19scfvg01s-igg4hinge-cd28tm-41bb-zeta-p2a-dhfrdm-t2a-egfrt_bghpolya_pbnp (SEQ ID NO: 97), vector pj06001:s1-61-il2mp_her2tg-t2a-il15_rbgpolya_2x3hsa_ef1al_hucd19scfvg01s-igg4hinge-cd28tm-41bb-zeta-p 2a-dhfrdm-t2a-egfrt_bghpolya_pbnp (SEQ ID NO: 98), vector pj06002:s1-61-il2mp_her2tg-t2a-scil12_rbgpolya_2x3hsa_ef1al_hucd19scfvg01s-igg4hinge-cd28tm-41bb-zeta-p2a-dhfrdm-t2a-egfrt_bghpolya_pbnp (SEQ ID NO: 99), vector pj0600 9:s1-61-il2mp_her2tg-t2a-dr-il186-12_rbgpolya_2x3hsa_ef1al_hucd19scfvg01s-igg4hinge-cd28tm-41bb-zeta-p2a-dhfrdm-t2a-egfrt_bghpolya_pbnp (SEQ ID NO: 101), and vector pj06637-s1-61-il2mp_her2tg_t2a_il15_pbnp (SEQ ID NO: 136). DETAILED DESCRIPTION OF THE INVENTION

[0032] Various therapies developed in recent decades involve the engineering of immune cells that express recombinant receptors that bind to antigens on unwanted cell types (e.g., cancer cells, virus-infected cells (also referred to herein as target cells)). Binding of the recombinant receptor to the target antigen activates T cells, which can then destroy the cells to which they have bound. Chimeric antigen receptor (CAR) cell therapy is one such treatment.

[0033] Chimeric antigen receptor (CAR) therapy involves modifying immune cells to express synthetic receptors designed to target cells, such as cancer cells or virus-infected cells. While CAR therapy has shown considerable success in treating a variety of cancers, challenges remain. For example, CAR T cells can exhibit suboptimal proliferation, functional unresponsiveness or exhaustion, impaired memory differentiation, and / or limited persistence, limiting their ability to induce or maintain remission in patients. Therefore, strategies to improve the efficacy of CAR T cells are needed, especially in situations involving prolonged antigen exposure, which can lead to functional unresponsiveness and exhaustion.

[0034] The present disclosure provides artificial expression constructs comprising a cytokine transgene under the control of an inducible promoter for enhancing the function of immune cells (e.g., T cells or chimeric antigen receptor (CAR)-T cells). The inducible cytokine transgenes disclosed herein enhance the efficacy of immune cells (e.g., T cells or CAR-T cells) to enhance killing, immune cell proliferation, and / or cytokine production. In certain embodiments, the cytokine transgene encodes a soluble cytokine transgene. In certain embodiments, the cytokine transgene encodes transforming growth factor beta 2-7m (TGFβ2-7m), mini TGFβ2-7m* (mTGFβ2-7m*), interleukin-21 (IL21), interleukin-15 (IL15), single-chain interleukin-12 (scIL12), decoy-resistant interleukin-18 (DR-IL18), or interleukin-36γ (IL36γ).

[0035] In certain embodiments, inducible expression of TGFβ2-7m, mTGFβ2-7m*, IL21, IL15, scIL12, DR-IL18, or IL36γ is used to enhance immune cell-mediated cell killing.

[0036] In certain embodiments, inducible expression of TGFβ2-7m, mTGFβ2-7m*, IL21, IL15, scIL12, DR-IL18, or IL36γ is used to enhance T cell proliferation.

[0037] In certain embodiments, inducible expression of TGFβ2-7m, mTGFβ2-7m*, IL21, IL15, scIL12, DR-IL18, or IL36γ is used to enhance cytokine production, hi certain embodiments, cytokine production includes IFNγ production and / or TNFα production.

[0038] In certain embodiments, inducible expression of IL21 is used to enhance immune cell-mediated cell killing, hi certain embodiments, inducible expression of IL15 is used to enhance immune cell-mediated cell killing.

[0039] In certain embodiments, inducible expression of IL21 is used to enhance immune cell proliferation, hi certain embodiments, inducible expression of IL15 is used to enhance immune cell proliferation.

[0040] In certain embodiments, inducible expression of scIL12 is used to enhance cytokine production. In certain embodiments, inducible expression of DR-IL18 is used to enhance cytokine production. In certain embodiments, inducible expression of IL36γ is used to enhance cytokine production. In certain embodiments, inducible expression of TGFβ2-7m or mTGFβ2-7m* is used to enhance cytokine production.

[0041] In certain embodiments, inducible expression of scIL12 is used to enhance IFNγ production. In certain embodiments, inducible expression of DR-IL18 is used to enhance IFNγ production. In certain embodiments, inducible expression of IL36γ is used to enhance IFNγ production. In certain embodiments, inducible expression of TGFβ2-7m or mTGFβ2-7m* is used to enhance IFNγ production.

[0042] In certain embodiments, inducible expression of scIL12 is used to enhance TNFα production. In certain embodiments, inducible expression of DR-IL18 is used to enhance TNFα production. In certain embodiments, inducible expression of IL36γ is used to enhance TNFα production.

[0043] In certain embodiments, a combination of inducible expression of IL21 and inducible expression of IL15, scIL12, DR-IL18, IL36γ, TGFβ2-7m or mTGFβ2-7m* is utilized.

[0044] In certain embodiments, a combination of inducible expression of IL15 and inducible expression of IL21, scIL12, DR-IL18, IL36γ, TGFβ2-7m or mTGFβ2-7m* is utilized.

[0045] As used herein, the terms "enhance" and "augment" are used interchangeably.

[0046] In some examples, the artificial expression construct comprising the cytokine transgene is encoded by the sequence set forth in SEQ ID NO: 96, 97, 98, 99 or 101.

[0047] In certain embodiments, the artificial expression construct comprising a cytokine transgene comprises an iSynPro promoter and encodes the cytokine transgene. In certain embodiments, the artificial expression construct encoding the cytokine transgene further comprises or encodes a skip sequence and a regulatory mechanism. In certain embodiments, the iSynPro promoter comprises S1-61 (SEQ ID NO: 8). In certain embodiments, the iSynPro promoter comprises S1-61 (SEQ ID NO: 8) operably linked to the IL2 minimal promoter (SEQ ID NO: 138).

[0048] Additionally, the present disclosure provides systems and methods for genetically modifying immune cells to express a recombinant receptor. In some embodiments, the artificial expression construct comprising a cytokine transgene may further comprise a sequence encoding the recombinant receptor. In another embodiment, the sequence encoding the recombinant receptor and the cytokine transgene may be present on separate artificial expression constructs. In certain embodiments, the recombinant receptor is under the control of a second promoter. In certain embodiments, the artificial expression construct comprising a recombinant receptor comprises a promoter and the recombinant receptor or encodes a promoter and the recombinant receptor. In certain embodiments, the artificial expression construct comprising a recombinant receptor comprises or encodes a promoter, the recombinant receptor, a skip sequence, and a control mechanism. In certain embodiments, the artificial expression construct comprising a recombinant receptor comprises or encodes a promoter, the recombinant receptor, a first skip sequence, a selection cassette (e.g., dihydrofolate reductase double mutant (DHFRdm)), a second skip sequence, and a transduction marker (EGFRt). In certain embodiments, the recombinant receptor comprises a CAR or a recombinant T cell receptor (eTCR). In certain embodiments, the recombinant receptor comprises an extracellular portion that binds to a target antigen, a transmembrane domain, and an intracellular portion.

[0049] In certain embodiments, the artificial expression construct comprising a cytokine transgene and a recombinant receptor comprises or encodes an iSynPro promoter, Her2tG, a first 2A skip sequence, a cytokine transgene, an EF1a(L) promoter, a recombinant receptor, a second 2A skip sequence, DHFRdm, a third 2A skip sequence, and EGFRt. In certain embodiments, the artificial expression construct comprising a cytokine transgene and a recombinant receptor comprises or encodes an S1-61 iSynPro promoter, Her2tG, a first 2A skip sequence, a cytokine transgene, an EF1a(L) promoter, a CAR, a second 2A skip sequence, DHFRdm, a third 2A skip sequence, and EGFRt. In certain embodiments, the cytokine transgene encodes a soluble cytokine. In certain embodiments, the cytokine transgene encodes TGFβ2-7m, mTGFβ2-7m*, IL-21, IL-15, scIL12, DR-IL18, or IL36γ. In a specific embodiment, the CAR is an anti-CD19 CAR. See Figure 2 for a schematic of an exemplary artificial expression construct.

[0050] Thus, the present disclosure provides a system for enhancing killing, proliferation, and / or cytokine production of immune cells bearing recombinant receptors. Immune cells bearing the recombinant receptors of the present disclosure can be engineered to target various target cells (e.g., cancer cells, viruses, bacteria, fungi) that express a target antigen by engineering the extracellular domain to include a binding domain that binds to the target antigen.

[0051] Various aspects of the present disclosure are described in further detail below under the following headings: (i) inducible expression of cytokine transgenes; (ii) immune cells; (iii) cell sample collection and cell enrichment; (iv) genetic engineering techniques; (v) control mechanisms including tag cassettes, transduction markers, selection cassettes, and / or suicide switches; (vi) recombinant receptors; (vi-a) binding domains; (vi-b) transmembrane domains; (vi-c) intracellular effector domains; (vi-d) linkers; (vii) ex vivo produced cell preparations; (viii) compositions for targeted viral vectors and nanoparticles for in vivo cell modification; (ix) methods of use; (x) kits; (xi) exemplary embodiments; and (xii) conclusion. These headings are provided for organizational purposes only and are not intended to limit the scope or interpretation of the present disclosure.

[0052] (i) Inducible expression of cytokine transgenes Cytokines are small proteins (usually 5-25 kDa) that play an important role in cell signaling. They are released by cells and affect the behavior of other cells and sometimes the cell itself (e.g., T cells). Cytokines include, for example, chemokines, interferons, interleukins, lymphokines, and / or tumor necrosis factors. Cytokines can be produced by a variety of cell types, including immune cells such as macrophages, B lymphocytes, T lymphocytes, and / or mast cells, as well as endothelial cells, fibroblasts, and / or various stromal cells.

[0053] Cytokines can act through receptors. Cytokines are important in the immune system because they can regulate the balance between humoral and cellular immune responses and can modulate the maturation, proliferation, and responsiveness of specific cell populations. Some cytokines enhance or suppress the actions of other cytokines in complex ways.

[0054] Soluble cytokines can cross the endoplasmic reticulum membrane intact and be secreted from the cells that produced them, and they regulate inflammatory and immune events by functioning as agonists or antagonists of cytokine signaling.

[0055] Minimonomeric transforming growth factor β2-7m (mmTGFβ2-7m) is a recombinant transforming growth factor β2 (TGFβ2) based on the TGFβ2 backbone. mmTGFβ2-7m, referred to herein as TGFβ2-7m or huTGFβ2-7m, is a conformation of TGFβ that contains a finger region and a cystine knot rather than a heel helix (Kim et al., Structure 2019, 27(9): 1427-1442). Two substitutions within the molecule increase the charge of the loop that replaces the heel helix, and seven additional substitutions within this loop, connecting fingers 1-2 and 3-4, which contact the TGFβ receptor II (TβRII) (Hinck, Bioorg Med Chem 26(19):5239-5246 (2018)). TGFβ2-7m is described in further detail in U.S. Patent No. 11,091,523. In certain embodiments, another form of TGFβ2-7m, referred to herein as mTGFβ2-7m* or huTGFβ2-7m*, comprises the sequence set forth in SEQ ID NO:49. In certain embodiments, SEQ ID NO:49 has 96% sequence identity with TGFβ2-7m (SEQ ID NO:48), as described in U.S. Patent No. 11,091,523. In certain embodiments, mTGFβ2-7m* set forth in SEQ ID NO:49 comprises seven substitutions within the loop connecting fingers 1-2 and fingers 3-4, as described above, and a deletion of residues 52-71. In certain embodiments, mTGFβ2-7m* does not comprise the amino acid substitutions L51A, A54K, or C77S. The L51A and / or A54K substitutions increase charge, and C77S is a substitution that eliminates the disulfide bond forming ability of mTGFβ2-7m*.

[0056] Interleukin-21 (IL21) is a class I cytokine containing a four-alpha helix bundle. IL21 has a wide range of pleiotropic effects on both innate and adaptive immune responses. IL21 is produced by natural killer T cells, CD4(+) T cells, and Th17 cells. IL21 plays diverse roles in antitumor, antiviral, and inflammatory responses, as well as in autoimmune and inflammatory diseases (Spolski et al., Nat Rev Drug Discov 13, 379-395 (2014)).

[0057] Interleukin-15 (IL-15) is a cytokine that, in its mature form, is a glycoprotein of 14–15 kDa and contains a four-alpha helix bundle. IL-15 is produced by various cell types, including monocytes, macrophages, dendritic cells, keratinocytes, epidermal skin cells, fibroblasts, various epithelial cells, bone marrow stromal cells, and neurons. Many cell types, including natural killer cells and CD8(+) T cells, are responsive to IL-15 (Perera et al., Microbes Infect 14(3):247–261 (2012)).

[0058] Single-chain interleukin-12 (scIL12) is a modified IL12 in which the α chain (p35) and β chain (p40) of IL12 are fused via a GlySer linker (PCT Publication WO2015095249A1). In a specific embodiment, the GlySer linker comprises (Gly4Ser)3 (SEQ ID NO: 102). IL-12 is an interleukin naturally produced by dendritic cells, macrophages, neutrophils, and human B lymphoblastoid cells (NC-37) in response to antigen stimulation. IL-12 is composed of a four-alpha helix bundle.

[0059] Decoy-resistant (DR) interleukin-18 (IL18) is a modified IL18 protein that binds to the IL18 receptor (IL18Rα) and maintains IL18 function while evading IL-18-binding protein (IL-18BP) (Zhou et al., Nature 583(7817):609-614 (2020)). In other words, DR-IL18 retains its signaling ability but is not susceptible to inhibition by IL-18BP. IL-18 is a potent pro-inflammatory cytokine that induces interferon-γ (IFN-γ) production from Th1 cells, NK cells, and activated macrophages, especially in the presence of IL-12. Furthermore, IL-18 also plays a role in regulating T lymphocyte helper type I cell development and Fas-mediated cytotoxicity. Suppression of IL-18 activity is being investigated for the treatment of chronic inflammatory diseases such as Crohn's disease and rheumatoid arthritis. IL-18 acts by inducing heterodimerization of the two subunits of the IL-18 receptor (IL-18Rα and IL-18Rβ).

[0060] Interleukin-36γ (IL36γ; also known as IL1F9) is an isoform of IL36 and belongs to the interleukin-1 superfamily of cytokines. IL36 is expressed by various cell types, including T cells, keratinocytes, skin cells, lung cells, and intestinal cells. IL36γ has been suggested to regulate keratinocyte- and endothelial cell-mediated inflammatory responses, as well as the differentiation of Treg and Th9 cells. Furthermore, IL36γ has been implicated in systemic and inflammatory diseases, such as systemic lupus erythematosus, arthritis, and inflammatory bowel disease (Yuan et al., Front Immunol 10:2532 (2019)).

[0061] The cytokine transgene is placed under the control of an inducible synthetic promoter (iSynPro). The iSynPro promoter may contain a sequence containing a transcription factor response element (TRE), such as S1-17 (SEQ ID NO: 10), S1-37 (SEQ ID NO: 11), S1-4 (SEQ ID NO: 12), S1-1 (SEQ ID NO: 13), S1-3 (SEQ ID NO: 14), S1-42 (SEQ ID NO: 15), S1-61 (SEQ ID NO: 8 or 9), S1-62 (SEQ ID NO: 16), S1-15 (SEQ ID NO: 17), S1-2 (SEQ ID NO: 18), S1-27 (SEQ ID NO: 19), S1-8 (SEQ ID NO: 20), S1-30 (SEQ ID NO: 21), S1-33 ​​(SEQ ID NO: 22), S1-41 (SEQ ID NO: 23), S1-59 (SEQ ID NO: 24), S1-66 (SEQ ID NO: 25), S1-71 (SEQ ID NO: 26), S1-56 (SEQ ID NO: 27), S1-6 (SEQ ID NO: 28), S1-60 (SEQ ID NO: 29), S1-86 (SEQ ID NO: 30), S1-32 (SEQ ID NO: 31), S1-10 (SEQ ID NO: 32), S1-18 (SEQ ID NO: 33), S1-14 (SEQ ID NO: 34), S1-16 (SEQ ID NO: 35), S1-19 (SEQ ID NO: 36), S1-26 (SEQ ID NO: 37), S1-65 (SEQ ID NO: 38), S2-n1 (SEQ ID NO: 39), S4-n1 (SEQ ID NO: 40), S6-n1 (SEQ ID NO: 41), S1-325 (SEQ ID NO: 42), S1-60 (SEQ ID NO: 43), S2-274 (SEQ ID NO: 44), S2-310 (SEQ ID NO: 45), S1-367 (SEQ ID NO: 46), or S1-7 (SEQ ID NO: 47). In a specific embodiment, the iSynPro promoter comprises S1-61 (SEQ ID NO: 8 or 9).In certain embodiments, the iSynPro promoter comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46 or SEQ ID NO:47.

[0062] In certain embodiments, the iSynPro promoter comprises S1-61 (SEQ ID NO: 8 or 9). In certain embodiments, the iSynPro promoter comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 8 or 9.

[0063] In certain embodiments, the iSynPro promoter further comprises a minimal promoter. In certain embodiments, the iSynPro promoter comprises S1-61 operably linked to the minimal promoter. In certain embodiments, the minimal promoter comprises a minimal IL2 promoter (SEQ ID NO: 138).

[0064] Furthermore, functional truncations and functional variants of cytokine transgenes may be used. Functional truncations have fewer amino acid residues than the corresponding reference wild-type sequence but retain cytokine activity. Functional variants have one or more amino acid mutations compared to the corresponding reference wild-type sequence but retain cytokine activity.

[0065] (ii) Immune cells The present disclosure describes immune cells genetically modified to express a cytokine transgene (e.g., a soluble cytokine) that improves immune cell function. In certain embodiments, the immune cells of the present disclosure are further genetically modified to express a recombinant receptor, such as a CAR. The immune cells may be any cell whose cytokine transgene expression can be inducibly controlled by iSynPro. In certain embodiments, the immune cells may include lymphocytes, monocytes / macrophages, hematopoietic stem cells (HSCs), hematopoietic progenitor cells (HPCs), and / or a mixture of HSCs and HPCs (i.e., HSPCs). In certain embodiments, the immune cells include lymphocytes. In certain embodiments, the lymphocytes include T cells, B cells, natural killer (NK) cells, or NK-T cells.

[0066] Several types of T cell subsets have been identified, each with its own distinct functions. For example, most T cells have a T cell receptor (TCR), which exists as a complex of several proteins. The actual T cell receptor is composed of two distinct peptide chains, which are produced by independent genes, the T cell receptor α gene and the T cell receptor β gene (TCRα and TCRβ), respectively, and are called the αTCR chain and the βTCR chain.

[0067] Gamma delta T cells are a small subset of T cells that have a distinctive T cell receptor (TCR) on their surface. In gamma delta T cells, the TCR consists of one gamma chain and one delta chain. This group of T cells is less common than alpha beta T cells (representing only 2% of all T cells).

[0068] CD3 is expressed on all mature T cells. Activated T cells express 4-1BB (CD137), CD69, and CD25. CD5 and the transferrin receptor are also expressed on T cells.

[0069] T cells can be further classified as helper T cells (CD4+ T cells) and cytotoxic T cells (CTLs, CD8+ T cells), including cytolytic T cells. Helper T cells assist other white blood cells in immunological processes, such as the maturation of B cells into plasma cells and the activation of cytotoxic T cells and macrophages. Helper T cells are also known as CD4+ T cells because they express the CD4 protein on their surface. Helper T cells are activated upon presentation of peptide antigens by MHC class II molecules expressed on the surface of antigen-presenting cells (APCs). Once activated, helper T cells rapidly divide and secrete small proteins called cytokines, which regulate or support active immune responses.

[0070] Cytotoxic T cells can destroy virus-infected cells and tumor cells and are also involved in transplant rejection. Cytotoxic T cells are also known as CD8+ T cells because they express the CD8 glycoprotein on their surface. Cytotoxic T cells recognize their targets by binding to antigens bound to MHC class I molecules, which are present on the surface of almost all cells in the body.

[0071] "Central memory" T cells (or "TCM") are antigen-experienced CTLs that express CD62L or CCR-7 and CD45RO on their surface, but do not express CD45RA or have reduced CD45RA expression, compared to naive cells. In a specific embodiment, central memory cells are positive for CD62L, CCR7, CD25, CD127, CD45RO, and CD95, and have reduced CD45RA expression, compared to naive cells.

[0072] "Effector memory" T cells (or "TEMs") are antigen-experienced T cells that do not express CD62L or have reduced CD62L expression on their surface compared to central memory cells, and do not express CD45RA or have reduced CD45RA expression compared to naive cells. In certain embodiments, effector memory cells are negative for CD62L and CCR7 expression and positive or negative for CD28 and CD45RA expression compared to naive or central memory cells. Effector T cells are also positive for granzyme B and perforin compared to memory or naive T cells.

[0073] "Naive" T cells are T cells that have not experienced antigen and express CD62L and CD45RA but not CD45RO compared to central memory cells or effector memory cells. In certain embodiments, naive CD8+ T lymphocytes are characterized by the expression of phenotypic markers of naive T cells, including CD62L, CCR7, CD28, CD127, and CD45RA.

[0074] Natural killer cells (also known as NK cells, K cells, and killer cells) are activated in response to interferon or macrophage-derived cytokines. NK cells play a role in combating viral infections, whereas adaptive immune responses produce antigen-specific cytotoxic T cells that can eliminate infections. NK cells express CD8, CD16, and CD56, but not CD3.

[0075] NK cells include NK-T cells. NK-T cells are a specialized T cell population that express the semi-invariant T cell receptor (TCRαβ) and surface antigens typically associated with natural killer cells. NK-T cells contribute to antibacterial and antiviral immune responses and promote tumor-associated immunosurveillance or tumor-associated immunosuppression. Like natural killer cells, NK-T cells can induce cytotoxicity associated with perforin, Fas, and TNF. Activated NK-T cells can produce IFN-γ and IL-4. In certain embodiments, NK-T cells are CD3+ / CD56+.

[0076] Macrophages (and their precursor cells, monocytes) are present in all tissues of the body (possibly as microglia, Kupffer cells, and osteoclasts) and phagocytose apoptotic cells, pathogens, and other non-self components. Monocytes / macrophages express CD11b, F4 / 80; CD68; CD11c; IL-4Rα; and / or CD163.

[0077] Immature dendritic cells (i.e., preactivated dendritic cells) phagocytose peripheral antigens and other non-self components, become activated, migrate to T cell areas of lymphoid tissues, and present antigens to T cells. Dendritic cells express CD1a, CD1b, CD1c, CD1d, CD21, CD35, CD39, CD40, CD86, CD101, CD148, CD209, and CD-205.

[0078] Hematopoietic stem / progenitor cells, or HSPCs, refer to the combination of hematopoietic stem cells and hematopoietic progenitor cells.

[0079] Hematopoietic stem cells refer to undifferentiated hematopoietic cells that are capable of self-renewal in vivo or self-renewal in vitro and proliferation virtually indefinitely, and can differentiate into any other type of hematopoietic cell.

[0080] Hematopoietic progenitor cells are cells derived from hematopoietic stem cells or cells derived from fetal tissue that can further differentiate into mature cells. In certain embodiments, hematopoietic progenitor cells express CD24 lo Lin - CD117 + Hematopoietic progenitor cells. HPCs can differentiate into (i) myeloid progenitor cells, which ultimately give rise to monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes / platelets, or dendritic cells, or (ii) lymphoid progenitor cells, which ultimately give rise to T cells, B cells, and NK cells.

[0081] HSPCs may be positive for specific markers that are expressed at higher levels in HSPCs than in other types of hematopoietic cells. Such markers include, for example, CD34, CD43, CD45RO, CD45RA, CD59, CD90, CD109, CD117, CD133, CD166, HLA DR, or a combination thereof. Furthermore, HSPCs may be negative for markers that are expressed in other types of hematopoietic cells. Such markers include, for example, Lin, CD38, or a combination thereof. Preferably, HSPCs are CD34+ cells.

[0082] A statement that a cell or cell population is "positive" for or expresses a particular marker refers to the presence of a detectable particular marker on the surface of or within the cell. When referring to a cell surface marker, "positive" refers to the presence of cell surface expression, as detected by flow cytometry, for example, by staining with an antibody that specifically binds to the cell surface marker and detecting the antibody, at a level that is substantially higher than that detected by the same procedure under identical conditions using an isotype-matched control in the flow cytometry, and / or at a level that is substantially similar to that of cells known to be positive for that marker, and / or at a level that is substantially higher than that of cells known to be negative for that marker.

[0083] A statement that a cell or cell population is "negative" for a particular marker, or that a cell or cell population does not express the marker, refers to the absence of a substantially detectable particular marker on the surface of the cell or within the cell. When referring to a cell surface marker, "negative" refers to the absence of cell surface expression, as detected by, for example, flow cytometry, using staining with an antibody that specifically binds to the cell surface marker and detecting the antibody, and the staining is not detected in the flow cytometer at a level substantially higher than that detected by the same procedure under identical conditions using an isotype-matched control, and / or is detected at a level substantially lower than that of cells known to be positive for that marker, and / or is detected to a degree substantially similar to that of cells known to be negative for that marker.

[0084] The cells to be genetically modified according to the teachings of the present disclosure may be patient-derived (autologous) cells, allogeneic cells, in vivo cells, or ex vivo cells, as appropriate. In certain embodiments, the immune cells are lymphocytes. In certain embodiments, the lymphocytes are T cells, B cells, or NK cells. In certain embodiments, the T cells are CD4+ T cells or CD8+ T cells.

[0085] (iii) Cell sample collection and cell concentration Methods for collecting and concentrating samples are known to those skilled in the art. In certain embodiments, the cells are derived from a human, for example, from a patient receiving treatment. The cells may be derived from a cell line. In some embodiments, the cells are derived from a heterologous cell source, for example, from a mouse, rat, non-human primate, or pig.

[0086] In some embodiments, the T cells are derived from or isolated from a sample such as whole blood, peripheral blood mononuclear cells (PBMCs), white blood cells, bone marrow, thymus, tissue biopsy, tumor, lymph node, gut-associated lymphoid tissue, mucosa-associated lymphoid tissue, spleen, other lymphoid tissue, liver, lung, stomach, intestine, colon, kidney, pancreas, breast, bone, prostate, cervix, testes, ovaries, tonsils, or other organ, and / or cells derived from these organs. In certain embodiments, cells are obtained from the subject's circulating blood, e.g., by apheresis or leukapheresis. In certain embodiments, the sample includes lymphocytes, such as T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, HSCs, HPCs, HSPCs, red blood cells, and / or platelets; in some embodiments, the sample includes cells other than red blood cells and platelets and requires further processing.

[0087] In some embodiments, blood cells collected from a subject are washed, for example, to remove the plasma fraction and suspend the cells in an appropriate buffer or medium for subsequent processing. In certain embodiments, the cells are washed with phosphate-buffered saline (PBS). In some embodiments, the wash solution does not contain calcium and / or magnesium and / or contains a majority or no divalent cations. Washing can be performed using a semi-automated "flow-through" centrifuge (e.g., a Cobe 2991 cell processor, Baxter) according to the manufacturer's instructions. Tangential flow filtration (TFF) can also be performed. In certain embodiments, the washed cells can be resuspended in a variety of biocompatible buffers, such as Ca++ / Mg++-free PBS.

[0088] Isolation may involve one or more of a variety of cell preparation and separation steps, including separation based on one or more properties, such as size, density, sensitivity or resistance to particular reagents, and / or affinity for antibodies or other binding partners (e.g., immunoaffinity). In certain embodiments, isolation is performed sequentially and / or simultaneously in a single method using a single device or facility. In certain embodiments, isolation, culture, and / or recombination of various distinct populations begins with a single starting material, such as a single sample.

[0089] In certain embodiments, samples can be enriched for T cells using density-based cell separation and related techniques. For example, white blood cells can be separated from other cell types in peripheral blood by lysing red blood cells followed by centrifugation through a Percoll or Ficoll gradient.

[0090] In certain embodiments, a bulk T cell population that is not enriched for a particular type of T cell can be used. In certain embodiments, a selected type of T cell can be enriched and / or isolated by positive and / or negative selection using a cell marker. In positive selection, cells having a cell marker bound to a capture agent are obtained and used for further application. In negative selection, cells that do not bind to a capture agent, such as an antibody against a cell marker, are obtained and used for further application. In some examples, both the positively selected fraction and the negatively selected fraction can be obtained and used for further application. In certain embodiments, CD4+ T cells and / or CD8+ T cells are enriched from PBMCs.

[0091] The above cell separation does not necessarily achieve 100% enrichment or removal of a particular cell population or cells that express a particular marker. For example, positive selection or enrichment of a particular type of cell means increasing the number or proportion of such cells, but does not necessarily completely remove cells that do not express that marker. Similarly, negative selection, removal or elimination of a particular type of cell means reducing the number or proportion of such cells, but does not necessarily completely remove such cells.

[0092] In some instances, the separation step is performed multiple times, whereby the positively or negatively selected fraction from one step is subjected to another separation step, such as a subsequent positive or negative selection.

[0093] In some embodiments, antibodies or binding domains against cell markers can be attached to solid supports or matrices, such as magnetic or paramagnetic beads, to separate cells by positive and / or negative selection. For example, in some embodiments, immunomagnetic separation techniques (or affinity magnetic separation techniques) are used to separate or isolate cells and cell populations (reviewed in "Methods" in Molecular Medicine, vol. 58: Metastasis Research Protocols, Vol. 2: Cell Behavior In Vitro and In Vivo, p. 17-25 Edited by: SA Brooks and U. Schumacher (c) Humana Press Inc., Totowa, NJ). See also U.S. Patent Publication No. 4,452,773; U.S. Patent Publication No. 4,795,698; U.S. Patent Publication No. 5,200,084; and European Patent Publication No. 452342.

[0094] In some embodiments, affinity-based selection is performed by magnetically activated cell sorting (MACS) (Miltenyi Biotec, Auburn, CA). The MACS system can select magnetic particle-bound cells with high purity. In certain embodiments, MACS is operated in a mode that sequentially elutes non-target and target species after application of an external magnetic field. That is, cells bound to magnetic particles are retained while cells that do not bind to magnetic particles are eluted. After this first elution step, cells trapped by the magnetic field and prevented from elution are then released by some method that allows for elution and recovery. In certain embodiments, non-target cells are labeled and removed from the heterogeneous cell population.

[0095] In some embodiments, the cell populations described herein are collected and enriched (or removed) by flow cytometry, in which cells stained for multiple cell surface markers are entrained in a fluid stream for analysis. In some embodiments, the cell populations described herein are collected and enriched (or removed) by preparative (FACS) sorting. In some embodiments, the cell populations described herein are collected and enriched (or removed) using a microelectromechanical systems (MEMS) chip in combination with a FACS-based detection system (see, e.g., WO2010 / 033140; Cho et al. (2010) Lab Chip 10, 1567-1573; and Godin et al. (2008) J Biophoton. 1(5):355-376). In either case, cells can be labeled with multiple markers to isolate clearly defined cell subsets with high purity.

[0096] Cell markers for various T cell subpopulations are described above. In certain embodiments, specific T cell subpopulations, e.g., T cells that are positive for or highly express one or more cell surface markers, e.g., CCR7, CD45RO, CD8, CD27, CD28, CD62L, CD127, CD4, and / or CD45RA T cells, are isolated by positive or negative selection techniques.

[0097] CD3+CD28+ T cells can be expanded by positive selection using anti-CD3 / anti-CD28 conjugated magnetic beads (e.g., DYNABEADS® M-450 CD3 / CD28 T Cell Expander).

[0098] In certain embodiments, a CD8+ or CD4+ selection step is performed to separate CD4+ helper T cells from CD8+ cytotoxic T cells. These CD8+ and CD4+ populations can be further sorted into various subpopulations by positive or negative selection for markers expressed on, or relatively highly expressed in, one or more naive, memory, and / or effector T cell subpopulations.

[0099] In certain embodiments, a CD8+ and / or CD4+ selection step can be used to separate CD4+ helper T cells and CD8+ cytotoxic T cells from the negative fraction, in certain embodiments utilizing the TICLE method, described in more detail elsewhere herein.

[0100] Additionally, other cell types can be enriched based on known marker profiles and known techniques. For example, CD34+ HSCs, HSPs, and HSPCs can be enriched using anti-CD34 antibodies directly or indirectly coupled to magnetic microparticles in combination with a magnetic cell separator (e.g., the CliniMACS® Cell Separation System (Miltenyi Biotec, Bergisch Gladbach, Germany)).

[0101] (iv) Genetic engineering techniques The cell population is genetically modified (or engineered) to express a cytokine transgene and, in addition to the cytokine transgene, may also be genetically modified (or engineered) to express a recombinant receptor (e.g., a chimeric antigen receptor (CAR)) described herein. The desired genes (e.g., soluble cytokine transgenes and recombinant receptors) disclosed herein can be introduced into the cells by methods known in the art, including transfection, electroporation, microinjection, lipofection, calcium phosphate transfection, infection with viral or bacteriophage vectors containing the gene sequence, cell fusion, chromosomal gene transfer, microcell fusion gene transfer, spheroplast fusion, in vivo nanoparticle delivery, and the like. Various techniques for introducing foreign genes into cells are known in the art (see, e.g., Loeffler and Behr, 1993, Meth. Enzymol. 217:599-618; Cohen, et al., 1993, Meth. Enzymol. 217:618-644; Cline, 1985, Pharmac. Ther. 29:69-92), and such techniques may be used as long as they do not unduly disrupt the developmental and physiological functions necessary for the recipient cells. These techniques can be used to stably transfer foreign genes into cells so that the foreign genes are expressed in the cells and, in certain instances, are preferably heritable and expressed in their progeny.

[0102] The term "gene" refers to a nucleic acid sequence (this term is used interchangeably with "polynucleotide" or "nucleotide sequence"). A gene can include a cytokine transgene or encode a recombinant receptor. This definition includes various sequence polymorphisms, mutations, and / or sequence variants, and such changes do not substantially affect the function of the encoded cytokine transgene or recombinant receptor. The term "gene" may include not only the coding sequence but also regulatory regions such as promoters, enhancers, and termination regions. Furthermore, the term may include any introns and other DNA sequences spliced ​​from the mRNA transcript, as well as variants resulting from alternative splice sites. Gene sequences encoding these molecules may be DNA or RNA that direct the expression of an open reading frame within an artificial expression construct. These nucleic acid sequences may be DNA strand sequences transcribed into RNA or RNA sequences translated into protein. The nucleic acid sequences include both full-length nucleic acid sequences and subsequences derived from full-length proteins. These sequences may further include sequences that may be introduced to confer codon preference in a particular type of cell or degenerate codons from the native sequence. Portions of the complete gene sequences are also referenced throughout this disclosure, as would be understood by one of skill in the art.

[0103] Gene sequences comprising cytokine transgenes and / or gene sequences encoding recombinant receptors are provided herein, and these gene sequences can be readily produced by synthetic or recombinant methods from the relevant amino acid sequences based on other information provided herein. In some embodiments, the gene sequences encoding any of these sequences may have one or more restriction enzyme sites at the 5' and / or 3' end of the coding sequence to allow for easy excision of the gene sequences encoding these sequences and replacement with another gene sequence encoding a different sequence. In some embodiments, the gene sequences encoding the above sequences may be codon-optimized for expression in mammalian cells.

[0104] "Encode" refers to the property that a particular nucleotide sequence within a gene, such as a cDNA or mRNA, serves as a template for the synthesis of another macromolecule, such as a defined amino acid sequence. Thus, a gene encodes a protein when its corresponding mRNA is transcribed and translated to produce the protein in a cell or other biological system. A "gene sequence encoding a protein" includes all degenerate nucleotide sequences that encode the same amino acid sequence or an amino acid sequence of substantially similar form and function.

[0105] Multiple polynucleotide gene sequences encoding two or more portions of an expressed artificial expression construct can be operably linked to each other and to associated regulatory sequences. For example, a regulatory sequence can be operably linked to an exogenous nucleic acid sequence, thereby allowing the exogenous nucleic acid sequence to be expressed. In another example, a first nucleic acid sequence and a second nucleic acid sequence can be operably linked if they are positioned in a functional relationship. For example, a promoter is operably linked to a coding sequence if it affects the transcription or expression of the coding sequence. Typically, operably linked DNA sequences are contiguous and, if coding regions are necessary or useful, are linked in the same reading frame.

[0106] Promoters include general promoters, tissue-specific promoters, cell-specific promoters, and / or cytoplasm-specific promoters. Promoters can also include strong promoters, weak promoters, constitutive expression promoters, and / or inducible promoters. A constitutive promoter is a promoter that allows continuous transcription of a single associated gene or multiple associated genes. An inducible promoter induces expression in response to specific conditions, signals, or cellular events. For example, a promoter may be an inducible promoter that requires a specific ligand, small molecule, transcription factor, or hormone protein to induce transcription from the promoter.

[0107] Specific examples of constitutive promoters include the human elongation factor 1α promoter (EF1α, such as EF1α(s) and EF1α(L)), myeloproliferative sarcoma virus (MND) promoter, cytomegalovirus (CMV) promoter, simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV) promoter, human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukosis virus promoter, Epstein-Barr virus immediate early promoter, and Rous sarcoma virus promoter; as well as human gene promoters such as the actin promoter, myosin promoter, hemoglobin promoter, and creatine kinase promoter. In a specific embodiment, the recombinant receptor is under the control of EF1α (SEQ ID NO: 55).

[0108] A "vector" is a nucleic acid molecule capable of transporting another nucleic acid. A vector may be, for example, a plasmid (DNA or RNA plasmid), a transposon-based system, a cosmid, a bacterial artificial chromosome, a virus, or a phage. An "expression vector" is a vector that, when placed under appropriate circumstances, is capable of inducing the expression of a protein encoded by one or more genes incorporated into the expression vector.

[0109] " Lentivirus " refers to the virus of the retrovirus genus that can infect dividing and non-dividing cells. Some examples of lentivirus include HIV (human immunodeficiency virus: including HIV type 1 and HIV type 2); equine infectious anemia virus; feline immunodeficiency virus (FIV); bovine immunodeficiency virus (BIV); and simian immunodeficiency virus (SIV).

[0110] Lentiviral vectors are vectors derived from at least a portion of the lentiviral genome, and include, in particular, self-inactivating lentiviral vectors such as those described in Milone et al., Mol. Ther. 17(8): 1453-1464 (2009). Other examples of lentiviral vectors that may be used clinically include the LENTIVECTOR® gene delivery technology available from Oxford BioMedica and the LENTIMAX® gene delivery technology available from Lentigen. TM and vector systems. In addition, non-clinical lentiviral vectors are available and known to those skilled in the art. In certain embodiments, lentiviruses or lentiviral vectors are used to genetically engineer cells to express an artificial expression construct.

[0111] A "retrovirus" is a virus with an RNA genome. A "gammaretrovirus" refers to a virus belonging to the Retroviridae family. Exemplary gammaretroviruses include murine stem cell virus, murine leukemia virus, feline leukemia virus, feline sarcoma virus, and avian reticuloendotheliosis virus.

[0112] Retroviral vectors can also be used (see Miller, et al., 1993, Meth. Enzymol. 217:581-599). In such embodiments, the gene to be expressed is cloned into the retroviral vector and delivered into cells. In certain embodiments, the retroviral vector contains all the cis-acting sequences required for packaging and integration of the viral genome, namely, (a) long terminal repeats (LTRs) or portions thereof located at both ends of the vector, (b) primer binding sites for minus-strand and plus-strand DNA synthesis, and (c) packaging signals required for integration of genomic RNA into virions. Further details regarding retroviral vectors can be found in Boesen et al., 1994, Biotherapy 6:291-302; Clowes et al., 1994, J. Clin. Invest. 93:644-651; Kiem et al., 1994, Blood 83:1467-1473; Salmons and Gunzberg, 1993, Human Gene Therapy 4:129-141; and Grossman and Wilson, 1993, Curr. Opin. in Genetics and Devel. 3:110-114. Adenoviruses, adeno-associated viruses (AAV), and alphaviruses can also be used.Kozarsky and Wilson, 1993, Current Opinion in Genetics and Development 3:499-503, Rosenfeld, et al., 1991, Science 252:431-434; Rosenfeld, et al., 1992, Cell 68:143-155; Mastrangeli, et al., 1993, J. Clin. Invest. See Walsh, et al., 1993, Proc. Soc. Exp. Bioi. Med. 204:289-300; and Lundstrom, 1999, J. Recept. Signal Transduct. Res. 19: 673-686. Other gene delivery methods include the use of mammalian artificial chromosomes (Vos, 1998, Curr. Op. Genet. Dev. 8:351-359); liposomes (Tarahovsky and Ivanitsky, 1998, Biochemistry (Mosc) 63:607-618); ribozymes (Branch and Klotman, 1998, Exp. Nephrol. 6:78-83); and triple-helical DNA (Chan and Glazer, 1997, J. Mol. Med. 75:267-282).

[0113] There are numerous viral vectors available that are suitable for the present disclosure, including those identified for human gene therapy (see Pfeifer and Verma, 2001, Ann. Rev. Genomics Hum. Genet. 2:177). Methods for using retroviral and lentiviral vectors and packaging them into mammalian host cells to transduce them with viral particles containing transgenes are described, for example, in U.S. Patent Publication No. 8,119,772; Walchli et al., 2011, PLoS One 6:327930; Zhao et al., 2005, J. Immunol. 174:4415; Engels et al., 2003, Hum. Gene Ther. 14:1155; Frecha et al., 2010, Mol. Ther. 18:1748; and Verhoeyen et al., 2009, Methods Mol. Biol. 506:97. Furthermore, retroviral and lentiviral vector constructs and expression systems thereof are commercially available.

[0114] Targeted genetic engineering methods may also be used.CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) / Cas (CRISPR-associated protein) nuclease system is a recombinant nuclease system used in bacterial genetic engineering.Information about CRISPR-Cas system and its components can be found in, for example, U.S. Patent Publication No. 8697359, U.S. Patent Publication No. 8771945, U.S. Patent Publication No. 8795965, U.S. Patent Publication No. 8865406, U.S. Patent Publication No. 8871445, U.S. Patent Publication No. 8889356, U.S. Patent Publication No. 8889418, U.S. Patent Publication No. 8895308, U.S. Patent Publication No. 8906616 ... Nos. 8,932,814, 8,945,839, 8,993,233, and 8,999,641 and related applications; and WO2014 / 018423, WO2014 / 093595, WO2014 / 093622, WO2014 / 093635, WO2014 / 093655, WO2014 / 093661, WO2014 / 093694, WO2014 / 093701, WO2014 / 093709, WO2014 / 093712, WO2014 / 093718, WO2014 / 145599, WO2014 / 204723, WO2014 / 204724 , WO2014 / 204725, WO2014 / 204726, WO2014 / 204727, WO2014 / 204728, WO2014 / 204729, WO2015 / 065964, WO2015 / 089351, WO2015 / 089354, WO2015 / 089364, WO2015 / 089419, WO2015 / 089427, WO2015 / 089462, WO2015 / 089465, WO2015 / 089473, WO2015 / 089486, WO2016205711, WO2017 / 106657 and WO2017 / 127807 and related applications thereof.

[0115] Certain embodiments use zinc finger nucleases (ZFNs) as gene editing agents. ZFNs are a type of site-specific nuclease engineered to bind and cleave DNA at specific locations.For further information regarding ZFNs and ZFNs useful within the teachings of the present disclosure, see, e.g., U.S. Patent Publication Nos. 6,534,261; 6,607,882; 6,746,838; 6,794,136; 6,824,978; 6,866,997; 6,933,113; 6,979,539; 7,013, 219; U.S. Patent Publication No. 7,030,215; U.S. Patent Publication No. 7,220,719; U.S. Patent Publication No. 7,241,573; U.S. Patent Publication No. 7,241,574; U.S. Patent Publication No. 7,585,849; U.S. Patent Publication No. 7,595,376; U.S. Patent Publication No. 6,903,185; U.S. Patent Publication No. 6,479,626; U.S. Patent Publication No. 2003 / 0232410 and U.S. Patent Publication No. 2009 / 0203140, and Gaj et al., Nat Methods, 2012, 9(8):805-7;Ramirez et al., Nucl Acids Res, 2012, 40(12):5560-8;Kim et al., Genome Res, 2012, 22(7): 1327-33;Urnov et al., Nature Reviews Genetics, 2010, 11 :636-646;Miller, et al. Nature biotechnology 25, 778-785 (2007);Bibikova, et al. Science 300, 764 (2003);Bibikova, et al. Genetics 161, 1169-1175 (2002);Wolfe, et al. Annual review of biophysics and biomolecular structure 29, 183-212 (2000);Kim,et al. Proceedings of the National Academy of Sciences of the United States of America 93, 1156-1160 (1996); and Miller, et al. The EMBO journal 4, 1609-1614 (1985).

[0116] In certain embodiments, the gene editing agent can be a transcription activator-like effector nuclease (TALEN), which is a fusion protein comprising a transcription activator-like effector (TALE) DNA-binding protein and a DNA cleavage domain. For more information regarding TALENs, see U.S. Patent Publication No. 8,440,431; U.S. Patent Publication No. 8,440,432; U.S. Patent Publication No. 8,450,471; U.S. Patent Publication No. 8,586,363; and U.S. Patent Publication No. 8,697,853; as well as Joung and Sander, Nat Rev Mol Cell Biol, 2013, 14(1):49-55; Beurdeley et al., Nat Commun, 2013, 4: 1762; Scharenberg et al., Curr Gene Ther, 2013, 13(4):291-303; Gaj et al., Nat Methods, 2012, 9(8):805-7; Miller, et al. Nature biotechnology 29, 143-148 (2011); Christian, et al. Genetics 186, 757-761 (2010); Boch, et al. Science 326, 1509-1512 (2009); and Moscou, & Bogdanove, Science 326, 1501 (2009).

[0117] Nanoparticles capable of selectively genetically modifying target cells in vivo have been reported and can be used within the teachings of the present disclosure. In certain embodiments, the nanoparticles may be those described in WO2014153114, WO2017181110, and WO201822672.

[0118] (v) a control mechanism including a tag cassette, a transduction marker, a selection cassette, and / or a suicide switch; In certain embodiments, an artificial expression construct can include or encode one or more tag cassettes and / or transduction markers. Tag cassettes and transduction markers can be used in vitro, in vivo, and / or ex vivo to activate, promote growth, detect, enrich, isolate, track, remove, and / or eliminate genetically modified cells. A "tag cassette" refers to a unique synthetic peptide sequence attached to, fused to, or incorporated as part of an artificial expression construct, which specifically binds a cognate binding molecule (e.g., a ligand, antibody, or other binding partner) and thereby utilizes its binding properties to activate, promote growth, detect, enrich, isolate, track, remove, and / or eliminate the tagged protein and / or cells expressing the tagged protein. Transduction markers can also be used for the same purpose, but they are derived from naturally occurring molecules and are often expressed using skipping elements (or skip sequences) that separate the transduction marker from other components within the expressed molecule.

[0119] Examples of tag cassettes that bind to cognate binding molecules include His tag (HHHHHH; SEQ ID NO: 103), Flag tag (DYKDDDDK; SEQ ID NO: 104), Xpress tag (DLYDDDDK; SEQ ID NO: 105), Avi tag (GLNDIFEAQKIEWHE; SEQ ID NO: 106), calmodulin tag (KRRWKKNFIAVSAANRFKKISSSGAL; SEQ ID NO: 107), polyglutamic acid tag, HA tag (YPYDVPDYA; SEQ ID NO: 108), Myc tag (EQKLISEEDL; SEQ ID NO: 109), Strep tag (referring to the conventional STREP® tag (WRHPQFGG; SEQ ID NO: 110)), STREP® tag II (WSHPQFEK; SEQ ID NO: 111 (IBA Institut fur Bioanalytik GmbH, Germany); see, for example, U.S. Patent Publication No. 7,981,632), Softag 1 (SLAELLNAGLGGS; SEQ ID NO: 112), Softag 3 (TQDPSRVG; SEQ ID NO: 113), and V5 tag (GKPIPNPLLGLDST; SEQ ID NO: 114).

[0120] Binding molecules that specifically bind to and form complexes with the tag cassette sequences disclosed herein are commercially available. For example, His tag antibodies are commercially available from manufacturers such as Life Technologies, Pierce Antibodies, and GenScript. Flag tag antibodies are commercially available from manufacturers such as Pierce Antibodies, GenScript, and Sigma-Aldrich. Xpress tag antibodies are commercially available from manufacturers such as Pierce Antibodies, Life Technologies, and GenScript. Avi tag antibodies are commercially available from manufacturers such as Pierce Antibodies, IsBio, and Genecopoeia. Calmodulin tag antibodies are commercially available from manufacturers such as Santa Cruz Biotechnology, Abcam, and Pierce Antibodies. HA tag antibodies are commercially available from manufacturers such as Pierce Antibodies, Cell Signal, and Abcam. Myc tag antibodies are commercially available from manufacturers such as Santa Cruz Biotechnology, Abcam, and Cell Signal. Strep tag antibodies are commercially available from manufacturers such as Abcam, Iba, and Qiagen.

[0121] The transduction marker may be selected from at least one of truncated CD19 (tCD19; see Budde et al., Blood 122: 1660, 2013); truncated human EGFR (tEGFR or EGFRt; see Wang et al., Blood 118: 1255, 2011); a cell surface localization polypeptide tag based on truncated human HER2 (referred to as Her2tG); the ECD of human CD34; and / or RQR8, which combines a target epitope from the CD34 antigen (see Fehse et al., Mol. Therapy 1(5 Pt 1); 448-456, 2000) and a target epitope from the CD20 antigen (see Philip et al., Blood 124: 1277-1278). In certain embodiments, the cells are genetically modified to express EGFRt. In certain embodiments, the cells are genetically modified to express Her2tG.

[0122] In certain embodiments, the selection cassette allows for positive or negative selection of a desired cell population. Negative selection is a method of eliminating some types of cells while leaving the desired type of cells. Positive selection is a method of targeting a desired cell population and retaining only the desired cells.

[0123] The selection cassette can encode (a) a protein that confers resistance to an antibiotic or other toxin, (b) a protein that complements an auxotrophic deficiency, or (c) a protein that supplies an important nutrient unavailable from complex media, such as a gene encoding D-alanine racemase for Bacillus. The number of selection systems used to recover transformed cells is not particularly limited. In certain embodiments, the positive selection cassette comprises a gene for resistance to neomycin, hygromycin, ampicillin, puromycin, phleomycin, zeomycin, blasticidin, or viomycin. In certain embodiments, the selection cassette comprises a DHFR (dihydrofolate reductase) gene or a DHFR double mutant (DHFRdm) gene that confers resistance to methotrexate (MTX), O 6 The gene may include the MGMT P140K gene, which confers resistance to BG / BCNU, the HPRT (hypoxanthine phosphoribosyltransferase) gene, which is responsible for the conversion of specific bases (aminopterin, hypoxanthine, thymidine) contained in the HAT selection medium, or other genes responsible for the detoxification of several types of drugs. In a specific embodiment, the selection agent may be neomycin, hygromycin, puromycin, phleomycin, zeomycin, blasticidin, viomycin, ampicillin, or O. 6 Examples include BG / BCNU, MTX, tetracycline, aminopterin, hypoxanthine, thymidine kinase, DHFR, Gln synthetase, or ADA.

[0124] In certain embodiments, the selection cassette comprises DHFRdm and the selection agent comprises MTX. In certain embodiments, the selection method does not require a selection cassette to obtain a highly purified cell population.

[0125] In certain embodiments, the negative selection cassette contains a gene that converts a substrate contained in the culture medium into a substance toxic to the cell expressing it. Examples of such molecules include the diphtheria toxin (DTA) antidote gene (Yagi et al., Anal Biochem. 214(1):77-86, 1993; Yanagawa et al., Transgenic Res. 8(3):215-221, 1999) and the herpesvirus thymidine kinase gene (HSV TK), which is sensitive to the presence of ganciclovir or FIAU. The HPRT gene may also be used for negative selection by adding 6-thioguanine (6TG) to the culture medium. Poly(A) transcription termination sequences from various sources may also be used for positive and negative selection, the most common being those derived from SV40 poly(A) or poly(A) derived from eukaryotic genes (e.g., bovine growth hormone or rabbit β-globin).

[0126] In certain embodiments, the artificial expression construct may include a polynucleotide encoding a self-cleaving polypeptide or a skip sequence. In certain embodiments, the polynucleotide encoding the self-cleaving polypeptide is located between the cytokine transgene and the polynucleotide encoding the regulatory mechanism. In certain embodiments, the polynucleotide encoding the self-cleaving polypeptide is located between the polynucleotide encoding the recombinant receptor and the polynucleotide encoding the selection cassette. In certain embodiments, the polynucleotide encoding the self-cleaving polypeptide is located between the polynucleotide encoding the selection cassette and the polynucleotide encoding the transduction marker (e.g., EGFRt). Exemplary self-cleaving polypeptides include the 2A peptide (P2A) from porcine teschovirus-1, the 2A peptide (T2A) from Thosea asigna virus, the 2A peptide (E2A) from equine rhinitis A virus, the 2A peptide (F2A) from foot-and-mouth disease virus, or variants thereof. Furthermore, exemplary nucleic acid and amino acid sequences of 2A peptides are described, for example, in Kim et al. (PLOS One 6: e18556 (2011)). In certain embodiments, the cells are genetically modified to contain a self-cleaving polypeptide. In certain embodiments, the self-cleaving polypeptide comprises T2A. In certain embodiments, the 2A sequence does not leave a 2A signature. In certain embodiments, the 2A sequence adds an amino acid to a protein (also referred to as a 2A signature). The 2A sequence is a peptide sequence that induces ribosomal skipping during translation. Cleavage is triggered by ribosomal skipping of the peptide bond between proline and glycine in the 2A sequence. This ribosomal skipping adds an extra amino acid to the C-terminus of the upstream protein, and this extra amino acid has an unknown effect on the function of the protein.

[0127] The regulatory mechanisms may be included in multiple copies in the artificial expression construct, or may be expressed as separate molecules using skip sequences. For example, the artificial expression construct may have one, two, three, four, or five tag cassettes and / or one, two, three, four, or five transduction markers may be expressed. For example, embodiments of the present disclosure may include an artificial expression construct having two Myc tag cassettes, a cassette containing a His tag and an HA tag, a cassette containing an HA tag and a Softag1 tag, or a cassette containing a Myc tag and an SBP tag. Exemplary transduction markers and cognate pairs are described in U.S. Patent Publication No. 13 / 463,247.

[0128] One advantage of incorporating at least one regulatory mechanism into an artificial expression construct is that cells expressing the artificial expression construct administered to a subject can be expanded or eliminated by using a cognate binding molecule for the tag cassette. In certain embodiments, the present disclosure provides methods for eliminating engineered cells expressing the artificial expression construct by using an antibody specific for the tag cassette, by using a cognate binding molecule specific for the regulatory mechanism, or by using a second engineered cell that has specificity for the regulatory mechanism and expresses the artificial expression construct. Elimination of engineered cells can be achieved using a elimination agent specific for the regulatory mechanism. For example, when using EGFRt, an anti-EGFRt binding domain (e.g., an antibody or scFv) fused or conjugated to a cytotoxic reagent (e.g., a toxin or radioactive metal) can be used, or an anti-EGFRt / anti-CD3 bispecific scFv or anti-EGFRt CAR T cells can be used. Similarly, when Her2tG is used, an anti-Her2tG binding domain fused to or conjugated to a cytotoxic agent may be used.

[0129] In certain embodiments, a polynucleotide encoding an iCaspase 9 construct (iCasp9) may be inserted into an artificial expression construct as a suicide switch.

[0130] In certain embodiments, engineered cells expressing the artificial expression construct may be detected or tracked in vivo by using an antibody (e.g., an anti-tag antibody) that specifically binds to the regulatory mechanism, or by other cognate binding molecules that specifically bind to the regulatory mechanism, where the binding partner of the regulatory mechanism is conjugated to a fluorescent dye, radioactive tracer, iron oxide nanoparticle, or other imaging agent known in the art so that it can be detected by X-ray, CT scan, MRI scan, PET scan, ultrasound, flow cytometry, near-infrared imaging system, or other imaging method (see, e.g., Yu, et al., Theranostics 2:3, 2012).

[0131] Thus, engineered cells expressing at least one regulatory mechanism within an artificial expression construct can, for example, be more easily identified, isolated, sorted, induced to grow, tracked, and / or removed than engineered cells that do not incorporate a tag cassette.

[0132] (vi) recombinant receptor In certain embodiments, the recombinant receptor is or comprises a binding domain that binds to a target antigen, and is expressed from a cell by artificially introducing into the cell a nucleic acid encoding the recombinant receptor. The recombinant receptor may be, for example, a CAR, a T cell receptor (TCR), or a CAR / TCR hybrid.

[0133] CARs contain several distinctive components that enable, for example, genetically engineered cells (e.g., T cells) to recognize and kill target cells, such as cancer cells. These components include at least an extracellular portion and an intracellular portion. The extracellular portion contains a binding domain that specifically binds to a marker selectively displayed on the surface of unwanted cells. Upon binding of the binding domain to such a marker, the intracellular portion activates the genetically engineered cell to destroy the bound cell. CARs can further include a transmembrane domain that links the extracellular portion to the intracellular portion, as well as other components that can enhance the function of the recombinant receptor. For example, incorporating a spacer sequence and / or one or more linker sequences into a CAR can provide additional conformational flexibility to the recombinant receptor, often enhancing the ability of the binding domain to bind to a marker on the target cell.

[0134] TCRs are protein complexes found on the surface of T cells that recognize antigen fragments as peptides bound to major histocompatibility complex molecules. TCRs can be recombined to form eTCRs, which bind to specific antigens. TCRs are heterodimeric fusion proteins, typically comprising an α chain and a β chain. Each chain comprises a variable region (Vα and Vβ) and a constant region (Cα and Cβ). In certain embodiments, the eTCR does not contain the native TCR variable region, but does contain the native TCR constant region.

[0135] (vi-a) Binding domain In certain instances, the recombinant receptor comprises a binding domain that binds to a target antigen selectively displayed on the surface of undesired cells. The binding domain comprises a substance that binds to a cell marker to form a complex. The selection of the binding domain may depend on the type and number of cell markers that define the surface of the target cell. Examples of binding domains include cell marker ligands, receptor ligands, antibodies, peptides, peptide aptamers, receptors (e.g., T cell receptors), and combinations thereof, as well as recombinant fragments or recombinant formats thereof.

[0136] As understood by those skilled in the art, a full-length antibody comprises two heavy chains and two light chains. Each heavy chain comprises a variable region and a first, second, and third constant regions, while each light chain comprises a variable region and a constant region. Mammalian heavy chains are classified as α, δ, ε, γ, or μ chains, while mammalian light chains are classified as λ or κ chains. Immunoglobulins containing α, δ, ε, γ, or μ heavy chains are classified as immunoglobulin (Ig) A, IgD, IgE, IgG, or IgM, respectively. Full-length antibodies are "Y" shaped. The stem of a Y-shaped full-length antibody is composed of the second and third constant regions of each of the two heavy chains (IgE and IgM additionally comprise a fourth constant region), and a disulfide bond (interchain bond) is formed at the hinge region. The gamma, alpha, and delta heavy chains have a constant region consisting of three immunoglobulin domains linked in tandem (serialized) and a hinge region that provides flexibility; the mu and epsilon heavy chains have a constant region consisting of four immunoglobulin domains. The second and third constant regions are called the "CH2 domain" and "CH3 domain," respectively. Each arm of a full-length Y-shaped antibody consists of one heavy chain variable region and one constant region linked to one light chain, which also consists of a variable region and a constant region. The light and heavy chain variable regions are responsible for antigen binding.

[0137] Light and heavy chain variable regions contain a "framework" region sandwiched between three hypervariable regions, also called "complementarity-determining regions" or "CDRs."

[0138] CDR combinations may be numbered using, for example, Kabat numbering (Kabat et al. (1991) "Sequences of Proteins of Immunological Interest," 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md.) ("Kabat" numbering scheme); Chothia (Al-Lazikani et al. (1997) JMB 273:927-948 ("Chothia" numbering scheme)); Martin (Abinandan et al. (2008) Mol Immunol. 45:3832-3839 ("Martin" numbering scheme)); Gelfand (Gelfand and Kister (1995) Proc Natl Acad Sci USA. 92:10884-10888; Gelfand et al. (1998) Protein Eng. 11:1015-1025; Gelfand et al. (1996) Proc Natl Acad Sci USA. 93:3675-3678; Gelfand et al. (1998) J Comput Biol. 5:467-477 ("Gelfand" numbering scheme); Contact (MacCallum et al. (1996) J. Mol. Biol. 262:732-745 (Contact numbering scheme)); IMGT (Lefranc et al. (2003) Dev Comp Immunol 27(1):55-77 ("IMGT" numbering scheme)); AHo (Honegger and Pluckthun (2001) J Mol Biol 309(3):657-670 ("AHo" numbering scheme)); North (North et al. (2011) J Mol Biol. 406(2):228-256 ("North" numbering scheme); or other numbering schemes.Various software programs and bioinformatics tools can be used to determine CDR sequences. Examples of such software programs and bioinformatics tools include ABodyBuilder (Leem et al. (2016) MAbs 8(7):1259-1268), PIGSPro (Lepore et al. (2017) Nucleic Acids Res 45(W1):W17-W23), Kotai Antibody Builder (Yamashita et al. (2014) Bioinformatics 30(22):3279-3280), Rosetta Antibody (Weitzner et al. (2017) Nature Protocols 12:401-416), Paratome (Kunik et al. (2012) Nucleic Acids Res 40: W521-W524), and Antibody i-Patch (Krawczyk et al. (2013) Protein Eng Des Sel 26(10):621-629), and proABC-2 (Ambrosetti et al. (2020) Bioinformatics 36(20):5107-5108).

[0139] The sequences of the framework regions of various light and heavy chains are relatively conserved across species, including humans. The framework region of an antibody, comprised of multiple framework regions connected in the light and heavy chains that make up the antibody, plays a role in positioning and aligning the CDRs in three-dimensional space. The CDRs are primarily responsible for binding to an antigen epitope. The CDRs of each chain are generally designated CDR1, CDR2, and CDR3, numbered sequentially from the N-terminus, and are generally identified by the chain in which they are located. Thus, the CDRs present in the variable domain of an antibody's heavy chain are designated CDRH1, CDRH2, and CDRH3, while the CDRs present in the variable domain of an antibody's light chain are designated CDRL1, CDRL2, and CDRL3. Different antibodies have different CDRs (i.e., different binding sites for various antigens). While the CDRs differ from antibody to antibody, only a few amino acid positions within each CDR are directly involved in antigen binding. These amino acid positions within the CDRs are called specificity determining residues (SDRs).

[0140] "V H " or "VH" refers to the heavy chain variable region of an immunoglobulin. L " or "VL" refers to the variable region of an immunoglobulin light chain.

[0141] Antibodies that specifically bind to an antigen can be produced using methods for obtaining monoclonal antibodies, phage display techniques, methods for producing human or humanized antibodies, or methods using transgenic animals or plants that have been genetically modified to produce human antibodies. Phage display libraries of partially or fully synthetic antibodies are available, and these libraries can be screened for antibodies or fragments thereof that can bind to the target antigen. Phage display libraries of human antibodies are also available. Once the amino acid or polynucleotide sequences encoding the antibodies have been identified, these sequences can be isolated and / or sequenced. Many related antibodies are known and commercially available.

[0142] In some embodiments, the antibodies specifically bind to a surface molecule on a cancer cell or a virally infected cell and do not cross-react with non-specific components such as bovine serum albumin or other unrelated antigens.

[0143] "Antibody fragment" refers to at least a portion of an antibody that retains the ability to specifically bind to an antigen. Examples of antibody fragments include Fab, Fab', F(ab'), Fv fragments, single-chain variable (scFv) antibody fragments, disulfide-linked Fvs (sdFv), Fd fragments comprising a VH domain and a CH1 constant domain, linear antibodies, single-domain antibodies (e.g., sdAb (VL or VH)), camelid heavy chain variable domains only (VHH), multispecific antibodies formed from antibody fragments (e.g., a bivalent fragment comprising two Fab fragments linked at the hinge region by a disulfide bridge), and isolated CDR or other epitope-binding fragments of antibodies (Harlow et al., 1999, in Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, in Antibodies: A Laboratory Manual, Cold Spring Harbor, NY; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426). Antigen-binding fragments can also be incorporated into single domain antibodies, maxibodies, minibodies, nanobodies, intrabodies, diabodies, triabodies, tetrabodies, v-NARs, and bis-scFvs (see, e.g., Hollinger and Hudson (2005) Nature Biotechnology 23:1126-1136).

[0144] In certain embodiments, the binding domain may comprise a humanized form of a non-human (e.g., murine) antibody or an antigen-binding fragment thereof. Humanized antibodies include antibodies in which the framework and constant regions of one or more human immunoglobulin variable regions are fused to the binding region (e.g., CDRs) of an animal (non-human) immunoglobulin. Such humanized antibodies are designed to avoid an immune response against the non-human antibody from which the binding region is derived while retaining the binding specificity of the non-human antibody from which the binding region is derived. In certain embodiments, the binding domain may comprise a fully human antibody or antibody fragment thereof, where the entire molecule is of human origin or contains amino acid sequences identical to the human form of the antibody or immunoglobulin.

[0145] "scFv" refers to a recombinant fusion protein that comprises a VH and VL derived from an antibody linked by a linker and can be expressed as a single polypeptide chain. scFvs retain the specificity of the original intact antibody. In certain embodiments, the linker linking the variable regions may comprise a glycine-serine linker, such as the glycine-serine linkers set forth in SEQ ID NOS: 102 or 122-135, or glycine-serine linkers described elsewhere herein. In certain embodiments, scFvs may comprise a VL variable region and a VH variable region in any order, for example, relative to the N-terminus and C-terminus of the polypeptide. The scFv may comprise a VL-linker-VH or a VH-linker-VL.

[0146] Additionally, recombinant receptors include TCRs (including recombinant TCRs) that can be used alone or as binding domains within a CAR (e.g., CAR / TCR hybrids). For example, numerous TCR sequences that bind to specific antigen fragments are known and publicly available.

[0147] A TCR for use with a particular antigen can be identified, for example, by isolating T cells that bind to a particular antigen / MHC complex and determining the sequence of the TCR chain that binds to this antigen / MHC complex. The TCR gene encoding the TCR can be readily cloned, for example, by 5' RACE using primers corresponding to sequences specific to the TCR α chain gene and the TCR β chain gene.

[0148] In certain embodiments, after sequencing, it may be necessary to combine the TCR α and TCR β chains (i.e., perform analysis of the combined TCR chains). If necessary, various methods can be used to combine the TCR chains. For example, in silico, TCR chains can be combined using computer-based methods, such as immunological gene alignment software available from IMGT, JOINSOLVER, VDJSolver, SoDA, or iHMMune-align, or other similar tools for annotating VDJ gene segments. Assays such as PairSEQ® (Adaptive Biotechnologies Corp., Seattle, WA) have also been developed.

[0149] In certain embodiments, the recombinant TCR includes a single-chain T cell receptor (scTCR) comprising a Vα / β chain and a Cα / β chain (e.g., Vα-Cα, Vβ-Cβ, Vα-Vβ) or a Vα-Cα pair, a Vβ-Cβ pair, or a Vα-Vβ pair specific for a target of interest (e.g., a peptide-MHC complex).

[0150] In certain embodiments, a CAR / TCR hybrid comprises a CAR component and a TCR component. For example, a CAR / TCR hybrid may comprise a TCR binding domain and a CAR-specific intracellular signaling domain. This configuration allows antigen recognition similar to that of a TCR and utilizes intracellular signaling similar to that of a CAR.

[0151] Cancer antigens are proteins produced by cancer cells, and viral antigens are proteins produced by virus-infected cells. The binding domain of the recombinant receptor disclosed herein can be selected to bind to a cancer antigen or a viral antigen. In some embodiments, the cancer antigen or viral antigen is selectively expressed or overexpressed on cancer cells or infected cells compared to other cells of the same tissue type. In some embodiments, the cancer antigen or viral antigen is a cell surface molecule present on cancer cells or virus-infected cells, and is substantially absent on normal tissues or its expression is restricted to nonessential normal tissues.

[0152] In certain embodiments, the cancer antigen or viral antigen is selectively expressed by cancer cells or virally infected cells, respectively. "Selectively expressed" means that the antigen is found at least 25%, 35%, 45%, 55%, 65%, 75%, 85%, 95%, 96%, 97%, 98%, 99% or 100% more frequently in cells of the target type than in non-target cells.

[0153] Exemplary cancer antigens include carcinoembryonic antigen (CEA), prostate-specific antigen, prostate stem cell antigen (PSCA), PSMA, Her2 / neu, estrogen receptor, progesterone receptor, ephrinB2, CD19, CD20, CD22, CD23, CD123, CS-1, CE7, ROR1, mesothelin, c-Met, GD-2, MAGE A3 TCR, EGFR, EGFRvIII, EphA2, IL13Ra2, L1CAM, oaGD2, GD2, B7H3, CD33, FITC, VAR2CSA, MUC16, PD-L1, ERBB2, folate receptor (FOLR), CD56; glypican 2, disialoganglioside, EpCam, L1-CAM, Lewis Examples include Y, WT-1, tyrosinase-related protein 1 (TYRP1 / gp75); GD2, B-cell maturation antigen (BCMA), CD24, SV40 T, carbonic anhydrase IX (CAIX); and CD133. Other examples are known to those skilled in the art. In certain embodiments, a binding domain that specifically binds to CD19 is utilized.

[0154] In certain embodiments, the binding domain that binds to the cancer antigen comprises an scFv. In a specific embodiment, the scFv includes huCD19(G01S) scFv, muCD19(FMC63) scFv, CD20(Leu 16) scFv, CD22(m971) scFv, B7H3(hBRCA84D) scFv, L1CAM(CE7) scFv, EGFR scFv, EGFRVIII(806) scFv, EphA2(2A4) scFv, EpHA2(4H5) scFv, FITC(E2) scFv, GD2(hu3F8) scFv, Her2 (Herceptin) scFv, IL13Ra2(hu08)VlVh scFv, IL13Ra2 hu08 VhV1 scFv, IL13Ra2(hu07)VhV1 scFv, IL13Ra2(hu07)VhV1 Examples of the amino acid sequences include the amino acid sequences of scFv, oaGD2(8B6) VlVh, ROR1(R12) scFv, CD33(h2H12) VhVl scFv, CD33(h2H12) VlVh scFv, mesothelin (P4) scFv, VAR2CSA(ID1-DBL2Xb) scFv, and IL13Ra2 (IL13 zetakine).

[0155] In a specific embodiment, the huCD19(G01S) scFv with GMCSFss is It contains the sequence shown in MLLLVTSLLLCELPHPAFLLIPEVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGISWNSGRIGYADSVKGRFTISRDNAKNSLFLQMNSLRAEDTAVYYCARDQGYHYYDSAEHAFDIWGQGTVVTVSSGGGGSGGGGSGGGGSQSALTQPRSVSGFPGQSVTISCTGTTSDDVSWYQQHPGKAPQLMLYDVSKRPSGVPHRFSGSRSGRAASLIISGLQTEDEADYFCSSYAGRYNSVLFGGGTKLTVL (sequence number 115).

[0156] In a specific embodiment, the huCD19(G01S) scFv is It contains the sequence shown in EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGISWNSGRIGYADSVKGRFTISRDNAKNSLFLQMNSLRAEDTAVYYCARDQGYHYYDSAEHAFDIWGQGTVVTVSSGGGGSGGGGSGGGGSQSALTQPRSVSGFPGQSVTISCTGTTSDDVSWYQQHPGKAPQLMLYDVSKRPSGVPHRFSGSRSGRAASLIISGLQTEDEADYFCSSYAGRYNSVLFGGGTKLTVL (sequence number 116).

[0157] In a specific embodiment, the muCD19(FMC63) scFv is It includes the sequence shown in MLLLVTSLLLCELPHPAFLLIPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS (SEQ ID NO: 117).

[0158] In a specific embodiment, the CD19 scFv is It contains the sequence shown in DIQMTQTTSSLSALGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS (SEQ ID NO: 118).

[0159] In a specific embodiment, the CD33(h2H12) VhVl scFv is It contains the sequence shown in QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYDINWVRQAPGQGLEWIGWIYPGDGSTKYNEKFKAKATLTADTSTSTAYMELRSLRSDDTAVYYCASGYEDAMDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTINCKASQDINSYLSWFQQKPGKAPKTLIYRANRLVDGVPSRFSGSGSGQDYTLTISSLQPEDFATYYCLQYDEFPLTFGGGTKVEIK (sequence number 119).

[0160] In a specific embodiment, the CD33(h2H12) VlVh scFv is It contains the sequence shown in DIQMTQSPSSLSASVGDRVTINCKASQDINSYLSWFQQKPGKAPKTLIYRANRLVDGVPSRFSGSGSGQDYTLTISSLQPEDFATYYCLQYDEFPLTFGGGTKVEIKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTNYDINWVRQAPGQGLEWIGWIYPGDGSTKYNEKFKAKATLTADTSTSTAYMELRSLRSDDTAVYYCASGYEDAMDYWGQGTTVTVSS (SEQ ID NO: 120).

[0161] In certain embodiments, the CD33 scFv (e.g., CD33(h2H12) VhVl scFv and / or CD33(h2H12) VlVh scFv) comprises a granulocyte-macrophage colony-stimulating factor (GM-CSF) signal sequence. In certain embodiments, the GM-CSF signal sequence comprises the sequence set forth in MLLLVTSLLLCELPHPAFLLIP (SEQ ID NO: 121).

[0162] In certain embodiments, the binding domain that binds to CD20 may be based on the binding domain of SP32 (ab64088), EP459Y (ab78237), rIGEL / 773 (ab219329), ocrelizumab, rituximab, ofatumumab, obinutuzumab, ibritumomab, or tositumomab.

[0163] Exemplary viral antigens include coronavirus antigens: spike (S) protein; cytomegalovirus antigens: envelope glycoprotein B and CMV pp65; Epstein-Barr virus antigens: EBV EBNAI, EBV P18, and EBV P23; hepatitis antigens: hepatitis B virus S protein, M protein, and L protein, hepatitis B virus pre-S antigen, HBCAGδ, HBV HBE, hepatitis C virus RNA, HCV NS3, and HCV NS4; herpes simplex virus antigens: immediate early protein and glycoprotein D; HIV antigens: gene products of the gag gene, pol gene, and env gene, e.g., HIV gp32, HIV gp41, HIV gp120, HIV gp160, HIV P17 / 24, HIV P24, HIV P55 GAG, HIV P66 POL, HIV TAT, HIV Examples of viral antigens include GP36, Nef protein, and reverse transcriptase; influenza antigens: hemagglutinin and neuraminidase; Japanese encephalitis virus antigens: E protein, ME protein, ME-NS1 protein, NS1 protein, NS1-NS2A protein, and 80%E protein; measles virus antigens: measles virus fusion protein; rabies virus antigens: rabies virus glycoprotein and rabies virus nucleoprotein; respiratory syncytial virus antigens: RSV fusion protein and M2 protein; rotavirus antigens: VP7sc; rubella virus antigens: E1 protein and E2 protein; and varicella-zoster virus antigens: gpI and gpII. For further examples of viral antigens, see Fundamental Virology, Second Edition, eds. Fields, BN and Knipe, DM (Raven Press, New York, 1991). In certain embodiments, binding domains that bind to viral antigens can be used.

[0164] In certain embodiments, bacterial antigens include antigens expressed by bacteria, hi certain embodiments, fungal antigens include antigens expressed by fungi, and hi certain embodiments, arthropod antigens include antigens expressed by arthropods.

[0165] In certain embodiments, the binding domain binds to an epitope that activates an immune cell. In certain embodiments, the epitope that activates an immune cell is a part of an antigen. In certain embodiments, the epitope that activates an immune cell may be expressed by immune cells in the negative fraction of a sample and / or may be a binding domain of a multispecific binding molecule (also called a chemical adaptor). The epitope that activates an immune cell is a part of a molecule (e.g., a part of a protein) that activates an immune cell when a binding domain expressed by the immune cell binds to the epitope. The epitope that activates an immune cell may be present on anything that can bind to a recombinant receptor on a T cell and link the bound T cell to an activating cell (e.g., PBMC). In certain examples, the chemical adaptor is an antibody.

[0166] In certain embodiments, the epitope that activates an immune cell may be a B cell ligand, such as CD1d, CD5, CD19, CD20, CD21, CD22, CD23 / FcεRII, CD24, CD25 / IL-2Rα, CD27 / TNFRSF7, CD32, CD34, CD35, CD38, CD40(TNFRSF5), CD44, CD45, CD45.1, CD45.2, CD54(ICAM-1), CD69, CD72, CD79, CD80, CD84 / SLAMF5, LFA-1, CALLA, BCMA, B cell receptor (BCR), IgM, IgD, B220 / CD45R, C1q R1 / CD93, CD84 / SLAMF5, BAFF R / TNFRSF13C, B220 / CD45R, B7-1 / CD80, B7-2 / CD86, TNFSF7, TNFRSF5, ENPP-1, HVEM / TNFRSF14, BLIMP 1 / PRDM1, CXCR4, DEP-1 / CD148 or EMMPRIN / CD147.

[0167] Other epitopes that activate immune cells can be found, for example, on natural killer T (NKT) cells, natural killer cells (also known as K cells or killer cells), tumor-infiltrating lymphocytes (TILs), bone marrow-infiltrating lymphocytes (MILs), MAIT cells, macrophages, monocytes, and / or dendritic cells. These cells and exemplary cell surface antigens are described elsewhere herein.

[0168] In certain embodiments, the immune cell-activating epitope is a hapten. A hapten can be any small molecule that, when combined with a larger carrier, such as a protein, elicits the production of antibodies that specifically bind to the hapten (free or bound to the carrier). Haptens include peptides, other larger chemical entities, and aptamers. In some embodiments, the hapten can be any of the haptens provided in the hapten database accessible on the World Wide Web at the following URL: crdd.osdd.net / raghava / haptendb / . In certain embodiments, the hapten is tethered to an activating cell. In certain embodiments, when the immune cell-activating epitope is a hapten, the cell expressing the recombinant receptor comprises a binding domain that binds to the hapten. In certain embodiments, the binding domain that binds to the hapten can be the binding domain of the recombinant receptor. In certain embodiments, the binding domain that binds to the hapten can be a binding domain that is not present in the recombinant receptor.

[0169] In certain embodiments, the epitope that activates immune cells may be a binding domain of a multispecific binding molecule (also called a chemical adaptor). In certain embodiments, the multispecific binding molecule comprises at least two binding domains, at least one of which is an immune cell-activating epitope that binds to T cells expressing the recombinant receptor and at least one of which binds to immune cells in the negative fraction. Multispecific binding molecules useful for activating T cells are described elsewhere herein. In certain embodiments, the multispecific binding molecule comprises a bispecific antibody. In certain embodiments, the multispecific binding molecule comprises an antibody. An antibody can be considered a multispecific binding molecule because the antigen-binding domain of the antibody binds to the antigen and the Fc portion interacts with immune cells in the negative fraction.

[0170] (vi-b) Transmembrane domain As described herein, the transmembrane domain in the recombinant receptor serves to link the extracellular portion with the intracellular portion through the cell membrane, and can tether the expressed molecule to the cell membrane in the engineered cell.

[0171] The transmembrane domain may be naturally occurring and / or synthetic. If naturally occurring, the transmembrane domain may be derived from a membrane-bound or transmembrane protein. The transmembrane domain may include at least the transmembrane region of the α, β, or ζ chain of the T cell receptor, CD28, CD27, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, or TLR9. In certain embodiments, the transmembrane domain comprises at least one of, for example, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, IL2Rβ, IL2Rγ, IL7Rα, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDl 1d, ITGAE, CD103, ITGAL, CDl 1a, ITGAM, CDl 1b, ITGAX, CDl It may also include the transmembrane domain of lc, ITGB1, CD29, ITGB2, CD18, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, LyL08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​PAG / Cbp, NKG2D, or NKG2C. In certain embodiments, various human hinges can be used as well, such as human Ig (immunoglobulin) hinges (e.g., IgG4 hinges and IgD hinges), GS linkers (e.g., GS linkers described herein), KIR2DS2 hinges, CD8a hinges, etc.

[0172] In certain embodiments, the transmembrane domain has a three-dimensional structure, typically 15 to 30 amino acids long, that is thermodynamically stable in the cell membrane and can include an α-helix, a β-barrel, a β-sheet, a β-helix, or any combination thereof.

[0173] The transmembrane domain may include one or more additional amino acids adjacent to the transmembrane region, for example, one or more amino acids within the extracellular region of the recombinant receptor (e.g., up to 15 amino acids of the extracellular region) and / or one or more additional amino acids within the intracellular region of the recombinant receptor (e.g., up to 15 amino acids of the intracellular portion). In one embodiment, the transmembrane domain may be derived from the same protein as the protein from which the signaling domain, costimulatory domain, or hinge domain is derived. In another embodiment, the transmembrane domain is derived from a protein separate from the protein from which the other domains of the recombinant receptor are derived. In some cases, the transmembrane domain may be selected or modified by amino acid substitutions that allow each domain of the recombinant receptor to avoid binding to transmembrane domains from the same or different surface membrane proteins, thereby minimizing interactions with other unintended members of the receptor complex. In a specific embodiment, the transmembrane domain is encoded by a nucleic acid sequence encoding the transmembrane domain of CD28 (SEQ ID NO: 67, 68, 69, or 70). In a specific embodiment, the transmembrane domain comprises the amino acid sequence of the transmembrane domain of CD28 (SEQ ID NO: 64, 65, or 66).

[0174] (vi-c) Intracellular effector domain The intracellular effector domain of a recombinant receptor is responsible for activating the cell in which the recombinant receptor is expressed. Thus, the term "effector domain" is intended to include a portion of the intracellular domain sufficient to transduce an activating signal. The effector domain can directly or indirectly promote a biological or physiological response of the cell when it receives an appropriate signal. In certain embodiments, the effector domain is part of a protein or protein complex that receives a signal when the recombinant receptor binds to a target molecule, or it binds directly to the target molecule, thereby inducing a signal from the effector domain. The effector domain may directly promote a cellular response if it contains one or more signaling domains or signaling motifs, such as immunoreceptor tyrosine-based activation motifs (ITAMs). In another embodiment, the effector domain promotes a cellular response indirectly by associating with one or more additional proteins that directly promote the cellular response, such as a costimulatory domain.

[0175] The effector domain can activate at least one function of the modified cell upon binding of the modified cell to a cell marker expressed by a cancer cell. Activation of the modified cell can include one or more of differentiation, proliferation, and / or activation, or other effector functions. In certain embodiments, the effector domain can include an intracellular signaling moiety comprising a T cell receptor and a costimulatory domain, and the costimulatory domain can include a cytoplasmic sequence derived from a coreceptor or costimulatory molecule.

[0176] The effector domain may comprise one, two, or more intracellular signaling moieties (e.g., receptor signaling domains, cytoplasmic signaling sequences, etc.), costimulatory domains, or combinations thereof. Exemplary effector domains include signaling and / or stimulatory domains selected from 4-1BB (CD137), CARD11, CD3γ, CD3δ, CD3ε, CD3ζ, CD27, CD28, CD79A, CD79B, DAP10, FcRα, FcRβ (FcεR1b), FcRγ, Fyn, HVEM (LIGHTR), ICOS, LAG3, LAT, Lck, LRP, NKG2D, NOTCH1, pTα, PTCH2, OX40, ROR2, Ryk, SLAMF1, Slp76, TCRα, TCRβ, TRIM, Wnt, Zap70, and any combination thereof.In certain embodiments, exemplary effector domains include CD86, FcγRIIa, DAP12, CD30, CD40, PD-1, lymphocyte function-associated antigen 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds to CD83, CDS, ICAM-1, GITR, BAFFR, SLAMF7, NKp80 (KLRF1), CD127, CD160, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, ITGAM, CD11b, ITGAX, CD11c, and ITGB1. , CD29, ITGB2, CD18, ITGB7, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​GADS, PAG / Cbp, NKp44, NKp30, NKp46, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, and TLR9. In certain embodiments, the effector domain comprises the signaling domain of CD3ζ.

[0177] The stimulatory intracellular signaling subsequence may comprise an iTAM. Examples of iTAMs comprising a major cytoplasmic signaling sequence include those derived from CD3γ, CD3δ, CD3ε, CD3ζ, CD5, CD22, CD66d, CD79a, CD79b, common FcRγ (FCER1G), FcγR11a, FcRβ (Fcε Rib), DAP10, or DAP12. In certain embodiments, variants of CD3ζ retain at least one, two, three, or all of the ITAM regions.

[0178] In certain embodiments, the effector domain comprises a cytoplasmic portion that associates with a cytoplasmic signaling protein, the cytoplasmic signaling protein being a lymphocyte receptor or signaling domain thereof, a protein containing multiple ITAMs, a costimulatory domain, or any combination thereof.

[0179] Further examples of intracellular signaling moieties include the cytoplasmic sequences of the CD3 zeta chain and / or coreceptors that act in concert with this to initiate subsequent signaling upon association with the binding domain.

[0180] The costimulatory domain is a domain whose activation is required for efficient lymphocyte response to binding to a cell marker. Several molecules can be substituted as intracellular signaling moieties or costimulatory domains. Examples of costimulatory domains include CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, and B7-H3, as well as ligands that specifically bind to CD83. For example, it has been demonstrated that CD27 costimulation enhances the proliferation, effector function, and survival of human CAR-T cells in vitro, and enhances the persistence and anti-cancer activity of human T cells in vivo (Song et al. Blood. 2012; 119(3):696-706). Further examples of such costimulatory domain molecules include CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDlld, ITGAE, CD103, ITGAL, CDlla, ITGAM, and CD1 lb, ITGAX, CDllc, ITGB1, CD29, ITGB2, CD18, ITGB7, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), NKG2D, CEACAM1, CRTAM, Ly9 (CD229), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, and CD19a. In certain embodiments, the costimulatory domain comprises the signaling domain of 4-1BB.

[0181] In certain embodiments, the nucleic acid sequence encoding the intracellular signaling moiety comprises a sequence encoding CD3ζ (SEQ ID NO: 80 or 81) and a sequence encoding a variant of the signaling moiety of 4-1BB (SEQ ID NO: 74, 75, or 76). In certain embodiments, the amino acid sequence of the intracellular signaling moiety comprises an intracellular signaling moiety comprising a variant of CD3ζ (SEQ ID NO: 77, 78, or 79) and a portion of 4-1BB (SEQ ID NO: 71, 72, or 73).

[0182] The intracellular portion may be a member of the Wnt signaling pathway (e.g., LRP, Ryk, or ROR2), a NOTCH signaling pathway (e.g., NOTCH1, NOTCH2, NOTCH3, or NOTCH4), a Hedgehog signaling pathway (e.g., PTCH or SMO), a receptor tyrosine kinase (RTK) (e.g., epidermal growth factor (EGF) receptor family, fibroblast growth factor (FGF) receptor family, hepatocyte growth factor (HGF) receptor family, insulin receptor (IR) family, platelet-derived growth factor (PDGF) receptor family, vascular endothelial growth factor (VEGF) receptor family, tropomyosin receptor kinase (Trk) receptor family, ephrin (Eph) receptor family, AXL receptor family, leukocyte tyrosine kinase (LTK) receptor family, tyrosine kinase with immunoglobulin-like and EGF-like domains 1 (TIE) receptor family, receptor tyrosine kinase-like orphan (ROR) receptor family, discoidin domain (DDR) receptor family, rearranged during transfection (RET) receptor family, tyrosine protein kinase-like (PTK7) receptor family, related to receptor tyrosine kinase (RYK) receptor family, or muscle-specific kinase (MuSK) receptor family); G protein-coupled receptors (GPCRs) (Frizzled or Smoothened); serine / threonine kinase receptors (BMPR or TGFR); or cytokine receptors (IL1R, IL2R, IL7R, or IL15R).

[0183] (vi-d) Linker The linker in a recombinant receptor can be any part of the recombinant receptor, as long as it can serve to connect two components or domains of the recombinant receptor. In certain embodiments, the linker can provide flexibility to the various components of the recombinant receptor. The linker may further comprise a spacer region and a linking amino acid. In certain instances, when a more rigid linker is required, a proline-rich linker can be used. While a linker may serve the sole purpose of linking components, many linkers also serve other purposes besides linking, such as multimerization domains.

[0184] A spacer is used to provide appropriate distance from and / or flexibility relative to other components of the recombinant receptor. As described herein, in certain embodiments, the length of the spacer is customized to enable binding to target cells and inducing cell destruction. In certain embodiments, the length of the spacer can be selected depending on the location of the epitope of the cell marker, the affinity of the binding domain for the epitope, and / or the ability of the binding domain to induce cell destruction after binding to the target.

[0185] Typical spacers include spacers of 10 to 250 amino acids in length, spacers of 10 to 200 amino acids in length, spacers of 10 to 150 amino acids in length, spacers of 10 to 100 amino acids in length, spacers of 10 to 50 amino acids in length, and spacers of 10 to 25 amino acids in length.

[0186] In certain embodiments, the spacer is 5, 8, 10, 12, 14, 20, 21, 26, 27, 45, 50, or 75 amino acids in length, and these spacers are considered short spacers.

[0187] In certain embodiments, the spacer is 76, 90, 100, 110, 120, 125, 128, 131, 135, 140, 150, 160, 170, or 179 amino acids in length. Spacers of such lengths are considered medium-length spacers.

[0188] In certain embodiments, the spacer is 180, 190, 200, 210, 212, 214, 216, 218, 220, 228, 230, 240, 250, 260, or 270 amino acids in length. Spacers of such lengths are considered long spacers.

[0189] Exemplary spacers include the entire length or a portion of an immunoglobulin hinge region. The immunoglobulin hinge region may be a wild-type immunoglobulin hinge region or a modified wild-type immunoglobulin hinge region. In a specific embodiment, the immunoglobulin hinge region is a human immunoglobulin hinge region. As used herein, the term "wild-type immunoglobulin hinge region" refers to the amino acid sequence of the hinge located between and connecting the CH1 and CH2 domains of the heavy chain in the upper and middle parts of a natural antibody (in the case of IgG, IgA, and IgD), or the amino acid sequence of the hinge located between and connecting the CH1 and CH3 domains of the heavy chain (in the case of IgE and IgM).

[0190] The immunoglobulin hinge region may be that of IgG, IgA, IgD, IgE, or IgM. The IgG hinge region may be that of IgG1, IgG2, IgG3, or IgG4. Sequences derived from IgG1, IgG2, IgG3, IgG4, or IgD may each be used alone, in combination with the entire CH2 region or a portion thereof, in combination with the entire CH3 region or a portion thereof, or in combination with the entire CH2 region or a portion thereof and the entire CH3 region or a portion thereof. In a specific embodiment, the IgG4 hinge region comprises the sequence set forth in SEQ ID NO: 58.

[0191] Other examples of hinge regions that can be used in the recombinant receptors described herein include those found in the extracellular domains of type 1 membrane proteins, such as CD8α, CD4, CD28, and CD7, whether wild-type or variants thereof.

[0192] In certain embodiments, the spacer comprises the hinge region of the interdomain region (stalk region) of a type II C-type lectin or the hinge region of the stalk region of a cluster of differentiation (CD) molecule. The "stalk region" of a type II C-type lectin or CD molecule refers to a portion of the extracellular domain (ECD) located between the C-type lectin-like domain (CTLD) (e.g., similar to the CTLD of a natural killer cell receptor) and the hydrophobic portion (transmembrane domain) in the type II C-type lectin or CD molecule. For example, the extracellular domain of human CD94 (GenBank Accession No. AAC50291.1) corresponds to amino acid residues 34-179, whereas the CTLD corresponds to amino acid residues 61-176. The stalk region of the human CD94 molecule comprises amino acid residues 34-60, and is therefore located between the hydrophobic portion (transmembrane domain) and the CTLD (see Boyington et al., Immunity 10:15, 1999; for descriptions of other stalk regions, see also Beavil et al., Proc. Nat'l. Acad. Sci. USA 89:153, 1992; and Figdor et al., Nat. Rev. Immunol. 2:11, 2002). These type II and C lectin or CD molecules may have additional linking amino acids between the stalk region and the transmembrane domain or between the stalk region and the CTLD (see below). In another example, the 233 amino acid long human NKG2A protein (GenBank Accession No. P26715.1) has a hydrophobic portion (transmembrane domain) consisting of amino acids 71 ​​to 93 and an extracellular domain consisting of amino acids 94 to 233. The CTLD of the human NKG2A protein comprises amino acids 119 to 231, and the stalk region comprises amino acids 99 to 116, and this stalk region may be flanked by additional linking amino acids.Other type II C-type lectins or CD molecules, or their extracellular ligand-binding domains, stalk regions and CTLDs, are also known in the art (e.g., see GenBank Accession Nos. NP 001993.2; AAH07037.1; NP 001773.1; AAL65234.1; and CAA04925.1 for the sequences and descriptions of human CD23, human CD69, human CD72, human NKG2A, and human NKG2D, respectively).

[0193] The linker can, for example, link the VL and VH of antibody-derived binding domains in an scFv, and can function as a linking amino acid between components of the recombinant receptor.

[0194] The linker may be flexible, rigid, or semi-rigid, depending on the desired function of the linker. The linker may further comprise a linking amino acid. For example, in certain embodiments, the linker provides flexibility and space for conformational movement between the various components of the recombinant receptor. A commonly used flexible linker is the Gly-Ser linker. In certain embodiments, the linker sequence comprises a repeating sequence of glycine and serine, e.g., 1 to 10 (Gly x Ser y ) n where x and y are independently integers of 0 to 10, excluding the case where both x and y are 0, and n is an integer of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. A specific example is a repeat sequence consisting of (Gly4Ser) n (SEQ ID NO: 122), (Gly3Ser) n (Gly4Ser) n (SEQ ID NO: 123), (Gly3Ser) n (Gly2Ser) n (SEQ ID NO: 124), or (Gly3Ser) nIn certain embodiments, the linker is (Gly4Ser) (SEQ ID NO: 126), (Gly4Ser) (SEQ ID NO: 102), (Gly4Ser) (SEQ ID NO: 128), (Gly4Ser) (SEQ ID NO: 129), (Gly3Ser) (SEQ ID NO: 130), (Gly3Ser) (SEQ ID NO: 131), (Gly2Ser) (SEQ ID NO: 132), (Gly2Ser) GGSGGGSGGSG (SEQ ID NO: 133), GGSGGGSGSG (SEQ ID NO: 134), or GGSGGGSG (SEQ ID NO: 135).

[0195] In certain embodiments, the linker region is (GGGGS) n (SEQ ID NO: 122), where n is an integer, including 1, 2, 3, 4, 5, 6, 7, 8, 9 or more. In certain embodiments, the spacer is (EAAAK) n (SEQ ID NO: 137), where n is an integer including 1, 2, 3, 4, 5, 6, 7, 8, 9 or more integers.

[0196] In some cases, a flexible linker may not be able to maintain the distance or position of recombinant receptors required for a particular application. In such cases, a rigid or semi-rigid linker may be useful. An example of a rigid or semi-rigid linker is a proline-rich linker. In certain embodiments, a proline-rich linker is a peptide sequence that has more proline residues than would be expected to be present in the sequence by chance alone. In certain embodiments, a proline-rich linker is a linker whose sequence is composed of at least 30%, at least 35%, at least 36%, at least 39%, at least 40%, at least 48%, at least 50%, or at least 51% proline residues. Specific examples of proline-rich linkers include fragments of salivary proline-rich proteins (PRPs).

[0197] The linker may be susceptible to cleavage, such as acid-induced cleavage, light-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage, disulfide bond cleavage, etc. (cleavable linker). Alternatively, the linker may be substantially resistant to cleavage (e.g., stable linker or non-cleavable linker). In some embodiments, the linker is a charge-prone linker, a hydrophilic linker, or a dicarboxylic acid-based linker.

[0198] The linking amino acid may be a linker that can be used to link sequences when a spacer is not required and / or desirable to provide distance. For example, the linking amino acid may be a short amino acid sequence that can be used to link intracellular costimulatory signaling moieties. In certain embodiments, the linking amino acid is 9 amino acids or less in length (e.g., 2, 3, 4, 5, 6, 7, 8, or 9 amino acids in length). In certain embodiments, a glycine-serine doublet can be used as a suitable linking amino acid linker. In certain embodiments, a single amino acid, such as alanine or glycine, can be used as a suitable linking amino acid.

[0199] In certain embodiments, the recombinant receptor may contain a multimerization domain. The biological activity of a protein depends on its tertiary and quaternary structure. Quaternary structure requires physical and chemical interactions with other protein subunits or polypeptides. A "multimerization domain" is a domain that allows two or more proteins (monomers) to interact with each other through covalent and / or non-covalent bonds. The presence of a multimerization domain in a protein allows the proteins to interact to form dimers, trimers, tetramers, pentamers, hexamers, heptamers, etc., depending on the number of units / monomers incorporated into the multimer.

[0200] (vii) Ex vivo produced cell preparations In certain embodiments, the genetically modified cells can be harvested from the culture medium, washed, concentrated, and mixed with a carrier in a therapeutically effective amount. Exemplary carriers include saline, buffered saline, normal saline, water, Hank's solution, Ringer's solution, Normosol-R (Abbott Labs), PLASMA-LYTE A® (Baxter Laboratories, Morton Grove, IL), and combinations thereof.

[0201] In certain embodiments, human serum albumin (HSA) or other human serum components or fetal bovine serum can be added to the carrier. In certain embodiments, the carrier for infusion contains buffered saline supplemented with 5% HSA or dextrose. Other tonicity agents include polyhydric sugar alcohols, including trihydric or higher hydric sugar alcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol, and mannitol.

[0202] The carrier may contain a buffer such as a citrate buffer, a succinate buffer, a tartrate buffer, a fumarate buffer, a gluconate buffer, an oxalate buffer, a lactate buffer, an acetate buffer, a phosphate buffer, a histidine buffer and / or a trimethylamine salt.

[0203] Stabilizers refer to a wide variety of additives that can have various functions, from preventing cell adhesion to the container wall to bulking agents. Typical stabilizers include polyhydric sugar alcohols; amino acids such as arginine, lysine, glycine, glutamine, asparagine, histidine, alanine, ornithine, L-leucine, 2-phenylalanine, glutamic acid, and threonine; organic sugars or sugar alcohols such as lactose, trehalose, stachyose, mannitol, sorbitol, xylitol, ribitol, myo-inositol, galactitol, glycerol, and cyclitols (e.g., inositol); PEG; amino acid polymers; urea, glutathione, and thioctophosphate. These include sulfur-containing reducing agents such as acids, sodium thioglycolate, thioglycerol, α-monothioglycerol, and sodium thiosulfate; low molecular weight polypeptides (i.e., polypeptides with fewer than 10 residues); proteins such as HSA, bovine serum albumin, gelatin, and immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; monosaccharides such as xylose, mannose, fructose, and glucose; disaccharides such as lactose, maltose, and sucrose; trisaccharides such as raffinose; and polysaccharides such as dextran.

[0204] Where necessary or beneficial, the formulation may also include a local anesthetic such as lidocaine to ease pain at the site of the injection.

[0205] Exemplary preservatives include phenol, benzyl alcohol, m-cresol, methylparaben, propylparaben, octadecyldimethylbenzylammonium chloride, benzalkonium halides, hexamethonium chloride, alkylparabens (e.g., methylparaben and propylparaben), catechol, resorcinol, cyclohexanol, and 3-pentanol.

[0206] The therapeutically effective amount of cells contained in the formulation is 10 2 More than 10 3 More than 10 4 More than 10 5More than 10 6 More than 10 7 More than 10 8 More than 10 9 More than 10 10 More than 10 11 The number may be more than one.

[0207] In the formulations disclosed herein, the cells are typically contained in a volume of 1 L or less, 500 ml or less, 250 ml or less, or 100 ml or less. Thus, the density of administered cells is typically 10 4 Density greater than 10 7 Density greater than 10 8 The density is greater than 1 / ml.

[0208] In certain embodiments, the formulation may include one or more types of genetically modified cells (e.g., modified T cells, modified NK cells, or modified stem cells), or the formulation may include different types of genetically modified cells (e.g., a combination of T cells, NK cells, and / or stem cells).

[0209] Different types of genetically modified cells or cell subsets (e.g., modified T cells, modified NK cells, and / or modified stem cells) can be provided in various ratios, such as a 1:1:1 ratio, a 2:1:1 ratio, a 1:2:1 ratio, a 1:1:2 ratio, a 5:1:1 ratio, a 1:5:1 ratio, a 1:1:5 ratio, a 10:1:1 ratio, a 1:10:1 ratio, a 1:1:10 ratio, a 2:2:1 ratio, a 1:2:2 ratio, a 2:1:2 ratio, a 5:5:1 ratio, a 1:5:5 ratio, a 5:1:5 ratio, a 10:10:1 ratio, a 1:10:10 ratio, a 10:1:10 ratio, etc. These ratios can also apply to numbers of cells expressing the same cytokine transgene and / or recombinant receptor, or different cytokine transgenes and / or recombinant receptors. When only two types of cells are combined, or when the formulation contains only two types of expressed cytokine transgene components, the ratio may be any combination of two values ​​that can be created from the combinations of the three values ​​above. In some embodiments, the combined cell population is tested in vitro, in vivo, and / or ex vivo for efficacy and / or cell proliferation, and a ratio of cells that provides efficacy and / or cell proliferation is selected. Specific embodiments include a 1:1 ratio of CD4 T cells to CD8 T cells.

[0210] The cell-based formulations disclosed herein can be prepared for administration by, for example, injection, infusion, perfusion, or lavage, and can be further formulated for bone marrow, intravenous, intradermal, intraarterial, intralymph node, intralymphatic, intraperitoneal, intralesional, intraprostatic, intravaginal, intrarectal, intrathecal, intratumoral, intramuscular, intravesical, and / or subcutaneous injection.

[0211] (viii) Compositions for targeting viral vectors and nanoparticles for modifying cells in vivo Targeted viral vectors and / or nanoparticles can also be used to genetically modify immune cells in vivo or ex vivo. Viral vectors that can be used to deliver artificial expression constructs to cells are described elsewhere herein, and many targeted viral vectors (e.g., pseudotyped viral vectors) are known in the art.

[0212] Exemplary cell-targeting nanoparticles include nanoparticles with cell-targeting ligands (e.g., CD3, CD4, CD8, CD34) attached to their surface, which are selectively taken up by selected cell types due to the cell-targeting ligands attached to their surface. The nanoparticles then deliver genetically modified components to express cytokine transgenes and recombinant receptors.

[0213] Exemplary nanoparticles include liposomes (tiny vesicles with at least one lipid bilayer surrounding an aqueous core, forming concentric spheres), liposomal nanoparticles (liposomal structures used to encapsulate smaller nanoparticles within their cores), and lipid nanoparticles (liposome-like structures that lack the continuous lipid bilayer characteristic of liposomes). Other polymer-based nanoparticles and porous nanoparticles composed of materials capable of forming a porous network can also be used. Exemplary materials include metals, transition metals, and metalloids (e.g., lithium, magnesium, zinc, aluminum, and silica).

[0214] Nanoparticles intended for in vivo delivery and cellular uptake may have an uncharged or negatively charged coating, and the size of the nanoparticles may be 130 nm or less. The size of the nanoparticles can be measured using conventional techniques, such as dynamic light scattering and / or electron microscopy. In certain embodiments, the nanoparticles may be those described in WO2014153114, WO2017181110, and WO201822672.

[0215] The therapeutically effective amount of vector and / or nanoparticles contained in the formulation may be in the range of 0.1-5 μg / kg or 0.5-1 μg / kg. In another example, the dose may be 1 μg / kg, 30 μg / kg, 90 μg / kg, 150 μg / kg, 500 μg / kg, 750 μg / kg, 0.1-5 mg / kg, or 0.5-1 mg / kg. In another example, the dose may be 1 mg / kg, 10 mg / kg, 30 mg / kg, 50 mg / kg, 70 mg / kg, 100 mg / kg, 300 mg / kg, 500 mg / kg, 700 mg / kg, 1000 mg / kg, or more.

[0216] (ix) How to use The methods disclosed herein include treating a subject (human, non-human primate, companion animal (dog, cat, reptile, bird, etc.), livestock (horse, cow, goat, pig, chicken, etc.), or research animal (monkey, rat, mouse, fish, etc.)) with a formulation disclosed herein. Treating a subject includes delivering a therapeutically effective amount. A therapeutically effective amount includes an amount that can provide effective, prophylactic, and / or therapeutic treatment.

[0217] An "effective amount" is the amount of a formulation required to induce a desired physiological change in a subject. For example, an effective amount can provide enhanced immunogenic anti-cancer or anti-infectious disease effects. Effective amounts are often administered for research purposes. The effective amount disclosed herein is an amount that can induce a statistically significant effect in an animal model or in vitro assay relevant to assessing the development or progression of cancer or infectious disease. The immunogenic formulation can be provided in an effective amount, which stimulates an immune response.

[0218] "Prophylactic treatment" includes treatment administered to a subject who does not exhibit signs or symptoms of cancer or an infectious disease, or who exhibits only early signs or symptoms of cancer or an infectious disease, with the intent of mitigating or reducing the risk of the cancer or infectious disease developing further. Thus, prophylactic treatment functions as a treatment to prevent cancer or an infectious disease. In certain embodiments, prophylactic treatment inhibits, delays, or prevents the development of metastases from the primary cancer tumor site. In certain embodiments, prophylactic treatment inhibits, delays, or prevents bacterial, viral, fungal, parasitic, or arthropod infections.

[0219] "Therapeutic treatment" includes treatment administered to a subject who exhibits symptoms or signs of cancer or an infectious disease with the intent of reducing or eliminating the signs or symptoms of the cancer or infectious disease. Therapeutic treatment may prevent, control, or eliminate the presence or activity of the cancer or infectious disease and / or may prevent, control, or eliminate the side effects of the cancer or infectious disease.

[0220] An effective amount, functioning as a prophylactic treatment or a therapeutic treatment are not mutually exclusive, and in certain embodiments, an administered dose may accomplish more than one treatment.

[0221] In certain embodiments, a therapeutically effective amount enhances the killing ability, proliferation, and / or cytokine production of immune cells. These effects can provide anti-cancer and / or anti-infectious disease effects. Anti-cancer effects include reducing the number of cancer cells, reducing the number of metastases, reducing tumor volume, extending life expectancy, inducing chemotherapy sensitivity or radiosensitivity in cancer cells, suppressing angiogenesis in the vicinity of cancer cells, suppressing cancer cell proliferation, suppressing tumor growth, preventing or reducing metastasis, extending the lifespan of a subject, suppressing cancer-related pain, and / or reducing cancer recurrence or recurrence after treatment. Anti-infectious disease effects include reducing the amount or level of infectious pathogens, reducing fatigue, reducing loss of appetite, suppressing weight loss, reducing fever, reducing night sweats, reducing chills, reducing aches and pains, reducing diarrhea, reducing bloating, reducing abdominal pain, reducing rash, reducing cough, and / or reducing runny nose.

[0222] A "tumor" is a swelling or lesion formed by the abnormal proliferation of cells (called neoplastic or neoplastic cells). "Tumor cells" are abnormal cells that grow by rapid and unregulated cell proliferation and continue to grow even after the stimulus to initiate new proliferation has ceased. A tumor is a partial or complete loss of the structural organization and functional coordination of normal tissue, usually forming a identifiable mass of tissue that may be benign, premalignant, or malignant.

[0223] In certain embodiments, immune cells obtained from a subject are screened for responsiveness to expression of an inducible cytokine transgene. The screening method includes introducing a genetic construct containing a sequence encoding an inducible cytokine, as described elsewhere herein, into a plurality of immune cells obtained from the subject; and measuring enhanced function of the resulting immune cells. In certain embodiments, enhanced function of the immune cells includes enhanced cell-killing ability, enhanced proliferation, and / or enhanced cytokine production. In certain embodiments, enhanced cytokine production includes IFNγ production and / or TNFα production. Methods for screening immune cells obtained from a subject for responsiveness to inducible cytokine expression may include cell-killing and proliferation assays, such as MTT cell proliferation assay, trypan blue assay, carboxyfluorescein succinimidyl ester (CFSE) assay, Ki67, XTT assay, BrdU assay, flow cytometry, and DNA quantification; or cytokine production assays, such as quantitative PCR and immunodetection methods (e.g., enzyme-linked immunosorbent assay). The method of screening immune cells obtained from a subject for responsiveness to inducible cytokine expression can be performed in vitro or in vivo.

[0224] In certain embodiments, an inducible cytokine can be selected for administration to a subject that elicits increased, enhanced function of immune cells compared to a baseline, which in certain embodiments includes the function of the immune cells before introduction of the genetic construct or the function of the immune cells after introduction of a control (non-cytokine-expressing) genetic construct.

[0225] In certain embodiments, a combination of cytokines can be selected for administration to a subject based on the screening results. For example, if a first cytokine increases cell killing ability and proliferation, and a second cytokine increases cytokine production in a first subject, these first cytokine and second cytokine can be selected for administration to the first subject. Alternatively, if a third cytokine increases cell killing ability, a fourth cytokine increases proliferation, and a fifth cytokine increases cytokine production in a second subject, these third cytokine, fourth cytokine, and fifth cytokine can be selected for administration to the second subject.

[0226] In certain embodiments, any combination of cytokines can be administered. In certain embodiments, the combination of cytokines that enhances cell killing, proliferation, and cytokine production comprises selecting at least one of mTGFβ2-7m*, TGFβ2-7m, IL15, scIL-12, DR-IL18, and IL-36γ, and at least one of mTGFβ2-7m*, TGFβ2-7m, scIL-12, DR-IL18, and IL-36γ. In certain embodiments, the combination of cytokines that enhances cell killing, proliferation, and cytokine production comprises IL15, and at least one of mTGFβ2-7m*, TGFβ2-7m, scIL-12, DR-IL18, and IL-36γ. In certain embodiments, the combination of cytokines that enhances cell killing, proliferation and cytokine production comprises IL21 in addition to at least one of mTGFβ2-7m*, TGFβ2-7m, scIL-12, DR-IL18 and IL-36γ.

[0227] When administering, the therapeutically effective amount (also referred to herein as "dosage") can be initially estimated based on the results of in vitro assays and / or animal model studies.This information can be used to more accurately determine the dosage that is useful for the intended subject.The actual dosage that is administered to a specific subject can be determined by a physician, veterinarian or researcher, taking into account parameters such as physical and physiological factors, such as target, body weight, disease severity, type of cancer or infectious disease, stage of cancer or infectious disease, previous therapeutic intervention or concurrent therapeutic intervention, subject's idiopathic disease and route of administration.

[0228] The therapeutically effective dose of the cell-based formulation is 10 4 ~10 9 pcs / kg body weight or 10 3 ~10 11 The therapeutically effective amount to be administered may be 10 2 More than 10 cells 3 More than 10 cells 4 More than 10 cells 5 More than 10 cells 6 More than 10 cells 7 More than 10 cells 8 More than 10 cells 9 More than 10 cells 10 More than 10 cells, or 11 Examples include more than 100 cells.

[0229] The therapeutically effective amount of vector and / or nanoparticles contained in the formulation may be in the range of 0.1-5 μg / kg or 0.5-1 μg / kg. In another example, the dose may be 1 μg / kg, 30 μg / kg, 90 μg / kg, 150 μg / kg, 500 μg / kg, 750 μg / kg, 0.1-5 mg / kg, or 0.5-1 mg / kg. In another example, the dose may be 1 mg / kg, 10 mg / kg, 30 mg / kg, 50 mg / kg, 70 mg / kg, 100 mg / kg, 300 mg / kg, 500 mg / kg, 700 mg / kg, 1000 mg / kg, or more.

[0230] A therapeutically effective amount can be achieved by a single administration or multiple administrations during the course of a treatment regimen (e.g., daily, every other day, every third day, every fourth day, every fifth day, every sixth day, weekly, every two weeks, every three weeks, monthly, every two months, every three months, every four months, every five months, every six months, every seven months, every eight months, every nine months, every ten months, every eleven months, or yearly). In certain embodiments, treatment protocols may be determined according to clinical trial protocols or FDA-approved treatment protocols.

[0231] A therapeutically effective amount can be administered, for example, by injection, infusion, perfusion, or lavage. Routes of administration include intravenous bolus, intradermal, intraarterial, intraperitoneal, intranodal, intralymphatic, intraperitoneal, intralesional, intraprostatic, intravaginal, intrarectal, topical, intrathecal, intratumoral, intramuscular, intravesical, and / or subcutaneous administration.

[0232] In certain embodiments, the formulations and / or compositions of the present disclosure are administered to a patient in conjunction with (e.g., before, simultaneously with, or after) a related treatment, and the number of related treatments is not particularly limited. In certain embodiments, the cells of the present disclosure may be used in conjunction with chemotherapy; photoirradiation; immunosuppressants such as cyclosporine, azathioprine, methotrexate, mycophenolate, FK506, etc.; antibodies; other immunoablative agents such as CAM PATH; anti-CD3 antibodies; other antibody treatments; cytotoxins; fludarabine; cyclosporine; FK506; rapamycin; mycophenolic acid; steroids; FR901228; cytokines; or radiation.

[0233] In certain embodiments, the formulations and / or compositions of the present disclosure may be administered in combination with any number of chemotherapeutic agents.Examples of chemotherapeutic agents include alkylating agents; alkyl sulfonates; aziridines; ethyleneimines and methylmelamines; nitrogen mustards; nitrosoureas; antibiotics; antimetabolites; folic acid analogs; purine analogs; pyrimidine analogs; androgens; adrenal suppressants; folic acid supplements; platinum analogs; retinoic acid derivatives; and pharmaceutically acceptable salts, acids, or derivatives of any of the above chemotherapeutic agents.The definition of chemotherapeutic agents further includes antihormonal agents that regulate or inhibit the action of hormones on tumors, and such antihormonal agents include antiestrogens and antiandrogens; and pharmaceutically acceptable salts, acids, or derivatives of any of these antihormonal agents. Additionally, if deemed appropriate, a combination of chemotherapy agents may be administered, including CHOP therapy, a combination of cyclophosphamide (Cytoxan®), doxorubicin (hydroxydoxorubicin), vincristine (Oncovin®), and prednisone.

[0234] In some embodiments, the chemotherapeutic agent is administered simultaneously with the administration of the formulations and / or compositions of the present disclosure, or within one week of administering the formulations and / or compositions of the present disclosure. In other embodiments, the chemotherapeutic agent is administered 1 to 4 weeks, 1 week to 1 month, 1 week to 2 months, 1 week to 3 months, 1 week to 6 months, 1 week to 9 months, or 1 week to 12 months after administration of the modified cells or nucleic acids of the present disclosure. In other embodiments, the chemotherapeutic agent is administered at least one month before administration of the cells or nucleic acids of the present disclosure. In some embodiments, the methods of the present disclosure further comprise administering two or more chemotherapeutic agents.

[0235] A variety of additional therapeutic agents may be used in combination with the formulations described herein. For example, potentially useful additional therapeutic agents include PD-1 inhibitors such as nivolumab (Opdivo®), pembrolizumab (Keytruda®), pembrolizumab, pidilizumab, and atezolizumab; and CTLA-4 inhibitors such as ipilimumab (Yervoy®).

[0236] Additional therapeutic agents suitable for use in combination with the present disclosure include abiraterone acetate, apalutamide, bicalutamide, cabazitaxel, Casodex (bicalutamide), degarelix, docetaxel, enzalutamide, Erleada® (apalutamide), flutamide, goserelin acetate, Jevtana® (cabazitaxel), leuprolide acetate, Lupron® (leuprolide acetate), Lupron Depot (leuprolide acetate), Lupron These include Depot-Ped (leuprolide acetate), mitoxantrone hydrochloride, Nilandron® (nilutamide), nilutamide, Provenge® (sipuleucel-T), radium-223 dichloride, sipuleucel-T, Taxotere (docetaxel), Viadur (leuprolide acetate), Xofigo (radium-223 dichloride), Xtandi (enzalutamide), Zoladex (goserelin acetate), or Zytiga (abiraterone acetate).

[0237] In further embodiments, the formulations and / or compositions of the present disclosure can be administered with an anti-inflammatory agent. Anti-inflammatory agents or anti-inflammatory drugs include steroids and glucocorticoids, as well as nonsteroidal anti-inflammatory drugs (NSAIDs). Exemplary analgesics include acetaminophen, oxycodone, tramadol, and propoxyphene hydrochloride. Exemplary biological response modifiers include molecules directed against cell surface markers (e.g., CD4 and CD5); cytokine inhibitors such as TNF antagonists (e.g., etanercept (Enbrel®), adalimumab (Humira®), and infliximab (Remicade®)); chemokine inhibitors; and adhesion molecule inhibitors. Biological response modifiers also include monoclonal antibodies and recombinant molecules. Exemplary disease-modifying antirheumatic drugs (DMARDs) include azathioprine, cyclophosphamide, cyclosporine, methotrexate, penicillamine, leflunomide, sulfasalazine, hydroxychloroquine, gold preparations (oral formulation (auranofin) and intramuscular injection), and minocycline.

[0238] In certain embodiments, the formulations and / or compositions of the present disclosure are administered in combination with a cytokine, which may be, for example, the same as the cytokine encoded by the transgene or may be different from the cytokine encoded by the transgene. Examples of cytokines that can be co-administered include interferons such as interferon alpha, interferon beta, and interferon gamma; colony-stimulating factors (CSFs) such as macrophage CSF (M-CSF), granulocyte-macrophage CSF (GM-CSF), and granulocyte CSF (G-CSF); interleukins (ILs) such as IL-1, IL-1 alpha, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, scIL12, IL-15, IL18, DR-IL18, IL21, and IL36 gamma; tumor necrosis factors such as TNF-alpha, TNF-beta, mTGF beta 2-7m*, and TGF beta 2-7m; and other polypeptide factors such as LIF and kit ligand (KL). In the context of these combination therapies, the term "cytokine" also includes naturally occurring proteins or proteins from recombinant cell culture and biologically active equivalents of the native-sequence cytokines.

[0239] (x) Kit The present disclosure further includes kits. The kits may include various components for carrying out the methods disclosed herein. For example, depending on the embodiment of the method to be carried out, the kit may include a nucleic acid encoding a cytokine transgene disclosed herein under the control of an iSynPro promoter; a nucleic acid encoding a recombinant receptor disclosed herein; a nucleic acid encoding Her2tg; a skip sequence; a nucleic acid encoding an scFv; a nucleic acid encoding a VL; a nucleic acid encoding a VH; a nucleic acid encoding a transmembrane domain; a nucleic acid encoding an intracellular effector domain; a nucleic acid encoding an EGFRt; a nucleic acid encoding a selection cassette (e.g., DHFRdm); methotrexate; cells (e.g., immune cells, T cells, CD4 T cells, CD8 T cells, etc.); T cells, B cells, natural killer (NK) cells, NK-T cells, monocytes / macrophages, lymphocytes, hematopoietic stem cells (HSCs), hematopoietic progenitor cells (HPCs), and / or mixtures of HSCs and HPCs (i.e., HSPCs), untransduced T cells, T cells transduced with an artificial expression construct described herein; cell lines; tissue samples (e.g., peripheral blood mononuclear cells (PBMCs), leukocytes, bone marrow, thymus, tissue biopsy samples, tumors, lymph nodes, gut-associated lymphoid tissue, mucosa-associated lymphoid tissue, spleen, other lymphoid tissues, liver, lung, stomach, intestine, colon, kidney, pancreas, breast, bone, prostate, cervix, testis, ovary, tonsils, or other organs, and / or are cells derived from these organs); gene expression components (e.g., expression genes provided by vectors (e.g., lentiviral or retroviral vectors), CRISPR components, ZFNs, TALENs, MegaTALs, targeted viral vectors and / or targeted nanoparticles); cell formulation or cell activation components (e.g., saline, buffered saline, phosphate buffered saline (PBS)); biocompatible buffers (Ca++ / Mg++-free PBS, saline, water, Hank's solution, Ringer's solution); T cell stimulatory epitopes (e.g., anti-CD3 / anti-CD28 conjugated beads, OKT3, TGN1412);The cells may include one or more of the following: compositions for initiating culture (RPMI, non-essential amino acids, sodium pyruvate, penicillin / streptomycin, EBV-transformed non-dividing lymphoblastoid cells (LCL), IL-21, human serum albumin (HSA) or other human serum components or fetal bovine serum, dextrose, stabilizers, preservatives); components for combination therapy (e.g., local anesthetics, chemotherapeutic agents, immunosuppressants, anti-inflammatory agents); fluorescently tagged antibodies; PCR amplification sequences; cytokines (e.g., IL-2, IL-7, IL-15, IL-21); culture vessels; reference levels; transgenic animals; primer pairs; GAPDH; enzyme-linked immunosorbent assay (ELISA) for IFN-γ; culture plates, etc.;

[0240] The following exemplary embodiments and examples are provided to illustrate specific embodiments of the present disclosure. Those skilled in the art, having reference to this disclosure, will recognize that various modifications can be made to the specific embodiments disclosed herein while still obtaining the same or similar results without departing from the spirit and scope of the present disclosure.

[0241] (xi) Exemplary Embodiments 1. An artificial expression construct comprising a cytokine encoding sequence under the regulatory control of a promoter comprising a sequence having at least 95% sequence identity to any one of SEQ ID NOs: 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46 or 47. 2. The artificial expression construct of embodiment 1, wherein the promoter comprises a sequence having at least 98% sequence identity to any one of SEQ ID NOs: 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, or 47. 3. The artificial expression construct of embodiment 1 or 2, wherein the promoter comprises a sequence having at least 99% sequence identity to any one of SEQ ID NOs: 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46 or 47. 4. An artificial expression construct according to any one of embodiments 1 to 3, wherein the promoter comprises a sequence as set forth in any one of SEQ ID NOs: 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46 or 47. 5. An artificial expression construct described in any one of embodiments 1 to 4, wherein the promoter further comprises a minimal promoter. 6. The artificial expression construct of embodiment 5, wherein said minimal promoter comprises a minimal promoter of IL2. 7. The artificial expression construct of embodiment 5 or 6, wherein the minimal promoter comprises the sequence set forth in SEQ ID NO: 138 or a sequence having at least 95% sequence identity to the sequence set forth in SEQ ID NO: 138. 8. The artificial expression construct of any one of embodiments 1 to 7, wherein the cytokine comprises transforming growth factor beta 2-7m (TGFβ2-7m), mini TGFβ2-7m* (mTGFβ2-7m*), interleukin 21 (IL21), interleukin 15 (IL15), single-chain interleukin 12 (scIL12), decoy-resistant interleukin 18 (DR-IL18), or interleukin 36γ (IL36γ). 9. An artificial expression construct according to any one of embodiments 1 to 8, wherein the cytokine is encoded by the sequence set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6 or SEQ ID NO:7, or by a sequence having at least 90% sequence identity to the sequence set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6 or SEQ ID NO:7. 10. The artificial expression construct of any one of embodiments 1 to 9, wherein the cytokine comprises the sequence set forth in SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52 or SEQ ID NO: 54, or a sequence having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52 or SEQ ID NO: 54. 11. An artificial expression construct according to any one of embodiments 1 to 10, further comprising a regulatory mechanism. 12. The artificial expression construct of embodiment 11, wherein the control mechanism comprises a transduction marker. 13. The artificial expression construct of embodiment 12, wherein the transduction marker comprises Her2tG. 14. An artificial expression construct according to any one of embodiments 1 to 13, further comprising a first skip sequence. 15. The artificial expression construct of embodiment 14, wherein the first skip sequence encodes a 2A self-cleaving polypeptide. 16. The artificial expression construct of embodiment 15, wherein the 2A self-cleaving polypeptide comprises T2A, P2A, E2A, or F2A. 17. An artificial expression construct according to any one of embodiments 1 to 16, having at least 95% sequence identity with the sequence set forth in SEQ ID NO: 136. 18. An artificial expression construct according to any one of embodiments 1 to 17, having at least 98% sequence identity with the sequence set forth in SEQ ID NO: 136. 19. An artificial expression construct according to any one of embodiments 1 to 18, having at least 99% sequence identity with the sequence set forth in SEQ ID NO: 136. 20. An artificial expression construct according to any one of embodiments 1 to 19, having the sequence set forth in SEQ ID NO: 136. 21. An artificial expression construct according to any one of embodiments 1 to 20, further comprising a sequence encoding a recombinant receptor comprising a binding domain within its extracellular portion that binds to an antigen expressed on the surface of a target cell. 22. The artificial expression construct of embodiment 21, wherein the sequence encoding the recombinant receptor is operably linked to a second promoter. 23. The artificial expression construct of embodiment 22, wherein the second promoter comprises an EF1α(L) promoter, an EF1α(s) promoter, a myeloproliferative sarcoma virus (MND) promoter, a cytomegalovirus (CMV) promoter, a simian virus 40 (SV40) early promoter, a mouse mammary tumor virus (MMTV) promoter, a human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, a MoMuLV promoter, an avian leukosis virus promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter, an actin promoter, a myosin promoter, a hemoglobin promoter, or a creatine kinase promoter. 24. An artificial expression construct described in any one of embodiments 21 to 23, wherein the binding domain is part of the extracellular portion. 25. An artificial expression construct described in any one of embodiments 21 to 24, wherein the target cells include cells infected with bacteria, viruses, fungi, parasites, or arthropods, or cancer cells. 26. An artificial expression construct described in any one of embodiments 21 to 25, wherein the recombinant receptor further comprises an intracellular portion. 27. The artificial expression construct of embodiment 26, wherein the intracellular portion comprises the signaling domain of CD3ζ and / or the signaling domain of 4-1BB. 28. An artificial expression construct described in embodiment 26 or 27, wherein the intracellular portion is linked to the extracellular portion via a transmembrane domain. 29. The artificial expression construct of embodiment 28, wherein the transmembrane domain comprises the transmembrane domain of CD28. 30. An artificial expression construct according to any one of embodiments 21 to 28, further comprising a second control mechanism. 31. The artificial expression construct of embodiment 30, wherein the second control mechanism comprises a selection cassette. 32. The artificial expression construct of embodiment 31, wherein the selection cassette comprises a dihydrofolate reductase double mutant (DHFRdm). 33. An artificial expression construct according to any one of embodiments 30 to 32, further comprising a third control mechanism. 34. The artificial expression construct of embodiment 33, wherein the third control mechanism comprises a transduction marker. 35. The artificial expression construct of embodiment 34, wherein the transduction marker comprises epidermal growth factor receptor (EGFRt) or truncated CD19 (tCD19). 36. An artificial expression construct according to any one of embodiments 21 to 35, further comprising a second skip sequence. 37. The artificial expression construct of embodiment 36, wherein the second skip sequence encodes a 2A self-cleaving polypeptide. 38. The artificial expression construct of embodiment 37, wherein the 2A-skipping self-cleaving polypeptide comprises T2A, P2A, E2A, or F2A. 39. An artificial expression construct described in any one of embodiments 34 to 38, comprising a second skip sequence at the 5' end of the selection cassette and a third skip sequence at the 5' end of the transduction marker. 40. An artificial expression construct according to any one of embodiments 1 to 39, having at least 90% sequence identity with the sequence set forth in SEQ ID NO: 96, 97, 98, 99 or 101. 41. An artificial expression construct according to any one of embodiments 1 to 40, having a sequence as set forth in SEQ ID NO: 96, 97, 98, 99 or 101. 42. A system for enhancing immune cell function, comprising: a first artificial expression construct encoding a cytokine under the regulatory control of a first promoter comprising a sequence having at least 95% sequence identity to any one of SEQ ID NOs: 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46 or 47; a second artificial expression construct comprising a sequence encoding a recombinant receptor comprising a binding domain within its extracellular portion that binds to an antigen expressed on the surface of a target cell; Including, the system. 43. The system of embodiment 42, wherein the first promoter comprises a sequence having at least 98% sequence identity to any one of SEQ ID NOs: 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46 or 47. 44. The system of embodiment 42 or 43, wherein the first promoter comprises a sequence having at least 99% sequence identity to any one of SEQ ID NOs: 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46 or 47. 45. The system of any one of embodiments 42 to 44, wherein the first promoter comprises an array set forth in any one of SEQ ID NOs: 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46 or 47. 46. ​​A system described in any one of embodiments 42 to 45, wherein the first promoter further comprises a minimal promoter. 47. A system described in any one of embodiments 42 to 46, wherein the minimal promoter comprises a minimal promoter of IL2. 48. A system described in any one of embodiments 42 to 47, wherein the minimal promoter comprises the sequence set forth in SEQ ID NO: 138 or a sequence having at least 95% sequence identity to the sequence set forth in SEQ ID NO: 138. 49. The system of any one of embodiments 42 to 48, wherein the cytokine comprises transforming growth factor beta 2-7m (TGFβ2-7m), mini TGFβ2-7m* (mTGFβ2-7m*), interleukin 21 (IL21), interleukin 15 (IL15), single-chain interleukin 12 (scIL12), decoy-resistant interleukin 18 (DR-IL18), or interleukin 36γ (IL36γ). 50. A system described in any one of embodiments 42 to 49, wherein the cytokine is encoded by a sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 7, or by a sequence having at least 90% sequence identity to a sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 7. 51. The system described in any one of embodiments 42 to 50, wherein the cytokine comprises a sequence set forth in SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52 or SEQ ID NO: 54, or a sequence having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52 or SEQ ID NO: 54. 52. A system described in any one of embodiments 42 to 51, wherein the first artificial expression construct further comprises a control mechanism. 53. The system of embodiment 52, wherein the control mechanism comprises a transduction marker. 54. The system described in embodiment 53, wherein the transduction marker comprises Her2tG. 55. A system described in any one of embodiments 42 to 54, wherein the first artificial expression construct further comprises a first skip sequence. 56. The system of embodiment 55, wherein the first skip sequence comprises a 2A skip sequence. 57. The system described in embodiment 56, wherein the 2A skip sequence encodes a T2A self-cleaving polypeptide, a P2A self-cleaving polypeptide, an E2A self-cleaving polypeptide, or an F2A self-cleaving polypeptide. 58. A system described in any one of embodiments 42 to 57, wherein the sequence encoding the recombinant receptor is operably linked to a second promoter. 59. The system described in embodiment 58, wherein the second promoter comprises an EF1α(L) promoter, an EF1α(s) promoter, an MND promoter, a CMV promoter, an SV40 early promoter, an MMTV promoter, an HIV LTR promoter, a MoMuLV promoter, an avian leukosis virus promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter, an actin promoter, a myosin promoter, a hemoglobin promoter, or a creatine kinase promoter. 60. A system described in any one of embodiments 42 to 59, wherein the second artificial expression construct further comprises a second control mechanism. 61. The system of embodiment 60, wherein the second control mechanism comprises a selection cassette. 62. The system described in embodiment 61, wherein the selection cassette comprises a dihydrofolate reductase double mutant (DHFRdm). 63. A system described in any one of embodiments 60 to 62, wherein the second artificial expression construct further comprises a third control mechanism. 64. The system described in embodiment 63, wherein the third control mechanism comprises a transduction marker. 65. The system described in embodiment 64, wherein the transduction marker comprises a truncated epidermal growth factor receptor (EGFRt) or tCD19. 66. A system described in any one of embodiments 42 to 65, wherein the second artificial expression construct further comprises a second skip sequence. 67. The system of embodiment 66, wherein the second skip sequence comprises a 2A skip sequence. 68. The system described in embodiment 67, wherein the 2A skip sequence encodes a T2A self-cleaving polypeptide, a P2A self-cleaving polypeptide, an E2A self-cleaving polypeptide, or an F2A self-cleaving polypeptide. 69. A system described in any one of embodiments 64 to 68, wherein the second artificial expression construct comprises a second skip sequence at the 5' end of the selection cassette and a third skip sequence at the 5' end of the transduction marker. 70. A system described in any one of embodiments 42 to 69, wherein the binding domain is part of the extracellular portion. 71. A system described in any one of embodiments 42 to 70, wherein the target cells include cells infected with bacteria, viruses, fungi, parasites or arthropods, or cancer cells. 72. A system described in any one of embodiments 42 to 71, wherein the recombinant receptor further comprises an intracellular portion. 73. The system described in embodiment 72, wherein the intracellular portion comprises the signaling domain of CD3ζ and / or the signaling domain of 4-1BB. 74. The system of embodiment 72 or 73, wherein the intracellular portion is linked to the extracellular portion via a transmembrane domain. 75. The system described in embodiment 74, wherein the transmembrane domain comprises the transmembrane domain of CD28. 76. A system described in any one of embodiments 42 to 75, wherein the first artificial expression construct and the second artificial expression construct are present on a single artificial expression construct. 77. A system described in any one of embodiments 42 to 75, wherein the first artificial expression construct and the second artificial expression construct are present on separate artificial expression constructs. 78. Nanoparticles encapsulating an artificial expression construct according to any one of embodiments 1 to 41 or a system according to any one of embodiments 42 to 77. 79. A cell genetically modified to express an artificial expression construct described in any one of embodiments 1 to 41 or a system described in any one of embodiments 42 to 77. 80. The cells described in embodiment 79, which are autologous cells obtained from the subject or cells allogeneic to the subject. 81. A cell described in embodiment 79 or 80, which is an in vivo cell or an ex vivo cell. 82. A cell described in any one of embodiments 79 to 81, which is an immune cell. 83. The cell described in embodiment 82, wherein the immune cell is a lymphocyte. 84. The cells described in embodiment 83, wherein the lymphocytes comprise T cells, B cells, natural killer (NK) cells, or NK-T cells. 85. The cell described in any one of embodiments 79 to 84, which is a T cell selected from CD3+ T cells, CD4+ T cells, CD8+ T cells, central memory T cells, effector memory T cells and / or naive T cells. 86. A cell described in any one of embodiments 79 to 85, which is a CD8+ T cell. 87. A cell described in any one of embodiments 79 to 85, which is a CD4+ T cell. 88. A cell population genetically modified to express an artificial expression construct described in any one of embodiments 1 to 41 or a system described in any one of embodiments 42 to 77. 89. The cell population described in embodiment 88, comprising autologous cells obtained from the subject or cells allogeneic to the subject. 90. The cell population described in embodiment 88 or 89, which is an in vivo cell population or an ex vivo cell population. 91. A cell population described in any one of embodiments 88 to 90, wherein the cells are immune cells. 92. The cell population described in embodiment 91, wherein the immune cells are lymphocytes. 93. The cell population described in embodiment 92, wherein the lymphocytes comprise T cells, B cells, natural killer (NK) cells, or NK-T cells. 94. A cell population described in any one of embodiments 88 to 93, comprising CD4+ T cells and / or CD8+ T cells. 95. A formulation comprising (i) a cell genetically modified to express an artificial expression construct described in any one of embodiments 1 to 41 or a system described in any one of embodiments 42 to 77, and (ii) a pharmaceutically acceptable carrier. 96. A method for genetically modifying immune cells to have enhanced function, comprising: 42. A method comprising contacting an immune cell with an artificial expression construct according to any one of embodiments 1 to 41, comprising a sequence encoding a cytokine. 97. The method of embodiment 96, wherein the enhanced function comprises enhanced cytotoxicity. 98. The method of embodiment 97, wherein the encoded cytokine comprises TGFβ2-7m, mTGFβ2-7m*, IL21, IL15, scIL12, DR-IL18, or IL36γ. 99. The method of embodiment 97 or 98, wherein the encoded cytokine comprises IL21 or IL15. 100. The method of any one of embodiments 96 to 99, wherein the enhanced function comprises enhanced proliferation. 101. The method of embodiment 9100, wherein the encoded cytokine comprises TGFβ2-7m, mTGFβ2-7m*, IL21, IL15, scIL12, DR-IL18, or IL36γ. 102. The method of embodiment 94 or 95, wherein the encoded cytokine comprises IL21 or IL15. 103. The method of any one of embodiments 96 to 102, wherein the enhanced function comprises enhanced cytokine production. 104. The method of embodiment 103, wherein the enhanced cytokine production comprises IFNγ. 105. The method of embodiment 104, wherein the encoded cytokine comprises scIL12, DR-IL18, IL36γ, TGFβ2-7m, or mTGFβ2-7m*. 106. A method according to any one of embodiments 103 to 105, wherein the enhanced cytokine production comprises the production of TNFα. 107. The method of embodiment 106, wherein the encoded cytokine comprises scIL12, DR-IL18, or IL36γ. 108. The method of any one of embodiments 96 to 107, wherein the artificial expression construct further comprises a sequence encoding a recombinant receptor comprising a binding domain that binds to an antigen expressed on the surface of a target cell. 109. The method of embodiment 108, wherein the sequence encoding the recombinant receptor is operably linked to a second promoter. 110. The method of embodiment 109, wherein the second promoter comprises an EF1α(L) promoter, an EF1α(s) promoter, a myeloproliferative sarcoma virus (MND) promoter, a cytomegalovirus (CMV) promoter, a simian virus 40 (SV40) early promoter, a mouse mammary tumor virus (MMTV) promoter, a human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, a MoMuLV promoter, an avian leukosis virus promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter, an actin promoter, a myosin promoter, a hemoglobin promoter, or a creatine kinase promoter. 111. The method of any one of embodiments 96 to 110, further comprising contacting the immune cells with an artificial expression construct comprising a sequence encoding a recombinant receptor comprising a binding domain that binds to an antigen expressed on the surface of a target cell. 112. The method described in embodiment 111, wherein the step of contacting the immune cells with the artificial expression construct described in any one of embodiments 1 to 41 and the step of contacting the immune cells with an artificial expression construct comprising a sequence encoding the recombinant receptor are performed simultaneously. 113. The method of embodiment 111, wherein the step of contacting the immune cells with the artificial expression construct described in any one of embodiments 1 to 41 and the step of contacting the immune cells with the artificial expression construct comprising a sequence encoding the recombinant receptor are performed at different times. 114. The method of any one of embodiments 96 to 113, wherein the artificial expression construct comprises the sequence set forth in SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99 or SEQ ID NO: 101, or comprises a sequence having 90% sequence identity to the sequence set forth in SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99 or SEQ ID NO: 101. 115. A method for treating a subject in need of treatment, comprising administering to the subject in need of treatment a therapeutically effective amount of an artificial expression construct described in any one of embodiments 1 to 41, a system described in any one of embodiments 42 to 77, a nanoparticle described in embodiment 78, or a formulation described in embodiment 95, thereby treating the subject. 116. The method of embodiment 115, wherein the subject in need of treatment has cancer or an infectious disease. 117. The method of embodiment 115 or 116, wherein the step of administering the therapeutically effective amount comprises administering intravesically, intravenously, intradermally, intraarterially, parenterally, intranodal, intralymphatic, intraperitoneally, intralesionally, intraprostatically, intravaginally, intrarectally, topically, intrathecally, intratumorally, intramuscularly, or subcutaneously.

[0242] (xii) Conclusion The nucleic acid and amino acid sequences provided herein are represented by the abbreviations used for nucleotide bases and amino acid residues as set forth in 37 CFR §§ 1.831-1.835 and as set forth in WIPO Standard ST.26 (effective July 1, 2022). Although only one strand is shown for each nucleic acid sequence, the complementary strand, if applicable, is also included in the embodiments.

[0243] Variants of the sequences disclosed and cited herein are also encompassed by this application. Guidelines for determining which amino acid residues can be substituted, inserted, or deleted without losing biological activity can be found using computer programs well known in the art, such as DNASTAR. TM Software (Madison, Wisconsin, USA) can be used to find amino acid changes. The amino acid changes in the protein variants disclosed herein are preferably conservative amino acid changes, i.e., substitutions of amino acids with similarly charged amino acids or with uncharged amino acids. Conservative amino acid changes include substitutions with members of a family of amino acids whose side chains are related.

[0244] Suitable conservative substitutions of amino acids in peptides or proteins are known to those skilled in the art, and such conservative substitutions can usually be made without altering the biological activity of the resulting molecule. Those skilled in the art will be familiar with the fact that substituting a single amino acid in a non-critical region of a polypeptide usually does not substantially alter the biological activity (see, for example, Watson et al., Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin / Cummings Pub. Co., p. 224). Naturally occurring amino acids are typically classified into conservative substitution families, specifically: Group 1: alanine (Ala), glycine (Gly), serine (Ser), and threonine (Thr); Group 2 (acidic): aspartic acid (Asp) and glutamic acid (Glu); Group 3 (acidic; also classified as polar, negatively charged residues and their amides): asparagine (Asn), glutamine (Gln), Asp, and Glu; Group 4: Gln and Asn; Group 5 (basic; also classified as polar, positively charged residues): arginine (Arg), lysine (Lys), and histidine (His); Group 6 (large aliphatic, nonpolar residues): isoleucine (Ile), leucine (Leu), and arginine (Arg). Group 11 (aliphatic): Gly, Ala, Val, Leu, and Ile; Group 10 (small aliphatic nonpolar or slightly polar aliphatic residues): Ala, Ser, Thr, Pro, and Gly; and Group 12 (sulfur-containing residues): Met and Cys. Further information can be found in Creighton (1984) Proteins, W.H. Freeman and Company.

[0245] In making such changes, the hydropathic index of amino acids may be taken into consideration. The importance of the hydropathic index of amino acids in conferring interactive biological function on a protein is widely understood in the art (Kyte and Doolittle, 1982, J. Mol. Biol. 157(1), 105-32). Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics (Kyte and Doolittle, 1982). The hydrophobicity index of each amino acid is Ile (+4.5); Val (+4.2); Leu (+3.8); Phe (+2.8); Cys (+2.5); Met (+1.9); Ala (+1.8); Gly (-0.4); Thr (-0.7); Ser (-0.8); Trp (-0.9); Tyr (-1.3); Pro (-1.6); His (-3.2); glutamic acid (-3.5); Gln (-3.5); aspartic acid (-3.5); Asn (-3.5); Lys (-3.9); and Arg (-4.5).

[0246] It is well known in the art that substitution of a particular amino acid with another amino acid having a similar hydrophobicity index or hydrophobicity index will result in a protein with similar biological activity, i.e., a protein with equivalent biological functionality. When making such changes, substitution of amino acids with hydrophobicity indices within ±2 is preferred, substitution of amino acids with hydrophobicity indices within ±1 is particularly preferred, and substitution of amino acids with hydrophobicity indices within ±0.5 is even more particularly preferred. Furthermore, it is well known in the art that substitution of similar amino acids can be effectively carried out based on hydrophilicity.

[0247] As detailed in U.S. Patent Publication No. 4,554,101, each amino acid residue is assigned a hydrophilicity value, which is as follows: Arg (+3.0); Lys (+3.0); aspartic acid (+3.0±1); glutamic acid (+3.0±1); Ser (+0.3); Asn (+0.2); Gln (+0.2); Gly (0); Thr (-0.4); Pro (-0.5±1); Ala (-0.5); His (-0.5); Cys (-1.0); Met (-1.3); Val (-1.5); Leu (-1.8); Ile (-1.8); Tyr (-2.3); Phe (-2.5); Trp (-3.4). It is well known that a particular amino acid can be substituted with another amino acid having a similar hydrophilicity value, and that such substitutions will result in biologically equivalent proteins, and in particular immunologically equivalent proteins. When making such changes, substitutions of amino acids with hydrophilicity values ​​within ±2 are preferred, substitutions of amino acids with hydrophilicity values ​​within ±1 are particularly preferred, and substitutions of amino acids with hydrophilicity values ​​within ±0.5 are even more particularly preferred.

[0248] As outlined above, amino acid substitutions may be made on the basis of the relative similarity of the amino acid side-chain substituents, e.g., their hydrophobicity, hydrophilicity, charge, size, etc. Also, as described elsewhere herein, variants of a gene sequence include codon-optimized variants, sequence polymorphisms, splice variants, and / or mutations that do not have a statistically significant effect on the function of the encoded product.

[0249] Variants of the protein, nucleic acid and gene sequences disclosed herein also include sequences that have at least 70% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity or at least 99% sequence identity to a protein, nucleic acid or gene sequence disclosed herein.

[0250] "Percent sequence identity" refers to the relatedness of two or more sequences, as determined by comparing the sequences. In the art, "identity" also means the degree of relatedness between protein, nucleic acid, or gene sequences, as determined by the matching between strings of protein, nucleic acid, or gene sequences. "Identity" (often referred to as "similarity") can be readily calculated using known methods, including those described in Computational Molecular Biology (Lesk, AM, ed.) Oxford University Press, NY (1988); Biocomputing: Informatics and Genome Projects (Smith, DW, ed.) Academic Press, NY (1994); Computer Analysis of Sequence Data, Part I (Griffin, AM, and Griffin, HG, eds.) Humana Press, NJ (1994); Sequence Analysis in Molecular Biology (Von Heijne, G., ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov, M. and Devereux, J., eds.) Oxford University Press, NY (1992). Methods to determine identity are designed to give the best match between the sequences tested. Methods for determining identity and similarity are codified in publicly available computer programs. Sequence alignments and identity calculations may be performed using the Megalign program within the LASERGENE suite of bioinformatics computing software (DNASTAR, Madison, Wis.).Multiple sequence alignment can also be performed using the Clustal format alignment method (Higgins and Sharp CABIOS, 5, 151-153 (1989) using default parameters (gap penalty=10, gap length penalty=10)). Further relevant programs include the GCG program suite (Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison, Wisconsin); BLASTP, BLASTN, BLASTX (Altschul, et al., J. Mol. Biol. 215:403-410 (1990)); DNASTAR (DNASTAR, Inc., Madison, Wisconsin); and the FASTA program incorporating the Smith-Waterman algorithm (Pearson, Comput. Methods Genome Res., [Proc. Int. Symp.] (1994), Meeting Date 1992, pp. 111-20. Editor(s): Suhai, Sandor. Publisher: Plenum, New York, NY). In this disclosure, when sequence analysis software is used for analysis, the analysis results are interpreted as being based on the "default values" that serve as the basis for the program. In this specification, "default values" refers to a set of values ​​or parameters that are pre-registered in the software when the software is initialized.

[0251] Variants also include nucleic acid molecules that hybridize to the sequences disclosed herein under stringent hybridization conditions and have the same function as the reference sequences. Exemplary stringent hybridization conditions include overnight incubation at 42°C in a solution containing 50% formamide, 5xSSC (750mM NaCl, 75mM trisodium citrate), 50mM sodium phosphate (pH 7.6), 5xDenhardt's solution, 10% dextran sulfate, and 20µg / ml denatured fragment-treated salmon sperm DNA, followed by washing the filter with 0.1xSSC at 50°C. The stringency of hybridization and signal detection can be changed mainly by adjusting the concentration of formamide (lowering the percentage of formamide results in lower stringency), salt conditions, or temperature. For example, moderately stringent conditions include overnight incubation at 37°C in a solution containing 6x SSPE (20x SSPE = 3M NaCl; 0.2M NaH2PO4; 0.02M EDTA, pH 7.4), 0.5% SDS, 30% formamide, and 100 μg / ml blocking salmon sperm DNA, followed by a wash at 50°C with 1x SSPE and 0.1% SDS. Even lower stringency can be achieved by performing stringent post-hybridization washes at a high salt concentration (e.g., 5x SSC). These conditions can be varied by adding and / or substituting alternative blocking reagents used to reduce background in hybridization experiments. Common blocking reagents include Denhardt's reagent, BLOTTO, heparin, denatured salmon sperm DNA, and commercially available proprietary formulations. The addition of certain blocking reagents may require some modification of the hybridization conditions described above due to compatibility issues.

[0252] "Specifically binds" means that it does not bind significantly to other molecules or components in the relevant environmental sample, but does bind to 10 5 M -1"High affinity" refers to the binding of a binding domain (e.g., a binding domain) to its cognate binding molecule with an affinity or Ka (i.e., the equilibrium binding constant for a particular binding interaction, expressed in units of 1 / M) equal to or greater than 10. Binding domains may also be classified as "high affinity" or "low affinity." In certain embodiments, a "high affinity" binding domain has an affinity of at least 10 7 M -1 , at least 10 8 M -1 , at least 10 9 M -1 , at least 10 10 M -1 , at least 10 11 M -1 , at least 10 12 M -1 or at least 10 13 M -1 In certain embodiments, a "low affinity" binding domain refers to a binding domain with a Ka of 10 7 M -1 Below, 10 6 M -1 Less than or equal to 10 5 M -1 Alternatively, affinity refers to the equilibrium dissociation constant (Kd) (units: M) of a particular binding interaction (e.g., 10 -5 M~10 -13M). In certain embodiments, a binding domain may have "enhanced affinity," which indicates that a selected or engineered binding domain exhibits stronger binding to its cognate binding molecule than the wild-type (or parent) binding domain. For example, enhanced affinity may be due to a higher K (equilibrium binding constant) for the cognate binding molecule than the reference binding domain, a lower K (dissociation constant) for the cognate binding molecule than the reference binding domain, or a lower dissociation rate (Koff) for the cognate binding molecule than the reference binding domain. A variety of assays are known for detecting binding domains that specifically bind to particular cognate binding molecules and for measuring binding affinity, such as Western blots, ELISAs, and BIACORE analyses (see also, e.g., Scatchard, et al., 1949, Ann. NY Acad. Sci. 51:660; and U.S. Pat. Nos. 5,283,173, 5,468,614, or similar publications).

[0253] Unless otherwise indicated, the present disclosure can be practiced using conventional techniques in immunology, molecular biology, microbiology, cell biology, and recombinant DNA. These methods are described in the following publications: Sambrook, et al., Molecular Cloning: A Laboratory Manual, 2nd Edition (1989); F.M. Ausubel, et al., eds., Current Protocols in Molecular Biology, (1987); the series Methods IN Enzymology (Academic Press, Inc.); M. MacPherson, et al., PCR: A Practical Approach, IRL Press at Oxford University Press (1991); MacPherson et al., eds. PCR 2: Practical Approach, (1995); Harlow and Lane, eds. Antibodies, A Laboratory Manual, (1988); and R.I. Freshney, ed., Animal Cell Culture (1987).

[0254] As will be understood by those skilled in the art, each embodiment disclosed herein comprises, consists essentially of, or consists of the specific components, steps, materials, or ingredients described. Accordingly, the terms "comprise" or "comprising" should be interpreted to mean "comprise, consist essentially of, or consist of." The transitional phrase "comprising" means the inclusion of, but is not limited to, any unrecited component, step, material, or ingredient, even if in greater amounts. The transitional phrase "consisting of" excludes all unrecited components, steps, materials, or ingredients. The transitional phrase "consisting essentially of" limits the scope of an embodiment to the recited components, steps, materials, or ingredients, as well as those components, steps, materials, or ingredients that do not materially affect the embodiment. A significant effect refers to a statistically significant decrease in killing, proliferation, and / or cytokine production of immune cells bearing the recombinant receptor, as described herein.

[0255] Unless otherwise indicated, all numerical values ​​in the specification and claims expressing quantities or properties of materials, such as molecular weight or reaction conditions, are to be construed in all instances as modified by the term "about." Accordingly, unless otherwise indicated, the numerical parameters set forth in the specification and appended claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. Without intending to limit the scope of the doctrine of equivalents to the scope of the claims, each numerical parameter should, at the very least, be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. More specifically, the term "about," when used in conjunction with a stated value or range, has the meaning reasonably interpreted by one of ordinary skill in the art, i.e., within ±20% of the stated value; ±19% of the stated value; ±18% of the stated value; ±17% of the stated value; ±16% of the stated value; ±15% of the stated value; ±14% of the stated value; ±13% of the stated value; ±12% of the stated value; ±11% of the stated value; ±10% of the stated value; ±9% of the stated value; ±8% of the stated value; ±7% of the stated value; ±6% of the stated value; ±5% of the stated value; ±4% of the stated value; ±3% of the stated value; ±2% of the stated value; or ±1% of the stated value.

[0256] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations and approximate ranges, the numerical values ​​set forth in the specific examples are reported as precisely as possible, however, all numerical values ​​inherently contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0257] As used in describing the present invention (particularly in the claims that follow), the terms "a," "an," "the," and similar designators are intended to encompass both the singular and the plural unless otherwise indicated or the context clearly dictates otherwise. Numerical ranges recited herein are intended as a shorthand way of referring individually to each numerical value within the range. Unless otherwise indicated, each numerical value is described herein as if it were individually described herein. Any methods described herein can be performed in any suitable order unless otherwise indicated or the context clearly dictates otherwise. The use of any examples or illustrative language (e.g., "etc.") provided herein is for the purpose of illustrating the invention only and does not limit the scope of the invention as recited in the claims. No term used herein should be construed as indicating any non-claimed element essential to the practice of the invention.

[0258] Groupings of other elements of the invention or of various embodiments of the invention disclosed herein should not be construed as limiting the invention. Members of each group may be described herein or in the claims individually or in combination with other members of the group or other elements described herein. It is anticipated that one or more members of a group may be added to another group, or one or more members may be deleted from a group, for reasons of convenience and / or patentability. When such additions or deletions are made, the specification includes groups that are constructed to satisfy the recitation of all Markush groups set forth in the appended claims.

[0259] Specific embodiments of the present invention are described herein, including the best mode known to the inventors for carrying out the invention. Of course, those skilled in the art will readily recognize from a review of the foregoing detailed description that the embodiments described herein can be modified in various ways. The inventors anticipate that such modifications may be accommodated by those skilled in the art, and intend that the invention may be practiced in ways other than those specifically described herein. Accordingly, the present invention includes all modifications of the subject matter recited in the appended claims and all equivalents of the subject matter to the fullest extent permitted by applicable law. Furthermore, the present invention includes all combinations of the above-described elements in any and all variations thereof, unless expressly stated otherwise or the context clearly dictates otherwise.

[0260] Additionally, throughout this specification, various patents, publications, journal articles, and other documents are cited (the "references"). Each reference cited herein is individually incorporated herein by reference for the teachings thereof.

[0261] Finally, the embodiments of the invention disclosed herein are to be considered illustrative of the principles of the invention. Other modifications may be employed within the scope of the invention. Thus, by way of example, but not of limitation, alternative configurations of the invention may be utilized in accordance with the teachings herein. Therefore, the invention is not to be limited to what is precisely as shown and described herein.

[0262] The details set forth herein are by way of example only and are intended to illustrate preferred embodiments of the invention, are presented to provide what is believed to be the most useful, and to facilitate an understanding of the principles and conceptual aspects of various embodiments of the invention. In this regard, no structural details of the invention have been described in more detail than is necessary for a fundamental understanding of the invention, and those skilled in the art will be able to readily understand how to actually embody several forms of the invention by perusing the description of the invention with reference to the drawings and / or examples.

[0263] The definitions and explanations used in this disclosure are intended to control future interpretations unless a clear and unambiguous change is made in the examples, or unless the interpretation becomes meaningless or substantially meaningless due to the meaning of the terms. If the definition of a term becomes meaningless or substantially meaningless due to the interpretation of the term, please refer to the definition of the term in a dictionary known to those skilled in the art, such as Webster's Dictionary (3rd Edition) or Oxford Dictionary of Biochemistry and Molecular Biology (Eds. Attwood T et al., Oxford University Press, Oxford, 2006).

Claims

1. 1. An artificial expression construct comprising a cytokine encoding sequence under the regulatory control of a promoter comprising a sequence having at least 98% sequence identity to any one of SEQ ID NOs: 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46 or 47.

2. 2. The artificial expression construct of claim 1, wherein the promoter comprises a sequence having at least 99% sequence identity to any one of SEQ ID NOs: 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46 or 47.

3. 2. The artificial expression construct of claim 1, wherein the promoter comprises a sequence set forth in any one of SEQ ID NOs: 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46 or 47.

4. 2. The artificial expression construct of claim 1, wherein the promoter further comprises a minimal promoter.

5. 5. The artificial expression construct of claim 4, wherein the minimal promoter comprises a minimal promoter of IL2.

6. 5. The artificial expression construct of claim 4, wherein the minimal promoter comprises the sequence set forth in SEQ ID NO: 138 or a sequence having at least 98% sequence identity to the sequence set forth in SEQ ID NO:

138.

7. 2. The artificial expression construct of claim 1, wherein the cytokine comprises transforming growth factor beta 2-7m (TGFβ2-7m), mini TGFβ2-7m* (mTGFβ2-7m*), interleukin 21 (IL21), interleukin 15 (IL15), single-chain interleukin 12 (scIL12), decoy-resistant interleukin 18 (DR-IL18), or interleukin 36γ (IL36γ).

8. 2. The artificial expression construct of claim 1, wherein the cytokine is encoded by a sequence set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6 or SEQ ID NO:7, or by a sequence having at least 90% sequence identity to a sequence set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6 or SEQ ID NO:

7.

9. 2. The artificial expression construct of claim 1, wherein the cytokine comprises the sequence set forth in SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52 or SEQ ID NO: 54, or a sequence having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52 or SEQ ID NO:

54.

10. 10. The artificial expression construct of claim 1, further comprising a control mechanism.

11. 11. The artificial expression construct of claim 10, wherein the control mechanism comprises a transduction marker.

12. 12. The artificial expression construct of claim 11, wherein the transduction marker comprises Her2tG.

13. 2. The artificial expression construct of claim 1, further comprising a first skip sequence.

14. 14. The artificial expression construct of claim 13, wherein the first skip sequence encodes a 2A self-cleaving polypeptide.

15. 15. The artificial expression construct of claim 14, wherein the 2A self-cleaving polypeptide comprises T2A, P2A, E2A, or F2A.

16. 2. The artificial expression construct of claim 1, having at least 95% sequence identity with the sequence set forth in SEQ ID NO:

136.

17. 2. The artificial expression construct of claim 1, having at least 98% sequence identity with the sequence set forth in SEQ ID NO:

136.

18. 2. The artificial expression construct of claim 1, having at least 99% sequence identity with the sequence set forth in SEQ ID NO:

136.

19. 2. The artificial expression construct of claim 1, having the sequence set forth in SEQ ID NO:

136.

20. 10. The artificial expression construct of claim 1, further comprising a sequence encoding a recombinant receptor comprising a binding domain within its extracellular portion that binds to an antigen expressed on the surface of a target cell.

21. 21. The artificial expression construct of claim 20, wherein the sequence encoding the recombinant receptor is operably linked to a second promoter.

22. 22. The artificial expression construct of claim 21, wherein the second promoter comprises an EF1α(L) promoter, an EF1α(s) promoter, a myeloproliferative sarcoma virus (MND) promoter, a cytomegalovirus (CMV) promoter, a simian virus 40 (SV40) early promoter, a mouse mammary tumor virus (MMTV) promoter, a human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, a MoMuLV promoter, an avian leukosis virus promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter, an actin promoter, a myosin promoter, a hemoglobin promoter, or a creatine kinase promoter.

23. 21. The artificial expression construct of claim 20, wherein the binding domain is part of the extracellular portion.

24. 21. The artificial expression construct of claim 20, wherein the target cell comprises a cell infected with a bacterium, a virus, a fungus, a parasite, or an arthropod, or a cancer cell.

25. 21. The artificial expression construct of claim 20, wherein the recombinant receptor further comprises an intracellular portion.

26. 26. The artificial expression construct of claim 25, wherein the intracellular portion comprises the signaling domain of CD3ζ and / or the signaling domain of 4-1BB.

27. 26. The artificial expression construct of claim 25, wherein the intracellular portion is linked to the extracellular portion via a transmembrane domain.

28. 28. The artificial expression construct of claim 27, wherein the transmembrane domain comprises the transmembrane domain of CD28.

29. 21. The artificial expression construct of claim 20, further comprising a second control mechanism.

30. 30. The artificial expression construct of claim 29, wherein the second control mechanism comprises a selection cassette.

31. 31. The artificial expression construct of claim 30, wherein the selection cassette comprises a dihydrofolate reductase double mutant (DHFRdm).

32. 30. The artificial expression construct of claim 29, further comprising a third control mechanism.

33. 33. The artificial expression construct of claim 32, wherein the third control mechanism comprises a transduction marker.

34. 34. The artificial expression construct of claim 33, wherein the transduction marker comprises epidermal growth factor receptor (EGFRt) or truncated CD19 (tCD19).

35. 21. The artificial expression construct of claim 20, further comprising a second skip sequence.

36. 36. The artificial expression construct of claim 35, wherein the second skip sequence encodes a 2A self-cleaving polypeptide.

37. 37. The artificial expression construct of claim 36, wherein the 2A-skipping self-cleaving polypeptide comprises T2A, P2A, E2A, or F2A.

38. 34. The artificial expression construct of claim 33, comprising a second skip sequence at the 5' end of the selection cassette and a third skip sequence at the 5' end of the transduction marker.

39. 2. The artificial expression construct of claim 1, having at least 90% sequence identity with the sequence set forth in SEQ ID NO: 96, 97, 98, 99 or 101.

40. 2. The artificial expression construct of claim 1, having the sequence set forth in SEQ ID NO: 96, 97, 98, 99 or 101.

41. A system for enhancing immune cell function, comprising: a first artificial expression construct encoding a cytokine under the regulatory control of a first promoter comprising a sequence having at least 98% sequence identity to any one of SEQ ID NOs: 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46 or 47; a second artificial expression construct comprising a sequence encoding a recombinant receptor comprising a binding domain within its extracellular portion that binds to an antigen expressed on the surface of a target cell; Including, the system.

42. 42. The system of claim 41, wherein the first promoter comprises a sequence having at least 99% sequence identity to any one of SEQ ID NOs: 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46 or 47.

43. 42. The system of claim 41, wherein the first promoter comprises a sequence set forth in any one of SEQ ID NOs: 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46 or 47.

44. 42. The system of claim 41, wherein the first promoter further comprises a minimal promoter.

45. The system of claim 44, wherein the minimal promoter comprises an IL2 minimal promoter.

46. 45. The system of claim 44, wherein the minimal promoter comprises the sequence set forth in SEQ ID NO: 138 or a sequence having at least 98% sequence identity to the sequence set forth in SEQ ID NO:

138.

47. 42. The system of claim 41, wherein the cytokine comprises transforming growth factor beta 2-7m (TGFβ2-7m), mini TGFβ2-7m* (mTGFβ2-7m*), interleukin 21 (IL21), interleukin 15 (IL15), single-chain interleukin 12 (scIL12), decoy-resistant interleukin 18 (DR-IL18), or interleukin 36γ (IL36γ).

48. 42. The system of claim 41, wherein the cytokine is encoded by a sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 7, or by a sequence having at least 90% sequence identity to a sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO:

7.

49. 42. The system of claim 41, wherein the cytokine comprises a sequence set forth in SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52 or SEQ ID NO: 54, or a sequence having at least 90% sequence identity to a sequence set forth in SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52 or SEQ ID NO:

54.

50. 42. The system of claim 41, wherein the first artificial expression construct further comprises a control mechanism.

51. 51. The system of claim 50, wherein the control mechanism comprises a transduction marker.

52. 52. The system of claim 51, wherein the transduction marker comprises Her2tG.

53. 42. The system of claim 41, wherein the first artificial expression construct further comprises a first skip sequence.

54. 54. The system of claim 53, wherein the first skip sequence comprises a 2A skip sequence.

55. The system of claim 54, wherein the 2A skip sequence encodes a T2A self-cleaving polypeptide, a P2A self-cleaving polypeptide, an E2A self-cleaving polypeptide, or an F2A self-cleaving polypeptide.

56. 42. The system of claim 41, wherein the sequence encoding the recombinant receptor is operably linked to a second promoter.

57. 57. The system of claim 56, wherein the second promoter comprises an EF1α(L) promoter, an EF1α(s) promoter, an MND promoter, a CMV promoter, an SV40 early promoter, an MMTV promoter, an HIV LTR promoter, a MoMuLV promoter, an avian leukosis virus promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter, an actin promoter, a myosin promoter, a hemoglobin promoter, or a creatine kinase promoter.

58. 42. The system of claim 41, wherein the second artificial expression construct further comprises a second control mechanism.

59. 59. The system of claim 58, wherein the second control mechanism comprises a selection cassette.

60. 60. The system of claim 59, wherein the selection cassette comprises a dihydrofolate reductase double mutant (DHFRdm).

61. 59. The system of claim 58, wherein the second artificial expression construct further comprises a third control mechanism.

62. 62. The system of claim 61, wherein the third control mechanism comprises a transduction marker.

63. 63. The system of claim 62, wherein the transduction marker comprises a truncated epidermal growth factor receptor (EGFRt) or tCD19.

64. 42. The system of claim 41, wherein the second artificial expression construct further comprises a second skip sequence.

65. 65. The system of claim 64, wherein the second skip sequence comprises a 2A skip sequence.

66. The system of claim 65, wherein the 2A skip sequence encodes a T2A self-cleaving polypeptide, a P2A self-cleaving polypeptide, an E2A self-cleaving polypeptide, or an F2A self-cleaving polypeptide.

67. 63. The system of claim 62, wherein the second artificial expression construct comprises a second skip sequence at the 5' end of the selection cassette and a third skip sequence at the 5' end of the transduction marker.

68. 42. The system of claim 41, wherein the binding domain is part of the extracellular portion.

69. 42. The system of claim 41, wherein the target cells comprise cells infected with bacteria, viruses, fungi, parasites or arthropods, or cancer cells.

70. 42. The system of claim 41, wherein the recombinant receptor further comprises an intracellular portion.

71. The system of claim 70, wherein the intracellular portion comprises the signaling domain of CD3ζ and / or the signaling domain of 4-1BB.

72. 71. The system of claim 70, wherein the intracellular portion is linked to the extracellular portion via a transmembrane domain.

73. The system of claim 72, wherein the transmembrane domain comprises the transmembrane domain of CD28.

74. 42. The system of claim 41, wherein the first artificial expression construct and the second artificial expression construct are present on a single artificial expression construct.

75. 42. The system of claim 41, wherein the first artificial expression construct and the second artificial expression construct are present on separate artificial expression constructs.

76. 42. A nanoparticle encapsulating an artificial expression construct according to claim 1 or a system according to claim 41.

77. 42. A cell genetically engineered to express the artificial expression construct of claim 1 or the system of claim 41.

78. 78. The cell of claim 77, which is an autologous cell obtained from the subject or a cell allogeneic to the subject.

79. 78. The cell of claim 77, which is an in vivo cell or an ex vivo cell.

80. 78. The cell of claim 77, which is an immune cell.

81. 81. The cell of claim 80, wherein the immune cell is a lymphocyte.

82. 82. The cell of claim 81 , wherein the lymphocyte comprises a T cell, a B cell, a natural killer (NK) cell, or an NK-T cell.

83. The cell of claim 77, which is a T cell selected from a CD3+ T cell, a CD4+ T cell, a CD8+ T cell, a central memory T cell, an effector memory T cell and / or a naive T cell.

84. The cell described in claim 77, which is a CD8+ T cell.

85. 78. The cell of claim 77, which is a CD4+ T cell.

86. A cell population genetically modified to express the artificial expression construct of claim 1 or the system of claim 41.

87. 87. The cell population of claim 86, comprising autologous cells obtained from a subject or cells allogeneic to the subject.

88. 87. The cell population of claim 86, which is an in vivo cell population or an ex vivo cell population.

89. 87. The cell population of claim 86, wherein the cells are immune cells.

90. 90. The cell population of claim 89, wherein the immune cells are lymphocytes.

91. 91. The cell population of claim 90, wherein the lymphocytes comprise T cells, B cells, natural killer (NK) cells, or NK-T cells.

92. 87. The cell population of claim 86, comprising CD4+ T cells and / or CD8+ T cells.

93. A formulation comprising: (i) a cell genetically modified to express the artificial expression construct of claim 1 or the system of claim 41; and (ii) a pharmaceutically acceptable carrier.

94. 1. A method of genetically modifying immune cells to have enhanced function, comprising:

10. A method comprising contacting an immune cell with the artificial expression construct of claim 1 having a sequence encoding a cytokine.

95. 95. The method of claim 94, wherein the enhanced function comprises enhanced cytotoxicity.

96. 96. The method of claim 95, wherein the encoded cytokine comprises TGFβ2-7m, mTGFβ2-7m*, IL21, IL15, scIL12, DR-IL18, or IL36γ.

97. 96. The method of claim 95, wherein the encoded cytokine comprises IL21 or IL15.

98. 95. The method of claim 94, wherein the enhanced function comprises enhanced proliferation.

99. 99. The method of claim 98, wherein the encoded cytokine comprises TGFβ2-7m, mTGFβ2-7m*, IL21, IL15, scIL12, DR-IL18, or IL36γ.

100. 99. The method of claim 98, wherein the encoded cytokine comprises IL21 or IL15.

101. 95. The method of claim 94, wherein the enhanced function comprises enhanced cytokine production.

102. 102. The method of claim 101, wherein the enhanced cytokine production comprises IFNγ.

103. 103. The method of claim 102, wherein the encoded cytokine comprises scIL12, DR-IL18, IL36γ, TGFβ2-7m, or mTGFβ2-7m*.

104. 102. The method of claim 101, wherein the enhanced cytokine production comprises production of TNFα.

105. The method of claim 104, wherein the encoded cytokine comprises scIL12, DR-IL18, or IL36γ.

106. 95. The method of Claim 94, wherein the artificial expression construct further comprises a sequence encoding a recombinant receptor comprising a binding domain that binds to an antigen expressed on the surface of a target cell.

107. 107. The method of claim 106, wherein the sequence encoding the recombinant receptor is operably linked to a second promoter.

108. The method of claim 107, wherein the second promoter comprises an EF1α(L) promoter, an EF1α(s) promoter, a myeloproliferative sarcoma virus (MND) promoter, a cytomegalovirus (CMV) promoter, a simian virus 40 (SV40) early promoter, a mouse mammary tumor virus (MMTV) promoter, a human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, a MoMuLV promoter, an avian leukosis virus promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter, an actin promoter, a myosin promoter, a hemoglobin promoter, or a creatine kinase promoter.

109. 95. The method of Claim 94, further comprising contacting the immune cells with an artificial expression construct comprising a sequence encoding a recombinant receptor comprising a binding domain that binds to an antigen expressed on the surface of a target cell.

110. 110. The method of Claim 109, wherein the steps of contacting the immune cells with the artificial expression construct of Claim 1 and contacting the immune cells with an artificial expression construct comprising a sequence encoding the recombinant receptor are performed simultaneously.

111. 110. The method of Claim 109, wherein the steps of contacting the immune cells with the artificial expression construct of Claim 1 and contacting the immune cells with an artificial expression construct comprising a sequence encoding the recombinant receptor are performed at different times.

112. 95. The method of claim 94, wherein the artificial expression construct comprises the sequence set forth in SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99 or SEQ ID NO: 101, or a sequence with 90% sequence identity to the sequence set forth in SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99 or SEQ ID NO:

101.

113. 100. A method of treating a subject in need of treatment, comprising administering to the subject in need of treatment a therapeutically effective amount of the artificial expression construct of claim 1, the system of claim 41, the nanoparticle of claim 76, or the formulation of claim 93, thereby treating the subject.

114. 114. The method of claim 113, wherein the subject in need of treatment has cancer or an infectious disease.

115. 114. The method of claim 113, wherein administering the therapeutically effective amount comprises administering intravesically, intravenously, intradermally, intraarterially, parenterally, intranodal, intralymphatic, intraperitoneally, intralesionally, intraprostatically, intravaginally, intrarectally, topically, intrathecally, intratumorally, intramuscularly, or subcutaneously.

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