Cell, method for expressing protein using cell, expression construct, and transformed cell

Abstract

To provide cells capable of converting signal transduction pathways derived from interactions between protein molecules, detecting signals with enhanced sensitivity, and converting the detected signals into other signals, a method for expressing protein using cells, an expression construct, and a transformed cell.SOLUTION: The foregoing problem is solved by a cell comprising, in the cell membrane: a first fusion membrane protein molecule comprising at least a part of the intracellular domain of interferon α / β receptor 1, a first transmembrane domain, and a first extracellular domain; and a second fusion membrane protein molecule comprising at least a part of the intracellular domain of interferon α / β receptor 2, a second transmembrane domain, and a second extracellular domain, where the first extracellular domain and the second extracellular domain are capable of binding to each other directly or indirectly.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a cell, a method for expressing a protein using the cell, an expression construct, and a transformed cell. [Background technology]

[0002] Various extracellular and intracellular signal transduction is mediated by specific interactions between two or more molecules. NanoLuc (registered trademark, sometimes written as Nluc), a type of luciferase, is widely used as a reporter for various biological signals because of its small molecular weight, strong and stable enzymatic activity (Non-Patent Document 1).

[0003] In order to detect protein-protein interactions, NanoBiT, a complementation reporter using a two-subunit system consisting of a large subunit called LgBiT and a small subunit called SmBiT, has been developed (Non-Patent Document 2). A subunit that can be fused to a target protein is bound to LgBiT or SmBiT. When these subunits bind to a target protein, LgBiT and SmBiT come into close proximity to form a functional enzyme that gives a luminescent signal. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Hall MP, et al., Engineered luciferase reporter from a deep sea shrimp utilizing a novel imidazopyrazinone substrate, ACS Chem Biol. 7, 1848 (2012) [Non-Patent Document 2] Dixon AS, et al., NanoLuc Complementation Reporter Optimized for Accurate Measurement of Protein Interactions in Cells, ACS Chem Biol. 11, 400 (2016) Summary of the Invention [Problem to be solved by the invention]

[0005] When detecting protein-protein interactions using the method described in Non-Patent Document 2, the luminescence signal may be insufficient depending on the type and amount of the target protein.

[0006] The present inventors have come up with the idea of ​​using the intracellular domains of interferon α and β receptors to convert the signal to the JAK-STAT signaling pathway and enhance the signal when detecting protein-protein interactions. The present inventors have also come up with the idea of ​​expressing not only proteins involved in signal detection but also other proteins such as growth factors to control the functions of co-cultured cells.

[0007] Specifically, the present invention provides cells in which the expression level of a protein involved in signal detection is improved, a method for expressing a protein using the cells, an expression construct, and a transformed cell. [Means for solving the problem]

[0008] The present invention includes the following aspects. [1] A first fusion membrane protein molecule comprising at least a portion of the intracellular domain of interferon α and β receptor 1, a first transmembrane domain, and a first extracellular domain; A cell comprising in its cell membrane a second fusion membrane protein molecule comprising at least a portion of the intracellular domain of interferon alpha and beta receptor 2, a second transmembrane domain, and a second extracellular domain, A cell, wherein the first extracellular domain and the second extracellular domain are capable of binding directly or indirectly. [2] The cell described in [1], wherein the first transmembrane domain has an amino acid sequence derived from the transmembrane domain of the interferon alpha and beta receptor 1. [3] The cell described in [2], wherein the first fusion membrane protein molecule further contains a portion of the extracellular domain of interferon alpha and beta receptor 1, and the portion of the extracellular domain is located between the first extracellular domain and the first transmembrane domain. [4] The cell described in any one of [1] to [3], wherein the first extracellular domain and the second extracellular domain are capable of binding to a target substance via a target substance sandwiched between them. [5] The cell described in [4], wherein the target substance is a complex protein to which two or more proteins are bound. [6] The cell described in [4], wherein the target substance comprises a cytokine, an antibody or an affibody. [7] The cells described in [6], wherein the cytokine is at least one of interferon-gamma, interleukin-1, interleukin-2, interleukin-4, interleukin-6, interleukin-10, interleukin-12, interleukin-17, interleukin-18, tumor necrosis factor-alpha, and transforming growth factor-beta. [8] The cell of claim 1, in which endogenous interferon alpha and beta receptor 1 are knocked out. [9] The cell according to any one of [1] to [8], wherein the cell is a HEK293T cell (a cell derived from a human fetal kidney cell), a HEK293 cell (a cell derived from a human fetal kidney cell), a HeLa cell (a cell derived from a human cervical cancer), an A549 cell (a cell derived from a human lung cancer), an MCF-7 cell (a cell derived from a human breast cancer), or an A431 cell (a cell derived from a human epithelial cell carcinoma).

[10] The cell according to any one of [1] to [9], further comprising an interferon response element (ISRE) and an ISRE-inducible expression unit comprising at least one nucleotide sequence encoding a protein.

[11] A cell described in

[10] , in which multiple ISRE-inducible expression units are introduced, and the nucleotide sequences encoding the proteins contained in the multiple ISRE-inducible expression units are different from each other.

[12] The cell described in

[10] or

[11] , wherein the ISRE-inducible expression unit contains four or more of the interferon response elements.

[13] The cell described in any one of

[10] to

[12] , wherein the nucleotide sequence encodes a protein involved in a signal-emitting reaction.

[14] The cell according to

[13] , wherein the protein involved in the reaction that emits the signal is one of luciferase, green fluorescent protein, red fluorescent protein, phosphatase and peroxidase.

[15] The cell described in any one of

[10] to

[12] , wherein the nucleotide sequence encodes at least one protein selected from molecules that control cell proliferation, activation, and differentiation and molecules that control cell-cell interactions.

[16] The cell described in

[15] , wherein the molecule that controls the proliferation is interleukin-2.

[17] The cell described in

[15] , wherein the molecule that controls the differentiation of the cell is one of interferon-gamma, interleukin-4, interleukin-6, interleukin-10, interleukin-12, interleukin-17, interleukin-23, tumor necrosis factor-alpha, and transforming growth factor-beta.

[18] The cell described in

[15] , wherein the molecule that controls the activation of the cell is at least one of costimulatory molecules that promote activation, such as B7-1, B7-2, CD40, and 4-1BB ligand, and costimulatory molecules that suppress activation, such as CTLA4, PD-L1, and galectin-9.

[19] The cell described in

[15] , wherein the molecule regulating intercellular interaction is a cell membrane-bound anti-CD4-scFv-TM, anti-CD4-CCR6-scFv-TM, or ZZ-affibody-TM.

[20] The first extracellular domain and the second extracellular domain are capable of binding to a target substance via a target substance sandwiched therebetween; The cell according to any one of

[10] to

[19] , wherein the target substance comprises a protein having the same structure as the protein.

[21] Using the cell according to any one of [1] to

[20] , directly or indirectly binding the first extracellular domain to the second extracellular domain in the cell; and bringing at least a portion of the intracellular domain of the interferon α and β receptor 1 and at least a portion of the intracellular domain of the interferon α and β receptor 2 into close proximity through the binding, thereby causing a nucleotide sequence encoding a downstream protein to be transcribed via an interferon response element present in the cell, thereby expressing the protein.

[22] A first expression construct comprising: a first promoter for expression functional in a cell; and a nucleotide sequence operably linked to the first promoter, the nucleotide sequence encoding a first fusion membrane protein molecule comprising at least a portion of an intracellular domain of interferon alpha and beta receptor 1, a first transmembrane domain, and a first extracellular domain; An expression construct, wherein the first extracellular domain is capable of binding to a target substance other than interferon α and β.

[23] The expression construct described in

[22] , wherein the nucleotide sequence encoding the first transmembrane domain is a nucleotide sequence encoding the transmembrane domain of the interferon alpha and beta receptor 1.

[24] The expression construct described in

[23] , wherein the first fusion membrane protein molecule further comprises a portion of the extracellular domain of interferon alpha and beta receptor 1, and a nucleotide sequence encoding the portion of the extracellular domain is located between the nucleotide sequence encoding the first extracellular domain and the nucleotide sequence encoding the first transmembrane domain.

[25] further comprising a second expression construct comprising a second promoter for expression functional in a cell, and a nucleotide sequence operably linked to the second promoter, the nucleotide sequence encoding a second fusion membrane protein molecule comprising at least a portion of the intracellular domain of interferon alpha and beta receptor 2, a second transmembrane domain, and a second extracellular domain; The expression construct according to any one of

[22] to

[24] , wherein the first extracellular domain and the second extracellular domain are capable of binding directly or indirectly to each other.

[26] The expression construct described in

[25] , wherein the first extracellular domain and the second extracellular domain are capable of binding to the target substance via the target substance sandwiched between them.

[27] The expression construct according to

[26] , wherein the target substance is a complex protein in which two or more proteins are bound.

[28] The expression construct described in

[26] , wherein the target substance comprises a cytokine, an antibody or an affibody.

[29] The expression construct according to

[28] , wherein the cytokine is at least one of interferon-gamma, interleukin-1, interleukin-2, interleukin-4, interleukin-6, interleukin-10, interleukin-12, interleukin-17, interleukin-18, tumor necrosis factor-alpha, and transforming growth factor-beta.

[30] The expression construct according to any one of

[22] to

[29] , wherein the expression construct is a plasmid vector-based expression construct.

[31] A transformed cell into which the expression construct according to any one of

[22] to

[30] has been introduced. Effect of the Invention

[0009] According to the above aspects, it is possible to provide a cell that can convert the transmission pathway of a signal brought about by an intermolecular protein interaction, detect the signal with higher sensitivity, and further convert it into another signal, a method for expressing a protein using the cell, an expression construct, and a transformed cell. [Brief description of the drawings]

[0010] [Figure 1] FIG. 1 is a schematic diagram of a cell according to one embodiment. [Diagram 2] FIG. 1 is a schematic diagram of a cell according to one embodiment. [Diagram 3] FIG. 1 is a schematic diagram (IFNARRS method) showing signal transduction via IFNAR1 and IFNAR2 by specific binding of HER2-EX (extracellular domain) to anti-HER2 affibody. [Figure 4] FIG. 1 shows fusion membrane proteins used in the Examples (IFNARRS method). [Diagram 5] 1 is a graph showing luciferase activity according to the combination of fusion membrane proteins used in an example (IFNARRS method). [Figure 6] 1 is a graph showing luciferase activity dependent on the specific binding of HER2-EX and anti-HER2 affibody by the combination of fusion membrane proteins used in the Examples (IFNARRS method). [Figure 7] FIG. 1 shows expression vectors according to the number of ISREs in an example (IFNARRS method) (A), and a graph of luciferase activity using the same (B). [Figure 8] FIG. 2 is a schematic diagram showing luciferase activity due to the proximity of SmBiT and LgBiT caused by specific binding between HER2-EX and an anti-HER2 affibody, as a comparative example (NanoBiT method). [Figure 9] FIG. 1 shows a fusion membrane protein used as a comparative example (NanoBiT method). [Figure 10] 1 is a graph showing luciferase activity by combination of fusion membrane proteins used as a comparative example (NanoBiT method). [Figure 11] 1 is a graph showing the luciferase activity of a combination of fusion membrane proteins used as an example (IFNARRS method) and a comparative example (NanoBiT method). [Figure 12]Schematic diagram showing signaling through IFNAR1 and IFNAR2 upon binding of the extracellular domain of PD-L1, a ZZ-affibody (which binds to the Fc region of an antibody), and an anti-PD-L1 antibody. [Figure 13] FIG. 1 shows fusion membrane proteins used in the examples. [Figure 14] Figure 1 is a graph showing luciferase activity dependent on specific binding of anti-PD-L1 antibodies to PD-L1. [Figure 15] (A) shows the binding of PD1 to PD-L1 using PD1-EX and anti-HER2 affibody fusion protein via a short linker peptide, and (B) shows that the inhibition of PD1 to PD-L1 binding by anti-PD-L1 antibody is specific. [Figure 16] Figure 1 shows a graph showing the inhibition of PD1-PD-L1 binding by anti-PD-L1 antibody or anti-GST antibody (negative control) as measured by luciferase activity. [Figure 17] FIG. 1 shows fusion membrane proteins used in the Examples to examine whether signals are transmitted only by the intracellular domains of IFNAR2 and IFNAR1. [Figure 18] FIG. 1 is a graph showing that luciferase activity signals exclusively through the intracellular domains of IFNAR2 and IFNAR1. [Figure 19] FIG. 1 is a schematic diagram showing a comparison between the NanoBiT method and the IFNARRS method using an anti-HER2 affibody dimer as a target substance. [Figure 20] FIG. 1 shows fusion membrane proteins used in the Examples (IFNARRS method). [Figure 21] FIG. 1 shows a fusion membrane protein used as a comparative example (NanoBiT method). [Figure 22] 1 is a graph showing luciferase activity when an anti-HER2 affibody dimer was used as a target substance in a comparative example (NanoBiT method). [Figure 23] The luciferase activity is shown by comparing the NanoBiT method and the IFNARRS method using an anti-HER2 affibody dimer as a target substance. [Figure 24] FIG. 1 is a schematic diagram comparing the detection of IFNGR1 dimerization by IFNγ using the NanoBiT method (A) and the IFNARRS method (B) with IFNγ as the target substance. [Diagram 25] FIG. 1 shows fusion membrane proteins used in the Examples (IFNARRS method). [Figure 26] FIG. 1 shows a fusion membrane protein used as a comparative example (NanoBiT method). [Figure 27] This is a graph showing that in the fusion membrane proteins used as comparative examples (NanoBiT method), IFNγ-dependent IFNGR1 dimerization could not be detected in any combination. [Figure 28] 1 is a graph showing luciferase activity comparing IFNγ-dependent IFNGR1 dimerization using the NanoBiT method and the IFNARRS method. [Figure 29] FIG. 1 is a schematic diagram illustrating the use of the IFNARRS method to detect, activate, and promote proliferation of activated T cells that secrete IFNγ. [Diagram 30] FIG. 1 is a schematic diagram of the IFNARRS method using the extracellular domains of IFNGR1 and IFNGR2. [Diagram 31] FIG. 1 shows fusion membrane proteins used in the examples. [Diagram 32] 1 is a graph showing IFNγ-dependent luciferase activity by combination of fusion membrane proteins used in the examples. [Diagram 33] FIG. 1 shows the fusion membrane proteins used in the examples and the IFNγ-dose-dependent increase in sensitivity due to ISRE×7-IFNG. [Diagram 34] 1 is a graph showing IFNγ-dependent luciferase activity of cells (multiple stable expression lines obtained by transfection and drug selection) used in the examples. [Diagram 35] FIG. 1 shows the structure of the sgRNA-expressing donor plasmid used in the Examples and an overview of the genomic structural changes associated with knockout of endogenous IFNAR1 by homologous recombination. [Diagram 36]FIG. 1 shows luciferase activity in each cell line of the Examples upon stimulation with IFNα or IFNγ. [Figure 37] FIG. 1 shows changes in luciferase activity by stimulation with IFNα or IFNγ before and after knockout of endogenous IFNAR1 according to an example. [Figure 38] FIG. 1 is a schematic diagram showing signal transduction via IFNAR1 and IFNAR2 when IL-2 is used as a target substance. [Figure 39] FIG. 1 shows fusion membrane proteins used in the examples. [Diagram 40] FIG. 1 is a graph showing luciferase activity by IL-2 stimulation in each combination of fusion membrane protein molecules used in the Examples (top), and increased IL-2 sensitivity and activation by coexpression of IL2RA subunits (bottom). [Diagram 41] FIG. 1 shows luciferase activity in response to IL-2 in the cell lines used in the Examples. [Diagram 42] FIG. 1 shows luciferase activity for each cytokine in the cell lines used in the Examples. [Diagram 43] This is a diagram showing the ISRE-IL-2 expression vector used in the Examples and the detection of IL-2 secreted by GRkS-974 cells in response to IFNγ stimulation in each cell line by 2LRk-134 cells. [Diagram 44] FIG. 1 shows the secretion of IL-2 in GRkS-974 cells used in the Examples upon stimulation with each cytokine. [Diagram 45] 13 is a microscopic image showing the proliferation of CTLL-2 cells when co-cultured with GRkS-974 cells. [Diagram 46] This is a graph showing the number of CTLL-2 cells when co-cultured with GRkS-974 cells. [Figure 47]The figure shows the vector constructs used to generate lentiviruses for expression in Jurkat cells: NY-ESO-1 peptide-specific TCR (T cell receptor) complex, CD8α-EGFP, NFAT-RE (Nuclear Factor of Activated T Cells-Response Element)-NLucP, and anti-NY-ESO-1-scFv-28BBZ (CD28-4-1BB-CD3ζ). [Figure 48] FIG. 1 shows luciferase activity upon stimulation with PMA and calcium ionophore or anti-CD3ε antibody, or upon administration of NY-ESO-1 peptide, in co-culture of GRkS-974 cells with Jurkat-CD8 / anti-ESO-TCR cells. [Figure 49] FIG. 1 is a schematic diagram illustrating the application of the IFNARRS method to detection of activated T cells secreting IFNγ (NLucP), signal enhancement (IFNγ) and cell proliferation stimulation (IL-2). [Figure 50] FIG. 13 shows luciferase activity after stimulation with anti-CD3ε antibody in coculture of GRkS-974 cells and Jurkat-CD8 / anti-ESO-TCR cells, and after forced expression of B7-1 or anti-CD3ε-scFv by transfection. [Figure 51] FIG. 1 shows luciferase activity upon administration of NY-ESO-1 peptide in a co-culture of GRkS-974 cells and Jurkat-CD8 / anti-ESO-TCR cells. [Figure 52] FIG. 1 shows the expression vectors used in the examples. [Figure 53] FIG. 1 shows a part of the expression vector used in the Examples and luciferase activity due to IFNγ-dependent induction of expression of B7-1 and anti-CD3ε-scFv. [Figure 54] FIG. 1 shows a comparison of firefly luciferase activity between ISRE-dependent and GAS (gamma interferon activation site)-dependent promoters. [Figure 55]FIG. 1 shows activation of firefly luciferase by IFNα stimulation of 293T cells stably expressing RIG-I and ISRE-firefly luciferase. [Figure 56] FIG. 1 shows luciferase activity in GRkS-974 cells in response to various cytokines. [Figure 57] As a comparative experiment, the image shows a plate in which activation of a given number of Jurkat-CD8 / anti-ESO-TCR cells treated with PMA / Ca was detected by ELISA assay. [Figure 58] As a comparative experiment, the figure shows the results (expressed as IFNγ concentration) of statistical processing of absorbance detected by ELISA assay for activation of a given number of Jurkat-CD8 / anti-ESO-TCR cells treated with PMA / Ca. [Figure 59] As a comparative experiment, the activation of a given number of Jurkat-CD8 / anti-ESO-TCR cells treated with PMA / Ca was detected by IFNARRS using GRkS-974 cells. [Figure 60] As a comparative experiment, the images show activation of a given number of Jurkat-CD8 / anti-ESO-TCR cells treated with PMA / Ca or anti-CD3ε antibody detected by ELISpot. [Figure 61] As a comparative experiment, the figure shows the results of statistical processing of the number of spots detected by ELISpot for activation of a given number of Jurkat-CD8 / anti-ESO-TCR cells treated with PMA / Ca or anti-CD3ε antibody. [Figure 62] As a comparative experiment, the activation of a certain number of Jurkat-CD8 / anti-ESO-TCR cells treated with PMA / Ca or anti-CD3ε antibody was detected by IFNARRS using GRkS-974 cells. [Figure 63] 13 is an image showing the results of a dot blot assay using anti-B7-1 antibody for GRkS-974 cells, GRkSB-043 cells, and GRkSBZ-188 cells. [Figure 64]RT-PCR images showing the expression of anti-CD3 ε-scFv in GRkS-974, GRkSB-043 and GRkSBZ-188 cells. [Figure 65] FIG. 1 shows specific binding of IGHG1 to surface ZZ-TM of GRkSB-043 cells. [Figure 66] FIG. 1 shows specific binding of anti-HER2-scFv-IGHG1 to surface ZZ-TM of GRkSB-043 cells. [Figure 67] This figure shows the construct for expressing ZZ-TM on the surface of GRkSB-043 cells, and the constructs of each molecule used to confirm the functional expression of this ZZ (binding to the Fc region of an antibody). [Figure 68] FIG. 1 shows luciferase activity when Jurkat-NFAT-Nluc cells were stimulated with PMA / Ca, anti-CD3ε antibody, or the like. [Figure 69] FIG. 13 shows luciferase activity when Jurkat-NFAT-Nluc / anti-ESO-28BBZ cells were cocultured with HEK293T cells to which NY-ESO-1 peptide had been added. [Figure 70] FIG. 1 shows luciferase activity when GRkS-974 cells, GRkSB-043 cells, and GRkSBZ-188 cells were cocultured with Jurkat cells and stimulated with IFNγ. [Figure 71] FIG. 1 shows luciferase activity when GRkS-974 cells, GRkSB-043 cells, and GRkSBZ-188 cells were cocultured with Jurkat cells or Jurkat-NFAT-NLuc cells and stimulated with IFNγ. [Figure 72] FIG. 1 shows luciferase activity when GRkS-974 cells, GRkSB-043 cells, and GRkSBZ-188 cells were cocultured with Jurkat-NFAT-NLuc cells in the presence or absence of an anti-CD3ε antibody. [Figure 73] FIG. 1 shows that GRkSBZ-188 cells have dendritic cell-like effects in response to IFNγ. [Figure 74]The figure shows luciferase activity in GRk-281 cells co-cultured with Jurkat-NFAT-Nluc / anti-ESO-28BBZ cells transfected with NY-ESO-1 cDNA and administered with or without NY-ESO-1 peptide. The lower panel shows that endogenous IFNAR1 was knocked out. [Figure 75] Luciferase activity in GRkh-117 cells co-cultured with Jurkat-NFAT-Nluc / anti-ESO-28BBZ cells transfected with NY-ESO-1 cDNA and administered with or without NY-ESO-1 peptide. The lower panel shows that endogenous IFNAR1 was knocked out. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] [First embodiment] <cell> (cell 1) In one embodiment of the present invention, the cell comprises, in its cell membrane, a first fusion membrane protein molecule comprising at least a portion of the intracellular domain of interferon alpha and beta receptor 1 (hereinafter sometimes referred to as IFNAR1), a first transmembrane domain, and a first extracellular domain, and a second fusion membrane protein molecule comprising at least a portion of the intracellular domain of interferon alpha and beta receptor 2 (hereinafter sometimes referred to as IFNAR2), a second transmembrane domain, and a second extracellular domain, wherein the first extracellular domain and the second extracellular domain are capable of binding directly or indirectly.

[0012] An overview of a cell in one embodiment of the present invention will be described with reference to FIG. 1. FIG. 1 shows a schematic diagram of a cell in this embodiment. In FIG. 1, the cell 1 includes a first fusion membrane protein molecule 11, a second fusion membrane protein molecule 12, and an ISRE-inducible expression unit 13. The first fusion membrane protein molecule 11 includes an intracellular domain 111, a first transmembrane domain 112, and a first extracellular domain 113 of IFNAR1. The second fusion membrane protein molecule 12 includes an intracellular domain 121, a second transmembrane domain 122, and a second extracellular domain 123 of IFNAR2.

[0013] Examples of cell 1 include HEK293T cells (human fetal kidney cell-derived cells), HEK293 cells (human fetal kidney cell-derived cells), HeLa cells (human cervical cancer-derived cells), A549 cells (human lung cancer-derived cells), MCF-7 cells (human breast cancer-derived cells), and A431 cells (human epithelial cell carcinoma-derived cells). The function of endogenous IFNAR1 in cell 1 has been lost by a method such as knockout using Cas9.

[0014] The first fusion membrane protein molecule 11 and the second fusion membrane protein molecule 12 penetrate the cell membrane 14, and the intracellular domain 111 of IFNAR1 and the intracellular domain 121 of IFNAR2 are located inside the cell membrane 14. The intracellular domain 111 of IFNAR1 and the intracellular domain 121 of IFNAR2 preferably include their entire sequences, but may be missing parts thereof as long as they function as the intracellular domains of IFNAR1 and IFNAR2.

[0015] A first extracellular domain 113 and a second extracellular domain 123 are located outside the cell membrane 14. In this embodiment, the first extracellular domain 113 includes a first protein 113a and a protein 113b derived from a part of the extracellular domain of IFNAR1. The protein 113b is located between the first protein 113a and the first transmembrane domain 112. The second extracellular domain 123 includes a second protein 123a and a protein 123b derived from a part of the extracellular domain of IFNAR2. The protein 123b is located between the second protein 123a and the second transmembrane domain 122. In this embodiment, the first protein 113a and the second protein 123a are proteins to be investigated for direct binding or not.

[0016] The first extracellular domain 113 and the second extracellular domain 123 may consist only of the first protein 113a and the second protein 123a, respectively. That is, the first extracellular domain 113 and the second extracellular domain 123 may consist only of proteins that can be directly or indirectly bound to each other.

[0017] A first transmembrane domain 112 and a second transmembrane domain 122 are located in the portion that penetrates the cell membrane 14. The first and second transmembrane domains 112 and 122 may have amino acid sequences derived from IFNAR1 and IFNAR2, respectively, or may have amino acid sequences derived from other membrane protein molecules. The case where the transmembrane domains are derived from other membrane protein molecules will be described later in the embodiments.

[0018] The ISRE-inducible expression unit (DNA) 13 includes an interferon responsive element (hereinafter sometimes referred to as ISRE), a minimal promoter, and a nucleotide sequence encoding a protein. As the ISRE, a 15-nucleotide sequence such as GGGAAAGTGAAACTA (SEQ ID NO: 93) can be used.

[0019] The ISRE-inducible expression unit 13 is preferably one that has a mammalian expression vector as a basic structure. For example, a plasmid in which the CMV promoter of pAAV-CMV Vector (manufactured by Takara Bio Inc.) has been removed and an ISRE and a minimal promoter have been introduced can be used.

[0020] In this embodiment, an example will be described in which the protein encoded by the nucleotide sequence is luciferase, but the present invention is not limited thereto. The protein encoded by the nucleotide sequence may be any protein involved in a reaction that generates a signal. Examples of signals include luminescence and fluorescence. For example, examples of proteins encoded by the nucleotide sequence include luciferase, green fluorescent protein, red fluorescent protein, phosphatase, and peroxidase. Examples of luciferase include, but are not limited to, Nluc (manufactured by Promega) and NlucP (manufactured by Promega). Examples of red fluorescent proteins include, but are not limited to, DsRed.

[0021] The number of ISREs contained in a nucleotide sequence encoding one protein is preferably 4 or more. The number of ISREs contained in a nucleotide sequence encoding one protein may be 7 or more, or 10 or more. The upper limit of the number of ISREs is, for example, 15.

[0022] Hereinafter, the IFNAR reconstitution system (hereinafter, sometimes referred to as IFNARRS) in the case where the first protein 113a and the second protein 123a directly bind will be described. When the first extracellular domain 113 and the second extracellular domain 123 bind, the intracellular domain 111 of IFNAR1 and the intracellular domain 121 of IFNAR2 in the cell approach each other, and TYK2 / JAK1 kinase is activated, followed by activation of STAT1, STAT2, and IRF9. The activated STAT1, STAT2, and IRF9 bind to a specific region of the ISRE of the ISRE-inducible expression unit 13. Since the minimal promoter and the ISRE function together as a promoter, the nucleotide sequence encoding the protein downstream of the ISRE and the minimal promoter of the ISRE-inducible expression unit 13 is transcribed, and the protein, in this case, luciferase, is expressed.

[0023] The expressed luciferase reacts with a substrate to generate a luminescent signal. Since the endogenous IFNAR1 of the cell 1 has lost its function as described above, the luminescent signal is specific to the binding between the first extracellular domain 113 and the second extracellular domain 123. By detecting this luminescent signal, the binding between the first extracellular domain 113 and the second extracellular domain 123 can be detected.

[0024] According to this embodiment, various target proteins are designed as the extracellular domains of the first and second fusion membrane protein molecules, respectively, so that direct binding of the target proteins can be detected with high sensitivity.

[0025] [Second embodiment] (cell 1') In the first embodiment, the case where the first extracellular domain and the second extracellular domain are directly bound to each other is described, whereas in the present embodiment, the case where the first extracellular domain and the second extracellular domain are indirectly bound to each other, that is, via a target substance, is described. In the present embodiment, the description of the same configuration as in the first embodiment is omitted.

[0026] The outline of a cell in one embodiment of the present invention will be described with reference to FIG. 2. FIG. 2 shows a schematic diagram of a cell in this embodiment. In FIG. 2, a cell 1' includes a first fusion membrane protein molecule 21, a second fusion membrane protein molecule 22, and an ISRE-inducible expression unit 23. In FIG. 2, only a part of the ISRE-inducible expression unit 23 is shown. The first fusion membrane protein molecule 21 includes a first intracellular domain 211, a first transmembrane domain 212, and a first extracellular domain 213. The first intracellular domain 211 includes an intracellular domain 211a of IFNAR1 and a part 211b of the intracellular domain of interferon gamma receptor 1 (hereinafter, sometimes referred to as IFNGR1). The second fusion membrane protein molecule 22 includes a second intracellular domain 221, a second transmembrane domain 222, and a second extracellular domain 223. The second intracellular domain 221 includes an intracellular domain 221a of IFNAR2 and a portion 221b of the intracellular domain of IFNGR1.

[0027] In this embodiment, an IFNAR regeneration system will be described in which the target substance 15 is interferon gamma (hereinafter, sometimes referred to as IFNγ), and the first and second extracellular domains 213 and 223 are the extracellular domains of IFNGR1. When the first and second extracellular domains 213 and 223 bind to the extracellular target substance 15 (here, a dimer of IFNγ), the intracellular domain 211a of IFNAR1 and the intracellular domain 221a of IFNAR2 are brought into close proximity within the cell. This activates TYK2 / JAK1 kinase, followed by activation of STAT1, STAT2, and IRF9, which then bind to a specific region of ISRE. The minimal promoter and ISRE function together as a promoter, so that the nucleotide sequence encoding the protein downstream of the ISRE and the minimal promoter of the ISRE-inducible expression unit 23 is transcribed, and the protein, i.e., luciferase, is expressed.

[0028] The expressed luciferase reacts with a substrate to generate a luminescent signal, which can be detected to detect interferon-γ.

[0029] The protein encoded by the nucleotide sequence contained in the ISRE-inducible expression unit 23 may be any of the proteins involved in the reaction that emits a signal described in the first embodiment. When the nucleotide sequence encoding the protein involved in the reaction that emits these signals is contained in the ISRE-inducible expression unit 23, the target substance 15 can be detected by detecting the signal corresponding to each of them.

[0030] In another aspect, the ISRE-inducible expression unit 23 may contain genes involved in the regulation of various cellular functions, genes that recognize interacting cells, etc. For example, the ISRE-inducible expression unit 23 may contain a nucleotide sequence encoding at least one protein of a molecule that controls cell proliferation, activation, and differentiation, and a molecule that controls cell-cell interactions.

[0031] A cell 1' into which an ISRE-inducible expression unit 23 containing a nucleotide sequence encoding a molecule that controls cell proliferation is introduced expresses the molecule that controls proliferation when a target substance 15 is present outside the cell. Thus, when a cell that produces the target substance 15 is co-cultured with the cell 1', proliferation of the cell that produces the target substance 15 can be promoted. The molecule that controls proliferation is not particularly limited, but an example thereof is interleukin-2, which promotes proliferation.

[0032] A cell 1' into which an ISRE-inducible expression unit 23 containing a nucleotide sequence encoding a molecule that controls cell differentiation is introduced expresses a molecule that controls cell differentiation when a target substance 15 is present outside the cell. Thus, when a cell that produces the target substance 15 is co-cultured with the cell 1', the cell differentiation of the cell that produces the target substance 15 can be controlled. Molecules that control cell differentiation include, but are not limited to, IFNγ, interleukin-2 (also referred to as IL-2), interleukin-4 (also referred to as IL-4), interleukin-6 (also referred to as IL-6), interleukin-10 (also referred to as IL-10), interleukin-12 (also referred to as IL-12), interleukin-17 (also referred to as IL-17), interleukin-23 (also referred to as IL-23), tumor necrosis factor-α (also referred to as TNFα), and transforming growth factor-β (also referred to as TGFβ).

[0033] The cell 1' into which the ISRE-inducible expression unit 23 containing a nucleotide sequence encoding a molecule that controls cell activation expresses a molecule that controls cell activation when the target substance 15 is present outside the cell. Thus, by co-culturing a cell that produces the target substance 15 with the cell 1', the activation of the cell that produces the target substance 15 can be controlled. Examples of costimulatory molecules that promote activation include, but are not limited to, costimulatory molecules that promote the activation of B7-1, B7-2, CD40, 4-1BB ligand, and the like, and co-inhibitory molecules that suppress the activation of CTLA4, PD-L1, galectin-9, and the like.

[0034] Molecules for controlling cell-cell interactions are not particularly limited, but examples include the use of single-chain variable fragments (also called scFv) or affibodies that function similarly to antibodies. They can select (target) interacting cells from cells co-cultured with cell 1', act on the target cells (activation, proliferation promotion, differentiation promotion, etc.), and receive feedback from the target cells (signal transduction molecules such as kinases or phosphatases can be fused to the intracellular domain of scFv-TM). Expression of anti-CD4-scFv-TM (cell membrane-bound type) or anti-CD4-CCR6-TM can inhibit CD4 + It can target T cells and Th17 cells. In addition, by expressing ZZ-Affibody-TM (cell membrane-bound type), it can be freely targeted by co-culturing with an antibody that recognizes molecules expressed on the cell surface of the target cell (because the Fc region of immunoglobulin IgG binds to ZZ-Affibody).

[0035] Furthermore, the protein encoded by the nucleotide sequence contained in the ISRE-inducible expression unit 23 may be a protein having the same structure as the target substance 15. For example, when IFNγ is the target substance 15, when IFNγ binds to the first and second extracellular domains 213 and 223, the intracellular domain 211a of IFNAR1 and the intracellular domain 221a of IFNAR2 are brought into close proximity, and IFNγ encoded by the nucleotide sequence contained in the ISRE-inducible expression unit 23 is expressed via the ISRE by the above-mentioned mechanism. This makes it possible to enhance the detection signal.

[0036] A plurality of ISRE-inducible expression units 23 may be introduced into one cell, and the nucleotide sequences encoding proteins contained in the plurality of ISRE-inducible expression units 23 may be different from each other. For example, an ISRE-inducible expression unit including a nucleotide sequence encoding a protein involved in a signal-emitting reaction and an ISRE-inducible expression unit including a nucleotide sequence encoding a cell growth factor may both be introduced into one cell. The types of ISRE-inducible expression units introduced into one cell may be determined depending on the purpose, and may be, for example, three or more types, or five or more types.

[0037] In another aspect, the ISRE-inducible expression unit 23 may contain multiple nucleotide sequences encoding proteins. For example, one ISRE-inducible expression unit 23 may contain multiple units containing nucleotide sequences encoding different proteins, with the ISRE, a minimal promoter, and a nucleotide sequence encoding the protein being considered as one unit. Also, one ISRE-inducible expression unit 23 may be configured to be able to express multiple nucleotide sequences encoding proteins as one unit protein linked by a self-cleaving peptide.

[0038] The target substance 15 can be appropriately selected depending on the purpose, and is not particularly limited. For example, the target substance 15 may include a cytokine, an antibody, or an affibody. The target substance 15 may also be a complex protein in which two or more proteins are bound. The two or more bound proteins may be the same or different from each other.

[0039] When the target substance 15 is a cytokine, examples of the target substance 15 include ligands of the class I cytokine receptor family such as IL-2, IL-4, IL-6, and IL-12, ligands of the class II cytokine receptor family such as IFNγ and IL-10, ligands of the immunoglobulin superfamily such as IL-1 and IL-18, ligands of the TNF (Tumor Necrosis Factor) receptor family such as TNFα and FasL, and ligands of the TGF-β receptor family such as TGF-β. In addition, examples of the target substance 15 include ligands of the IL-17 receptor family such as IL-17A-F, which are not classified into general cytokine receptor families due to their unique structure and properties.

[0040] When the target substance 15 is a cytokine, the number of fusion membrane proteins that bind to the target substance 15 is not limited to two, and may be three or more. For example, when the target substance is IFNγ, in the case where interferon gamma receptor 2 (hereinafter, sometimes referred to as IFNGR2) is incorporated into a dimer of IFNGR1 to form a tetramer, the first fusion membrane protein may have the extracellular domain of IFNGR2, and the second fusion membrane protein may have the extracellular domain of IFNGR1. When IFNγ is present outside the cell, the second fusion membrane protein binds to the dimer of IFNγ, and the first fusion membrane protein is further incorporated into the two adjacent second fusion membrane proteins to form a tetramer. This brings the intracellular domain of IFNAR1 and the intracellular domain of IFNAR2 into close proximity within the cell, and the protein encoded by the nucleotide sequence contained in the ISRE-inducible expression unit 23 is expressed via the ISRE by the above-mentioned mechanism. In many cytokine receptors, two different molecules are brought into close proximity by the binding of the cytokine, and a signal is often transmitted. As shown in Figure 30 (in the case of IFNγ) and Figure 38 (in the case of IL-2), it is expected that by linking the extracellular domain of each different cytokine receptor to the intracellular domain of IFNAR1 / 2 and an arbitrary transmembrane domain, it will be possible to create a system that can detect most cytokines specifically and with high sensitivity.

[0041] According to this embodiment, by designing proteins capable of binding to a target substance as the extracellular domains of the first and second fusion membrane protein molecules, respectively, the target substance can be detected with high sensitivity.

[0042] Furthermore, by co-culturing the cells of this embodiment with cells that produce a target substance, it is possible to control the proliferation, activation, differentiation, and intercellular interactions of the cells that produce the target substance.

[0043] [Third embodiment] <Methods for expressing proteins> A method for expressing a protein according to this embodiment includes using a cell described in the first and second embodiments, directly or indirectly binding the first extracellular domain to the second extracellular domain in the cell, and bringing at least a portion of the intracellular domain of interferon α and β receptor 1 and at least a portion of the intracellular domain of interferon α and β receptor 2 into close proximity through said binding, thereby transcribing a nucleotide sequence encoded downstream of the interferon response element present in the cell, thereby expressing the protein.

[0044] The cell, the first extracellular domain, the second extracellular domain, the intracellular domain of interferon α and β receptor 1, the intracellular domain of interferon α and β receptor 2, the interferon response element, and the nucleotide sequence encoded downstream are as described in the first and second embodiments, and therefore will not be described here.

[0045] In this embodiment, when the first extracellular domain and the second extracellular domain are directly bound to each other, various target proteins are designed as the extracellular domains of the first and second fusion membrane protein molecules, respectively, so that the direct binding of the target proteins can be detected with high sensitivity.

[0046] In this embodiment, when the first extracellular domain and the second extracellular domain are indirectly bound to each other, in other words, when the target substance is sandwiched between them, the target substance can be detected with high sensitivity by designing proteins in which the first extracellular domain and the second extracellular domain are capable of binding to the target substance as the extracellular domains of the first and second fusion membrane protein molecules, respectively.

[0047] Furthermore, by carrying out the method of expressing the protein of this embodiment while co-culturing the cells of this embodiment with cells that produce a target substance, it is possible to control the proliferation, activation, and differentiation of the cells that produce the target substance, as well as cell-to-cell interactions.

[0048] [Fourth embodiment] <Expression construct> The expression construct in this embodiment includes a first expression construct including a first promoter for expression that functions within a cell, and a nucleotide sequence operably linked to the first promoter, the nucleotide sequence encoding a first fusion membrane protein molecule including at least a portion of the intracellular domain of interferon α and β receptor 1, a first transmembrane domain, and a first extracellular domain, wherein the first extracellular domain is capable of binding to a target substance other than interferon α and β.

[0049] The expression construct in this embodiment further includes a second expression construct comprising a second promoter for expression functional within a cell, and a nucleotide sequence operably linked to the second promoter, the nucleotide sequence encoding a second fusion membrane protein molecule comprising at least a portion of the intracellular domain of interferon alpha and beta receptor 2, a second transmembrane domain, and a second extracellular domain, wherein the first extracellular domain and the second extracellular domain can be directly or indirectly bound to each other.

[0050] The expression construct in this embodiment is an expression construct for producing cells 1 and 1' in the first and second embodiments. Therefore, when the expression construct in this embodiment includes the first expression construct, the first fusion membrane protein molecule can be expressed in cells 1 and 1'. When the expression construct in this embodiment further includes the second expression construct, the first fusion membrane protein molecule and the second fusion membrane protein molecule can be expressed in cells 1 and 1'.

[0051] The first and second fusion membrane protein molecules are as explained in the above embodiment, and therefore explanation thereof will be omitted.

[0052] The promoter in the expression construct of this embodiment is a nucleotide sequence that causes the first or second fusion membrane protein molecule linked downstream to be expressed in a cell, and a promoter contained in a known vector can be applied. The promoter and the nucleotide sequence encoding a protein are "operably linked" so that the promoter can regulate the transcription and / or translation of the nucleotide sequence encoding the protein.

[0053] A promoter generally means a DNA sequence capable of regulating protein translation or RNA transcription. In this embodiment, a promoter means a transcriptional regulatory sequence capable of inducing the translation of a target fusion protein. The promoter of this embodiment can be appropriately adopted depending on the type of cell into which the expression construct is introduced, and examples thereof include the following promoters.

[0054] Examples of promoter sequences operable in cells include a cytomegalovirus (CMV)-derived promoter, a human polypeptide chain elongation factor EF-1α (EF) promoter, a cytomegalovirus enhancer and a chicken β-actin (CAG)-derived promoter, and the like.

[0055] In the expression construct of this embodiment, a polyadenylation sequence is preferably provided as a transcription termination sequence downstream of the coding region of the fusion protein to be expressed. Examples of such known transcription termination sequences include bovine growth hormone terminator, SV40-derived polyadenylation sequence, HIV-1 polyA, β-globin polyA, HSV TK polyA, polyoma virus polyA, etc.

[0056] The expression construct of this embodiment may contain an antibiotic resistance gene commonly used by those skilled in the art as a selection marker, such as a puromycin, blasticidin S, hygromycin B, or zeocin resistance gene.

[0057] The expression construct of this embodiment can be prepared in a variety of forms, and can be prepared, for example, based on a known expression vector such as a plasmid vector.

[0058] The expression vector is not particularly limited, and for example, mammalian expression vectors such as pEF-BOS, pCAGGS, and pcDNA, as well as vectors modified from these, etc. Among these, pcDNA is preferred.

[0059] As described above, the first and second expression constructs use a constitutive and strong expression promoter such as CMV, and are composed of a backbone such as pcDNA. Therefore, in cells containing the first and second expression constructs, proteins are constantly expressed by transcription from the DNA contained in the first and second expression constructs. The ISRE-inducible expression unit is also an expression construct, but unlike the first and second expression constructs, it contains an ISRE-minimal promoter. Therefore, the protein encoded by the DNA contained in the ISRE-inducible expression unit is not usually expressed in cells, but its expression is induced by a stimulus as described above. In this respect, the first and second expression constructs are different from the ISRE-inducible expression unit.

[0060] [Fifth embodiment] <Transformed cells> The transformed cell in this embodiment is genetically transfected with the expression construct described in the fourth embodiment.

[0061] Examples of the cells into which the gene is introduced include HEK293T cells (cells derived from human fetal kidney cells), HEK293 cells (cells derived from human fetal kidney cells), HeLa cells (cells derived from human cervical cancer), A549 cells (cells derived from human lung cancer), MCF-7 cells (cells derived from human breast cancer), and A431 cells (cells derived from human epithelial cell carcinoma), and HEK293T cells are preferred. The endogenous IFNAR of the cells into which the gene is introduced has lost its function by a method such as knockout using Cas9. The function of the endogenous IFNAR may be performed before or after the gene introduction into the cells of the expression construct described in the fourth embodiment.

[0062] Genetic introduction of the expression construct described in the fourth embodiment into cells can be performed by a conventional method. For example, the expression construct described in the fourth embodiment is first transfected into cells and used transiently (used after 24 to 48 hours). This verifies whether the created expression construct can be used. If the expression construct can be used, it can be transfected into cells together with a drug resistance gene and drug selection can be performed to establish a stable cell line.

[0063] The transformed cell into which the expression construct has been introduced expresses the first and second fusion membrane protein molecules described in the first and second embodiments within the cell. Therefore, according to the transformed cell of this embodiment, various target proteins can be designed as the extracellular domains of the first and second fusion membrane protein molecules, respectively, to detect direct or indirect binding of the target protein with high sensitivity. In addition, by co-culturing the transformed cell of this embodiment with cells that produce a target substance, it is possible to control the proliferation, activation, and differentiation of the cells that produce the target substance, as well as cell-cell interactions.

[0064] Although each embodiment has been described in detail above with reference to the drawings, the specific configuration is not limited to the above and various design modifications are possible. In addition, two or more of the above-described embodiments may be appropriately combined. EXAMPLES

[0065] The present invention will now be described in more detail with reference to examples, but the present invention is not limited to the following examples.

[0066] (Cell culture and reagents) Human fetal kidney cell-derived HEK293T cells were cultured in Dulbecco's modified Eagle's medium (Fujifilm Wako Chemicals, part number 044-29765) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific, part number 26140079). Jurkat cells (human T cell line) were cultured in RPMI1640 medium supplemented with 10% fetal bovine serum. CTLL-2 cells (gift from Professor Nagahiro Minato, Kyoto University) were cultured in RPMI1640 medium (Fujifilm Wako Chemicals, part number 189-02025) supplemented with 10% fetal bovine serum and 50 μM 2-mercaptoethanol. The antibodies used were recombinant human IFNγ (FUJIFILM Wako Chemicals, CYT-206), IL-2 (Kyowa Pharmaceutical Co., Ltd., product code 6399), phorbol 12-myristate 13-acetate (also known as PMA, Sigma-Aldrich, product code P1585), calcium ionophore (Sigma-Aldrich, product code C7522), and anti-GST antibody (Santa Cruz Biotechnology, product code sc-138), as well as anti-B7-1 (Proteintech, product code 65083-1-Ig) antibody. The anti-PD-L1 (27A2) antibody and anti-CD3ε (OKT3) antibody used were those described in Iwasaki M, et al. Expression and function of PD-1 in human gammadelta T cells that recognize phosphoantigens. Eur J Immunol. 41, 345 (2011) and Miyagawa F, et al. Essential contribution of germline-encoded lysine residues in Jgamma1.2 segment to the recognition of nonpeptide antigens by human gammadelta T cells. J Immunol. 167, 6773 (2001).

[0067] (cDNA cloning and vector constructs) Total mRNA from human cells was prepared by the standard AGPC (acid guanidinium thiocyanate-phenol-chloroform) method, and cDNA was synthesized using oligo(dT)18 primer and ProtoScript II reverse transcriptase (New England Biolabs Inc.) as described in Hayashi H, Kubo Y, Izumida M, Matsuyama T. Efficient viral delivery of Cas9 into human safe harbor. Sci Rep. 10, 21474 (2020). IFNAR1, IFNAR2, IFNGR1 and IFNGR2 genes were amplified using PrimeSTAR Max DNA Polymerase (Takara Bio Inc.) with cDNA derived from HEK293T cells as a template. Jurkat cell-derived cDNA was used to amplify IFNG, IL-2, IL2RA, IL2RB and IL2RG. Human B cell-derived cDNA was used to amplify the B7-1 gene.

[0068] Double-stranded DNA of TCR α and β chains for SmBiT, LgBiT, PD-L1, PD-1, and anti-NY-ESO-1, and anti-CDε-scFv genes were purchased from Integrated DNA Technologies. NlucP expression vector with minimal promoter (NlucP / MinP) was purchased from Promega. HER2 (Part No. 16257), CD8α-EGFP (Part No. 86051), TCRα / β / CD3ε / ζ (Part No. 89347), Cas9 (Part No. 52961), pSLCAR-CD19-28z (Part No. 135991), and pSLCAR-CD19-BBz (Part No. 135992) expression vectors were purchased from Addgene. IHGH1 (Part No. IRAK174M03) was provided by Riken BRC. GAS-Luc plasmid was provided by Stark-GR. The DNA sequence information of anti-NY-ESO-1-scFv (3M4E5) was provided by Ochi-T.

[0069] The DNA primers used to amplify each gene were IFNAR1-TM-IN-394 Forward (SEQ ID NO: 1), IFNAR1-TM-IN-394 Reverse (SEQ ID NO: 2), IFNAR1-TM-IN-231 Forward (SEQ ID NO: 3), IFNAR1-TM-IN-231 Reverse (SEQ ID NO: 4), IFNGR1-EX3 Forward (SEQ ID NO: 5), IFNGR1-EX3 Reverse (SEQ ID NO: 6), IFNGR2-EX5 Forward (SEQ ID NO: 7), IFNGR2-EX5 Reverse (SEQ ID NO: 8), IFNG(full-length) Forward (SEQ ID NO: 9), IFNG(full-length) Reverse (SEQ ID NO: 10), IL-2(full-length) Forward (SEQ ID NO: 11), IL-2(full-length) Reverse (SEQ ID NO: 12), IL2RA(full-length) Forward (SEQ ID NO: 13), and IL2RA(full-length). The following antibodies were used: IL2RB-EX Forward (SEQ ID NO: 15), IL2RB-EX Reverse (SEQ ID NO: 16), IL2RG-EX Forward (SEQ ID NO: 17), IL2RG-EX Reverse (SEQ ID NO: 18), B7-1(full-length) Forward (SEQ ID NO: 19), B7-1(full-length) Reverse (SEQ ID NO: 20), and ANTI-CD3ε-scFv-TM (0.35 kb fragment for RT-PCR) Forward (SEQ ID NO: 94), and ANTI-CD3ε-scFv-TM (0.35 kb fragment for RT-PCR) Reverse (SEQ ID NO: 95).

[0070] The DNA sequences encoding the fusion membrane proteins shown in FIG. 4 are (1) HER2-EX-IFNAR1-TM-IN-360 (SEQ ID NO: 21), (2) HER2-EX-IFNAR1-TM-IN-394 (SEQ ID NO: 22), (3) HER2-EX-IFNAR2-TM-IN-183 (SEQ ID NO: 23), (4) HER2-EX-IFNAR2-TM-IN-231 (SEQ ID NO: 24), (5) Anti-HER2-Af-IFNAR1-TM-IN-360 (SEQ ID NO: 25), (6) Anti-HER2-Af-IFNAR1-TM-IN-394 (SEQ ID NO: 26), (7) Anti-HER2-Af-IFNAR2-TM-IN-183 (SEQ ID NO: 27), and (8) Anti-HER2-Af-IFNAR2-TM-IN-231 (SEQ ID NO: 28).

[0071] The DNA sequences of the expression vectors shown in Figure 7 are 4xISRE-Nluc (sequence number 29), 6xISRE-Nluc (sequence number 30), 7xISRE-Nluc (sequence number 31), 10xISRE-Nluc (sequence number 32) and 15xISRE-Nluc (sequence number 33), respectively.

[0072] The DNA sequences encoding the fusion membrane proteins shown in Figure 9 are (1) HER2-EX-SmBiT (sequence number 34), (2) HER2-EX-Linker-SmBiT (sequence number 35), (3) HER2-EX-LgBiT (sequence number 36), (4) HER2-EX-Linker-LgBiT (sequence number 37), (5) Anti-HER2-SmBiT (sequence number 38), (6) Anti-HER2-Linker-SmBiT (sequence number 39), (7) Anti-HER2-LgBiT (sequence number 40), and (8) Anti-HER2-Linker-LgBiT (sequence number 41).

[0073] The DNA sequences encoding the fusion membrane proteins shown in Figure 13 are ZZ Af-IFNAR1-TM-IN-394 (SEQ ID NO: 42), PD-L1-EX-IFNAR2-TM-IN-231 (SEQ ID NO: 43), HER2-EX-IFNAR1-TM-IN-394 (SEQ ID NO: 44), and PD1-EX-Linker-anti-HER2-AF (SEQ ID NO: 45), respectively.

[0074] The DNA sequences encoding the fusion membrane proteins shown in Figure 17 are (1) HER2-EX-TM-IFNAR2-IN-265 (sequence number 46), (2) HER2-EX-TM-IFNAR1-IN-458 (sequence number 47), (3) HER2-EX-TM-IFNAR1-IN-D51 (sequence number 48), (4) HER2-EX-TM-IFNAR1-IN-D43 (sequence number 49), (5) HER2-EX-TM-IFNAR1-IN-D41 (sequence number 50), (6) HER2-EX-TM-IFNAR1-IN-D62 (sequence number 51), (7) HER2-EX-TM-IFNAR1-IN-D61 (sequence number 52) and (8) HER2-EX-TM-IFNAR1-IN-D71 (sequence number 53), respectively.

[0075] The DNA sequences encoding the fusion membrane proteins shown in Figures 20 and 21 are (1) HER2-EX-TM-IFNAR2-IN-265 (sequence number 54), (6) HER2-EX-TM-IFNAR1-IN-D62 (sequence number 55), Anti-HER2-Af-Dimer (sequence number 56), (1) HER2-EX-TM-SmBiT (sequence number 57), (2) HER2-EX-TM-Linker-SmBiT (sequence number 58), (3) HER2-EX-TM-LgBiT (sequence number 59) and (4) HER2-EX-TM-Linker-LgBiT (sequence number 60), respectively.

[0076] The DNA sequences encoding the fusion membrane proteins shown in Figures 25 and 26 are (1) IFNGR1-EX-TM-IFNAR2-IN-265 (sequence number 61), (2) IFNGR1-EX-TM-IFNAR1-IN-D62 (sequence number 62), (1) IFNGR1-EX-TM-SmBiT (sequence number 63), (2) IFNGR1-EX-TM-Linker-SmBiT (sequence number 64), (3) IFNGR1-EX-TM-LgBiT (sequence number 65) and (4) IFNGR1-EX-TM-Linker-LgBiT (sequence number 66), respectively.

[0077] The DNA sequences encoding the fusion membrane proteins shown in Figures 31 and 33 are (1) IFNGR1-EX1-IFNAR2-TM-IN-231 (SEQ ID NO: 67), (2) IFNGR1-EX2-IFNAR2-TM-IN-231 (SEQ ID NO: 68), (3) IFNGR1-EX3-IFNAR2-TM-IN-231 (SEQ ID NO: 69), and (4) IFNGR1-EX3-IFNAR2-TM- (5) IFNGR2-EX4-IFNAR1-TM-IN-394 (SEQ ID NO: 71), (6) IFNGR2-EX5-IFNAR1-TM-IN-394 (SEQ ID NO: 72), (7) IFNGR2-EX5-IFNAR1-TM-IN-431 (SEQ ID NO: 73) and (8) IFNGR2-EX5-IFNAR1-TM-IN-D83 (SEQ ID NO: 74). The DNA sequence of the expression vector shown in Figure 33 is 7xISRE-IFNG (SEQ ID NO: 75).

[0078] The DNA sequence of the sgRNA expression plasmid shown in FIG. 35 is sgRNA (SEQ ID NO: 76), target DNA sequence (SEQ ID NO: 77), Left arm-P2A-Puro r (SEQ ID NO: 78) and Right arm (SEQ ID NO: 79).

[0079] The DNA sequences encoding the fusion membrane proteins shown in Figure 39 are IL2RA (sequence number 80), (1) IL2RB-EX-IFNAR2-TM-IN-231 (sequence number 81), (2) IL2RB-EX-IFNAR2-TM-IN-240 (sequence number 82), (3) IL2RB-EX-IFNAR1-TM-IN-427 (sequence number 83), (4) IL2RB-EX-IFNAR1-TM-IN-431 (sequence number 84), (5) IL2RG-EX-IFNAR2-TM-IN-240 (sequence number 85), (6) IL2RG-EX-IFNAR1-TM-IN-427 (sequence number 86), and (7) IL2RG-EX-IFNAR1-TM-IN-D83 (sequence number 87), respectively.

[0080] The DNA sequence of the IL-2 expression vector shown in Figure 43 is as shown in SEQ ID NO:88.

[0081] The DNA sequence of the construct used to prepare the viral vector shown in Figure 47 is anti-NY-ESO-1-TCRα-E2A-TCRβ-P2A-CD3ε-T2A-CD3ζ (sequence number 89).

[0082] The DNA sequences of the expression vectors shown in Figures 52 and 53 are anti-CD3ε-scFv-Sc (SEQ ID NO: 90), anti-CD3ε-scFv-TM (SEQ ID NO: 91), and B7-1 (SEQ ID NO: 92), respectively. All constructs were verified by sequencing.

[0083] (Establishment of cell lines (clones) that stably express related genes) To establish HEK293T cells stably expressing IFNγ, five pcDNA expression vectors containing IFNGR1-EX2-IFNAR2-TM-IN, IFNGR2-EX-IFNAR1-TM-IN, 15xISRE-Nluc, 7xISRE-IFNγ, and a hygromycin B resistance gene downstream of the CMV promoter were co-transfected (Total DNA: 400 ng, 10:10:10:10:1, 1.2 μL of Lipofectamine 2000 (Thermo Fisher Scientific)).

[0084] After selection with 0.5 mg / mL hygromycin B for 2 weeks, a stable cell line, GR-535, was established that showed the highest signal / background ratio (IFNγ-dependent Nluc activation) among several stable clones (FIG. 34).

[0085] To eliminate IFNα-dependent activation in GR-535 cells, the endogenous IFNAR1 gene was knocked out. A Cas9 expression plasmid and a plasmid expressing sgRNA targeting the IFNAR1 gene and carrying a donor dsDNA sequence for homologous recombination were co-transfected (Total DNA: 300 ng, 1:2, 1.2 μL Lipofectamine 2000). This plasmid was designed to express a puromycin resistance gene after successful homologous recombination (Figure 35). After selection with 2.8 μg / mL puromycin for 2 weeks, a cell line GRk-281 that was unresponsive to IFNα and showed the highest signal / background ratio (IFNγ-dependent Nluc activation) by knocking out endogenous IFNAR1 was selected from several stable clones and established (Figures 36 and 37).

[0086] To produce IL-2 in response to IFNγ, GRk-281 cells were further co-transfected with a plasmid expressing 10xISRE-IL-2 and a blasticidin S resistance gene (total DNA: 300 ng, 60:1, 1.2 μL of Lipofectamine 2000). After 3 weeks of selection with 64 μg / mL blasticidin S, a cell line GRkS-974 was established that showed the highest signal / background ratio (IFNγ-dependent Nluc activation) among several stable clones.

[0087] The GRkS-974 cells were further co-transfected with 10xISRE-B7-1-, 10xISRE-anti-CD3ε-scFv-TM-, and Zeocin resistance gene-expressing plasmids (Total DNA: 400ng, 1.5:1.5:1, 1.2μL of Lipofectamine 2000). After selection with 120μg / mL Zeocin for 3 weeks, a cell line GRkSB-043 was established that expresses B7-1 and anti-CD3ε antibodies in response to IFNγ. GRkS-974 cells were also co-transfected with 10xISRE-B7-1-, 10xISRE-anti-CD3ε-scFv-TM-, ZZ-TM-, and Zeocin resistance gene-expressing plasmids (Total DNA: 400ng, 1:1:1:1, 1.2μL of Lipofectamine 2000). After 3 weeks of selection with 125 μg / mL Zeocin, we established a cell line, GRkSBZ-188, which expresses B7-1 and anti-CD3ε antibodies in response to IFNγ and constitutively expresses ZZ-TM. The expressed or knocked-out genes and biological characteristics of these stable cell lines are shown in Table 1.

[0088] [Table 1]

[0089] To establish HEK293T stable cells capable of detecting IL-2, five pcDNA expression vectors containing IL-2Rα, IL-2Rβ-EX-IFNAR2-TM-IN, IL-2Rγ-EX-IFNAR1-TM-IN, 10×ISRE-NlucP and hygromycin B resistance genes under the CMV promoter, respectively, were co-transfected.

[0090] After 2 weeks of selection with hygromycin B as described above, the stable cell line 2LR-398 was established, which showed the highest signal / background ratio (IL-2-dependent Nluc activation) among several stable clones (Figure 41). The endogenous IFNAR1 gene of 2LR-398 cells was knocked out as described above. After puromycin selection as described above, the cell line 2LRk-134 was established, which showed the highest signal / noise ratio (IL-2-dependent Nluc activation) among several stable clones, by knocking out the endogenous IFNAR1 (Figure 42).

[0091] Jurkat cells were infected with recombinant lentiviruses expressing RetroNectin (Takara Bio, product number t100A), CD8α-EGFP, and a puromycin resistance gene as described above.

[0092] After selection with 1.3 μg / mL puromycin, fluorescent CD8α-EGFP expressing cells were isolated. Jurkat cells expressing CD8α-EGFP were further infected with recombinant lentivirus to introduce anti-NY-ESO-1-TCRα / β, CD3ε / ζ, and blasticidin resistance genes. The variable region of TCRα / β in the pLentivirus vector for expressing TCRα / β / CD3ε / ζ was replaced with anti-NY-ESO-1-TCRα / β (SEQ ID NO: 89). After selection with 20 μg / mL blasticidin S, the expression of anti-NY-ESO-1-TCRα / β was confirmed by the production of IFNγ in HEK293T cells loaded with NY-ESO-1 peptide. For the experiments, Jurkat cells expressing CD8α-GFP, anti-NY-ESO-1-TCRα / β, and CD3ε / ζ (Jurkat-CD8 / anti-ESO-TCR cells) were used.

[0093] The promoter activity of NFAT-RE has been widely used to evaluate the activation of T cell receptors. Jurkat cells were infected with recombinant lentivirus to express NlucP under the control of 6xNFAT-RE (SEQ ID NO: 96) and a constitutive Zeocin resistance gene. After selection with 400 μg / mL Zeocin, Jurkat-NFAT-Nluc cells were established that showed PMA / Ca (100 ng / mL PMA and 300 ng / mL calcium ionophore)-dependent Nluc activation. To express anti-NY-ESO-1-scFv-28BBZ, anti-NY-ESO-1-scFv was fused to 28BBZ in the CAR-T construct. Jurkat-NFAT-Nluc cells were infected with recombinant lentivirus expressing anti-NY-ESO-1-scFv-28BBZ and a blasticidin resistance gene. After selection with 20 μg / mL blasticidin S, Jurkat-NFAT-RE-NlucP / anti-NY-ESO-1-scFv-28BBZ cells (Jurkat-NFAT-Nluc / anti-ESO-28BBZ cells) showing Nluc activation against NY-ESO-1 peptide-loaded HEK293T cells were established.

[0094] (Addition of peptides) Highly purified NY-ESO-1 peptide (SLLMWITQC) was purchased from GenScript Biotech, Inc. NY-ESO-1 peptide was immunized with endogenous MHC class I and HLA-A molecules of HEK293T cells by incubation with 1–20 μM of NY-ESO-1 peptide for 12 h.

[0095] (Transient transfection and Nluc assay) HEK293T cells (2 × 10 4 1000 x 1000 / well) was plated on a 96-well plate, and 12 hours later, each plasmid DNA (40 ng in total) was introduced using 0.2 μL of Lipofectamine 2000. 24 hours after the introduction, the cells were stimulated with the reagents indicated for each experiment for 12 hours, and luciferase activity was measured by adding the substrate of Nano-Glo® Luciferase Assay System (Promega, product number N1110).

[0096] (IL-2 production assay) To measure the IL-2 concentration in the medium, 10 μL of medium was added to 40 μL of 2LRk-134 cells seeded in a round-bottom 96-well plate 12 hours before measurement. Instead of medium, 10 μL of each purified IL-2 (0, 3, 10, 30 or 100 U / mL) was used for calibration. Nluc luciferase assay was performed 12 hours after the addition of medium, and the IL-2 concentration in the medium was calculated using a third-order polynomial equation with reference to the standard material.

[0097] IL-2 production was also assessed by CTLL-2 cells, which proliferate in response to IL-2. GRkS-974 cells (40 cells in 20 μL) were seeded in round-bottom 96-well plates, and CTLL-2 cells (100 cells in 40 μL) were added 12 hours after seeding of HEK293T-GRkS-974 cells. Co-cultured cells were stimulated with 30-300 ng / mL IFNγ for 48 hours, and CTLL-2 proliferation was quantitatively assessed by microscopic observation (Figure 45) and CellTiter-Glo® assay (Promega, part number G7570) (Figure 46).

[0098] (Firefly luciferase assay) A GAS (gamma interferon activation site)-containing promoter region or 4xISRE was placed in front of the firefly luciferase gene as a reporter. Firefly luciferase reporter plasmids (GAS-FireflyLuc or ISRE-FireflyLuc, 40 ng each) were transfected into HEK293T cells (2 × 10 per well) that had been plated in advance on a 96-well plate using Lipofectamine 3000 (Invitrogen). 4 After 24 hours, the cells were stimulated with 1000 pg / mL of IFNγ or IFNα for 12 hours. Firefly luciferase activity was measured.

[0099] ELISpot Assay To detect activated T cells, ELISpot assays were performed using 96-well plates precoated with monoclonal anti-human IFNγ antibody (R&D Systems, part number EL285) according to the manufacturer's instructions: In 96-well plates, the indicated numbers of Jurkat-CD8 / anti-ESO-TCR cells were stimulated with 100 ng / mL PMA and 300 ng / mL calcium ionophore (PMA / Ca), 100 ng / mL anti-CD3ε antibody, or medium alone (-) for 13 h, and secreted IFNγ was captured on a PVDF membrane, detected with another anti-human IFNγ antibody, and visualized with alkaline phosphatase and BCIP / NBT.

[0100] (Dot blot assay) GRk-974, GRkSB-043, and GRkSBZ-188 cells were cultured in 24-well plates (1x10 5The cells were placed in a 100x100mm x 100mm PBS (100x100mm x 100mm HUVECs) and treated with IFNα for 24 hours. The cell lysates were extracted with 150mL of GLO lysis buffer (Promega, part number E2661) and 5μg of protein was spotted onto a nitrocellulose membrane. B7-1 protein was detected with anti-B7-1 antibody (Proteintech, part number 65083-1-Ig) at a 1:500 dilution. Visualization was performed with HRP-conjugated anti-mouse IgG antibody and ECL reagent.

[0101] (ELISA Assay) An ELISA assay was performed to measure IFNγ secreted by activated T cells. Specifically, Jurkat-CD8 / anti-ESO-TCR cells were treated with 100ng / mL PMA and 300ng / mL calcium ionophore (PMA / Ca) or medium alone (-) for 13 hours in a 96-well plate using PeproTech's part number 900-K27. The supernatant was collected and added to a 96-well plate precoated with a monoclonal anti-human IFNγ antibody. IFNγ secreted into the medium was captured and detected with another anti-human IFNγ antibody and visualized with HRP and TMB. The color increase was measured at 404 nm using a plate reader. Calibration was performed using a series of purified IFNγ, and IFNγ concentrations were calculated using a third-order polynomial equation.

[0102] (RT-PCR) GRk-974, GRkSB-043, and GRkSBZ-188 cells were cultured in 24-well plates (1x10 5The cells were placed in a 100-ml 100-ml tube (1000 ml / well) and treated with IFNα for 24 hours. Total RNA was prepared from the cells using the acid phenol-guanidinium thiocyanate method. Reverse transcription was performed from 1 μg of total RNA at 42°C for 60 minutes using ProtoScript II Reverse Transcriptase (NEW ENGLAND BIOLABS, product number M0368L), and PCR was performed using LightCycler 1.5 (ABI) with anti-CD3ε-scFv-TM (0.35 kb fragment for RT-PCR) Forward (SEQ ID NO: 94) and anti-CD3ε-scFv-TM (0.35 kb fragment for RT-PCR) Reverse (SEQ ID NO: 95) and KOD-FX-NEO DNA Polymerase (Toyobo, product number KFX-201). The reaction conditions were 94°C for 2 minutes, followed by denaturation at 98°C for 10 seconds and extension at 68°C for 30 seconds for 30 cycles.

[0103] (statistical analysis) Quantitative data were analyzed using Student's T-test with two-tailed p values. The n for each analysis is indicated in the figure legends. A value of p<0.01 was considered statistically significant.

[0104] (Experimental Example 1) We investigated whether the extracellular domains of IFNAR1 and IFNAR2 could be replaced with other proteins of interest without impairing the intracellular functions of IFNAR1 and IFNAR2, which activate TYK2 / JAK1 and STAT1 / STAT2 / IRF9 and induce the expression of ISGs via the ISRE.

[0105] The extracellular domain (EX) of HER2 and anti-HER2 affibody were used as model binding proteins. These model binding proteins were fused to a part of the extracellular domain, transmembrane domain, and intracellular domain (TM-IN) of IFNAR1 and IFNAR2 as shown in Figure 3. Four fusion membrane protein molecules ((2), (4), (6), and (8)) for validation of the IFNAR1 and IFNAR2 signal reconstitution system were selected from the eight in Figure 4 as they showed higher signal / noise.

[0106] As shown in Figure 5, when co-transfected with the 4xISRE-NlucP plasmid into HEK293T cells, the fusion membrane protein molecules (2), (4), (6), and (8) containing short extracellular domains of IFNAR1 or IFNAR2 showed higher signal-to-noise ratios than the fusion membrane protein molecules (1), (3), (5), and (7) containing long extracellular domains of IFNAR1 or IFNAR2.

[0107] (Experimental Example 2) To confirm that the specific binding of HER2-EX to anti-HER2 affibody is associated with signal transduction via IFNAR1 and IFNAR2, the following experiment was performed.

[0108] HEK293T cells transfected with the 7×ISRE-Nluc plasmid were transfected with four possible combinations of fusion membrane protein molecules (2), (4), (6), and (8) containing short extracellular domains of IFNAR1 or IFNAR2.

[0109] In Figure 6, luciferase activity reflects the specific binding of HER2-EX to anti-HER2 affibody. As shown in Figure 6, only the combination of membrane fusion protein molecules (2) + (8) and the combination of membrane fusion protein molecules (6) + (4) showed significantly high Nluc activity via ISRE. In addition, using each expression vector shown in Figure 7(A), it was confirmed that the intensity of Nluc activity (mean ± SD of three independent experiments) increased depending on the number of ISREs in the promoter due to the specific binding of the extracellular domain of HER2 to anti-HER2 affibody (Figure 7(B)).

[0110] As a comparative example, the Nluc assay was performed using the NanoBiT method. Specifically, as shown in Figures 8 and 9, the same HER2-EX or anti-HER2 affibody as in the IFNARRS method was fused with SmBiT or LgBiT. The fusion membrane protein molecules (2), (4), (6), and (8) in Figure 9 contain a flexible peptide linker between the extracellular domain of HER2 and SmBiT or LgBiT. As shown in Figure 10, Nluc activity was increased when this peptide linker was inserted. Although it is difficult to accurately compare the IFNARRS method and the NanoBiT method, the basic structure of the plasmid expressing each gene was the same, and the amount of plasmid DNA for introduction was also the same. The Nluc assay was also performed simultaneously with the same amount of substrate. The signal intensity of the IFNARRS method using 10xISRE was 24.6 times higher than the combination with the highest signal intensity ((6) + (4)) of the NanoBiT method (Figure 11) (average value ± sd of three independent experiments). * indicates significant difference from control (*p<0.0001).

[0111] (Experimental Example 3) By using ZZ-affibody, which binds to the Fc region of immunoglobulin IgG, instead of anti-HER2 affibody, it is possible to use antibodies against various molecules and apply them to binding assays. To confirm this, the following experiment was performed.

[0112] Specific binding of anti-PD-L1 antibodies to PD-L1 was detected by expressing a fusion membrane protein molecule of the extracellular domain of PD-L1 with the transmembrane and intracellular domains of IFNAR2 (PD-L1-EX-IFNAR2-TM-IN), a fusion membrane protein molecule of the transmembrane and intracellular domains of IFNAR1 with ZZ-Af-IFNAR1-TM-IN, and 10×ISRE-Nluc (mean ± SD of three independent experiments, Figure 12 and Figure 13). When PD-L1 antibodies were added to cells expressing these molecules, dose-dependent Nluc activity was detected compared to the control anti-GST antibody (Figure 14). * indicates significant difference from the control (*p<0.01, **p<0.001).

[0113] In addition, PD1-EX and anti-HER2 affibody fusion proteins were prepared via a short peptide linker (PD1-EX-Linker-anti-HER2-Af) as shown in Figure 15. PD1-EX-Linker-anti-HER2-Af, HER2-EX-IFNAR1-TM-IN-394, PD-L1-EX-IFNAR2-TM-IN-231, and 10xISRE-Nluc were transfected into HEK293T cells. As shown in Figure 16, Nluc activity was inhibited by anti-PD-L1 antibody in a dose-dependent manner. Meanwhile, the control anti-GST antibody had little effect (mean ± SD of three independent experiments). * indicates significant difference from the control (*p<0.001). These results indicate that IFNARRS can investigate the binding between various protein molecules.

[0114] (Experimental Example 4) We investigated whether the intracellular domains of IFNAR1 and IFNAR2, which do not contain their transmembrane regions, alone are sufficient to activate STAT1 / STAT2 through TYK2 / JAK1 and transmit ISRE-dependent signals.

[0115] The extracellular and transmembrane domains of HER2 (HER2-EX-TM) were fused to the intracellular domains of IFNAR1 and IFNAR2 (IFNAR1-IN and IFNAR2-IN) (Figure 17). Multiple deletion patterns of the intracellular domain of IFNAR1 were prepared because combining unrelated domains with the two domains may impair the function of each domain. As shown in Figure 18, (1) HER2-EX-TM-IFNAR2-IN-265 (265-515 amino acids) and (6) HER2-EX-TM-IFNAR1-IN-D62 (461-537 amino acids) were able to transduce JAK / STAT signaling with anti-HER2-IFNAR1-TM-IN-394 and anti-HER2-IFNAR2-TM-IN-231, respectively. This means that the intracellular domains of IFNAR1 and IFNAR2 alone are sufficient to transduce TYK2 / JAK1 and STAT1 / STAT2 signals.

[0116] Figure 19 shows a schematic diagram comparing the NanoBiT method and the IFNARRS method. For the IFNARRS method, the constructs of the fusion membrane protein molecule (1) and (6) and the anti-HER2 affibody dimer, whose functions were proven in Figure 18, are shown (Figure 20). For the NanoBiT method, luciferase activity was detected only when the construct shown in Figure 21 was co-transfected with the anti-HER2 affibody dimer, and the combination of (2) + (4) with the peptide linker inserted showed the strongest signal (Figure 22). * indicates a significant difference from the control (*p<0.001).

[0117] Figure 23 shows the data comparing the NanoBiT method and the IFNARRS method. The basic structure of the plasmid expressing each gene was the same, and the same amount of plasmid DNA was used for the transfection, and the same amount of Nluc substrate was used for the measurement. The signal intensities of the IFNARRS method using 7xISER and 10xISER ((1)+(6)) were 8.3-fold and 27.7-fold higher, respectively, than the combination with the highest signal intensity ((2)+(4)) when using the NanoBiT method (mean ± sd of three independent experiments). * indicates a significant difference from the control (*p<0.01, **p<0.0001). Furthermore, the specificity (signal / noise) was 22.0-fold and 40.9-fold, respectively, which was higher than the 8.4-fold obtained with NanoBiT. These results confirmed that the IFNARRS method has a higher sensitivity than the NanoBiT method.

[0118] (Experimental Example 5) The IFNγ receptor is composed of two distinct chains, the high affinity IFNGR1 and the low affinity IFNGR2. When dimeric IFNγ and IFNGR1 bind, an IFNGR1 dimer is formed, which incorporates two IFNGR2 molecules. The resulting receptor tetramer activates JAK1 / JAK2, followed by STAT1 homodimer-dependent signaling. In the following, in Experimental Examples 5-6, we created a stable cell line for detecting IFNγ.

[0119] The intracellular domains of IFNAR1 and IFNAR2 were fused to the extracellular and transmembrane domains of IFNGR1 (FIGS. 24 and 25). When co-transfected with 7×ISRE-NlucP or 10×ISRE-NlucP, IFNγ induced strong ISRE-dependent Nluc activity (mean ± sd of three independent experiments, FIG. 28). * indicates significant difference from control (*p<0.01, **p<0.0001). Dimerization of extracellular IFNGR1 induced by IFNγ dimers is thought to bring the intracellular domains of IFNAR1 and IFNAR2 into close proximity, activating TYK2 / JAK1 and STAT1 / STAT2-dependent signals.

[0120] As a comparative example, a NanoBiT experiment was performed. LgBiT or SmBiT was fused to the same IFNGR1 extracellular and transmembrane domains as in the case of IFNAR1 and IFNAR2. At that time, a linker peptide was either included or not included (Figure 26). However, regardless of whether or not the linker peptide was inserted, no increase in Nluc activity was observed by the addition of IFNγ in any combination (Figure 27).

[0121] Figure 28 shows the data comparing the NanoBiT method and the IFNARRS method. The basic structure of the plasmid expressing each gene is the same as when the intracellular domains of IFNAR1 and IFNAR2 were used, and the same amount of plasmid DNA was used for transfection and the same amount of Nluc substrate was used for measurement. In the IFNARRS method, IFNγ induced strong Nluc activity, but in the NanoBiT method, there was no increase in Nluc activity due to IFNγ. This shows that the aggregation (clustering) of IFNγ-dependent receptor molecules is detected by the IFNARRS method but not by the NanoBiT method.

[0122] (Experimental Example 6) Considering the superiority of the IFNARRS method over the NanoBiT method, we verified whether the IFNARRS method can be used to detect activated T cells secreting IFNγ. As mentioned above, IFNγ induces IFNGR1 and IFNGR2 tetramers to transmit signals. On the other hand, GAS (gamma interferon activation site)-dependent promoter activity is weaker than that of ISRE-dependent promoters (Figures 54 to 55, * indicates significant difference from control (*p<0.01, **p<0.00001)). Therefore, as shown in Figures 29 and 30, we created molecules in which the extracellular domains of IFNGR1 and IFNGR2 were fused to the transmembrane and intracellular domains of IFNAR2 and IFNAR1, respectively. The cell line GRkS-974, which stably expresses these molecules, responded only to IFNγ and significantly increased luciferase activity via ISRE (Figure 56). * indicates significant difference from the control (*p<0.01, **p<0.001, ***p<0.0001). Furthermore, it is expected that increasing the expression of IL-2, B7-1, and anti-CD3ε-scFv-TM via this ISRE will promote the proliferation and activation of co-cultured T cells (interaction between GRKS974 and activated T cells in FIG. 29).

[0123] As IFNARRS constructs, different lengths of extracellular domains of IFNGR1 and IFNGR2 were fused to the transmembrane and intracellular domains of IFNAR2 and IFNAR1, respectively (Figure 31). These were transfected into HEK293T cells to determine which combination was optimal for transiently activating Nluc activity in an IFNγ-dependent manner 24 hours after transfection (Figure 32). Judging from the transient luciferase activity, the combinations (4) IFNR1-EX3-IFNAR2-TM-IN-240 and (8) IFNR2-EX5-IFNAR1-TM-IN-D83 were the best in terms of signal / noise ratio. Furthermore, co-transfection with 7×ISRE-IFNG enhanced the signal (Figure 33).

[0124] To obtain a stable cell line, expression plasmids of the fusion membrane protein molecule (4) + (8) shown in Figure 33, 15xISRE-NlucP, 7xISRE-IFNγ, and hygromycin B resistance gene were introduced into HEK293T cells. After selection with hygromycin B, the stable cell line GR-535, which showed the highest signal / noise ratio (IFNγ-dependent Nluc activation) among several stable cell lines, was established (Figure 34). Furthermore, to eliminate IFNα-dependent activation in GR-535 cells, the endogenous IFNAR1 gene was introduced into a Cas9 expression plasmid and a plasmid carrying donor dsDNA expressing sgRNA targeting the endogenous IFNAR1 gene. The plasmid was designed so that the puromycin resistance gene was expressed if homologous recombination was successful and the endogenous IFNAR1 gene was knocked out (Figure 35). After puromycin selection, we established the cell line GRk-281, which was IFNα-insensitive and showed the highest signal-to-noise ratio (IFNγ-dependent Nluc activation) by knocking out endogenous IFNAR1 (Figures 36 and 37).

[0125] (Experimental Example 7) The high-affinity IL-2 receptor is composed of IL2Rα / β / γ subunits, which have an intracellular signaling domain that activates JAK1 / JAK3 and STAT5 homodimer signals. To confirm the usefulness of IFNARRS for the analysis of ligand-dependent receptor signaling, we created a chimeric receptor between IFNAR1 / IFNR2 and IL-2. As shown in Figure 38, the extracellular domain of IL2Rβ / γ subunits was fused to the transmembrane and intracellular domains of IFNAR2 and IFNAR1, respectively. In this case, fusion proteins were created in several patterns (Figure 39). Among them, the combination of the fusion membrane protein molecule (2) (IL2Rβ-EX-IFNAR2-TM-IN-240) and the fusion membrane protein molecule (7) (IL2Rγ-EX-IFNAR1-TM-IN-D83) was optimal for IL-2-dependent activation, and the signal was further enhanced by coexpression with the IL2RA subunit (Figure 40).

[0126] To obtain stable cell lines, expression plasmids of the fusion membrane protein molecules (2) and (7), IL2RA, 10xISRE-Nluc, and hygromycin B resistance gene were introduced into HEK293T cells. After selection with hygromycin B, the stable cell line 2LR-398, which showed the highest signal-to-noise ratio (IL-2-dependent Nluc activation) among several stable cell lines, was selected and established (Figure 41). To eliminate IFNα-dependent activation in 2LR-398 cells, the endogenous IFNAR1 gene was knocked out in the same manner as above. After selection with puromycin, the cell line 2LRk-134, which did not respond to IFNα and had the highest IL-2-stimulated Nluc activity among several stable cell lines, was established by knocking out the endogenous IFNAR1 (Figure 42). 2LRk-134 cells responded only to IL-2 in a dose-dependent manner (1-100U / mL) except at a very high concentration (1000pg / mL IFNγ), and did not respond to other cytokines (mean ± sd of three independent experiments). * indicates significant difference from control (*p<0.01, **p<0.0001). The luciferase intensity for 1000pg / mL IFNγ was almost the same as that for 1U / mL IL-2 production. Thus, IL-2 concentrations of 1U / mL or higher can be estimated for any sample using 2LRk-134 cells. Table 2 shows the expressed or knocked-out genes and biological characteristics of the stable cell lines.

[0127] [Table 2]

[0128] (Experimental Example 8) To produce IL-2 in response to IFNγ, 10xISRE-IL-2 and blasticidin S resistance genes were further introduced into the expression plasmid of GRk-281 cells. After selection with blasticidin S, the cell line GRkS-974, which showed the highest signal / noise ratio (IFNγ-dependent IL-2 production) among several stable cell lines, was established (Figure 43). The GRkS-974 cells secreted a large amount of IL-2 in a dose- and time-dependent manner in response to 10-1000 pg / mL IFNγ (Figure 44). The detection sensitivity for IFNγ was sufficient to detect normal activated T cells.

[0129] As shown in Figure 56, luciferase activity was not affected by other cytokines except high doses of TNFα (1000 pg / mL). The intensity of luciferase activity was limited (3.4-fold) compared to the same dose of IFNγ (210-fold), and the sensitivity to obtain an adequate response was 1 / 100 of that of IFNγ. Therefore, IFNγ appears to be the only practical ligand to activate the chimeric receptor.

[0130] To further evaluate the specificity and sensitivity of IL-2 secreted by GRkS-974 in response to IFNγ, mouse CTLL-2 cells, which show IL-2-dependent proliferation, were used to confirm the results. When CTLL-2 cells were co-cultured with GRkS-974 cells, CTLL-2 cells significantly proliferated in a dose-dependent manner in response to 30 to 300 pg / mL of IFNγ (Figures 45 to 46, in Figure 46, * indicates significant difference from the control (*p<0.01)). CTLL-2 cells did not proliferate by IFNγ treatment, which means that CTLL-2 cells responded to IL-2 secreted by GRkS-974 cells stimulated by IFNγ.

[0131] We investigated whether GRkS-974 cells could actually detect T cells activated by various stimuli. In addition to CD8α-EGFP and TCRα / β specific to MHC class I with NY-ESO-1 peptide antigen, CD3ε and CD3ζ were sequentially introduced into Jurkat cells by infection with the respective recombinant lentiviruses. The vector for producing the recombinant virus was created by introducing the construct shown in Figure 47. Jurkat cells expressing CD8α and anti-NY-ESO-1-TCRα / β were co-cultured with GRkS-974 cells and stimulated with PMA and calcium ionophore, or anti-CD3ε antibody. GRkS-974 cells showed a significant increase in luciferase activity in response to these stimuli (Figure 48 left). When GRkS-974 cells were preincubated with NY-ESO-1 peptide to confer endogenous MHC class I, co-culture with Jurkat cells expressing CD8α and anti-NY-ESO-1-TCR increased luciferase activity in a NY-ESO-1 peptide-dose-dependent manner (Figure 48, right). Figure 49 shows the characteristics of GRkS-974 cells stably expressing the indicated molecules.

[0132] An experiment was carried out to detect Jurkat-CD8 / anti-ESO-TCR cells activated with PMA / Ca by IFNARRS using GRkS-974 cells. Since ELISA and ELISpot assays are commonly used to detect activated T cells, an experiment was carried out to detect Jurkat-CD8 / anti-ESO-TCR cells activated with PMA / Ca by ELISA as a comparative experiment (Figure 57). Compared with ELISA, IFNARRS had a higher detection sensitivity for Jurkat-CD8 / anti-ESO-TCR cells activated with PMA / Ca (Figures 58-59, * indicates a significant difference from the control (*p<0.0001)).

[0133] As shown in Figure 60, a comparative experiment was performed using a given number of Jurkat-CD8 / anti-ESO-TCR cells treated with PMA / Ca or anti-CD3ε antibody. PMA / Ca stimulation induced a much stronger response than anti-CD3ε antibody stimulation. In both IFNARRS and ELISpot using GRkS-974 cells, Jurkat cells activated with PMA / Ca showed a significantly higher response than 10 2 The number of Jurkat cells activated with anti-CD3ε antibody was as low as 10 cells / ml, but only in the IFNARRS of the present invention, the number of Jurkat cells activated with anti-CD3ε antibody was 10 4 were detected (Figures 61 to 62, * indicates a significant difference from the control (*p<0.01, *p<0.0001)).

[0134] In general, when T cells are strongly activated with PMA / Ca, the ELISpot assay can detect single T cells as clear spots. However, when T cells are activated with weak stimuli such as anti-CD3ε antibody, only a small amount of IFNγ is secreted from the T cells. 4 It is difficult to detect clear spots in an ELISpot assay using a relatively large number of T cells, i.e., 10 cells / mL. The IFNARRS of the present invention is a clear spot obtained by detecting 10 T cells activated with an anti-CD3ε antibody. 4 In other words, the IFNARRS of the present invention shows almost the same sensitivity as ELISpot and is superior to ELISpot in detecting T cells activated by weaker stimuli.

[0135] (Experimental Example 9) T cells, such as cytotoxic T cells and Th1 cells, secrete IFNγ and IL-2 when activated. Stable GRkS-974 cells secrete large amounts of IL-2 in response to 10-1000pg / mL IFNγ in a dose-dependent manner. Recently, it has been reported that several costimulatory molecules act on the activation and proliferation of naive T cells by dendritic cells (DCs). Therefore, Jurkat cells expressing TCR specific to the NY-ESO-1 peptide were nonspecifically activated with anti-CD3ε antibody or specifically activated with HEK293T cells bearing MHC class I to which the NY-ESO-1 peptide was applied. In this case, the effect of the B7-1 costimulatory molecule was also examined.

[0136] When Jurkat cells were stimulated with anti-CD3ε antibody, B7-1 significantly enhanced anti-CD3ε antibody or anti-CD3ε-scFv-Sc-dependent activation (Figure 50). Expression of B7-1 also enhanced NY-ESO-1 peptide-dependent activation (Figure 51). * indicates significant difference from control (*p<0.01, **p<0.001). Therefore, we created plasmids expressing B-7, secreted anti-CD3ε-scFv (anti-CD3ε-scFv-Sc), and cell membrane-bound anti-CD3ε-scFv (anti-CD3ε-scFv-TM) under the 10×ISRE-dependent promoter (Figure 52). When these were transiently transfected and B7-1 and anti-CD3ε-scFv-TM were co-expressed in an ISRE-dependent manner by IFNγ stimulation, luciferase activity increased synergistically (Figure 53). * indicates significant difference from the control (*p<0.01). Therefore, it was considered that GRkS-974 cells can be used to activate and proliferate T cells like DCs (FIG. 29).

[0137] To verify the findings from the above transient expression and to confirm the effect of simple co-culture of B7-1 and anti-CD3ε-scFv-TM, two stable cell lines (GRkSB-043 and GRkSBZ-188) were established expressing B7-1 and anti-CD3ε-scFv-TM in response to IFNγ. Both GRkSB-043 and GRkSBZ-188 cells expressed B7-1 and anti-CD3ε-scFv-TM in response to IFNγ (Figures 63 and 64). Furthermore, in GRkSBZ-188 cells, functional ZZ-affibodies that bind to the Fc region of IGHG1 (immunoglobulin heavy constant gamma 1) of NlucP-IGHG1 and anti-HER2-affibody-IGHG1 were expressed on the cell surface, in addition to B7-1 and anti-CD3ε-scFv-TM (Figures 65 to 67, * indicates significant difference from the control (*p<0.0001, **p<0.000001)).

[0138] Consistent with the results of the transient experiment (Figure 50), IFNγ treatment further promoted Nluc activation in GRkSB-043 and GRkSBZ-188 cells by co-culture with Jurkat cells, but activation was unchanged in GRkS-974 (Figure 70, * indicates significant difference from control (*p<0.01, **p<0.001)). This effect was due to the activation of Jurkat cells by expression of B7-1 and anti-CD3ε-scFv-TM in GRkSB-043 and GRkSBZ-188 cells, but not in GRkS-974 cells.

[0139] To investigate the activation of TCR in T cells, NFAT-RE (Nuclear Factor of Activated T Cells-Response Element) using NlucP gene as a reporter is commonly used. In this example, Jurkat cells stably expressing NlucP under the control of 6xNFAT-RE (Jurkat-NFAT-NlucP) were used to evaluate the effect of Jurkat cells by the expression of B7-1 and anti-CD3ε-scFv-TM in HEK293T cells (Figures 68-69, * indicates significant difference from the control (*p<0.001, **p<0.00001)). When co-cultured with Jurkat-NFAT-Nluc instead of Jurkat cells used in Figure 70, GRkSB-043 cells and GRkSBZ-188 cells showed a significant increase in luciferase activity, but GRkS-974 cells did not show such an increase (Figure 71, * indicates a significant difference from the control (***p<0.0001)).

[0140] The difference between Jurkat-NFAT-Nluc cells and Jurkat cells reflected the Nluc activity from NFAT-Nluc, indicating B7-1 and anti-CD3ε-scFv-TM dependent activation of Jurkat-NFAT-Nluc cells. Furthermore, GRkSBZ-188 cells expressing ZZ-affibody enhanced the activation of Jurkat-NFAT-Nluc cells by recruiting anti-CD3ε antibody to the cell surface (Figure 72, * indicates significant difference from control (***p<0.0001)). By utilizing a specific antibody that recognizes the target T cells, GRkSBZ-188 cells can enhance their activation (Figure 73).

[0141] Finally, a stable cell line, GRkh-117 cells, was established by knocking out endogenous HLA-A, HLA-B, HLA-C, and endogenous IFNAR1 in GR-535 cells. Each HLA genotype of interest was transfected and re-expressed to present specific antigens with the HLA molecules. Table 3 shows the expressed or HLA knocked-out genes and biological characteristics of GRkh-117 cells. First, the lower diagrams of Figures 74 to 75 show that endogenous IFNAR1 is knocked out in both GRk-281 cells and GRkh-117 cells. Next, in contrast to the GRk-281 cells in the upper panel of Figure 74, the HLA knockout GRkh-117 cells in the upper panel of Figure 75 did not show any reaction to the transfected NY-ESO-1 cDNA or NY-ESO-1 peptide administration because they have no endogenous HLA in Jurkat cells expressing anti-NY-ESO-1-scFv-28BBZ (Figure 47). However, the reaction to the peptide-HLA-A2 complex was restored when the HLA-A2 gene was simultaneously transfected. These results indicate the possibility of activating T cells corresponding to individual HLA molecules.

[0142] [Table 3] [Explanation of symbols]

[0143] 1,1'...cell, 11,21...first fusion membrane protein molecule, 12,22...second fusion membrane protein molecule, 13,23...ISRE-inducible expression unit, 14...cell membrane, 15...target substance, 111...intracellular domain of IFNAR1, 112,212...first transmembrane domain, 113,213...first extracellular domain, 121...intracellular domain of IFNAR2, 122,222...second transmembrane domain, 123,223...second extracellular domain, 211...first intracellular domain, 221...second intracellular domain

Claims

1. a first fusion membrane protein molecule comprising at least a portion of the intracellular domain of interferon alpha and beta receptor 1, a first transmembrane domain, and a first extracellular domain; A cell comprising in its cell membrane a second fusion membrane protein molecule comprising at least a portion of the intracellular domain of interferon alpha and beta receptor 2, a second transmembrane domain, and a second extracellular domain, A cell, wherein the first extracellular domain and the second extracellular domain are capable of binding directly or indirectly.

2. The cell of claim 1 , wherein the first transmembrane domain has an amino acid sequence derived from the transmembrane domain of the interferon alpha and beta receptor 1.

3. The cell of claim 2, wherein the first fusion membrane protein molecule further comprises a portion of the extracellular domain of interferon alpha and beta receptor 1, the portion of the extracellular domain being located between the first extracellular domain and the first transmembrane domain.

4. The cell of claim 1 , wherein the first extracellular domain and the second extracellular domain are capable of binding to a target substance via a target substance sandwiched between them.

5. The cell according to claim 4 , wherein the target substance is a complex protein to which two or more proteins are bound.

6. The cell of claim 4 , wherein the target substance comprises a cytokine, an antibody, or an affibody.

7. The cell according to claim 6, wherein the cytokine is at least one of interferon gamma, interleukin-1, interleukin-2, interleukin-4, interleukin-6, interleukin-10, interleukin-12, interleukin-17, interleukin-18, tumor necrosis factor-alpha, and transforming growth factor-beta.

8. The cell of claim 1 , wherein endogenous interferon alpha and beta receptor 1 are knocked out.

9. The cell according to claim 1, wherein the cell is a HEK293T cell (human fetal kidney cell-derived cell), a HEK293 cell (human fetal kidney cell-derived cell), a HeLa cell (human cervical cancer-derived cell), an A549 cell (human lung cancer-derived cell), an MCF-7 cell (human breast cancer-derived cell), or an A431 cell (human epithelial cell carcinoma-derived cell).

10. The cell of claim 1, further comprising an interferon responsive element (ISRE) and an ISRE-inducible expression unit comprising at least one nucleotide sequence encoding a protein.

11. The cell of claim 10, wherein a plurality of ISRE-inducible expression units are introduced, and the nucleotide sequences contained in the plurality of ISRE-inducible expression units are different from each other.

12. The cell of claim 10, wherein the ISRE-inducible expression unit comprises four or more interferon response elements.

13. The cell of claim 11 , wherein the nucleotide sequence encodes a protein involved in a signaling response.

14. The cell according to claim 13 , wherein the protein involved in the reaction that emits the signal is one of luciferase, green fluorescent protein, red fluorescent protein, phosphatase and peroxidase.

15. The cell of claim 10 , wherein the nucleotide sequence encodes at least one protein of a molecule that controls cell proliferation, activation and differentiation and a molecule that controls cell-cell interactions.

16. The cell according to claim 15, wherein the proliferation-regulating molecule is interleukin-2.

17. The cell according to claim 15, wherein the molecule that controls cell differentiation is one of interferon gamma, interleukin-4, interleukin-6, interleukin-10, interleukin-12, interleukin-17, interleukin-23, tumor necrosis factor-alpha, and transforming growth factor-beta.

18. The cell according to claim 15, wherein the molecule that controls the activation of the cell is at least one of costimulatory molecules that promote activation, such as B7-1, B7-2, CD40, and 4-1BB ligand, and co-inhibitory molecules that suppress activation, such as CTLA4, PD-L1, and galectin-9.

19. The cell according to claim 15, wherein the molecule that controls intercellular interactions is a cell membrane-bound anti-CD4-scFv-TM, anti-CD4-CCR6-scFv-TM, or ZZ-affibody-TM.

20. the first extracellular domain and the second extracellular domain are capable of binding to a target substance via a target substance sandwiched therebetween; The cell of claim 10 , wherein the target substance comprises a protein having the same structure as the protein.

21. Using the cell according to claim 1, directly or indirectly binding the first extracellular domain to the second extracellular domain in the cell; and bringing at least a portion of the intracellular domain of the interferon α and β receptor 1 and at least a portion of the intracellular domain of the interferon α and β receptor 2 into close proximity through the binding, thereby causing a nucleotide sequence encoded downstream of the interferon response element present in the cell to be transcribed, thereby expressing a protein.

22. a first expression construct comprising a first promoter for expression functional in a cell, and a nucleotide sequence operably linked to said first promoter, the nucleotide sequence encoding a first fusion membrane protein molecule comprising at least a portion of an intracellular domain of interferon alpha and beta receptor 1, a first transmembrane domain, and a first extracellular domain; An expression construct, wherein the first extracellular domain is capable of binding to a target substance other than interferon α and β.

23. 23. The expression construct of claim 22, wherein the nucleotide sequence encoding the first transmembrane domain is a nucleotide sequence encoding the transmembrane domain of interferon alpha and beta receptor 1.

24. 23. The expression construct of claim 22, wherein the first fusion membrane protein molecule further comprises a portion of the extracellular domain of interferon alpha and beta receptor 1, and a nucleotide sequence encoding the portion of the extracellular domain is located between the nucleotide sequence encoding the first extracellular domain and the nucleotide sequence encoding the first transmembrane domain.

25. a second expression construct comprising a second promoter for expression functional in the cell, and a nucleotide sequence operably linked to the second promoter, the nucleotide sequence encoding a second fusion membrane protein molecule comprising at least a portion of the intracellular domain of interferon alpha and beta receptor 2, a second transmembrane domain, and a second extracellular domain; The expression construct of claim 22 , wherein the first extracellular domain and the second extracellular domain are capable of binding directly or indirectly.

26. The expression construct of claim 25 , wherein the first extracellular domain and the second extracellular domain are capable of binding to the target substance via the target substance.

27. The expression construct of claim 26, wherein the target substance is a complex protein in which two or more proteins are bound.

28. 27. The expression construct of claim 26, wherein the targeting agent comprises a cytokine, an antibody, or an affibody.

29. 29. The expression construct of claim 28, wherein the cytokine is at least one of interferon gamma, interleukin-1, interleukin-2, interleukin-4, interleukin-6, interleukin-10, interleukin-12, interleukin-17, interleukin-18, tumor necrosis factor-alpha, and transforming growth factor-beta.

30. 23. The expression construct of claim 22, wherein the expression construct is a plasmid vector-based expression construct.

31. A transformed cell into which the expression construct according to any one of claims 22 to 30 has been introduced.

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