Inducible programmed cell death 1 (PD1)-cytokine chimeras for enhancing immune cell function

Abstract

This disclosure describes an artificial expression construct containing a PD1:cytokine chimeric transgene under the control of an inducible promoter. The artificial expression construct of this disclosure can be used to enhance the function of immune cells (e.g., CAR-T cells) that have recombinant receptors. The inducible PD1:cytokine chimeric transgene disclosed herein enhances the potency of immune cells (e.g., CAR-T cells) that have recombinant receptors, thereby enhancing killing, proliferation of immune cells, and / or cytokine production.

Description

Technical Field

[0001] Cross-reference of related applications This application claims the priority of U.S. Provisional Patent Application No. 63 / 496,360, filed on April 14, 2023, which is hereby incorporated by reference in its entirety as if fully set forth herein.

[0002] Sequence listing reference The Sequence Listing accompanying this application is provided in XML format rather than as a hard copy, and this Sequence Listing is hereby incorporated by reference in this specification. The name of the XML file containing this Sequence Listing is 31I7118.xml. The size of this file is 200,704 bytes, it was created on April 12, 2024, and was electronically submitted via Patent Center.

[0003] The present disclosure provides an artificial expression construct comprising a programmed cell death 1 (PD1): cytokine chimeric transgene under the control of an inducible promoter for enhancing the function of immune cells (e.g., CAR-T cells). The PD1: cytokine chimeric transgene disclosed herein enhances the function of immune cells at the tumor site by enhancing (i) killing of target cells by immune cells, (ii) proliferation of immune cells, and / or (iii) production of cytokines in the environment of immune cells.

Background Art

[0004] According to the World Health Organization, cancer is one of the leading causes of death worldwide, and cancer deaths reached nearly 10 million in 2020. Conventional treatments such as surgery, chemotherapy, and / or radiotherapy have existed for over a century, but they affect not only cancer cells but also normal cells. Furthermore, surgery, chemotherapy, and radiotherapy have significant side effects and sometimes cause permanent side effects.

[0005] In recent years, more targeted cancer therapies have been developed. Targeted therapies primarily identify and utilize specific molecular changes and / or specific immunophenotypic changes observed in cancer cells to specifically target them. For example, many cancer cells selectively express markers on their cell surface, and such markers may be used as targets for antibody-based therapies.

[0006] Genetic engineering techniques for immune system cells have advanced significantly, making it possible to target and kill undesirable cells such as cancer cells. Many of these immune cells are T cells that have been genetically engineered to express recombinant receptors, such as chimeric antigen receptors (CARs). CARs are proteins with several characteristic components that enable genetically modified immune cells to recognize and kill target types of cells. These components include at least an extracellular and an intracellular portion, which are expressed as a single protein or in a form assembled into functional units. The extracellular portion contains a binding domain that specifically binds to markers (e.g., antigens) selectively present on the surface of undesirable cells such as cancer cells. When the binding domain binds to such a marker, a signal is transmitted from the intracellular portion to the immune cell, causing the cell to be destroyed. CARs may further include a transmembrane domain that can link the extracellular portion to the intracellular portion.

[0007] Other components of a CAR can also be used to enhance its function. For example, spacers can often provide greater flexibility to the CAR's three-dimensional structure, enhancing the binding ability of the binding domain to target cell markers, and consequently increasing its cytolytic effect. The appropriate length of the spacer in a particular CAR depends on numerous factors, including the requirement of how close or far the target marker is from the surface of the cell membrane of undesirable cells.

[0008] While CAR T cells have achieved considerable success in treating various cancers, challenges remain. For example, while CAR T cells have been successful in treating humoral tumors, their application to solid tumors faces challenges. Furthermore, CAR T cells may exhibit suboptimal proliferation, functional unresponsiveness or exhaustion, memory differentiation disorders, 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, particularly in situations involving prolonged antigen exposure that can lead to functional unresponsiveness and exhaustion. [Overview of the project] [Problems that the invention aims to solve]

[0009] In chimeric antigen receptor (CAR) T cell therapy for solid tumors, the aforementioned drawbacks necessitate enhancing CAR T cells with transgenes that can boost their efficacy. Generally, programmed cell death ligand 1 (PD-L1) is upregulated in cells within solid tumors, and interaction with endogenous programmed cell death 1 (PD1) expressed on the surface of CAR T cells leads to inhibition of CAR T cells, loss of effector function, and cellular dysfunction. On the other hand, by utilizing the upregulation of PD-L1, secreted CAR T cell efficacy-enhancing molecules can be released. These secreted enhancing molecules can improve the effectiveness of CAR T cells by, for example, maintaining the memory CAR T cell pool, promoting the proliferation and survival of memory and naive T cells, and further recruiting immunostimulatory cell types into the tumor microenvironment. [Means for solving the problem]

[0010] In certain embodiments, the secreted CAR T cell efficacy-enhancing molecules disclosed herein include PD1 linked to an immunostimulatory cytokine (referred to as the "PD1:cytokine chimera"). Because the immunostimulatory cytokine is linked to PD1 and PD-L1 is upregulated in tumor cells, this immunostimulatory cytokine can be maintained within the tumor microenvironment. Furthermore, the expression of this PD1:cytokine chimera can be regulated by a promoter that can be induced immediately after CAR activation and signaling. These characteristics allow for the restriction of PD1:cytokine chimera secretion within the tumor microenvironment, further enhancing the localization effect of the artificial expression constructs of this disclosure. In certain embodiments, the A99L mutation is utilized in the PD1 portion of the PD1:cytokine chimera to enhance binding to PD-L1.

[0011] In certain embodiments, an artificial expression construct is provided that includes a PD1:cytokine chimeric transgene under the control of an inducible promoter. As described herein, the PD1:cytokine chimeras of this disclosure can be used to enhance the function of immune cells (e.g., T cells or chimeric antigen receptor (CAR) T cells), etc. The inducible PD1:cytokine chimeric transgenes disclosed herein can enhance the efficacy of immune cells (e.g., T cells or CAR-T cells) to increase killing, proliferation of immune cells, and / or cytokine production.

[0012] In certain embodiments, the PD1:cytokine chimeric transgene of the Disclosure encodes a soluble PD1:cytokine chimera. In certain embodiments, the PD1:cytokine chimeric transgene of the Disclosure encodes a recombinant protein comprising (i) PD1 and (ii) interleukin 21 (IL21), interleukin 15, interleukin 15 mutant protein N72D (IL15(N72D)), interleukin 18 (IL18), decoy-resistant interleukin 18 (DR-IL18 or drIL18), interleukin 12 (IL12), or interferon α (IFNα). PD1 means wild-type PD1 or PD1(A99L) mutant.

[0013] In certain embodiments, the inducible expression of PD1:IL21, PD1:IL15(N72D), PD1:drIL18, PD1:IL12, or PD1:IFNα is utilized to enhance immune cell-mediated cell killing. In certain embodiments, the inducible expression of PD1:IL12, PD1:IL15(N72D), PD1:drIL18, and PD1:IL21 is utilized to enhance immune cell-mediated cell killing.

[0014] In certain embodiments, T cell proliferation is enhanced by utilizing the inducible expression of PD1:IL21, PD1:IL15(N72D), PD1:drIL18, PD1:IL12, or PD1:IFNα.

[0015] In certain embodiments, cytokine production is enhanced by utilizing the inducible expression of PD1:IL21, PD1:IL15(N72D), PD1:drIL18, PD1:IL12, or PD1:IFNα.

[0016] In certain embodiments, inducible expression of PD1:IFNα is utilized to enhance IFNγ production.

[0017] In certain embodiments, inducible expression of PD1:IFNα is utilized to enhance TNFα production.

[0018] In certain embodiments, inducible expression of PD1:IL12 is used in combination with inducible expression of PD1:IL15(N72D), PD1:drIL18, and PD1:IL21. In certain embodiments, inducible expression of PD1:IL21 is used in combination with inducible expression of PD1:IL15(N72D), PD1:drIL18, PD1:IL12, and / or PD1:IFNα. In certain embodiments, inducible expression of PD1:IL15(N72D) is used in combination with inducible expression of PD1:IL21, PD1:drIL18, PD1:IL12, and / or PD1:IFNα. In certain embodiments, inducible expression of PD1:drIL18 is used in combination with inducible expression of PD1:IL21, PD1:IL15(N72D), PD1:IL12, and / or PD1:IFNα. In certain embodiments, inducible expression of PD1:IL12 is used in combination with inducible expression of PD1:IL21, PD1:IL15(N72D), PD1:drIL18 and / or PD1:IFNα. In certain embodiments, inducible expression of PD1:IFNα is used in combination with inducible expression of PD1:IL21, PD1:IL15(N72D), PD1:drIL18 and / or PD1:IL12.

[0019] In certain embodiments, PD1 and cytokines are linked via a linker. This linker may be a flexible linker, such as a glycineserine linker. In certain embodiments, the glycineserine linker contains the sequence shown in SEQ ID NO: 128. In certain embodiments, the glycineserine linker contains the sequence GGG.

[0020] In certain embodiments, the expression of the PD1:cytokine chimeric transgene of this disclosure is under the control of a promoter. In certain embodiments, the promoter is the iSynPro promoter. In certain embodiments, the iSynPro promoter comprises S1-61 (SEQ ID NO: 17 or 18). In certain embodiments, the iSynPro promoter comprises S1-61 (SEQ ID NO: 17). In certain embodiments, the artificial expression construct comprising the PD1:cytokine chimeric transgene of this disclosure further comprises or encodes skip sequences and regulatory mechanisms.

[0021] In certain embodiments, the immune cells include T cells. In certain embodiments, the immune cells can be recombinant to express a recombinant receptor. In certain embodiments, the artificial expression construct encoding the recombinant receptor may be included in an artificial expression construct comprising the PD1:cytokine chimeric transgene of the Disclosure, or in a separate artificial expression construct from the one comprising the PD1:cytokine chimeric transgene of the Disclosure. In certain embodiments, the artificial expression construct encoding the recombinant receptor includes a promoter and the recombinant receptor, or encodes a promoter and the recombinant receptor. In certain embodiments, the artificial expression construct encoding the recombinant receptor includes a promoter, the recombinant receptor, a skip sequence, and a regulatory mechanism, or encodes them. In certain embodiments, the recombinant receptor includes a CAR or recombinant T cell receptor (eTCR). In certain embodiments, the recombinant receptor includes an extracellular portion that binds to a target antigen, a transmembrane domain, and an intracellular portion.

[0022] Accordingly, this disclosure provides a system that influences the killing of immune cells, the proliferation of immune cells, and / or the production of cytokines by immune cells. The immune cells may include recombinant receptor-expressing immune cells that can be recombined to target various target cells expressing a target antigen (e.g., cancer cells, viruses, bacteria, fungi) by recombining their extracellular domains to include a binding domain that binds to a target antigen. [Brief explanation of the drawing]

[0023] Some of the drawings submitted in this application are considered easier to understand in color. The applicant considers color versions of these drawings to be part of the original application and reserves the right to submit color images of the drawings in subsequent proceedings.

[0024] [Figure 1] Schematic diagrams of various PD1:cytokine chimeric transgenes are shown (in this specification, they may be referred to as "Staplekine," and sometimes "Staplekine" is used instead of "PD1" in construct names). These PD1:cytokine chimeras utilize the A99L mutant programmed cell death 1 (PD1(A99L)) receptor. This receptor is known to strongly interact with its ligand, PDL1, which is a ligand that is normally upregulated in tumor-derived tissues. Five examples of immunostimulatory signals that can be added to PD1(A99L) via a short linker are presented: interleukin (IL) 21, IL 15 (N72D), decoy-resistant IL 18 (DR-18 or drIL 18), IL 12, and IFNα. By adding these immunostimulatory signals to the PD1(A99L) receptor, cytokine signaling can be maintained in local environments with high PDL1 expression, thereby enhancing the efficacy and function of recombinant T cells.

[0025] [Figure 2]The structure of a construct to facilitate T cell production and screening is shown. The PD1(A99L):cytokine chimeric transgene is driven under the regulation of an inducible promoter (e.g., S1-61) of a chimeric antigen receptor (CAR), enabling the controlled release of the encoded immunomodulatory protein after the CAR has associated with its target antigen. As shown in the design of this construct, it employs a dual promoter construct format, featuring an inducible promoter that drives the PD1:cytokine chimeric transgene and a constitutive expression promoter (e.g., EF1a (long chain) promoter) that drives the expression of CAR, double mutant dihydrofolate reductase (DHFRdm), and cleaved epidermal growth factor receptor (EGFRt), with CAR and DHFRdm separated by a 2A skip sequence, and DHFRdm and EGFRt also separated by a 2A skip sequence. This inducible promoter can further drive the gene expression of a cell surface localized polypeptide tag based on cleaved human HER2 (Her2tG), which can then be linked to the PD1 cytokine chimeric transgene via a 2A skip sequence.

[0026] [Figure 3]Effector T cell lines were manufactured by introducing the above construct and evaluated by detection of EGFRt, and it was found that these effector T cell lines maintained a high CD19CAR positivity rate. Specifically, after manufacturing cells using the TICLE manufacturing method, the CAR positivity rate of CD4+ T cells was investigated by staining for EGFRt and analyzing by flow cytometry. Briefly, the TICLE method used herein includes the steps of: obtaining CD8+ fraction, CD4+ fraction, and negative fraction from a sample (e.g., peripheral blood mononuclear cells); introducing the above artificial expression construct into CD8+ cells or CD4+ cells using electroporation or viral transduction; co-culturing the obtained transduced cells with the CD4+ fraction or CD8+ fraction and negative fraction; and co-culturing in a gas-permeable vessel in a medium supplemented with 7.5 U / mL IL2, 20 ng / mL IL4, 10 ng / mL IL7, and 20 ng / mL IL21. Based on the high EGFRt staining positivity rate for CD19CAR transgenes or CD19CAR+iSynPro PD1:IFNα cytokine transgenes, CD4+ cells into which each delivered construct was introduced were enriched.

[0027] [Figure 4]This study demonstrates cell killing and cell proliferation. CD4+ cells were highly enriched in a different donor based on the expression of CD19CAR alone or CD19CAR+iSynPro sPD1(A99L)-G3-IFNα (i.e., PD1:IFNα) and subjected to a repeated killing assay using the neuroblastoma cell line Be2-mCherry-CD19t. Effectors and targets were co-cultured for 72 hours, followed by two additional stimulations at 144 and 216 hours. Effectors and targets were set to various E:T ratios. As shown in the figure, 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 difference between each evaluated group was determined using the killing score (sum of the difference in Be2 cell killing between CD19CAR alone and CD19CAR+iSynPro PD1:IFNα). In CD4+ cells, phasing was enhanced by iSynPro PD1:IFNα compared to CD19CAR alone, suggesting that PD1:IFNα can improve the function of CAR T cells. 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 evaluated compared to CD19CAR alone using the same scoring method as above. Compared to CD19CAR alone, T cell proliferation was enhanced by PD1:IFNα.

[0028] [Figure 5] This study demonstrates cytokine production. Supernatants were collected 24 hours after initiation of co-cultures of CD19CAR CD4+ cells or CD19CAR+iSynPro sPD1(A99L)-G3-IFNα (i.e., PD1:IFNα) CD4+ cells and Be2-mCherry-CD19t target cells in a 1:1 E:T ratio. The cytokine concentrations of interferon-gamma (IFNg) and tumor necrosis factor-alpha (TNFα), which have effector functions, were evaluated. iSynPro PD1:IFNα enhanced the production of both IFNg and TNFα.

[0029] [Figure 6]This study demonstrates target killing by donor-derived CD4+ cells. CD4+ cell lines were constructed using a library of equimolar mixtures of four dual-promoter plasmids (PD1(A99L):IL-12, PD1(A99L):IL15(N72D), PD1(A99L):drIL18, and PD1(A99L):IL-21) using a method referred to herein as the TICLE method. Using these effector cells, an Incucyte-based target killing assay was performed with the neuroblastoma cell line Be2-CD19t-mCherry, repeatedly stimulating the target with multiple effector:target ratios for a total of two stimulations. The persistence rate of the Be2 signal, measured after the first and second stimulations, is plotted. Cells with the above library showed enhanced T-cell target killing at all E:T ratios, both after the first and second stimulations. "Venus" represents a library of PD1 cytokine chimeras, including PD1(A99L):IL-12, PD1(A99L):IL15(N72D), PD1(A99L):drIL18, and PD1(A99L):IL-21.

[0030] [Figure 7] This study demonstrates target killing by donor-derived CD8+ cells. CD8+ cell lines were constructed using a library of equimolar mixtures of four dual-promoter plasmids (PD1(A99L):IL-12, PD1(A99L):IL15(N72D), PD1(A99L):drIL18, and PD1(A99L):IL-21) using a method referred to herein as the TICLE method. Using these effector cells, an Incucyte-based target killing assay was performed with the neuroblastoma cell line Be2-CD19t-mCherry, repeatedly stimulating the target with multiple effector:target ratios for a total of two stimulations. The persistence rate of the Be2 signal, measured after the first and second stimulations, is plotted. Cells containing the PD1:cytokine chimera library enhanced T cell target killing at all E:T ratios, both during the first and second stimulations.

[0031] [Figure 8]The sequences supporting this disclosure are shown below: sdnPD1(A99L)(SEQ ID NO: 1), IL21(SEQ ID NO: 2), IL15(N72D)(SEQ ID NO: 3), DR-IL18(SEQ ID NO: 4), IL12(SEQ ID NO: 5), IFNα(SEQ ID NO: 6), the code sequence of PD1:IL21(PD1(A99L) and G4S×3 linker and IL21)(SEQ ID NO: 7), the code sequence of PD1:IL15(N72D)(PD1(A99L) and G4S×3 linker and IL15(N72D))(SEQ ID NO: 8), the code sequence of PD1:drIL18(PD1(A99L) and G4S×3 linker and drIL18)(Sequence No. 9), the coding sequence of PD1:IL12 (PD1(A99L) and G4S×3 linker and IL12) (SEQ ID NO: 10), the coding sequence of PD1:IFNα (PD1(A99L) and G×3 linker and IFNα) (SEQ ID NO: 11), the amino acid sequence of PD1:IL21 (PD1(A99L) and G4S×3 linker and IL21) (SEQ ID NO: 12), the amino acid sequence of PD1:IL15(N72D) (PD1(A99L) and G4S×3 linker and IL15(N72D)) (SEQ ID NO: 13), PD1:drIL18 (PD1(A99L) and G4S×3 linker and dr The amino acid sequence of IL18) (SEQ ID NO: 14), the amino acid sequence of PD1:IL12 (PD1(A99L) and G4S×3 linker and IL12) (SEQ ID NO: 15), the amino acid sequence of PD1:IFNα (PD1(A99L) and G×3 linker and IFNα) (SEQ ID NO: 16), S1-61-version 1 (SEQ ID NO: 17), S1-61-version 2 (SEQ ID NO: 18), S1-17 (SEQ ID NO: 19), S1-37 (SEQ ID NO: 20), S1-4 (SEQ ID NO: 21), S1-1 (SEQ ID NO: 22), S1-3 (SEQ ID NO: 23), S1-42 (SEQ ID NO: 24), S1- 62 (SEQ ID NO: 25), S1-15 (SEQ ID NO: 26), S1-2 (SEQ ID NO: 27), S1-27 (SEQ ID NO: 28), S1-8 (SEQ ID NO: 29), S1-30 (SEQ ID NO: 30), S1-33 ​​(SEQ ID NO: 31), S1-41 (SEQ ID NO: 32), S1-59 (SEQ ID NO: 33), S1-66 (SEQ ID NO: 34), S1-71 (SEQ ID NO: 35), S1-56 (SEQ ID NO: 36), S1-6 (SEQ ID NO: 37), S1-60 (SEQ ID NO: 38), S1-86 (SEQ ID NO: 39), S1-32 (SEQ ID NO: 40), S1-10 (SEQ ID NO: 41), S1-18 (SEQ ID NO: 42),S1-14 (SEQ ID NO: 43), S1-16 (SEQ ID NO: 44), S1-19 (SEQ ID NO: 45), S1-26 (SEQ ID NO: 46), S1-65 (SEQ ID NO: 47), S2-n1 (SEQ ID NO: 48), S4-n1 (SEQ ID NO: 49), S6-n1 (SEQ ID NO: 50), S1-325 (SEQ ID NO: 51), S1-60 (SEQ ID NO: 52), S2-274 (SEQ ID NO: 53), S2-310 (SEQ ID NO: 54), S1-367 (SEQ ID NO: 55), S1-7 (SEQ ID NO: 56), EF1a promoter (long chain; Ef1a-HTLV hybrid) (SEQ ID NO: 57), Core insulator - version 1 (SEQ ID NO: 58), Core insulator - version 2 (SEQ ID NO: 59), IgG4 hinge-A ESKYGPPCPPCP (SEQ ID NO: 60), IgG4 hinge coding sequence-A (SEQ ID NO: 61), IgG4 hinge coding sequence-B (SEQ ID NO: 62), IgG4 hinge coding sequence-C (SEQ ID NO: 63), IgG4-CH2 domain coding sequence (SEQ ID NO: 64), IgG4-CH3 coding sequence (SEQ ID NO: 65), CD28 transmembrane domain-A (SEQ ID NO: 66), CD28 transmembrane domain-B (SEQ ID NO: 67), CD28 transmembrane domain-C (SEQ ID NO: 68), CD28™ coding sequence-A (SEQ ID NO: 69), CD28™ coding sequence-B (SEQ ID NO: 70), CD28™ coding sequence-C (SEQ ID NO: 71), CD28™ coding sequence-D (SEQ ID NO: 72), 4-1B B co-stimulatory domain-A (SEQ ID NO: 73), 4-1BB co-stimulatory domain-B (SEQ ID NO: 74), 4-1BB co-stimulatory domain-C (SEQ ID NO: 75), 4-1BB signaling domain coding sequence-A (SEQ ID NO: 76), 4-1BB signaling domain coding sequence-B (SEQ ID NO: 77), 4-1BB signaling domain coding sequence-C (SEQ ID NO: 78), CD3ζ signaling domain-A (SEQ ID NO: 79), CD3ζ signaling domain-B (SEQ ID NO: 80), CD3ζ signaling domain-C (SEQ ID NO: 81), CD3ζ signaling domain coding sequence-A (SEQ ID NO: 82), CD3ζ signaling domain coding sequence-B (SEQ ID NO: 83), Thosea asigna virus 2A (T2A) peptide-v1 (SEQ ID NO: 84), Thosea asigna virus 2A (T2A) peptide-v2 (SEQ ID NO: 85),Thosea asigna virus 2A (T2A) peptide-v3 (SEQ ID NO: 86), porcine tesiovirus-1 2A (P2A) peptide (SEQ ID NO: 87), equine rhinitis A virus (ERAV) 2A (E2A) peptide (SEQ ID NO: 88), foot-and-mouth disease virus 2A (F2A) peptide (SEQ ID NO: 89), T2A code sequence-v1 (SEQ ID NO: 90), T2A code sequence-v2 (SEQ ID NO: 91), EGFRt-version 1 (SEQ ID NO: 92), EGFRt-version 2 (SEQ ID NO: 93), EGFRt code sequence (SEQ ID NO: 94), tCD19 code sequence (SEQ ID NO: 95), HER2tG (excluding start codon) )(SEQ ID NO: 96), DHFRdm(SEQ ID NO: 97), vector s1-61-il2mp_her2tg-t2a-sdnpd1a99l-g4s3-il21_rbgpolya_2x3hsa_ef1al_hucd19scfvg01s-igg4hinge-cd28tm-41bb-zeta-p2a-dhfrdm-t2a-egfrt_bghpolya_pbnp(SEQ ID NO: 98), vector s1-61-il2mp_her2tg-t2a-sdnpd1199-g4s3 -il15n72d_rbgpolya_2x3hs-a_ef1a7-3_hucd19scfvg01s-igg4hinge-cd28tm-41bb-zeta-p2a-dhfrdm-t2a-egfrt_bghpolya_pbn p (SEQ ID NO: 99), vector s1-61-il2mp_her2tg-t2a-sdnpd1199-g4s3-dr-il186-12_rbgpolya_2x3hs-a_ef1a7-3_hucd19scfvg01s-igg4hi nge-cd28tm-41bb-zeta-p2a-dhfrdm-t2a-egfrt_bghpolya_pbnp (SEQ ID NO: 100), vector s1-61-il2mp_her2tg-t2a-sdnpd1a99l-g4s3-il12_rbgpolya_2x3hsa_ef1al_hucd19scfvg01s-igg4hinge-cd28tm-41bb-zeta-p2a-dhfrdm-t2a-egfrt_bghpolya_pbnp (SEQ ID NO: 101),Vector s1-61-il2mp_her2tg-t2a-sdnpd1a99l-g3-iFNα5878_rbgpolya_2x3hsa_ef1al_hucd19scfvg01s-igg4hinge-cd28tm-41bb-zeta-p2a-dhfrdm-t2a-egfrt_bghpolya_pbnp (SEQ ID NO: 102), and vector s1-61-il2mp_her2tg_t2a_pd1a99l-g4s3-il12_pbnp (SEQ ID NO: 103). [Modes for carrying out the invention]

[0032] Over the past several decades, various therapies have been developed to construct immune cells that express recombinant receptors that bind to antigens on undesirable types of cells (e.g., cancer cells or virus-infected cells, also referred to herein as “target cells”). When recombinant receptors bind to target antigens, immune cells are activated, and these immune cells can destroy the bound cells. Chimeric antigen receptor (CAR) cell therapy is one such therapy.

[0033] Chimeric antigen receptor (CAR) therapy involves recombining immune cells to express synthetic receptors designed to target cells such as cancer cells or virus-infected cells. While CAR therapy has achieved considerable success in treating various cancers, challenges remain. For example, CAR T cells have been successful in treating humoral tumors, but their application to solid tumors faces challenges. Furthermore, CAR T cells may exhibit suboptimal proliferation, functional unresponsiveness or exhaustion, memory differentiation disorders, and / or limited persistence, limiting their ability to induce or maintain remission in patients. Therefore, strategies are needed to improve the efficacy of CAR T cells, particularly in situations involving prolonged antigen exposure that can lead to functional unresponsiveness and exhaustion.

[0034] In CAR T cell therapy for solid tumors, the aforementioned drawbacks necessitate enhancing CAR T cells with transgenes that can boost their efficacy. Generally, programmed death ligand 1 (PD-L1) is upregulated in cells within solid tumors, and interaction with endogenous PD1 expressed on the surface of CAR T cells leads to inhibition of CAR T cells, loss of effector function, and cellular dysfunction. On the other hand, by utilizing the upregulation of PD-L1, secreted CAR T cell efficacy-enhancing molecules can be released. These secreted enhancing molecules can improve the effectiveness of CAR T cells by, for example, maintaining the memory CAR T cell pool, promoting the proliferation and survival of memory T cells and naive T cells, and further recruiting immunostimulatory cell types into the tumor microenvironment.

[0035] In certain embodiments, the secreted CAR T cell efficacy-enhancing molecules disclosed herein include PD1 linked to an immunostimulatory cytokine (referred to as the "PD1:cytokine chimera"). Because the immunostimulatory cytokine is linked to PD1 and PD-L1 is upregulated in tumor cells, this immunostimulatory cytokine can be maintained within the tumor microenvironment. Furthermore, the expression of this PD1:cytokine chimera can be regulated by a promoter that can be induced immediately after CAR activation and signaling. These characteristics allow for the restriction of PD1:cytokine chimera secretion within the tumor microenvironment, further enhancing the localization effect of the artificial expression constructs of this disclosure. In certain embodiments, the A99L mutation is utilized in the PD1 portion of the PD1:cytokine chimera to enhance binding to PD-L1.

[0036] In certain embodiments, an artificial expression construct is provided that includes a programmed cell death 1 (PD1):cytokine chimeric transgene under the control of an inducible promoter. As described herein, the PD1:cytokine chimeras of this disclosure can be used to enhance the function of immune cells (e.g., T cells or chimeric antigen receptor (CAR) T cells), etc. The inducible PD1:cytokine chimeric transgenes disclosed herein can enhance the potency of immune cells (e.g., T cells or CAR-T cells) to increase killing, proliferation of immune cells, and / or cytokine production.

[0037] In certain embodiments, the PD1:cytokine chimeric transgene of the Disclosure encodes a soluble PD1:cytokine chimeric transgene. In certain embodiments, the PD1:cytokine chimeric transgene of the Disclosure encodes a recombinant protein comprising (i) wild-type PD1 or PD1 A99L variant (PD1(A99L)) and (ii) interleukin 21 (IL21), interleukin 15, interleukin 15 mutant protein N72D (IL15(N72D)), interleukin 18 (IL18), decoy-resistant interleukin 18 (also called DR-IL18 or drIL18), interleukin 12 (IL12), or interferon α (IFNα). PD1 means wild-type PD1 or PD1(A99L) variant. In certain embodiments, PD1 is wild-type PD1. In certain embodiments, PD1 is PD1(A99L) variant.

[0038] In certain embodiments, the inducible expression of PD1:IL21, PD1:IL15(N72D), PD1:drIL18, PD1:IL12, or PD1:IFNα is utilized to enhance immune cell-mediated cell killing. In certain embodiments, the inducible expression of PD1:IL12, PD1:IL15(N72D), PD1:drIL18, and PD1:IL21 is utilized to enhance immune cell-mediated cell killing. In certain embodiments, the inducible expression of PD1:IFNα is utilized to enhance immune cell-mediated cell killing.

[0039] In certain embodiments, T cell proliferation is enhanced by utilizing the inducible expression of PD1:IL21, PD1:IL15(N72D), PD1:drIL18, PD1:IL12, or PD1:IFNα.

[0040] In certain embodiments, cytokine production is enhanced by utilizing the inducible expression of PD1:IL21, PD1:IL15(N72D), PD1:drIL18, PD1:IL12, or PD1:IFNα. In certain embodiments, cytokine production is enhanced by utilizing the inducible expression of PD1:IFNα. In certain embodiments, cytokine production includes the production of IFNγ and / or TNFα.

[0041] In certain embodiments, inducible expression of PD1:IFNα is utilized to enhance IFNγ production.

[0042] In certain embodiments, inducible expression of PD1:IFNα is utilized to enhance TNFα production.

[0043] In this specification, the terms “enhancement” and “strengthening” are used interchangeably.

[0044] In certain embodiments, inducible expression of PD1:IL12 is used in combination with inducible expression of PD1:IL15(N72D), PD1:drIL18, and PD1:IL21. In certain embodiments, inducible expression of PD1:IL21 is used in combination with inducible expression of PD1:IL15(N72D), PD1:drIL18, PD1:IL12, and / or PD1:IFNα. In certain embodiments, inducible expression of PD1:IL15(N72D) is used in combination with inducible expression of PD1:IL21, PD1:drIL18, PD1:IL12, and / or PD1:IFNα. In certain embodiments, inducible expression of PD1:drIL18 is used in combination with inducible expression of PD1:IL21, PD1:IL15(N72D), PD1:IL12, and / or PD1:IFNα. In certain embodiments, inducible expression of PD1:IL12 is used in combination with inducible expression of PD1:IL21, PD1:IL15(N72D), PD1:drIL18 and / or PD1:IFNα. In certain embodiments, inducible expression of PD1:IFNα is used in combination with inducible expression of PD1:IL21, PD1:IL15(N72D), PD1:drIL18 and / or PD1:IL12. In certain embodiments, inducible expression of PD1:IL15 is used in combination with inducible expression of PD1:IL21, PD1:drIL18, PD1:IL12 and / or PD1:IFNα.

[0045] In certain embodiments, PD1 and a stimulating cytokine are linked via a linker. This linker may be a flexible linker, such as a glycineserine linker. In certain embodiments, the glycineserine linker contains the sequence shown in SEQ ID NO: 128. In certain embodiments, the glycineserine linker contains the sequence GGG.

[0046] In some examples, the artificial expression constructs of this disclosure, including the PD1:cytokine chimeric transgene, are encoded by the sequences shown in SEQ ID NOs. 7, 8, 9, 10, or 11.

[0047] In certain embodiments, the artificial expression construct of the present disclosure comprising a PD1:cytokine chimeric transgene comprises a promoter encoding the PD1:cytokine chimeric transgene. In certain embodiments, the promoter comprises an iSynPro promoter and a minimal promoter of IL2. In certain embodiments, the artificial expression construct of the present disclosure encoding the PD1:cytokine chimeric transgene further comprises or encodes skip sequences and regulatory mechanisms. In certain embodiments, the iSynPro promoter comprises S1-61 (SEQ ID NO: 17).

[0048] Furthermore, this disclosure provides systems and methods for genetically modifying immune cells to express recombinant receptors. In some embodiments, the artificial expression construct of this disclosure, which includes a PD1:cytokine chimeric transgene, may further include a sequence encoding the recombinant receptor. In another embodiment, the sequence encoding the recombinant receptor and the PD1:cytokine chimeric transgene may reside 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 of this disclosure includes or encodes a promoter and a recombinant receptor. In certain embodiments, the artificial expression construct of this disclosure includes or encodes a promoter, a recombinant receptor, a skip sequence, and a regulatory mechanism. In certain embodiments, the artificial expression construct of this disclosure includes or encodes a promoter, a recombinant receptor, a first skip sequence, a selection cassette (e.g., a dihydrofolate reductase double mutant (DHFRdm)), a second skip sequence, and a transduction marker (EGFRt). In certain embodiments, the recombinant receptor includes a CAR or 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 of this disclosure, comprising a PD1:cytokine chimeric transgene and a recombinant receptor, comprises or encodes an inductive promoter, a PD1:cytokine chimeric transgene, a 2A skip sequence, a constitutive promoter, and a recombinant receptor. In certain embodiments, the artificial expression construct of this disclosure, comprising a PD1:cytokine chimeric transgene and a recombinant receptor, comprises or encodes an inductive promoter, a PD1:cytokine chimeric transgene, a first 2A skip sequence, Her2tG, a constitutive promoter, a recombinant receptor, a second 2A skip sequence, DHFRdm, a third 2A skip sequence, and EGFRt. In certain embodiments, the inductive promoter comprises an iSynPro promoter. In certain embodiments, the inductive promoter comprises an iSynPro promoter and a minimal promoter. In certain embodiments, the minimal promoter comprises a minimal promoter of IL2.

[0050] In certain embodiments, the artificial expression construct of this disclosure, comprising a PD1:cytokine chimeric transgene and a recombinant receptor, comprises or encodes an iSynPro promoter, a PD1:cytokine chimeric transgene, a first 2A skip sequence, Her2tG, 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 of this disclosure, comprising a PD1:cytokine chimeric transgene and a recombinant receptor, comprises or encodes an S1-61 iSynPro promoter, a PD1:cytokine chimeric transgene, a first 2A skip sequence, Her2tG, an EF1a(L) promoter, a CAR, a second 2A skip sequence, DHFRdm, a third 2A skip sequence, and EGFRt. In certain embodiments, the PD1:cytokine chimeric transgene of this disclosure encodes a soluble PD1:cytokine chimera. In certain embodiments, the PD1:cytokine chimeric transgenes of this disclosure encode PD1:IL21, PD1:IL15(N72D), PD1:drIL18, PD1:IL12, or PD1:IFNα. In certain embodiments, the CAR is an anti-CD19 CAR. See Figure 2 for a schematic diagram of an exemplary artificial expression construct.

[0051] Accordingly, this disclosure provides systems and methods for improving the killing of immune cells expressing recombinant receptors, the proliferation of immune cells expressing recombinant receptors, and / or the production of cytokines by immune cells expressing recombinant receptors. Immune cells expressing recombinant receptors can be recombined to target various target cells expressing target antigens (e.g., cancer cells, viruses, bacteria, fungi, parasites, or arthropods) by recombining their extracellular domains to include a binding domain that binds to the target antigen.

[0052] The various constructs described herein may be used to tailor treatment to specific patients or patient groups. For example, while we do not wish to be bound by any theory, cytokine release syndrome generally occurs when the immune system response becomes excessively aggressive. Therefore, in some cases, it may be useful to administer cells treated with an artificial expression construct that shows lower responsiveness compared to existing CAR-T cell therapies such as CD19CAR, or cells treated with an artificial expression construct that, while not the best transcription factor transgene for killing by recombinant receptor immune cells, proliferating recombinant receptor immune cells, and enhancing cytokine production by recombinant receptor immune cells, has some effect. That is, the response may be more responsive than that of natural cells, but further tuned. In another embodiment, it may be useful to obtain a stepwise or hierarchical response by administering a series of cells or combinations of cells treated with a combination of artificial expression constructs. For example, the stimulating dose or number of administrations in the T cell activation process may be partially modified depending on the needs of the patient or patient group. In another embodiment, two or more transcription factor transgenes may be used.

[0053] Various aspects of this disclosure are described below with further detail. These various aspects of this disclosure are described in accordance with the following items: (i) Inducible expression of PD1: cytokine chimeric transgenes; (ii) Immune cells; (iii) Recovery of cell samples and enrichment of cells; (iv) Genetic engineering techniques; (v) Regulatory 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) Cell formulations manufactured ex vivo; (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 systematic purposes only and do not limit the scope or interpretation of this disclosure.

[0054] (i) PD1: Inducible expression of cytokine chimeric transgenes In certain embodiments, the PD1:cytokine chimera comprises wild-type PD1 or PD1(A99L) linked to an immunostimulatory cytokine via a linker. PD1(A99L) is a programmed cell death 1 protein with the A99L mutation. The A99L mutation in PD-1 has been shown to have higher binding affinity to the PD-1 receptor than the wild type (Lazar-Molnar et al., Proc Natl Acad Sci US A. 105(30):10483-10488 (2008)).

[0055] Cytokines are small proteins (usually 5-25 kDa) that play a crucial role in cellular signaling. Cytokines are released by cells and influence the behavior of other cells, and sometimes even the behavior of the cytokine-releasing cell itself (e.g., T cells). Examples of cytokines include chemokines, interferons, interleukins, lymphokines, and / or tumor necrosis factors. Cytokines can be produced by various types of cells, 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.

[0056] Cytokines can act via receptors. They are important in the immune system because they can regulate the balance between humoral and cellular immune responses and modulate the maturation, proliferation, and responsiveness of specific cell populations. Some cytokines enhance or suppress the effects of other cytokines in complex ways.

[0057] Soluble cytokines can completely cross the endoplasmic reticulum membrane and be secreted from the cells that produced them. Soluble cytokines modulate inflammatory and immune events by functioning as agonists or antagonists in cytokine signaling.

[0058] Interleukin-21 (IL21) is a class I cytokine with a 4-α-helix bundle. IL21 has broad, multifaceted effects on both innate and adaptive immune responses. IL21 is produced by natural killer T cells, CD4(+) T cells, and Th17 cells. IL21 plays various 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)). PD1(A99L):IL21 is a PD1:cytokine chimera containing PD1(A99L) linked to IL21 via a linker.

[0059] Interleukin-15 (IL15) is an inflammatory cytokine with a structure similar to IL2. IL15 is secreted by mononuclear phagocytic cells in response to viral infection. Diseases associated with IL15 include severe combined immunodeficiency and X-linked primary cutaneous T-cell non-Hodgkin lymphoma. Pathways associated with IL15 include the MIF-mediated glucocorticoid regulation / TGFβ pathway. IL15(N72D) is an IL15 molecule in which the 72nd amino acid is substituted from asparagine to aspartic acid. This mutation results in a 4-5 times increase in biological activity compared to the native molecule. PD1(A99L):IL15 is a PD1:cytokine chimera containing PD1(A99L) linked to IL15 via a linker.

[0060] Decoy-resistant (DR) interleukin-18 (IL18) (DR-IL18 or drIL18) is a modified IL18 protein that binds to the IL18 receptor (IL18Rα) and maintains IL18 function, but can evade IL-18-binding protein (IL-18BP) (Zhou et al., Nature 583(7817):609-614 (2020)). In other words, DR-IL18 maintains signaling ability but is unaffected by 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, particularly in the presence of IL-12. Furthermore, IL-18 also plays a role in regulating the development of T lymphocyte helper type I cells and in Fas-mediated cytotoxicity. Suppression of IL-18 activity is being studied in 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). PD1(A99L):drIL18 is a PD1:cytokine chimera containing PD1(A99L) linked to drIL18 via a linker.

[0061] 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 4-α-helix bundle. PD1(A99L):IL12 is a PD1:cytokine chimera containing PD1(A99L) linked to IL12 via a linker.

[0062] Interferon-alpha (IFNα) is a cytokine produced by cells of the innate immune system in response to viral infections and other environmental stressors. IFNα is a type I interferon known to modulate both innate and adaptive immunity. PD1(A99L):IFNα is a PD1:cytokine chimera containing PD1(A99L) linked to interferon-alpha (IFNα) via a linker.

[0063] PD1: The cytokine chimeric transgene is under the control of the inducible synthesis promoter (iSynPro). iSynPro promoters are S1-17 (SEQ ID NO: 19), S1-37 (SEQ ID NO: 20), S1-4 (SEQ ID NO: 21), S1-1 (SEQ ID NO: 22), S1-3 (SEQ ID NO: 23), S1-42 (SEQ ID NO: 24), S1-61 Version 1 (SEQ ID NO: 17), S1-61 Version 2 (SEQ ID NO: 18), S1-62 (SEQ ID NO: 25), S1-15 (SEQ ID NO: 26), S1-2 (SEQ ID NO: 27), S1-27 (SEQ ID NO: 28), S1-8 (SEQ ID NO: 29), S1-30 (SEQ ID NO: 30), S1-33 ​​(SEQ ID NO: 31), S1-41 (SEQ ID NO: 32), S1-59 (SEQ ID NO: 33), S1-66 (SEQ ID NO: 34), S1-71 (SEQ ID NO: 35), S1-56 (SEQ ID NO: 36), S1-6 (SEQ ID NO: 37), S1 iSynPro may also have S1-60 (sequence number 38), S1-86 (sequence number 39), S1-32 (sequence number 40), S1-10 (sequence number 41), S1-18 (sequence number 42), S1-14 (sequence number 43), S1-16 (sequence number 44), S1-19 (sequence number 45), S1-26 (sequence number 46), S1-65 (sequence number 47), S2-n1 (sequence number 48), S4-n1 (sequence number 49), S6-n1 (sequence number 50), S1-325 (sequence number 51), S1-60 (sequence number 52), S2-274 (sequence number 53), S2-310 (sequence number 54), S1-367 (sequence number 55), or S1-7 (sequence number 56), any one of these. In certain embodiments, iSynPro includes S1-61 (sequence number 17 or sequence number 18).In a particular embodiment, the iSynPro promoter has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with 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, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, or SEQ ID NO: 56.

[0064] In certain embodiments, iSynPro includes S1-61 (sequence number 17 or sequence number 18). In certain embodiments, iSynPro has sequence identity of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% with sequence number 17 or sequence number 18.

[0065] In certain embodiments, one of the above iSynPro sequences is combined with a minimal promoter. In certain embodiments, the minimal promoter includes the IL2 minimal promoter. In certain embodiments, the IL2 minimal promoter includes the sequence represented by ACATTTTGACACCCCCATAATATTTTTCCAGAATTAACAGTATAAATTGCATCTCTTGTTCAAGAGTTCCCTATCACTCTCTTTAATCACTACTCACAGTAACCTCAACTCCTG (SEQ ID NO: 138).

[0066] Furthermore, functional cleavage products and functional variants of the PD1:cytokine chimeric transgene may be used. Functional cleavage products have fewer amino acid residues than the corresponding reference wild-type sequence but retain the activity of the PD1:cytokine chimeric transgene. Functional variants have one or more amino acid mutations compared to the corresponding reference wild-type sequence but retain the activity of the PD1:cytokine chimeric transgene.

[0067] (ii) Immune cells This disclosure describes immune cells that have been genetically modified to inducibly express a PD1:cytokine chimeric transgene (e.g., soluble PD1:cytokine chimeric) that enhances the function of immune cells. In certain embodiments, the immune cells of this disclosure are further genetically modified to express a recombinant receptor such as CAR. The immune cells may be any cells in which the expression of the PD1:cytokine chimeric transgene can be inducibly controlled by the above-mentioned inducible promoter (e.g., iSynPro). In certain embodiments, immune cells may include lymphocytes, monocytes / macrophages, hematopoietic stem cells (HSCs), hematopoietic progenitor cells (HPCs), and / or mixtures of HSCs and HPCs (i.e., HSPCs). In certain embodiments, immune cells include lymphocytes. In certain embodiments, lymphocytes may include T cells, B cells, natural killer (NK) cells, or NK-T cells.

[0068] Several types of T cell subsets have been identified, each possessing characteristic 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.

[0069] γδ T cells are a small subset of T cells that possess a characteristic T cell receptor (TCR) on their surface. In γδ T cells, the TCR consists of one γ chain and one δ chain. This group of T cells is not as commonly found as αβ T cells (they make up only 2% of all T cells).

[0070] 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.

[0071] T cells can be further classified into helper T cells (CD4+ T cells) and cytotoxic T cells (CTLs, CD8+ T cells), with cytotoxic T cells including cytolytic T cells. Helper T cells play a role in assisting other white blood cells in immunological processes, performing functions 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 by the 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 that are responsible for regulating or supporting active immune responses.

[0072] Cytotoxic T cells can destroy virus-infected cells and tumor cells and are also involved in transplant rejection. Because they express the CD8 glycoprotein on their surface, cytotoxic T cells are also known as CD8+ T cells. Cytotoxic T cells recognize their targets by binding to antigens associated with MHC class I molecules, which are present on the surface of almost all cells in the body.

[0073] "Central memory" T cells (or "TCMs") refer to CTLs that have experienced an antigen and, compared to naive cells, express CD62L or CCR-7 and CD45RO on their surface, but do not express CD45RA or have reduced CD45RA expression. In certain embodiments, central memory cells are positive for CD62L, CCR7, CD25, CD127, CD45RO, and CD95 expression and have reduced CD45RA expression compared to naive cells.

[0074] "Effector memory" T cells (or "TEM") refer to T cells that have experienced an antigen and, compared to central memory cells, either do not express CD62L on their surface or have reduced CD62L expression, and compared to naive cells, either do not express CD45RA or have reduced CD45RA expression. 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. Additionally, effector T cells are positive for granzyme B and perforin compared to memory or naive T cells.

[0075] "Naive" T cells are T cells that have not experienced an antigen and, compared to central memory cells or effector memory cells, express CD62L and CD45RA but not CD45RO. In certain embodiments, naive CD8+ T lymphocytes are characterized by the expression of naive T cell phenotypic markers, such as CD62L, CCR7, CD28, CD127, and CD45RA.

[0076] Natural killer cells (also known as NK cells, K cells, or killer cells) are activated in response to interferon or macrophage-derived cytokines. NK cells play a role in suppressing viral infections, while the adaptive immune response produces antigen-specific cytotoxic T cells that can eliminate infections. NK cells express CD8, CD16, and CD56, but not CD3.

[0077] NK cells include NK-T cells. NK-T cells are a specialized population of T cells 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 immune surveillance 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+.

[0078] Macrophages (and their precursor cells, monocytes) are present in all tissues of the body (sometimes 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.

[0079] Immature dendritic cells (i.e., pre-activated dendritic cells) phagocytose peripheral antigens and other non-self components to become activated, then migrate to T cell regions in lymphoid tissues and present antigens to T cells. Dendritic cells express CD1a, CD1b, CD1c, CD1d, CD21, CD35, CD39, CD40, CD86, CD101, CD148, CD209, and DEC-205.

[0080] Hematopoietic stem / progenitor cells, or HSPCs, refer to a combination of hematopoietic stem cells and hematopoietic progenitor cells.

[0081] Hematopoietic stem cells refer to undifferentiated hematopoietic cells that are capable of self-renewal in vivo or self-renewal in vitro and can proliferate virtually without limit, and can differentiate into any other type of hematopoietic cell.

[0082] Hematopoietic progenitor cells are cells derived from hematopoietic stem cells or cells derived from embryonic tissue that can further differentiate into mature cells. In certain embodiments, hematopoietic progenitor cells are CD24 lo Lin - CD117 + Hematopoietic progenitor cells. HPCs can (i) differentiate into myeloid progenitor cells, ultimately generating monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes / platelets, or dendritic cells, or (ii) differentiate into lymphoid progenitor cells, ultimately generating T cells, B cells, and NK cells.

[0083] HSPCs may be positive for specific markers whose expression levels are increased in HSPCs compared to other types of hematopoietic cells. Examples of such markers include CD34, CD43, CD45RO, CD45RA, CD59, CD90, CD109, CD117, CD133, CD166, HLA DR, or combinations thereof. Furthermore, HSPCs may be negative for markers expressed in other types of hematopoietic cells. Examples of such markers include Lin, CD38, or combinations thereof. HSPCs are preferably CD34+ cells.

[0084] The description of a cell or cell population as "positive" for a particular marker, or as a cell or cell population expressing a particular marker, means that a detectable particular marker is present on the cell surface or inside the cell. When referring to cell surface markers, "positive" means that there is cell surface expression that can be detected by flow cytometry, for example, by staining with an antibody that specifically binds to the cell surface marker and detecting that antibody, and that the staining is detected at substantially a higher level in flow cytometry than staining detected using the same procedure under the same conditions with an isotype-matched control, and / or at substantially the same level as staining of cells known to be positive for that marker, and / or at substantially a higher level than staining of cells known to be negative for that marker.

[0085] The description of a cell or cell population as "negative" for a particular marker, or as a cell or cell population not expressing a marker, means that the particular marker is substantially absent from the cell surface or inside the cell. When referring to cell surface markers, "negative" means that there is no cell surface expression detectable by flow cytometry, for example, by staining with an antibody that specifically binds to the cell surface marker and detecting that antibody; the stain is substantially not detected at a higher level in flow cytometry than staining detected using the same procedure under the same conditions with an isotype-matched control, and / or is substantially detected at a lower level than staining cells known to be positive for that marker, and / or is substantially detected at the same level as staining cells known to be negative for that marker.

[0086] Cells whose genes are modified in accordance with the teachings of this disclosure may be patient-derived cells (autologous cells), cells of the same species as the patient, in vivo cells, or ex vivo cells, where 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.

[0087] (iii) Recovery of cell samples and concentration of cells Methods for recovering and concentrating samples are known to those skilled in the art. In certain embodiments, the cells are derived from humans, for example, from a patient receiving treatment. The cells may also be derived from a cell line. In some embodiments, the cells are obtained from a heterologous cell source, for example, from a mouse, rat, non-human primate, or pig.

[0088] In some embodiments, T cells are derived from or isolated from samples such as whole blood, peripheral blood mononuclear cells (PBMCs), leukocytes, bone marrow, thymus, tissue biopsy specimens, tumors, lymph nodes, intestinal lymphoid tissue, mucosa-associated lymphoid tissue, spleen, other lymphoid tissues, liver, lungs, stomach, intestines, colon, kidneys, pancreas, breasts, bones, prostate, cervix, testes, ovaries, tonsils, or other organs, and / or cells derived from these organs. In certain embodiments, cells derived from the circulating blood of the subject are obtained, for example, by apheresis or leukocyte apheresis. In certain embodiments, the sample includes lymphocytes such as T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, HSCs, HPCs, HSPCs, erythrocytes, and / or platelets, and in some embodiments, the sample includes cells other than erythrocytes and platelets that require further processing.

[0089] In some embodiments, blood cells recovered from the subject are washed, for example, to remove the plasma fraction and to suspend the cells in a suitable buffer or medium for the next processing step. In certain embodiments, the cells are washed with phosphate-buffered saline (PBS). In some embodiments, the washing solution does not contain calcium and / or magnesium and / or contains a large proportion of divalent cations or no divalent cations at all. Washing can be performed using a semi-automatic "flow-through" centrifuge (e.g., Cobe 2991 cell processor, Baxter) according to the manufacturer's instructions. Tangential flow filtration (TFF) may also be performed. In certain embodiments, the washed cells can be resuspended in various types of biocompatible buffers, such as Ca++ / Mg++-free PBS.

[0090] Isolation may include one or more of various cell preparation and cell separation steps, which may include separation based on one or more characteristics such as size, density, sensitivity or resistance to specific reagents, and / or affinity to antibodies or other binding partners (e.g., immunoaffinity). In certain embodiments, isolation is performed sequentially and / or simultaneously in a single method using one apparatus or equipment. In certain embodiments, isolation, culture, and / or recombination of various different populations are initiated from a single starting material, such as a single sample.

[0091] In certain embodiments, a sample can be enriched with T cells using density-based cell separation methods and related techniques. For example, leukocytes can be separated from other types of cells in peripheral blood by lysing erythrocytes and then centrifugating using a Percoll or Ficoll concentration gradient.

[0092] In certain embodiments, a bulk T cell population that is not enriched with a specific type of T cell can be used. In certain embodiments, selected types of T cells can be enriched and / or isolated by positive and / or negative selection using cell markers. Positive selection involves obtaining cells with a cell marker bound to a capture agent for use in the following applications. Negative selection involves obtaining cells that are not bound to a capture agent, such as an antibody against the cell marker, for use in the following applications. In some examples, both positively selected and negatively selected fractions can be obtained and used in the following applications. In certain embodiments, CD4+ T cells and / or CD8+ T cells are enriched from PBMCs.

[0093] The cell isolation described above does not necessarily require the enrichment or removal of 100% of a specific cell population or cells expressing 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 require the complete removal of cells that do not express that marker. Similarly, negative selection, removal, or elimination of a particular type of cell means decreasing the number or proportion of such cells, but does not require the complete removal of such cells.

[0094] In some cases, the separation process is performed multiple times, so that the positively selected or negatively selected fractions from one process are processed in another separation process, such as a subsequent positive or negative selection.

[0095] In some embodiments, antibodies or binding domains against cell markers can be conjugated to solid carriers or solid 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, pp. 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.

[0096] In some embodiments, affinity-based selection is performed by magnetically activated cell sorting (MACS) (Milteny Biotech, Auburn, California). The MACS system can select cells bound to magnetic particles with high purity. In certain embodiments, MACS is operated in a mode that sequentially elutes non-target and target species after an external magnetic field is applied. That is, cells bound to magnetic particles are retained in place while cells that did not bind to magnetic particles are eluted. Next, once this first elution step is complete, cells that were trapped by the magnetic field and prevented from eluting are released in some way that allows for elution and recovery. In certain embodiments, non-target cells are labeled and removed from heterogeneous cell populations.

[0097] In some embodiments, the cell populations described herein are recovered and concentrated (or removed) by flow cytometry, in which cells stained with multiple cell surface markers are analyzed in a fluid flow. In some embodiments, the cell populations described herein are recovered and concentrated (or removed) by preparative (FACS) sorting. In some embodiments, the cell populations described herein are recovered and concentrated (or removed) by using a combination of a microelectromechanical system (MEMS) chip and 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 any case, cells can be labeled with multiple markers to isolate a clearly defined subset of cells with high purity.

[0098] Cell markers for various T cell subpopulations are as described above. In certain embodiments, specific T cell subpopulations, such as 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 selection techniques or negative selection techniques.

[0099] CD3+CD28+ T cells can be positively selected and proliferated using anti-CD3 / anti-CD28 bound magnetic beads (e.g., DYNABEADS® (Life Technologies, Norway) M-450 CD3 / CD28 T Cell Expander).

[0100] In certain embodiments, CD4+ helper T cells and CD8+ cytotoxic T cells are separated by a CD8+ or CD4+ selection step. Such CD8+ and CD4+ populations can then be further sorted into various subpopulations by positive or negative selection of markers expressed in one or more naive T cell subpopulations, memory T cell subpopulations and / or effector T cell subpopulations, or markers that are relatively highly expressed in these T cell subpopulations.

[0101] 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, the TICLE method, which is described in more detail separately herein, is used.

[0102] Furthermore, other types of cells can also be enriched based on known marker profiles and known techniques. For example, CD34+ HSCs, HSPs, and HSPCs can be enriched using a combination of anti-CD34 antibodies directly or indirectly conjugated to magnetic microparticles and a magnetic cell separator (e.g., CliniMACS® cell separation system (Mirteny Biotech, Bergisch Gladbach, Germany)).

[0103] (iv) Genetic engineering techniques Cell populations may be genetically modified (or genetically modified) to express a PD1:cytokine chimera, and in addition, they may be genetically modified (or genetically modified) to further express a recombinant receptor (e.g., a chimeric antigen receptor (CAR)) as described herein. The desired genes disclosed herein (e.g., soluble PD1:cytokine chimera transgene and recombinant receptor) can be introduced into cells by methods known in the art, such as transfection, electroporation, microinjection, lipofection, transfection by calcium phosphate, infection with a viral vector or bacteriophage vector containing a gene sequence, cell fusion, gene transfer by chromosome, micronucleus cell fusion, spheroplast fusion, and delivery by in vivo nanoparticles. In this field, various techniques are known for introducing foreign genes into cells (see, for example, 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 the developmental and physiological functions necessary for the recipient cells are not excessively disrupted. These techniques can be used to stably introduce foreign genes into cells so that they are expressed in the cells, and in certain cases, preferably heritable, so that they are also expressed in their offspring cells.

[0104] The term “gene” refers to a nucleic acid sequence (this term is used synonymously with “polynucleotide” or “nucleotide sequence”). A gene may contain a PD1: cytokine chimeric transgene or encode a recombinant receptor. The definition of this term includes various sequence polymorphisms, mutations, and / or sequence variants, such changes that do not substantially affect the function of the PD1: cytokine chimeric transgene or the recombinant receptor it encodes. The term “gene” may include regulatory regions such as promoters, enhancers, and termination regions, as well as coding sequences. Furthermore, this term may include any introns and other DNA sequences spliced ​​from mRNA transcripts, as well as variants arising from alternative splice sites. Gene sequences encoding these molecules may be DNA or RNA that induces 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 proteins. These nucleic acid sequences include both full-length nucleic acid sequences and partial sequences derived from full-length proteins. These sequences may further include sequences that may be introduced to confer codon selectivity in certain types of cells, or degenerate codons of native sequences. A portion of the complete gene sequence is also referenced throughout this disclosure, as will be understood by those skilled in the art.

[0105] Gene sequences containing PD1 cytokine chimeric transgenes and / or gene sequences encoding recombinant receptors are provided herein, and these gene sequences can be readily prepared by synthetic or recombinant methods from relevant amino acid sequences based on other information provided herein. In some embodiments, gene sequences encoding any of these sequences may have one or more restriction enzyme sites at the 5' and / or 3' ends of the coding sequence so that the gene sequences encoding these sequences can be readily cleaved and readily substituted with other gene sequences encoding other sequences. In some embodiments, the gene sequences encoding the above sequences may be codon-optimized for expression in mammalian cells.

[0106] "Code" refers to the property of a specific nucleotide sequence within a gene, such as cDNA or mRNA, to function as a template for synthesizing another macromolecule, such as a defined amino acid sequence. Therefore, if mRNA corresponding to a gene is transcribed and translated to produce a protein in a cell or other biological system, that gene codes for this protein. A "protein-coding gene sequence" includes any degenerate nucleotide sequence that codes for the same amino acid sequence or an amino acid sequence having a substantially similar form and function.

[0107] Multiple polynucleotide gene sequences encoding two or more portions of an expressed artificial expression construct can be operably linked to each other and can also be linked to relevant regulatory sequences. For example, a regulatory sequence and an exogenous nucleic acid sequence may be functionally linked, thereby enabling the expression of the exogenous nucleic acid sequence. In another example, if a first nucleic acid sequence and a second nucleic acid sequence are arranged to be functionally related, these first and second nucleic acid sequences may be operably linked. For example, if a promoter affects the transcription or expression of a coding sequence, this promoter is operably linked to this coding sequence. Typically, operably linked DNA sequences are contiguous, and coding regions are linked within the same reading frame if a coding region is needed or useful.

[0108] Examples of promoters include general promoters, tissue-specific promoters, cell-specific promoters, and / or cytoplasm-specific promoters. Other examples of promoters include strong promoters, weak promoters, constitutive expression promoters (i.e., constitutive promoters), and / or inducible promoters. A constitutive promoter is a promoter that enables the continuous transcription of a single or multiple 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. In certain embodiments, the inducible promoter includes the iSynPro and minimal promoter described above. In certain embodiments, the minimal promoter includes the IL2 minimal promoter.

[0109] Specific examples of constitutive promoters include the human elongation factor 1α promoter (EF1α such as EF1α(s) and EF1α(L)), the myeloproliferative sarcoma virus (MND) promoter, the cytomegalovirus (CMV) promoter, the Simian virus 40 (SV40) early promoter, the mouse mammary cancer virus (MMTV) promoter, the human immunodeficiency virus (HIV) long-chain terminal repeat (LTR) promoter, the MoMuLV promoter, the avian leukemia virus promoter, the Epstein-Barr virus very early promoter, and the Rous sarcoma virus promoter; as well as human gene promoters such as the actin promoter, myosin promoter, hemoglobin promoter, and creatine kinase promoter. In certain embodiments, the recombinant receptor is under the control of EF1α (SEQ ID NO: 57).

[0110] A "vector" is a nucleic acid molecule capable of transporting another nucleic acid. A vector may be, for example, a plasmid (DNA plasmid 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 conditions, can induce the expression of a protein encoded by one or more genes incorporated within the expression vector.

[0111] "Lentiviruses" refer to viruses of the retrovirus genus that can infect both dividing and non-dividing cells. Some examples of lentiviruses include HIV (human immunodeficiency virus: including HIV1 and HIV2); equine infectious anemia virus; feline immunodeficiency virus (FIV); bovine immunodeficiency virus (BIV); and simian immunodeficiency virus (SIV).

[0112] 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 commercialized by Oxford BioMedica (UK) and the LENTIMAX vector system commercialized by Lentigen. Furthermore, non-clinical lentiviral vectors are also available and are known to those skilled in the art. In certain embodiments, lentiviruses or lentiviral vectors are used to recombinate cells to express an artificial expression construct.

[0113] A "retrovirus" is a virus that has an RNA genome. A "gammaretrovirus" refers to a virus belonging to the Retroviridae family. Examples of gammaretroviruses include mouse stem cell virus, mouse leukemia virus, feline leukemia virus, feline sarcoma virus, and reticuloendotheliosis virus.

[0114] 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 a retroviral vector and delivered into the cell. In certain embodiments, the retroviral vector contains all the cis-acting sequences required for packaging and incorporating the viral genome, namely (a) long-terminal repeat sequences (LTRs) or a portion thereof located at both ends of the vector, (b) primer binding sites for minus and plus-strand DNA synthesis, and (c) packaging signals required for incorporating the genomic RNA into the virion. 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 (AAVs), 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-helix DNA (Chan and Glazer, 1997, J. Mol. Med. 75:267-282).

[0115] Numerous viral vectors are suitable for use in this disclosure and are available, 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, as well as methods for packaging them into cells for transduction of mammalian host cells 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 their expression systems are commercially available.

[0116] Targeted genetic engineering methods may be used. The CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) / Cas (CRISPR-related protein) nuclease system is a recombinant nuclease system used in genetic engineering using bacterial systems. Information regarding the CRISPR-Cas system and its components can be found, for example, in 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, U.S. Patent Publication No. U.S. Patent Publication No. 8932814, U.S. Patent Publication No. 8945839, U.S. Patent Publication No. 8993233 and U.S. Patent Publication No. 8999641 and related applications thereto; 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 This is described in / 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.

[0117] In certain embodiments, zinc finger nucleases (ZFNs) are used as gene editing agents. ZFNs are a type of site-specific nuclease that has been recombinant to bind to and cleave DNA at specific locations.For further information regarding ZFNs and ZFNs useful within the scope of the teachings of this disclosure, see, for example, U.S. Patent Publication No. 6,534,261; U.S. Patent Publication No. 6,607,882; U.S. Patent Publication No. 6,746,838; U.S. Patent Publication No. 6,794,136; U.S. Patent Publication No. 6,824,978; U.S. Patent Publication No. 6,866,997; U.S. Patent Publication No. 6,933,113; U.S. Patent Publication No. 6,979,539; U.S. Patent Publication No. 7,013, U.S. Patent Publication No. 219; U.S. Patent Publication Nos. 7,030,215; U.S. Patent Publication Nos. 7,220,719; U.S. Patent Publication Nos. 7,241,573; U.S. Patent Publication Nos. 7,241,574; U.S. Patent Publication Nos. 7,585,849; U.S. Patent Publication Nos. 7,595,376; U.S. Patent Publication Nos. 6,903,185; U.S. Patent Publication Nos. 6,479,626; U.S. Patent Publication Nos. 2003 / 0232410 and U.S. Patent Publication Nos. 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 See also 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).

[0118] In certain embodiments, activator-like effector nucleases (TALENs) can be used as gene editing agents. A TALEN is a fusion protein containing an activator-like effector (TALE) DNA-binding protein and a DNA cleavage domain. For further information on TALEN, please refer to U.S. Patent Publications 8,440,431; 8,440,432; 8,450,471; 8,586,363; and 8,697,853; as well as Joung and Sander, Nat Rev Mol Cell Biol, 2013, 14(l):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 See 186, 757-761 (2010); Boch, et al. Science 326, 1509-1512 (2009); and Moscow, & Bogdanove, Science 326, 1501 (2009).

[0119] Nanoparticles capable of selectively genetically modifying target cells in vivo have been reported and can be used within the scope of the teachings of this disclosure. In certain embodiments, the nanoparticles may be those described in WO2014153114, WO2017181110 and WO201822672.

[0120] (v) Control mechanism including tag cassette, transduction marker, selection cassette and / or suicide switch In certain embodiments, an artificial expression construct may 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 proliferation, detect, concentrate, isolate, track, remove, and / or eliminate genetically modified cells. A “tag cassette” refers to a unique synthetic peptide sequence that is attached to, fused to, or incorporated as part of an artificial expression construct, and whose binding properties allow for the specific binding of a cognitive-binding molecule (e.g., ligand, antibody, or other binding partner) to the tagged protein and / or cells expressing the tagged protein. Transduction markers can also be used for the same purposes, although they are derived from natural molecules and are often expressed using skipping elements (or skip sequences) that allow for the separation of the transduction marker from other components within the expressed molecule.

[0121] Examples of tag cassettes that bind to cognitive-binding molecules include His tag (HHHHHH; SEQ ID NO: 104), Flag tag (DYKDDDDK; SEQ ID NO: 105), Xpress tag (DLYDDDDK; SEQ ID NO: 106), Avi tag (GLNDIFEAQKIEWHE; SEQ ID NO: 107), calmodulin tag (KRRWKKNFIAVSAANRFKKISSSGAL; SEQ ID NO: 108), polyglutamic acid tag, HA tag (YPYDVPDYA; SEQ ID NO: 109), Myc tag (EQKLISEEDL; SEQ ID NO: 110), Strep tag (referring to the conventional STREP® tag (WRHPQFGG; SEQ ID NO: 111)), STREP® tag II (WSHPQFEK; SEQ ID NO: 112 (IBA Institut fur Bioanalytik, Germany); see, for example, U.S. Patent Publication No. 7,981,632), Softag Examples include 1 (SLAELLNAGLGGS; SEQ ID NO: 113), Softag 3 (TQDPSRVG; SEQ ID NO: 114), and V5 tag (GKPIPNPLLGLDST; SEQ ID NO: 115).

[0122] Binding molecules that specifically bind to the tag cassette sequences disclosed herein to form complexes are commercially available. For example, His-tagged antibodies are commercially available from manufacturers such as Life Technologies, Pierce Antibodies, and GenScript. Flag-tagged antibodies are commercially available from manufacturers such as Pierce Antibodies, GenScript, and Sigma-Aldrich. Xpress-tagged antibodies are commercially available from manufacturers such as Pierce Antibodies, Life Technologies, and GenScript. Avi-tagged antibodies are commercially available from manufacturers such as Pierce Antibodies, IsBio, and Genecopoeia. Calmodulin-tagged antibodies are commercially available from manufacturers such as Santa Cruz Biotechnology, Abcam, and Pierce Antibodies. HA-tagged antibodies are commercially available from manufacturers such as Pierce Antibodies, Cell Signal, and Abcam. Myc-tagged antibodies are commercially available from manufacturers such as Santa Cruz Biotechnology, Abcam, and Cell Signal. Strep-tagged antibodies are commercially available from manufacturers such as Abcam, Iba, and Qiagen.

[0123] The transduction marker may be selected from at least one of the following: 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 (called Her2tG); human CD34 ECD; and / or RQR8 combining a target epitope derived from the CD34 antigen (see Fehse et al, Mol. Therapy 1(5 Pt 1); 448-456, 2000) and a target epitope derived from the CD20 antigen (see Philip et al, Blood 124: 1277-1278). In certain embodiments, cells are genetically modified to express EGFRt. In certain embodiments, cells are genetically modified to express Her2tG.

[0124] In certain embodiments, the selection cassette allows for positive or negative selection of a desired cell population. Negative selection is a method of removing some types of cells while retaining the desired type. Positive selection is a method of targeting a desired cell population and retaining only the desired cells.

[0125] The selection cassette may encode (a) a protein that confers resistance to antibiotics or other toxins, (b) a protein that compensates for nutritional deficiencies, or (c) a protein that supplies essential nutrients unavailable from the complex medium, such as a gene encoding D-alanine racemase for Bacillus. The number of selection systems used to harvest transformed cells is not particularly limited. In certain embodiments, the positive selection cassette includes a resistance gene to neomycin, hygromycin, ampicillin, puromycin, phleomycin, zeomycin, blastocydin, or biomycin. In certain embodiments, the selection cassette includes a DHFR (dihydrofolate reductase) gene or a DHFR double mutant (DHFRdm) gene that confers resistance to methotrexate (MTX), O 6 The selective agent may include the MGMT P140K gene responsible for resistance to BG / BCNU, the HPRT (hypoxanthine phosphoribosyltransferase) gene responsible for the conversion of specific bases (aminopterin, hypoxanthine, thymidine) contained in HAT selective medium, or other genes responsible for detoxification of several types of drugs. In certain embodiments, the selective agent may be neomycin, hygromycin, puromycin, phleomycin, zeomycin, blastosidine, biomycin, ampicillin, O 6 Examples include BG / BCNU, MTX, tetracycline, aminopterin, hypoxanthine, thymidine kinase, DHFR, Gln synthase, or ADA.

[0126] In certain embodiments, the selection cassette contains DHFRdm, and the selector contains MTX. In certain embodiments, the selection method does not require a selection cassette to obtain a highly purified cell population.

[0127] In certain embodiments, the negative selection cassette includes a gene that converts a substrate present in the culture medium into a toxic substance for the cell expressing it. Examples of such molecules include the diphtheria toxin (DTA) detoxification 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) that is sensitive to the presence of ganciclovir or FIAU. Alternatively, the HPRT gene may be used as a negative selection by adding 6-thioguanine (6TG) to the culture medium. Furthermore, polyA transcription termination sequences of various origins may be used for positive and negative selection, the most standard being those derived from SV40 polyA or polyA derived from eukaryotic cell genes (such as bovine growth hormone or rabbit β-globin).

[0128] In certain embodiments, the artificial expression construct may include a polynucleotide or skip sequence encoding a self-cleaving polypeptide. In certain embodiments, the polynucleotide encoding the self-cleaving polypeptide is located between the PD1: cytokine chimeric 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 2A peptide (P2A) derived from porcine tesiovirus-1, 2A peptide (T2A) derived from Thosea asigna virus, 2A peptide (E2A) derived from equine rhinitis A virus, 2A peptide (F2A) derived from foot-and-mouth disease virus, or variants thereof. Furthermore, exemplary nucleic acid and amino acid sequences of 2A peptides are also described, for example, by Kim et al. (PLOS One 6: e18556 (2011)). In certain embodiments, cells are genetically modified to contain a self-cleaving polypeptide. In certain embodiments, the self-cleaving polypeptide contains T2A. In certain embodiments, the 2A sequence leaves no trace of 2A. In certain embodiments, the 2A sequence adds an amino acid to the protein (also called a trace of 2A). The 2A sequence is a peptide sequence that induces ribosome skipping during translation. The cleavage is triggered by ribosome skipping of the peptide bond between proline and glycine in the 2A sequence. This ribosome 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 protein's function.

[0129] The control mechanism may be included in multiple copies in the artificial expression construct, or it may be expressed as a separate molecule 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 cognitive pairs are described in U.S. Patent Publication 13 / 463,247.

[0130] One advantage of incorporating at least one regulatory mechanism into the artificial expression construct is that, by using a cognitive-binding molecule to the tag cassette, the number of cells expressing the artificial expression construct administered to a target can be increased or decreased. In certain embodiments, the disclosure provides a method for removing modified cells expressing an artificial expression construct by using an antibody specific to the tag cassette, by using a cognitive-binding molecule specific to the regulatory mechanism, or by using a second modified cell that is specific to the regulatory mechanism and expresses the artificial expression construct. The removal of modified cells may be carried out using a removal agent specific to the regulatory mechanism. For example, when using EGFRt, an anti-EGFRt binding domain (e.g., an antibody or scFv) fused to or bound to a cytotoxic reagent (e.g., a toxin or radioactive metal) may be used, or anti-EGFRt / anti-CD3 bispecific scFv or anti-EGFRt CAR T cells may be used. Similarly, when using Her2tG, an anti-Her2tG binding domain fused to a cytotoxic reagent or an anti-Her2tG binding domain conjugated to a cytotoxic reagent may be used.

[0131] In certain embodiments, a polynucleotide encoding the iCaspase9 construct (iCasp9) may be inserted into the artificial expression construct as a suicide switch.

[0132] In certain embodiments, modified cells expressing an artificial expression construct may be detected or tracked in vivo by using an antibody that specifically binds to the regulatory mechanism (e.g., an anti-tagged antibody) or by other cognitive-binding molecules that specifically bind to the regulatory mechanism, the binding partner of this regulatory mechanism being conjugated to a fluorescent dye, radiotracer, iron oxide nanoparticles, or other contrast 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, for example, Yu, et al., Theranostics 2:3, 2012).

[0133] Therefore, modified cells expressing at least one regulatory mechanism within an artificial expression construct can be, for example, more easily identified, isolated, sorted, allowed to grow, tracked, and / or removed than modified cells without a tag cassette.

[0134] (vi) Recombinant receptors In certain embodiments, the recombinant receptor is either a binding domain that binds to a target antigen or contains a binding domain that binds to a target antigen, and the recombinant receptor is expressed from cells by artificially introducing the nucleic acid encoding the recombinant receptor into the cells. The recombinant receptor may be, for example, a CAR, a T cell receptor (TCR), or a CAR / TCR hybrid.

[0135] CARs include several characteristic components that enable genetically modified 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 includes a binding domain that specifically binds to a marker selectively presented on the surface of undesirable cells. When the binding domain binds to such a marker, the intracellular portion activates the genetically modified cell, which then destroys the cell to which it has bound. CARs may further include a transmembrane domain that connects the extracellular portion to the intracellular portion, and other components that can enhance the function of the recombinant receptor. For example, incorporating spacer sequences and / or one or more linker sequences into the CAR can provide further flexibility to the three-dimensional structure of the recombinant receptor, often thereby enhancing the binding ability of the binding domain to markers on target cells.

[0136] (vi-a) Joint domain In certain cases, recombinant receptors include a binding domain that binds to a target antigen selectively presented on the surface of undesirable cells. The binding domain contains a substance that binds to a cell marker to form a complex. The choice of binding domain may depend on the type and number of cell markers defining the surface of the target cell. Examples of binding domains include ligands for cell markers, ligands for receptors, antibodies, peptides, peptide aptamers, receptors (e.g., T cell receptors), and combinations thereof, as well as recombinant fragments or recombinant formats thereof.

[0137] As is understood by those skilled in the art, conventional antibodies consist of two heavy chains and two light chains. Each heavy chain consists of a variable region and a first, second, and third constant region, while each light chain consists of a variable region and a constant region. Mammalian heavy chains are classified as α, δ, ε, γ, or μ chains, and 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 shaped like a "Y". The stem of a Y-shaped full-length antibody is composed of the second and third constant regions (and a fourth constant region in IgE and IgM) of each of the two heavy chains linked together, with a disulfide bond (interchain bond) formed at the hinge. The γ, α, and δ heavy chains have a constant region in which three immunoglobulin domains are linked in tandem (series) and a hinge region that provides flexibility; the μ and ε heavy chains have a constant region consisting of four immunoglobulin domains. The second and third constant regions are called the "CH2 domain" and the "CH3 domain," respectively. Each arm of a Y-shaped full-length antibody consists of a variable region and a first constant region of a heavy chain linked to a light chain, which consists of a variable region and a constant region. The light chain variable region and the heavy chain variable region are responsible for binding to the antigen.

[0138] The light chain variable region and the heavy chain variable region contain a "framework" region that is sandwiched between three hypervariable regions, also called the "complementarity determination region" or "CDR".

[0139] The combination of CDRs is, 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. The numbering scheme may be determined based on 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 Examples include Des Sel 26(10):621-629) and proABC-2 (Ambrosetti et al. (2020) Bioinformatics 36(20):5107-5108).

[0140] The sequences of various light and heavy chain framework regions are relatively conserved across biological species, including humans. The framework region of an antibody is a combination of multiple framework regions within the light and heavy chains that constitute the antibody, and it plays a role in arranging and aligning the CDRs in three-dimensional space. CDRs are primarily responsible for binding to the antigen's epitope. The CDRs of each chain are generally numbered sequentially from the N-terminus and called CDR1, CDR2, and CDR3, and are generally identified by the chain on which they are located. Therefore, CDRs in the variable domain of the antibody's heavy chain are called CDRH1, CDRH2, and CDRH3, while those in the variable domain of the antibody's light chain are called CDRL1, CDRL2, and CDRL3. If the antibody has different specificities (i.e., different binding sites for various antigens), the CDRs it possesses will also differ. While the CDRs differ from antibody to antibody, only a limited number of amino acid positions within each CDR are directly involved in antigen binding. These amino acid positions within a CDR are called specificity-determining residues (SDRs).

[0141] "V H " or "VH" refers to the heavy chain variable region of immunoglobulin. L "VL" or "VL" refers to the variable region of the light chain of immunoglobulin.

[0142] Antibodies that specifically bind to an antigen can be produced using methods for obtaining monoclonal antibodies, phage display, methods for producing human antibodies or humanized antibodies, or methods using transgenic animals or plants genetically modified to produce human antibodies. Phage display libraries of partially synthesized or totally synthesized antibodies are available, and from these libraries, antibodies or fragments capable of binding to a target antigen can be screened. Phage display libraries of human antibodies are also available. Once the amino acid or polynucleotide sequence encoding the antibody is identified, these sequences can be isolated and / or sequenced. Numerous related antibodies are known and commercially available.

[0143] In some embodiments, the antibody specifically binds to surface molecules of cancer cells or virus-infected cells and does not cross-react with nonspecific components such as bovine serum albumin or other unrelated antigens.

[0144] An "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 fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, single-chain variable (scFv) antibody fragments, disulfide-linked Fv (sdFv), Fd fragments containing a VH domain and a CH1 constant domain, linear antibodies, single-domain antibodies (e.g., sdAb(VL or VH)), domains consisting only of the heavy chain variable region of camelids (VHH), multispecific antibodies formed from antibody fragments (e.g., a bivalent fragment containing two Fab fragments linked at a hinge region by disulfide crosslinking), and epitope-binding fragments of isolated CDRs or other antibodies (Harlow et al., 1999, published in *Using Antibodies: A Laboratory Manual*, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, published 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, for example, Hollinger and Hudson (2005) Nature Biotechnology 23:1126-1136).

[0145] In certain embodiments, the binding domain may include a humanized form of a non-human (e.g., mouse) antibody or an antigen-binding fragment thereof. Examples of humanized antibodies include antibodies in which the framework region and constant region of the variable region of one or more human immunoglobulins are fused to the binding region (e.g., CDR) of an animal (non-human) immunoglobulin. Such humanized antibodies are designed to avoid immune responses to the non-human antibody while retaining the binding specificity of the non-human antibody from which the binding region originates. In certain embodiments, the binding domain may include a fully human antibody or an antibody fragment thereof, either entirely derived from a human molecule or containing the same amino acid sequence as the human form of the antibody or immunoglobulin.

[0146] "scFv" refers to a recombinant fusion protein that contains antibody-derived VH and VL regions linked by a linker and can be expressed as a single-chain polypeptide. scFv retains the specificity of the original intact antibody. In certain embodiments, the linker linking the variable regions may include a glycineserine linker, such as the glycineserine linker described separately herein, for example, SEQ ID NOs: 123-136. In certain embodiments, scFv may contain the VL variable region and the VH variable region in any order, for example, relative to the N-terminus and C-terminus of the polypeptide, and scFv may contain VL-linker-VH or VH-linker-VL.

[0147] Furthermore, recombinant receptors include TCRs (including recombinant TCRs) that can be used alone, or TCRs (including recombinant TCRs) that can be used as binding domains within CARs (e.g., CAR / TCR hybrids). For example, many TCR sequences that bind to specific antigen fragments are known and publicly available.

[0148] TCRs for use with specific antigens can be identified, for example, by isolating T cells that bind to a specific 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 the 5'RACE method using primers corresponding to sequences specific to the TCRα chain gene and sequences specific to the TCRβ chain gene.

[0149] In certain embodiments, it may be necessary to combine the TCRα and TCRβ chains after sequencing (i.e., analyze the combined TCR chains). Various methods can be used to combine the TCR chains as needed. For example, computer-aided methods such as immunological gene alignment software available from IMGT, JOINSOLVER, VDJSolver, SoDA, or iHMMune-align, or other similar tools for VDJ gene segment annotation, may be used to combine the TCR chains in silico. Assays such as PairSEQ® (Adaptive Biotechnologies Corp., Seattle, Washington) have also been developed.

[0150] In certain embodiments, recombinant TCRs include single-chain T cell receptors (scTCRs) that are specific to a target of interest (e.g., a peptide-MHC complex) and contain Vα / β chains and Cα / β chains (e.g., Vα-Cα, Vβ-Cβ, Vα-Vβ) or Vα-Cα pairs, Vβ-Cβ pairs, or Vα-Vβ pairs.

[0151] In certain embodiments, the CAR / TCR hybrid includes components of both a CAR and a TCR. For example, the CAR / TCR hybrid may include a TCR binding domain and a CAR-specific intracellular signaling domain. Such a configuration enables antigen recognition similar to that of a TCR and allows for the utilization of intracellular signaling similar to that of a CAR.

[0152] Cancer antigens are proteins produced by cancer cells, and viral antigens are proteins produced by virus-infected cells. The binding domains of recombinant receptors disclosed herein can be selected to bind to cancer antigens or viral antigens. In some embodiments, cancer antigens or viral antigens are selectively expressed or overexpressed on cancer cells or infected cells compared to other cells of the same type of tissue. In some embodiments, cancer antigens or viral antigens are cell surface molecules present on cancer cells or virus-infected cells that are substantially absent on normal tissues or whose expression is restricted to normal tissues where it is not significant.

[0153] In certain embodiments, a cancer antigen or viral antigen is selectively expressed by cancer cells or virus-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 the target cell type than in non-target cells.

[0154] Examples of cancer antigens include carcinoembryonic antigen (CEA), prostate-specific antigen, prostate stem cell antigen (PSCA), PSMA, Her2 / neu, estrogen receptor, progesterone receptor, ephrin B2, 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.

[0155] In certain embodiments, the binding domain that binds to the cancer antigen includes scFv. In a specific embodiment, scFv is 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)VhVl Contains 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, or IL13Ra2 (IL13 zetakin).

[0156] In a particular embodiment, huCD19(G01S) scFv having GMCSFss is Contains the sequence indicated by MLLLVTSLLLCELPHPAFLLIPEVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGISWNSGRIGYADSVKGRFTISRDNAKNSLFLQMNSLRAEDTAVYYCARDQGYHYYDSAEHAFDIWGQGTVVTVSSGGGGSGGGGSGGGGSQSALTQPRSVSGFPGQSVTISCTGTTSDDVSWYQQHPGKAPQLMLYDVSKRPSGVPHRFSGSRSGRAASLIISGLQTEDEADYFCSSYAGRYNSVLFGGGTKLTVL (sequence code 116).

[0157] In a particular embodiment, huCD19(G01S) scFv is This sequence includes the sequence indicated by EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGISWNSGRIGYADSVKGRFTISRDNAKNSLFLQMNSLRAEDTAVYYCARDQGYHYYDSAEHAFDIWGQGTVVTVSSGGGGSGGGGSGGGGSQSALTQPRSVSGFPGQSVTISCTGTTSDDVSWYQQHPGKAPQLMLYDVSKRPSGVPHRFSGSRSGRAASLIISGLQTEDEADYFCSSYAGRYNSVLFGGGTKLTVL (sequence code 117).

[0158] In a particular embodiment, muCD19(FMC63) scFv is Contains the sequence indicated by MLLLVTSLLLCELPHPAFLLIPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS (sequence number 118).

[0159] In a particular embodiment, CD19 scFv is This sequence includes the sequence represented by DIQMTQTTSSLSALGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS (Sequence ID 119).

[0160] In a particular embodiment, CD33(h2H12) VhVl scFv is Includes the sequence indicated by QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYDINWVRQAPGQGLEWIGWIYPGDGSTKYNEKFKAKATLTADTSTSTAYMELRSLRSDDTAVYYCASGYEDAMDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTINCKASQDINSYLSWFQQKPGKAPKTLIYRANRLVDGVPSRFSGSGSGQDYTLTISSLQPEDFATYYCLQYDEFPLTFGGGTKVEIK (sequence number 120).

[0161] In a particular embodiment, CD33(h2H12) VlVh scFv is This sequence includes the sequence indicated by DIQMTQSPSSLSASVGDRVTINCKASQDINSYLSWFQQKPGKAPKTLIYRANRLVDGVPSRFSGSGSGQDYTLTISSLQPEDFATYYCLQYDEFPLTFGGGTKVEIKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTNYDINWVRQAPGQGLEWIGWIYPGDGSTKYNEKFKAKATLTADTSTSTAYMELRSLRSDDTAVYYCASGYEDAMDYWGQGTTVTVSS (Sequence ID 121).

[0162] In certain embodiments, CD33 scFv (e.g., CD33(h2H12) VhVl scFv and / or CD33(h2H12) VlVh scFv) includes a signal sequence for granulocyte-macrophage colony-stimulating factor (GM-CSF). In certain embodiments, the signal sequence for GM-CSF includes the sequence shown in MLLLVTSLLLCELPHPAFLLIP (SEQ ID NO: 122).

[0163] In certain embodiments, the binding domain that binds to CD20 may be based on the binding domains of SP32 (ab64088), EP459Y (ab78237), rIGEL / 773 (ab219329), ocrelizumab, rituximab, ofatumumab, obinutuzumab, ibritumomab, or tositumomab.

[0164] Exemplary viral antigens include: coronavirus antigen: spike (S) protein; cytomegalovirus antigen: envelope glycoprotein B and CMV pp65; Epstein-Barr virus antigen: EBV EBNAI, EBV P18 and EBV P23; hepatitis antigen: S protein, M protein and L protein of hepatitis B virus, pre-S antigen of hepatitis B virus, HBCAGδ, HBV HBE, hepatitis C virus RNA, HCV NS3 and HCV NS4; herpes simplex virus antigen: initial protein and glycoprotein D; HIV antigen: gene products of 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 include GP36, Nef protein and reverse transcriptase; influenza antigen: hemagglutinin and neuraminidase; Japanese encephalitis virus antigen: E protein, ME protein, ME-NS1 protein, NS1 protein, NS1-NS2A protein and 80%E protein; measles virus antigen: measles virus fusion protein; rabies virus antigen: rabies virus glycoprotein and rabies virus nucleoprotein; RSV antigen: RSV fusion protein and M2 protein; rotavirus antigen: VP7sc; rubella virus antigen: E1 protein and E2 protein; and varicella-zoster virus antigen: 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 may be used.

[0165] In certain embodiments, the bacterial antigen includes an antigen expressed by a bacterium. In certain embodiments, the antigen is expressed by cells associated with a bacterial infection. Exemplary bacteria include Bacillus anthrax, Gram-negative bacilli, Chlamydia, Diphtheriae, Haemophilus influenzae, Helicobacter pylori, Malaria, Mycobacterium tuberculosis, Pertussis toxin, Streptococcus pneumoniae, Rickettsia, Staphylococcus, Streptococcus, and Neisseria tetanus.

[0166] Specific examples of bacterial antigens include anthrax antigens such as the protective antigen of Bacillus anthrax; Gram-negative bacilli antigens such as lipopolysaccharides; Haemophilus influenzae antigens such as capsular polysaccharides; diphtheria antigens such as diphtheria toxin; Mycobacterium tuberculosis antigens such as mycolic acid, heat shock protein 65 (HSP65), and antigen 85A, a major secreted protein of 30 kDa; pertussis toxin antigens such as hemagglutinin, partactin, FIM2, FIM3, and adenylyl cyclase; pneumococcal antigens such as pneumomolesin and capsular polysaccharides of Streptococcus pneumoniae; rickettsiae antigens such as rompA; streptococcal antigens such as M protein; and tetanus antigens such as tetanus toxin.

[0167] In certain embodiments, the fungal antigen includes an antigen expressed by a fungus. In certain embodiments, the antigen is expressed by cells associated with a fungal infection. Exemplary fungi include Candida, Coccidioides, Cryptococcus, Histoplasma, Leishmania, Plasmodium, protozoa, parasites, schistosomiasis, tinea, Toxoplasma, and Trypanosoma cruzi.

[0168] Further specific examples of fungal antigens include coccidioides antigens such as globular antigens; cryptococcus antigens such as capsular polysaccharides; histoplasma antigens such as heat shock protein 60 (HSP60); leishmania antigens such as gp63 and lipophosphoglycan; Plasmodium falciparum antigens such as merozoite surface antigens, sporozoite surface antigens, perisporozoite antigens, genital / genital surface antigens, protozoan antigens and other parasitic antigens (such as intraerythrozoite antigen pf 155 / RESA); schistosomiasis antigens such as glutathione-S-transferase and paramyosin; tinea fungal antigens such as trichophytin; toxoplasma antigens such as SAG-1 and p30; and Trypanosoma cruzi antigens such as antigens of 75-77 kDa and 56 kDa.

[0169] In certain embodiments, arthropod antigens include antigens expressed by arthropods. In certain embodiments, parasitic antigens include antigens expressed by parasites.

[0170] In certain embodiments, the binding domain binds to an epitope that activates immune cells. In certain embodiments, the epitope that activates immune cells is part of an antigen. In certain embodiments, the epitope that activates immune cells may be expressed by immune cells in the negative fraction of a sample and / or may be the binding domain of a multispecific binding molecule (also called a chemical adapter). The epitope that activates immune cells is part of a molecule (e.g., part of a protein) that activates the immune cell when a binding domain expressed by the immune cell binds to this epitope. The epitope that activates immune cells may be present in something that can bind to a recombinant receptor on a T cell and link the T cell it binds to to an activating cell (e.g., a PBMC). In certain examples, the chemical adapter is an antibody.

[0171] In certain embodiments, the epitope that activates immune cells may be a B cell ligand, and this B cell ligand may be 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 These are 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.

[0172] Other epitopes that activate immune cells can be found, for example, in 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 separately herein.

[0173] In certain embodiments, the epitope that activates immune cells is a hapten. A hapten can be any small molecule that, when combined with a larger carrier such as a protein, induces the production of antibodies that specifically bind to the hapten (either in a free state or bound to a carrier). Examples of haptens include peptides, other larger chemicals, and aptamers. In some embodiments, the hapten can be any hapten provided in the hapten database accessible from the World Wide Web at URL:crdd.osdd.net / raghava / haptendb / . In certain embodiments, the hapten is tethered to a cell having activating properties. In certain embodiments, if the epitope that activates immune cells is a hapten, the cell expressing the recombinant receptor includes a binding domain that binds to the hapten. In certain embodiments, the binding domain that binds to the hapten may be the binding domain of the recombinant receptor. In certain embodiments, the binding domain that binds to the hapten may be a binding domain that is not present on the recombinant receptor.

[0174] In certain embodiments, the epitope that activates immune cells may be the binding domain of a multispecific binding molecule (also called a chemical adapter). In certain embodiments, the multispecific binding molecule comprises at least two binding domains, at least one of which is an immune cell activation epitope that binds to T cells expressing recombinant receptors, and at least one binding domain that binds to immune cells in the negative fraction. Multispecific binding molecules useful for T cell activation are described separately herein. In certain embodiments, the multispecific binding molecule includes a bispecific antibody. In certain embodiments, the multispecific binding molecule includes an antibody. Since the antigen-binding domain of the antibody binds to the antigen and the Fc portion interacts with immune cells in the negative fraction, the antibody can be considered a multispecific binding molecule.

[0175] (vi-b) Transmembrane domain As described herein, the transmembrane domain within recombinant receptors plays a role in bridging the cell membrane and connecting the extracellular and intracellular components. In modified cells, the transmembrane domain can tether expressed molecules to the cell membrane.

[0176] The transmembrane domain may be of natural origin and / or synthetic origin. If of natural origin, the transmembrane domain may be derived from a membrane-bound protein and / or from a transmembrane protein. The transmembrane domain may include at least the transmembrane region of the α, β, or ζ chain of a 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 domains include, 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, IL7R a, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDl ld, ITGAE, CD103, ITGAL, CDl la, ITGAM, CDl lb, ITGAX, CDl It may also contain transmembrane regions 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 also be used, such as human Ig (immunoglobulin) hinges (e.g., IgG4 hinges or IgD hinges), GS linkers (e.g., the GS linkers described herein), KIR2DS2 hinges, and CD8a hinges.

[0177] In certain embodiments, the transmembrane domain has a thermodynamically stable three-dimensional structure, typically 15-30 amino acids long, within the cell membrane. The structure of the transmembrane domain can include α-helix, β-barrel, β-sheet, β-helix, or any combination thereof.

[0178] The transmembrane domain may contain 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 in 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 in the intracellular portion). In one embodiment, the transmembrane domain may originate from the same protein as the protein from which the signaling domain, costimulatory domain, or hinge domain originates. In another embodiment, the transmembrane domain may originate from a protein different from the protein from which the other domains of the recombinant receptor originate. In some cases, transmembrane domains may be selected such that each domain of the recombinant receptor avoids binding to transmembrane domains derived from the same or different surface membrane proteins, or the transmembrane domains may be modified by amino acid substitutions that allow for such avoidance, thereby minimizing interactions with other unintended members of the receptor complex. In certain embodiments, the transmembrane domain is the transmembrane domain of CD28 (e.g., encoded by SEQ ID NOs. 69, 70, 71, or 72). In certain embodiments, the transmembrane domain of CD28 has the sequence shown in SEQ ID NOs: 66, 67, or 68.

[0179] (vi-c) Intracellular effector domain The intracellular effector domain of a recombinant receptor is responsible for the activation of cells expressing the recombinant receptor. Therefore, the term “effector domain” means that it contains a portion of the intracellular domain sufficient for transduction of the activation signal. The effector domain can directly or indirectly promote a cellular biological or physiological response when it receives the appropriate signal. In certain embodiments, the effector domain is part of a signaling protein or protein complex, or directly binds to the target molecule, when the recombinant receptor binds to the target molecule, resulting in signal induction 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 an immune receptor-activated tyrosine motif (ITAM). In another embodiment, the effector domain indirectly promotes a cellular response by associating with one or more other proteins that directly promote a cellular response, such as a co-stimulatory domain.

[0180] The effector domain can activate at least one function of the modified cell when the modified cell binds to a cellular marker expressed by the cancer cell. Activation of the modified cell may include one or more of the following: differentiation, proliferation, and / or activation, or other effector functions. In certain embodiments, the effector domain may include an intracellular signaling moiety comprising a T cell receptor and a costimulatory domain, the costimulatory domain may include an intraplasmic sequence derived from a co-receptor or costimulatory molecule.

[0181] The effector domain may contain one, two, or more intracellular signaling regions (e.g., receptor signaling domains or cytoplasmic signaling sequences), co-stimulatory domains, or combinations thereof. Exemplary effector domains include signaling and 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 ligands that specifically bind to CD86, FcγRIIa, DAP12, CD30, CD40, PD-1, lymphocyte function-associated antigen 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, CD83, CDDS, 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 ITG. Examples of signaling and co-stimulatory domains selected from B1, 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 includes the signaling domain of CD3ζ.

[0182] Stimulative intracellular signaling sub-sequences may contain iTAMs. Examples of iTAMs containing major cytoplasmic signaling sequences include those derived from CD3γ, CD3δ, CD3ε, CD3ζ, CD5, CD22, CD66d, CD79a, CD79b, common FcRγ (FCER1G), FcγRlla, FcRβ (Fcε Rib), DAP10, or DAP12. In certain embodiments, variants of CD3ζ retain at least one, two, three, or all of the ITAM regions.

[0183] In certain embodiments, the effector domain comprises an intracytoplasmic portion that associates with an intracytoplasmic signaling protein, the intracytoplasmic signaling protein being a lymphocyte receptor or its signaling domain, a protein containing multiple ITAMs, a co-stimulatory domain, or any combination thereof.

[0184] Further examples of intracellular signaling regions include the cytoplasmic sequence of the CD3ζ chain and / or co-receptors that, in cooperation with it, initiate subsequent signaling after association with the binding domain.

[0185] Co-stimulatory domains are domains whose activation is required for an efficient lymphocyte response to binding to cellular markers. Several molecules can be replaced as either intracellular signaling regions or co-stimulatory domains. Examples of co-stimulatory domains include ligands that specifically bind to 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 CD83. For example, CD27 co-stimulation has been demonstrated to enhance the proliferation, effector function, and survival of human CAR-T cells in vitro, and to enhance 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 co-stimulatory 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 CDl Examples include lb, ITGAX, CDllc, ITGBl, 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, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, and CD19a. In certain embodiments, the co-stimulatory domain includes a signaling domain between 4 and 1BB.

[0186] In certain embodiments, the intracellular signaling moiety comprises CD3ζ (encoded, for example, by SEQ ID NO: 82 or 83) and a variant of 4-1BB (encoded, for example, by SEQ ID NO: 76, 77, or 78). In certain embodiments, CD3ζ has the sequence shown in SEQ ID NO: 79, 80, or 81. In certain embodiments, 4-1BB has the sequence shown in SEQ ID NO: 73, 74, or 75.

[0187] The intracellular portion includes the Wnt signaling pathway (e.g., LRP, Ryk, or ROR2), the NOTCH signaling pathway (e.g., NOTCH1, NOTCH2, NOTCH3, or NOTCH4), the Hedgehog signaling pathway (e.g., PTCH or SMO), receptor tyrosine kinases (RTKs) (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 The system may further include one or more proteins selected from the following: 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 smoothed); serine / threonine kinase receptors (BMPR or TGFR); and cytokine receptors (IL1R, IL2R, IL7R, or IL15R).

[0188] (vi-d) Linker Linkers within recombinant receptors can be any part of the recombinant receptor, as long as they can serve the role of connecting two components or domains of the recombinant receptor. In certain embodiments, linkers can provide flexibility to various components of the recombinant receptor. Linkers may further include spacer regions and linking amino acids. In certain examples, if a more rigid linker is required, proline-rich linkers can be used. While linkers may serve only the purpose of linking components, many linkers also serve other purposes besides linking, such as being multimerizing domains.

[0189] Spacers are used to provide an appropriate distance from other components of the recombinant receptor and / or to provide flexibility compared to other components. As described herein, in certain embodiments, the length of the spacer is customized so that it can bind to target cells and induce cell disruption. In certain embodiments, the length of the spacer may be selected depending on the location of the epitope of the cell marker, the affinity of the binding domain to the epitope, and / or the cell disruption-inducing ability of the binding domain after binding to the target.

[0190] Typical spacers include spacers with lengths of 10-250 amino acids, 10-200 amino acids, 10-150 amino acids, 10-100 amino acids, 10-50 amino acids, or 10-25 amino acids.

[0191] In certain embodiments, the spacers are 5 amino acid lengths, 8 amino acid lengths, 10 amino acid lengths, 12 amino acid lengths, 14 amino acid lengths, 20 amino acid lengths, 21 amino acid lengths, 26 amino acid lengths, 27 amino acid lengths, 45 amino acid lengths, 50 amino acid lengths, or 75 amino acid lengths. Spacers of such lengths are considered short spacers.

[0192] In certain embodiments, the spacers are 76 amino acid lengths, 90 amino acid lengths, 100 amino acid lengths, 110 amino acid lengths, 120 amino acid lengths, 125 amino acid lengths, 128 amino acid lengths, 131 amino acid lengths, 135 amino acid lengths, 140 amino acid lengths, 150 amino acid lengths, 160 amino acid lengths, 170 amino acid lengths, or 179 amino acid lengths. Spacers of such lengths fall under the category of medium-length spacers.

[0193] In certain embodiments, the spacers are 180 amino acid lengths, 190 amino acid lengths, 200 amino acid lengths, 210 amino acid lengths, 212 amino acid lengths, 214 amino acid lengths, 216 amino acid lengths, 218 amino acid lengths, 220 amino acid lengths, 228 amino acid lengths, 230 amino acid lengths, 240 amino acid lengths, 250 amino acid lengths, 260 amino acid lengths, or 270 amino acid lengths. Spacers of such lengths are considered long spacers.

[0194] Exemplary spacers include the entire length or portion of the hinge region of an immunoglobulin. The hinge region of the immunoglobulin may be the hinge region of a wild-type immunoglobulin or a modified wild-type immunoglobulin hinge region. In certain embodiments, the hinge region of the immunoglobulin is the hinge region of a human immunoglobulin. In this specification, “wild-type immunoglobulin hinge region” refers to the amino acid sequence of a hinge located between the CH1 and CH2 domains of the heavy chain in the upper and middle portions of a native antibody (for IgG, IgA, and IgD), or the amino acid sequence of a hinge located between the CH1 and CH3 domains of the heavy chain (for IgE and IgM).

[0195] The hinge region of the immunoglobulin may be the hinge region of IgG, IgA, IgD, IgE, or IgM. The hinge region of IgG may be the hinge region of IgG1, IgG2, IgG3, or IgG4. Sequences derived from IgG1, IgG2, IgG3, IgG4, or IgD can each be used alone, in combination with all or part of a CH2 region, in combination with all or part of a CH3 region, or in combination with all or part of a CH2 region and all or part of a CH3 region. In certain embodiments, the hinge region of IgG4 includes the sequence shown in Sequence ID No. 60.

[0196] Other examples of hinge regions that can be used in recombinant receptors described herein include hinge regions located in the extracellular domains of type I membrane proteins such as CD8α, CD4, CD28, and CD7, where these type I membrane proteins may be wild-type or variants thereof.

[0197] In certain embodiments, the spacer includes a hinge region of the interdomain region (stalk region) of a type II C lectin, or a hinge region of the stalk region of a cluster of differentiation (CD) molecule. The “stalk region” of a type II C lectin or CD molecule refers to a portion of the extracellular domain (ECD) located between the C 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 lectin or CD molecule. For example, the extracellular domain of human CD94 (GenBank accession number AAC50291.1) corresponds to amino acid residues 34-179, while the CTLD corresponds to amino acid residues 61-176. Since the stalk region of the human CD94 molecule contains amino acid residues 34-60, it is located between the hydrophobic portion (transmembrane domain) and the CTLD (see Boyington et al., Immunity 10:15, 1999; for further descriptions of the stalk region, see 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 C-type lectins or CD molecules may also have additional linking amino acids between the stalk region and the transmembrane domain or between the stalk region and the CTLD (as described later). In another example, the 233-amino acid-length human NKG2A protein (GenBank accession number: P26715.1) has a hydrophobic region (transmembrane domain) consisting of amino acids 71-93 and an extracellular domain consisting of amino acids 94-233. The CTLD of the human NKG2A protein contains amino acids 119-231, and the stalk region contains amino acids 99-116, and this stalk region may have additional linked amino acids adjacent to it.Other type II C-type lectins or CD molecules, or extracellular ligand-binding domains, stalk regions and CTLDs thereof are also known in the art (for example, for the sequences and descriptions of human CD23, human CD69, human CD72, human NKG2A and human NKG2D, see GenBank accession numbers: NP 001993.2; AAH07037.1; NP 001773.1; AAL65234.1; and CAA04925.1 respectively).

[0198] Linkers can, for example, in scFv, link the VL and VH of antibody-derived binding domains and function as linker amino acids between components of a recombinant receptor.

[0199] The linker can be any of a flexible linker, a rigid linker, or a semi-rigid linker, depending on the desired function of the linker. The linker may further contain linker amino acids. For example, in certain embodiments, the linker provides flexibility and space for the conformational movement between various components of the recombinant receptor. A commonly used flexible linker is the Gly-Ser linker. In certain embodiments, the linker sequence contains a repetitive sequence of glycine and serine, for example, 1 to 10 (Gly[[ID=⑨]] x [[ID=⑩]]Ser[[ID=⑪]] y [[ID=⑫]])[[ID=⑬]] n [[ID=⑭]]such as a repetitive sequence consisting of, where x and y are independently integers from 0 to 10, provided that the case where both x and y are 0 is excluded, and n is an integer of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. Specific examples include (Gly4Ser)[[ID=⑮]] n [[ID=⑯]](SEQ ID NO: 12), (Gly3Ser)[[ID=⑰]] n [[ID=⑱]](Gly4Ser)[[ID=⑲]] n [[ID=⑳]](SEQ ID NO: 124), (Gly3Ser)[[ID=㉑]] n [[ID=㉒]](Gly2Ser)[[ID=㉓]] n [[ID=㉔]](SEQ ID NO: 125), or (Gly3Ser) [[ID=㉕]] n(Gly4Ser)1 (SEQ ID NO: 126) is an example. In certain embodiments, the Gly-Ser linker is (Gly4Ser)4 (SEQ ID NO: 127), (Gly4Ser)3 (SEQ ID NO: 128), (Gly4Ser)2 (SEQ ID NO: 129), (Gly4Ser)1 (SEQ ID NO: 130), (Gly3Ser)2 (SEQ ID NO: 131), (Gly3Ser)1 (SEQ ID NO: 132), (Gly2Ser)2 (SEQ ID NO: 133), (Gly2Ser)1, GGSGGGSGGSG (SEQ ID NO: 134), GGSGGGSGSG (SEQ ID NO: 135), or GGSGGGSG (SEQ ID NO: 136). Various Gly-Ser linkers can be used to link PD1 to immunostimulatory cytokines, as described separately herein. In certain embodiments, the glycineserine linker includes the sequence shown in SEQ ID NO: 128. In certain embodiments, the glycineserine linker includes the sequence GGG.

[0200] In certain embodiments, the linker region is (GGGGS) n (Sequence ID 123), 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 (Sequence code 137), where n is an integer that includes 1, 2, 3, 4, 5, 6, 7, 8, 9 or more.

[0201] In some cases, flexible linkers may not be able to maintain the distance and position of recombinant receptors required for specific applications. In such cases, rigid or semi-rigid linkers may be useful. Examples of rigid or semi-rigid linkers include proline-rich linkers. In certain embodiments, a proline-rich linker is a peptide sequence that has more proline residues than would be expected to be included in the sequence by chance alone. In certain embodiments, a proline-rich linker is a linker in which proline residues account for 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% of the total in its sequence. A specific example of a proline-rich linker is a fragment of salivary proline-rich protein (PRP).

[0202] The linker may be sensitive to cleavage such as acid-inducible cleavage, photo-inducible cleavage, peptidase-inducible cleavage, esterase-inducible cleavage, or disulfide bond cleavage (a cleavable linker). Alternatively, the linker may be substantially resistant to cleavage (e.g., a stable linker or an uncleavable linker). In some embodiments, the linker is a readily charged linker, a hydrophilic linker, or a dicarboxylic acid-based linker.

[0203] Linked amino acids may be linkers that can be used to link sequences together when it is not necessary to use spacers to create distance, and / or when it is undesirable to use spacers to create distance. For example, linked amino acids may be short amino acid sequences that can be used to link intracellular co-stimulatory signaling regions. In certain embodiments, linked amino acids are 9 amino acid lengths or less (e.g., 2, 3, 4, 5, 6, 7, 8, or 9 amino acid lengths). In certain embodiments, a glycine-serine doublet can be used as a suitable linked amino acid linker. In certain embodiments, a single amino acid, such as alanine or glycine, can be used as a suitable linked amino acid.

[0204] In certain embodiments, recombinant receptors may contain a multimerizing domain. The biological activity of a protein depends on its tertiary and quaternary structures. The quaternary structure requires physical and chemical interactions with other protein subunits or polypeptides. A "multimerizing domain" is a domain that causes two or more proteins (monomers) to interact with each other via covalent and / or non-covalent bonds. The presence of a multimerizing domain in a protein can cause protein interactions to occur, potentially forming dimers, trimers, tetramers, pentamers, hexamers, heptamers, and so on, depending on the number of units / monomers incorporated into the multimer.

[0205] (vii) Cell therapies manufactured by ExVivo In certain embodiments, genetically modified cells can be recovered from the culture medium, washed, concentrated, and mixed with a carrier in a therapeutically effective amount. Exemplary carriers include saline, buffered saline, saline, water, Hanks' solution, Ringer's solution, Normosol-R (Abbott Labs), PLASMA-LYTE A® (Baxter Laboratories, Morton Grove, Illinois), and combinations thereof.

[0206] In certain embodiments, human serum albumin (HSA) or other human serum components or fetal bovine serum may be added to the carrier. In certain embodiments, the carrier for infusion contains buffered saline supplemented with 5% HSA or dextrose. Other isotonic agents include polyhydric sugar alcohols, including trihydric sugar alcohols or sugar alcohols with a higher valency, such as glycerin, erythritol, arabitol, xylitol, sorbitol, and mannitol.

[0207] The carrier may contain buffers such as citrate buffer, succinate buffer, tartaric acid buffer, fumarate buffer, gluconate buffer, oxalate buffer, lactate buffer, acetate buffer, phosphate buffer, histidine buffer and / or trimethylamine salts.

[0208] Stabilizers refer to a wide range of additives that can have various functions, from additives that can prevent cells from adhering to the walls of a container to fillers. 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 cyclitol (e.g., inositol); PEG; amino acid polymers; urea, glutathione, thioctose. Examples include acids, sulfur-containing reducing agents such as 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.

[0209] If necessary or beneficial, the formulation may contain a local anesthetic, such as lidocaine, to relieve pain at the injection site.

[0210] Examples of 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.

[0211] The therapeutically effective amount of cells contained in the formulation is 10 2 Numbers exceeding 10 3 Numbers exceeding 10 4 Numbers exceeding 10 5 Numbers exceeding 10 6 Numbers exceeding 10 7 Numbers exceeding 10 8 Numbers exceeding 10 9 Numbers exceeding 10 10 A number greater than 10 11 The number may exceed one.

[0212] In the formulations disclosed herein, cells are typically contained in volumes of 1 L or less, 500 ml or less, 250 ml or less, or 100 ml or less. Therefore, the density of cells administered is typically 10 4 Density exceeding 10 7 Density exceeding 10 particles / ml, or 10 8 The density is greater than particles / ml.

[0213] In certain embodiments, the formulation may contain one or more genetically modified cells (e.g., modified T cells, modified NK cells, or modified stem cells). Alternatively, the formulation may contain different types of genetically modified cells (e.g., a combination of T cells, NK cells, and / or stem cells).

[0214] Different types of genetically modified cells or cell subsets (e.g., modified T cells, modified NK cells, and / or modified stem cells) can be supplied in various ratios, such as 1:1:1, 2:1:1, 1:2:1, 1:1:2, 5:1:1, 1:5:1, 1:1:5, 10:1:1, 1:10:1, 1:1:10, 2:2:1, 1:2:2, 2:1:2, 5:5:1, 1:5:5, 5:1:5, 10:10:1, 1:10:10, and 10:1:10. These ratios can also be applied to the number of cells expressing the same PD1:cytokine chimeric transgene and / or recombinant receptor, or different PD1:cytokine chimeric transgenes and / or recombinant receptors. When only two types of cells are combined, or when the components of the PD1:cytokine chimeric transgene expressed in the formulation consist of only two combinations, these ratios may be combinations of two values ​​that can be created from the three numerical combinations described above. In some embodiments, the combined cell population is tested for efficacy and / or cell proliferation in vitro, in vivo, and / or ex vivo, and the ratio of cells that yields efficacy and / or cell proliferation is selected.

[0215] The cell-based formulations disclosed herein can be prepared, for example, for administration by injection, infusion, perfusion, or lavage. These formulations can be further formulated for bone marrow injection, intravenous injection, intradermal injection, intraarterial injection, intra-lymph node injection, intralymphatic injection, intraperitoneal injection, intrafocal injection, intraprostatic injection, intravaginal injection, intrarectal injection, intrathecal injection, intratumor injection, intramuscular injection, intravesical injection, and / or subcutaneous injection.

[0216] (viii) Compositions for targeted viral vectors and nanoparticles for in vivo cell modification Immune cells can also be genetically modified in vivo or ex vivo using target-directed viral vectors and / or nanoparticles. Viral vectors that can be used to deliver artificial expression constructs to cells are described separately herein, and target-directed viral vectors (e.g., pseudotype viral vectors) are well known in the art.

[0217] An exemplary example of cell-targeting nanoparticles is a nanoparticle with cell-targeting ligands (e.g., CD3, CD4, CD8, CD34) bound to its surface. Because the cell-targeting ligands are bound to the surface of these nanoparticles, they are selectively taken up by selected types of cells. Next, the nanoparticles deliver genetically modified components, leading to the expression of PD1: cytokine chimeric transgenes and recombinant receptors.

[0218] Exemplary nanoparticles include liposomes (tiny vesicles in which at least one lipid bilayer surrounding an aqueous core forms concentric spheres), liposome nanoparticles (liposome structures used to encapsulate even smaller nanoparticles within their core), and lipid nanoparticles (liposome-like structures that lack the continuous lipid bilayer characteristic of liposomes). Other polymer nanoparticles and porous nanoparticles composed of materials capable of forming porous networks can also be used. Exemplary materials include metals, transition metals, and metalloids (e.g., lithium, magnesium, zinc, aluminum, and silica).

[0219] Nanoparticles intended for in vivo delivery and uptake into cells may have an uncharged or negatively charged coating, and the size of these nanoparticles may be 130 nm or less. The dimensions 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.

[0220] The therapeutically effective amount of vectors 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 other examples, doses include 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 other examples, doses include 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 higher.

[0221] (ix) How to use The methods disclosed herein include treating subjects (humans, non-human primates, companion animals (dogs, cats, reptiles, birds, etc.), livestock (horses, cattle, goats, pigs, chickens, etc.), or research animals (monkeys, rats, mice, fish, etc.)) using the formulations disclosed herein. Treatment of subjects includes delivering a therapeutically effective dose. A therapeutically effective dose includes an amount that can provide an effective dose, a prophylactic treatment, and / or a therapeutic treatment.

[0222] An "effective dose" is the amount of formulation required to induce a desired physiological change in a subject. For example, an effective dose may provide enhanced immunogenic anticancer or immunogenic anti-infective effects (e.g., anti-infective effects against infections caused by viruses, bacteria, fungi, parasites, or arthropods). Effective doses are often administered for research purposes. The effective doses disclosed herein are amounts that can produce a statistically significant effect in animal models or in vitro assays related to the evaluation of the development or progression of cancer or infection. Immunogenic formulations can be provided in effective doses that stimulate an immune response.

[0223] "Prophylactic measures" include measures taken for subjects who do not show signs or symptoms of cancer or infection, or who show only early signs or symptoms of cancer or infection, with the aim of reducing or mitigating the risk of further progression of that cancer or infection. Thus, prophylactic measures function as measures to prevent cancer or infection. In certain embodiments, prophylactic measures suppress, delay, or prevent the development of metastases from a primary cancer tumor site. In certain embodiments, prophylactic measures suppress, delay, or prevent infections caused by bacteria, viruses, fungi, parasites, or arthropods.

[0224] "Therapeutic treatment" includes treatments performed on subjects exhibiting symptoms or signs of cancer or infection with the aim of reducing or eliminating the symptoms or signs of cancer or infection. Therapeutic treatment can suppress, control or eliminate the presence or activity of cancer or infection, and / or suppress, control or eliminate side effects of cancer or infection.

[0225] The functions of an effective dose, a prophylactic treatment, or a therapeutic treatment are not mutually exclusive, and in certain embodiments, two or more treatments may be performed depending on the administered dose.

[0226] In certain embodiments, the therapeutically effective dose enhances the cytotoxicity of immune cells, the proliferation of immune cells, and / or the production of cytokines by immune cells. These effects can provide anticancer and / or anti-infective effects. Anticancer effects include a reduction in the number of cancer cells, a reduction in the number of metastases, a reduction in tumor volume, an extension of life expectancy, induction of chemotherapy or radiosensitivity in cancer cells, suppression of angiogenesis near cancer cells, suppression of cancer cell proliferation, suppression of tumor growth, prevention or reduction of metastasis, extension of the lifespan of the subject, suppression of cancer-related pain, and / or a reduction in cancer recurrence or relapse after treatment. Anti-infective effects include a reduction in the amount or level of infectious pathogens, a reduction in fatigue, a reduction in loss of appetite, suppression of weight loss, a reduction in fever, a reduction in night sweats, a reduction in chills, a reduction in pain and soreness, a reduction in diarrhea, a reduction in bloating, a reduction in abdominal pain, a reduction in rash, a reduction in cough, and / or a reduction in runny nose.

[0227] A tumor is a swelling or lesion formed by the abnormal proliferation of cells (called neoplastic cells or tumor cells). Tumor cells are abnormal cells that proliferate rapidly and uncontrolledly and continue to grow even after the stimulus that initiated new proliferation has ceased. Tumors are characterized by a partial or complete loss of the structural organization and functional coordination of normal tissue, and usually form a recognizable tissue mass, which may be benign, premalignant, or malignant.

[0228] In certain embodiments, immune cells obtained from a subject are screened for their responsiveness to the expression of an inducible PD1:cytokine chimera transgene. The screening method includes the steps of introducing a gene construct containing a sequence encoding an inducible PD1:cytokine chimera, as described separately herein, into a group of immune cells obtained from a subject; and measuring the enhanced function of the resulting immune cells. In certain embodiments, the enhanced function of the immune cells includes enhanced cell killing, enhanced cell proliferation, and / or cytokine production. In certain embodiments, enhanced cytokine production includes the production of IFNγ and / or TNFα. Methods for screening immune cells obtained from a subject for their responsiveness to the secretion of inducible cytokines may include cell killing and proliferation assays such as MTT cell proliferation assays, trypan blue assays, carboxyfluorescein succinimimidyl (CFSE) assays, Ki67, XTT assays, BrdU assays, flow cytometry, and DNA quantification; or cytokine production assays such as quantitative PCR and immunoassays (e.g., enzyme-linked immunosorbent assays). Methods for screening immune cells obtained from subjects for their responsiveness to inducible cytokine expression can be performed in vitro or in vivo.

[0229] In certain embodiments, an inducible PD1:cytokine chimeric transgene that elicits enhanced immune cell function compared to baseline may be selected for administration to a subject. In certain embodiments, baseline includes the function of immune cells before introduction of the artificial expression construct of this disclosure, or the function of immune cells after introduction of a control artificial expression construct (which does not express the PD1:cytokine chimeric).

[0230] In certain embodiments, combinations of PD1:cytokine chimeric transgenes can be selected and administered to a subject based on screening results. For example, if in a first subject a first PD1:cytokine chimera increases cell-killing ability and proliferation, and a second PD1:cytokine chimera increases cytokine production, these first and second PD1:cytokine chimeras may be selected and administered to the first subject. Alternatively, if in a second subject a third PD1:cytokine chimera increases cell-killing ability, a fourth PD1:cytokine chimera increases proliferation, and a fifth PD1:cytokine chimera increases cytokine production, these third, fourth, and fifth PD1:cytokine chimeras may be selected and administered to the second subject.

[0231] In certain embodiments, any combination of PD1:cytokine chimeric transgenes can be administered. In certain embodiments, a combination of PD1:cytokine chimeric transgenes that enhances cell-killing ability, proliferation, and / or cytokine production includes selecting at least one of PD1:IL21, PD1:IL15(N72D), PD1:drIL18, PD1:IL12, and PD1:IFNα, and at least another one of PD1:IL21, PD1:IL15(N72D), PD1:drIL18, PD1:IL12, and PD1:IFNα. In certain embodiments, a combination of PD1:cytokine chimeric transgenes that enhances cell-killing includes at least one of PD1:IL21, PD1:IL15(N72D), PD1:drIL18, and PD1:IL12, and PD1:IFNα. In certain embodiments, the combination of PD1:cytokine chimeric transgenes that enhances cell killing includes at least one of PD1:IL15(N72D), PD1:drIL18, PD1:IL12, and PD1:IFNα, and PD1:IL21. In certain embodiments, the combination of PD1:cytokine chimeric transgenes that enhances cell killing includes at least one of PD1:IL21, PD1:drIL18, PD1:IL12, and PD1:IFNα, and PD1:IL15(N72D). In certain embodiments, the combination of PD1:cytokine chimeric transgenes that enhances cell killing includes at least one of PD1:IL21, PD1:IL15(N72D), PD1:IL12, and PD1:IFNα, and PD1:drIL18. In certain embodiments, the combination of PD1:cytokine chimeric transgenes that enhance cell killing includes at least one of PD1:IL21, PD1:IL15(N72D), PD1:drIL18, and PD1:IFNα, and PD1:IL12.

[0232] When administering a drug, the therapeutically effective dose (also referred to herein as “dose”) can first be estimated based on the results of in vitro assays and / or animal model studies. Using such information, a more accurate determination of a useful dose for the target subject can be made. The actual dose administered to a particular subject can be determined by a physician, veterinarian, or researcher, taking into account parameters such as the target, body weight, disease severity, type of cancer or infection, stage of cancer or infection, past or concomitant therapeutic interventions, the subject’s idiopathic disease, and physical and physiological factors such as the route of administration.

[0233] The effective therapeutic dose of cell-based formulations is 10 4 ~10 9 pieces / kg body weight or 10 3 ~10 11 It may also be 10 cells / kg body weight. The effective therapeutic dose to be administered is 10 2 More than one cell, 10 3 More than one cell, 10 4 More than one cell, 10 5 More than one cell, 10 6 More than one cell, 10 7 More than one cell, 10 8 More than one cell, 10 9 More than one cell, 10 10 More than one cell, or 10 11 This includes a number of cells exceeding one.

[0234] The therapeutically effective amount of vectors 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 other examples, doses include 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 other examples, doses include 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 higher.

[0235] The therapeutically effective dose can be achieved with one or more doses during a course of treatment regimen (e.g., daily, every other day, every three days, every four days, every five days, every six days, once a week, 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 once a year). In particular embodiments, the treatment protocol may be determined according to a clinical trial protocol or an FDA-approved treatment protocol.

[0236] The therapeutically effective dose can be administered, for example, by injection, infusion, perfusion, or lavage. Routes of administration include intravenous bolus, intradermal, intra-arterial, intraperitoneal, intra-lymph node, intra-lymphatic, intraperitoneal, intrafocal, intraprostatic, intravaginal, intrarectal, topical, intrathecal, intratumoral, intramuscular, intravesical, and / or subcutaneous.

[0237] In certain embodiments, the formulations and / or compositions of the Disclosure are administered to a patient in combination with relevant therapeutic methods (e.g., before, simultaneously with, or after performing the relevant therapeutic methods), the number of such relevant therapeutic methods is not particularly limited. In certain embodiments, the cells of the Disclosure may be used in combination with chemotherapy; phototherapy; immunosuppressants such as cyclosporine, azathioprine, methotrexate, mycophenolate, FK506; antibodies; other immunosuppressants such as CAM PATH; anti-CD3 antibodies; other antibody therapies; cytotoxins; fludarabine; cyclosporine; FK506; rapamycin; mycophenolate; steroids; FR901228; cytokines; or radiation therapy.

[0238] In certain embodiments, the formulations and / or compositions of the Disclosure may be administered concomitantly with chemotherapeutic agents, and the number of chemotherapeutic agents used concomitantly is not particularly limited. 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 depressants; 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 a chemotherapeutic agent further includes antihormone agents that control or suppress the effects of hormones on tumors, such antihormone agents include anti-estrogens and anti-androgens; and pharmaceutically acceptable salts, acids, or derivatives of any of these antihormone agents. Furthermore, if deemed appropriate, a combination of chemotherapy agents may be administered, including CHOP therapy, which is a combination of cyclophosphamide (Citoxane®, Ingenus Pharmaceuticals, Tampa, Florida), doxorubicin (hydroxydoxorubicin), vincristine (Oncovin), and prednisone.

[0239] In some embodiments, the chemotherapeutic agent is administered concurrently with the administration of the formulations and / or compositions of the Disclosure, or within one week after the administration of the formulations and / or compositions of the 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 the administration of the modified cells or nucleic acids of the Disclosure. In other embodiments, the chemotherapeutic agent is administered at least one month before the administration of the cells or nucleic acids of the Disclosure. In some embodiments, the method of the Disclosure further includes administering two or more chemotherapeutic agents.

[0240] Various 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®, Bristol-Myers Squibb Company, New York, NY), pembrolizumab (Keytruda®, Merck Sharp & Dohme, Loway, NJ), pembrolizumab, pizilizumab, and atezolizumab; and CTLA-4 inhibitors such as ipilimumab (Yervoy®, Bristol-Myers Squibb Company).

[0241] In further embodiments, the formulations and / or compositions of the present disclosure may be administered together with an analgesic. Exemplary analgesics include acetaminophen, oxycodone, tramadol, and propoxifene hydrochloride. The formulations and / or compositions of the present disclosure may also be administered together with an anti-inflammatory agent. Examples of anti-inflammatory agents include steroids, glucocorticoids, and non-steroidal anti-inflammatory drugs (NSAIDs). In further embodiments, the formulations and / or compositions of the present disclosure may be administered together with a bio-response modifier. Examples of bio-response modifiers include molecules that are directional to cell surface markers (e.g., CD4 and CD5); cytokine inhibitors such as TNF antagonists (e.g., etanercept (Enbrel®, Immunex Corporation, Seattle, Washington)), adalimumab (Humira®, AbbVie Biotechnology, North Chicago, Illinois), and infliximab (Remicade®, Janssen Biotech, Horsham Township, Pennsylvania); chemokine inhibitors; and adhesion molecule inhibitors. In some embodiments, the bio-response modifiers may include monoclonal antibodies and recombinant molecules. The formulations and / or compositions of this disclosure may also be administered together with disease-modifying antirheumatic drugs. Examples of disease-modifying antirheumatic drugs (DMARDs) include azathioprine, cyclophosphamide, cyclosporine, methotrexate, penicillamine, leflunomide, sulfasalazine, hydroxychloroquine, gold preparations (oral preparations (auranofin) and intramuscular injection), and minocycline. In further embodiments, the formulations and / or compositions of the present disclosure may be administered together with an anti-infective agent (e.g., an antiviral agent).Examples of antiinfective agents include ribavirin, 4'-fluorouridine, and small molecule drugs such as TMC353121, AVG-388, valacyclovir, acyclovir, cidofovir, foscarnet, ganciclovir, valganciclovir, penciclovir, famciclovir, idoxuridine, trifluorothymidine, vidarabine, fomivirsen, amantadine, rimantadine, zanamivir, oseltamivir, lamivudine, adefovir dipivoxil, entecavir, terbivudine, and Examples include levudine, podophyllotoxin, imiquimod, interferon, preconalil, maraviroc, enfuvirtide, zidovudine, didanosine, zalcitabine, stabudine, lamivudine, emtricitabine, tenofovir disoproxil fumarate, nevirapine, delavirdin, efavirenz, raltegravir, saquinavir, indinavir, ritonavir, nelfinavir, amprenavir, fosamprenavir, lopinavir, atazanavir, tipranavir, and darunavir.

[0242] In certain embodiments, the formulations and / or compositions of the Disclosure are administered in combination with cytokines. Cytokines that can be co-administered with the formulations and / or compositions of the Disclosure include, for example, interferons such as interferon-α, interferon-β, and interferon-γ; 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-1α, 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, drIL18, IL21, and IL36γ; tumor necrosis factors such as TNFα, TNFβ, and TGFβ2-7m; and other polypeptide factors such as LIF and kit ligand (KL). In relation to these combination therapies, the term “cytokine” also includes naturally occurring proteins or proteins derived from recombinant cell cultures, as well as biologically active equivalents of cytokines with naturally occurring sequences.

[0243] (x) KitThis disclosure further includes a kit. The kit may include various components for carrying out the methods disclosed herein. For example, depending on the aspect of the method being carried out, the kit may include: a nucleic acid encoding the PD1 cytokine chimeric transgene of this disclosure under the control of the iSynPro promoter; a minimal promoter of IL2; a constitutive promoter; a nucleic acid encoding the recombinant receptor disclosed herein; a nucleic acid encoding Her2tg; a skip sequence; a nucleic acid encoding scFv; a nucleic acid encoding VL; a nucleic acid encoding VH; a nucleic acid encoding the transmembrane domain; a nucleic acid encoding the intracellular effector domain; a nucleic acid encoding EGFRt; a nucleic acid encoding a selective cassette (e.g., DHFRdm); methotrexate; cells (e.g., immune cells, T cells, CD4+ T cells, CD8+ 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, and T cells transduced with the artificial expression constructs described herein. Cells); cell lines; tissue samples (e.g., peripheral blood mononuclear cells (PBMCs), leukocytes, bone marrow, thymus, tissue biopsy samples, tumors, lymph nodes, intestinal lymphoid tissue, mucosa-associated lymphoid tissue, spleen, other lymphoid tissue, liver, lung, stomach, intestines, colon, kidney, pancreas, breast, bone, prostate, cervix, testes, ovaries, tonsils, or other organs, and / or cells derived from these organs); gene expression components (e.g., provided by vectors (e.g., lentiviral vectors or retroviral vectors)). Genes for expression, CRISPR components, ZFNs, TALENs, MegaTAL, targeted viral vectors and / or targeted nanoparticles); cell formulation components or cell activation components (e.g., physiological saline, buffered physiological saline, phosphate-buffered saline (PBS)); biocompatible buffers (Ca++ / Mg++-free PBS, physiological saline, water, Hanks' solution, Ringer's solution); T cell stimulating epitopes (e.g., anti-CD3 / anti-CD28 binding beads, OKT3, TGN1412);The culture may include one or more of the following: a composition for initiating culture (RPMI, non-essential amino acids, sodium pyruvate, penicillin / streptomycin, EBV-transformed non-mitotic 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, anti-infective agents); antibodies tagged with fluorescent molecules; sequences for PCR amplification; PD1: cytokine chimeric transgenes (e.g., PD1:IL21, PD1:IL15(N72D), PD1:drIL18, PD1:IL12, PD1:IFNα); culture vessels; reference levels; transgenic animals; primer pairs; GAPDH; enzyme-linked immunosorbent assay (ELISA) of IFN-γ; culture plates, etc.

[0244] The following exemplary embodiments and examples are provided to illustrate specific embodiments of the Disclosure. Those skilled in the art who have referred to the Disclosure will understand that various modifications can be made to the specific embodiments disclosed herein, and that such modifications will not depart from the essence and scope of the Disclosure and will result in similar or comparable outcomes.

[0245] (xi) Exemplary Embodiment 1. An artificial expression construct comprising a sequence encoding programmed cell death 1 (PD1):cytokine chimera, wherein the PD1:cytokine chimera comprises PD1 linked to a cytokine. 2. The artificial expression construct according to Embodiment 1, wherein the cytokine is an immunostimulatory cytokine. 3. The artificial expression construct according to Embodiment 2, wherein the immunostimulatory cytokine comprises interleukin (IL) 21, IL 15, N72D mutant IL 15 (IL 15 (N72D)), IL 18, decoy-resistant IL 18 (drIL 18), IL 12, or interferon α (IFN α). 4. The artificial expression construct according to any one of Embodiments 1 to 3, wherein the sequence encoding the PD1:cytokine chimera is under the regulatory control of a promoter, and the promoter comprises a sequence having at least 95% sequence identity with any one of SEQ ID NOs: 17 to 56, and a minimal promoter. 5. The artificial expression construct according to Embodiment 4, wherein the promoter comprises a sequence having at least 98% sequence identity with any one of sequence numbers 17 to 56, and a minimal promoter. 6. The artificial expression construct according to Embodiment 4 or 5, wherein the promoter comprises a sequence having at least 99% sequence identity with any one of Sequence IDs 17 to 56, and a minimal promoter. 7. The artificial expression construct according to any one of embodiments 4 to 6, wherein the promoter comprises the sequence shown in any one of sequence numbers 17 to 56 and a minimal promoter. 8. The artificial expression construct according to any one of embodiments 4 to 7, wherein the minimum promoter is the minimum promoter of IL2. 9. The artificial expression construct according to Embodiment 8, wherein the minimum promoter of IL2 includes the sequence shown in SEQ ID NO: 138. 10. The aforementioned PD1:cytokine chimera PD1 (PD1:IL21) linked to interleukin (IL)21, PD1 connected to IL15 (PD1: IL15), PD1 linked to the N72D mutant IL15 (PD1:IL15(N72D)), PD1 connected to IL18 (PD1: IL18), PD1 linked to decoy resistance IL18 (PD1:drIL18), PD1 connected to IL12 (PD1:IL12), or PD1 linked to interferon-alpha (PD1:IFNα) An artificial expression construct according to any one of embodiments 1 to 9, including the above. 11. The artificial expression construct according to any one of Embodiments 1 to 10, wherein the PD1 is a PD1 having an A99L mutation. 12. The artificial expression construct according to any one of Embodiments 1 to 10, wherein the PD1 is wild-type PD1. 13. The artificial expression construct according to any one of Embodiments 1 to 12, wherein the PD1 is linked to the cytokine via a protein linker. 14. The artificial expression construct according to Embodiment 13, wherein the protein linker is a flexible linker. 15. The artificial expression construct according to Embodiment 14, wherein the flexible linker is a Gly-Ser linker. 16. The artificial expression construct according to Embodiment 15, wherein the Gly-Ser linker contains the sequence shown in SEQ ID NO: 128 or the sequence GGG. 17. The artificial expression construct according to any one of Embodiments 1 to 16, wherein the PD1:cytokine chimera is encoded by the sequence shown in SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 11, or is encoded by a sequence having at least 90% sequence identity with SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 11. 18. The artificial expression construct according to any one of Embodiments 1 to 17, wherein the PD1:cytokine chimera contains the sequence shown in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 or SEQ ID NO: 16, or contains a sequence having at least 90% sequence identity with the sequence shown in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 or SEQ ID NO: 16. 19. The artificial expression construct according to any one of Embodiments 1 to 18, further comprising a control mechanism. 20. The artificial expression construct according to Embodiment 19, wherein the control mechanism encodes a transduction marker. 21. The artificial expression construct according to Embodiment 20, wherein the transduction marker contains Her2tG. 22. An artificial expression construct according to any one of embodiments 1 to 21, further comprising a first skip sequence. 23. The artificial expression construct according to Embodiment 22, wherein the first skip sequence encodes a 2A autocleaving polypeptide. 24. The artificial expression construct according to Embodiment 23, wherein the 2A self-cleaving polypeptide comprises T2A, P2A, E2A, or F2A. 25. An artificial expression construct according to any one of Embodiments 1 to 24, comprising a promoter, a control mechanism, a skip sequence, and a sequence shown in any one of Sequence IDs 1 to 11, in the direction from the 5' end to the 3' end, including the sequence shown in any one of Sequence IDs 17 to 56 and the sequence shown in Sequence ID 138. 26. An artificial expression construct according to any one of Embodiments 1 to 25, comprising a promoter including the sequence shown in any one of Sequence IDs 17 to 56 and the sequence shown in Sequence ID 138, a sequence encoding a transduction marker, a skip sequence, and the sequence shown in any one of Sequence IDs 1 to 11, in the direction from the 5' end to the 3' end. 27. An artificial expression construct according to any one of Embodiments 1 to 26, having at least 95% sequence identity with the sequence shown in Sequence ID No. 103. 28. An artificial expression construct according to any one of Embodiments 1 to 27, having at least 98% sequence identity with the sequence shown in Sequence ID No. 103. 29. An artificial expression construct according to any one of Embodiments 1 to 28, having at least 99% sequence identity with the sequence shown in Sequence ID No. 103. 30. An artificial expression construct according to any one of embodiments 1 to 29, having the sequence shown in Sequence ID No. 103. 31. An artificial expression construct according to any one of Embodiments 1 to 30, further comprising a sequence encoding a recombinant receptor, wherein the recombinant receptor comprises a binding domain that binds to a target antigen. 32. The artificial expression construct according to Embodiment 31, wherein the sequence encoding the recombinant receptor is operably linked to a second promoter. 33. The artificial expression construct according to Embodiment 32, wherein the second promoter comprises the EF1α(L) promoter, the EF1α(s) promoter, the myeloproliferative sarcoma virus (MND) promoter, the cytomegalovirus (CMV) promoter, the Simian virus 40 (SV40) early promoter, the mouse mammary cancer virus (MMTV) promoter, the human immunodeficiency virus (HIV) long-chain terminal repeat (LTR) promoter, the MoMuLV promoter, the avian leukemia virus promoter, the Epstein-Barr virus very early promoter, the Rous sarcoma virus promoter, the actin promoter, the myosin promoter, the hemoglobin promoter, or the creatine kinase promoter. 34. The artificial expression construct according to Embodiment 31, wherein the binding domain is part of the extracellular portion. 35. An artificial expression construct according to any one of embodiments 31 to 34, wherein the target antigen is expressed on the surface of target cells. 36. The artificial expression construct according to Embodiment 35, wherein the target cells include cells infected with bacteria, viruses, fungi, parasites, or arthropods, or cancer cells. 37. The artificial expression construct according to Embodiment 35 or 36, wherein the target cells include cancer cells. 38. An artificial expression construct according to any one of embodiments 31 to 37, wherein the recombinant receptor further comprises an intracellular portion. 39. The artificial expression construct according to Embodiment 38, wherein the intracellular portion comprises a CD3ζ signaling domain and / or a 4-1BB signaling domain. 40. The artificial expression construct according to embodiment 38 or 39, wherein the intracellular portion is linked to the extracellular portion via a transmembrane domain. 41. The artificial expression construct according to Embodiment 40, wherein the transmembrane domain includes the transmembrane domain of CD28. 42. An artificial expression construct according to any one of embodiments 31 to 41, further comprising a second control mechanism. 43. The artificial expression construct according to embodiment 42, wherein a second control mechanism codes a selection cassette. 44. The artificial expression construct according to Embodiment 43, wherein the selected cassette comprises a dihydrofolate reductase double mutant (DHFRdm). 45. An artificial expression construct according to any one of embodiments 42 to 44, further comprising a third control mechanism. 46. ​​The artificial expression construct according to Embodiment 45, wherein a third control mechanism encodes a transduction marker. 47. The artificial expression construct according to Embodiment 46, wherein the transduction marker comprises epidermal growth factor receptor (EGFRt) or cleaved CD19 (tCD19). 48. An artificial expression construct according to any of embodiments 31 to 47, further comprising a second skip sequence. 49. The artificial expression construct according to Embodiment 48, wherein the second skip sequence encodes a 2A autocleaving polypeptide. 50. The artificial expression construct according to Embodiment 49, wherein the 2A skipped autocleavage polypeptide comprises T2A, P2A, E2A, or F2A. 51. An artificial expression construct according to any one of embodiments 31 to 50, wherein the selection cassette includes a second skip sequence at its 5' end and the transduction marker includes a third skip sequence at its 5' end. 52. An artificial expression construct according to any one of Embodiments 1 to 51, comprising a first promoter including the sequence shown in any one of SEQ ID NOs: 17 to 56 and the sequence shown in SEQ ID NO: 138, a first regulatory mechanism, a first skip sequence, the sequence shown in any one of SEQ ID NOs: 1 to 11, a second promoter, a sequence encoding a recombinant receptor, a second skip sequence, a second regulatory mechanism, a third skip sequence, and a third regulatory mechanism, in the direction from the 5' end to the 3' end. 53. An artificial expression construct according to any one of Embodiments 1 to 52, comprising, in the direction from the 5' end to the 3' end, a first promoter including the sequence shown in any one of SEQ ID NOs: 17 to 56 and the sequence shown in SEQ ID NO: 138, a sequence encoding a first transduction marker, a first skip sequence, a sequence shown in any one of SEQ ID NOs: 1 to 11, a second promoter, a sequence encoding a recombinant receptor, a second skip sequence, a sequence encoding a selection cassette, a third skip sequence, and a sequence encoding a second transduction marker. 54. An artificial expression construct according to any one of Embodiments 1 to 53, having at least 90% sequence identity with the sequence shown in SEQ ID NOs: 98, 99, 100, 101, or 102. 55. An artificial expression construct according to any one of Embodiments 1 to 54, having the sequence shown in SEQ ID NOs: 98, 99, 100, 101, or 102. 56. A system for enhancing the function of immune cells, A first artificial expression construct encoding a PD1:cytokine chimera under the regulatory control of the first promoter, A second artificial expression construct containing a sequence encoding a recombinant receptor and Includes, The first promoter comprises a minimal promoter and a sequence having at least 95% sequence identity with any of sequence numbers 17-56. The PD1 cytokine chimera comprises PD1 linked to a cytokine, The recombinant receptor includes a binding domain that binds to an antigen expressed on the surface of a target cell. system. 57. The system according to embodiment 56, wherein the cytokine is an immunostimulatory cytokine. 58. The system according to Embodiment 57, wherein the immunostimulatory cytokine comprises interleukin (IL) 21, IL 15, N72D mutant IL 15 (IL 15 (N72D)), IL 18, decoy-resistant IL 18 (drIL 18), IL 12, or interferon α (IFN α). 59. The system according to any one of embodiments 56 to 58, wherein the first promoter comprises a sequence having at least 98% sequence identity with any one of SEQ ID NOs: 17 to 56 and a minimal promoter. 60. The system according to any one of embodiments 56 to 59, wherein the first promoter comprises a sequence having at least 99% sequence identity with any one of SEQ ID NOs: 17 to 56 and a minimal promoter. 61. The system according to any one of embodiments 56 to 60, wherein the first promoter comprises a sequence shown in any one of SEQ ID NOs: 17 to 56 and a minimal promoter. 62. The system according to any one of embodiments 56 to 61, wherein the minimal promoter comprises the minimal promoter of IL2. 63. The system according to embodiment 62, wherein the minimal promoter of IL2 comprises the sequence shown in SEQ ID NO: 138. 64. The PD1:cytokine chimera is PD1 linked to interleukin (IL) 21 (PD1:IL21), PD1 linked to IL15 (PD1:IL15), PD1 linked to N72D mutant IL15 (PD1:IL15(N72D)), PD1 linked to IL18 (PD1:IL18), PD1 linked to decoy-resistant IL18 (PD1:drIL18), PD1 linked to IL12 (PD1:IL12), or PD1 linked to interferon α (PD1:IFNα) The system according to any one of embodiments 56 to 63. 65. The system according to any one of embodiments 56 to 64, wherein the PD1 is PD1 having an A99L mutation. 66. The system according to any one of embodiments 56 to 64, wherein the PD1 is wild-type PD1. 67. The system according to any one of embodiments 56 to 66, wherein the PD1 is linked to the cytokine via a protein linker. 68. The system according to embodiment 67, wherein the protein linker is a flexible linker. 69. The system according to embodiment 68, wherein the flexible linker is a Gly-Ser linker. 70. The system according to Embodiment 69, wherein the Gly-Ser linker comprises the sequence shown in Sequence ID No. 128 or the sequence GGG. 71. The system according to any one of embodiments 56 to 70, wherein the PD1:cytokine chimera is encoded by the sequence shown in SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11, or by a sequence having at least 90% sequence identity with SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11. 72. The system according to any one of embodiments 56 to 71, wherein the PD1:cytokine chimera includes the sequence shown in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16, or includes a sequence having at least 90% sequence identity with the sequence shown in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16. 73. The system according to any one of embodiments 56 to 72, wherein the first artificial expression construct further includes a control mechanism. 74. The system according to embodiment 73, wherein the control mechanism codes for a transduction marker. 75. The system according to embodiment 74, wherein the transduction marker includes Her2tG. 76. The system according to any one of embodiments 56 to 75, wherein the first artificial expression construct further comprises a first skip sequence. 77. The system according to embodiment 76, wherein the first skip sequence encodes a 2A self-cleaving polypeptide. 78. The system according to embodiment 77, wherein the 2A self-cleaving polypeptide comprises T2A, P2A, E2A, or F2A. 79. The system according to any one of embodiments 56 to 78, wherein the sequence encoding the recombinant receptor is operably linked to a second promoter. 80. The system according to any one of embodiments 56 to 79, 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, a bird leukemia virus promoter, an Epstein-Barr virus very early promoter, a Roussarcoma virus promoter, an actin promoter, a myosin promoter, a hemoglobin promoter, or a creatine kinase promoter. 81. The system according to any one of embodiments 56 to 80, wherein the second artificial expression construct further includes a second control mechanism. 82. The system according to embodiment 81, wherein the second control mechanism codes the selected cassette. 83. The system according to Embodiment 82, wherein the selection cassette comprises a dihydrofolate reductase double mutant (DHFRdm). 84. The system according to any one of embodiments 56 to 83, wherein the second artificial expression construct further includes a third control mechanism. 85. The system according to embodiment 84, wherein a third control mechanism encodes a transduction marker. 86. The system according to Embodiment 85, wherein the transduction marker comprises cleaved epidermal growth factor receptor (EGFRt) or tCD19. 87. The system according to any one of embodiments 56 to 86, wherein the second artificial expression construct further comprises a second skip sequence. 88. The system according to embodiment 87, wherein the second skip sequence encodes a 2A self-cleaving polypeptide. 89. The system according to embodiment 88, wherein the 2A self-cleaving polypeptide comprises T2A, P2A, E2A, or F2A. 90. The system according to any one of embodiments 56 to 89, wherein the second artificial expression construct includes 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. 91. The system according to any one of embodiments 56 to 90, wherein the binding domain is part of the extracellular portion. 92. The system according to any one of embodiments 56 to 91, wherein the target cells include cells infected with bacteria, viruses, fungi, parasites, or arthropods, or cancer cells. 93. The system according to any one of embodiments 56 to 92, wherein the target cells include cancer cells. 94. The system according to any one of embodiments 56 to 93, wherein the recombinant receptor further comprises an intracellular portion. 95. The system according to Embodiment 94, wherein the intracellular portion includes a CD3ζ signaling domain and / or a 4-1BB signaling domain. 96. The system according to embodiment 94 or 95, wherein the intracellular portion is connected to the extracellular portion via a transmembrane domain. 97. The system according to embodiment 96, wherein the transmembrane domain includes a transmembrane domain of CD28. 98. The system according to any one of embodiments 56 to 97, wherein the first artificial expression construct and the second artificial expression construct reside on a single artificial expression construct. 99. The system according to any one of embodiments 56 to 97, wherein the first artificial expression construct and the second artificial expression construct reside on separate artificial expression constructs. 100. (i) The first artificial expression construct, (ii) Second artificial expression construct and Includes, The first artificial expression construct includes a first promoter containing the sequence shown in any one of SEQ ID NOs. 17-56 and the sequence shown in SEQ ID NO. 138, a first regulatory mechanism, a first skip sequence, and the sequence shown in any one of SEQ ID NOs. 1-11, in the direction from the 5' end to the 3' end. The second artificial expression construct includes, from the 5' end to the 3' end, a second promoter, a sequence encoding a recombinant receptor, a second skip sequence, a second regulatory mechanism, a third skip sequence, and a third regulatory mechanism. The system according to any one of embodiments 56 to 99. 101. Nanoparticles encapsulating an artificial expression construct according to any of Embodiments 1 to 55 or a system according to any of Embodiments 56 to 100. 102. Cells genetically modified to express the artificial expression construct described in any of Embodiments 1 to 55 or the system described in any of Embodiments 56 to 100. 103. The cells according to Embodiment 102, which are autologous cells obtained from the subject or cells of the same species as the subject. 104. Cells according to embodiment 102 or 103, which are in vivo or ex vivo cells. 105. A cell according to any of embodiments 102 to 104, which is an immune cell. 106. The cell according to embodiment 105, wherein the immune cell is a lymphocyte. 107. The cells according to Embodiment 106, wherein the lymphocytes include T cells, B cells, natural killer (NK) cells, or NK-T cells. 108. A cell according to any of embodiments 102 to 107, 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. 109. A cell according to any of embodiments 102 to 108, which is a CD8+ T cell. 110. A cell according to any of embodiments 102 to 109, which is a CD4+ T cell. 111. A population of cells genetically modified to express the artificial expression construct described in any of Embodiments 1 to 55 or the system described in any of Embodiments 56 to 100. 112. A population of cells according to Embodiment 111, comprising autologous cells obtained from a subject or cells of the same species as the subject. 113. A population of cells according to Embodiment 111 or 112, which is an in vivo or ex vivo cell population. 114. A population of cells according to any one of embodiments 111 to 113, wherein the cells are immune cells. 115. The cell population according to Embodiment 114, wherein the immune cells are lymphocytes. 116. The cell population according to Embodiment 115, wherein the lymphocytes include T cells, B cells, natural killer (NK) cells, or NK-T cells. 117. A population of cells according to any one of embodiments 111 to 116, comprising CD4+ T cells and / or CD8+ T cells. 118. A formulation comprising (i) cells genetically modified to express an artificial expression construct according to any of Embodiments 1 to 55 or a system according to any of Embodiments 56 to 100, and (ii) a pharmaceutically acceptable carrier. 119. A method for genetically modifying immune cells to have enhanced function, A method comprising the step of contacting immune cells with an artificial expression construct according to any one of embodiments 1 to 55 having a sequence encoding a cytokine chimera. 120. The method according to Embodiment 119, wherein the enhanced function includes enhanced cell-killing ability. 121. The PD1:cytokine chimera described above is PD1 (PD1:IL21) linked to interleukin (IL)21, PD1 connected to IL15 (PD1: IL15), PD1 linked to the N72D mutant IL15 (PD1:IL15(N72D)), PD1 connected to IL18 (PD1: IL18), PD1 linked to decoy resistance IL18 (PD1:drIL18), PD1 connected to IL12 (PD1:IL12), or PD1 linked to interferon-alpha (PD1:IFNα) The method according to Embodiment 120, including the method described above. 122. The method according to Embodiment 121, wherein the PD1 is a PD1 having the A99L mutation. 123. The method according to Embodiment 121, wherein the PD1 is wild-type PD1. 124. The method according to any one of embodiments 121 to 123, wherein the coded PD1:cytokine chimera comprises PD1:IFNα. 125. The method according to Embodiment 119, wherein the enhanced function includes enhanced proliferation. 126. The PD1:cytokine chimera described above is PD1 (PD1:IL21) linked to interleukin (IL)21, PD1 connected to IL15 (PD1: IL15), PD1 linked to the N72D mutant IL15 (PD1:IL15(N72D)), PD1 connected to IL18 (PD1: IL18), PD1 linked to decoy resistance IL18 (PD1:drIL18), PD1 connected to IL12 (PD1:IL12), or PD1 linked to interferon-alpha (PD1:IFNα) The method according to Embodiment 125, including the method described above. 127. The method according to Embodiment 126, wherein the coded PD1:cytokine chimera comprises PD1:IFNα. 128. The method according to Embodiment 119, wherein the enhanced function includes enhanced cytokine production. 129. The method according to Embodiment 128, wherein the enhanced cytokine production includes IFNγ. 130. The PD1:cytokine chimera described above is PD1 (PD1:IL21) linked to interleukin (IL)21, PD1 connected to IL15 (PD1: IL15), PD1 linked to the N72D mutant IL15 (PD1:IL15(N72D)), PD1 connected to IL18 (PD1: IL18), PD1 linked to decoy resistance IL18 (PD1:drIL18), PD1 connected to IL12 (PD1:IL12), or PD1 linked to interferon-alpha (PD1:IFNα) The method according to Embodiment 129, including the method described in Embodiment 129. 131. The method according to Embodiment 130, wherein the PD1 is a PD1 having the A99L mutation. 132. The method according to Embodiment 130, wherein the PD1 is wild-type PD1. 133. The method according to any one of embodiments 130 to 132, wherein the coded PD1:cytokine chimera comprises PD1:IFNα. 134. The method according to Embodiment 128, wherein the enhanced cytokine production includes the production of TNFα. 135. The PD1:cytokine chimera described above is PD1 (PD1:IL21) linked to interleukin (IL)21, PD1 connected to IL15 (PD1: IL15), PD1 linked to the N72D mutant IL15 (PD1:IL15(N72D)), PD1 connected to IL18 (PD1: IL18), PD1 linked to decoy resistance IL18 (PD1:drIL18), PD1 connected to IL12 (PD1:IL12), or PD1 linked to interferon-alpha (PD1:IFNα) The method according to Embodiment 134, including the method described above. 136. The method according to Embodiment 135, wherein the PD1 is a PD1 having the A99L mutation. 137. The method according to embodiment 135, wherein the PD1 is wild-type PD1. 138. The method according to any one of embodiments 135 to 137, wherein the coded PD1:cytokine chimera comprises PD1:IFNα. 139. The method according to any one of embodiments 119 to 138, wherein the artificial expression construct further comprises a sequence encoding a recombinant receptor having a binding domain that binds to an antigen expressed on the surface of a target cell. 140. The method according to embodiment 139, wherein the sequence encoding the recombinant receptor is operably linked to a second promoter. 141. The method according to Embodiment 140, wherein the second promoter comprises the EF1α(L) promoter, the EF1α(s) promoter, the myeloproliferative sarcoma virus (MND) promoter, the cytomegalovirus (CMV) promoter, the Simian virus 40 (SV40) early promoter, the mouse mammary cancer virus (MMTV) promoter, the human immunodeficiency virus (HIV) long-chain terminal repeat (LTR) promoter, the MoMuLV promoter, the avian leukemia virus promoter, the Epstein-Barr virus very early promoter, the Rous sarcoma virus promoter, the actin promoter, the myosin promoter, the hemoglobin promoter, or the creatine kinase promoter. 142. The method according to any one of embodiments 119 to 141, further comprising the step of contacting the immune cells with an artificial expression construct comprising a sequence encoding a recombinant receptor including a binding domain that binds to an antigen expressed on the surface of a target cell. 143. The method according to Embodiment 142, wherein the steps of contacting the immune cells with the artificial expression construct described in any of Embodiments 1 to 55 and contacting the immune cells with the artificial expression construct containing the sequence encoding the recombinant receptor are performed simultaneously. 144. The method according to Embodiment 142, wherein the steps of contacting the immune cells with the artificial expression construct described in any of Embodiments 1 to 55 and contacting the immune cells with the artificial expression construct containing the sequence encoding the recombinant receptor are performed at different time points. 145. The method according to any one of Embodiments 119 to 144, wherein the artificial expression construct includes the sequence shown in SEQ ID NOs. 98, 99, 100, 101, or 102, or includes a sequence having 90% sequence identity with the sequence shown in SEQ ID NOs. 98, 99, 100, 101, or 102. 146. A method of treating a subject that requires treatment, A method comprising the step of treating a subject in need of treatment by administering a therapeutically effective amount of an artificial expression construct according to any of Embodiments 1 to 55, a system according to any of Embodiments 56 to 100, nanoparticles according to Embodiment 101, or a formulation according to Embodiment 118 to the subject. 147. The method according to embodiment 146, wherein the subject requiring treatment has cancer or an infectious disease. 148. The method according to Embodiment 146 or 147, wherein the administration of the therapeutically effective dose includes intravesical, intravenous, intradermal, intra-arterial, parenteral, intra-lymph node, intra-lymphatic, intraperitoneal, intrafocal, intraprostatic, vaginal, intrarectal, topical, intrathecal, intratumoral, intramuscular, or subcutaneous administration. 149. Recombinant protein containing programmed cell death 1 (PD1) and cytokines. 150. The recombinant protein according to Embodiment 149, wherein the cytokine is an immunostimulatory cytokine. 151. The recombinant protein according to Embodiment 150, wherein the immunostimulatory cytokine comprises interleukin (IL) 21, IL 15, N72D mutant IL 15 (IL 15 (N72D)), IL 18, decoy-resistant IL 18 (drIL 18), IL 12, or interferon α (IFN α). 152. PD1 (PD1:IL21) linked to interleukin (IL)21, PD1 connected to IL15 (PD1: IL15), PD1 linked to the N72D mutant IL15 (PD1:IL15(N72D)), PD1 connected to IL18 (PD1: IL18), PD1 linked to decoy resistance IL18 (PD1:drIL18), PD1 connected to IL12 (PD1: IL12), PD1 linked to interferon-alpha (PD1:IFNα), A99L mutation PD1 linked to IL12 (PD1(A99L))(PD1(A99L):IL21), PD1(A99L) connected to IL15 (PD1(A99L):IL15), PD1(A99L) (PD1(A99L):IL15(N72D)) linked to the N72D mutant IL15, PD1(A99L) connected to IL18 (PD1(A99L):IL18), PD1(A99L)(PD1(A99L):drIL18) linked to decoy resistance IL18, PD1(A99L) connected to IL12 (PD1(A99L):IL12), or PD1(A99L) (PD1(A99L):IFNα) linked to interferon-alpha Recombinant protein according to Embodiment 151, including the above. 153. The recombinant protein according to any one of embodiments 149 to 152, wherein the PD1 is linked to the cytokine via a protein linker. 154. The recombinant protein according to Embodiment 153, wherein the protein linker is a flexible linker. 155. The recombinant protein according to Embodiment 154, wherein the flexible linker is a Gly-Ser linker. 156. The recombinant protein according to Embodiment 155, wherein the Gly-Ser linker comprises the sequence shown in Sequence ID No. 128 or the sequence GGG. 157. A recombinant protein according to any of embodiments 149 to 156, encoded by the sequence shown in SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11, or encoded by a sequence having at least 90% sequence identity with SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11. 158. A recombinant protein according to any one of embodiments 149 to 157, comprising the sequence shown in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16, or comprising a sequence having at least 90% sequence identity with the sequence shown in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16.

[0246] (xii) Conclusion In this specification, the terms “recombinant protein” and “chimera” are used interchangeably.

[0247] The nucleic acid sequences and amino acid sequences provided herein are represented by abbreviations used for nucleotide bases and amino acid residues, as defined in U.S. Patent Law Enforcement Rule 37 CFR Rules 1.831-1.839 and also shown in WIPO Standard ST.26 (effective July 1, 2022). Although only one type of strand is shown for each nucleic acid sequence, complementary strands are also included in the embodiments where appropriate.

[0248] Variants of sequences disclosed herein and cited sequences are also included in this application. Indicators for determining which amino acid residues can be substituted, inserted, or deleted without loss of biological activity are computer programs well known in the art, such as DNASTAR. TM This can be discovered using software (Madison, Wisconsin, USA). The amino acid changes of the protein variants disclosed herein are preferably conservative amino acid changes, i.e., substitutions between amino acids with similar charge or substitutions between uncharged amino acids. Conservative amino acid changes include substitutions of side chains by members of the related amino acid family.

[0249] Appropriate conservative substitutions of amino acids in peptides or proteins are known to those skilled in the art, and such substitutions can usually be made without altering the biological activity of the resulting molecule. Those skilled in the art will know that substituting a single amino acid in a non-essential region of a polypeptide usually does not substantially alter its biological activity (see, for example, Watson et al. Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin / Cummings Pub. Co., p. 224). Natural amino acids are typically classified into conserved 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 negative-charged residues and their amides): asparagine (Asn), glutamine (Gln), Asp, and Glu; Group 4: Gln and Asn; Group 5: (basic; also classified as polar positive-charged residues): arginine (Arg), lysine (Lys), and histidine (His); Group 6: (large aliphatic nonpolar residues): isoleucine (Ile), leucine (Leu Groups 10 (nonpolar small aliphatic residues or slightly polar small 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, WH Freeman and Company.

[0250] When making such changes, the hydrophobicity index of amino acids may be taken into consideration. The importance of the hydrophobicity index of amino acids in conferring biological functions that interact with each other on proteins is widely understood in this art (Kyte and Doolittle, 1982, J. Mol. Biol. 157(1), 105-32). Each amino acid has been assigned a hydrophobicity index based on its hydrophobicity and charge properties (Kyte and Doolittle, 1982). The hydrophobicity index for each amino acid is as follows: 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).

[0251] It is well known in the art that substituting a specific amino acid with another amino acid having a similar hydrophobic index or degree of hydrophobicity can yield a protein with similar biological activity, i.e., a protein with biologically equivalent functionality. When making such a change, substitutions between amino acids with hydrophobic indexes within ±2 are preferred, substitutions between amino acids with hydrophobic indexes within ±1 are particularly preferred, and substitutions between amino acids with hydrophobic indexes within ±0.5 are even more particularly preferred. Furthermore, it is well known in the art that substitutions between similar amino acids can be effectively carried out based on their hydrophilicity.

[0252] As detailed in U.S. Patent 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 specific amino acids can be substituted with other amino acids that have a similar hydrophilicity value, and that such substitutions can yield biologically equivalent proteins, and in particular, immunologically equivalent proteins. When making such changes, substitutions between amino acids with hydrophilicity values ​​within ±2 are preferred, substitutions between amino acids with hydrophilicity values ​​within ±1 are particularly preferred, and substitutions between amino acids with hydrophilicity values ​​within ±0.5 are even more preferred.

[0253] As outlined earlier, amino acid substitutions may be made based on the relative similarity of the substituents on the amino acid side chains, for example, their hydrophobicity, hydrophilicity, charge, size, etc. Furthermore, as separately described herein, gene sequence variants include codon-optimization variants, sequence polymorphisms, splice variants, and / or mutations that do not have a statistically significant effect on the function of the encoded product.

[0254] Variants of proteins, nucleic acids, and gene sequences disclosed herein also include sequences having 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 with the proteins, nucleic acids, or gene sequences disclosed herein.

[0255] "Sequence identity (%)" refers to the relationship between two or more sequences measured by comparing them. In this technology, "identity" also means the degree of association between protein sequences, nucleic acid sequences, or gene sequences, measured by matching between protein sequence chains, nucleic acid sequence chains, or gene sequence chains. "Identity" (often called "similarity") can be easily 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). The methods for measuring identity are designed to obtain the best match between the sequences being tested. Methods for measuring identity and similarity are systematized in publicly available computer programs. Sequence alignment and identity calculations may be performed using the Megalign program (DNASTAR, Madison, Wisconsin), which is included in the LASERGENE suite of bioinformatics computing software.Multiple alignment of sequences can also be performed using the Clustal alignment method (Higgins and Sharp CABIOS, 5, 151-153 (1989), using default parameters (gap penalty = 10, gap length penalty = 10)). Related 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, Madison, Wisconsin); and the FASTA program incorporating the Smith-Waterman algorithm (Pearson, Comput. Methods Genome Res., [Proc. Int. Symp.] (1994), Meeting Date 1992, 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 program's default values. In this specification, "default values" means a set of numerical values ​​or parameters pre-registered in the software during software initialization.

[0256] The 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 incubation overnight at 42°C in a solution containing 50% formamide, 5×SSC (750mM NaCl, 75mM trisodium citrate), 50mM sodium phosphate (pH 7.6), 5× Denhardt's solution, 10% dextran sulfate, and fragment-treated 20 μg / ml denatured salmon sperm DNA, followed by washing the filter at 50°C with 0.1×SSC. Modifications to the stringency of hybridization and signal detection are primarily achieved by adjusting the formamide concentration (lower formamide percentages result in lower stringency), salt conditions, or temperature. For example, moderately high stringency conditions include incubation at 37°C overnight in a solution containing 6×SSPE (20×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 washing at 50°C with 1×SSPE and 0.1% SDS. Further lower stringency can be achieved by washing after stringent hybridization with a higher salt concentration (e.g., 5×SSC). The aforementioned conditions can be varied in various ways by adding and / or substituting other blocking reagents used to reduce the background of the hybridization experiment. Common blocking reagents include Denhardt's reagent, BLOTTO, heparin, denatured salmon sperm DNA, and commercially available proprietary formulations. When adding specific blocking reagents, it may be necessary to modify some of the aforementioned hybridization conditions due to compatibility issues.

[0257] "To bind" means that it does not significantly associate with other molecules or components in the relevant environmental sample, but 10 5 M -1 Equivalent or greater affinity or Ka This refers to the association of a binding domain (e.g., a binding domain of a particular binding domain) with its cognitive binding molecule at an equilibrium association constant of a specific binding interaction, expressed in units of 1 / M. Binding domains may be classified as "high affinity" or "low affinity." In a particular embodiment, a "high affinity" binding domain has 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 K a This refers to a binding domain having . In a particular embodiment, the "low affinity" binding domain is 10 7 M -1 The following 10 6 M -1 The following or 10 5 M -1 The following K a This refers to a binding domain that has [a specific characteristic]. Alternatively, affinity is the equilibrium dissociation constant (K) of a particular binding interaction. d )(Unit: M)(For example, 10 -5 M~10 -13 M) may be defined as follows: In certain embodiments, the binding domain may have “enhanced affinity,” meaning that the selected or recombinant binding domain exhibits stronger binding to its cognitive-binding molecule than the wild-type (or parent) binding domain. For example, enhanced affinity is K to its cognitive-binding molecule. a This may be due to the (equilibrium association constant) being higher than that of the reference binding domain, and the K for the cognitive binding molecule d This may be due to the (dissociation constant) being lower than that of the reference binding domain, and the dissociation rate (K) relative to the cognitive binding molecule. offThis may be due to the binding domain being lower than the reference binding domain. Various assays are known for detecting binding domains that bind to specific cognitive binding molecules and for measuring binding affinity, including Western blotting, ELISA, and BIACORE® analysis (see also, for example, Scatchard, et al., 1949, Ann. NY Acad. Sci. 51:660; and U.S. Patents No. 5,283,173, U.S. Patent No. 5,468,614 or similar publications).

[0258] Unless otherwise stated, this disclosure can be carried out using prior art in immunology, molecular biology, microbiology, cell biology, and recombinant DNA. These methods are described in the following publications: for example, Sambrook, et al. Molecular Cloning: A Laboratory Manual, 2nd Edition (1989); FM 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 RI Freshney, ed. Animal Cell Culture (1987).

[0259] As those skilled in the art will understand, each embodiment disclosed herein includes, substantially consists of, or comprises the specific components, processes, materials, or ingredients described herein. Therefore, the terms “includes” or “contains” should be interpreted as “includes, substantially consists of, or comprises.” The “includes” transition means, but is not limited to, the inclusion of components, processes, materials, or ingredients not described herein, even in large quantities. The “consists of” transition excludes all components, processes, materials, or ingredients not described herein. The “substantially consists of” transition limits the scope of the embodiment to the components, processes, materials, or ingredients described herein, and components, process, materials, or ingredients that do not materially affect the embodiment. A material effect means an effect that statistically significantly reduces the killing of immunocytoplasmic receptor-containing immune cells, the proliferation of immunocytoplasmic receptor-containing immune cells, and / or the production of cytokines by immunocytoplasmic receptor-containing immune cells, as described herein.

[0260] Unless otherwise stated, in this specification and in the claims, all numerical values ​​representing the quantity or properties of materials, such as molecular weight and reaction conditions, are construed in all cases as being modified with the term "approximately." Therefore, unless otherwise stated, the numerical parameters described herein and in the appended claims are approximations that will vary depending on the desired properties to be obtained by the present invention. Without limiting the scope of the claim range and equivalence principle, each numerical parameter should be interpreted at least in light of the reported significant figures and with normal rounding. To be more precise, the term “approximately” when used with a given number or range has a meaning that can be reasonably interpreted by a person skilled in the art, namely, a range of ±20% of the given number; a range of ±19% of the given number; a range of ±18% of the given number; a range of ±17% of the given number; a range of ±16% of the given number; a range of ±15% of the given number; a range of ±14% of the given number; a range of ±13% of the given number; a range of ±12% of the given number; a range of ±11% of the given number; a range of ±10% of the given number; a range of ±9% of the given number; a range of ±8% of the given number; a range of ±7% of the given number; a range of ±6% of the given number; a range of ±5% of the given number; a range of ±4% of the given number; a range of ±3% of the given number; a range of ±2% of the given number; or a range of ±1% of the given number, indicating that the number or range is somewhat greater or less than the given number or range.

[0261] While the numerical ranges and parameters representing the broad scope of this invention are approximations and approximate ranges, the numerical values ​​described in the specific examples are reported as accurately as possible. However, all numerical values ​​inherently contain certain errors that inevitably arise due to the standard deviation associated with each test measurement.

[0262] In the description of this invention (particularly in the description of the following claims), “a,” “an,” “the,” and similar demonstrative pronouns are to be interpreted as encompassing both singular and plural unless otherwise stated or the context explicitly indicates otherwise. The numerical ranges described herein are intended to be a simplified way of referring individually to each numerical value within that range. Unless otherwise stated, each numerical value is described herein as if it were described individually. Unless otherwise stated or the context explicitly indicates otherwise, any method described herein may be carried out in any suitable order. Any use of any examples provided herein, or any language indicating examples (e.g., “etc.”), is for the sole purpose of illustrating the invention in detail and does not limit the scope of the invention as described in the claims. Terms described herein should not be interpreted as referring to non-claimed components essential for carrying out the invention.

[0263] The grouping of other components of the present invention disclosed herein or the grouping of various embodiments of the present invention should not be construed as limiting the present invention. Members of each group may be described individually in this specification or in the claims, or they may be described in this specification or in the claims in combination with other members of the groups described herein or other components. For convenience and / or patentability reasons, it is anticipated that one or more members of one group may be added to another group, or one or more members may be removed from a group. In the event of such additions or deletions, this specification includes groups configured to satisfy the description of all Markush groups described in the appendix claims.

[0264] Specific embodiments of the present invention are described herein, including embodiments that the inventors consider to be the best mode for carrying out the invention. Naturally, those skilled in the art will readily understand, by carefully reading the above detailed description, that the embodiments described herein can be modified in various ways. The inventors anticipate that those skilled in the art may appropriately adopt such modifications, and intend that the present invention may be carried out in ways other than those specifically described herein. Accordingly, the present invention includes, to the extent possible within the scope of applicable law, all modifications from the subject matter of the present invention as described in the appended claims and all equivalents of the subject matter of the present invention. Furthermore, unless otherwise stated or unless the context expresses otherwise, all combinations of the aforementioned components in any modification are also included in the present invention.

[0265] Furthermore, this specification draws reference to various patents, publications, journal articles, and other documents (references herein). Each reference cited herein constitutes part of this specification, and the teachings cited therein are incorporated herein by reference.

[0266] Finally, embodiments of the present invention disclosed herein are to be interpreted as illustrating the principles of the present invention. Other modifications may be adopted within the scope of the present invention. Therefore, as an example, other configurations of the present invention may be used in accordance with the teachings herein, but are not limited thereto. Accordingly, the present invention is not strictly limited to what is expressed and described herein.

[0267] The details described herein are illustrative and intended solely to illustrate preferred embodiments of the present invention, provided to offer what is considered most useful and to facilitate an understanding of the principles and conceptual aspects of various embodiments of the present invention. In this regard, no further details of the structure of the present invention are described beyond what is necessary for a basic understanding of the present invention, and those skilled in the art will be able to easily understand how some forms of the present invention can be actually implemented by carefully reading the description of the present invention with reference to the drawings and / or examples.

[0268] The definitions and descriptions used in this disclosure are intended to control future interpretations, unless they are explicitly and expressly changed 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 does not make sense or substantially meaningless due to the interpretation of the terms, please refer to the definition of the term from a dictionary known to those skilled in the art, such as Webster's Dictionary (3rd edition) or the Oxford Dictionary of Biochemistry and Molecular Biology (Eds. Attwood T et al., Oxford University Press, Oxford, 2006).

Claims

1. It is an artificial expression construct, Inducible promoters including SEQ ID NOs. 17 and 138, Programmed cell death 1 (PD1): Sequences encoding cytokine chimeras and Includes, The PD1:cytokine chimera is an artificial expression construct comprising PD1 linked to a cytokine selected from interferon-alpha (IFNα), interleukin (IL) 21, IL15, N72D mutant IL15 (IL15(N72D)), IL18, decoy-resistant IL18 (drIL18), and IL12.

2. An artificial expression construct comprising a sequence encoding programmed cell death 1 (PD1):cytokine chimera, wherein the PD1:cytokine chimera comprises PD1 linked to a cytokine.

3. The artificial expression construct according to claim 2, wherein the cytokine is an immunostimulatory cytokine.

4. The artificial expression construct according to claim 3, wherein the immunostimulatory cytokine comprises interleukin (IL) 21, IL15, N72D mutant IL15 (IL15(N72D)), IL18, decoy-resistant IL18 (drIL18), IL12, or interferon α (IFNα).

5. The artificial expression construct according to claim 2, wherein the sequence encoding the PD1:cytokine chimera is under the regulatory control of a promoter, and the promoter comprises a sequence having at least 95% sequence identity with any one of sequence numbers 17 to 56, and a minimal promoter.

6. The artificial expression construct according to claim 5, wherein the promoter comprises a sequence having at least 98% sequence identity with any one of sequence numbers 17 to 56, and a minimal promoter.

7. The artificial expression construct according to claim 5, wherein the promoter comprises a sequence having at least 99% sequence identity with any one of sequence numbers 17 to 56, and a minimal promoter.

8. The artificial expression construct according to claim 5, wherein the promoter comprises the sequence shown in any one of sequence numbers 17 to 56 and a minimal promoter.

9. The artificial expression construct according to claim 5, wherein the minimum promoter is the minimum promoter of IL2.

10. The artificial expression construct according to claim 9, wherein the minimum promoter of IL2 includes the sequence shown in SEQ ID NO:

138.

11. The aforementioned PD1: cytokine chimera PD1 (PD1: IL21) linked to interleukin (IL) 21, PD1 connected to IL15 (PD1: IL15), PD1 linked to N72D mutant IL15 (PD1: IL15 (N72D)), PD1 connected to IL18 (PD1: IL18), PD1 linked to decoy-resistant IL18 (PD1: drIL18), PD1 connected to IL12 (PD1: IL12), or PD1 linked to interferon-alpha (PD1: IFNα) The artificial expression construct according to claim 2, comprising:

12. The artificial expression construct according to claim 2, wherein the PD1 is PD1 having the A99L mutation.

13. The artificial expression construct according to claim 2, wherein the PD1 is wild-type PD1.

14. The artificial expression construct according to claim 2, wherein the PD1 is linked to the cytokine via a protein linker.

15. The artificial expression construct according to claim 14, wherein the protein linker is a flexible linker.

16. The artificial expression construct according to claim 15, wherein the flexible linker is a Gly-Ser linker.

17. The artificial expression construct according to claim 16, wherein the Gly-Ser linker comprises the sequence shown in Sequence ID No. 128 or the sequence GGG.

18. The artificial expression construct according to claim 2, wherein the PD1:cytokine chimera is encoded by the sequence shown in SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11, or by a sequence having at least 90% sequence identity with SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO:

11.

19. The artificial expression construct according to claim 2, wherein the PD1:cytokine chimera includes the sequence shown in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16, or includes a sequence having at least 90% sequence identity with the sequence shown in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO:

16.

20. The artificial expression construct according to claim 2, further comprising a control mechanism.

21. The artificial expression construct according to claim 20, wherein the control mechanism encodes a transduction marker.

22. The artificial expression construct according to claim 21, wherein the transduction marker comprises Her2tG.

23. The artificial expression construct according to claim 2, further comprising a first skip sequence.

24. The artificial expression construct according to claim 23, wherein the first skip sequence encodes a 2A autocleaving polypeptide.

25. The artificial expression construct according to claim 24, wherein the 2A self-cleaving polypeptide comprises T2A, P2A, E2A, or F2A.

26. The artificial expression construct according to claim 2, comprising a promoter, a control mechanism, a skip sequence, and a sequence shown in any one of sequence numbers 1 to 11, extending from the 5' end to the 3' end, including the sequence shown in any one of sequence numbers 17 to 56 and the sequence shown in sequence number 138.

27. The artificial expression construct according to claim 2, comprising a promoter including the sequence shown in any one of SEQ ID NOs: 17 to 56 and the sequence shown in SEQ ID NO: 138, a sequence encoding a transduction marker, a skip sequence, and the sequence shown in any one of SEQ ID NOs: 1 to 11, in the direction from the 5' end to the 3' end.

28. The artificial expression construct according to claim 2, having at least 95% sequence identity with the sequence shown in Sequence ID No.

103.

29. The artificial expression construct according to claim 2, having at least 98% sequence identity with the sequence shown in Sequence ID No.

103.

30. The artificial expression construct according to claim 2, having at least 99% sequence identity with the sequence shown in Sequence ID No.

103.

31. The artificial expression construct according to claim 2, having the sequence shown in Sequence ID No.

103.

32. The artificial expression construct according to claim 2, further comprising a sequence encoding a recombinant receptor, wherein the recombinant receptor comprises a binding domain that binds to a target antigen.

33. The artificial expression construct according to claim 32, wherein the sequence encoding the recombinant receptor is operably linked to a second promoter.

34. The artificial expression construct according to claim 33, 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 cancer virus (MMTV) promoter, a human immunodeficiency virus (HIV) long-chain terminal repeat (LTR) promoter, a MoMuLV promoter, a bird leukemia virus promoter, an Epstein-Barr virus very early promoter, a Rous sarcoma virus promoter, an actin promoter, a myosin promoter, a hemoglobin promoter, or a creatine kinase promoter.

35. The artificial expression construct according to claim 32, wherein the binding domain is part of the extracellular portion.

36. The artificial expression construct according to claim 32, wherein the target antigen is expressed on the surface of target cells.

37. The artificial expression construct according to claim 36, wherein the target cells include cells infected with bacteria, viruses, fungi, parasites, or arthropods, or cancer cells.

38. The artificial expression construct according to claim 36, wherein the target cells include cancer cells.

39. The artificial expression construct according to claim 32, further comprising an intracellular portion of the recombinant receptor.

40. The artificial expression construct according to claim 39, wherein the intracellular portion comprises a CD3ζ signaling domain and / or a 4-1BB signaling domain.

41. The artificial expression construct according to claim 39, wherein the intracellular portion is connected to the extracellular portion via a transmembrane domain.

42. The artificial expression construct according to claim 41, wherein the transmembrane domain comprises a transmembrane domain of CD28.

43. The artificial expression construct according to claim 32, further comprising a second control mechanism.

44. The artificial expression construct according to claim 43, wherein the second control mechanism codes a selection cassette.

45. The artificial expression construct according to claim 44, wherein the selection cassette comprises a dihydrofolate reductase double mutant (DHFRdm).

46. The artificial expression construct according to claim 43, further comprising a third control mechanism.

47. The artificial expression construct according to claim 46, wherein a third control mechanism encodes a transduction marker.

48. The artificial expression construct according to claim 47, wherein the transduction marker comprises epidermal growth factor receptor (EGFRt) or cleaved CD19 (tCD19).

49. The artificial expression construct according to claim 32, further comprising a second skip sequence.

50. The artificial expression construct according to claim 49, wherein the second skip sequence encodes a 2A autocleaved polypeptide.

51. The artificial expression construct according to claim 50, wherein the 2A skip self-cleaving polypeptide comprises T2A, P2A, E2A, or F2A.

52. The artificial expression construct according to claim 32, wherein the selection cassette includes a second skip sequence at its 5' end and the transduction marker includes a third skip sequence at its 5' end.

53. The artificial expression construct according to claim 2, comprising a first promoter including the sequence shown in any one of SEQ ID NOs: 17 to 56 and the sequence shown in SEQ ID NO: 138, in the direction from the 5' end to the 3' end; a first regulatory mechanism; a first skip sequence; the sequence shown in any one of SEQ ID NOs: 1 to 11; a second promoter; a sequence encoding a recombinant receptor; a second skip sequence; a second regulatory mechanism; a third skip sequence; and a third regulatory mechanism.

54. The artificial expression construct according to claim 2, comprising, in the direction from the 5' end to the 3' end, a first promoter including the sequence shown in any one of SEQ ID NOs. 17 to 56 and the sequence shown in SEQ ID NO. 138, a sequence encoding a first transduction marker, a first skip sequence, a sequence shown in any one of SEQ ID NOs. 1 to 11, a second promoter, a sequence encoding a recombinant receptor, a second skip sequence, a sequence encoding a selection cassette, a third skip sequence, and a sequence encoding a second transduction marker.

55. The artificial expression construct according to claim 2, having at least 90% sequence identity with the sequence shown in sequence number 98, 99, 100, 101, or 102.

56. The artificial expression construct according to claim 2, having the sequence shown in sequence number 98, 99, 100, 101, or 102.

57. A system for enhancing the function of immune cells, A first artificial expression construct encoding a PD1:cytokine chimera under the regulatory control of the first promoter, A second artificial expression construct containing a sequence encoding a recombinant receptor and Includes, The first promoter comprises a sequence having at least 95% sequence identity with any of sequence numbers 17 to 56 and a minimal promoter. The PD1:cytokine chimera comprises PD1 linked to a cytokine, The recombinant receptor includes a binding domain that binds to an antigen expressed on the surface of a target cell. system.

58. The system according to claim 57, wherein the cytokine is an immunostimulatory cytokine.

59. The system according to claim 58, wherein the immunostimulatory cytokine comprises interleukin (IL) 21, IL15, N72D mutant IL15 (IL15(N72D)), IL18, decoy-resistant IL18 (drIL18), IL12, or interferon α (IFNα).

60. The system according to claim 57, wherein the first promoter comprises a sequence having at least 98% sequence identity with any one of sequence numbers 17 to 56, and a minimal promoter.

61. The system according to claim 57, wherein the first promoter comprises a sequence having at least 99% sequence identity with any one of sequence numbers 17 to 56, and a minimal promoter.

62. The system according to claim 57, wherein the first promoter comprises the sequence shown in any one of sequence numbers 17 to 56 and a minimal promoter.

63. The system according to claim 57, wherein the minimum promoter includes the minimum promoter of IL2.

64. The system according to claim 63, wherein the minimum promoter of IL2 includes the sequence shown in sequence number 138.

65. The aforementioned PD1: cytokine chimera PD1 (PD1: IL21) linked to interleukin (IL) 21, PD1 connected to IL15 (PD1: IL15), PD1 linked to N72D mutant IL15 (PD1: IL15 (N72D)), PD1 connected to IL18 (PD1: IL18), PD1 linked to decoy-resistant IL18 (PD1: drIL18), PD1 connected to IL12 (PD1: IL12), or PD1 linked to interferon-alpha (PD1: IFNα) The system according to claim 57, including the system described in claim 57.

66. The system according to claim 57, wherein the PD1 is a PD1 having the A99L mutation.

67. The system according to claim 57, wherein the PD1 is wild-type PD1.

68. The system according to claim 57, wherein the PD1 is linked to the cytokine via a protein linker.

69. The system according to claim 68, wherein the protein linker is a flexible linker.

70. The system according to claim 69, wherein the flexible linker is a Gly-Ser linker.

71. The system according to claim 70, wherein the Gly-Ser linker comprises the sequence shown in Sequence ID No. 128 or the sequence GGG.

72. The system according to claim 57, wherein the PD1:cytokine chimera is encoded by the sequence shown in SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11, or by a sequence having at least 90% sequence identity with SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO:

11.

73. The system according to claim 57, wherein the PD1:cytokine chimera includes the sequence shown in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16, or includes a sequence having at least 90% sequence identity with the sequence shown in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO:

16.

74. The system according to claim 57, wherein the first artificial expression construct further includes a control mechanism.

75. The system according to claim 74, wherein the control mechanism codes for a trait introduction marker.

76. The system according to claim 75, wherein the transduction marker comprises Her2tG.

77. The system according to claim 57, wherein the first artificial expression construct further comprises a first skip sequence.

78. The system according to claim 77, wherein the first skip sequence encodes a 2A self-cleaving polypeptide.

79. The system according to claim 78, wherein the 2A self-cleaving polypeptide comprises T2A, P2A, E2A, or F2A.

80. The system according to claim 57, wherein the sequence encoding the recombinant receptor is operably linked to a second promoter.

81. The system according to claim 57, wherein the second promoter includes 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, a bird leukemia virus promoter, an Epstein-Barr virus very early promoter, a Roussarcoma virus promoter, an actin promoter, a myosin promoter, a hemoglobin promoter, or a creatine kinase promoter.

82. The system according to claim 57, wherein the second artificial expression construct further includes a second control mechanism.

83. The system according to claim 82, wherein the second control mechanism codes the selected cassette.

84. The system according to claim 83, wherein the selection cassette comprises a dihydrofolate reductase double mutant (DHFRdm).

85. The system according to claim 57, wherein the second artificial expression construct further includes a third control mechanism.

86. The system according to claim 85, wherein a third control mechanism codes for a transduction marker.

87. The system according to claim 86, wherein the transduction marker comprises cleaved epidermal growth factor receptor (EGFRt) or tCD19.

88. The system according to claim 57, wherein the second artificial expression construct further comprises a second skip sequence.

89. The system according to claim 88, wherein the second skip sequence encodes a 2A self-cleaving polypeptide.

90. The system according to claim 89, wherein the 2A self-cleaving polypeptide comprises T2A, P2A, E2A, or F2A.

91. The system according to claim 57, wherein the second artificial expression construct includes 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.

92. The system according to claim 57, wherein the binding domain is part of the extracellular portion.

93. The system according to claim 57, wherein the target cells include cells infected with bacteria, viruses, fungi, parasites, or arthropods, or cancer cells.

94. The system according to claim 57, wherein the target cells include cancer cells.

95. The system according to claim 57, wherein the recombinant receptor further comprises an intracellular portion.

96. The system according to claim 95, wherein the intracellular portion includes a CD3ζ signaling domain and / or a 4-1BB signaling domain.

97. The system according to claim 95, wherein the intracellular portion is connected to the extracellular portion via a transmembrane domain.

98. The system according to claim 97, wherein the transmembrane domain includes a transmembrane domain of CD28.

99. The system according to claim 57, wherein the first artificial expression construct and the second artificial expression construct reside on a single artificial expression construct.

100. The system according to claim 57, wherein the first artificial expression construct and the second artificial expression construct reside on separate artificial expression constructs.

101. (i) The first artificial expression construct, (ii) The second artificial expression construct and Includes, The first artificial expression construct includes a first promoter containing the sequence shown in any one of SEQ ID NOs. 17-56 and the sequence shown in SEQ ID NO. 138, a first regulatory mechanism, a first skip sequence, and the sequence shown in any one of SEQ ID NOs. 1-11, in the direction from the 5' end to the 3' end. The second artificial expression construct includes, from the 5' end to the 3' end, a second promoter, a sequence encoding a recombinant receptor, a second skip sequence, a second regulatory mechanism, a third skip sequence, and a third regulatory mechanism. The system according to claim 57.

102. Nanoparticles encapsulating the artificial expression construct described in claim 2 or the system described in claim 57.

103. Cells genetically modified to express the artificial expression construct described in claim 2 or the system described in claim 57.

104. The cell according to claim 103, which is an autologous cell obtained from the subject or a cell of the same species as the subject.

105. The cell according to claim 103, which is an in vivo cell or an ex vivo cell.

106. The cell according to claim 103, which is an immune cell.

107. The cell according to claim 106, wherein the immune cell is a lymphocyte.

108. The cells according to claim 107, wherein the lymphocytes include T cells, B cells, natural killer (NK) cells, or NK-T cells.

109. The cell according to claim 103, 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.

110. The cell according to claim 103, which is a CD8+ T cell.

111. The cell according to claim 103, which is a CD4+ T cell.

112. A population of cells genetically modified to express the artificial expression construct described in claim 2 or the system described in claim 57.

113. A population of cells according to claim 112, comprising autologous cells obtained from a subject or cells of the same species as the subject.

114. The cell population according to claim 112, which is an in vivo cell population or an ex vivo cell population.

115. The population of cells according to claim 112, wherein the cells are immune cells.

116. The group of cells according to claim 115, wherein the immune cells are lymphocytes.

117. The population of cells according to claim 116, wherein the lymphocytes include T cells, B cells, natural killer (NK) cells, or NK-T cells.

118. The cell population according to claim 112, comprising CD4+ T cells and / or CD8+ T cells.

119. A formulation comprising (i) cells genetically modified to express the artificial expression construct described in claim 2 or the system described in claim 57, and (ii) a pharmaceutically acceptable carrier.

120. A method for genetically modifying immune cells to have enhanced function, A method comprising the step of contacting immune cells with an artificial expression construct according to claim 2, which has a sequence encoding a cytokine chimera (PD1).

121. The method according to claim 120, wherein the enhanced function includes enhanced cell-killing ability.

122. The aforementioned coded PD1:cytokine chimera PD1 (PD1: IL21) linked to interleukin (IL) 21, PD1 connected to IL15 (PD1: IL15), PD1 linked to N72D mutant IL15 (PD1: IL15 (N72D)), PD1 connected to IL18 (PD1: IL18), PD1 linked to decoy-resistant IL18 (PD1: drIL18), PD1 connected to IL12 (PD1: IL12), or PD1 linked to interferon-alpha (PD1: IFNα) The method according to claim 121, including the method according to claim 121.

123. The method according to claim 122, wherein the PD1 is a PD1 having the A99L mutation.

124. The method according to claim 122, wherein the PD1 is wild-type PD1.

125. The method according to claim 122, wherein the coded PD1:cytokine chimera comprises PD1:IFNα.

126. The method according to claim 120, wherein the enhanced function includes enhanced growth.

127. The aforementioned coded PD1:cytokine chimera PD1 (PD1: IL21) linked to interleukin (IL) 21, PD1 connected to IL15 (PD1: IL15), PD1 linked to N72D mutant IL15 (PD1: IL15 (N72D)), PD1 connected to IL18 (PD1: IL18), PD1 linked to decoy-resistant IL18 (PD1: drIL18), PD1 connected to IL12 (PD1: IL12), or PD1 linked to interferon-alpha (PD1: IFNα) The method according to claim 126, including the method described in claim 126.

128. The method according to claim 127, wherein the coded PD1:cytokine chimera comprises PD1:IFNα.

129. The method according to claim 120, wherein the enhanced function includes enhanced cytokine production.

130. The method according to claim 129, wherein the enhanced cytokine production includes IFNγ.

131. The aforementioned coded PD1:cytokine chimera PD1 (PD1: IL21) linked to interleukin (IL) 21, PD1 connected to IL15 (PD1: IL15), PD1 linked to N72D mutant IL15 (PD1: IL15 (N72D)), PD1 connected to IL18 (PD1: IL18), PD1 linked to decoy-resistant IL18 (PD1: drIL18), PD1 connected to IL12 (PD1: IL12), or PD1 linked to interferon-alpha (PD1: IFNα) The method according to claim 130, including the method described in claim 130.

132. The method according to claim 131, wherein the PD1 is a PD1 having the A99L mutation.

133. The method according to claim 131, wherein the PD1 is wild-type PD1.

134. The method according to claim 131, wherein the coded PD1:cytokine chimera comprises PD1:IFNα.

135. The method according to claim 129, wherein the enhanced cytokine production includes the production of TNFα.

136. The aforementioned coded PD1:cytokine chimera PD1 (PD1: IL21) linked to interleukin (IL) 21, PD1 connected to IL15 (PD1: IL15), PD1 linked to N72D mutant IL15 (PD1: IL15 (N72D)), PD1 connected to IL18 (PD1: IL18), PD1 linked to decoy-resistant IL18 (PD1: drIL18), PD1 connected to IL12 (PD1: IL12), or PD1 linked to interferon-alpha (PD1: IFNα) The method according to claim 135, including the method described in claim 135.

137. The method according to claim 136, wherein the PD1 is a PD1 having the A99L mutation.

138. The method according to claim 136, wherein the PD1 is wild-type PD1.

139. The method according to claim 136, wherein the coded PD1:cytokine chimera comprises PD1:IFNα.

140. The method according to claim 120, wherein the artificial expression construct further comprises a sequence encoding a recombinant receptor having a binding domain that binds to an antigen expressed on the surface of a target cell.

141. The method according to claim 140, wherein the sequence encoding the recombinant receptor is operably linked to a second promoter.

142. The method according to claim 141, 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 cancer virus (MMTV) promoter, a human immunodeficiency virus (HIV) long-chain terminal repeat (LTR) promoter, a MoMuLV promoter, a bird leukemia virus promoter, an Epstein-Barr virus very early promoter, a Rous sarcoma virus promoter, an actin promoter, a myosin promoter, a hemoglobin promoter, or a creatine kinase promoter.

143. The method according to claim 120, further comprising the step of contacting the immune cells with an artificial expression construct comprising a sequence encoding a recombinant receptor including a binding domain that binds to an antigen expressed on the surface of a target cell.

144. The method according to claim 143, wherein the steps of contacting the immune cells with the artificial expression construct according to claim 2 and contacting the immune cells with the artificial expression construct containing the sequence encoding the recombinant receptor are performed simultaneously.

145. The method according to claim 143, wherein the steps of contacting the immune cells with the artificial expression construct according to claim 2 and contacting the immune cells with the artificial expression construct containing the sequence encoding the recombinant receptor are performed at different time points.

146. The method according to claim 120, wherein the artificial expression construct includes the sequence shown in SEQ ID NO: 98, 99, 100, 101, or 102, or includes a sequence having 90% sequence identity with the sequence shown in SEQ ID NO: 98, 99, 100, 101, or 102.

147. A method of treating a subject that requires treatment, A method comprising the step of treating a subject in need of treatment by administering a therapeutically effective amount of the artificial expression construct according to claim 2, the system according to claim 57, the nanoparticles according to claim 102, or the formulation according to claim 119 to the subject.

148. The method according to claim 147, wherein the subject requiring the treatment has cancer or an infectious disease.

149. The method according to claim 147, wherein the administration of the therapeutically effective amount includes administration by intravesical, intradermal, intraarterial, parenteral, intralymph node, intralymphatic, intraperitoneal, intrafocal, intraprostatic, vaginal, intrarectal, topical, intrathecal, intratumoral, intramuscular, or subcutaneous.

150. A recombinant protein containing programmed cell death 1 (PD1) and cytokines.

151. The recombinant protein according to claim 150, wherein the cytokine is an immunostimulatory cytokine.

152. The recombinant protein according to claim 151, wherein the immunostimulatory cytokine comprises interleukin (IL) 21, IL15, N72D mutant IL15 (IL15(N72D)), IL18, decoy-resistant IL18 (drIL18), IL12, or interferon α (IFNα).

153. PD1 (PD1: IL21) linked to interleukin (IL) 21, PD1 connected to IL15 (PD1: IL15), PD1 linked to N72D mutant IL15 (PD1: IL15 (N72D)), PD1 connected to IL18 (PD1: IL18), PD1 linked to decoy-resistant IL18 (PD1: drIL18), PD1 connected to IL12 (PD1: IL12), PD1 linked to interferon-alpha (PD1: IFNα), IL12-linked A99L mutation PD1 (PD1(A99L)) (PD1(A99L): IL21), PD1(A99L) connected to IL15 (PD1(A99L): IL15), PD1(A99L) linked to the N72D mutation IL15 (PD1(A99L):IL15(N72D)), PD1(A99L) connected to IL18 (PD1(A99L): IL18), PD1(A99L):drIL18 linked to decoy-resistant IL18, PD1(A99L) connected to IL12 (PD1(A99L): IL12), or PD1(A99L) linked to interferon-alpha (PD1(A99L): IFNα) The recombinant protein according to claim 150, comprising:

154. The recombinant protein according to claim 150, wherein the PD1 is linked to the cytokine via a protein linker.

155. The recombinant protein according to claim 154, wherein the protein linker is a flexible linker.

156. The recombinant protein according to claim 155, wherein the flexible linker is a Gly-Ser linker.

157. The recombinant protein according to claim 156, wherein the Gly-Ser linker comprises the sequence shown in Sequence ID No. 128 or the sequence GGG.

158. The recombinant protein according to claim 150, which is encoded by the sequence shown in SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11, or by a sequence having at least 90% sequence identity with SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO:

11.

159. The recombinant protein according to claim 150, comprising the sequence shown in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16, or comprising a sequence having at least 90% sequence identity with the sequence shown in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16.

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