Immune control method of cells for transplantation

Abstract

The purpose is to provide a method for suppressing rejection by NK cells in cells or a tissue for transplantation. Provided is a method for producing cells or a tissue for transplantation, the method comprising a step for expressing, on the surface of cells for transplantation or cells constituting a tissue for transplantation, at least one membrane-type stimulating antibody specific to an inhibitory receptor for NK cells. Preferably, the method comprises a step of expressing a membrane-type stimulating antibody specific to each of one or more, for example, three or more, for example, four inhibitory receptors selected from the group consisting of NKG2A, KIR2DL1, KIR2DL3, KIR3DL1 and LILRB1. The present invention further provides pluripotent stem cells and T cells or progenitor cells thereof in which a gene encoding the membrane-type stimulating antibody is introduced or expressed. The present invention further provides an antibody specific to an inhibitory receptor for NK cells.

Description

Method for immune regulation of transplant cells

[0001] The present disclosure relates to a method for suppressing NK cell-mediated rejection of cells or tissues for transplantation.

[0002] Currently, regenerative medicine is primarily based on allogeneic transplantation. However, this inevitably leads to the problem of immune rejection by the recipient. The primary mechanism of immune rejection is when the immune system recognizes HLA that differs from the recipient's as foreign. Therefore, HLA-matched transplants are recommended. The iPS Cell Stock Project provided by the iPS Cell Research Foundation employs a strategy of transplanting regenerated tissue derived from HLA haplotype homozygous strains into HLA heterozygous recipients. If these iPS cells are used as materials, they can be used as "ready-to-ship cell preparations" in cases where the HLA is matched. However, to cover 70% of the Japanese population, 70 types of homozygous strains would be required, making it far from being universally applicable.

[0003] To avoid HLA-induced rejection, it has been proposed to use HLA-knockout cells as universal cells for allogeneic transplantation. Transplanting HLA-knockout cells can significantly reduce T cell-mediated rejection. However, it is known that NK cells detect the loss of HLA class I molecules and attack them. Suppressing this NK cell attack has been the key to creating universal transplant cells.

[0004] "Technologies that have been proposed to prevent rejection by NK cells despite a lack of HLA include a strategy to prevent rejection by NK cells by forcibly expressing HLA-E and stimulating the inhibitory receptor NKG2A (Non-Patent Document 1: Nat. Biotech, 35:765, 2017), and the use of iPS cells that retain only one HLA-C (Non-Patent Document 2: Cell Stem Cell, 24:566, 2019). However, these are clearly insufficient, as they only address a portion of the inhibitory receptors.

[0005] In addition to the above, other methods that have been reported include a method of expressing CD47, a "don't eat-me signal" (Non-Patent Document 3), a method of expressing CD47, PD-L1, and HLA-G in HLA-deficient cells (Non-Patent Document 4), and a method of knocking out CD155, a ligand for the NK cell activating receptor DNAM1 (Non-Patent Document 5).

[0006] Some of the present inventors have proposed a method for preparing cultured cells or tissues for transplantation, which includes, when there is a mismatch between the cultured cells or tissues for transplantation and a specific HLA of the recipient, expressing a specific HLA molecule in the cultured cells or tissues for transplantation based on information about the mismatch (Patent Document 1).

[0007] NK cells are known to randomly express several types of inhibitory receptors (Non-Patent Document 6). None of the methods proposed in the prior art can be said to provide sufficient control.

[0008] JP 2019-004702 A, WO 2020 / 022512, WO 2022 / 065444

[0009] Nat. Biotech, 35:765, 2017Cell Stem Cell. 24:566, 2019Nat. Biotechnol. 37:252-258, 2019Proc Natl Acad Sci USA 116: 10441-10446, 2019Nat Biomed Eng 5: 429-440, 2021Blood 2008, 112: 2366-2380

[0010] The present disclosure aims to provide a method for avoiding or suppressing attack by recipient NK cells against transplanted cells or tissues during allogeneic cell or tissue transplantation. The present disclosure also aims to provide a membrane-type antibody useful for such a method and a vector for expressing the membrane-type antibody. The present disclosure also aims to provide pluripotent stem cells useful for producing cells or tissues for transplantation. The present disclosure further aims to provide T cells or their precursors that are suitable for use in transplantation.

[0011] The present disclosure provides a method for producing cells or tissues for transplantation, which comprises the step of expressing one or more membrane-type stimulatory antibody molecules specific to NK cell inhibitory receptors on the surface of the cells or tissues for transplantation.

[0012] The present application provides a membrane-type stimulatory antibody specific to an NK cell inhibitory receptor, which has an antigen-binding site specific to the NK cell inhibitory receptor and a transmembrane domain.

[0013] The present disclosure also provides a vector for producing cells or tissues for transplantation, which comprises one or more genes encoding membrane-type stimulatory antibody molecules specific to an inhibitory receptor on NK cells.

[0014] The present disclosure also provides pluripotent stem cells into which one or more membrane-type stimulatory antibody molecules specific to NK cell inhibitory receptors have been genetically introduced or expressed. The present disclosure further provides transplantable cells and transplantable tissues induced to differentiate from such pluripotent stem cells. Examples of transplantable tissues include tissue fragments, organoids, and organs. Examples of pluripotent stem cells include ES cells and iPS cells. An example, but not limited to, is the ES cell line SEES3.

[0015] The present disclosure also provides T cells or T cell precursors for transplantation, which express one or more membrane-type stimulatory antibody molecules specific to an NK cell inhibitory receptor on their cell surface. The T cells or T cell precursors may be those differentiated from pluripotent stem cells into which a membrane-type stimulatory antibody specific to an NK cell inhibitory receptor has been genetically introduced or expressed, or may be T cells obtained from a living body and allowed to express a membrane-type stimulatory antibody specific to an NK cell inhibitory receptor.

[0016] The pluripotent stem cells or T cells provided in the present disclosure into which one or more membrane-bound stimulatory antibody molecules specific to an NK cell inhibitory receptor have been genetically introduced or expressed may have a cassette deck structure containing a cassette tape gene encoding a marker protein in its genome so that the marker protein can be expressed. Such cassette deck structures are disclosed in Patent Documents 2 and 3.

[0017] Specifically, the present application provides the following: [1] A method for producing cells or tissues for transplantation, comprising the step of expressing one or more membrane-type stimulatory antibodies specific to an NK cell inhibitory receptor on the surface of cells constituting the cells or tissues for transplantation. [2] The method described in [1], comprising the step of introducing genes encoding one or more membrane-type stimulatory antibodies specific to an NK cell inhibitory receptor into cells constituting the cells or tissues for transplantation, or into precursor cells thereof, or expressing the membrane-type stimulatory antibodies. [3] The method described in [1] or [2], wherein the NK cell inhibitory receptor is selected from the group consisting of NKG2A, KIR2DL1, KIR2DL3, KIR3DL1, and LILRB1. [4] The method described in any of [1] to [3], wherein the membrane-type stimulatory antibody has an antigen-binding site and a transmembrane site of an antibody specific to an NK cell inhibitory receptor. [5] The method of [4], wherein the antigen-binding site of the antibody specific to an NK cell inhibitory receptor is an scFv. [6] The method of any of [1] to [5], wherein the cells constituting the cells or tissue for transplantation are cells lacking any or all of the HLAs. [7] The method of any of [4] to [6], wherein the transmembrane site is that of a classical class I HLA or a non-classical class I HLA. [8] The method of [7], wherein the transmembrane site is that of HLA-E. [9] The method of any of [1] to [8], wherein the cells or tissue for transplantation are cells or tissue induced from pluripotent stem cells.

[10] The method of [9], wherein the cells or tissue for transplantation are cells or tissue obtained by inducing differentiation from pluripotent stem cells lacking any or all of the HLAs.

[11] The method of any of [1] to

[10] , wherein the cells for transplantation are T cells.

[12] The method according to any one of [1] to

[11] , comprising the step of introducing into pluripotent stem cells genes encoding one or more membrane-type stimulatory antibodies specific to an NK cell inhibitory receptor, or expressing the membrane-type stimulatory antibodies.

[0018]

[13] A membrane-type stimulating antibody specific to an NK cell inhibitory receptor.

[14] The membrane-type stimulating antibody according to

[13] , wherein the NK cell inhibitory receptor is selected from the group consisting of NKG2A, KIR2DL1, KIR2DL3, KIR3DL1, and LILRB1.

[15] The membrane-type stimulating antibody according to

[13] or

[14] , wherein the membrane-type stimulating antibody has an antigen-binding site of an antibody specific to an NK cell inhibitory receptor and a transmembrane site.

[16] The membrane-type stimulating antibody according to

[15] , wherein the antigen-binding site of the antibody specific to an NK cell inhibitory receptor is an scFv.

[17] A nucleic acid molecule encoding the membrane-type stimulating antibody specific to an NK cell inhibitory receptor according to any one of

[13] to

[16] .

[18] A vector comprising the nucleic acid molecule according to

[17] .

[0019]

[19] A pluripotent stem cell into which one or more membrane-type stimulatory antibodies specific to an NK cell inhibitory receptor have been genetically introduced or expressed.

[20] The pluripotent stem cell of

[19] , wherein the NK cell inhibitory receptor is selected from the group consisting of NKG2A, KIR2DL1, KIR2DL3, KIR3DL1, and LILRB1.

[21] The pluripotent stem cell of

[19] or

[20] , wherein the pluripotent stem cell is a cell obtained by induction from a donor with a homozygous HLA haplotype.

[22] The pluripotent stem cell of any of

[19] to

[21] , wherein the pluripotent stem cell lacks any or all HLA.

[23] The pluripotent stem cell according to any one of

[19] to

[22] , wherein the membrane-binding stimulatory antibody has an antigen-binding site of an antibody specific to an inhibitory receptor of NK cells and a transmembrane site.

[24] The pluripotent stem cell according to

[23] , wherein the antigen-binding site of the antibody specific to an inhibitory receptor of NK cells is an scFv.

[25] The pluripotent stem cell according to any one of

[19] to

[24] , wherein the pluripotent stem cell is derived from the SEES3 strain.

[0020]

[26] A T cell for transplantation or its precursor cell, expressing one or more membrane-type stimulatory antibodies specific to an NK cell inhibitory receptor on the cell surface.

[27] The T cell or its precursor cell according to

[26] , wherein the NK cell inhibitory receptor is selected from the group consisting of NKG2A, KIR2DL1, KIR2DL3, KIR3DL1, and LILRB1.

[28] The T cell or its precursor cell according to

[26] or

[27] , wherein the membrane-type stimulatory antibody has an antigen-binding site and a transmembrane site of an antibody specific to an NK cell inhibitory receptor.

[29] The T cell or its precursor cell according to

[28] , wherein the antigen-binding site of the antibody specific to an NK cell inhibitory receptor is an scFv.

[30] The T cell or its precursor cell according to any of

[26] to

[29] , wherein the T cell has a homozygous HLA haplotype.

[31] The T cell or precursor cell thereof according to any one of

[26] to

[30] , which is deficient in any or all of HLA.

[32] The T cell or precursor cell thereof according to any one of

[26] to

[31] , which is induced to differentiate from a pluripotent stem cell.

[33] The T cell or precursor cell thereof according to

[32] , wherein the pluripotent stem cell is a SEES3 strain.

[0021]

[34] A membrane-stimulating antibody specific for an NK cell inhibitory receptor, comprising an antigen-binding site having any one selected from the following combinations of light chain hypervariable regions and heavy chain hypervariable regions, and a transmembrane region:

[35] The membrane-stimulating antibody according to

[34] , wherein the antigen-binding site is an scFv.

[36] The membrane-stimulating antibody according to

[35] , which is a polypeptide having any of the following sequences:

[37] A nucleic acid sequence encoding the membrane-type stimulatory antibody according to any one of

[34] to

[36] .

[38] A vector having the nucleic acid sequence according to

[37] .

[39] A pluripotent stem cell comprising one or more genes encoding the membrane-type stimulatory antibody according to any one of

[34] to

[38] , or expressing one or more of said membrane-type stimulatory antibodies.

[40] The pluripotent stem cell according to

[39] , which is an ES cell.

[41] The pluripotent stem cell according to

[40] , which is a SEES3 strain.

[0022] More specifically, the present application provides the following: (1) A method for producing cells or tissues for transplantation, comprising the step of expressing, on the surface of cells constituting the cells or tissues for transplantation, three or more membrane-type stimulatory antibodies specific for three or more NK cell inhibitory receptors selected from the group consisting of NKG2A, KIR2DL1, KIR2DL3, KIR3DL1, and LILRB1. (2) The method according to (1), in which four membrane-type stimulatory antibodies specific for the NK cell inhibitory receptors NKG2A, KIR2DL1, KIR2DL3, and KIR3DL1, respectively, are expressed. (3) The method according to (1) or (2), in which the membrane-type stimulatory antibodies have the antigen-binding site of the antibody specific for the NK cell inhibitory receptor and a transmembrane site. (4) The method according to (3), in which the antigen-binding site of the antibody specific for the NK cell inhibitory receptor is an scFv. (5) The method according to any one of (1) to (4), wherein the cells constituting the transplant cells or tissue for transplant are cells in which any or all of the HLAs have been deleted. (6) The method according to any one of (3) to (5), wherein the transmembrane site is that of a classical class I molecular type HLA or a non-classical class I molecular type HLA. (7) The method according to (6), wherein the transmembrane site is that of HLA-E. (8) The method according to any one of (1) to (7), wherein the transplant cells or tissue are cells or tissue induced from pluripotent stem cells. (9) The method according to (8), wherein the transplant cells or tissue are cells or tissue obtained by inducing differentiation from pluripotent stem cells in which any or all of the HLAs have been deleted. (10) The method according to any one of (1) to (9), wherein the transplant cells are T cells. (11) The method according to any one of (1) to (10), wherein the membrane-stimulating antibody has an antigen-binding site comprising light chain CDR1, light chain CDR2, and light chain CDR3 of the light chain hypervariable region and heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 of the heavy chain variable region in a combination of a light chain hypervariable region and a heavy chain hypervariable region selected from the table below.

[0023] (12) A pluripotent stem cell into which three or more membrane-bound stimulatory antibodies specific for three or more NK cell inhibitory receptors selected from the group consisting of NKG2A, KIR2DL1, KIR2DL3, KIR3DL1, and LILRB1 have been genetically introduced or expressed. (13) The pluripotent stem cell according to (12), into which four membrane-bound stimulatory antibodies specific for the NK cell inhibitory receptors NKG2A, KIR2DL1, KIR2DL3, and KIR3DL1, respectively, have been genetically introduced or expressed. (14) The pluripotent stem cell according to (12) or (13), which is a cell obtained by induction from a donor with a homozygous HLA haplotype. (15) The pluripotent stem cell according to any one of (12) to (14), which is a cell in which any or all of HLA has been deleted. (16) The pluripotent stem cell according to any one of (12) to (15), wherein the membrane-type stimulating antibody has the antigen-binding site and a transmembrane site of an antibody specific to an inhibitory receptor of NK cells. (17) The pluripotent stem cell according to (16), wherein the antigen-binding site of the antibody specific to an inhibitory receptor of NK cells is an scFv. (18) The pluripotent stem cell according to any one of (12) to (17), wherein the pluripotent stem cell is derived from the SEES3 strain. (19) The pluripotent stem cell according to any one of (12) to (18), wherein the membrane-type stimulating antibody has an antigen-binding site comprising light chain CDR1, light chain CDR2, and light chain CDR3 of the light chain hypervariable region and heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 of the heavy chain variable region, in any one combination of light chain hypervariable region and heavy chain hypervariable region shown in the table below.

[0024] (20) A T cell for transplantation or its precursor cell, on the cell surface of which are expressed three or more membrane-type stimulatory antibodies specific for three or more NK cell inhibitory receptors selected from the group consisting of NKG2A, KIR2DL1, KIR2DL3, KIR3DL1, and LILRB1. (21) A T cell for transplantation or its precursor cell according to (20), on which are expressed four membrane-type stimulatory antibodies specific for the NK cell inhibitory receptors NKG2A, KIR2DL1, KIR2DL3, and KIR3DL1, respectively. (22) A T cell for transplantation or its precursor cell according to (20) or (21), in which the membrane-type stimulatory antibody has the antigen-binding site and transmembrane site of an antibody specific for an NK cell inhibitory receptor. (23) The T cell for transplantation or its precursor cell according to (22), wherein the antigen-binding site of the antibody specific to an inhibitory receptor of NK cells is scFv. (24) The T cell for transplantation or its precursor cell according to any of (20) to (23), wherein the cells constituting the cells for transplantation or the cells for transplantation tissue are cells in which any or all of HLA has been deleted. (25) The T cell for transplantation or its precursor cell according to any of (20) to (24), wherein the transmembrane site is that of a classical class I molecule-type HLA or a non-classical class I molecule-type HLA. (26) The T cell for transplantation or its precursor cell according to (25), wherein the transmembrane site is that of HLA-E. (27) The T cell for transplantation or its precursor cell according to any of (20) to (26), wherein the cells or tissue for transplantation are cells or tissue induced from pluripotent stem cells. (28) The T cell for transplantation or its precursor cell according to (27), wherein the cell or tissue for transplantation is a cell or tissue obtained by inducing differentiation from a pluripotent stem cell in which any or all of HLA has been deleted. (29) The T cell for transplantation or its precursor cell according to any of (20) to (28), wherein the membrane-type stimulatory antibody comprises an antigen-binding site comprising light chain CDR1, light chain CDR2, and light chain CDR3 of the light chain hypervariable region and heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 of the heavy chain variable region in a combination of a light chain hypervariable region and a heavy chain hypervariable region selected from the table below.

[0025] (30) An antibody specific for an inhibitory receptor of NK cells, having an antigen-binding site comprising light chain CDR1, light chain CDR2, and light chain CDR3 of the light chain hypervariable region and heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 of the heavy chain variable region in any one of the combinations of light chain hypervariable region and heavy chain hypervariable region shown in the table below. (31) The antibody specific to an NK cell inhibitory receptor according to (30), which is a membrane-stimulating antibody further comprising a transmembrane domain. (32) The antibody according to (31), whose antigen-binding domain is an scFv. (33) The membrane-stimulating antibody according to (32), which is a polypeptide having any of the following sequences: (34) A nucleic acid encoding the antibody according to any one of (30) to (33). (35) A vector comprising the nucleic acid molecule according to (34).

[0026] Schematic diagram of membrane-type antibody structures having tetramers, dimers, and scFvs as antigen-binding sites. Overview of reporter assay. Time axis of reporter assay. Overview of CD107a assay. Time axis of CD107a assay. Reporter assay results for membrane-type anti-KIR2DL1 antibodies whose antigen-binding sites are tetramers or dimers. Reporter assay results for membrane-type anti-KIR2DL3 antibodies whose antigen-binding sites are tetramers or dimers. Reporter assay results for two types of membrane-type anti-NKG2A antibodies whose antigen-binding sites are dimers. Reporter assay results for three types of membrane-type anti-LILRB1 antibodies whose antigen-binding sites are dimers. Reporter assay results for membrane-type anti-KIR2DL1 antibodies whose antigen-binding sites are dimers and scFv-type. Reporter assay results for membrane-type anti-NKG2A antibodies whose antigen-binding sites are dimers and scFv-type. Reporter assay results for a membrane-type anti-LILRB1 antibody whose antigen-binding site is a dimer and scFv type. Reporter assay results for a membrane-type bound antibody whose antigen-binding site is an scFv type anti-NKG2A-9-1-7 and whose transmembrane domain is PILR or HLA-E. The relationship between the inhibitory receptors of NK cells focused on in Example 4 and the ligands that recognize them. FACS profile when effector NK cells were selected from PBMCs of a healthy person with a C1 / C1, Bw4- profile. CD107a assay results for a membrane-type bound antibody whose antigen-binding site is an scFv type anti-NKG2A clone 9-1-7 and whose transmembrane domain is PILR or HLA-E. Reporter assay results for two clones (9-1-7, 32-1-7) of membrane-stimulating anti-NKG2A antibodies whose antigen-binding site is an scFv-type anti-NKG2A antibody and whose membrane-binding region is HLA-E. Reporter assay results for an scFv-type anti-KIR2DL1 antibody (KIR9) whose antigen-binding site is an scFv-type anti-KIR2DL1 antibody whose membrane-binding region is HLA-E. Reporter assay results for an scFv-type anti-KIR2DL3 antibody (KIR13, KIR37) whose antigen-binding site is an scFv-type anti-KIR2DL3 antibody whose membrane-binding region is HLA-E. Relationship between NK cell inhibitory receptors and ligands focused on in Example 7(1). FACS profile when NK cells for CD107a assay were selected from PBMCs of C1 / C1, Bw4- healthy subjects.

[0039] Figure 1 shows the relationship between NK cell inhibitory receptors and ligands focused on in Example 7(2) in the CD107a assay results for membrane-stimulatory NKG2A antibody clones 9-1-7 and 32-1-7. FACS profile of NK cells selected for CD107a assay from C2 / C2 and Bw4-PBMCs. CD107a assay results for membrane-stimulatory anti-KIR2DL1 antibody clone KIR9. The relationship between NK cell inhibitory receptors and ligands focused on in Example 7(3). FACS profile of NK cells selected for CD107a assay from C1 / C1 and Bw4-PBMCs. CD107a assay results for membrane-stimulatory anti-KIR2DL3 antibody clones KIR13 and KIR37. The relationship between NK cell inhibitory receptors and ligands focused on in Example 7(4). FACS profile when NK cells for CD107a assay were selected from C1 / C1, Bw4+ PBMCs. CD107a assay results for membrane-stimulatory anti-KIR3DL1 antibody clones KIR34, KIR69, KIR103, and KIR114. FACS profile when NK cells for CD107a assay were selected from C1 / C1, Bw4+ PBMCs. CD107a assay results for membrane-stimulatory anti-KIR3DL1 antibody clones KIR34, KIR69, KIR103, and KIR114. Bw4 trimer was used as a positive control. Relationship between NK cell inhibitory receptors and ligands focused on in Example 8. FACS profile when NK cells for CD107a assay were selected from C2 / C2, Bw4- PBMCs.

[0033] Figure 1 shows the results of a CD107a assay using regenerated CTLs co-expressing a membrane-stimulatory anti-NKG2A antibody and a membrane-stimulatory anti-KIR2DL1 antibody. An overview of the NK cell killing assay. The relationship between the NK cell inhibitory receptor and its ligand, which is the focus of Example 9. The FACS profile when NK cells for the killing assay were selected from PBMCs obtained from a C1 / C1, Bw4- healthy volunteer. The results of the killing assay of Example 9 (cell death rate). The results of the killing assay of Example 9 (cell viability rate). The relationship between the NK cell inhibitory receptor and its ligand, which is the focus of Example 10. The FACS profile when NK cells for the killing assay were selected from C2 / C2, Bw4- PBMCs.Results of the killing assay of Example 10 (cell death rate). Results of the killing assay of Example 10 (cell viability rate). Results of PCR electrophoresis of the ES cell genome into which a gene for a membrane-stimulating antibody whose antigen-binding site is an scFv anti-KIR2DL1 (KIR9) antibody and whose transmembrane domain is derived from HLA-E has been introduced. Schematic diagram of the process leading up to the production of super-universal regenerative CTLs. Marker gene introduction process and expression confirmation for luciferase gene-introduced strains. Schematic diagram of the gene introduction sequence. Marker gene expression analysis after introduction of an NK inhibitory antibody gene sequence into ES cells. NK inhibitory antibody marker gene expression analysis of super-universal regenerative CTLs after induction of differentiation from super-universal ES cells. Schematic diagram of the system for evaluating the killing activity of super-universal regenerative CTLs. HLA. ＋ Killing analysis results of no mAb regenerated CTL. HLA ＋ Killing analysis results of 4mAb regenerative CTLs. Killing analysis results of HLA-dKO no mAb regenerative CTLs. Killing analysis results of HLA-dKO 4mAb regenerative CTLs (super-generative regenerative CTLs). Schematic diagram of CD107a assay to measure the inhibitory effect of super-generative regenerative CTLs on NK activity. Schematic diagram of CD107a assay time axis. C2 / C2 Bw4 － NK cell licensing environment. C2 / C2 Bw4 － FACS profile of NK cells. C2 / C2 Bw4 － CD107a assay results when NK cells and regenerated CTLs were co-cultured. C1 / C1 Bw4 － NK cell licensing environment. C1 / C1 Bw4 － FACS profile of NK cells. C1 / C1 Bw4 － CD107a assay results when NK cells and regenerated CTLs were co-cultured. C1 / C1 Bw4 ＋ NK cell licensing environment. C1 / C1 Bw4 ＋ FACS profile of NK cells. C1 / C1 Bw4 ＋ CD107a assay results when NK cells and regenerated CTLs were co-cultured. C1 / C2 Bw4 ＋ NK cell licensing environment. C1 / C2 Bw4 ＋FACS profile of NK cells. C1 / C2 Bw4 ＋ CD107a assay results when NK cells and regenerated CTLs were co-cultured.

[0027] The present disclosure provides a method for producing cells or tissues for transplantation, which includes a step of expressing one or more membrane-type stimulatory antibody molecules specific to an inhibitory receptor on an NK cell on the surface of cells constituting the cells or tissue for transplantation.

[0028] In this disclosure, an "antibody" is an immunoglobulin molecule that binds to an antigen. An immunoglobulin molecule is composed of one or more tetramers of two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. The light chains of an immunoglobulin can be of type kappa or lambda. Immunoglobulin molecules may be of any type (including, but not limited to, IgA, IgG, IgE, IgD, or IgM, as well as their subtypes). The amino-terminal portion of each chain contains a variable region of about 100 to about 110 amino acids primarily responsible for antigen recognition via the complementarity-determining regions (CDRs) contained therein. The carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function.

[0029] The CDRs are interspersed with more conserved regions called frame regions (FRs). Each light chain variable region (LCVR, also known as VL) and heavy chain variable region (HCVR, also known as VH) is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The three CDRs of the light chain are referred to as "CDR-L1, CDR-L2, and CDR-L3," and the three CDRs of the heavy chain are referred to as "CDR-H1, CDR-H2, and CDR-H3." The CDRs contain most of the residues that form specific interactions with the antigen.

[0030] As used herein, the term "monoclonal antibody" (mAb) refers to an antibody derived from a single copy or clone, including, for example, any eukaryotic, prokaryotic, or phage clone. The mAbs of the present disclosure preferably exist in a homogeneous or substantially homogeneous population. An intact mAb contains two heavy chains and two light chains. Monoclonal antibodies can be produced, for example, by hybridoma technology, recombinant technology, phage display technology, synthetic technology, such as CDR grafting, or a combination of such techniques or other techniques known in the art.

[0031] In the present disclosure, the phrase "humanized antibody" refers to a monoclonal antibody that has a combination of the heavy and light chain CDRs provided in the present disclosure and that has been generated and / or engineered so that the framework regions surrounding the CDRs have framework sequences that are substantially similar or identical to those of human variants. Antibody humanization is well known in the art. The humanized antibodies provided in the present disclosure may be intentionally modified compared to the native sequence, for example, in the constant region, to alter effector or biofunctional properties or biophysical properties (such as stability, developability, and / or solubility, among others).

[0032] A "framework region" or "framework sequence" refers to any one of framework regions 1-4. Engineered human antibodies and antigen-binding sites thereof encompassed by the present disclosure include molecules in which any one or more of framework regions 1-4 are substantially or fully human (i.e., any possible combination of individual substantially or fully human framework regions 1-4 is present). For example, this includes molecules in which framework region 1 and framework region 2, framework region 1 and framework region 3, framework regions 1, 2, and 3, etc., are substantially or fully human. A substantially human framework is one that has at least about 80% sequence identity to known human germline framework sequences. Germline sequences for human frameworks can be obtained from ImMunoGeneTics (IMGT) or from "The 20 Immunoglobulin Facts Book" by Marie-Paule Lefranc and Gerard Lefranc (Academic Press, 2001, ISBN 012441351).

[0033] The sequences of CDRs can be defined by various methods, including the IMGT definition (Lefranc et al., 2003, Dev Comparat Immunol 27:55-77), Kabat (Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md.), and Chothia (Al-Lazikani et al., 1997, J. Mol. Biol 273:927-948).

[0034] In the present disclosure, a "membrane-type antibody" refers to an antibody that is bound to a cell membrane or the membrane of a cell-derived vesicle or artificial vesicle. In the present disclosure, a "stimulatory antibody" refers to an antibody that acts as an agonist against a specific antigen, in this case, an NK cell inhibitory receptor, and has the ability to stimulate the activity of the NK cell inhibitory receptor. In the present disclosure, a "membrane-type stimulatory antibody" refers to an antibody that has both the properties of a membrane-type antibody and a stimulatory antibody.

[0035] When antibody molecules are secreted and in a soluble form, they often function to inhibit signal transduction. However, when soluble antibodies are expressed on the surface of the cell membrane as membrane-bound antibodies, they often function as stimulatory antibodies. In the present disclosure, a stimulatory antibody specific to an inhibitory receptor of NK cells is used in the form of a membrane-bound antibody.

[0036] To obtain a membrane-type stimulatory antibody molecule specific to an NK cell inhibitory receptor, first, an antibody specific to the target NK cell inhibitory receptor is obtained by a conventional method, and then the antigen-binding site of the obtained antibody is fused with a transmembrane domain to form a membrane-type antibody. The membrane-type antibody can be expressed on the cell membrane surface, and an antibody that specifically induces stimulation of the target NK cell inhibitory receptor can be selected.

[0037] In one embodiment, an animal is immunized with a protein of the inhibitory receptor of the target NK cell to obtain antibody-producing B cells. Among the obtained antibody-producing B cells, B cells that produce antibodies that bind to the target receptor but do not bind to other similar receptors are sorted using FACS. The affinity of the obtained antibodies for the target receptor is examined, and those with high affinity for the target receptor are selected. The selected antibodies are monocloned using standard methods, and a transmembrane site is attached to the monoclonal antibody to make it a membrane-type antibody. After making it a membrane-type antibody, it is then confirmed whether the antibody can induce stimulation of the target receptor.

[0038] To confirm whether a membrane-bound antibody is a stimulatory antibody against an inhibitory receptor on NK cells, reporter cells expressing the target inhibitory receptor are prepared. Examples of reporter cells include those that express an inhibitory receptor or a complex with the inhibitory receptor and emit fluorescence when bound to an antibody that exhibits agonistic activity against the receptor or complex (Hisashi Arase et al., Science (2002) Vol. 296, Issue 5571 pp. 1323-1326).

[0039] In the present specification and claims, the NK cell inhibitory receptor is not particularly limited as long as it is a known inhibitory receptor. Examples include NKG2A, KIR2DL1, KIR2DL3, KIR3DL1, and LILRB1, which are inhibitory receptors that have been reported to be important in NK cell-mediated rejection reactions, particularly in attacking HLA-deficient cells. (Blood 2008, 112: 2366-2380)

[0040] In the present disclosure, some of the NK cell receptors exhibit activity only after forming a complex with other membrane proteins. For example, NKG2A functions as an inhibitory receptor when it forms a complex with CD94. On the other hand, NKG2C functions as an activating receptor when it forms a complex with CD94. In the present disclosure, when an antibody against a certain protein is described, if the protein exhibits some activity only after forming a complex with another protein, it is understood to mean an antibody against the protein complex.

[0041] In the present application, the membrane-type stimulatory antibody molecules to be expressed on the cell surface may be an appropriate combination of membrane-type stimulatory antibody molecules against not only one but also two, three, four, or five of these five inhibitory receptors. Furthermore, other membrane-type stimulatory antibody molecules may be expressed in combination with one or more membrane-type stimulatory antibody molecules specific to NKG2A, KIR2DL1, KIR2DL3, KIR3DL1, and / or LILRB1.

[0042] In the present application, a plurality of membrane-bound stimulatory antibody molecules specific to three or more, more preferably four or more, inhibitory receptors selected from the group consisting of NKG2A, KIR2DL1, KIR2DL3, KIR3DL1, and LILRB1 are expressed on the cell surface. For example, four membrane-bound stimulatory antibody molecules specific to NKG2A, KIR2DL1, KIR2DL3, and KIR3DL1, respectively, are expressed on the cell.

[0043] In the present disclosure, a "membrane-stimulating antibody" is a polypeptide comprising an antibody antigen-binding site and a transmembrane region. The antibody antigen-binding site is a fragment comprising at least one heavy chain and one light chain hypervariable region contained in the Fab region of the antibody molecule. The antibody antigen-binding site may be a tetramer comprising two identical heavy chains and two identical light chains, a dimer comprising one heavy chain and one light chain, or a single-chain Fv (scFv) in which the hypervariable regions of the heavy chain and light chain are linked together. ScFv is particularly preferred as the antigen-binding site used in the membrane-stimulating antibody of the present disclosure. Methods for obtaining scFv from antibodies are known, and they can be obtained by conventional methods.

[0044] The present disclosure provides a membrane-stimulatory antibody specific to an inhibitory receptor on NK cells. In a membrane-stimulatory antibody, the antigen-binding site of the antibody and the transmembrane domain may be linked in any form, and are not limited thereto. If desired, when fusing the antigen-binding site of the antibody with the transmembrane domain, the transmembrane domain may be fused immediately downstream of the antigen-binding site of the antibody. Any amino acid sequence may be freely set as a linker between the fragment containing the antigen-binding site and the transmembrane domain. Examples of linkers include amino acid sequences with a length of about 2 to 10 amino acids.

[0045] For example, when the antigen-binding site of an antibody is a tetramer, the antigen-binding site can be a fragment comprising the Fab region of the antibody through the region after the hinge region of the Fc region, specifically the VL and CL regions of the light chain, the VH and CH1 regions of the heavy chain, and the hinge region connecting the heavy chains downstream of the CH1 region (just before the CH2 region). A membrane-type antibody can be obtained by fusing a transmembrane region downstream of the heavy chain hinge region.

[0046] Furthermore, when the antigen-binding site of an antibody is a dimer, a fragment containing the Fab region and the region immediately preceding the hinge region of the antibody, specifically the VL and CL regions of the light chain, the VH and CH1 regions of the heavy chain, and the region immediately preceding the hinge region connecting the heavy chains downstream of the CH1 region, can be used. A transmembrane region can be fused to such a fragment.

[0047] When the antigen-binding site of the antibody is an scFv, the Fab region of the light chain and the region just before the hinge region of the heavy chain are connected by a linker sequence commonly used in producing scFv to form the scFv. A conventionally known method for producing scFv antibodies may be used, and is disclosed, for example, in Protein Engineering, Design and Selection, Volume 6, Issue 8, 1993, Pages 989-995. The upstream side of the scFv may be either a light chain or a heavy chain.

[0048] As the membrane-stimulating antibody, a polypeptide containing an scFv and a transmembrane region is particularly preferably used.

[0049] The transmembrane region contained in the membrane-stimulating antibody of the present disclosure may be derived from a natural polypeptide or may be artificially designed. The transmembrane region derived from a natural polypeptide can be obtained from any membrane-bound or transmembrane protein. Examples of transmembrane regions include, but are not limited to, ADORA2A, CCR5, CCR8, CD133, CD151, CD20, CD28, CD37, CD81, Claudin-1, Claudin-18.1, Claudin-18.2, Claudin-4, Claudin-6, Claudin-9, CXCR3, CXCR4 ... CCR5, CCR8, CD133, CD151, CD20, CD28, CD37, CD81, CCR5, CCR8, CCR5, CCR8, CCR5, CCR8, CCR5, CCR8, CCR5, CCR8, CCR5, CCR8, CCR5, CCR5, CCR8, CCR5, CCR5, CCR5, CCR5, CCR5, CCR5, CCR5, CCR5, CCR5, CCR5, CCR5, CCR5, CCR5, CCR5, CCR5, CCR5, CCR5, CCR5, CCR5, CCR5, CCR5, CCR5, CCR5, CCR5, CCR5, CCR5, CCR5, CCR5, CCR5, CCR5, CCR5, CCR5, CCR5, CCR5, CCR5, CCR5, CCR5, CCR5, C Examples of such transmembrane domains include XCR5, GCGR, GLP1R, GPRC5B, GPRC5D, GPR43, GPR56, GPR64, GPR87, SLC1A2, STEAP1, and PILRα, as well as transmembrane domains derived from natural proteins of classical HLA class I molecules (HLA-A, B, and C) and non-classical HLA class I molecules (HLA-E, F, G, etc.). Among these, transmembrane domains of classical and non-classical HLA class I molecules, particularly HLA-E, are preferably used, and it has been confirmed in the Examples that the transmembrane domain of HLA-E is preferably used, for example.

[0050] Examples of membrane-stimulating antibodies provided by the present disclosure include those containing the combinations shown in the table below as the light chain hypervariable region and heavy chain hypervariable region of the antigen-binding site. Furthermore, examples of membrane-stimulating antibodies having an scFv as the antigen-binding site and an HLA-E transmembrane region as the transmembrane region include those shown in the table below.

[0051]

[0052] The present disclosure further provides membrane-stimulating antibodies having an antigen-binding site and a transmembrane region having CDR-L1, CDR-L2, and CDR-L3, and CDR-H1, CDR-H2, and CDR-H3, respectively, identified by a conventional method from the combination of light chain hypervariable region and heavy chain hypervariable region sequences in Table 8. Methods for identifying CDR sequences are well known to those skilled in the art, as described above, and CDR sequences may be identified by the IMGT definition, Kabat definition, Chothia definition, or any other known method.

[0053] The membrane-stimulating antibody of the present disclosure may have a humanized sequence having, as an antigen-binding site, CDR-L1, CDR-L2, and CDR-L3, CDR-H1, CDR-H2, and CDR-H3, which are identified by standard methods from the light chain hypervariable region and heavy chain hypervariable region sequences in Table 8, respectively, and may also have a transmembrane region.

[0054] The present disclosure further provides a nucleic acid encoding a membrane-type stimulatory antibody specific for an inhibitory receptor of NK cells. The nucleic acid may be DNA or mRNA.

[0055] When the antigen-binding site of the membrane-stimulating antibody of the present disclosure is a tetramer containing two heavy chains and two light chains, a nucleic acid is provided that combines a nucleic acid encoding a polypeptide containing a light chain hypervariable region with a nucleic acid encoding a polypeptide containing a heavy chain hypervariable region, a portion of the constant region, and a transmembrane region.When the antigen-binding site is an scFv, it may be provided as a single nucleic acid encoding a polypeptide containing the scFv and the transmembrane region.

[0056] The targets for expressing the membrane-type stimulatory antibody that serves as a ligand for the NK cell inhibitory receptor can be any cell type that is targeted for attack by NK cells, as well as their tissues or small organs (organoids), or structures such as extracellular secretory endoplasmic reticulum (exosomes) or artificial vesicles that can activate the NK cell inhibitory receptor by expressing the membrane-type stimulatory antibody protein in their surrounding environment. When NK cell inhibitory receptors are stimulated by expressing the antibody in a lipid bilayer membrane such as exosomes or artificial endoplasmic reticulum, it is preferable that the exosomes or artificial endoplasmic reticulum be one that is specifically directed toward the tissue to be transplanted.

[0057] There are no particular limitations on the cells for transplantation, and they include not only cells currently used in transplantation therapy, but also cells that will be used in transplantation therapy in the future. Examples of cells for transplantation include, but are not limited to, hematopoietic stem cells, T cells, endothelial cells, and organ primordium cells. Examples of tissues for transplantation include tissue fragments, organoids, and organs. Both the cells for transplantation and the tissues for transplantation may be cells or tissues obtained from another person, or cultured cells or tissues. Cultured cells or tissues may be those induced to differentiate from pluripotent stem cells.

[0058] Furthermore, in the present specification and claims, the terms "cells for transplantation" and "tissues for transplantation" are intended to include not only cells that have differentiated to a state that can be used for transplantation as is, but also their precursor cells, for example, precursor cells that have been induced to differentiate from pluripotent stem cells.

[0059] The membrane-stimulatory antibody of the present disclosure can be expressed on the cell membrane by introducing a polypeptide comprising the antigen-binding site of the antibody and a transmembrane region, or a nucleic acid encoding the polypeptide, into cells for transplantation or cells constituting tissue for transplantation or their precursor cells.

[0060] The method for expressing the membrane-stimulatory antibody of the present disclosure in cells is not particularly limited, and known methods can be used. When a plasmid vector is introduced, for example, calcium phosphate coprecipitation, PEG method, electroporation, microinjection, lipofection, etc. can be used. When a viral vector is used, the vector can be introduced into cells by infecting the cells with the vector using an appropriate method depending on the viral vector.

[0061] mRNA, plasmid DNA, or the protein itself may be encapsulated in an endoplasmic reticulum-like structure, such as a liposome or exosome, and then introduced into cells by electroporation or lipofection to express a membrane-type antibody or transport the introduced protein onto the cell membrane. When encapsulating these antibodies in vesicles or artificial vesicles derived from cells expressing the antibodies, or when using vesicles expressed on a membrane to prevent malfunction, the antibodies can also be expressed in transplant cells by using them as carriers that are directed toward the target to be protected.

[0062] The present disclosure also provides vesicles encapsulating a nucleic acid or polypeptide of the present disclosure.

[0063] The present disclosure provides an expression vector for expressing a nucleic acid of the present disclosure in a cell. The vector of the present disclosure includes at least a nucleic acid encoding a membrane-stimulating antibody and a promoter. If desired, the vector may also include transcriptional and translational regulatory sequences, ribosome binding sites, enhancers, replication origins, poly(A) addition signals, selectable marker genes, and the like.

[0064] The vector used in the present application may be appropriately selected from vectors used in genetic recombination, and examples thereof include vectors such as viruses, plasmids, and artificial chromosomes. Examples of viral vectors include retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, and Sendai viral vectors. Examples of artificial chromosome vectors include human artificial chromosomes (HAC), yeast artificial chromosomes (YAC), and bacterial artificial chromosomes (BAC, PAC). Plasmids for mammalian cells may be used as the plasmid. Commercially available vectors may be appropriately selected and used depending on the purpose.

[0065] Examples of promoters that can be used include an EF1α promoter, a CAG promoter, a ubiquitin promoter, an SRα promoter, an SV40 promoter, an LTR promoter, a CMV (cytomegalovirus) promoter, an RSV (Rous sarcoma virus) promoter, an MMLV (Moloney murine leukemia virus) LTR, an HSV-TK (herpes simplex virus thymidine kinase) promoter, a TCR Vα gene promoter, and a TCR Vβ gene promoter.

[0066] A vector containing the nucleic acid of the present disclosure may contain only one type of nucleic acid encoding a membrane-type stimulatory antibody specific to an NK cell inhibitory receptor, or may contain nucleic acids encoding two or more types of membrane-type stimulatory antibodies. When containing nucleic acids encoding two or more types of membrane-type stimulatory antibodies specific to an NK cell inhibitory receptor, the vector may be configured so that each polypeptide is expressed, and the polypeptides may be expressed in any order.

[0067] In order to permanently protect cells or tissues for transplantation from attack by NK cells of the recipient, it is preferable to constitutively express the membrane-type stimulatory antibody of the present disclosure. In the case of therapies involving transplantation of cells or tissues derived from pluripotent stem cells, cell preparations such as CAR-T cells, or cells or other organs such as allogeneic bone marrow cells, it may be preferable to constitutively protect the cells or tissues for transplantation from attack by NK cells.

[0068] When it is desired to temporarily protect cells or tissues for transplantation from attack by recipient NK cells, i.e., when it is acceptable for the expression of membrane-type inhibitory antibodies to disappear and be attacked by the recipient after the cells or tissues for transplantation have exerted their effects, or when the cells or tissues are used in a therapy that expects this, it is preferable to transiently express the membrane-type stimulatory antibodies of the present disclosure. Examples of cells that transiently express the membrane-type stimulatory antibodies of the present disclosure include cells and tissues derived from pluripotent stem cells, cell preparations such as CAR-T cells, and cell grafts of allogeneic bone marrow cells and other organs. Therapies that involve transient expression include, but are not limited to, immune cell therapies using regenerative T cells that are not intended to be permanently established, allogeneic CAR-T cells or bone marrow cells, and cell therapies using mesenchymal stem cells and their progenitor cells that exert their effects through immunomodulation or paracrine regulation.

[0069] First, in order to constitutively express a membrane-stimulatory antibody, one method is to use pluripotent stem cells, which are made from differentiated cells derived from ES / iPS cells or a cell population composed of such cells, by arranging the antibody gene so that it is expressed under the control of a tissue-independent ubiquitous promoter sequence such as ubiquitin, EF1-α, or CAG, and then incorporating the gene into the chromosome using a retroviral or lentiviral vector, and then differentiating these into cells to be transplanted, such as T cells, to be used as a therapeutic cell preparation.

[0070] On the other hand, whether in autologous, allogeneic, or tissue cells, adenovirus vectors can be used to transiently express membrane-bound antibodies without incorporating them into the chromosomes of target cells. Furthermore, there are methods for expressing membrane-bound antibodies by directly introducing mRNA, plasmid DNA, or proteins into target cells or cells via vesicle-like structures using electroporation or lipofection.

[0071] Furthermore, when using exosomes encapsulated in the vesicles of these cells or expressed on the vesicle membrane, or artificial vesicles into which antibody proteins have been properly incorporated, it is preferable to impart a mechanism for targeting to the transplant recipient, which may allow them to be used as a safe cell-free therapeutic agent that can avoid signal induction of unintended NK cells. The type of expression form may be appropriately selected depending on the purpose of transplantation, the type of cells or tissue to be transplanted, etc.

[0072] When the cells or tissues to be transplanted are cells or tissues induced to differentiate from pluripotent stem cells, the membrane-type stimulatory antibodies of the present disclosure may be transfected or expressed into pluripotent stem cells and then induced to differentiate, or may be transfected or expressed into cells constituting the cells or tissues induced to differentiate from pluripotent stem cells. Furthermore, the antibodies may be transfected or expressed into progenitor cells that are currently being induced to differentiate from pluripotent stem cells into cells or tissues that can be used for transplantation. Many methods for inducing differentiation from pluripotent stem cells into various cells have been proposed, and a method may be selected appropriately depending on the cell type of interest. Furthermore, methods for inducing differentiation of various progenitor cells induced from pluripotent stem cells into the desired mature cells are known to those skilled in the art.

[0073] In the present disclosure, when cells expressing a membrane-type stimulatory antibody are transplanted, it is preferable to knock out the HLA of the transplant cells to avoid graft rejection. If a certain level of HLA matching is confirmed between the donor of the transplant cells and the patient receiving the transplant, and if immunosuppressive treatment is administered in advance, or if measures are taken such as generating and returning regulatory T cells that recognize the transplanted cells to the body, a certain degree of immunosuppressive effect can be expected, and by expressing the membrane-type stimulatory antibody of the present disclosure in such cells, safer transplantation can be performed.

[0074] The iPS cell stock project is currently being vigorously promoted in Japan. In this project, for example, HLA haplotype homozygous iPS cells are generated from donors homozygous for HLA-A, HLA-B, and HLA-DR, and stocked in order of increasing frequency. Plans are also underway to perform genome editing on the haplotype homozygous iPS cell stock to stock iPS cells in which HLA-A / B / CIITA have been knocked out. This plan aims to reduce the risk of rejection by NK cells by preserving HLA-C, HLA-E, and other HLA-related genes. The project aims to confirm safety and distribute stable iPS cells to research and medical institutions for widespread use in regenerative medicine. Cells obtained from this iPS cell stock may be used as iPS cells for use in the present disclosure. Furthermore, pluripotent stem cells made versatile by knocking out HLAs other than those mentioned above using genome editing technology may also be used.

[0075] To stably express the membrane-stimulatory antibody of the present disclosure in pluripotent stem cells, a gene for the membrane-stimulatory antibody may be positioned so that it is expressed under the control of a tissue-independent ubiquitous promoter sequence such as ubiquitin, EF1-α, or CAG, and the gene for the membrane-stimulatory antibody may be integrated into the chromosomes of the pluripotent stem cells using a retroviral or lentiviral vector. By previously incorporating a nucleic acid encoding the membrane-stimulatory antibody of the present disclosure into an iPS cell stock with HLA homozygous or partial or complete HLA knockout, the pluripotent stem cells can be differentiated into various cells for use in transplantation.

[0076] Whether cells constituting the cells or tissues express the desired membrane-type stimulatory antibody can be confirmed by conventionally known methods such as ELISA, flow cytometry, and ELISPOT.

[0077] The cells expressing the membrane-type stimulatory antibody specific to the inhibitory receptor of NK cells of the present disclosure stimulate the inhibitory receptor of NK cells of the recipient during transplantation, thereby preventing the transplanted cells or tissue from being attacked by NK cells. Therefore, by testing the HLA of the recipient before transplantation therapy, the inhibitory receptor of NK cells that is easily activated can be predicted, and the membrane-type stimulatory antibody specific to the inhibitory receptor of NK cells that is easily activated can be expressed in the cells or endoplasmic reticulum to be transplanted, or in the cells that constitute the tissue to be transplanted.

[0078] Furthermore, whether cells expressing the membrane-stimulatory antibodies of the present disclosure can avoid attack by NK cells can be confirmed by, as shown in the Examples, reporter assays of cells expressing the target inhibitory receptor of interest, or by assessing the suppression of CD107a expression, an NK cell degranulation marker. Furthermore, signal strength and duration can also be assessed by observing immunosynapse formation with cells expressing the target antigen and related signals. Using these methods, it is possible not only to evaluate the quality of cell preparations before transplantation, but also to evaluate the efficacy and safety of administered patient biological samples in advance.

[0079] The present application will be further described in detail by the following examples, which are for illustrative purposes only and are not intended to limit the invention of the present disclosure in any way.

[0080] The test methods for each example are described below. (A) Preparation of NK cell inhibitory receptor-specific membrane-type antibodies. I) Preparation of NK cell inhibitory receptor-specific antibodies. <Anti-NKG2A antibodies> 1: Because immunization was required as a complex of NKG2A and CD94, mouse B16 cells were transfected with NKG2A and CD94 in the pME18S vector using PEI (Polysciences) as a transfection reagent, and mice were immunized with the transfectants. Titermax Gold (TiterMax) was used as an adjuvant during immunization. 2: Mouse spleens were removed, and all spleen cells were hemolyzed to prepare hybridomas. 3: Hybridomas that recognized NKG2A and CD94 transfectants but not NKG2C and CD94 transfectants were screened. 4: The BCR gene sequence was analyzed from specifically binding hybridoma clones. 5: Soluble antibodies were prepared and identified that specifically bound to NKG2A+CD94 expressed in human 293T cells. 6: The VH+VL regions of the obtained antibodies, as well as a portion of the constant region and the transmembrane region, were used to construct membrane-bound antibody sequences. 7: Plasmid vectors containing DNA encoding the obtained membrane-bound antibodies were prepared and used in assays.

[0081] <Anti-KIR antibodies> 1: Fc-fusions were prepared by linking the Fc region of human IgG to the extracellular domain of KIR molecules, and mice were immunized with each KIR-Fc-fusion molecule. Titermax Gold (TiterMax) was used as an adjuvant. 2: The spleens of immunized mice were removed, hemolyzed, and splenocytes were collected. B cells that specifically bound to the target KIR-Fc-fusion were sorted. For example, when obtaining antibodies specifically binding to KIR2DL1, we selected those that bound only to KIR2DL1, but not to KIR2DL3 / KIR2DS1 / 2DS2 / 2DS3 / 2DS4 / 2DS5. Single-cell RNA sequencing / repertoire analysis was performed to analyze the obtained antibody genes. 3: Sequence information showing an increase in two or more BCR clones was prioritized, and IgGs were selected. 4: Soluble antibodies were produced from the selected antibodies and confirmed to bind specifically to each KIR molecule expressed in human 293T cells. 5: The VH+VL regions of the clones that specifically bound to the target KIR were used to create a membrane-type antibody sequence.

[0082] II) Preparation of Membrane-Bound Antibody Sequences From the obtained antibodies, antigen-binding sites of tetramer, dimer, and scFv structures were prepared, and these were linked to transmembrane regions to express membrane-bound antibodies on the membrane of human 293T cells. Each structure is shown in Figure 1. The tetramers were prepared by constructing an expression vector containing a nucleic acid sequence encoding the human immunoglobulin κ chain (light chain) of the obtained antibody, and a nucleic acid sequence encoding a protein comprising the heavy chain antigen recognition region (V region) of the obtained antibody, followed by the CH1 region to CH2 region or the region immediately following the hinge (AH), and a transmembrane region downstream thereof, and further downstream containing a gene encoding EGFP as a marker. These vectors were then expressed in 293T cells by lipofection. A plasmid vector was used as the expression vector.

[0083] The dimer was prepared by preparing an expression vector containing a nucleic acid sequence encoding the human immunoglobulin κ chain (light chain) of the obtained antibody, and a nucleic acid sequence encoding a protein comprising the sequence (BH) of the Fc region following the heavy chain antigen recognition region (V region) of the obtained antibody up to the hinge and the transmembrane region downstream of that, and further downstream of that a gene encoding EGFP as a marker, and expressing both vectors in 293T cells by lipofection.

[0084] The scFv was obtained by constructing an expression vector containing a nucleic acid sequence encoding a protein in which the light chain hypervariable region and heavy chain hypervariable region were linked with a linker in accordance with standard methods, the human IgG antibody heavy chain Fc region up to the hinge (BH) was linked downstream of the heavy chain hypervariable region, and a transmembrane region was linked downstream of that, and further downstream of that a gene encoding EGFP as a marker was created, and expressing it in 293T cells by lipofection.

[0085] III) Lipofection of membrane-bound stimulatory antibodies into cells (preparation of stimulator cells) Viafect (Promega) was used as the lipofection reagent. The cocktails listed in the table below were prepared and incubated at room temperature for 15 minutes. They were then added to subconfluent 293T cells in a 6-well plate or 10 cm dish and incubated at 37°C with 5% CO2. The medium was replaced on Day 1, and 293T cells were harvested on Day 2. The GFP+ fraction of 293T cells was then used in the reporter assay shown below in (B) using a FACS Aria II (BD).

[0086]

[0087] (B) Reporter Assay Method I) Preparation of Reporter Cells An overview of the reporter assay is shown in Figure 2. Reporter cells were generated from CT237 cells, a mouse T cell line, which express GFP in an NFAT-dependent manner via ITAM upon stimulation (Hisashi Arase et al., Science (2002) Vol. 296, Issue 5571 pp. 1323-1326). The resulting cells were then transfected with KIR / NKG2A / LILR molecules to serve as reporter cells for stimulatory antibodies. NKG2A molecules were co-expressed with CD94. Candidate membrane-binding stimulatory antibodies were transfected into 293T cells by lipofection, and the GFP+ fraction, a marker gene, was sorted to identify cells expressing stimulatory membrane-binding antibodies (stimulator cells). After 24 hours of co-culture, the reporter cells were analyzed for GFP expression by flow cytometry to assess the stimulatory potency of the membrane-binding stimulatory antibodies (Figure 2). During the measurement, BV421-anti-mouse CD45 (Biolegend) was used to distinguish between mouse cells, CT237, and human cells, 293T.

[0088] II) Reporter Assay Procedure The reporter assay timeline is shown in Figure 3. Stimulator cells obtained by lipofection in (A) III) above were co-cultured with reporter cells for the reporter assay. Co-culture (Day 2) 293T cells were harvested, and the GFP+ fraction of 293T was sorted using a FACS Aria II (BD). 1x10^4 cells / 50μl / well were seeded into a 96-well plate for use as a stimulator. After cell counting, reporter cells were seeded into a 96-well plate at 1x10^4 cells / 50μl / well (stimulator:reporter ratios of 0.3:1, 1:1, and 3:1). After seeding, the cells were co-cultured for 24 hours at 37°C with 5% CO2.

[0089] Reporter assay (Day 3) The signal strength of the inhibitory antibody was evaluated by detecting GFP in the reporter cells using a FACS Canto II (BD). To distinguish between mouse CT237 cells and human 293T cells, BV421-anti-mouse CD45 (Biolegend) was diluted 1 / 200. Propidium iodide (PI) was also added at 1 / 1000 for FACS analysis to remove dead cells.

[0090] (C) CD107a assay (verification of NK cell suppression effect) The CD107a assay is an assay to examine whether cells expressing membrane-binding stimulatory antibodies on the T cell membrane actually have the function of suppressing NK cells. An overview of the CD107a assay is shown in Figure 4.

[0091] I) Construction of retroviral vectors for membrane-stimulating antibody expression. Day 0: 1 mL / well of 0.1% gelatin PBS(-) was added to a 12-well plate and incubated at 37°C for at least 30 minutes. After washing with PBS-, platE cells (S. Morita et al., Gene Therapy (2000) 7, 1063-1066) were seeded at 0.3 x 10^6 / mL / well into a 24-well plate. After 6 hours at 37°C in 5% CO2, the cocktails listed in the table below were prepared and incubated at room temperature for 15 minutes. The cocktails were then added to the platE cells and incubated at 37°C in 5% CO2.

[0092] platE medium: DMEM high-Glucose(nacalaitesque), puromycin: 1μg / ml (gibco), blasticidin: 10μg / ml (invivogen), FCS: 10%

[0093] gag / pol RRE: viral structural protein VSV-G: Vesicular Stomatitis Virus Glycoprotein (for envelope) REV: viral structural protein

[0094] Day 1: Change of plate medium. PG13 cells (A. Dusty Miller et al., Biotechniques. 1989 October; 7(9): 980-990.) were seeded onto a new plate at 3000 cells / mL / well (24-well plate). PG13 medium: DMEM high-glucose (nacalaitesque), gentamycin: 50 μg / mL (Gibco), FCS: 10%

[0095] Days 2-5: The platE supernatant was collected using a syringe and passed through a 0.45 μm filter. 0.4 μL of 10 mg / mL polybrene was added to 0.5 mL of the collected virus-containing supernatant. The mixture was kept on ice for 15 minutes (mixing by inversion every 5 minutes), then placed at room temperature for 15 minutes (mixing by inversion every 5 minutes). The PG13 supernatant was discarded and replaced with the platE-virus supernatant. Spin-infection was performed at 32°C, 2500 rpm, and 90 minutes. After 60 minutes of incubation at 37°C in 5% CO2, the mixture was replaced with 1 mL of PG13 medium and incubated at 37°C in 5% CO2. This process was repeated daily for 4 days.

[0096] After the 4-day infection process, once PG13 cells reached confluence, the supernatant was either used directly or collected in a cryotube, flash-frozen in liquid nitrogen, and stored at -80°C. When needed, the cells were thawed and cultured in PG13 cell medium. The culture supernatant containing the retroviral vector containing the membrane-bound antibody was used for gene transfer.

[0097] II) NK cell suppression effect verification method by CD107a expression level analysis (CD107a assay)

[0098] We induced regenerated CTLs (cytotoxic T lymphocytes, also known as CD8 T cells) from pluripotent stem cells using standard methods, and then used retroviral vectors to express membrane-bound stimulatory antibodies, which have scFv antibodies as their antigen-binding sites.

[0099] The regenerated CTL cells were used as virtual donors (targets) and NK cells sorted from peripheral blood of healthy individuals were used as virtual recipients (effectors) in co-culture, and the expression level of the degranulation marker CD107a on NK cells was analyzed after 6 hours. The time axis of the CD107a assay is shown in Figure 5.

[0100] Regenerated CTL stimulation: Day 7: Anti-CD3 Ab: 1 μg / ml (Invitrogen), Anti-CD28 Ab: 1 μg / ml (Invitrogen), Retronectin: 5 μg / ml (Takara Bio) were adjusted with PBS (-) and coated onto a non-treated 24-well plate at 300 μl / well. After incubation at 37°C for at least 1 hour, the plate was washed with PBS (-). Regenerated CTLs were suspended in the following reCTL medium and added to each well at 5 x 10^5 cells / mL / well. The plate was then incubated at 37°C and 5% CO2. *reCTL medium: 20% FCS / αMEM + IL7: 5 ng / ml (Peprotech) + IL21: 10 ng / ml (Peprotech) + IL-2: 10 ng / mL (Peprotech) + VitC: 100 μM (Nacalaitesque).

[0101] Day 5 - Gene transfection: As a preliminary preparation, a 24-well plate (non-treated) was coated with 300 μL / well of PBS(-) + 10 ng / μL of RetroNectin. After incubation at 37°C for at least 1 hour, the plate was washed twice with PBS(-). 500 μL / well of retroviral supernatant was added (2000 G, 32°C, 2 hr) to allow the retrovirus to adsorb to the wells. After washing twice with PBS(-), an equal volume of reCTL stimulated on Day 7 was added to each well, and spin-infection was performed at 1000 G, 32°C, 10 min. The plate was then incubated at 37°C under 5% CO2.

[0102] Thereafter, half of the medium was replaced with reCTL medium every 2-3 days.

[0103] Day 0: NK cell isolation. PBMCs collected from healthy volunteers were treated with the necessary FACS antibodies, placed on ice for 20 minutes, and washed. The cells were then collected in a FACS tube and sorted using a FACS Aria II. The list of antibodies used for sorting is shown below.

[0104] NK cell collection tubes were centrifuged and suspended at approximately 5x10^5 cells / well in the following NK cell culture medium, and then seeded into a 24-well plate (NK medium: 20% FCS, PC / SM 1%:aMEM: 1mL + IL2: 20ng / ml (peprotech) + IL15: 10ng / ml (peprotech) + VitC: 100uM (nacalaitesque)). The plate was left at 37℃, 5% CO2 until Day 5.

[0105] FACS Antibody List

[0106] - Stimulation of regenerated CTLs: Anti-CD3 Ab: 1 μg / ml (Invitrogen), Anti-CD28 Ab: 1 μg / ml (Invitrogen), Retronectin: 5 μg / ml (Takara Bio) were adjusted with PBS (-), and 300 μl of each solution was coated onto a non-treated 24-well plate at the required number of wells and incubated at 37°C for at least 1 hour.

[0107] For sorting of membrane-type transfected cells, cells were collected in FACS tubes, centrifuged, and then sorted using a FACS Aria with 1 / 1000 PI added to remove dead cells. After centrifugation of the collected tubes, the coated wells were washed with PBS(-), suspended in reCTL medium, and added to the coated wells at 5x10^5 cells / mL / well and incubated at 37°C under 5% CO2.

[0108] Day 2: Release of stimulation from regenerated CTLs The cell culture medium was collected from each well, centrifuged, suspended in 1 mL / well of reCTL medium, and plated onto a new 24-well plate (normal).

[0109] Day 5: Co-culture of NK cells and regenerated CTLs. 40 μl / well of NK cells from healthy donors were added as effectors, 50 μl / well of membrane-bound antibody-expressing regenerated CTLs were added as targets, and 10 μl / well of an antibody cocktail (20% FCS PC / SM 1% aMEM containing APC-anti-CD107a antibody at 1 / 20 (Biolegend) + Golgistop at 1 / 150 (BD Biosciences) + GolgiPlug at 1 / 100 (BD Biosciences)) was added to a 96-well V-bottom plate. The effector:target ratio was adjusted appropriately, with the cell number adjusted accordingly. After mixing the cells and antibody cocktail, the plate was incubated at 1200 rpm for 5 min and then 6 hr at 37°C in 5% CO2. After 5 minutes at 1200 rpm, the supernatant was discarded, and BV421-CD3 (1 / 200), PE-Cy7-CD56 (1 / 200), and Fixable viability dye efluor780 (eBioscience) (1 / 1000) were added. After incubation on ice for 20 minutes, the cells were washed. The cells were then loaded into a FACS tube and analyzed for CD107a expression using Aria (BD).

[0110] Structure of the antigen-binding site of membrane-associated stimulatory antibodies: comparison of tetramers and dimers (1) Anti-KIR2DL1 antibodies. 293T cells expressing membrane-associated antibodies with tetrameric and dimeric structures were prepared for anti-KIR2DL1 antibody clones 1-6-4 and 1-8-1, and the stimulatory potency of each membrane-associated stimulatory antibody was compared by reporter assay. The transmembrane domain of mouse PILRa was used as the transmembrane domain. Each membrane-type antibody is referred to as anti-2DL1-6-4-CH2mIg (membrane-type tetramer of clone 1-6-4, up to the CH2 region), anti-2DL1-6-4-BHmIg (membrane-type dimer of clone 1-6-4, up to the region before the hinge region), anti-2DL1-8-1-CH2mIg (membrane-type tetramer of clone 1-8-1, up to the CH2 region), and anti-2DL1-8-1-BHmIg (membrane-type dimer of clone 1-8-1, up to the region before the hinge region).

[0111] The results are shown in Figure 6. The antigen-binding site of the membrane-stimulatory anti-KIR2DL1 antibody stimulated reporter cells better in the dimer form than in the tetramer form.

[0112] (2) Anti-KIR2DL3 antibodies. 293T cells expressing membrane-bound antibodies with tetrameric and dimeric antigen-binding sites were prepared for the anti-KIR2DL3 antibody clones 1-3-3 and 5-2-9, respectively, and the stimulatory potency of each membrane-bound antibody was compared by reporter assay. The transmembrane domain of mouse PILRa was used as the transmembrane domain. Each membrane-type antibody is referred to as anti-2DL3-1-3-3-AHmIg (membrane-type tetramer of clone 1-3-3, post-hinge), anti-2DL3-1-3-3-BHmIg (membrane-type dimer of clone 1-6-4, up to the hinge region), anti-2DL3-5-2-9-AHmIg (membrane-type tetramer of clone 5-2-9, up to the CH2 region), and anti-2DL3-5-2-9-BHmIg (membrane-type dimer of clone 5-2-9, up to the hinge region). The results are shown in Figure 7. Regarding the antigen-binding site of the membrane-type stimulatory anti-KIR2DL3 antibody, the dimer stimulated reporter cells better than the tetramer.

[0113] (3) Comparison of Anti-NKG2A and Anti-LILRB1 Clones (Dimers) Two anti-NKG2A clones, 9-1-7 and 32-1-7, and three anti-LILRB1 clones, 31-3, 15-1, and 2644, were used to express dimeric membrane-bound antibodies with antigen-binding sites using the sequence up to the pre-hinge region (before the BH, SS-binding site) and the transmembrane region of mouse PILRa. The stimulatory potency of each antibody was compared by reporter assay. The results are shown in Figures 8 and 9.

[0114] Among the two anti-NKG2A antibody clones, 9-1-7 and 32-1-7, 9-1-7 showed a stronger stimulatory potency. Among the three anti-LILRB1 antibody clones, 31-3, 15-1, and 2644, all clones stimulated reporter cells at a stimulator:reporter ratio of 3:1, but clone 31-3 was found to stimulate reporter cells most strongly at a low ratio.

[0115] Structure of the antigen-binding site of membrane-bound stimulatory antibodies: Comparison of stimulatory potency between dimer and scFv structures. The stimulatory potency of membrane-bound antibodies with dimer and scFv structures as the antigen-binding site of anti-KIR2DL1 antibody 1-8-1, anti-NKG2A antibody 9-1-7, and anti-LILRB1 antibody 31-3 was compared. Using 293T cells expressing membrane-bound antibodies with dimer or scFv antigen-binding sites derived from the anti-KIR2DL1 antibody 1-8-1, anti-NKG2A antibody 9-1-7, and anti-LILRB1 antibody 31-3 clones, and with the transmembrane domain of mouse PILRa as the transmembrane domain, the stimulatory potency of dimers and scFvs was compared. The results are shown in Figures 10 to 12.

[0116] As shown in Figure 10, for the anti-KIR2DL1 antibody, membrane-type antibodies with scFv as the antigen-binding site exhibited stronger stimulatory potency than membrane-type antibodies with dimers. As shown in Figure 11, for the anti-NKG2A antibody, the scFv structure stimulated reporter cells comparable to the dimer structure. As shown in Figure 12, for the anti-LILRB1 antibody, membrane-type antibodies with scFv as the antigen-binding site exhibited stronger stimulatory potency than membrane-type antibodies with dimers. Based on these results, we decided to adopt the scFv antibody structure as the antigen-binding site for membrane-type stimulatory antibodies.

[0117] Selection of transmembrane domain To determine whether mouse PILR or human HLA-E transmembrane domains are appropriate as transmembrane domains, we first investigated whether reporter cells could be stimulated using the human HLA-E transmembrane domain. In Example 2, we confirmed that reporter cells could be stimulated using a membrane-bound antibody containing an anti-NKG2A antibody scFv as the antigen-binding site and mouse PILR as the transmembrane domain.

[0118] Membrane-stimulating antibodies containing the antigen-binding domain (scFv) prepared from the anti-NKG2A antibody clone 9-1-7 prepared in Example 2 and either human HLA-E or mouse PILR as the transmembrane domain were prepared, and reporter assays were performed. The results are shown in Figure 13. The membrane-stimulating antibody using the transmembrane domain of human HLA-E was able to stimulate reporter cells in the same way as the membrane-stimulating antibody using mouse PILR.

[0119] A CD107a assay was carried out using cells in which the membrane-type stimulatory antibody used in Example 3 was expressed in regenerated CTLs induced from pluripotent stem cells as targets and NK cells as effectors.

[0120] The regenerated CTLs used as target cells in this example were T cells induced by standard methods from HLA-homozygous iPSCs (I14s04 iPSCs) provided by the Center for iPS Cell Research and Application (CiRA) and from iPSCs (dKO iPSCs) in which HLA-class I and II were double-knocked out (dKO) of I14s04 iPSCs. The double knockout of HLA-class I and II was performed using the same method as the double knockout in ES cells described in Example 6. These regenerated CTLs were retrovirally transfected with a membrane-binding stimulatory anti-NKG2A antibody and used as target cells. This test system focuses on the relationship between the NK cell inhibitory receptor NKG2A and the HLA-E receptor it recognizes (Figure 14).

[0121] NK cell effector cells have HLA types C1 / C1, Bw4 - PBMCs from healthy volunteers were analyzed using CD14 - CD19 - CD3 - CD56 + KIR2DL3 - NKG2A + The NK cells fraction was sorted by FACS and used (FIG. 15). Effector:Target=1:1=10,000 cells:10,000 cells (n=3). The results are shown in FIG.

[0122] Target cells expressing a membrane-type anti-NKG2A-scFv antibody that uses the mouse PILR transmembrane domain in its transmembrane domain nonspecifically activated NK cells (Fig. 16, second from the left).On the other hand, target cells expressing a membrane-type anti-NKG2A-scFv that uses the human HLA-E transmembrane domain did not activate NK cells (Fig. 16, third from the left).

[0123] Furthermore, NK cell activation was suppressed against target cells in which anti-NKG2A-scFv was expressed in HLA-deficient iPS cell-derived regenerated CTLs (first panel from the right in Figure 16). Based on this result, the transmembrane domain of HLA-E was used as the transmembrane domain of the membrane-stimulating antibody used to verify the inhibitory effect of NK cells.

[0124] Production of membrane-stimulating antibodies with scFv-type antigen-binding sites specific for NK cell inhibitory receptors (1) Membrane-stimulating anti-NKG2A antibodies. B16 cells were transfected with NKG2A and CD94, and the transfectants were immunized in mice. Antibodies that recognized NKG2A and CD94 transfectants but not NKG2C and CD94 transfectants were screened. Antibody genes were analyzed to identify NKG2A-specific clones 9-1-7 and 32-1-7. From the resulting clones, membrane-stimulating antibodies were produced that contained scFv antigen-binding sites and the transmembrane domain of HLA-E. The light chain hypervariable region-linker-heavy chain hypervariable region-human IgG prehinge sequence-transmembrane domain were connected in this order. An endoplasmic reticulum targeting signal (residues 1-24) was also attached upstream of the light chain hypervariable region. The sequences of each antibody are shown below.

[0125] NKG2A-9-1-7 (SEQ ID NO: 1)

[0126] NKG2A-9-1-7 light chain hypervariable region (SEQ ID NO: 2) LEDILLTQSPAILSVSPGERVSFSCRASQSIGTSIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDVAYYYCQQSNSWPIFTFGSGTKLEIKR NKG2A-9-1-7 heavy chain hypervariable region (SEQ ID NO: 3) EVQLVESGGGLVKPGGSLKLSCAASGFAFSDYDMSWVRQTPEKRLEWVAYISSGGGSTYYPDTMKGRFTISRDNAKNTLYLQMSSLKSEDTAMYYCVRHGNRYDDAMDYWGQGTSVTVSS

[0127] NKG2A-32-1-7 (SEQ ID NO: 4) NKG2A-32-1-7 light chain hypervariable region (SEQ ID NO: 5) LEDIQMTQTTSSLSASLGDRVTISCSASQGISNYLNWYQQKPDGTVKLLIYYTSSLYSGVPSRFSGSGSGTDYSLTISNLEPEDIATYYCQHYSKLPWTFGGGTKLEIKR NKG2A-32-1-7 heavy chain hypervariable region (SEQ ID NO: 6) EVQLVESGGGLVKPGGSLKLSCAASGFTFSDYYMYWVRQTPEKRLEWVATISEGGSYTYYPDSVKGRFTISRDNAKNNLYLQMSSLKSEDTAMYYCARGGVIGILRPPFAYWGQGTLVTVSA

[0128] Of the above, linker sequence (boxed text, SEQ ID NO: 7): GSTSGSGKPGSGEGS Up to the hinge of Fc (double underlined, SEQ ID NO: 8): ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHT Transmembrane region of HLA-E (wavy line, SEQ ID NO: 9): TIPIVGIIAGLVLLGSVVSGAVVAAVIWRKKSSGGKGGSYSKAEWSDSAQGSESHSL*

[0129] Comparison of stimulatory potency between anti-NKG2A antibody clones in scFv structure. Two of the resulting membrane-stimulatory anti-NKG2A antibody clones (9-1-7 and 32-1-7) were expressed in 293T cells and used as stimulators in reporter assays. The stimulatory potency of the inhibitory antibodies was assessed by detecting GFP in NKG2A reporter cells after 24 hours of co-culture at a stimulator:reporter ratio of 1:1. The results are shown in Figure 17. Both scFv clones were confirmed to be capable of binding to NKG2A. Furthermore, 9-1-7 had a stronger stimulatory potency.

[0130] (2) Membrane-Stimulating Anti-KIR2DL1 Antibody. Antibodies that bind only to KIR2DL1 but not to KIR2DL3 / KIR2DS1 / 2DS2 / 2DS3 / 2DS4 / 2DS5 were sorted from KIR-immunized mice, and the antibody genes obtained by single-cell RNAseq / repertoire analysis were analyzed. Of the 2,000 sorted, scRNAseq data was obtained for 1,000. IgG clones with two or more BCR clones were selected, and 12 clones that bound to KIR were identified. From these, KIR9, a clone specific for KIR2DL1, was isolated. A membrane-stimulating antibody with an scFv-type antigen-binding site and an HLA-E transmembrane domain was produced from the resulting KIR9. The amino acid sequence of the membrane-stimulating antibody is shown below. KIR2DL1 (KIR9) (SEQ ID NO: 10): KIR2DL1 (KIR9) light chain hypervariable region (SEQ ID NO: 11) LEDIVLTQSPATLSVTPGDSVSLSCRASQSISNHLHWYHQKSHESPRLLIKYASQSISGIPSRFSGSGSGTDFTLSINSVETEDFGMYFCQQSNSWPPLTFGAGTKLELKR KIR2DL1 (KIR9) heavy chain hypervariable region (SEQ ID NO: 12) DVQLVESGGGLVQPGGSRTLSCAASGFTFSSFGMHWVRQAPEKGLEWVAYISSGSRTIYYADTVKGRFTISRDNPKNTLFLQMTSLRSEDTAMYYCARNDAYDYGFAYWGQGTLVTVSA The sequences of the linker, the region before the Fc hinge, and the HLA-E transmembrane region are SEQ ID NOs: 7, 8, and 9, respectively. An endoplasmic reticulum targeting signal (positions 1-24) is attached upstream of the light chain hypervariable region.

[0131] Stimulation by membrane-bound stimulatory anti-KIR2DL1 antibody The obtained membrane-bound stimulatory anti-KIR2DL1 antibody (KIR9) was expressed in 293T cells, and reporter assays were performed using this as a stimulator. The stimulatory strength of the inhibitory antibody was evaluated by detecting GFP in reporter cells 24 hours after co-culture at a stimulator:reporter ratio of 1:1. The results are shown in Figure 18. The membrane-bound stimulatory anti-KIR2DL1 antibody (KIR9) bound to KIR2DL1 and induced stimulation.

[0132] (3) Membrane-stimulatory anti-KIR2DL3 antibodies. From mice immunized with KIR, antibodies that bind only to KIR2DL3 but not to KIR2DL1 / KIR2DS1 / 2DS2 / 2DS3 / 2DS4 / 2DS5 were sorted, and the antibody genes obtained by single cell RNAseq / repertoire analysis were analyzed.

[0133] Of the 2,000 clones sorted, scRNA-seq data was obtained for 1,000. When IgG clones with two or more BCR clones were selected, 12 clones bound to KIR were identified. From these, clone number 13, specific for KIR2DL3, was identified (KIR13). 27 new clones with fewer than two IgG clones were identified, and clone number 37 was also identified as specific for KIR2DL3 (KIR37).

[0134] From the obtained KIR13 and KIR37, a membrane-stimulating antibody was produced that has an scFv-type antigen-binding site and an HLA-E transmembrane domain. The amino acid sequence of the membrane-stimulating antibody is shown below.

[0135] KIR13 (SEQ ID NO: 13): KIR13 light chain hypervariable region (SEQ ID NO: 14) LEDIVMTQAAFSNPVTLGTSASISCRSSKSLLHSTGITYLYWYLQKPGQSPQLLIYQMSNLASGVPDRFSSSGSGTDFTLRISRVEAEDVGVYYCAQKLELPWTFGGGTKLEIKR KIR13 heavy chain hypervariable region (SEQ ID NO: 15) DVQLQESGPGLVKPSQSLSLTCAVTGYSITSDYAWNWIRQFPGNKLEWMGYIIYSGSTRYNPSLKSRISITRDTSKNQFFLQLNSVTTEDTATYYCARWGYYGYLDYWGQGTTLTVSS The sequences of the linker, the region before the Fc hinge, and the HLA-E transmembrane region are SEQ ID NOs: 7, 8, and 9, respectively. An endoplasmic reticulum targeting signal (positions 1-24) is attached upstream of the light chain hypervariable region.

[0136] KIR37 (SEQ ID NO: 16): KIR37 light chain hypervariable region (SEQ ID NO: 17) LEDIVLTQSPATLSVTPGDSVSLSCRASQSLSNNLHWYQQKSHESPRLLIKYASQSISGIPSRFSGSGSGTDFTLSINSVETEDFGMYFCQQSNSWPFTFGSGTNLEIKR KIR37 heavy chain hypervariable region (SEQ ID NO: 18) EVKLVESGGGLVQPGGSLKLSCAASGFTFSSYTMSWIRQTPEKRLEWVAYISSGGGSTYYPDTVKGRFTISRDNAKNTLYLQMSSLKSEDTAMYYCARRLDPYWYFDVWGAGTTVTVSS The sequences of the linker, the region before the Fc hinge, and the HLA-E transmembrane region are SEQ ID NOs: 7, 8, and 9, respectively. An endoplasmic reticulum targeting signal (positions 1-24) is attached upstream of the light chain hypervariable region.

[0137] Stimulation by membrane-binding stimulatory anti-KIR2DL3 antibodies. Two of the obtained membrane-binding stimulatory anti-KIR2DL3 antibody clones, KIR13 and KIR37, were expressed in 293T cells and used as stimulators in reporter assays. The stimulatory strength of the inhibitory antibodies was assessed by detecting GFP in reporter cells 24 hours after co-culture at a stimulator:reporter ratio of 1:1. The results are shown in Figure 19. While both membrane-binding stimulatory anti-KIR2DL3 antibody clones, KIR13 and KIR37, stimulated KIR2DL3, KIR37 induced stronger stimulation. (4) From mice immunized with the membrane-stimulatory anti-KIR3DL1 antibody 3DL1-Fc, we sorted B cells that bound only to KIR3DL1 but not to KIR2DL1 / 2DL3 / 2DS1 / 2DS2 / 2DS3 / 2DS4 / 2DS5 / 3DS1 / 3DL2, and analyzed the antibodies using single-cell RNA sequencing / repertoire analysis. Of 41 clones examined, 14 recognized KIR3DL1. Among these, four clones specific for KIR3DL1 were identified (KIR34, KIR69, KIR103, and KIR114).

[0138] From the obtained KIR34, KIR69, KIR103, and KIR114, membrane-stimulating antibodies were produced that have an scFv-type antigen-binding site and an HLA-E transmembrane domain. The amino acid sequences of the membrane-stimulating antibodies are shown below.

[0139] KIR34 (SEQ ID NO: 19): KIR34 light chain hypervariable region (SEQ ID NO: 20) LEDVLMTQTPLSLPVSLGDQASISCRSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPWTFGGGTKLEIKR KIR34 heavy chain hypervariable region (SEQ ID NO: 21) QIQLVQSGPELKKPGETVKISCKASGYTFTNYGMNWVKQAPGKGLKWMGWINTYTGEPTYADDFKGRFAFSLETSASTAYLQINNLKNEDMATYFCAGNRYDRGNYAMDYWGQGTSVTVSS The sequences of the linker, the region before the Fc hinge, and the HLA-E transmembrane region are SEQ ID NOs: 7, 8, and 9, respectively. An endoplasmic reticulum targeting signal (positions 1-24) is attached upstream of the light chain hypervariable region.

[0140] KIR69 (SEQ ID NO: 22): KIR69 light chain hypervariable region (SEQ ID NO: 23) LEDIVMTQSPATLSVTPGDRVSLSCRASQSISDYLHWYQQKSHESPRLLIKYASQSISGIPSRFSGSGSGSDFTLSINSVEPEDVGVYYCQNGHSFPPTFGGGTKLEIKR KIR69 heavy chain hypervariable region (SEQ ID NO: 24) QVQLKESGPGLVAPSQSLSITCTVSGFSLTGYGVNWVRQPPGKGLEWLGMIWGDGSTDYNSALKSRLSINKDNSKSQVFLKMNSLQTDDTARYYCAREPIYDGYYVGAMDYWGQGTSVTVSS

[0141] KIR103 (SEQ ID NO: 25): KIR103 light chain hypervariable region (SEQ ID NO: 26) LEDIVMTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNDYSYPLTFGAGTKLELKR KIR103 heavy chain hypervariable region (SEQ ID NO: 27) QIQLVQSGPELKKPGETVKISCKASGYTFTDYSMHWVKQAPGKGLKWMGWINTETGEPTYADDFKGRFAFSLETSASTAYLQINNLKNEDTATYFCAPWLLRIAYWGQGTLVTVSA

[0142] KIR114 (SEQ ID NO: 28): KIR114 light chain hypervariable region (SEQ ID NO: 29): LEDIQMTQSPASLSASVGETVTITCRASENIYSYLAWYQQKQGKSPQLLVYNAKTLAEGVPSRFSGSGSGTQFSLKINSLQPEDFGSYYCQHHYGTATFGSGTKLEIKR KIR114 heavy chain hypervariable region (SEQ ID NO: 30): QGQMQQSGAELVKPGASVKLSCKTSGFTFSSSYISWLKQKPGQSLEWIAWIYAGTGGTSYNQKFTDKAQLTVDTSSSTAYMQFSSLTTEDSAIYYCARHGYDYAFAYWGQGTLVTVSA

[0143] In all of these, the sequences of the linker, the region up to the Fc hinge, and the HLA-E transmembrane region are SEQ ID NOs: 7, 8, and 9, respectively. An endoplasmic reticulum targeting signal (positions 1-24) is attached upstream of the light chain hypervariable region.

[0144] Generation of T cells expressing membrane-bound stimulatory antibodies specific for NK cell inhibitory receptors, with both HLA class I and class II knockouts. HLA class I and class II were knocked out in pluripotent stem cells using genome editing, and T cells were induced from the knockout cells. ES cells (SEES3-10) provided by the National Center for Child Health and Development were used as pluripotent stem cells. The b2m gene was targeted to knock out HLA class I expression, and the CIITA gene was targeted to knock out HLA class II expression. The sequences of the crRNAs that recognize the target sequences of each gene are as follows:

[0145] b2m gene: GGCCGAGAUGUCUCGCUCCGGUUUUAGAGCUAUGCU (SEQ ID NO: 31) CIITA gene: GCUGAACUGGUCGCAGUUGAGUUUUAGAGCUAUGCU (SEQ ID NO: 32)

[0146] An RNP complex solution (Table 12) was prepared for 4x10^5 cells of SEES3-10, mixed with the cell suspension, and then electroporation (Nepa Gene) was performed. RNP complex composition table

[0147] The genome-edited ES cells were analyzed for HLA-class I and class II expression by flow cytometry, and the fraction that was negative for both was isolated and cultured to establish HLA-I / II double knockout (dKO) ES cells (clone: ​​SEES3-10-dKO-2).

[0148] To differentiate ES cells into T cells, the established SEES3-10-dKO-2 cells were transfected with an HLA-A*02:01-restricted NY-ESO-1 antigen-specific TCR, resulting in the establishment of SEES3-10-dKO-2-NY6 cells. Subsequently, CD8+ T cells induced to differentiate from SEES3-10-dKO-2-NY6 cells by standard methods or CD8+ T cells induced to differentiate from SEES3-10-NY8 cells without HLA knockout were used as target cells for the CD107a assay. The antibodies used in flow cytometry are listed below (Table 13).

[0149] List of FACS antibodies used to obtain dKO cells

[0150] The membrane-type stimulatory antibodies were expressed in each CD8-positive T cell using a retroviral vector.

[0151] The inhibitory effect on NK cell activity was confirmed by a CD107a assay. ES cells (SEES3-10-NY8 (ESCs) and SEES3-10-dKO-2-NY6 (dKO ESCs)) derived from ES cells obtained in Example 6 and knocked out HLA-class I and class II using genome editing were used. Regenerated T cells expressing each membrane-type stimulatory antibody obtained in Example 5 were used as target cells, and the NK cell fraction bearing the inhibitory receptor to which each membrane-type stimulatory antibody specifically binds was used as effector cells for a CD107a assay.

[0152] (1) Test to confirm the induction of stimulation of the inhibitory receptor NKG2A (Figure 20). Membrane-bound stimulatory antibodies expressed in target cells: NKG2A-specific membrane-bound stimulatory antibodies scNKG2A9-1-7 and scNKG2A32-1-7 obtained in Example 5(1). NK cell fraction used as effector: NK cells sorted from PBMC obtained from a healthy C1 / C1, Bw4- volunteer in the CD14-CD19-CD3-CD56+KIR2DL3-NKG2A+ fraction were used (Figure 21). Effector: Target = 1:1 = 5000 cells: 5000 cells (n = 3). The results are shown in Figure 22.

[0153] We succeeded in suppressing NK cell activation by expressing a membrane-bound antibody bearing an scFv specific for the NKG2A receptor in HLA-dKO ES cell line-derived regenerated CTLs. Of the two clones, 32-1-7 had stronger inhibitory potency (Figure 22, first panel from the right).

[0154] (2) Test to confirm the induction of stimulation of the inhibitory receptor KIR2DL1 (Figure 23). Membrane-type stimulatory antibody expressed in target cells: KIR2DL1-specific membrane-type stimulatory antibody KIR9 obtained in Example 5(2). NK cell fraction used as effector: NK cells sorted from C2 / C2, Bw4- PBMCs (purchased from Lonza (21TL258433)) in the CD14-CD19-CD3-CD56+NKG2A- fraction were used (Figure 24). Effector: Target = 1:1 = 5000 cells: 5000 cells (n = 3). The results are shown in Figure 25.

[0155] By expressing the membrane-bound stimulatory antibody KIR9 in HLA-dKO ES cell line-derived regenerated CTLs, we succeeded in suppressing NK cell activation (Figure 25, right end). In Figure 25, "autoT" refers to T cells isolated and expanded from PBMCs.

[0156] (3) Test to confirm the induction of stimulation of the inhibitory receptor KIR2DL3 (Figure 26). Membrane-type stimulatory antibodies expressed in target cells: KIR2DL3-specific membrane-type stimulatory antibodies KIR13 and KIR37 obtained in Example 5(3). NK cell fraction used as effector: NK cells sorted from PBMC obtained from a healthy C1 / C1, Bw4- volunteer in the CD14-CD19-CD3-CD56+ NKG2A- KIR2DL3+ fraction were used (Figure 27). Effector: Target = 1:1 = 5000 cells: 5000 cells (n = 3). The results are shown in Figure 28.

[0157] By expressing the membrane-type stimulatory antibody KIR37 specific to KIR2DL3 in HLA-dKO ES cell line-derived regenerated CTLs, NK cell activation was suppressed (right end of Figure 28).

[0158] (4) Test to confirm the induction of stimulation of the inhibitory receptor KIR3DL1 (Figure 29). Membrane-type stimulatory antibodies expressed in target cells: KIR3DL1-specific KIR34, KIR69, KIR103, and KIR114 obtained in Example 5(4). NK cell fraction used as effector: NK cells sorted from PBMCs of C1 / C1, Bw4+ mice into the CD14-CD19-CD3-CD56+ NKG2A- KIR2DL3- fraction were used (Figure 30). Effector: Target = 1:1 = 10,000 cells: 10,000 cells (n = 3). Prior to analysis, CD107a expression levels on 3DL1+ NK cells were assessed using the BV421-KIR3DL1 antibody. The results are shown in Figure 31.

[0159] NK cells from HLA-Bw4+ individuals are educated to recognize stimuli from their own HLA-Bw4 during differentiation. After maturation, NK cells attack cells lacking Bw4. Expression of the membrane-bound stimulatory antibody KIR69 specific for KIR3DL1 in HLA-dKO ES cell line-derived regenerated CTLs, i.e., CTLs lacking Bw4, suppressed the activation of NK cells that recognize and attack HLA-Bw4 loss (Figure 31, third panel from the right). (5) Confirmation of the induction of stimuli for the inhibitory receptor KIR3DL1. To determine whether the inhibitory effect of the anti-KIR3DL1 antibody was adequate, we prepared Bw4 trimers that are expected to function as Bw4, and performed experiments similar to (4). The preparation of Bw4 trimers was based on the work of Lenart I et al. (2022) Front. Immunol. 13:902135.

[0160] Membrane-type stimulatory antibodies expressed in target cells: KIR3DL1-specific KIR34, KIR69, KIR103, and KIR114 obtained in Example 5(4) were each expressed using retroviruses. Target cells expressing the Bw4 trimer using retroviruses were also obtained. NK cell fraction used as effector: NK cells in the CD14-CD19-CD3-CD56+ NKG2A- KIR2DL3- fraction were sorted from C1 / C1 and Bw4+ PBMCs and used (Figure 32). CD107a expression levels were assessed on 3DL1+ NK cells using the BV421-KIR3DL1 antibody prior to analysis. Effector: Target = 1:1 = 10,000 cells: 10,000 cells. The results are shown in Figure 33.

[0161] The NK cell suppression effect of Bw4 trimer was confirmed against regenerated CTLs derived from an HLA-dKO ES cell line (rightmost panel in Figure 33). The suppression effect of the anti-KIR3DL1-scFv antibody, as determined in Example 7(4), was confirmed to be equal to or greater than that of the positive control Bw4 trimer. The effect of KIR69 was particularly strong (fourth panel from the right in Figure 33).

[0162] NK cell-inhibitory effect of cells coexpressing a membrane-bound stimulatory antibody specific for the inhibitory receptor NKG2A and a membrane-bound stimulatory antibody specific for KIR2DL1 (Figure 34). In Example 7, the membrane-bound stimulatory antibodies NKG2A-9-1-7 and KIR9 (KIR2DL1-specific antibody) each independently suppressed the activation of NK cells expressing their respective target inhibitory receptors. We used a CD107a assay to verify whether coexpression of these antibodies suppresses the activation of NK cell populations that are not selected by inhibitory receptors.

[0163] Membrane-type stimulatory antibodies expressed in target cells: NKG2A-specific membrane-type stimulatory antibody scNKG2A9-1-7 obtained in Example 5(1) and KIR2DL1-specific membrane-type stimulatory antibody KIR9 obtained in Example 5(2). NK cell fraction used as effector: CD14-CD19-CD3-CD56+ NK cells sorted from PBMCs of C2 / C2, Bw4- (purchased from Lonza Inc. (21TL132832)) were used (Figure 35). Effector: Target = 1:1 = 6000 cells:6000 cells (n = 5). The results are shown in Figure 36.

[0164] By expressing the NKG2A-specific membrane-bound stimulatory antibody scNKG2A9-1-7 and the KIR2DL1-specific membrane-bound stimulatory antibody scKIR2DL1 in HLA-dKO ES cell line-derived regenerated CTLs, we succeeded in suppressing the activity of all C2 / C2 Bw4- NK cells (Figure 36, right end).The results of Example 8 suggest that the expression of four membrane-bound inhibitory antibodies (NKG2A, KIR2DL1, 2DL3, and 3DL1) may be able to suppress the activation of all NK cells.

[0165] Inhibition of NK cell killing by regenerated CTLs expressing NK cell inhibitory receptor-specific membrane-bound stimulatory antibodies. A killing assay was performed to examine whether regenerated CTLs expressing the KIR2DL3-specific membrane-bound stimulatory antibodies scKIR13 or scKIR37 could avoid NK cell killing. A schematic diagram of the killing assay is shown in Figure 37. Regenerated CTLs expressing membrane-bound inhibitory antibodies were used as targets, and NK cells sorted from peripheral blood of healthy donors were used as effectors. After coculture, fixable viability dye efluor780 (eBioscience) was added at 1 / 1000 to compare the increase or decrease in the percentage of dead cells among target cells. Simultaneously, changes in the percentage of viable cells were also compared by tracking the increase or decrease in the green fluorescent marker of the regenerated CTLs. The relationship between the inhibitory receptors and ligands focused on in this study is shown in Figure 38.

[0166] Membrane-bound stimulatory antibody expressed in target cells: membrane-bound stimulatory antibody whose antigen-binding site is an scFv-type KIR2DL3-specific antibody (KIR13 or KIR37) and whose transmembrane domain is HLA-E. NK cell fraction used as effector: NK cells sorted from PBMC obtained from a C1 / C1, Bw4- healthy volunteer were used as the CD14-CD19-CD3-CD56+ NKG2A- KIR2DL3+ fraction (Figure 39). Effector: Target = 1:1 = 5000 cells: 5000 cells (n = 3). The results of the killing assay are shown in Figure 40, and the changes in viable cell rate are shown in Figure 41.

[0167] In Figure 40, when focusing on dead cells, the proportion of dead cells was significantly increased in dKO-ES cells (third from the right), but in comparison, dKO-ES cells expressing scKIR13 or KIR37 (second from the right, far right) were able to avoid attack by NK cells. The avoidance of attack by NK cells was at the same level as cells without HLA knockout.

[0168] Furthermore, when focusing on live cells, Figure 41 shows that dKO-ES cells expressing scKIR13 or KIR37 (second from the right, far right) were able to suppress NK cell attack to a certain extent compared to dKO-ES cells (third from the right). Of the two membrane-binding stimulatory antibodies, scKIR37 had a stronger NK cell-suppressing effect.

[0169] We investigated whether regenerated CTLs expressing membrane-bound stimulatory antibodies specific for the inhibitory receptor NKG2A and KIR2DL1 as target cells could evade killing by NK cells. The regenerated CTLs were differentiated from ES cells SEES3-10-NY8 (ES) or SEES3-10-dKO-2-NY6 (dKO ES). The assay method was the same as in Example 9.

[0170] Membrane-bound stimulatory antibodies expressed in target cells: NKG2A-specific membrane-bound stimulatory antibody scNKG2A-32-1-7 and KIR2DL1-specific membrane-bound stimulatory antibody scKIR2DL1(KIR9) (Figure 42). NK cell fraction used as effector: CD14-CD19-CD3-CD56+ NK cells sorted from PBMCs of C2 / C2, Bw4- (purchased from Lonza (21TL132832)) were used (Figure 43). Effector: Target = 1:1 = 4000 cells:4000 cells (n = 3). The changes in the percentage of dead cells in the killing assay are shown in Figure 44, and the changes in the percentage of live cells are shown in Figure 45.

[0171] Focusing on dead cells in Figure 44, compared to dKO-ES mock, regenerated CTLs expressing scNKG2A or KIR2DL1 (KIR9) (third and second from the right) and regenerated CTLs induced from dKO-ES cells expressing both (dKO ES mix, far right) were shown to be able to evade NK cell attack and suppress NK cell attack to the same extent as cells without HLA knockout. Focusing on live cells in Figure 45, compared to dKO-ES mock, regenerated CTLs expressing scNKG2A or KIR2DL1 (KIR9) (third and second from the right) and regenerated CTLs induced from dKO-ES cells expressing both (dKO ES mix, far right) were shown to be able to evade NK cell attack and suppress NK cell attack to the same extent as cells without HLA knockout.

[0172] Generation of ultra-versatile cells Generation of cells expressing the membrane-bound stimulatory antibody scKIR2DL1 (KIR9) HLA-dKO ES cells were transfected with a lentiviral vector using a ubiquitin promoter by spin infection (1800g, 60 min), and PCR was used to verify whether the transfection was successful.

[0173] Using the DNeasy Blood & Tissue Kit (QIAGEN), we expressed the membrane-associated stimulatory antibody scKIR2DL1 (KIR9) in SEES3-10 NY8 and SEES3-1- dKO-2 NY6. Genome extraction was performed from the resulting ES cells, and the following primers and Quick Taq DNA were used. R PCR was performed using HS DyeMix (TOYOBO) and scKIR2DL1 (KIR9) was identified by agarose gel electrophoresis. The PMX-scKIR9 vector was used as a positive control.

[0174] Primer for identifying scKIR2DL1(KIR9) Expected PCR product base pairs: 135bp

[0175] PCR reaction Stage1: 94℃ 2min 1cycle Stage2: 94℃ 30sec 59℃ 30sec 68℃ 12sec →35 cycle Stage3: 68℃ 10min 1cycle

[0176] The electrophoresis results of the obtained cells are shown in Figure 46. Since a band was observed at the target position in the scKIR2DL1 (KIR9) transfected cells, it was confirmed that the gene transfer was successful. Subsequently, by using a similar process to transfer the genes scNKG2A (32-1-7), 2DL3 (KIR37), and 3DL1 (KIR69), it was possible to produce ultra-versatile cells.

[0177] Preparation of super-universal cells Genes encoding membrane-stimulatory antibodies with the antigen-binding sites obtained in Example 5 that are scFvs of anti-NKG2A clone 32-1-7, anti-KIR2DL1 antibody (KIR9), anti-KIR2DL antibody (KIR37), and anti-KIR3DL1 antibody (KIR69) and that have the transmembrane site of HLA-E were used. We established luciferase-transfected cell lines for killing assays using HLA-expressing ES cells transfected with the NY-ESO-1-TCR gene (NY-ESO-1-TCR-ESCs) and their HLA-dKO counterparts (HLA-dKO NY-ESO-1-TCR-ESCs). We then established a 4mAb-expressing ES cell line (NY-ESO-1-TCR-luc-4mAb ESCs) expressing four membrane-bound stimulatory antibodies (4mAb), and a super-generic ES cell line (HLA-dKO NY-ESO-1-TCR-luc-4mAb ESCs). We then induced differentiation of each cell line according to standard procedures to generate regenerated CTLs (Figure 47).

[0178] (1) Generation of luciferase gene-expressing ES cells. NY-ESO-1-TCR-ESCs and HLA-dKO NY-ESO-1-TCR-ESCs were transfected with the luciferase gene by spin-infection (1800g, 60min) using a ubiquitin promoter-driven lentiviral vector (pUltra-Chili-luc) carrying the dtomato marker gene to establish the cell lines NY-ESO-1-TCR-luc-ESCs and HLA-dKO NY-ESO-1-TCR-luc-ESCs (Figure 48). venus and dtomato are marker genes that reflect the expression of NY-ESO-1 TCR and luciferase, respectively.

[0179] (2) Generation of ES cells expressing four membrane-bound stimulatory antibodies. The NY-ESO-1-TCR-luc-ESCs and HLA-dKO NY-ESO-1-TCR-luc-ESCs generated in (1) were transfected with four membrane-bound antibody genes by spin-infection (1800g, 60 min) using the CS-UbC lentiviral vector, which utilizes the ubiquitin promoter. Lentiviral vectors carrying the genes shown in Figure 49 were constructed, and the following cell lines were established, which highly expressed the tagBFP (tag blue fluorescent protein) and hNGFR (a transmembrane marker derived from the intramembrane signaling domain of hNGFR) marker genes (Figure 50). The amino acid sequences of the membrane-bound stimulatory antibodies specific for each NK cell inhibitory receptor are as described above. The amino acid sequences of tagBFP, hNGFR, and p2a are as follows: tagBFP is a marker gene that reflects the expression of anti-NKG2A and anti-KIR2DL3 antibodies, and hNGFR is a marker gene that reflects the expression of anti-KIR2DL1 and anti-KIR3DL1 antibodies. hNGFR-expressing cells were stained using APC-anti-human hNGFR antibody (Biolegend).

[0180] tagBFP: MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKVVEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTATQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALKLVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYCDLPSKLGHKLN* (SEQ ID NO: 35)

[0181] hNGFR: MDGPRLLLLLLLGVSLGGAKEACPTGLYTHSGECCKACNLGEGVAQPCGANQTVCEPCLDSVTFSDVVSATEPCKPCTECVGLQSMSAPCVEADDAVCRCAYGYYQDETTGRCEACRVCEAGSGLVFSCQDKQNTVCEECPDGTYSDEANHVDPCLPCTVCEDTERQLRECTRWADAECEEIPGRWITRSTPPEGSDSTAPSTQEPEAPPEQDLIASTVAGVVTTVMGSSQPVVTRGTTDNLIPVYCSILAVVVGLVAYIAFKRWNSCKQNKQGANS* (SEQ ID NO: 36)

[0182] GSG+p2a: GSGATNFSLLKQAGDVEENPGPGS (SEQ ID NO: 37)

[0183] 3) Induction of differentiation from ES cells to regenerative CTLs Using standard methods, ES cells were induced to differentiate into regenerative CTLs. After differentiation induction, expression of NK inhibitory antibody marker genes was confirmed, confirming the expression of tagBFP and hNGFR marker genes (Figure 51). Hereafter, among the regenerative CTLs obtained, HLA-dKO 4mAb regenerative CTLs are referred to as "super-universal regenerative CTLs."

[0184] Evaluation of antigen-specific killing activity of supergene regenerated CTLs as T cells. The supergene regenerated CTLs obtained in Example 12 were examined for their NY-ESO-1 antigen-specific killing activity. Target cells used were U266 cells expressing the NY-ESO-1 antigen and carrying the luciferase gene, or THP1 cells not expressing the NY-ESO-1 antigen. Regenerated CTLs were activated 5 days after stimulation with anti-CD3 / anti-CD28 antibodies (see "Regenerated CTL Stimulation"). After 24 hours of co-culture at E:T ratios of 1:3, 1:1, or 3:1, Bright-Glo (Promega) was added, and luminescence intensity was measured using GloMax (Promega) (Figure 52).

[0185] The % of lysis was calculated from the luminescence intensity obtained: 100x (1 - luminescence intensity of the co-culture of regenerated CTLs and target / luminescence intensity of target alone). The results confirmed that all regenerated CTLs possessed NY-ESO-1 antigen-specific killing activity. Furthermore, it was confirmed that the expression of four membrane-stimulatory anti-NK cell inhibitory receptor antibodies did not attenuate the killing activity of the regenerated CTLs (Figures 53-56).

[0186] Evaluation of suppression of NK activity by supergene regenerative CTLs (CD107a assay) To verify whether the supergene regenerative CTLs prepared in Example 12 inhibited the activation of recipient NK cells upon transplantation, a CD107a assay was performed using NK cells collected from healthy individuals with different HLA backgrounds as effector cells for hypothetical recipients (Figure 57). Figure 58 shows a schematic diagram of the CD107a assay timeline in this example. NK cells from healthy individuals' PBMCs were sorted and collected on day 0, and supergene regenerative CTLs were stimulated with anti-CD3 / anti-CD28 antibodies (see "Stimulation of Regenerative CTLs").

[0187] On day 2, the stimulation of the supergene regenerative CTLs was stopped, and on day 5, NK cells and the supergene regenerative CTLs were co-cultured. NK cell activation was assessed by analyzing the expression levels of CD107a after 6 hours.

[0188] (1) C2 / C2 Bw4 - Inhibitory effect on NK cells HLA-C C2 / C2 type and HLA-Bw4 negative (C2 / C2 Bw4 - When NK cells are used as effectors, NKG2A and KIR2DL1 on these NK cells can detect loss of HLA expression (Figure 59). - CD107a assay was performed using NK cells (purchased from Lonza Inc. (lot: 21TL258433)) collected from healthy individuals. - CD19 - CD3 - CD56 +The fraction was sorted to obtain effector NK cells, and the regenerated CTLs obtained in Example 12 were used as target cells. Co-culture was performed at an effector:target ratio of 1:1, 5000 cells:5000 cells (FIG. 60).

[0189] The results showed that HLA-deficient HLA-dKO no mAb regenerated CTLs activated NK cells more effectively than HLA-expressing HLA+ no mAb regenerated CTLs, whereas HLA-deficient supergenerated CTLs expressing 4 mAb (HLA-dKO 4 mAb regenerated CTLs) suppressed NK cell activation to the same level as HLA-expressing HLA+ no mAb regenerated CTLs (***; p<0.001, ns; not significant) (Figure 61).

[0190] (2) C1 / C1 Bw4 - Inhibitory effect on NK cells HLA-C is C1 / C1 type and HLA-Bw4 negative (C1 / C1 Bw4 - ) NK cells can detect the loss of HLA expression through the inhibitory receptors NKG2A and KIR2DL3 (Figure 62). HLA type is C1 / C1 Bw4 - A CD107a assay was performed using NK cells collected from a healthy individual. CD14-CD19-CD3-CD56+ NK cells were sorted to prepare effector NK cells (Figure 63). Target cells were regenerated from the regenerated CTLs obtained in Example 12. Effector:target cells were co-cultured at a ratio of 1:1 (5,000 cells:5,000 cells). HLA-deficient HLA-dKO no mAb regenerated CTLs activated NK cells more effectively than HLA-expressing HLA+ no mAb regenerated CTLs. Meanwhile, HLA-deficient, 4mAb-expressing super-generated CTLs (HLA-dKO 4mAb regenerated CTLs) suppressed NK cell activation to a level similar to that of HLA-expressing HLA+ no mAb regenerated CTLs (****; p<0.0001) (Figure 64).

[0191] (3) Suppressive effect on C1 / C1 Bw4+ NK cells. NK cells with HLA-C C1 / C1 type and HLA-Bw4 positive (C1 / C1 Bw4+) can detect the loss of HLA expression through the inhibitory receptors NKG2A, KIR2DL3, and KIR3DL1 (Figure 65). + A CD107a assay was performed using NK cells collected from healthy individuals. CD14-CD19-CD3-CD56+ NK cells were sorted to prepare effector NK cells (Figure 66), and the regenerated CTLs obtained in Example 12 were used as target cells. Effector:target = 1:1 = 5,000 cells:5,000 cells were co-cultured. Results showed that HLA-deficient HLA-dKO no mAb regenerated CTLs activated NK cells more effectively than HLA-expressing HLA+ no mAb regenerated CTLs, whereas HLA-deficient and 4mAb-expressing super-generated CTLs (HLA-dKO 4mAb regenerated CTLs) suppressed NK cell activation to a level similar to that of HLA-expressing HLA+ no mAb regenerated CTLs (****; p<0.0001) (Figure 67).

[0192] (4) Suppressive Effect on C1 / C2 Bw4+ NK Cells. NK cells with C1 / C2 HLA-C and Bw4 positivity (C1 / C2 Bw4+) express HLA expression, including NKG2A, KIR2DL1, KIR2DL3, and KIR3DL1, all NK cell inhibitory receptors that can detect HLA expression loss (Figure 68). A CD107a assay was performed using NK cells collected from healthy individuals with C1 / C2 Bw4+ HLA. CD14-CD19-CD3-CD56+ NK cells were sorted to prepare effector NK cells (Figure 69). Regenerated CTLs obtained in Example 12 were used as target cells. Effector:target cells were co-cultured at a ratio of 1:1 (5,000 cells:5,000 cells). The results showed that HLA-deficient HLA-dKO no mAb regenerated CTLs activated NK cells more effectively than HLA-expressing HLA+ no mAb regenerated CTLs, whereas HLA-deficient super-generated CTLs expressing 4 mAb (HLA-dKO 4 mAb regenerated CTLs) successfully suppressed NK cell activation to the same level as HLA-expressing HLA+ no mAb regenerated CTLs (****; p<0.0001, ns; not significant) (Figure 70).

[0193] As confirmed in this example, the supergene CTLs of the present invention suppressed the activation of NK cells collected from healthy individuals with different HLA backgrounds, regardless of HLA-C type or whether they were Bw4 positive or negative.

Claims

1. A method for producing cells or tissue for transplantation, comprising a step of expressing, on the surface of cells constituting the cells or tissue for transplantation, three or more membrane-type stimulatory antibodies that are specific to three or more NK cell inhibitory receptors selected from the group consisting of NKG2A, KIR2DL1, KIR2DL3, KIR3DL1, and LILRB1.

2. The method according to claim 1, wherein four membrane-type stimulatory antibodies specific for the NK cell inhibitory receptors NKG2A, KIR2DL1, KIR2DL3 and KIR3DL1, respectively, are expressed.

3. The method according to claim 1 or 2, wherein the membrane-type stimulatory antibody has the antigen-binding site and transmembrane site of an antibody specific to an inhibitory receptor of NK cells.

4. The method according to claim 3, wherein the antigen-binding site of the antibody specific for the inhibitory receptor of NK cells is an scFv.

5. The method according to any one of claims 1 to 4, wherein the cells for transplantation or the cells constituting the tissue for transplantation are cells in which any or all of the HLAs have been deleted.

6. The method according to any one of claims 3 to 5, wherein the transmembrane domain is a transmembrane domain of a classical class I HLA molecule or a non-classical class I HLA molecule.

7. The method according to claim 6, wherein the transmembrane domain is the transmembrane domain of HLA-E.

8. The method according to any one of claims 1 to 7, wherein the cells or tissue for transplantation are cells or tissue induced from pluripotent stem cells.

9. The method according to claim 8, wherein the cells or tissues for transplantation are cells or tissues obtained by inducing differentiation from pluripotent stem cells in which any or all of the HLAs have been deleted.

10. The method according to any one of claims 1 to 9, wherein the cells for transplantation are T cells.

11. The method according to any one of claims 1 to 10, wherein the membrane-stimulating antibody has an antigen-binding site comprising light chain CDR1, light chain CDR2, and light chain CDR3 of the light chain hypervariable region and heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 of the heavy chain variable region in a combination of a light chain hypervariable region and a heavy chain hypervariable region selected from the table below.

12. Pluripotent stem cells into which three or more membrane-type stimulatory antibodies specific for three or more NK cell inhibitory receptors selected from the group consisting of NKG2A, KIR2DL1, KIR2DL3, KIR3DL1, and LILRB1 have been genetically introduced or expressed.

13. The pluripotent stem cell according to claim 12, into which four membrane-type stimulatory antibodies specific to the NK cell inhibitory receptors NKG2A, KIR2DL1, KIR2DL3 and KIR3DL1, respectively, have been genetically introduced or expressed.

14. The pluripotent stem cells according to claim 12 or 13, which are cells obtained by induction from a donor with a homozygous HLA haplotype.

15. The pluripotent stem cells according to any one of claims 12 to 14, wherein the pluripotent stem cells are cells in which any or all of HLA has been deleted.

16. The pluripotent stem cell according to any one of claims 12 to 15, wherein the membrane-type stimulatory antibody has an antigen-binding site and a transmembrane site of an antibody specific to an inhibitory receptor of NK cells.

17. The pluripotent stem cell according to claim 16, wherein the antigen-binding site of the antibody specific to an inhibitory receptor of NK cells is an scFv.

18. The pluripotent stem cells according to any one of claims 12 to 17, which are derived from the SEES3 strain.

19. A pluripotent stem cell according to any one of claims 12 to 18, wherein the membrane-stimulating antibody has an antigen-binding site comprising light chain CDR1, light chain CDR2, and light chain CDR3 of the light chain hypervariable region and heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 of the heavy chain variable region in any one of the combinations of light chain hypervariable region and heavy chain hypervariable region shown in the table below.

20. A T cell for transplantation or its precursor cell, which expresses on its surface three or more membrane-type stimulatory antibodies specific to three or more NK cell inhibitory receptors selected from the group consisting of NKG2A, KIR2DL1, KIR2DL3, KIR3DL1, and LILRB1.

21. The T cells for transplantation or their precursor cells according to claim 20, which express four membrane-type stimulatory antibodies specific to the NK cell inhibitory receptors NKG2A, KIR2DL1, KIR2DL3 and KIR3DL1, respectively.

22. The T cells for transplantation or their precursor cells according to claim 20 or 21, wherein the membrane-type stimulatory antibody has the antigen-binding site and transmembrane site of an antibody specific to an inhibitory receptor of NK cells.

23. The T cells for transplantation or their precursor cells according to claim 22, wherein the antigen-binding site of the antibody specific to an inhibitory receptor of NK cells is an scFv.

24. The T cells for transplantation or their precursor cells according to any one of claims 20 to 23, wherein the cells for transplantation or the cells constituting the tissue for transplantation are cells in which any or all of the HLAs have been deleted.

25. The T cell for transplantation or its precursor cell according to any one of claims 20 to 24, wherein the transmembrane site is that of a classical class I molecular type HLA or a non-classical class I molecular type HLA.

26. The T cell for transplantation or its precursor cell according to claim 25, wherein the transmembrane domain is the transmembrane domain of HLA-E.

27. The T cells for transplantation or their precursor cells according to any one of claims 20 to 26, wherein the cells or tissue for transplantation are cells or tissue induced from pluripotent stem cells.

28. The T cells for transplantation or their precursor cells according to claim 27, wherein the cells or tissue for transplantation are cells or tissue obtained by inducing differentiation from pluripotent stem cells in which any or all of the HLAs have been deleted.

29. A T cell for transplantation or a precursor cell thereof according to any one of claims 20 to 28, wherein the membrane-type stimulatory antibody comprises an antigen-binding site comprising light chain CDR1, light chain CDR2, and light chain CDR3 of the light chain hypervariable region and heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 of the heavy chain variable region in a combination of a light chain hypervariable region and a heavy chain hypervariable region selected from the table below.

30. An antibody specific for an inhibitory receptor on NK cells, having an antigen-binding site comprising light chain CDR1, light chain CDR2, and light chain CDR3 of the light chain hypervariable region and heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 of the heavy chain variable region, in any one of the combinations of light chain hypervariable region and heavy chain hypervariable region shown in the table below.

31. The antibody specific to an inhibitory receptor of NK cells according to claim 30, which is a membrane-stimulating antibody further comprising a transmembrane domain.

32. The antibody of claim 31, wherein the antigen-binding site is an scFv.

33. The membrane-stimulating antibody of claim 32, which is a polypeptide having any of the following sequences:

34. A nucleic acid encoding an antibody according to any one of claims 30 to 33.

35. A vector comprising the nucleic acid molecule of claim 34.

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