Methods for evasion of immunological rejection using agonists against inhibitory KIR

Abstract

The present invention provides a substance having an activity that binds to an inhibitory KIR and inhibits the cell-killing activity of NK cells, which can be used in allogenic techniques relating to organ transplantation and cell therapy, and a method for preparing the same. Specifically, the invention relates to a substance capable of binding to inhibitory KIR and inhibiting the cell killing activity of NK cells, and a cell expressing the substance on the surface of the cell.

Description

Technical Field

[0001] The present invention relates to a substance having an activity of binding to an inhibitory KIR and inhibiting the cytotoxic activity of NK cells, a method for preparing the substance, a method for using the substance, and the like, which can be used in allogeneic technology related to organ transplantation and cell therapy. Background Art

[0002] Immunity is divided into natural immunity and acquired immunity. Natural immunity is a rapid immune response to a wide range of pathogens centered on immune cells such as natural killer cells (NK cells), macrophages, and granulocytes. On the other hand, acquired immunity is a selective and effective immune response centered on immune cells such as T cells, B cells, and dendritic cells.

[0003] Human pluripotent stem cells can treat a variety of diseases, but there is a huge limitation in the clinical use of human pluripotent stem cells, that is, the rejection of transplanted cells by the receptors due to differences in the major histocompatibility complex (MHC: Major Histocompatibility Antigen Complex).

[0004] The major MHCs are multicomponent cell surface molecules that mediate leukocyte or other cell-leukocyte interactions found in all vertebrates. The MHC gene family is divided into three groups, class I, class II, and class III, which are called human leukocyte antigens (HLA) in humans. HLA class I (HLA-I) proteins are expressed in all nucleated cells and consist of HLA class I heavy chains (or alpha chains) and beta 2 microglobulin (B2M). HLA class I proteins display peptides to CD8-positive cytotoxic T cells. Six HLA class I alpha chains have been identified, including three conventional (HLA-A, HLA-B, and HLA-C) alpha chains and three unconventional (HLA-E, HLA-F, and HLA-G) alpha chains. The specificity of the peptide bond to the peptide binding groove of the HLA class I molecule is determined by the alpha chain. Recognition of peptides displayed by HLA class I molecules by CD8-positive T cells mediates cellular immunity.

[0005] In transplantation, HLA class I proteins of the same origin are recognized as exogenous antigens, and the recognition of non-self HLA class I proteins is a major obstacle to the transplantation of pluripotent stem cells or the use of replacement therapies. Currently, HLA-identical or partially identical cells from HLA-type stem cell banks or pluripotent stem cell (iPSC) lines from various patient sources are being developed for transplantation, but the development of each consistent cell line requires huge costs, months of cell culture, highly trained personnel, and extensive validation of the final product, all of which must be performed after obtaining approval from regulatory authorities. In addition, each cell line may behave slightly differently in gene expression patterns, culture characteristics, differentiation ability, and inherited mutations, making it very difficult to apply them to every cell line.

[0006] Individualized stem cell preparation or HLA-diverse stem cell banks can address current transplantation issues, but require characterization of multiple cell lines, differentiation into therapeutic cell products, and approval for human administration. This time-consuming, technically difficult, and expensive process is a major factor preventing stem cell-based therapies from participating in clinical trials. Therefore, there is a need for more effective and less expensive cell-based therapies that are not hampered by rejection reactions.

[0007] T cells recognize HLA molecules on the cell surface through T cell receptors (TCR). HLA displays endogenous self-peptides in normal cells and non-self-peptides derived from viruses in infected cells, thereby adjusting the immune response and maintaining homeostasis.

[0008] NK (Natural Killer) cells have a self-other recognition function called "missing self", that is, they recognize their own HLA type and show cell killing activity to cells that do not express the same type of HLA (non-patent literature 1). In the missing self function, receptors such as NKG2 and KIR expressed on the surface of NK cells are responsible for identifying their own HLA type. Among these receptors, there are inhibitory receptors and active receptors. In one's own cells, HLA of the same type as NK cells will bind to inhibitory receptors on NK cells, inhibiting signal transmission to NK cells, thereby inhibiting the cell killing activity of NK cells and avoiding rejection by NK cells.

[0009] In addition, regarding inhibitory receptors on NK cells, it is known that even NK cells in one individual may include NK cell populations that express multiple inhibitory receptors. For example, a NK cell population is reported, which is included in human peripheral blood mononuclear cells (PBMCs), and about half of the NK cells identified by CD56 positive CD3 negative are NKG2A positive, and the remaining half are NKG2A negative, and express other inhibitory receptors of KIR and LILB (Non-Patent Document 2).

[0010] NKG2A (CD159) is a type of inhibitory receptor on the surface of NK cells, and recognizes HLA-E as a ligand. NKG2C / D / E are known as activating receptors corresponding to NKG2A (Non-Patent Document 3).

[0011] KIR (Killer cell immunoglobulin-like receptor) is a single transmembrane protein expressed in NK cells, NKT cells and a portion of T cells, recognizing HLA-A / B / C as the main ligand (Non-patent Document 2). In particular, recognition is based on the host's HLA type. For example, human NK cells of HLA-C1 / C1 (the type with the 77th amino acid of HLA-C as S and the 80th amino acid as N is called C1 type) will attack when sensing HLA-C2 (the type with the 77th amino acid of HLA-C as N and the 80th amino acid as K is called C2 type), and vice versa, they will also show cell killing activity by the same mechanism (Non-patent Document 1). This mechanism is called "Missing self". Among the molecules responsible for the missing self, if the host is from HLA-C1, KIR2DL2 / 3 functions as an inhibitory receptor; if the host is from HLA-C2, KIR2DL1 functions as an inhibitory receptor. Inhibitory receptors have ITIM (Immuno receptor Tyrosine-based inhibitor Motif) in their intracellular parts, form clusters by associating with HLA, and transmit inhibitory signals inside cells via adaptor molecules such as SHP-1 (Non-Patent Document 4).

[0012] As antibodies against KIR2DL1 / 2 / 3, there are Lirilumab (Lirilumab, BMS-986015, human IgG4) and mouse antibody Pan2D (NKVFS1, mouse IgG1) as anticancer agents in clinical trials. In addition, regarding antibodies against KIR3DL1, the antibodies described in the patent document (WO2018 / 148223A1) are also reported as candidate substances for anticancer agents. Cancer cells have a mechanism to escape cell killing activity by reducing the expression of activating ligands of receptors by immune cells or enhancing the expression of inhibitory ligands. HLA expressed on the surface of cancer cells can also escape the attack of NK cells by acting on the inhibitory receptors of NK cells and using the missing self function. Although it has been reported that the mechanism of action of lirelumab and Pan2D antibody is to bind to KIR2DL1 / 2 / 3, which are inhibitory KIRs, to block the association with HLA, thereby promoting the activation of NK cells against cancer cells (Non-Patent Document 5, Patent Document 1), there is no description of agonist activity against inhibitory KIRs.

[0013] So far, in order to solve the problem of immune cell rejection caused by HLA inconsistency between the host and the donor, Dr. David Russel et al. reported a method of using gene editing to delete B2M (beta-2 microglobulin), which is part of the protein composition of HLA, so that the expression of HLA on the cell membrane is inhibited to escape the recognition of CD8 positive T cells. This can obtain cells with HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, and HLA-G missing on the cell surface. On the other hand, it has been reported that in order to escape the rejection of NK cells, while deleting B2M, HLA-E (naturally a heterotrimer) is forced to be expressed as a single-chain protein on the cell surface of the same cell. This can obtain cells that do not express other HLAs but only HLA-E (hereinafter referred to as "universal cell technology"), thereby inhibiting the cell killing activity of NK cells (patent document 2).

[0014] As another immune rejection escape technique, there is a report that an agonist targeting CD45 widely expressed in NK cells and T cells is expressed on the cell surface, and the cell suppresses the cytotoxic activity of CD45-expressing immune cells (Patent Document 3).

[0015] Prior art literature

[0016] Patent Literature

[0017] Patent Document 1: International Publication No. WO2006 / 003179

[0018] Patent Document 2: International Publication No. WO2012 / 145384

[0019] Patent Document 3: International Publication No. WO2021 / 113853

[0020] Non-patent literature

[0021] Non-patent document 1: NK cell receptors: of missing sugar and missing self., P Parham et al., Curr Biol. 2000 Mar 9; 10(5): R195-7.

[0022] Non-patent literature 2: MHC class I-specific inhibitory receptors and their ligands structure diverse human NK-cell repertoires toward a balance of missing self-response, Yawata et al., Blood. 2008 Sep 15; 112(6): 2369-80.

[0023] Non-patent document 3: The CD94 / NKG2 family of receptors: from molecules and cells to clinical relevance. F. Borrego et al., Immunol Res. 2006; 35(3): 263-78.

[0024] Non-patent document 4: Negative signaling by inhibitory receptors: the NK cell paradigm. EO Long et al., Immunol Rev. 2008 Aug; 224: 70-84.

[0025] Non-patent document 5: Preclinical characterization of 1-7F9, a novel human anti-KIR receptor therapeutic antibody that augments natural killer-mediated killing of tumor cells. F Romagne et al., Blood. 2009 Sep 24; 114(13): 2667-77. Summary of the invention

[0026] Problems to be solved by the invention

[0027] The universal cell technology disclosed in Patent Document 1 has the following problems. Not all NK cells possessed by human individuals express NKG2A. For example, about half of the NK cells contained in human peripheral blood mononuclear cells (PBMC) and identified as CD56 positive and CD3 negative are NKG2A positive, and the remaining half are NKG2A negative, and express other inhibitory receptors of KIR and LILB. Therefore, even if the universal cell technology is used, there is a risk of being attacked by NKG2A negative NK cells.

[0028] Regarding the technology disclosed in Patent Document 2, since CD45 is expressed in T cells, for example, when CD45 agonists are carried in allogeneic CAR (Chimeric antigen receptor)-T cells, the risk of inhibiting their own functions and activities should be considered. In addition, since CD45, a target molecule expressed in a wide range of immune cells, is targeted, there are also side effects and the risk of being unable to escape by rejection even if the treated cells become cancerous. In summary, it is necessary to use an immune rejection escape technology for an agonist substance directed against a target directly associated with the properties of immune cells, which is not extensive but has the required minimum expression distribution.

[0029] The expression distribution of NKG2A and KIR on the surface of NK cells is in an inversely correlated relationship (non-patent document 2), and the KIR expression level is high in NK cells with low NKG2A expression. It can be seen that in universal cell technology, HLA-E introduced cells show effectiveness in NKG2A high-expressing cells, but do not show effectiveness in NK cells with low or non-expression of NKG2A and inhibitory KIR expression. On the other hand, since the ligand of inhibitory KIR is HLA-ABC, its direct introduction will not be able to take into account TCR escape, so HLA-type substances with agonist activity are required.

[0030] It has been reported that lirelumab (BMS-986015, human IgG4) and mouse antibody Pan2D (NKVFS1, mouse IgG1), which are known as antibodies against KIR, show an inhibitory signal that blocks natural ligand binding to inhibitory KIR, but their effects in the absence of ligand are unclear.

[0031] Technical solutions to solve problems

[0032] The inventors found that the lirelumab showed agonist activity against the inhibitory KIR of NK cells in the absence of a ligand. It was also found that cells expressing scFv (single-chain Fv) by introducing its gene sequence can escape the attack of NK cells. It was also found that Pan2D (NKVSF1) against KIR also showed agonist activity against inhibitory KIR in a soluble and cell surface manner. However, it was confirmed that lirelumab and Pan2D not only showed binding to inhibitory KIR, but also to active KIR, and acted as agonists against activated KIR (KIR2DS1, 2, 4, etc.), thereby enhancing the activity of NK cells.

[0033] Thus, it binds only to inhibitory KIR without contacting an activator, thereby obtaining an agonist binding substance for inhibitory KIR. As a result, through phage panning using a human antibody phage library, a substance that binds to inhibitory KIR2DL2 and KIR2DL3 and exhibits agonist activity but does not bind to KIR2DS1, KIR2DS2, and KIR2DS4, a substance that binds to KIR2DL1 and exhibits agonist activity but does not bind to KIR2DS1, KIR2DS2, and KIR2DS4, and a substance that binds to KIR3DL1 and exhibits agonist activity but does not bind to KIR3DS1 were identified, and further studies were conducted to complete the present invention.

[0034] That is, the present invention provides the following contents.

[0035] [1] A substance having an activity of binding to human inhibitory KIR and inhibiting the cytotoxic activity of NK cells.

[0036] [2] The substance according to [1], which is a polypeptide comprising a binding site for human inhibitory KIR.

[0037] [3] The substance according to [2], characterized in that the binding site of the substance for human inhibitory KIR contains amino acid sequences from the heavy chain variable region and light chain variable region of a monoclonal antibody or antigen-binding fragment that binds to human inhibitory KIR.

[0038] [4] The substance according to [1], wherein the binding activity of the substance to human inhibitory KIR is higher than the binding activity to the corresponding human active KIR.

[0039] [5] The substance according to [1], wherein the substance has a higher agonist activity against human inhibitory KIR than against the corresponding human active KIR.

[0040] [6] The substance according to [1], wherein the substance does not substantially exhibit agonist activity against the corresponding human active KIR.

[0041] [7] The substance according to [2], wherein the polypeptide is any one of the following polypeptides (1) to (3):

[0042] (1) A polypeptide whose binding site for KIR2DL2 and / or KIR2DL3 comprises a C1 epitope motif sequence (amino acid 77 is S and amino acid 80 is N) contained in HLA-C1 type HLA-Cw1, Cw3, Cw7, Cw8, Cw9, Cw10, Cw12, Cw14, Cw16, HLA-B46 or B73, and which lacks or weakens binding to TCR;

[0043] (2) a polypeptide whose binding site for KIR2DL1 comprises a C2 epitope motif sequence (amino acid 77 is N and amino acid 80 is K) contained in HLA-Cw2, Cw4, Cw5, Cw6, Cw15 or Cw17 belonging to HLA-C2 type, and which lacks or weakens the binding to TCR;

[0044] (3) A polypeptide whose binding site for KIR3DL1 comprises a Bw4 epitope motif sequence (amino acid 77 is N, amino acid 80 is I or T, and amino acid 83 is R) contained in HLA-B5, B27, B37, B38, B44, B49, B51, B52, B53, B57, B58, B59, B77, A23, A24, A25 or A32 belonging to HLA-Bw4 type, and in which the binding property to TCR is deleted or weakened.

[0045] [8] The substance according to [3], characterized in that the human inhibitory KIR is KIR2DL2 and / or KIR2DL3, and the binding site contains amino acid sequences from the heavy chain variable region and the light chain variable region of a monoclonal antibody or antigen-binding fragment that binds to KIR2DL2 and / or KIR2DL3.

[0046] [9] The substance according to [8], characterized in that the monoclonal antibody or antigen-binding fragment that binds to KIR2DL2 and / or KIR2DL3 competes with lirelumab, Pan2D, or a scFv consisting of any of the following amino acid sequences (i) to (x) in binding to KIR2DL2 and / or KIR2DL3:

[0047] (i) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 33 and the amino acid sequence of the light chain variable region of SEQ ID NO: 34;

[0048] (ii) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 35 and the amino acid sequence of the light chain variable region of SEQ ID NO: 36;

[0049] (iii) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 37 and the amino acid sequence of the light chain variable region of SEQ ID NO: 38;

[0050] (iv) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 39 and the amino acid sequence of the light chain variable region of SEQ ID NO: 40;

[0051] (v) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 41 and the amino acid sequence of the light chain variable region of SEQ ID NO: 42;

[0052] (vi) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 43 and the amino acid sequence of the light chain variable region of SEQ ID NO: 44;

[0053] (vii) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 45 and the amino acid sequence of the light chain variable region of SEQ ID NO: 46;

[0054] (viii) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 47 and the amino acid sequence of the light chain variable region of SEQ ID NO: 48;

[0055] (ix) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 49 and the amino acid sequence of the light chain variable region of SEQ ID NO: 50;

[0056] (x) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 51 and the amino acid sequence of the light chain variable region of SEQ ID NO: 52;

[0057] (xi) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 61 and the amino acid sequence of the light chain variable region of SEQ ID NO: 62; and,

[0058] (xii) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 63 and the amino acid sequence of the light chain variable region of SEQ ID NO: 64.

[0059] [9-2] The substance according to [9], characterized in that the substance competes with an scFv composed of an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 51 and the amino acid sequence of the light chain variable region of SEQ ID NO: 52.

[0060]

[10] The substance according to [8], characterized in that the binding site of the substance for KIR2DL2 and / or KIR2DL3 comprises a variable region containing amino acids arranged to be close to the following amino acids: 1 to 6 amino acids containing Asp at position 47 in the region of amino acids 44 to 49 in the amino acid sequence of KIR2DL2 of SEQ ID NO:6; 2 to 4 amino acids containing Gly at position 67 and Pro at position 68 in the region of amino acids 65 to 68; and 1 to 4 amino acids containing Asp at position 72 in the region of amino acids 70 to 73.

[0061]

[11] The substance according to

[10] , characterized in that, in the binding site of the substance for KIR2DL2 and / or KIR2DL3, the amino acid that interacts with Asp at position 47 of SEQ ID NO:6 is Tyr, the amino acid that interacts with Gly at position 67 of SEQ ID NO:6 is Gly, the amino acid that interacts with Pro at position 68 of SEQ ID NO:6 is Tyr, and the amino acid that interacts with Asp at position 72 of SEQ ID NO:6 is Thr, Asn, Gly, His, Ile, Ser, Val or Ala.

[0062]

[12] The substance according to

[11] , characterized in that the amino acids contained in the variable region defined in

[11] belong to CDR3.

[0063]

[13] The substance according to [8], which is a polypeptide comprising any one of the following binding sites (I) to (XII) as a binding site for KIR2DL2 and / or KIR2DL3, and may have one or two amino acid mutations in each CDR:

[0064] (I) a binding site comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO:33 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO:34;

[0065] (II) a binding site comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO:35 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO:36;

[0066] (III) a binding site comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 37 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 38;

[0067] (IV) a binding site comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO:39 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO:40;

[0068] (V) a binding site comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO:41 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO:42;

[0069] (VI) a binding site comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO:43 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO:44;

[0070] (VII) a binding site comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO:45 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO:46;

[0071] (VIII) a binding site comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO:47 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO:48;

[0072] (IX) a binding site comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO:49 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO:50;

[0073] (X) a binding site comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO:51 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO:52;

[0074] (XI) a binding site comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO:61 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO:62; and

[0075] (XII) A binding site comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2 and CDR3 of SEQ ID NO:63 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2 and CDR3 of SEQ ID NO:64.

[0076] [13-2] The substance according to

[13] , wherein the substance is a polypeptide comprising a binding site, wherein the binding site comprises a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2 and CDR3 of SEQ ID NO:51 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2 and CDR3 of SEQ ID NO:52.

[0077]

[14] The substance according to

[13] , wherein the C-terminal side and / or N-terminal side of each CDR may have one or two amino acid mutations.

[0078] [14-2] The substance according to

[13] or

[14] , wherein in the second variable region in (x), the 94th amino acid Thr of SEQ ID NO: 52 can be substituted with Asn, Gly, His, Ile, Ser, Val or Ala.

[0079]

[15] The substance according to

[13] , which is a polypeptide comprising any one of the following binding sites (I) to (XII) as a binding site for KIR2DL2 and / or KIR2DL3:

[0080] (I) a binding site comprising a combination of a first variable region comprising the amino acid sequence of SEQ ID NO: 33 and a second variable region comprising the amino acid sequence of SEQ ID NO: 34;

[0081] (II) a binding site comprising a combination of a first variable region comprising the amino acid sequence of SEQ ID NO: 35 and a second variable region comprising the amino acid sequence of SEQ ID NO: 36;

[0082] (III) a binding site comprising a combination of a first variable region comprising the amino acid sequence of SEQ ID NO: 37 and a second variable region comprising the amino acid sequence of SEQ ID NO: 38;

[0083] (IV) a binding site comprising a combination of a first variable region comprising the amino acid sequence of SEQ ID NO: 39 and a second variable region comprising the amino acid sequence of SEQ ID NO: 40;

[0084] (V) a binding site comprising a combination of a first variable region comprising the amino acid sequence of SEQ ID NO:41 and a second variable region comprising the amino acid sequence of SEQ ID NO:42;

[0085] (VI) a binding site comprising a combination of a first variable region comprising the amino acid sequence of SEQ ID NO: 43 and a second variable region comprising the amino acid sequence of SEQ ID NO: 44;

[0086] (VII) a binding site comprising a combination of a first variable region comprising the amino acid sequence of SEQ ID NO: 45 and a second variable region comprising the amino acid sequence of SEQ ID NO: 46;

[0087] (VIII) a binding site comprising a combination of a first variable region comprising the amino acid sequence of SEQ ID NO: 47 and a second variable region comprising the amino acid sequence of SEQ ID NO: 48;

[0088] (IX) a binding site comprising a combination of a first variable region comprising the amino acid sequence of SEQ ID NO: 49 and a second variable region comprising the amino acid sequence of SEQ ID NO: 50;

[0089] (X) a binding site comprising a combination of a first variable region comprising the amino acid sequence of SEQ ID NO: 51 and a second variable region comprising the amino acid sequence of SEQ ID NO: 52;

[0090] (XI) a binding site comprising a combination of a first variable region comprising the amino acid sequence of SEQ ID NO: 61 and a second variable region comprising the amino acid sequence of SEQ ID NO: 62; and

[0091] (XII) A binding site comprising a combination of a first variable region comprising the amino acid sequence of SEQ ID NO:63 and a second variable region comprising the amino acid sequence of SEQ ID NO:64.

[0092] [15-2] The substance according to

[15] , wherein the substance is a polypeptide comprising a binding site, wherein the binding site comprises a combination of a first variable region comprising the amino acid sequence of SEQ ID NO:51 and a second variable region comprising the amino acid sequence of SEQ ID NO:52.

[0093]

[16] The substance according to [8], characterized in that the binding activity of the substance against KIR2DL2 and / or KIR2DL3 is higher than the binding activity against KIR2DS1, KIR2DS2 and / or KIR2DS4.

[0094]

[17] The substance according to [8], characterized in that the agonist activity of the substance against KIR2DL2 and / or KIR2DL3 is higher than the agonist activity against KIR2DS1, KIR2DS2 and / or KIR2DS4.

[0095]

[18] The substance according to

[17] , characterized in that the substance does not substantially exhibit agonist activity against KIR2DS1, KIR2DS2 and / or KIR2DS4.

[0096]

[19] The substance according to

[17] , characterized in that the substance does not substantially bind to KIR2DS1, KIR2DS2 and KIR2DS4.

[0097]

[20] The substance according to claim 19, characterized in that the substance comprises an antigen-binding fragment obtained by the following method:

[0098] Step A: mixing a tagged substance containing a soluble protein of the extracellular domain of KIR2DL2 or KIR2DL3 into a suspension containing a polyclonal display body group, then bringing the suspension into contact with a carrier on which a substance that specifically binds to the tag portion is fixed, and recovering the display body bound to the carrier, thereby obtaining a display body group that displays an antigen binding site that binds to KIR2DL2 or KIR2DL3, wherein the polyclonal display body group includes an antigen binding site that exhibits binding to KIR2DL2 or KIR2DL3 displayed on the surface;

[0099] Step B: In the suspension containing the display body group obtained in step A, a labeled substance containing a soluble protein of the extracellular domain of KIR2DS1, KIR2DS2 and KIR2DS4 is mixed, and the labeled substance is brought into contact with a carrier on which a substance binding to the tag portion is fixed, and the display body not bound to the carrier is recovered, thereby removing the display body displaying the antigen binding site binding to KIR2DS1, KIR2DS2 and KIR2DS4 from the cell group;

[0100] Step C: performing gene analysis on the display obtained by the method comprising the above steps to identify the gene sequence encoding the antigen binding site; and

[0101] Step D: Allowing cells to express the gene containing the identified antigen binding site.

[0102]

[21] The substance according to

[19] , characterized in that the substance comprises an antigen-binding fragment obtained by the following method:

[0103] Step A: In a suspension containing a polyclonal display body group, a labeled substance containing a soluble protein of the extracellular domain of KIR2DL2 or KIR2DL3 and a soluble protein containing the extracellular domain of KIR2DS1, KIR2DS2 and KIR2DS4 that is not labeled are mixed, and then the suspension is brought into contact with a carrier on which a substance that specifically binds to the label portion is fixed, and the display body bound to the carrier is recovered, thereby obtaining a display body group that displays an antigen binding site that specifically binds to KIR2DL2 or KIR2DL3, wherein the polyclonal display body group includes an antigen binding site that exhibits binding to KIR2DL2 or KIR2DL3 displayed on the surface;

[0104] Step B: performing gene analysis on the display obtained by the method comprising the above steps to identify the gene sequence encoding the antigen binding site; and

[0105] Step C: Allowing cells to express the gene containing the identified antigen binding site.

[0106]

[22] The substance according to

[20] or

[21] , wherein the combination of the tag and the substance that binds to the tag is a combination of biotin-avidin.

[0107]

[23] The substance according to

[20] or

[21] , characterized in that the substance is obtained by a method further comprising the step of selecting a substance having agonist activity against KIR2DL2 or KIR2DL3.

[0108]

[24] The substance according to any one of [8] to

[23] , wherein the polypeptide is a monoclonal antibody or an antigen-binding fragment.

[0109]

[25] The substance according to

[24] is characterized in that the substance is a single-chain antibody or scFv in which the first variable region and the second variable region are connected by a GS linker or a G4S linker.

[0110]

[26] The substance according to any one of [8] to

[23] , wherein the polypeptide is a membrane protein having an extracellular domain comprising a binding site for KIR2DL2 and / or KIR2DL3 and a transmembrane domain.

[0111]

[27] The substance according to [3], characterized in that the inhibitory KIR is KIR3DL1, and the binding site contains amino acid sequences from the heavy chain variable region and the light chain variable region of a monoclonal antibody or antigen-binding fragment that binds to KIR3DL1.

[0112]

[28] The substance according to

[27] , characterized in that the monoclonal antibody or antigen-binding fragment that binds to KIR3DL1 competes with a scFv consisting of any one of the following amino acid sequences (xiii) to (xviii) in binding to KIR3DL1:

[0113] (xiii) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 56 and the amino acid sequence of the light chain variable region of SEQ ID NO: 55;

[0114] (xiv) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 137 and the amino acid sequence of the light chain variable region of SEQ ID NO: 138;

[0115] (xv) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 139 and the amino acid sequence of the light chain variable region of SEQ ID NO: 140;

[0116] (xvi) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 141 and the amino acid sequence of the light chain variable region of SEQ ID NO: 142;

[0117] (xvii) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 143 and the amino acid sequence of the light chain variable region of SEQ ID NO: 144; and

[0118] (xviii) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 145 and the amino acid sequence of the light chain variable region of SEQ ID NO: 146.

[0119]

[29] The substance according to

[27] , which is a polypeptide comprising any one of the following binding sites (XIII) to (XVIII) as a binding site for KIR3DL1, and each CDR may have 1 or 2 amino acid mutations:

[0120] (XIII) a binding site comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO:56 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO:55;

[0121] (XIV) a binding site comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 137 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 138;

[0122] (XV) a binding site comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 139 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 140;

[0123] (XVI) a binding site comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 141 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 142;

[0124] (XVII) a binding site comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 143 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 144; and

[0125] (XVIII) A binding site comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2 and CDR3 of SEQ ID NO:145 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2 and CDR3 of SEQ ID NO:146.

[0126]

[30] The substance according to

[29] , wherein the C-terminal side and / or N-terminal side of each CDR may have one or two amino acid mutations.

[0127]

[31] The substance according to

[27] , which is a polypeptide comprising any one of the following binding sites (XIII) to (XVIII) as a binding site for KIR3DL1:

[0128] (XIII) a binding site comprising a combination of a first variable region comprising the amino acid sequence of SEQ ID NO: 56 and a second variable region comprising the amino acid sequence of SEQ ID NO: 55;

[0129] (XIIII) a binding site comprising a combination of a first variable region comprising the amino acid sequence of SEQ ID NO: 137 and a second variable region comprising the amino acid sequence of SEQ ID NO: 138;

[0130] (XV) a binding site comprising a combination of a first variable region comprising the amino acid sequence of SEQ ID NO: 139 and a second variable region comprising the amino acid sequence of SEQ ID NO: 140;

[0131] (XVI) a binding site comprising a combination of a first variable region comprising the amino acid sequence of SEQ ID NO: 141 and a second variable region comprising the amino acid sequence of SEQ ID NO: 142;

[0132] (XVII) a binding site comprising a combination of a first variable region comprising the amino acid sequence of SEQ ID NO: 143 and a second variable region comprising the amino acid sequence of SEQ ID NO: 144; and

[0133] (XVIII) A binding site comprising a combination of a first variable region comprising the amino acid sequence of SEQ ID NO: 145 and a second variable region comprising the amino acid sequence of SEQ ID NO: 146.

[0134]

[32] The substance according to

[27] , characterized in that the binding activity of the substance against KIR3DL1 is higher than the binding activity against KIR3DS1.

[0135]

[33] The substance according to

[27] , characterized in that the agonist activity of the substance against KIR3DL1 is higher than the agonist activity against KIR3DS1.

[0136]

[34] The substance according to

[33] , characterized in that the substance does not substantially exhibit agonist activity against KIR3DS1.

[0137]

[35] The substance according to

[34] , characterized in that the substance does not substantially bind to KIR3DS1.

[0138]

[36] The substance according to

[35] , characterized in that the substance comprises an antigen-binding fragment obtained by a method comprising the following steps:

[0139] Step A: mixing a tagged substance containing a soluble protein of the extracellular domain of KIR3DL1 in a suspension containing a polyclonal display body group, and then bringing the suspension into contact with a carrier on which a substance that specifically binds to the tag portion is fixed, and recovering the display body bound to the carrier, thereby obtaining a display body group that displays an antigen binding site that binds to KIR3DL1, wherein the polyclonal display body group includes an antigen binding site that exhibits binding to KIR3DL1 and is displayed on the surface;

[0140] Step B: a labeled substance containing a soluble protein of the extracellular domain of KIR3DS1 is mixed in a suspension containing the display body group obtained in step A, and the labeled substance is brought into contact with a carrier on which a substance binding to the tag portion is fixed, and the display body not bound to the carrier is recovered, thereby removing the display body displaying the antigen binding site bound to KIR3DS1 from the cell group;

[0141] Step C: performing gene analysis on the display obtained by the method comprising the above steps to identify the gene sequence encoding the antigen binding site; and

[0142] Step D: Allowing cells to express the gene containing the identified antigen binding site.

[0143]

[37] The substance according to

[36] , characterized in that the substance comprises an antigen-binding fragment obtained by the following method:

[0144] Step A: In a suspension containing a polyclonal display body group, a labeled substance containing a soluble protein of the extracellular domain of KIR3DL1 and a soluble protein containing an unlabeled extracellular domain of KIR3DS1 are mixed, and then the suspension is brought into contact with a carrier on which a substance that specifically binds to the label portion is fixed, and the display body bound to the carrier is recovered, thereby obtaining a display body group that displays an antigen binding site that specifically binds to KIR3DL1, wherein the polyclonal display body group includes an antigen binding site that exhibits binding to KIR3DL1 and is displayed on the surface;

[0145] Step B: performing gene analysis on the display obtained by the method comprising the above steps to identify the gene sequence encoding the antigen binding site; and

[0146] Step C: Allowing cells to express the gene containing the identified antigen binding site.

[0147]

[38] The substance according to

[36] or

[37] , wherein the combination of the tag and the substance that binds to the tag is a combination of biotin-avidin.

[0148]

[39] The substance according to any one of

[36] to

[38] , characterized in that the substance is obtained by a method further comprising the step of selecting a substance having agonist activity against KIR3DL1.

[0149]

[40] The substance according to any one of

[27] to

[39] , wherein the polypeptide is a monoclonal antibody or an antigen-binding fragment.

[0150]

[41] The substance according to

[40] is characterized in that the substance is a single-chain antibody or scFv in which the first variable region and the second variable region are connected by a GS linker or a G4S linker.

[0151]

[42] The substance according to any one of

[27] to

[34] , wherein the polypeptide is a membrane protein having an extracellular domain including a binding site for KIR3DL1 and a transmembrane domain.

[0152]

[43] The substance according to [3], characterized in that the inhibitory KIR is KIR2DL1, and the binding site contains amino acid sequences from the heavy chain variable region and the light chain variable region of a monoclonal antibody or antigen-binding fragment that binds to KIR2DL1.

[0153]

[44] The substance according to

[43] , characterized in that the monoclonal antibody or antigen-binding fragment that binds to KIR2DL1 competes with Pan2D in binding to KIR2DL1.

[0154]

[45] The substance according to

[43] , characterized in that the monoclonal antibody or antigen-binding fragment that binds to KIR2DL1 competes with a scFv consisting of any one of the following amino acid sequences (ci) to (cxv) in binding to KIR2DL1:

[0155] (ci) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 107 and the amino acid sequence of the light chain variable region of SEQ ID NO: 108;

[0156] (cii) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 109 and the amino acid sequence of the light chain variable region of SEQ ID NO: 110;

[0157] (ciii) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 111 and the amino acid sequence of the light chain variable region of SEQ ID NO: 112;

[0158] (civ) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 113 and the amino acid sequence of the light chain variable region of SEQ ID NO: 114;

[0159] (cv) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 115 and the amino acid sequence of the light chain variable region of SEQ ID NO: 116;

[0160] (cvi) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 117 and the amino acid sequence of the light chain variable region of SEQ ID NO: 118;

[0161] (cvii) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 119 and the amino acid sequence of the light chain variable region of SEQ ID NO: 120;

[0162] (cviii) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 121 and the amino acid sequence of the light chain variable region of SEQ ID NO: 122;

[0163] (cviiii) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 123 and the amino acid sequence of the light chain variable region of SEQ ID NO: 124;

[0164] (cx) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 125 and the amino acid sequence of the light chain variable region of SEQ ID NO: 126;

[0165] (cxi) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 127 and the amino acid sequence of the light chain variable region of SEQ ID NO: 128;

[0166] (cxii) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 129 and the amino acid sequence of the light chain variable region of SEQ ID NO: 130;

[0167] (cxiii) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 131 and the amino acid sequence of the light chain variable region of SEQ ID NO: 132;

[0168] (cxiv) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 133 and the amino acid sequence of the light chain variable region of SEQ ID NO: 134; and

[0169] (cxv) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 135 and the amino acid sequence of the light chain variable region of SEQ ID NO: 136.

[0170]

[46] The substance according to

[43] , which is a polypeptide comprising any one of the following binding sites (CI) to (CXV) as a binding site for KIR2DL1, and each CDR may have 1 or 2 amino acid mutations:

[0171] (CI) a binding site comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 107 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 108;

[0172] (CII) a binding site comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 109 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 110;

[0173] (CIII) a binding site comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 111 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 112;

[0174] (CIV) a binding site comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 113 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 114 (preferably a variable region comprising an amino acid sequence of SEQ ID NO: 114);

[0175] (CV) a binding site comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 115 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 116;

[0176] (CVI) a binding site comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 117 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 118;

[0177] (CVII) a binding site comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 119 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 120;

[0178] (CVIII) a binding site comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 121 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 122;

[0179] (CVIIII) a binding site comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 123 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 124;

[0180] (CX) a binding site comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 125 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 126;

[0181] (CXI) a binding site comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 127 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 128;

[0182] (CXII) a binding site comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 129 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 130;

[0183] (CXIII) a binding site comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 131 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 132;

[0184] (CXIV) a binding site comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 133 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 134; and

[0185] (CXV) comprises a combined binding site of a first variable region comprising an amino acid sequence derived from CDR1, CDR2 and CDR3 of SEQ ID NO:135 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2 and CDR3 of SEQ ID NO:136.

[0186]

[47] The substance according to

[46] , wherein the C-terminal side and / or N-terminal side of each CDR may have one or two amino acid mutations.

[0187]

[48] ​​The substance according to

[46] , which is a polypeptide comprising any one of the following binding sites (CI) to (CXV) as a binding site for KIR2DL1:

[0188] (CI) a binding site comprising a combination of a first variable region comprising the amino acid sequence of SEQ ID NO: 107 and a second variable region comprising the amino acid sequence of SEQ ID NO: 108;

[0189] (CII) a binding site comprising a combination of a first variable region comprising the amino acid sequence of SEQ ID NO: 109 and a second variable region comprising the amino acid sequence of SEQ ID NO: 110;

[0190] (CIII) a binding site comprising a combination of a first variable region comprising the amino acid sequence of SEQ ID NO: 111 and a second variable region comprising the amino acid sequence of SEQ ID NO: 112;

[0191] (CIV) a binding site comprising a combination of a first variable region comprising the amino acid sequence of SEQ ID NO: 113 and a second variable region comprising the amino acid sequence of SEQ ID NO: 114 (preferably a variable region comprising the amino acid sequence of SEQ ID NO: 114);

[0192] (CV) a binding site comprising a combination of a first variable region comprising the amino acid sequence of SEQ ID NO: 115 and a second variable region comprising the amino acid sequence of SEQ ID NO: 116;

[0193] (CVI) a binding site comprising a combination of a first variable region comprising the amino acid sequence of SEQ ID NO: 117 and a second variable region comprising the amino acid sequence of SEQ ID NO: 118;

[0194] (CVII) a binding site comprising a combination of a first variable region comprising the amino acid sequence of SEQ ID NO: 119 and a second variable region comprising the amino acid sequence of SEQ ID NO: 120;

[0195] (CVIII) a binding site comprising a combination of a first variable region comprising the amino acid sequence of SEQ ID NO: 121 and a second variable region comprising the amino acid sequence of SEQ ID NO: 122;

[0196] (CVIIII) a binding site comprising a combination of a first variable region comprising the amino acid sequence of SEQ ID NO: 123 and a second variable region comprising the amino acid sequence of SEQ ID NO: 124;

[0197] (CX) a binding site comprising a combination of a first variable region comprising the amino acid sequence of SEQ ID NO: 125 and a second variable region comprising the amino acid sequence of SEQ ID NO: 126;

[0198] (CXI) a binding site comprising a combination of a first variable region comprising the amino acid sequence of SEQ ID NO: 127 and a second variable region comprising the amino acid sequence of SEQ ID NO: 128;

[0199] (CXII) a binding site comprising a combination of a first variable region comprising the amino acid sequence of SEQ ID NO: 129 and a second variable region comprising the amino acid sequence of SEQ ID NO: 130;

[0200] (CXIII) a binding site comprising a combination of a first variable region comprising the amino acid sequence of SEQ ID NO: 131 and a second variable region comprising the amino acid sequence of SEQ ID NO: 132;

[0201] (CXIV) a binding site comprising a combination of a first variable region comprising the amino acid sequence of SEQ ID NO: 133 and a second variable region comprising the amino acid sequence of SEQ ID NO: 134; and

[0202] (CXV) comprises a binding site comprising a first variable region comprising the amino acid sequence of SEQ ID NO:135 and a second variable region comprising the amino acid sequence of SEQ ID NO:136.

[0203]

[49] The substance according to

[43] , characterized in that the binding activity of the substance against KIR2DL1 is higher than the binding activity against KIR2DS1.

[0204]

[50] The substance according to

[43] , characterized in that the agonist activity of the substance against KIR2DL1 is higher than the agonist activity against KIR2DS1.

[0205]

[51] The substance according to

[50] , characterized in that the substance does not substantially exhibit agonist activity against KIR2DS1.

[0206]

[52] The substance according to

[51] , characterized in that the substance does not substantially bind to KIR2DS1, KIR2DS2 and KIR2DS4.

[0207]

[53] The substance according to

[52] , characterized in that the substance comprises an antigen-binding fragment obtained by the following method:

[0208] Step A: mixing a tagged substance containing a soluble protein of the extracellular domain of KIR2DL1 in a suspension containing a polyclonal display body group, and then bringing the suspension into contact with a carrier on which a substance that specifically binds to the tag portion is fixed, and recovering the display body bound to the carrier, thereby obtaining a display body group that displays an antigen binding site that binds to KIR2DL1, wherein the polyclonal display body group includes an antigen binding site that exhibits binding to KIR2DL1 and is displayed on the surface;

[0209] Step B: In the suspension containing the display body group obtained in step A, a labeled substance containing a soluble protein of the extracellular domain of KIR2DS1, KIR2DS2 and KIR2DS4 is mixed, and the labeled substance is brought into contact with a carrier on which a substance binding to the tag portion is fixed, and the display body not bound to the carrier is recovered, thereby removing the display body displaying the antigen binding site binding to KIR2DS1, KIR2DS2 and KIR2DS4 from the cell group;

[0210] Step C: performing gene analysis on the display obtained by the method comprising the above steps to identify the gene sequence encoding the antigen binding site; and

[0211] Step D: Allowing cells to express the gene containing the identified antigen binding site.

[0212]

[54] The substance according to

[52] , characterized in that the substance comprises an antigen-binding fragment obtained by a method comprising the following steps:

[0213] Step A: In a suspension containing a polyclonal display body group, a labeled substance containing a soluble protein of the extracellular domain of KIR2DL1 and a soluble protein containing the extracellular domain of KIR2DS1, KIR2DS2 and KIR2DS4 that is not labeled are mixed, and then the suspension is brought into contact with a carrier on which a substance that specifically binds to the tag portion is fixed, and the display body bound to the carrier is recovered, thereby obtaining a display body group that displays an antigen binding site that specifically binds to KIR2DL1, wherein the polyclonal display body group includes an antigen binding site that exhibits binding to KIR2DL1 and is displayed on the surface;

[0214] Step B: performing gene analysis on the display obtained by the method comprising the above steps to identify the gene sequence encoding the antigen binding site; and

[0215] Step C: Allowing cells to express the gene containing the identified antigen binding site.

[0216]

[55] The substance according to

[53] or

[54] , wherein the combination of the tag and the substance that binds to the tag is a combination of biotin-avidin.

[0217]

[56] The substance according to any one of

[53] to

[55] , characterized in that the substance is obtained by a method further comprising the step of selecting a substance having agonist activity against KIR3DL1.

[0218]

[57] The substance according to any one of

[43] to

[56] , wherein the polypeptide is a monoclonal antibody or an antigen-binding fragment.

[0219]

[58] The substance according to

[57] is characterized in that the substance is a single-chain antibody or scFv in which the first variable region and the second variable region are connected by a GS linker.

[0220]

[59] The substance according to any one of

[43] to

[56] , wherein the polypeptide is a membrane protein having an extracellular domain comprising a binding site for KIR2DL1 and a transmembrane domain.

[0221]

[60] A pharmaceutical composition comprising the substance described in any one of [1] to

[59] as an active ingredient.

[0222]

[61] The pharmaceutical composition according to

[60] , which is an immunorejection inhibitor.

[0223]

[62] A cell, wherein the substance having the activity of binding to an inhibitory KIR and inhibiting the cytotoxic activity of NK cells according to any one of [1] to

[59] is expressed on the cell surface.

[0224]

[63] The cell according to

[62] , wherein a ligand substance for NKG2A is also expressed on the cell surface.

[0225]

[64] The cell according to

[63] , wherein the ligand for NKG2A is HLA-E.

[0226]

[65] The cell according to

[62] , wherein the expression of at least one class I HLA on the cell surface is absent or weakened.

[0227]

[66] The cell according to

[65] , wherein the disappearance or attenuation of the expression of at least one class I HLA on the cell surface is caused by gene editing to modify the class I HLA, B2M, TAP1, TAP2, TAPBP regions on the genome.

[0228]

[67] The cell according to

[65] , wherein the cell is a cell line established from an individual in which HLA expression is hereditarily lost.

[0229]

[68] The cell according to

[62] , wherein a ligand substance for NKG2A is also expressed on the cell surface, and the expression of at least one class I HLA on the cell surface disappears or is weakened.

[0230]

[69] The cell according to

[62] , wherein the cell is a pluripotent stem cell, a T cell, a NK cell, a peripheral blood mononuclear cell (PBMC), a mesenchymal stem cell, a cardiomyocyte, a chondrocyte, a retinal cell, a hepatocyte, a kidney cell or a pancreatic islet cell.

[0231]

[70] The cell according to

[69] , wherein the pluripotent stem cell is an iPS cell or an ES cell.

[0232]

[71] The cell according to any one of

[62] to

[70] , wherein a chimeric receptor is also expressed on the cell surface.

[0233]

[72] The cell according to

[62] , wherein the inhibitory KIR is KIR2DL2 and / or KIR2DL3, and the substance having the activity of binding to KIR2DL2 and / or KIR2DL3 and inhibiting the cytotoxic activity of NK cells is the membrane protein described in

[26] .

[0234]

[73] The cell according to

[72] , characterized in that the membrane protein that binds to KIR2DL2 and / or KIR2DL3 comprises a binding site that competes with lirelumab, Pan2D, or a scFv consisting of any of the following amino acid sequences (i) to (xii) in binding to KIR2DL2 and / or KIR2DL3:

[0235] (i) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 33 and the amino acid sequence of the light chain variable region of SEQ ID NO: 34;

[0236] (ii) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 35 and the amino acid sequence of the light chain variable region of SEQ ID NO: 36;

[0237] (iii) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 37 and the amino acid sequence of the light chain variable region of SEQ ID NO: 38;

[0238] (iv) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 39 and the amino acid sequence of the light chain variable region of SEQ ID NO: 40;

[0239] (v) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 41 and the amino acid sequence of the light chain variable region of SEQ ID NO: 42;

[0240] (vi) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 43 and the amino acid sequence of the light chain variable region of SEQ ID NO: 44;

[0241] (vii) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 45 and the amino acid sequence of the light chain variable region of SEQ ID NO: 46;

[0242] (viii) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 47 and the amino acid sequence of the light chain variable region of SEQ ID NO: 48;

[0243] (ix) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 49 and the amino acid sequence of the light chain variable region of SEQ ID NO: 50;

[0244] (x) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 51 and the amino acid sequence of the light chain variable region of SEQ ID NO: 52;

[0245] (xi) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 61 and the amino acid sequence of the light chain variable region of SEQ ID NO: 62; and,

[0246] (xii) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 63 and the amino acid sequence of the light chain variable region of SEQ ID NO: 64.

[0247]

[74] The cell according to

[72] , wherein a membrane protein is expressed on the cell surface, wherein the membrane protein is a membrane protein comprising any one of the following binding sites (I) to (XII) as a binding site for KIR2DL2 and / or KIR2DL3, and each CDR may have one or two amino acid mutations:

[0248] (I) a binding site comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO:33 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO:34;

[0249] (II) a binding site comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO:35 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO:36;

[0250] (III) a binding site comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 37 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 38;

[0251] (IV) a binding site comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO:39 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO:40;

[0252] (V) a binding site comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO:41 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO:42;

[0253] (VI) a binding site comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO:43 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO:44;

[0254] (VII) a binding site comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO:45 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO:46;

[0255] (VIII) a binding site comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO:47 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO:48;

[0256] (IX) a binding site comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO:49 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO:50;

[0257] (X) a binding site comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO:51 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO:52;

[0258] (XI) a binding site comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO:61 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO:62; and

[0259] (XII) A binding site comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2 and CDR3 of SEQ ID NO:63 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2 and CDR3 of SEQ ID NO:64.

[0260]

[75] The cell according to

[74] , wherein the C-terminal side and / or N-terminal side of each CDR may have one or two amino acid mutations.

[0261]

[76] The cell according to

[62] , wherein the inhibitory KIR is KIR3DL1, and the substance having the activity of binding to KIR3DL1 and inhibiting the cytotoxic activity of NK cells is the membrane protein according to

[42] .

[0262]

[77] The cell according to

[76] , wherein the membrane protein that binds to KIR3DL1 comprises a binding site that competes with a scFv consisting of any one of the following amino acid sequences (xiii) to (xviii) in binding to KIR3DL1:

[0263] (xiii) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 56 and the amino acid sequence of the light chain variable region of SEQ ID NO: 55;

[0264] (xiv) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 137 and the amino acid sequence of the light chain variable region of SEQ ID NO: 138;

[0265] (xv) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 139 and the amino acid sequence of the light chain variable region of SEQ ID NO: 140;

[0266] (xvi) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 141 and the amino acid sequence of the light chain variable region of SEQ ID NO: 142;

[0267] (xvii) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 143 and the amino acid sequence of the light chain variable region of SEQ ID NO: 144; and

[0268] (xviii) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 145 and the amino acid sequence of the light chain variable region of SEQ ID NO: 146.

[0269]

[78] The cell according to

[75] , wherein a membrane protein is expressed on the cell surface, wherein the membrane protein is a membrane protein comprising any one of the following binding sites (XIII) to (XVIII) as a binding site for KIR3DL1, and each CDR may have one or two amino acid mutations:

[0270] (XIII) a binding site comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO:56 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO:55;

[0271] (XIV) a binding site comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 137 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 138 (preferably a variable region comprising an amino acid sequence of SEQ ID NO: 138);

[0272] (XV) a binding site comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 139 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 140;

[0273] (XVI) a binding site comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 141 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 142;

[0274] (XVII) a binding site comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 143 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 144; and

[0275] (XVIII) A binding site comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2 and CDR3 of SEQ ID NO:145 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2 and CDR3 of SEQ ID NO:146.

[0276]

[79] The cell according to

[78] , wherein the C-terminal side and / or N-terminal side of each CDR may have one or two amino acid mutations.

[0277]

[80] The cell according to

[62] , wherein the inhibitory KIR is KIR2DL1, and the substance having the activity of binding to KIR2DL1 and inhibiting the cytotoxic activity of NK cells is the membrane protein according to

[59] .

[0278]

[81] The cell according to

[80] , characterized in that the membrane protein that binds to KIR2DL1 contains a binding site that competes with Pan2D in binding to KIR2DL1.

[0279]

[82] The cell according to

[78] , wherein a membrane protein is expressed on the cell surface, wherein the membrane protein is a membrane protein comprising the following (P) binding site as a binding site for KIR2DL1, and each CDR may have 1 or 2 amino acid mutations:

[0280] (P) A binding site comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2 and CDR3 of SEQ ID NO:31 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2 and CDR3 of SEQ ID NO:32.

[0281]

[83] The cell according to

[82] , wherein the C-terminal side and / or N-terminal side of each CDR may have one or two amino acid mutations.

[0282]

[84] The cell according to

[80] , characterized in that the membrane protein that binds to KIR2DL1 contains a binding site that competes with the scFv consisting of any one of the following amino acid sequences (ci) to (cxv) in binding to KIR2DL1:

[0283] (ci) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 107 and the amino acid sequence of the light chain variable region of SEQ ID NO: 108;

[0284] (cii) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 109 and the amino acid sequence of the light chain variable region of SEQ ID NO: 110;

[0285] (ciii) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 111 and the amino acid sequence of the light chain variable region of SEQ ID NO: 112;

[0286] (civ) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 113 and the amino acid sequence of the light chain variable region of SEQ ID NO: 114;

[0287] (cv) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 115 and the amino acid sequence of the light chain variable region of SEQ ID NO: 116;

[0288] (cvi) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 117 and the amino acid sequence of the light chain variable region of SEQ ID NO: 118;

[0289] (cvii) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 119 and the amino acid sequence of the light chain variable region of SEQ ID NO: 120;

[0290] (cviii) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 121 and the amino acid sequence of the light chain variable region of SEQ ID NO: 122;

[0291] (cviiii) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 123 and the amino acid sequence of the light chain variable region of SEQ ID NO: 124;

[0292] (cx) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 125 and the amino acid sequence of the light chain variable region of SEQ ID NO: 126;

[0293] (cxi) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 127 and the amino acid sequence of the light chain variable region of SEQ ID NO: 128;

[0294] (cxii) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 129 and the amino acid sequence of the light chain variable region of SEQ ID NO: 130;

[0295] (cxiii) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 131 and the amino acid sequence of the light chain variable region of SEQ ID NO: 132;

[0296] (cxiv) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 133 and the amino acid sequence of the light chain variable region of SEQ ID NO: 134; and

[0297] (cxv) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 135 and the amino acid sequence of the light chain variable region of SEQ ID NO: 136.

[0298]

[85] The cell according to

[80] , wherein a membrane protein is expressed on the cell surface, wherein the membrane protein is a membrane protein comprising any one of the following binding sites (CI) to (CXV) as a binding site for KIR2DL1, and each CDR may have one or two amino acid mutations:

[0299] (CI) a binding site comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 107 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 108;

[0300] (CII) a binding site comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 109 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 110;

[0301] (CIII) a binding site comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 111 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 112;

[0302] (CIV) a binding site comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 113 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 114 (preferably a variable region comprising an amino acid sequence of SEQ ID NO: 114);

[0303] (CV) a binding site comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 115 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 116;

[0304] (CVI) a binding site comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 117 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 118;

[0305] (CVII) a binding site comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 119 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 120;

[0306] (CVIII) a binding site comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 121 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 122;

[0307] (CVIIII) a binding site comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 123 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 124;

[0308] (CX) a binding site comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 125 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 126;

[0309] (CXI) a binding site comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 127 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 128;

[0310] (CXII) a binding site comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 129 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 130;

[0311] (CXIII) a binding site comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 131 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 132;

[0312] (CXIV) a binding site comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 133 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 134; and

[0313] (CXV) comprises a combined binding site of a first variable region comprising an amino acid sequence derived from CDR1, CDR2 and CDR3 of SEQ ID NO:135 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2 and CDR3 of SEQ ID NO:136.

[0314]

[86] The cell according to

[85] , wherein the C-terminal side and / or N-terminal side of each CDR may have one or two amino acid mutations.

[0315]

[87] A pharmaceutical composition, the active ingredient of which contains the cell according to any one of

[62] to

[86] , wherein the risk of immune rejection in a subject to which the composition is administered or transplanted is reduced.

[0316]

[88] The pharmaceutical composition according to

[87] , which is an immunorejection inhibitor.

[0317]

[89] A method for reducing or inhibiting immune rejection of therapeutic cells by a subject, the method comprising the step of expressing the substance described in any one of [1] to

[59] on the cell surface of the therapeutic cells.

[0318]

[90] A method for producing therapeutic cells in which the risk of immune rejection in a subject is reduced or suppressed, the method comprising the step of expressing the substance described in any one of [1] to

[59] on the cell surface of the therapeutic cells.

[0319]

[91] A method for evaluating the immune rejection escape activity caused by the agonist activity of a test substance on inhibitory KIR2DL2 or KIR2DL3, the method comprising the following steps:

[0320] (A) a step of contacting NK cells from a subject homozygous for HLA-C type C1 / C1 and target cells from a subject homozygous for HLA-C type C2 / C2 in vitro in the presence of a test substance that binds to KIR2DL2 or KIR2DL3, wherein the expression type of HLA containing the Bw4 motif is the same in the donor of the NK cells and the donor of the target cells;

[0321] (B) After step A, a step of measuring the number of surviving target cells.

[0322]

[92] A method for evaluating the immune rejection escape activity caused by the agonist activity of a test substance on inhibitory KIR2DL1, the method comprising the following steps:

[0323] (A) a step of contacting NK cells from a subject homozygous for HLA-C type C2 / C2 and target cells from a subject homozygous for HLA-C type C1 / C1 in vitro in the presence of a test substance that binds to KIR2DL1, wherein the expression type of HLA containing the Bw4 motif of the donor of the NK cells and the donor of the target cells is the same;

[0324] (B) After step A, a step of measuring the number of surviving target cells.

[0325]

[93] A method for evaluating the immune rejection escape activity caused by the agonist activity of a test substance on inhibitory KIR3DL1, the method comprising the following steps:

[0326] (A) a step of contacting NK cells from a subject with HLA-Bw4 type positive / positive homozygous or positive / negative heterozygous and target cells from a subject with HLA-Bw4 type negative / negative homozygous in vitro in the presence of a test substance that binds to KIR3DL1, wherein the donor of the NK cells and the donor of the target cells have the same expression type of HLA-C type;

[0327] (B) After step A, a step of measuring the number of surviving target cells.

[0328]

[94] A method for screening a substance having immune rejection escape activity, the method comprising: in the method described in any one of

[91] to

[93] , selecting a test substance having a high number of surviving target cells in step B as a substance having immune rejection escape activity.

[0329]

[95] The method according to any one of

[91] to

[93] , characterized in that in step (A), the test substance is present in the form of a membrane protein expressed on the surface of the target cell.

[0330]

[96] A method for producing a substance capable of binding to human inhibitory KIR and inhibiting the activity of NK cells, the method comprising the following steps:

[0331] Step A: mixing a tagged substance of a soluble protein containing an extracellular domain of an inhibitory KIR in a suspension containing a polyclonal display body group, and then bringing the suspension into contact with a carrier on which a substance that specifically binds to the tag portion is fixed, and recovering the display body bound to the carrier, thereby obtaining a display body group that displays an antigen binding site that binds to the KIR, wherein the polyclonal display body group includes an antigen binding site that exhibits binding to the inhibitory KIR and is displayed on the surface;

[0332] Step B: In a suspension containing the display body group obtained in step A, a labeled substance containing a soluble protein of the extracellular domain of the active KIR corresponding to the inhibitory KIR is mixed, and the labeled substance is brought into contact with a carrier to which a substance bound to the label portion is fixed, and the display body that is not bound to the carrier is recovered, thereby removing the display body that displays the antigen binding site bound to the active KIR from the cell group;

[0333] Step C: performing gene analysis on the display obtained by the method comprising the above steps to identify the gene sequence encoding the antigen binding site; and

[0334] Step D: Allowing cells to express the gene containing the identified antigen binding site.

[0335]

[97] A method for producing a substance capable of binding to human inhibitory KIR and inhibiting the activity of NK cells, the method comprising the following steps:

[0336] Step A: In a suspension containing a polyclonal display body group, a labeled substance containing a soluble protein of the extracellular domain of an inhibitory KIR and a non-labeled soluble protein containing the extracellular domain of an active KIR corresponding to the inhibitory KIR are mixed, and then the suspension is brought into contact with a carrier on which a substance that specifically binds to the label portion is fixed, and the display body bound to the carrier is recovered, thereby obtaining a display body group that displays an antigen binding site that specifically binds to the inhibitory KIR, wherein the polyclonal display body group includes an antigen binding site that exhibits binding to the inhibitory KIR and is displayed on the surface;

[0337] Step B: performing gene analysis on the display obtained by the method comprising the above steps to identify the gene sequence encoding the antigen binding site; and

[0338] Step C: Allowing cells to express the gene containing the identified antigen binding site.

[0339]

[98] The method according to

[96] or

[97] , wherein the combination of the tag and the substance that binds to the tag is a combination of biotin-avidin.

[0340]

[99] The method according to any one of

[96] to

[98] , wherein the method further comprises the step of selecting a substance having agonist activity against the inhibitory KIR.

[0341]

[100] The method according to any one of

[96] to

[99] , wherein the inhibitory KIR is KIR2DL2 or KIR2DL3, and the corresponding active KIR is KIR2DS1, KIR2DS2 and KIR2DS4.

[0342]

[101] The method according to any one of

[96] to

[99] , wherein the inhibitory KIR is KIR3DL1 and the corresponding active KIR is KIR3DS1.

[0343]

[102] The method according to any one of

[96] to

[99] , wherein the inhibitory KIR is KIR2DL1, and the corresponding active KIR is KIR2DS1, KIR2DS2 and KIR2DS4.

[0344] This specification incorporates the disclosure of Japanese Patent Application No. 2022-170981 which is the basis of priority for this application.

[0345] Effects of the Invention

[0346] Substances that have the activity of binding to inhibitory KIR and inhibiting the cytotoxic activity of NK cells and cells expressing the substance inhibit the cytotoxic activity of NK cells, thereby being able to inhibit the rejection reaction of transplanted organs and therapeutic cells. Therefore, substances that have the activity of binding to inhibitory KIR and inhibiting the cytotoxic activity of NK cells and cells expressing the substance can be used as a new allogeneic technology that greatly changes the versatility of organ transplantation and cell therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0347] Figure 1-1 It is a graph showing the results of evaluating the cell-killing activity of NK92 expressing KIR2DL2 against K562 cells, and it is also a graph showing the agonist activity of lirelumab against inhibitory KIR2DL2.

[0348] Figure 1-2 It is a graph showing the results of evaluating the cell-killing activity of NK92 expressing KIR2DL2 against HLA-Cw3-K562 cells, and also a graph showing the agonist inhibitory activity of lirelumab against inhibitory KIR2DL2.

[0349] Figure 2The graph shows that the cell-killing activity against the KIR2DL2-NK92 cell line was attenuated when the anti-KIR antibodies lirelumab and Pan2D were expressed as scFv-membrane proteins.

[0350] Figure 3 This is a graph showing that a binding compound that binds to active KIR enhances the activity of an NK cell line expressing active KIR.

[0351] Figure 4 This is a graph showing the binding properties of KIR2DL2 / 3-specific binders newly obtained by phage panning to inhibitory and active KIRs.

[0352] Figure 5 This is a graph showing the agonist activity of KIR2DL2 / 3-specific binders newly obtained by phage panning against inhibitory KIR.

[0353] Figure 6 This is a graph showing the results of evaluating the agonist activity of KIR2DL2 / 3-specific binders using primary NK cells. Figure 6 A represents the results using NK cells having genes for KIR2DL2, KIR2DL3, KIR2DS1, and KIR2DS2; Figure 6 B shows the results using NK cells harboring the KIR2DL3 gene.

[0354] Figure 7 This is a diagram showing that the newly obtained binding compound binds to 2DL2 or 2DL3 and inhibits the activation of NK cells.

[0355] Figure 8 The graph shows the results of evaluating whether a binding compound (scFv) that exhibits binding properties to KIR2DL1 and KIR3DL1 changes the cytotoxic activity of NK cell lines expressing the respective receptors.

[0356] Fig. 9 is a schematic diagram of a membrane-bound scFv binder.

[0357] Fig.10A diagram showing a comparison of the full-length amino acid sequences of KIR2DL1*003 (SEQ ID NO: 4, extracellular region 1 to 222) with those of KIR2DL2*001 (SEQ ID NO: 6, extracellular region 1 to 224), KIR2DL2*003 (SEQ ID NO: 57, extracellular region 1 to 224), KIR2DL3*001 (SEQ ID NO: 8, extracellular region 1 to 224), KIR2DS1*002 (SEQ ID NO: 10, extracellular region 1 to 224), KIR2DS2*001 (SEQ ID NO: 12, extracellular region 1 to 224) and KIR2DS4*001 (SEQ ID NO: 14, extracellular region 1 to 224).

[0358] Fig.11 A diagram showing a comparison of the full-length amino acid sequences of KIR2DL2*003 (SEQ ID NO: 57, extracellular region 1-224), KIR2DL1*003 (SEQ ID NO: 4, extracellular region 1-224), KIR2DL3*001 (SEQ ID NO: 8, extracellular region 1-224), KIR2DS1*002 (SEQ ID NO: 10, extracellular region 1-224), KIR2DS2*001 (SEQ ID NO: 12, extracellular region 1-224) and KIR2DS4*001 (SEQ ID NO: 14, extracellular region 1-224) based on KIR2DL2*001 (SEQ ID NO: 6, extracellular region 1-224).

[0359] Fig.12 A diagram showing comparison of the full-length amino acid sequences of KIR3DL1*015 (SEQ ID NO: 16, extracellular region 1 to 319) with those of KIR3DL2*002 (SEQ ID NO: 59, extracellular region 1 to 319) and KIR3DS1*013 (SEQ ID NO: 60, extracellular region 1 to 319) based on KIR3DL1*015 (SEQ ID NO: 16, extracellular region 1 to 319).

[0360] Fig.13 It is a diagram showing the amino acid sequence of a CD8-derived signal peptide (SEQ ID NO: 23), a CD8-derived hinge (SEQ ID NO: 24), a G4S linker (SEQ ID NO: 25), a CD8-derived hinge (SEQ ID NO: 26), a CD8-derived cell membrane domain (SEQ ID NO: 27), and a CD8-derived intracellular domain (SEQ ID NO: 28).

[0361] Fig.14It is a diagram showing the amino acid sequence of lirelumab (1-7F9) VH (SEQ ID NO: 29) and the amino acid sequence of lirelumab (1-7F9) VL (SEQ ID NO: 30). The underlined sequence represents the sequence of CDR1 to 3.

[0362] Fig.15 It is a diagram showing the amino acid sequence of Pan2D VH (SEQ ID NO: 31) and the amino acid sequence of Pan2D VL (SEQ ID NO: 32). The underlined sequence represents the sequence of CDR1 to 3.

[0363] Fig.16 It is a diagram showing the amino acid sequence of a2DL2 / 3-DS1 VH (SEQ ID NO: 33) and the amino acid sequence of a2DL2 / 3-DS1 VL (SEQ ID NO: 34). The underlined sequence represents the sequence of CDR1 to 3.

[0364] Fig.17 It is a diagram showing the amino acid sequence of a2DL2 / 3-DS2 VH (SEQ ID NO: 35) and the amino acid sequence of a2DL2 / 3-DS2 VL (SEQ ID NO: 36). The underlined sequence represents the sequence of CDR1 to 3.

[0365] Fig.18 It is a diagram showing the amino acid sequence of a2DL2 / 3-DS3 VH (SEQ ID NO: 37) and the amino acid sequence of a2DL2 / 3-DS3 VL (SEQ ID NO: 38). The underlined sequence represents the sequence of CDR1 to 3.

[0366] Fig.19 It is a diagram showing the amino acid sequence of a2DL2 / 3-DS4 VH (SEQ ID NO: 39) and the amino acid sequence of a2DL2 / 3-DS4 VL (SEQ ID NO: 40). The underlined sequence represents the sequence of CDR1 to 3.

[0367] Fig. 20 It is a diagram showing the amino acid sequence of a2DL2 / 3-DS5 VH (SEQ ID NO: 41) and the amino acid sequence of a2DL2 / 3-DS5 VL (SEQ ID NO: 42). The underlined sequence represents the sequence of CDR1 to 3.

[0368] Fig.21It is a diagram showing the amino acid sequence of a2DL2 / 3-DS6 VH (SEQ ID NO: 43) and the amino acid sequence of a2DL2 / 3-DS6 VL (SEQ ID NO: 44). The underlined sequence represents the sequence of CDR1 to 3.

[0369] Fig. 22 It is a diagram showing the amino acid sequence of a2DL2 / 3-DS7 VH (SEQ ID NO: 45) and the amino acid sequence of a2DL2 / 3-DS7 VL (SEQ ID NO: 46). The underlined sequence represents the sequence of CDR1 to 3.

[0370] Fig.23 It is a diagram showing the amino acid sequence of a2DL2 / 3-DS8 VH (SEQ ID NO: 47) and the amino acid sequence of a2DL2 / 3-DS8 VL (SEQ ID NO: 48). The underlined sequence represents the sequence of CDR1 to 3.

[0371] Fig.24 It is a diagram showing the amino acid sequence of a2DL2 / 3-DS9 VH (SEQ ID NO: 49) and the amino acid sequence of a2DL2 / 3-DS9 VL (SEQ ID NO: 50). The underlined sequence represents the sequence of CDR1 to 3.

[0372] Fig.25 It is a diagram showing the amino acid sequence of a2DL2 / 3-DS10 VH (SEQ ID NO: 51) and the amino acid sequence of a2DL2 / 3-DS10 VL (SEQ ID NO: 52). The underlined sequence represents the sequence of CDR1 to 3.

[0373] Fig.26 It is a diagram showing the amino acid sequence of a3DL1-DS1 VL (SEQ ID NO: 55) and the amino acid sequence of a3DL1-DS1 VH (SEQ ID NO: 56). The underlined sequence represents the sequence of CDR1 to 3.

[0374] Figure 27-1 It is a diagram showing that CAR-T cells express KIR2DL2 / 3 specific binders, thereby inhibiting the cell killing activity of NK cells against CAR-T cells and maintaining the cell killing activity of CAR-T cells against target cells. Figure 27-1 A represents the cell killing activity of CAR-T cells in wells without NK cells added; Figure 27-1 B shows the cell killing activity of CAR-T cells in the wells where NK cells were added.

[0375] Figure 27-2This is a graph showing the number of PBMC-C-derived T cells in wells co-cultured with PBMC-A-derived NK cells.

[0376] Fig.28 The graph shows the agonist activity of mutants in which each amino acid in CDR3 of the variable region (VH) of the KIR2DL2 / 3-specific binder a2DL2 / 3-DS10 was substituted with alanine against inhibitory KIR.

[0377] Fig.29 The graph shows the agonist activity of CDR grafts obtained by grafting the CDR of a KIR2DL2 / 3-specific binder onto the constant regions of different scFvs against inhibitory KIR.

[0378] Fig.30 The graph shows the additive inhibitory effect of cells co-expressing a KIR2DL2 / 3-specific binding compound and HLA-E on the cytotoxic activity of NK cells expressing inhibitory KIR and NKG2A.

[0379] Fig.31 This is a graph showing the inhibitory effect of iPS cells that do not express class I HLA but express a KIR2DL2 / 3-specific binding substance on the cytotoxic activity of NK cells.

[0380] Figure 32-1 The figure shows the binding model diagram of the Fab fragment of a2DL2 / 3-DS10, a KIR2DL2 / 3 specific binder identified by X-ray crystal structure analysis, and KIR2DL2.

[0381] Figure 32-2 This figure shows the mapping of the epitope site of the KIR2DL2 / 3 specific binder a2DL2 / 3-DS10 identified by X-ray crystal structure analysis on KIR2DL2.

[0382] Fig.33 This is a diagram showing the results of verifying the competition between a2DL2 / 3-DS10 and each KIR2DL2 / 3 specific binder in an antigen binding assay with respect to the KIR2DL2 / 3 specific binder.

[0383] Fig.34 This is a graph showing the binding properties of KIR2DL1-specific binders or KIR3DL1-specific binders newly obtained by phage panning to inhibitory and active KIRs.

[0384] Fig.35 This is a graph showing the agonist activity of KIR2DL1-specific binders newly obtained by phage panning against inhibitory KIR.

[0385] Fig.36This is a graph showing the agonist activity of KIR3DL1-specific binders newly obtained by phage panning against inhibitory KIR.

[0386] Figure 37-1 The diagram shows the amino acid sequence of a2DL2 / 3-DS11 VH (SEQ ID NO: 61) and the amino acid sequence of a2DL2 / 3-DS11 VL (SEQ ID NO: 62).

[0387] Figure 37-2 It is a diagram showing the amino acid sequence of a2DL2 / 3-DS12 VH (SEQ ID NO: 63) and the amino acid sequence of a2DL2 / 3-DS12 VL (SEQ ID NO: 64).

[0388] Fig.38 The diagram shows the amino acid sequence of the 3DL2 signal peptide (SEQ ID NO: 65) and the amino acid sequence of the 3DS1 signal peptide (SEQ ID NO: 66).

[0389] Fig.39 The diagram shows the amino acid sequence of R99A (SEQ ID NO: 67), the amino acid sequence of V99A (SEQ ID NO: 68), the amino acid sequence of S100A (SEQ ID NO: 69), the amino acid sequence of G101A (SEQ ID NO: 70), the amino acid sequence of T102A (SEQ ID NO: 71), the amino acid sequence of T103A (SEQ ID NO: 72), the amino acid sequence of G104A (SEQ ID NO: 73), the amino acid sequence of G105A (SEQ ID NO: 74), the amino acid sequence of Y106A (SEQ ID NO: 75), the amino acid sequence of Y107A (SEQ ID NO: 76), the amino acid sequence of T108A (SEQ ID NO: 77), the amino acid sequence of G109A (SEQ ID NO: 78), the amino acid sequence of M110A (SEQ ID NO: 79), the amino acid sequence of D111A (SEQ ID NO: 80), and the amino acid sequence of V112A (SEQ ID NO: 81).

[0390] Figure 40-1 It is a diagram showing the amino acid sequence of a2DL2 / 3-DS1-2 VH (SEQ ID NO: 82) and the amino acid sequence of a2DL2 / 3-DS1-2 VL (SEQ ID NO: 83).

[0391] Figure 40-2It is a diagram showing the amino acid sequence of a2DL2 / 3-DS2-2 VH (SEQ ID NO: 84) and the amino acid sequence of a2DL2 / 3-DS2-2 VL (SEQ ID NO: 85).

[0392] Figure 40-3 It is a diagram showing the amino acid sequence of a2DL2 / 3-DS3-2 VH (SEQ ID NO: 86) and the amino acid sequence of a2DL2 / 3-DS3-2 VL (SEQ ID NO: 87).

[0393] Fig.41 It is a diagram showing the amino acid sequence of the peptide derived from the HLA-G signal peptide (SEQ ID NO: 88), the amino acid sequence of the G4S linker (SEQ ID NO: 89), the amino acid sequence of HLA-E (SEQ ID NO: 90), the amino acid sequence of the IL-2 signal peptide (SEQ ID NO: 91), the amino acid sequence of the His tag (SEQ ID NO: 92), and the amino acid sequence of the VH signal peptide (SEQ ID NO: 93).

[0394] Fig.42 It is a diagram showing the amino acid sequence of the Fab fragment H chain constant region (SEQ ID NO: 94), the amino acid sequence of the VL signal peptide (SEQ ID NO: 95), and the amino acid sequence of the Fab fragment L chain constant region (SEQ ID NO: 96).

[0395] Fig.43 It is a diagram showing the amino acid sequence of the GGGS linker (SEQ ID NO: 97), the amino acid sequence of the IgG Fc domain (SEQ ID NO: 98), the amino acid sequence of the GAA linker (GAA), and the amino acid sequence of the Avi tag (SEQ ID NO: 99).

[0396] Fig.44 It is a diagram showing the amino acid sequence of CA19CAR VL (SEQ ID NO: 100) and the amino acid sequence of CA19CAR VH (SEQ ID NO: 101).

[0397] Fig.45 The diagram shows the amino acid sequence of the CD8-derived cell membrane domain and intracellular domain (SEQ ID NO: 102), the amino acid sequence of the intracellular co-stimulatory molecule (SEQ ID NO: 103), and the amino acid sequence of the blue fluorescent protein (SEQ ID NO: 104).

[0398] Fig.46It is a diagram showing the amino acid sequence of the IL-21 fragment (SEQ ID NO: 105) and the amino acid sequence of the CD8 hinge (62aa) (SEQ ID NO: 106).

[0399] Figure 47-1 The diagram shows the amino acid sequence of a2DL1-DS1 VH (SEQ ID NO: 107) and the amino acid sequence of a2DL1-DS1 VL (SEQ ID NO: 108).

[0400] Figure 47-2 It is a diagram showing the amino acid sequence of a2DL1-DS2 VH (SEQ ID NO: 109) and the amino acid sequence of a2DL1-DS2 VL (SEQ ID NO: 110).

[0401] Figure 47-3 The diagram shows the amino acid sequence of a2DL1-DS3 VH (SEQ ID NO: 111) and the amino acid sequence of a2DL1-DS1 VL (SEQ ID NO: 112).

[0402] Figure 47-4 The diagram shows the amino acid sequence of a2DL1-DS4 VH (SEQ ID NO: 113) and the amino acid sequence of a2DL1-DS4 VL (SEQ ID NO: 114).

[0403] Figure 47-5 It is a diagram showing the amino acid sequence of a2DL1-DS5 VH (SEQ ID NO: 115) and the amino acid sequence of a2DL1-DS5 VL (SEQ ID NO: 116).

[0404] Figure 47-6 It is a diagram showing the amino acid sequence of a2DL1-DS6 VH (SEQ ID NO: 117) and the amino acid sequence of a2DL1-DS6 VL (SEQ ID NO: 118).

[0405] Figure 47-7 It is a diagram showing the amino acid sequence of a2DL1-DS7 VH (SEQ ID NO: 119) and the amino acid sequence of a2DL1-DS7 VL (SEQ ID NO: 120).

[0406] Figure 47-8 It is a diagram showing the amino acid sequence of a2DL1-DS8 VH (SEQ ID NO: 121) and the amino acid sequence of a2DL1-DS8 VL (SEQ ID NO: 122).

[0407] Figure 47-9It is a diagram showing the amino acid sequence of a2DL1-DS9 VH (SEQ ID NO: 123) and the amino acid sequence of a2DL1-DS9 VL (SEQ ID NO: 124).

[0408] Figure 47-10 It is a diagram showing the amino acid sequence of a2DL1-DS10 VH (SEQ ID NO: 125) and the amino acid sequence of a2DL1-DS10 VL (SEQ ID NO: 126).

[0409] Figure 47-11 It is a diagram showing the amino acid sequence of a2DL1-DS11 VH (SEQ ID NO: 127) and the amino acid sequence of a2DL1-DS11 VL (SEQ ID NO: 128).

[0410] Figure 47-12 It is a diagram showing the amino acid sequence of a2DL1-DS12 VH (SEQ ID NO: 129) and the amino acid sequence of a2DL1-DS12 VL (SEQ ID NO: 130).

[0411] Figure 47-13 It is a diagram showing the amino acid sequence of a2DL1-DS13 VH (SEQ ID NO: 131) and the amino acid sequence of a2DL1-DS13 VL (SEQ ID NO: 132).

[0412] Figure 47-14 It is a diagram showing the amino acid sequence of a2DL1-DS14 VH (SEQ ID NO: 133) and the amino acid sequence of a2DL1-DS14 VL (SEQ ID NO: 134).

[0413] Figure 47-15 It is a diagram showing the amino acid sequence of a2DL1-DS15 VH (SEQ ID NO: 135) and the amino acid sequence of a2DL1-DS15 VL (SEQ ID NO: 136).

[0414] Figure 48-1 It is a diagram showing the amino acid sequence of a3DL1-DS2 VH (SEQ ID NO: 137) and the amino acid sequence of a3DL1-DS2 VL (SEQ ID NO: 138).

[0415] Figure 48-2 It is a diagram showing the amino acid sequence of a3DL1-DS3 VH (SEQ ID NO: 139) and the amino acid sequence of a3DL1-DS3 VL (SEQ ID NO: 140).

[0416] Figure 48-3 The diagram shows the amino acid sequence of a3DL1-DS4 VH (SEQ ID NO: 141) and the amino acid sequence of a3DL1-DS4 VL (SEQ ID NO: 142).

[0417] Figure 48-4 It is a diagram showing the amino acid sequence of a3DL1-DS5 VH (SEQ ID NO: 143) and the amino acid sequence of a3DL1-DS5 VL (SEQ ID NO: 144).

[0418] Figure 48-5 It is a diagram showing the amino acid sequence of a3DL1-DS6 VH (SEQ ID NO: 145) and the amino acid sequence of a3DL1-DS6 VL (SEQ ID NO: 145). DETAILED DESCRIPTION

[0419] The present invention is described in detail below.

[0420] 1. Substances that have the activity of binding to human inhibitory KIR (Killer-cell immunoglobulin-like receptors) and inhibiting the cytotoxic activity of NK cells

[0421] The present invention provides a substance having an activity of binding to an inhibitory KIR and inhibiting the cell killing activity of NK cells. This substance is sometimes also referred to as a substance having an agonist activity for inhibitory KIR. That is, sometimes the activity of binding to an inhibitory KIR and inhibiting the cell killing activity of NK cells is referred to as agonist activity. In addition, in the present invention, a substance that binds to KIR is referred to as a conjugate, and a substance that binds to KIR and has the effect of activating a signal transmitted from the KIR to the NK cell is referred to as an agonist conjugate. That is, an agonist conjugate for an inhibitory KIR will show an effect of inhibiting the activity of NK cells, and an agonist conjugate for an active KIR will show an effect of activating NK cells.

[0422] KIR is expressed on the surface of NK cells and is related to the self-other recognition function of NK cells, which is "missing self". KIR has inhibitory KIR and active KIR, each with multiple subtypes. Signals from inhibitory KIR inhibit the cell killing activity of NK cells. That is, it has the following mechanism: when NK cells in a constant state recognize the surface antigens of target cells, if there is a signal input to the inhibitory receptor, it will be activated, thereby obtaining the activity of killing or destroying the recognized cells (hereinafter referred to as "NK cell cell killing activity"). In other words, when the target cell is a cell expressing the same type of HLA as the NK cell, the cell's HLA will bind to the inhibitory KIR on the NK cell, inhibiting the signal from being transmitted to the NK cell, thereby inhibiting the cell killing activity of the NK cell. As a result, cells expressing the same type of HLA as the NK cell can avoid being rejected by the NK cell.

[0423] On the other hand, active KIR recognizes HLA and peptides displayed on cells expressing the same type of HLA as NK cells, transmits activation signals to NK cells, and activates the cell-killing activity of NK cells. Unlike inhibitory KIR, the function of active KIR is to recognize and repel viral peptides displayed on HLA.

[0424] In the present invention, "binding" is also referred to as "recognition". In the present invention, "recognition" or "binding" means binding that is not non-specific adsorption. As a criterion for determining whether there is recognition or binding, for example, the dissociation constant (hereinafter referred to as "KD") can be cited. The KD value of the preferred antibody of the present invention relative to the antigen protein is 1×10 -5 M or less, 5×10 -6 M or less, 2×10 -6 M or less, 1×10 -6 The binding in the present invention can be measured or determined by a biomolecular interaction analysis system such as the SPR method and the BLI method, or by an ELISA method, a RIA method, etc. The binding between a substance expressed on the cell surface and a recombinant protein of an inhibitory KIR can be measured by a flow cytometric analysis method, etc.

[0425] The substance having the activity of binding to inhibitory KIR and inhibiting the cytotoxic activity of NK cells is a substance that binds to inhibitory KIR, activates inhibitory KIR, transmits an inhibitory signal into NK cells, and thereby inhibits the cytotoxic activity of NK cells.

[0426] As inhibitory KIR, KIR belonging to the KIR2DL subclass and the KIR3DL subclass can be cited. As KIR belonging to the KIR2DL subclass, KIR2DL1, KIR2DL2, KIR2DL3 and KIR2DL5 can be cited; as KIR belonging to the KIR3DL subclass, KIR3DL1, KIR3DL2 and KIR3DL3 can be cited. In addition, as subtypes of active KIR, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5 and KIR3DS1 can be cited. In addition, as a subtype with dual properties of inhibitory and active types, there is KIR2DL4. The ligands (HLA) that bind to these inhibitory KIR and active KIR are shown in Table 1.

[0427] [Table 1]

[0428]

[0429]

[0430] The inhibitory and active KIR of the present invention have various variants, and the embodiments of the present invention select and use variants with amino acid sequences with high expression frequencies. Therefore, the effects and effects of the present invention are not limited to individual variants, but are applicable to KIRs belonging to various subtypes, as well as cells and hosts with HLA types corresponding to the KIRs. KIR2DL1 used KIR2DL1*003 (SEQ ID NO:4, IPD Accession No.: KIR00003), KIR2DL2 used KIR2DL2*001 (SEQ ID NO:6, IPD Accession No.: KIR00010) and KIR2DL2*003 (SEQ ID NO:57, IPD Accession No.: KIR00012), KIR2DL3 used KIR2DL3*001 (SEQ ID NO:8, IPD Accession No.: KIR00014), KIR2DS1 used KIR2DS1*002 (SEQ ID NO:10, IPD Accession No.: KIR00034), KIR2DS2 used KIR2DS2*001 (SEQ ID NO:12, IPD Accession No.: KIR00037), and KIR2DS4 used KIR2DS4*001 (SEQ ID NO:13, IPD Accession No.: KIR00038). NO:14, IPD accession number: KIR00045), and KIR3DL1 used KIR3DL1*015 (SEQ ID NO:16, IPD accession number: KIR00102).

[0431] like Fig.11As shown, the amino acid sequences of KIR2DL2*003 and KIR2DL3*001 are highly identical to each other (especially the amino acid sequences of the extracellular regions are extremely identical), and the amino acid sequences of active KIRs KIR2DS1*002, KIR2DS2*001, and KIR2DS4*001 are also highly identical.

[0432] The substance of the present invention having the activity of binding to an inhibitory KIR and inhibiting the cell killing activity of NK cells is a substance having the activity of binding to at least one of the subclasses of the above-mentioned inhibitory KIR and inhibiting the cell killing activity of NK cells expressing the bound KIR. The inhibitory KIR to be bound is not particularly limited, and the substance of the present invention is preferably a substance having the activity of binding to KIR2DL1, KIR2DL2, KIR2DL3 and / or KIR3DL1 and inhibiting the cell killing activity of NK cells; more preferably, it is a substance having the activity of binding to KIR2DL1, KIR2DL2 and / or KIR2DL3 and inhibiting the cell killing activity of NK cells. The substance binds to the inhibitory KIR and exerts agonist activity.

[0433] KIR2DL1 recognizes the C2 epitope of the HLA-C type of MHC class I molecules (Cw2, Cw4, Cw5, Cw6, Cw15, Cw17).

[0434] KIR2DL2 and KIR2DL3 recognize the C1 epitope of the HLA-C type of MHC class I molecules (Cw1, Cw3, Cw7, Cw8, Cw9, Cw10, Cw12, Cw14, Cw16) and the C1 epitope of the HLA-B type (B46 and B73).

[0435] KIR3DL1 recognizes the Bw4 epitope (A23, A24, A25, A32, B5, B27, B37, B38, B44, B49, B51, B52, B53, B57, B58, B59, B77) of the MHC class I molecule HLA-B type.

[0436] KIR3DL2 recognizes A3 and A11 of the MHC class I molecules HLA-A.

[0437] The substance of the present invention having the activity of binding to inhibitory KIR and inhibiting the cell killing activity of NK cells has low activity in activating the cell killing activity of NK cells. For example, the activity of the substance of the present invention having the activity of binding to inhibitory KIR and inhibiting the cell killing activity of NK cells is higher than the activity of the substance binding to the corresponding active KIR and activating the cell killing activity of NK cells. Among them, regarding the active KIR corresponding to the inhibitory KIR, the active KIR corresponding to KIR2DL1, KIR2DL2 or KIR2DL3 is KIR2DS1, KIR2DS2, KIR2DS3 and KIR2DS4, and the particularly important KIR is KIR2DS1, KIR2DS2 and KIR2DS4. In addition, the active KIR corresponding to KIR3DL1 is KIR3DS1. The substance of the present invention having the activity of binding to inhibitory KIR and inhibiting the cell killing activity of NK cells is preferably not bound to the active KIR. That is, it will not substantially show agonist activity to the active KIR. Therefore, the substance having the activity of binding to the inhibitory KIR and inhibiting the cytotoxic activity of NK cells is preferably a substance that binds to: Fig.10 In the comparison of the amino acid sequences of KIR2DL2, KIR2DL3, KIR2DS1, KIR2DS2, and KIR2DS4 shown, there are few identical sequences, or there are partial sequences with ACCESS differences when the three-dimensional structures of the proteins are obtained.

[0438] The NK cells inhibited from activation by the substance of the present invention refer to NK cells in which the inhibitory KIR as a target is expressed on the cell surface. As such NK cells, for example, a blood sample of a subject having an HLA type corresponding to the inhibitory KIR, or a NK cell population separated from the blood can be used. In addition, NK cells in which the inhibitory KIR as a target is expressed by a genetic engineering method in a commercially available NK cell line can also be used.

[0439] The inhibitory KIR agonist activity of the substance of the present invention can be evaluated by the following method: measuring the cytotoxic activity of the following effector cells against the following target cells in the presence or absence of the test substance, and calculating the inhibition ratio of the cytotoxic activity in the presence of the test substance. As effector cells, NK cells expressing the inhibitory KIR of the above-mentioned object are used. As target cells, cells that do not express the HLA corresponding to the inhibitory KIR of the object are used. In the determination of the agonist activity, the test substance can be added to the culture medium in the form of a soluble type, and it can be expressed on the surface of the target cell in the form of a membrane protein described later.

[0440] For example, in the determination of the agonist activity of a substance that binds to KIR2DL2 / 3, NK cells expressing KIR2DL2 and / or KIR2DL3 under the HLA-C1 homotype or HLA-C1 / C2 heterozygous origin are used as effector cells, and cells that do not express the HLA-C1 type (for example, cells collected from subjects with the C2 / C2 homozygous HLA type, etc.) are used as target cells. It is preferred that both the effector cells and the target cells are consistent with the Bw4 type (negative homozygous for both, or positive homozygous for both).

[0441] In addition, for example, in the determination of the agonist activity of a substance that binds to KIR2DL1, NK cells expressing KIR2DL1 under the HLA-C2 homotype or HLA-C1 / C2 heterozygous origin are used as effector cells, and cells that do not express the HLA-C2 type (for example, cells collected from subjects with C1 / C1 homozygous HLA type, etc.) are used as target cells. It is preferred that both the effector cells and the target cells are consistent with the BW4 type (negative homozygous for both, or positive homozygous for both).

[0442] In addition, for example, in the determination of the agonist activity of a substance that binds to KIR3DL1, NK cells expressing KIR3DL1 under the HLA-Bw4 type are used as effector cells, and cells that do not express HLA-Bw4 type (for example, cells collected from a subject with a Bw4 positive / Bw4 negative homozygous HLA type, etc.) are used as target cells. It is preferred that both the effector cells and the target cells have the same HLA-C type (both C1 / C1 homozygous, both C2 / C2 homozygous, or both C1 / C2 heterozygous).

[0443] In addition, for example, in the determination of the agonist activity of a substance that binds to KIR3DL2, NK cells expressing KIR3DL2 under the HLA-A03, A11 type are used as effector cells, and cells that do not express HLA-A03, A11 type are used as target cells. It is preferred that both the effector cells and the target cells have the same HLA-C type (both C1 / C1 homozygous, both C2 / C2 homozygous, or both C1 / C2 heterozygous), and both have the same BW4 type (both negative homozygous, both positive homozygous, or both positive / negative heterozygous).

[0444] As a substance having the activity of binding to inhibitory KIR and inhibiting the cytotoxic activity of NK cells, a substance having a binding site for inhibitory KIR can be mentioned. As such a substance, a polypeptide having a binding site for inhibitory KIR and binding to inhibitory KIR can be mentioned.

[0445] Examples of polypeptides that have a binding site for inhibitory KIR and bind to inhibitory KIR include the following polypeptides (1) to (3).

[0446] (1) Antibodies or antigen-binding fragments targeting inhibitory KIR

[0447] Examples of antibodies against inhibitory KIR include lirelumab, Pan2D, and monoclonal antibodies transplanted with CDRs or variable regions included in scFv (i) to (x) described below. In the present invention, these antibodies are not limited, and include all antibodies that have the activity of binding to inhibitory KIR and inhibiting the cytotoxic activity of NK cells.

[0448] Antibodies having the activity of binding to inhibitory KIR and inhibiting the cell killing activity of NK cells and antigen-binding fragments binding to inhibitory KIR (hereinafter also referred to as "antibodies of the present invention, etc.") can be any of monoclonal antibodies and polyclonal antibodies. The isotype of the monoclonal antibody of the present invention is not particularly limited, and examples thereof include IgG such as IgG1, IgG2, IgG3, IgG4, IgM, IgA such as IgA, IgD, Ig, etc. The isotype and subclass of the monoclonal antibody can be determined, for example, by agar two-way immunodiffusion method, ELISA method, RIA method, etc. As monoclonal antibodies of the present invention, examples include: antibodies of non-human animal origin (non-human animal antibodies), human antibodies, chimeric antibodies (also called "chimeric antibodies"), humanized antibodies, etc., and preferably human antibodies can be used. The scope of the antibodies of the present invention also includes mutants of antibodies, for example, the scope of human antibodies also includes human mutant antibodies.

[0449] Examples of non-human animal antibodies include antibodies from vertebrates such as mammals and birds. Examples of mammal-derived antibodies include antibodies from rodents such as mouse antibodies and rat antibodies, and single-chain antibodies with only one variable region from camels, alpacas, and llamas. Examples of avian-derived antibodies include chicken antibodies.

[0450] Examples of chimeric antibodies include, but are not limited to, antibodies in which a variable region derived from a non-human animal antibody is linked to a constant region of a human antibody (human immunoglobulin).

[0451] Examples of humanized antibodies include, but are not limited to: antibodies formed by transplanting CDRs in the variable region of a non-human animal antibody into a human antibody (variable region of a human immunoglobulin); antibodies formed by transplanting a portion of the framework region of a non-human animal antibody in addition to the CDRs into a human antibody; antibodies formed by replacing one or two or more amino acids derived from any of the above non-human animal antibodies with human amino acids, etc.

[0452] CDR-grafted antibodies include, but are not limited to: antibodies or antigen-binding fragments in which the CDR in the variable region of a certain antibody or antigen-binding fragment is transplanted to the variable region of another immunoglobulin; antibodies or antigen-binding fragments in which, in addition to the CDR, a portion of the sequence of the framework region of the original antibody or antigen-binding fragment is transplanted to the variable region; antibodies or antigen-binding fragments in which one or more amino acids derived from any of the above antibodies or antigen-binding fragments are replaced by other amino acids, etc.

[0453] In the above-mentioned CDR transplantation, when the amino acids in the CDR are replaced with other amino acids, 1 to 6 amino acids selected from 1, 2 or 3 amino acids on the N-terminal side and / or C-terminal side of the CDR region can be replaced; more preferably, 1 to 4 amino acids selected from 2 amino acids on the N-terminal side and / or C-terminal side are replaced; further preferably, 2 amino acids selected from 1 amino acid on the N-terminal side and / or C-terminal side are replaced.

[0454] Antibodies can be prepared by various known methods. As a known method, they can be prepared by methods using hybridomas and cell immunization methods, or by genetic recombination methods. In addition, methods for obtaining human antibodies screened from human antibody phage libraries are also known. For example, a phage display method can be used in which the variable region of a human antibody is expressed on the surface of a phage as scFv to select phages that bind to an antigen. By analyzing the genes of the phages selected based on binding to the antigen, the DNA sequence encoding the variable region of the human antibody that binds to the antigen can be determined. Once the DNA sequence of the scFv that binds to the antigen is determined, an expression vector containing the sequence can be prepared, introduced into an appropriate host and expressed, thereby obtaining human antibodies [WO92 / 01047; WO92 / 20791; WO93 / 06213; WO93 / 11236; WO93 / 19172; WO95 / 01438; WO95 / 15388; Annu. Rev. Immunol (1994) 12, 433-455]. In addition, single-chain antibodies composed only of heavy chains are antibodies possessed by camels, alpacas, llamas, etc., and can be obtained by immunoblotting these animals or using phage libraries derived from these animals [for example, refer to David R. Maass et al. J Immunol Methods, (2007 Jul 31), 324 (1-2): 13-25; Lukas Roth et al., Methods Mol Biol. (2020); 2070: 173-189, etc.].

[0455] In the present invention, "antigen binding fragment" refers to a peptide composed of one or two variable regions and having binding activity with an antigen. As "antigen binding fragment", for example, but not limited to: scFv, Fab, F(ab')2, Fab', Fv, single-domain antibody (sdAb: single-domain antibody) and other antigen binding fragments. Antigen binding fragments are obtained by treating the full-length molecule of antibody protein with enzymes such as papain and pepsin. In addition, they can also be recombinant proteins produced in appropriate host cells using recombinant genes. When an antigen binding fragment having a heavy chain variable region and a light chain variable region is manufactured by genetic engineering methods, the heavy chain variable region and the light chain variable region contained in the monoclonal antibody or antigen binding fragment that binds to the target antigen (in the case of a single-chain antibody, there are also cases where only the heavy chain variable region exists), or the CDR1, CDR2 and CDR3 contained therein can be transplanted into the framework sequence of the desired antigen binding fragment. An antigen-binding fragment derived from a single-chain antibody consisting only of a heavy chain can be produced in the same manner as scFv and membrane-type scFv by using a display library of a phage or yeast having a gene encoding a variable region of a single-chain antibody derived from camel, alpaca, llama, etc. that binds to a target antigen, and introducing it into an expression configuration suitable for a single variable region [for example, see David R. Maass et al. J Immunol Methods, (2007 Jul 31); 3 24 (1-2): 13-25; Lukas Roth et al., Methods Mol Biol., (2020); 2070: 173-189, etc.]. In addition, in the case of transplanting a variable region or CDR, examples include, but are not limited to, an antigen-binding fragment in which one or more amino acids in the sequence of an original antibody, etc. are replaced with amino acids of the desired antigen-binding fragment. When the antigen-binding fragment of the present invention is a single chain such as scFv, when the variable region or CDR is transplanted from the original antibody or antigen-binding fragment to the desired antigen-binding fragment, it is known that there may be a situation where the binding property is maintained even if the heavy chain and light chain are replaced. The present invention includes such antigen-binding fragments in which the heavy chain variable region and light chain variable region are replaced [Effects of variable domain orientation on anti-HER2 single-chain variable fragment antibody expressed in the Escherichia coli cytoplasm, Ilkay Kocer et al., Biotechnol Prog., 2021Mar; 37(2): 33102].

[0456] Among them, scFv is preferred. scFv is an antigen-binding fragment obtained by connecting the heavy chain variable region and the light chain variable region with a polypeptide linker [Pluckthun A. The Pharmacology of Monoclonal Antibodies 113, Rosenburg and Moore eds., Springer Verlag, New York, 269-315 (1994); Nature Biotechnology (2005), 23, 1126-1136]. As the linker sequence, various sequences known as linker sequences for single-chain antibodies and scFv, such as artificial sequences, can be used. As artificial linker sequences, amino acid sequences rich in glycine and serine are known, for example, GS linkers composed of three amino acids of GSG or four amino acids of GSGS (SEQ ID NO: 3), and linker sequences represented by the formula (G4S)n can be cited. In the formula, n is 1 to 10, preferably n = 3 (GGGGSGGGGSGGGGS, SEQ ID NO: 25). The linker domain is also called a peptide linker, and a known peptide linker can be used as the extracellular linker domain. The sequence represented by the formula (G4S)n is called a G4S linker.

[0457] scFv binding to the target antigen can be obtained by using a phage display method [Nature Biotechnology (2005), 23, (9), p.1105-1116] in which a library of variable regions is expressed as a single-chain antibody (scFv) on the surface of a phage and phage binding to the antigen is selected. By analyzing the genes of phages selected for binding to the antigen, the DNA sequence encoding the variable region binding to the antigen can be determined. As long as the sequences of the variable regions of the heavy and light chains are clear, the sequences of the CDRs contained in the variable regions can also be determined. An expression vector having a sequence obtained by introducing these CDR and variable region sequences into the sequence configuration of the desired antibody or antigen-binding fragment is prepared, introduced into an appropriate host and expressed, thereby obtaining an antibody or antigen-binding fragment having the desired antigen-binding property [WO92 / 01047; WO92 / 20791; WO93 / 06213; WO93 / 11236; WO93 / 19172; WO95 / 01438; WO95 / 15388; Annu. Rev. Immunol (1994) 12, p. 433-455; Nature Biotechnology (2005) 23 (9), p. 1105-1116].

[0458] The CDR can be modified within the range that the agonist activity of the polypeptide of the present invention remains unchanged. That is, with respect to the CDR of the polypeptide of the present invention, it is sufficient to replace a specific amino acid residue with alanine to confirm whether the agonist activity against the inhibitory KIR is still maintained. For amino acids that retain the agonist activity against the inhibitory KIR even when substituted with alanine, it can be judged that they are not important for the agonist activity, and such amino acids in the CDR can be replaced with other amino acids, and the polypeptide with the amino acid replaced has the same reactivity as the polypeptide before the amino acid is replaced. When the amino acids in the CDR are replaced with other amino acids, it is preferred to replace 1 to 6 amino acids selected from 1, 2 or 3 amino acids on the N-terminal side and / or C-terminal side of the CDR region; more preferably, 1 to 4 amino acids selected from 2 amino acids on the N-terminal side and / or C-terminal side are replaced; and further preferably, 2 amino acids selected from 1 amino acid on the N-terminal side and / or C-terminal side are replaced.

[0459] As the scFv of the present invention against inhibitory KIR2DL2 or KIR2DL3, there can be mentioned an scFv consisting of any one of the following amino acid sequences (i) to (xii), wherein the heavy chain variable region and the light chain variable region can be linked, for example, by a GS linker, a G4S linker, or the like:

[0460] (i) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 33 and the amino acid sequence of the light chain variable region of SEQ ID NO: 34;

[0461] (ii) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 35 and the amino acid sequence of the light chain variable region of SEQ ID NO: 36;

[0462] (iii) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 37 and the amino acid sequence of the light chain variable region of SEQ ID NO: 38;

[0463] (iv) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 39 and the amino acid sequence of the light chain variable region of SEQ ID NO: 40;

[0464] (v) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 41 and the amino acid sequence of the light chain variable region of SEQ ID NO: 42;

[0465] (vi) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 43 and the amino acid sequence of the light chain variable region of SEQ ID NO: 44;

[0466] (vii) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 45 and the amino acid sequence of the light chain variable region of SEQ ID NO: 46;

[0467] (viii) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 47 and the amino acid sequence of the light chain variable region of SEQ ID NO: 48;

[0468] (ix) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 49 and the amino acid sequence of the light chain variable region of SEQ ID NO: 50;

[0469] (x) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 51 and the amino acid sequence of the light chain variable region of SEQ ID NO: 52;

[0470] (xi) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 61 and the amino acid sequence of the light chain variable region of SEQ ID NO: 62; and,

[0471] (xii) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 63 and the amino acid sequence of the light chain variable region of SEQ ID NO: 64.

[0472] In addition, as the scFv for inhibitory KIR3DL1 of the present invention, there can be mentioned an scFv consisting of any one of the following amino acid sequences (xiii) to (xviii), wherein the heavy chain variable region and the light chain variable region can be connected by, for example, a GS linker, a G4S linker, or the like:

[0473] (xiii) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 56 and the amino acid sequence of the light chain variable region of SEQ ID NO: 55;

[0474] (xiv) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 137 and the amino acid sequence of the light chain variable region of SEQ ID NO: 138;

[0475] (xv) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 139 and the amino acid sequence of the light chain variable region of SEQ ID NO: 140;

[0476] (xvi) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 141 and the amino acid sequence of the light chain variable region of SEQ ID NO: 142;

[0477] (xvii) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 143 and the amino acid sequence of the light chain variable region of SEQ ID NO: 144; and

[0478] (xviii) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 145 and the amino acid sequence of the light chain variable region of SEQ ID NO: 146.

[0479] As the scFv for inhibitory KIR2DL1 of the present invention, there can be mentioned an scFv consisting of any one of the following amino acid sequences (ci) to (cxv), wherein the heavy chain variable region and the light chain variable region can be connected by, for example, a GS linker, a G4S linker, etc.:

[0480] (ci) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 107 and the amino acid sequence of the light chain variable region of SEQ ID NO: 108;

[0481] (cii) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 109 and the amino acid sequence of the light chain variable region of SEQ ID NO: 110;

[0482] (ciii) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 111 and the amino acid sequence of the light chain variable region of SEQ ID NO: 112;

[0483] (civ) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 113 and the amino acid sequence of the light chain variable region of SEQ ID NO: 114;

[0484] (cv) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 115 and the amino acid sequence of the light chain variable region of SEQ ID NO: 116;

[0485] (cvi) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 117 and the amino acid sequence of the light chain variable region of SEQ ID NO: 118;

[0486] (cvii) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 119 and the amino acid sequence of the light chain variable region of SEQ ID NO: 120;

[0487] (cviii) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 121 and the amino acid sequence of the light chain variable region of SEQ ID NO: 122;

[0488] (cviiii) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 123 and the amino acid sequence of the light chain variable region of SEQ ID NO: 124;

[0489] (cx) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 125 and the amino acid sequence of the light chain variable region of SEQ ID NO: 126;

[0490] (cxi) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 127 and the amino acid sequence of the light chain variable region of SEQ ID NO: 128;

[0491] (cxii) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 129 and the amino acid sequence of the light chain variable region of SEQ ID NO: 130;

[0492] (cxiii) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 131 and the amino acid sequence of the light chain variable region of SEQ ID NO: 132;

[0493] (cxiv) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 133 and the amino acid sequence of the light chain variable region of SEQ ID NO: 134; and

[0494] (cxv) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 135 and the amino acid sequence of the light chain variable region of SEQ ID NO: 136.

[0495] In addition, polypeptides having a binding site for inhibitory KIR and binding to inhibitory KIR include polypeptides having a binding site composed of an amino acid sequence derived from an antibody or scFv that recognizes and binds to the above inhibitory KIR. Such polypeptides can take various forms, preferably, monoclonal antibodies, antigen-binding fragments such as scFv, or membrane proteins having an amino acid sequence of an antigen-binding fragment outside the cell.

[0496] The amino acid sequence derived from the antibody or scFv that recognizes and binds to inhibitory KIR includes the amino acid sequence of the site that binds to inhibitory KIR in the antibody or scFv that recognizes and binds to inhibitory KIR, and also includes an amino acid sequence formed by replacing, deleting, adding (addition includes insertion) (hereinafter collectively referred to as "mutation") one or more amino acids in the amino acid sequence of the site that binds to inhibitory KIR in the antibody or scFv that recognizes and binds to inhibitory KIR, and includes an amino acid sequence that has the activity of binding to inhibitory KIR and inhibiting the cell killing activity of NK cells. Among them, the "multiple" in "1 or more" refers to 2 to 10, preferably 7 or less, more preferably 5 or less, and further preferably 4, 3, 2 or 1.

[0497] As binding sites for inhibitory KIR, specifically include sites containing the following amino acid sequences: amino acid sequences derived from CDR1, CDR2 and CDR3 respectively contained in the light chain variable region and heavy chain variable region of the monoclonal antibody or scFv that binds to the inhibitory KIR; amino acid sequences derived from the variable region containing each CDR1, CDR2 and CDR3; or amino acid sequences derived from the light chain variable region and the heavy chain variable region. The amino acid sequence derived from the antibody or scFv for inhibitory KIR has the same meaning as above.

[0498] For example, a polypeptide having a binding site for inhibitory KIR2DL2 and / or KIR2DL3 and binding to inhibitory KIR2DL2 and / or KIR2DL3 has a combination of heavy chain variable regions and light chain variable regions shown in Table 2. In Table 2, "a2DL2 / 3" indicates that the binder binds to inhibitory KIR2DL2 and / or KIR2DL3.

[0499] [Table 2]

[0500]

[0501]

[0502]

[0503] * The CDR1, CDR2, and CDR3 regions in the table are represented by the range of amino acid numbers in the amino acid sequence of each variable region. The amino acid numbers are determined according to the IMGT definition.

[0504] In addition, the polypeptide having a binding site for inhibitory KIR2DL1 and binding to inhibitory KIR2DL1 has a combination of heavy chain variable regions and light chain variable regions shown in Table 3. In Table 3, "a2DL1" indicates that the binding substance binds to inhibitory KIR2DL1.

[0505] [Table 3]

[0506]

[0507]

[0508] In addition, the polypeptide having a binding site for inhibitory KIR3DL1 and binding to inhibitory KIR3DL1 has a combination of heavy chain variable regions and light chain variable regions shown in Table 4. In Table 4, "a3DL1" indicates that the binding substance binds to inhibitory KIR3DL1.

[0509] [Table 4]

[0510]

[0511]

[0512] * The CDR1, CDR2, and CDR3 regions in the table are represented by the range of amino acid numbers in the amino acid sequence of each variable region. The amino acid numbers are determined according to the IMGT definition.

[0513] As a form of the polypeptide of the present invention that has a binding site for inhibitory KIR2DL2 or KIR2DL3 and binds to inhibitory KIR, for example, there can be mentioned a polypeptide comprising any one of the following amino acid sequences (I) to (XII) (preferably a monoclonal antibody, an antigen-binding fragment, a scFv consisting of the amino acid sequence, or a membrane protein containing the scFv in the extracellular domain).

[0514] (I) an amino acid sequence comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 33 (preferably a variable region comprising the amino acid sequence of SEQ ID NO: 33) and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 34 (preferably a variable region comprising the amino acid sequence of SEQ ID NO: 34); (II) an amino acid sequence comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 35 (preferably a variable region comprising the amino acid sequence of SEQ ID NO: 35) and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 36 (preferably a variable region comprising the amino acid sequence of SEQ ID NO: 36);

[0515] (III) an amino acid sequence comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 37 (preferably a variable region comprising the amino acid sequence of SEQ ID NO: 37) and a second variable region comprising CDR1, CDR2, and CDR3 of SEQ ID NO: 38 (preferably a variable region comprising the amino acid sequence of SEQ ID NO: 38);

[0516] (IV) an amino acid sequence comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 39 (preferably a variable region comprising the amino acid sequence of SEQ ID NO: 39) and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 40 (preferably a variable region comprising the amino acid sequence of SEQ ID NO: 40);

[0517] (V) an amino acid sequence comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 41 (preferably a variable region comprising the amino acid sequence of SEQ ID NO: 41) and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 42 (preferably a variable region comprising the amino acid sequence of SEQ ID NO: 42);

[0518] (VI) an amino acid sequence comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 43 (preferably a variable region comprising an amino acid sequence of SEQ ID NO: 43) and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 44 (preferably a variable region comprising an amino acid sequence of SEQ ID NO: 44);

[0519] (VII) an amino acid sequence comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 45 (preferably a variable region comprising the amino acid sequence of SEQ ID NO: 45) and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 46 (preferably a variable region comprising the amino acid sequence of SEQ ID NO: 46);

[0520] (VIII) an amino acid sequence comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 47 (preferably a variable region comprising the amino acid sequence of SEQ ID NO: 47) and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 48 (preferably a variable region comprising the amino acid sequence of SEQ ID NO: 48);

[0521] (IX) an amino acid sequence comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 49 (preferably a variable region comprising an amino acid sequence of SEQ ID NO: 49) and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 50 (preferably a variable region comprising an amino acid sequence of SEQ ID NO: 50);

[0522] (X) an amino acid sequence comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 51 (preferably a variable region comprising the amino acid sequence of SEQ ID NO: 51) and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 52 (preferably a variable region comprising the amino acid sequence of SEQ ID NO: 52);

[0523] (XI) a binding site comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO:61 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO:62; and

[0524] (XII) A binding site comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2 and CDR3 of SEQ ID NO:63 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2 and CDR3 of SEQ ID NO:64.

[0525] In addition, as a form of the polypeptide of the present invention that has a binding site for inhibitory KIR3DL1 and binds to inhibitory KIR3DL1, for example, a polypeptide comprising any one of the following amino acid sequences (XIII) to (XVIII) (preferably a monoclonal antibody, an antigen-binding fragment, a scFv consisting of the amino acid sequence, and a membrane protein containing the scFv in the extracellular domain).

[0526] (XIII) an amino acid sequence comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 56 (preferably a variable region comprising the amino acid sequence of SEQ ID NO: 56) and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 55 (preferably a variable region comprising the amino acid sequence of SEQ ID NO: 55);

[0527] (XIV) an amino acid sequence comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 137 (preferably a variable region comprising the amino acid sequence of SEQ ID NO: 137) and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 138 (preferably a variable region comprising the amino acid sequence of SEQ ID NO: 138);

[0528] (XV) an amino acid sequence comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 139 (preferably a variable region comprising the amino acid sequence of SEQ ID NO: 139) and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 140 (preferably a variable region comprising the amino acid sequence of SEQ ID NO: 140);

[0529] (XVI) an amino acid sequence comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 141 (preferably a variable region comprising the amino acid sequence of SEQ ID NO: 141) and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 142 (preferably a variable region comprising the amino acid sequence of SEQ ID NO: 142);

[0530] (XVII) an amino acid sequence comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 143 (preferably a variable region comprising an amino acid sequence of SEQ ID NO: 143) and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 144 (preferably a variable region comprising an amino acid sequence of SEQ ID NO: 144); and

[0531] (XVIII) an amino acid sequence comprising a first variable region (preferably a variable region comprising the amino acid sequence of SEQ ID NO: 145) containing an amino acid sequence derived from CDR1, CDR2 and CDR3 of SEQ ID NO: 145, and a second variable region (preferably a variable region comprising the amino acid sequence of SEQ ID NO: 146) containing an amino acid sequence derived from CDR1, CDR2 and CDR3 of SEQ ID NO: 146.

[0532] In addition, as a form of the polypeptide of the present invention that has a binding site for inhibitory KIR2DL1 and binds to inhibitory KIR2DL1, for example, a polypeptide comprising the following amino acid sequence (P) (preferably a monoclonal antibody, an antigen-binding fragment, a scFv consisting of the amino acid sequence, a membrane protein containing the scFv in the extracellular domain).

[0533] (P) an amino acid sequence comprising a first variable region containing an amino acid sequence derived from CDR1, CDR2 and CDR3 of SEQ ID NO: 31 (preferably a variable region containing the amino acid sequence of SEQ ID NO: 31) and a second variable region containing an amino acid sequence derived from CDR1, CDR2 and CDR3 of SEQ ID NO: 32 (preferably a variable region containing the amino acid sequence of SEQ ID NO: 32).

[0534] In addition, as a form of the polypeptide of the present invention that has a binding site for inhibitory KIR2DL1 and binds to inhibitory KIR2DL1, for example, a polypeptide comprising any one of the following amino acid sequences (CI) to (CXV) (preferably a monoclonal antibody, an antigen-binding fragment, a scFv consisting of the amino acid sequence, and a membrane protein containing the scFv in the extracellular domain).

[0535] (CI) an amino acid sequence comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 107 (preferably a variable region comprising the amino acid sequence of SEQ ID NO: 107) and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 108 (preferably a variable region comprising the amino acid sequence of SEQ ID NO: 108);

[0536] (CII) an amino acid sequence comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 109 (preferably a variable region comprising the amino acid sequence of SEQ ID NO: 109) and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 110 (preferably a variable region comprising the amino acid sequence of SEQ ID NO: 110);

[0537] (CIII) an amino acid sequence comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 111 (preferably a variable region comprising the amino acid sequence of SEQ ID NO: 111) and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 112 (preferably a variable region comprising the amino acid sequence of SEQ ID NO: 112);

[0538] (CIV) an amino acid sequence comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 113 (preferably a variable region comprising an amino acid sequence of SEQ ID NO: 113) and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 114 (preferably a variable region comprising an amino acid sequence of SEQ ID NO: 114);

[0539] (CV) an amino acid sequence comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 115 (preferably a variable region comprising the amino acid sequence of SEQ ID NO: 115) and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 116 (preferably a variable region comprising the amino acid sequence of SEQ ID NO: 116);

[0540] (CVI) an amino acid sequence comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 117 (preferably a variable region comprising the amino acid sequence of SEQ ID NO: 117) and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 118 (preferably a variable region comprising the amino acid sequence of SEQ ID NO: 118);

[0541] (CVII) an amino acid sequence comprising a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 119 (preferably a variable region comprising the amino acid sequence of SEQ ID NO: 119) and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 120 (preferably a variable region comprising the amino acid sequence of SEQ ID NO: 120);

[0542] (CVIII) an amino acid sequence comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 121 (preferably a variable region comprising the amino acid sequence of SEQ ID NO: 121) and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 122 (preferably a variable region comprising the amino acid sequence of SEQ ID NO: 122);

[0543] (CVIIII) an amino acid sequence comprising a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 123 (preferably a variable region comprising an amino acid sequence of SEQ ID NO: 123) and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 124 (preferably a variable region comprising an amino acid sequence of SEQ ID NO: 124);

[0544] (CX) an amino acid sequence comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 125 (preferably a variable region comprising the amino acid sequence of SEQ ID NO: 125) and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 126 (preferably a variable region comprising the amino acid sequence of SEQ ID NO: 126);

[0545] (CXI) an amino acid sequence comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 127 (preferably a variable region comprising the amino acid sequence of SEQ ID NO: 127) and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 128 (preferably a variable region comprising the amino acid sequence of SEQ ID NO: 128);

[0546] (CXII) an amino acid sequence comprising a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 129 (preferably a variable region comprising the amino acid sequence of SEQ ID NO: 129) and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 130 (preferably a variable region comprising the amino acid sequence of SEQ ID NO: 130);

[0547] (CXIII) an amino acid sequence comprising a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 131 (preferably a variable region comprising the amino acid sequence of SEQ ID NO: 131) and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 132 (preferably a variable region comprising the amino acid sequence of SEQ ID NO: 132);

[0548] (CXIV) an amino acid sequence comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 133 (preferably a variable region comprising an amino acid sequence of SEQ ID NO: 133) and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 134 (preferably a variable region comprising an amino acid sequence of SEQ ID NO: 134); and

[0549] (CXV) comprises an amino acid sequence of a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2 and CDR3 of SEQ ID NO: 135 (preferably a variable region comprising an amino acid sequence of SEQ ID NO: 135) and a second variable region comprising an amino acid sequence derived from CDR1, CDR2 and CDR3 of SEQ ID NO: 136 (preferably a variable region comprising an amino acid sequence of SEQ ID NO: 136).

[0550] The CDR1, CDR2 and CDR3 of the heavy chain variable region and the CDR1, CDR2 and CDR3 of the light chain variable region may have amino acid mutations that do not lose reactivity with antigens. Such mutations include, for example, substitutions of one or two amino acids in the amino acid sequence of the CDR, preferably substitutions of one or two amino acids at the C-terminus and / or N-terminus of the amino acid sequence of the CDR.

[0551] Among them, in the above-mentioned polypeptides (I) to (XIII), (P) and (CI) to (CXV), the structure other than the variable region can be appropriately selected according to the form of the polypeptide to be selected (monoclonal antibody, antigen binding fragment, etc.). When the polypeptide is a single chain, it can be configured as "first variable region-linker sequence-second variable region" from the N-terminal side, or it can be configured as "second variable region-linker sequence-first variable region" in sequence. In addition, when the polypeptide is divided into a heavy chain and a light chain, the first variable region can be configured as a heavy chain and the second variable region can be configured as a light chain; the first variable region can also be configured as a light chain and the second variable region can be configured as a heavy chain.

[0552] In one aspect of the present invention, the present invention provides a substance having inhibitory KIR-specific agonist activity. "Inhibitory KIR-specific agonist activity" refers to an agonist activity against an inhibitory KIR, and the agonist activity against an active KIR corresponding to the inhibitory KIR is less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 15%, less than 10% or less than 5% of the agonist activity against the inhibitory KIR, and most preferably, it does not substantially show agonist activity against the active KIR. Among them, "active KIR corresponding to an inhibitory KIR" refers to an active KIR whose amino acid sequence of the extracellular domain shows a higher homology with the amino acid sequence of the extracellular domain of the inhibitory KIR of the object. For example, as active KIRs corresponding to KIR2DL2 / 3, KIR2DS1, KIR2DS2 or KIR2DS4 can be cited. The active KIR corresponding to KIR2DL1 is KIR2DS1, the active KIR corresponding to KIR2DL2 / 3 is KIR2DS2, and the active KIR corresponding to KIR3DL1 and KIR3DL2 is KIR3DS1.

[0553] Such an antibody can be obtained, for example, by contacting an antibody or antigen-binding fragment obtained by the above method that binds to an inhibitory KIR and exhibits agonist activity with an NK cell expressing the corresponding active KIR, measuring the cell killing activity of the NK cell against the target cell at this time, and selecting an antibody or antigen-binding fragment with a specified activity or less. In this agonist activity, the antibody or antigen-binding fragment can be added as a soluble protein, and can be expressed on the surface of the target cell in the form of a membrane protein described later. In addition, in the acquisition of such antibodies, antibodies that bind to an inhibitory KIR and have low binding to an active KIR can be obtained, for example, by expressing the variable region of the antibody as a single-chain antibody (scFv) on the surface of a phage, and selecting a phage that binds to the antigen by phage display [Nature Biotechnology (2005), 23, (9), p.1105-1116]. As a method for obtaining a binder that specifically binds to inhibitory KIR using phage display, for example, phage expressing scFv that binds to inhibitory KIR is recovered and concentrated from a human antibody phage library (positive selection), and then phage expressing scFv that binds to active KIR is removed from the obtained phage mixture (consumption), thereby selecting phage that expresses scFv that does not bind to active KIR. Among them, as an inhibitory or active KIR, a recombinant protein / peptide composed of an amino acid sequence of the extracellular region of each molecule, i.e., a region containing a partial sequence that is important for the agonist activity of inhibitory KIR, can be used. The recovery and concentration step or removal step of the phage can adopt various methods for selecting substances with binding or affinity. For example, one of two molecules that show strong binding (such as a combination of biotin and avidin) is bound to a KIR fragment (tagged KIR), and the other is bound to a carrier (such as magnetic beads, etc.). After the phage solution and the tagged KIR are mixed, they are brought into contact with the above-mentioned carrier, so that the phage bound to the tagged KIR is recovered and concentrated on the carrier side. In the positive selection, the inhibitory KIR is tagged to obtain phages concentrated and recovered on the carrier side. On the contrary, in the consumption, the active KIR is tagged, and the phages not bound to the carrier in the solution are recovered, so that phages that are not bound to the active KIR can be obtained. As another method, in the above-mentioned positive selection step, an untagged active KIR fragment is added in excess, so that phages with high binding specificity to the tagged KIR can be recovered and concentrated (exclusion (デセレクション)). These methods are repeated several times, or implemented in combination, to increase the proportion of scFvs with high binding specificity for inhibitory KIR.

[0554] Specifically, antibodies that bind to inhibitory KIR and do not bind to active KIR can be prepared by a method including the steps shown in the following (1) or (2), and the present invention provides such a manufacturing method, a substance manufactured by the manufacturing method, etc. Among them, "display-body" refers to a gene holder for cells having the function of displaying proteins and peptides encoded by genes introduced by a vector on the surface of a lipid membrane, for example, phages, yeast, cells, etc. can be cited. When a phage is used as a display, a known technique can be used as the above-mentioned phage display method or phage panning method. In addition, the method using yeast as a display can use a method known as a yeast display method (for example, reference Lukas Roth, et al., Methods Mol Biol, 2020, Vol. 207, pp. 173-189, doi: 10.1007 / 978-1-4939-9853-1_10, etc.).

[0555] (1) A method for producing a substance that binds to human inhibitory KIR and inhibits the activity of NK cells, the method comprising the following steps:

[0556] Step A: mixing a tagged substance of a soluble protein containing an extracellular domain of an inhibitory KIR in a suspension containing a polyclonal display body group, and then bringing the suspension into contact with a carrier on which a substance that specifically binds to the tag portion is fixed, and recovering the display body bound to the carrier, thereby obtaining a display body group that displays an antigen binding site that binds to the KIR, wherein the polyclonal display body group includes an antigen binding site that exhibits binding to the inhibitory KIR and is displayed on the surface;

[0557] Step B: In a suspension containing the display body group obtained in step A, a labeled substance containing a soluble protein of the extracellular domain of the active KIR corresponding to the inhibitory KIR is mixed, and the labeled substance is brought into contact with a carrier to which a substance bound to the label portion is fixed, and the display body not bound to the carrier is recovered, thereby removing the display body displaying the antigen binding site bound to the active KIR from the cell group;

[0558] Step C: performing gene analysis on the display obtained by the method comprising the above steps to identify the gene sequence encoding the antigen binding site; and

[0559] Step D: The cells are allowed to express the gene encoding the identified antigen binding site in an antibody or antigen binding fragment expression configuration.

[0560] (2) A method for producing a substance that binds to human inhibitory KIR and inhibits the activity of NK cells, the method comprising the following steps:

[0561] Step A: In a suspension containing a polyclonal display body group, a labeled substance containing a soluble protein of the extracellular domain of an inhibitory KIR and a soluble protein containing an unlabeled extracellular domain of an active KIR corresponding to the inhibitory KIR are mixed, and then the cell fluid is brought into contact with a carrier on which a substance that specifically binds to the label portion is fixed, and the display body bound to the carrier is recovered, thereby obtaining a display body group that displays an antigen binding site that specifically binds to the inhibitory KIR, wherein the polyclonal display body group includes an antigen binding site that exhibits binding to the inhibitory KIR and is displayed on the surface;

[0562] Step B: performing gene analysis on the display obtained by the method comprising the above steps to identify the gene sequence encoding the antigen binding site; and

[0563] Step C: The cells are allowed to express the gene encoding the identified antigen binding site in an expression configuration of the antibody or antigen binding fragment.

[0564] Steps AB of the above method (1) and step A of the above method (2) can be repeated multiple times respectively, or the two can be combined.

[0565] In the method (1) or (2), as the combination of the tag and the substance that binds to the tag, a combination of various known substances can be used, and an example of such combination is a combination of biotin-avidin.

[0566] Regarding the antibody or antigen-binding fragment (soluble or membrane protein) expressed in method (1) or method (2), the method may further include the step of selecting a substance having agonist activity against the inhibitory KIR according to the method described in the present specification.

[0567] In the above method, the inhibitory KIR may be KIR2DL2 or KIR2DL3, and the corresponding active KIR may be KIR2DS1, KIR2DS2, and KIR2DS4. In addition, the inhibitory KIR may be KIR3DL1, and the corresponding active KIR may be KIR3DS1. In addition, the inhibitory KIR may be KIR2DL1, and the corresponding active KIR may be KIR2DS1, KIR2DS2, and KIR2DS4.

[0568] A polyclonal display group that displays an antigen binding site that binds to an inhibitory KIR on the surface can be obtained or prepared by a known method. For example, a phage library that displays a variety of variable regions used in a phage display method used to obtain antibodies or a yeast library used in a yeast display method can be used. In addition, as another form, an animal can be immunized with an antigen protein containing an extracellular domain of an inhibitory KIR, and cells such as B cells with antigen-specific antibody genes can be recovered from the spleen cells of an animal that produces antibodies or antigen-binding fragments against the antigen, and the gene sequence encoding the variable region of the antibody gene of the cell can be amplified and introduced into a vector that displays the variable region on the lipid membrane surface in a specified configuration to prepare a display library. This method is described, for example, in David R. Maass et al. J Immunol Methods, (2007 Jul 31); 324 (1-2): 13-25; Lukas Roth et al., Methods Mol Biol., (2020); 2070: 173-189, etc. When the display object is a phage, a phagemid vector is used as the vector.

[0569] In method (1) or method (2), the cells expressing the antibody or antigen-binding fragment may be various cells such as animal cells and Escherichia coli, depending on the purpose. When expressed as a membrane protein, target cells used in agonist activity assays and transplant cells used for cell transplantation (including but not limited to blood cells, T cells, pluripotent stem cells, etc.) may be used.

[0570] An example of a substance having inhibitory KIR2DL2 / 3 specific agonist activity is any of the scFvs (i) to (xii) above, any of the polypeptides (I) to (XII) above, etc. An example of a substance having inhibitory KIR3DL1 specific agonist activity is any of the scFvs (xiii) above, any of the polypeptides (XIII) above, etc.

[0571] The substance having inhibitory KIR2DL1-specific agonist activity is, for example, any one of the scFvs (ci) to (cxv) above, any one of the polypeptides (CI) to (CXV) above, and the like.

[0572] In addition, as one form of the present invention, a polypeptide containing a binding site derived from a heavy chain variable region and a light chain variable region contained in a monoclonal antibody or antigen-binding fragment (competing binding antibody, etc.) that competes with the monoclonal antibody or antigen-binding fragment that binds to the inhibitory KIR when binding to the inhibitory KIR is provided. Such a competitive binding antibody, etc., recognizes the same epitope as the above-mentioned monoclonal antibody or scFv, or an epitope overlapping with the epitope, or an epitope located near the epitope and binds to it, so it is more likely to exhibit the same agonist activity as the above-mentioned monoclonal antibody or antigen-binding fragment to the inhibitory KIR. Therefore, from antibodies against inhibitory KIR, etc., multiple antibodies or antigen-binding fragments that competitively bind to antibodies or antigen-binding fragments that have been confirmed to have agonist activity in the examples of this specification are obtained, and antibodies or antigen-binding fragments that exhibit the desired agonist activity (especially inhibitory KIR-specific agonist activity) are selected from them, thereby obtaining a polypeptide having the desired agonist activity to the inhibitory KIR.

[0573] For example, the polypeptide of the present invention containing a binding site for inhibitory KIR2DL2 and / or KIR2DL3, when binding to inhibitory KIR2DL2 and / or KIR2DL3, contains a binding site derived from the heavy chain variable region and light chain variable region of a monoclonal antibody or antigen-binding fragment that competes with an scFv, lirelumab or Pan2D composed of any of the amino acid sequences in (i) to (xii) above.

[0574] For example, a polypeptide comprising a binding site for KIR2DL1 comprises binding sites derived from the heavy chain variable region and the light chain variable region of a monoclonal antibody or antigen-binding fragment that competes with Pan2D or (P) above when binding to KIR2DL1.

[0575] In addition, for example, a polypeptide containing a binding site for KIR2DL1 contains a binding site derived from the heavy chain variable region and light chain variable region of a monoclonal antibody or antigen-binding fragment that competes with an scFv composed of any one of the amino acid sequences (ci) to (cxv) above when binding to KIR2DL1.

[0576] In addition, for example, the polypeptide of the present invention containing a binding site for inhibitory KIR3DL1 contains a binding site derived from the heavy chain variable region and light chain variable region of a monoclonal antibody or antigen-binding fragment that competes with an scFv composed of any one of the amino acid sequences (xiii) to (xviii) above when binding to inhibitory KIR3DL1.

[0577] The epitope of inhibitory KIR recognized by the polypeptide containing a binding site for inhibitory KIR of the present invention can be analyzed by X-ray crystal structure analysis of a complex of the polypeptide containing a binding site for inhibitory KIR and inhibitory KIR. Epitope analysis using X-ray crystal structure analysis can be performed, for example, in the following manner: a complex of the polypeptide having a binding site for inhibitory KIR of the present invention and KIR is crystallized and subjected to X-ray structure analysis. In this case, a Fab fragment can be used as a polypeptide having a binding site for inhibitory KIR. Regarding the analysis, for example, a ribbon model is obtained to determine the region located near the Fab fragment (for example, located near It is sufficient to have an amino acid residue with a residue of ≤ 1.0, and the amino acid residue can be determined to be an amino acid that forms an epitope.

[0578] For example, the amino acids constituting the inhibitory KIR2DL2 epitope in the binding to α2DL2 / 3-DS10 (VH: SEQ ID NO: 51, VL: SEQ ID NO: 52) are Glu42 (amino acid 21 of SEQ ID NO: 6), His61 (amino acid 40 of SEQ ID NO: 6), Lys65 (amino acid 44 of SEQ ID NO: 6), Phe66 (amino acid 45 of SEQ ID NO: 6), Lys67 (amino acid 46 of SEQ ID NO: 6), Asp68 (amino acid 47 of SEQ ID NO: 6), Thr69 (amino acid 48 of SEQ ID NO: 6), Leu70 (amino acid 49 of SEQ ID NO: 6), Ser86 (amino acid 65 of SEQ ID NO: 6), Ile87 (amino acid 66 of SEQ ID NO: 6), Gly88 (amino acid 67 of SEQ ID NO: 6), Pro89 (amino acid 48 of SEQ ID NO: 6), and Ser69 (amino acid 49 of SEQ ID NO: 6). NO:6, amino acid 68), Met91 (amino acid 70), Gln92 (amino acid 71), Asp93 (amino acid 72), Leu94 (amino acid 73), Tyr101 (amino acid 80), Leu111 (amino acid 90). Among them, the amino acids that differ from the amino acid sequence of the extracellular domain of active KIR2DS1 are Lys65, Lys67, Gly88, Pro89 and Met91, the amino acid that differs from the amino acid sequence of the extracellular domain of KIR2DS2 is Phe66, and the amino acids that differ from the amino acid sequence of the extracellular domain of KIR2DS4 are Lys67, Asp68, Gln92 and Asp93. Therefore, as a KIR2DL2 / 3 specific binder, the interaction between these amino acids and the amino acids nearby is particularly important.

[0579] In the interaction analysis between a2DL2 / 3-DS10 and KIR2DL2, it was confirmed that Tyr at position 107 of the VH chain of a2DL2 / 3-DS10 (amino acid position 107 of SEQ ID NO: 51, hereinafter referred to as "DS10-VH-Y107") formed a hydrogen bond with Asp68 on KIR2DL2 and was close to Phe66. On the other hand, the interaction analysis of the active KIR corresponding to a2DL2 / 3-DS10 showed that the hydrogen bond between DS10-VH-Y107 and Asn68 (position 47 of SEQ ID NO: 14) of KIR2DS4 corresponding to Asp68 was weak or could not be formed, and sterically hindered and could not be close to Tyr66 (Tyr at position 45 of SEQ ID NO: 12) of KIR2DS2 corresponding to Phe66; further indicating that a2DL2 / 3-DS10 contributes to the property of not binding to KIR2DS2 and KIR2DS4. That is, the amino acid in the variable region close to Phe66 and Asp68 on KIR2DL2 is an amino acid that sterically hinders when the amino acid corresponding to Phe66 of the receptor is Tyr. Although it can form a hydrogen bond with Asp, the hydrogen bonding ability with Asn is weak or absent, and Tyr is preferred.

[0580] In the interaction analysis between a2DL2 / 3-DS10 and KIR2DL2, it was confirmed that Tyr at position 106 of the VH chain of a2DL2 / 3-DS10 (amino acid Tyr at position 106 of SEQ ID NO: 51, hereinafter referred to as "DS10-VH-Y106") formed a hydrogen bond with the main chain carbonyl group of Pro89 on KIR2DL2. On the other hand, the interaction analysis of the active KIR corresponding to a2DL2 / 3-DS10 showed that DS10-VH-Y106 could not form a hydrogen bond or a weak hydrogen bond with the main chain carbonyl group of Arg89 (Arg at position 68 of SEQ ID NO: 10) of KIR2DS1 corresponding to Pro89; further indicating that a2DL2 / 3-DS10 contributes to the property of not binding to KIR2DS1. That is, the amino acid in the variable region close to Pro89 on KIR2DL2 is an amino acid that can form a hydrogen bond with Pro but has a weak or absent ability to form a hydrogen bond with Arg, and is preferably Tyr.

[0581] In the interaction analysis between a2DL2 / 3-DS10 and KIR2DL2, it was confirmed that Gly at position 104 of the VH chain of a2DL2 / 3-DS10 (amino acid Gly at position 104 of SEQ ID NO: 51, hereinafter referred to as "DS10-VH-G104") formed a hydrogen bond with the imino group of the main chain of Gly88 on KIR2DL2 on its main chain carbonyl group. On the other hand, the interaction analysis of the active KIR corresponding to a2DL2 / 3-DS10 showed that due to steric hindrance, DS10-VH-G104 could not form a main chain hydrogen bond or the hydrogen bond was weak with Ser88 of KIR2DS1 corresponding to Gly88 (Ser at position 67 of SEQ ID NO: 10); further indicating that a2DL2 / 3-DS10 contributes to the property of not binding to KIR2DS1. That is, the amino acid in the variable region close to Gly88 on KIR2DL2 is an amino acid that can form a hydrogen bond with the main chain imino group of Gly but has a weak or absent ability to form a hydrogen bond with the main chain imino group of Ser, and is preferably Gly.

[0582] In the interaction analysis between a2DL2 / 3-DS10 and KIR2DL2, it was confirmed that Thr at position 94 of the VL chain of a2DL2 / 3-DS10 (amino acid Thr at position 94 of SEQ ID NO:52, hereinafter referred to as "DS10-VL-T94") formed a hydrogen bond with the side chain of Asp93 on KIR2DL2 at its main chain imino group. On the other hand, the interaction analysis of the active KIR corresponding to a2DL2 / 3-DS10 showed that DS10-VL-T94 could not form a hydrogen bond or a weak hydrogen bond with Val93 of KIR2DS4 corresponding to Asp93 (Val at position 72 of SEQ ID NO:14), which further indicated that a2DL2 / 3-DS10 contributes to the property of not binding to KIR2DS4. In addition, it was shown that the specificity of the interaction with KIR2DL2 is maintained even when DS10-VL-T94 is substituted with Asn, Gly, His, Ile, Ser, Val or Ala. That is, the amino acid in the variable region close to Asp93 on KIR2DL2 is an amino acid that can form a hydrogen bond with the side chain of Asp and has a weak or absent ability to form a hydrogen bond with Val, preferably Thr, Asn, Gly, His, Ile, Ser, Val or Ala, and more preferably Thr.

[0583] One form of the KIR2DL2 or KIR2DL3 specific agonist binder of the present invention is a substance (antibody or antigen-binding fragment) comprising a variable region that interacts with the following amino acids: 1 to 6 (preferably 3 to 6, more preferably 4 to 6, further preferably 5 or 6) amino acids at amino acid position 47 in the region including amino acids 44 to 49 in the amino acid sequence of KIR2DL2 of SEQ ID NO:6; 2 to 4 (preferably 2, 3 or 4) amino acids at amino acids 67 and 68 in the region including amino acids 65 to 68; and 1 to 4 (preferably 2, 3 or 4) amino acids at amino acid position 72 in the region including amino acids 70 to 73. Preferably, the substance is a binder comprising a variable region that interacts with the following amino acids: amino acids in the region of positions 45 to 47, the region of positions 45 to 48, the region of positions 44 to 47, the region of positions 45 to 48, the region of positions 44 to 48, the region of positions 45 to 49, or the region of positions 44 to 49 in the amino acid sequence of KIR2DL2 (SEQ ID NO: 6); amino acids in the region of positions 67 to 68, the region of positions 66 to 68, or the region of positions 65 to 68 of SEQ ID NO: 6; and amino acids in the region of positions 72 to 73, the region of positions 71 to 73, or the region of positions 70 to 73 of SEQ ID NO: 6.

[0584] In addition, one form of the KIR2DL2-specific agonist of the present invention is an antibody or antigen-binding fragment in which the amino acid interacting with KIR2DL2 in the variable region is the following amino acids: the amino acid interacting with Asp at position 47 of SEQ ID NO: 6 is Tyr, the amino acid interacting with Gly at position 67 of SEQ ID NO: 6 is Gly, the amino acid interacting with Pro at position 68 of SEQ ID NO: 6 is Tyr, and the amino acid interacting with Asp at position 72 of SEQ ID NO: 6 is Thr, Asn, Gly, His, Ile, Ser, Val or Ala. In such an antibody or antigen-binding fragment, preferably, in addition to the above amino acids, the amino acid close to Phe at position 45 of SEQ ID NO: 6 is Tyr; more preferably, all of the above amino acids are contained in CDR3 of the variable region. As such an antibody or antigen-binding fragment, preferably, it is characterized in that it has a CDR3 comprising an amino acid sequence consisting of positions 97 to 112 of SEQ ID NO:51 as a first variable region, and a CDR3 comprising an amino acid sequence in which Thr at position 94 in the amino acid sequence in which positions 89 to 97 of SEQ ID NO:52 can be replaced by Asn, Gly, His, Ile, Ser, Val or Ala as a second variable region; more preferably, it is characterized in that it comprises an amino acid sequence of SEQID NO:51 as a first variable region, and a CDR3 comprising an amino acid sequence in which Thr at position 94 in the amino acid sequence of SEQ ID NO:52 can be replaced by Asn, Gly, His, Ile, Ser, Val or Ala as a second variable region.

[0585] In addition, in the binding test with the antigen, the peptides containing the binding site for the inhibitory KIR are allowed to compete with each other to conduct a competition test, thereby analyzing the epitope. For example, the Fab fragment of the polypeptide different from the K562 cell line expressing the scFv of the polypeptide having the binding site for the inhibitory KIR is mixed with the recombinant KIR, and the K562 cell line expressing the scFv and the Fab fragment are allowed to compete with the recombinant KIR for binding. Whether the binding of the K562 cell line expressing the scFv to the recombinant KIR is hindered by competition can be used as an indicator to evaluate the epitope site of each binder. It should be noted that in the present invention, recombinant (Recombinant, Recombinant, Group Change) and soluble type refer to the same thing.

[0586] In addition, the polypeptide containing a binding site for inhibitory KIR of the present invention includes other polypeptides that recognize the epitope recognized by the polypeptide containing a binding site for inhibitory KIR analyzed by the above method. The other polypeptides compete with the polypeptide containing a binding site for inhibitory KIR of the present invention in binding to inhibitory KIR and have inhibitory selectivity.

[0587] The membrane protein of the present invention has an extracellular domain and a transmembrane domain containing a binding site for inhibitory KIR, so when expressed in a cell, it has a binding site for inhibitory KIR, and a polypeptide that recognizes and binds to inhibitory KIR can exist on the cell membrane surface. The membrane protein of the present invention may also include an intracellular domain.

[0588] The polypeptide having a binding site for the inhibitory KIR and binding to the inhibitory KIR is connected to the transmembrane domain via an extracellular linker domain.

[0589] Among them, the structural domain refers to a region present in a polypeptide, which is a region that folds into a specific structure independently of other regions. In membrane proteins, a polypeptide that has a binding site for an inhibitory KIR and binds to an inhibitory KIR can be called a binding domain. When the binding domain includes the first variable region and the second variable region derived from the heavy chain variable region and the light chain variable region of the monoclonal antibody or antigen-binding fragment described in (1) above, the two variable regions can adopt the linker sequence that can be adopted by the above-mentioned scFv. In addition, starting from the N-terminal side of the polypeptide, it can be configured in sequence as "first variable region-linker sequence-second variable region", or it can be configured in sequence as "second variable region-linker sequence-first variable region".

[0590] The transmembrane domain is a domain that has affinity for the lipid bilayer constituting the cell membrane, and examples thereof include but are not limited to transmembrane domains of HLA, CD28, CD3, CD8, CD19, CD4, or 4-1BB, and transmembrane sequences derived from various natural membrane proteins and artificially designed transmembrane sequences can be used. Preferably, the transmembrane domain (SEQ ID NO: 27) is derived from CD8α.

[0591] As an extracellular linker, the full length or a portion of the extracellular domain of HLA, CD28, CD8, CD19, CD3, CD4 or 4-1BB, etc. can be cited, but not limited to, various natural protein-derived linker sequences, hinge sequences (e.g., hinge sequences of immunoglobulins) and artificially designed flexible linker sequences can be used. Preferably, a CD8α-derived hinge sequence (SEQ ID NO: 26) with a length of 45 amino acids and a CD8α-derived hinge sequence (SEQ ID NO: 24) with a length of 66 amino acids are used.

[0592] The intracellular domain is necessary to maintain the stability of the scFv body, and a portion of the intracellular domain that does not participate in intracellular signaling of HLA, CD28, CD3, CD8, CD19, CD4 or 4-1BB, etc. (preferably less than 10 amino acids, less than 9 amino acids, less than 8 amino acids, less than 7 amino acids, less than 6 amino acids, less than 5 amino acids, less than 4 amino acids, less than 3 amino acids, less than 2 amino acids or 1 amino acid) can be used. However, in the case where it is necessary to promote the activation and inhibition of intracellular signals by binding to KIR, a portion of the intracellular domain that participates in intracellular signaling can also be used.

[0593] Examples of the membrane protein of the present invention include the following membrane proteins.

[0594] A membrane protein having an extracellular domain and a transmembrane domain comprising a binding site for KIR2DL2 and / or KIR2DL3, a membrane protein having an extracellular domain and a transmembrane domain comprising a binding site for KIR3DL1, a membrane protein having an extracellular domain and a transmembrane domain comprising a binding site for KIR2DL1, and the like.

[0595] (3) A polypeptide comprising a binding site for an inhibitory KIR derived from a sequence of the inhibitory KIR binding site in an HLA molecule which is a natural ligand of the inhibitory KIR, and wherein the binding property to TCR (T cell receptor) is deleted or weakened.

[0596] For example, when the inhibitory KIR is KIR2DL2 or KIR2DL3, a polypeptide can be used that contains the C1 epitope motif sequence (the 77th amino acid is S and the 80th amino acid is N) contained in HLA-Cw1, Cw3, Cw7, Cw8, Cw9, Cw10, Cw12, Cw14, Cw16 and HLA-B46 or B73 belonging to the HLA-C1 type, and contains a domain in which the region recognized by the TCR of the HLA molecule is mutated or deleted so that it is not recognized by the TCR.

[0597] For example, when the inhibitory KIR is KIR2DL1, a polypeptide can be used that contains the C2 epitope motif sequence (the 77th amino acid is N, the 80th amino acid is K) contained in HLA-Cw2, Cw4, Cw5, Cw6, Cw15, Cw17, etc. belonging to the HLA-C2 type, and contains a domain that is mutated or deleted in the region recognized by the TCR of the HLA molecule so that it is not recognized by the TCR.

[0598] For example, when the inhibitory KIR is KIR3DL1, a polypeptide containing the Bw4 epitope motif sequence (amino acid 77 is N, amino acid 80 is I or T, and amino acid 83 is R) contained in HLA-Bw4 type HLA-B5, B27, B37, B38, B44, B49, B51, B52, B53, B57, B58, B59, B77, A23, A24, A25, A32, etc., and containing a domain that is not recognized by TCR by mutating or deleting the region recognized by TCR of the HLA molecule can be used. Such a polypeptide can be in the form of a soluble ligand containing an amino acid sequence containing the above epitope motif sequence, or in the form of a membrane protein containing the epitope motif sequence in the extracellular region.

[0599] For example, when the inhibitory KIR is KIR3DL2, a polypeptide containing a domain that is not recognized by TCR can be used by mutating or deleting the region recognized by TCR of HLA-A3, A11, B27 free heavy chain or the HLA molecule. Such a polypeptide can be in the form of a soluble ligand or a membrane protein.

[0600] As a substance having agonist activity against inhibitory KIR, a low molecular weight compound that binds to inhibitory KIR and exhibits agonist activity against the KIR can also be cited. Such a compound can be selected from a compound library by the above-mentioned evaluation method for agonist activity against inhibitory KIR. In addition, for a compound group that has been subjected to derivative development according to the structure of the compound selected by this method, the binding activity and the above-mentioned agonist activity with the inhibitory KIR as the object are further measured, the structure-activity correlation is analyzed, and the derivative development is repeated, thereby obtaining a compound with better binding and / or agonist activity.

[0601] 2. Method for producing a substance having the activity of binding to inhibitory KIR and inhibiting the cytotoxic activity of NK cells

[0602] The polypeptide of the present invention having a binding site for inhibitory KIR and binding to inhibitory KIR can be prepared in the form of secretory protein (soluble protein) or membrane protein by recombination, genome editing, in vitro translation, chemical synthesis, peptide synthesis, etc.

[0603] For example, the polypeptide of the present invention can be produced by introducing a polynucleotide containing a nucleotide sequence encoding the amino acid sequence of the above-mentioned polypeptide into cells, culturing the cells, and recovering the polypeptide from the culture.

[0604] Regarding a polypeptide having a binding site for inhibitory KIR and binding to inhibitory KIR, and a membrane protein having a transmembrane domain, nucleotides encoding the amino acid sequence of each domain can be connected, and further functionally connected to elements such as a promoter, an enhancer, and a polyadenylation signal. In addition, in order to be present between the polypeptide bound to the inhibitory KIR and the transmembrane domain, the extracellular linker domain can be connected to the base sequence encoding the amino acid sequence of each domain and inserted into a vector so that it is expressed in a host cell. Here, functionally connected means that the element is connected so that its function is achieved. These nucleotides can be inserted into an expression vector, and the host cell is transformed by the vector, and the host cell is cultured and produced and recovered. The vector may also include nucleotides encoding a signal peptide that promotes host cell secretion of the above-mentioned polypeptide. In this case, the nucleotide encoding the signal peptide is previously connected to the nucleotide encoding the above-mentioned polypeptide in frame. After the above-mentioned polypeptide is produced, the signal peptide is removed, thereby obtaining the above-mentioned polypeptide as a mature protein. As a signal peptide, for example, a CD8 signal peptide can be used.

[0605] As long as the expression vector can be replicated in hosts such as animal cells, bacteria, and yeast, there is no particular limitation, and examples thereof include known plasmids, phages, and the like. As vectors for constructing expression vectors, examples thereof include: pcDNA (trademark) (Thermo Fisher Scientific), Flexi (registered trademark) vectors (Promega), pUC19, pUEX2 (produced by Amersham), pGEX-4T, pKK233-2 (produced by Pharmacia), pMAM-neo (produced by Clontech), and the like. As host cells, prokaryotic cells such as Escherichia coli and Bacillus subtilis can be used, and eukaryotic cells such as yeast and animal cells can also be used, preferably eukaryotic cells. For example, as animal cells, human embryonic kidney cell line HEK293 cells, Chinese hamster ovary (CHO) cells, and the like can be used. Expression vectors can be introduced into host cells by known methods to transform host cells. For example, electroporation, calcium phosphate precipitation, DEAE-dextran transfection, and the like can be cited. The produced antibodies can be purified using separation and purification methods commonly used for proteins. For example, affinity chromatography, other chromatography, filtration membranes, ultrafiltration membranes, salting out, dialysis, etc. can be appropriately selected and combined.

[0606] 3. Cells expressing on the cell surface a substance having an activity of binding to inhibitory KIR and inhibiting the cytotoxic activity of NK cells and methods for producing the same

[0607] As a cell expressing a substance having an activity that binds to inhibitory KIR and inhibits the cytotoxic activity of NK cells on the cell surface, it is preferred that the cell expresses a polypeptide having a binding site for inhibitory KIR and binding to inhibitory KIR and a membrane protein having a transmembrane domain as described in the above-mentioned "1(2)". The transmembrane domain of the membrane protein exists in a state spanning the cell membrane, and the polypeptide having a binding site for inhibitory KIR and binding to inhibitory KIR exists on the cell surface.

[0608] A nucleotide encoding a polypeptide having a binding site for an inhibitory KIR and binding to the inhibitory KIR can be introduced into a cell, and then expressed in the cell.

[0609] The method for introducing nucleotides may be any commonly used method without particular limitation. In the case of using a vector for introduction, the vector that can be used is not particularly limited, and examples thereof include lentiviral vectors, retroviral vectors, foamy virus vectors, adeno-associated virus vectors, and the like.

[0610] When expressing the above membrane protein, expression control elements such as enhancers and promoters are connected to the nucleotides encoding the above membrane protein, introduced into an expression vector, and the cells are transformed by the expression vector, so that the cells express the membrane protein. When introducing nucleotides, it is necessary to connect an appropriate promoter to the upstream of the nucleotides to be introduced in a form that can function. There is no limitation on the promoter, and viral promoters such as lentivirus, retrovirus, adenovirus, SV40 virus, cytomegalovirus, and promoters derived from mammalian cells can be used.

[0611] When the nucleotide encoding the membrane protein is introduced into cells, the expression vector may contain a selectable marker gene. As the selectable marker gene, a known marker gene such as thymidine kinase gene can be used.

[0612] The present invention also includes the DNA encoding the above membrane protein, and the RNA transcribed from the DNA.

[0613] In addition, the expression of the membrane protein of the present invention can also use genome editing technology. Genome editing technology can use various methods such as CRISPR-Cas, Zinc-Finger, TaLEN, etc., without limitation. As a method for preparing genetically modified cells using genome editing technology, for example, the method described in WO2018 / 018534 can effectively implement genome editing not only on ordinary cells, but also on pluripotent stem cells such as iPS cells.

[0614] As cells expressing the above-mentioned membrane protein, cells used for transplantation or for allogeneic transplantation for the purpose of cell therapy can be cited. These cells are called therapeutic cells. Cells expressing the above-mentioned membrane protein express on the surface a polypeptide having a binding site for inhibitory KIR and binding to inhibitory KIR, which binds to the inhibitory KIR of NK cells, causes the inhibitory KIR to work, and transmits inhibitory signals into NK cells, thereby inhibiting the cell killing activity of NK cells. As a result, immune rejection during transplantation can be suppressed, and immune rejection is not easily suffered, thereby reducing the risk of immune rejection.

[0615] The cells used for transplantation or allogeneic transplantation for cell therapy purposes are pluripotent stem cells, particularly ES cells or iPS cells. In these pluripotent stem cells, the membrane protein of the present invention can be expressed by the above-mentioned genome editing, or effective gene editing can be performed. In addition, mesenchymal stem cells (Mesencymal Stem Cell) and T cells, NK cells, macrophages, etc. derived from PBMC (peripheral blood mononuclear cells) as primary cells can also be cited. These cells collected from biological tissues can, for example, knock out HLA, or suppress NK activity by being modified to express KIR agonists. In addition, with respect to mesenchymal stem cells, effective therapeutic factors such as factors for cancer therapy can be modified to be expressed and secreted and transplanted; with respect to T cells and NK cells, cells with anti-cancer effects such as CAR-T / NK can be modified and used as therapeutic cells. Cells used for transplantation or allogeneic transplantation for cell therapy purposes can also include cardiomyocytes, chondrocytes, retinal cells, hepatocytes, kidney cells, islet cells, etc. These cells can be cells collected from donors, or various cells prepared by artificial differentiation induction from pluripotent stem cells. Regardless of the type of cell, as long as it expresses membrane proteins on the cell surface that can bind to the inhibitory KIR of NK cells, it can bind to the inhibitory KIR of NK cells, allowing the inhibitory KIR to work and transmit inhibitory signals to the NK cells, thereby inhibiting the cell killing activity of NK cells.

[0616] Regarding the cells of the present invention, the expression of at least one class I HLA on the cell surface may also disappear or weaken. The disappearance or weakening of the expression of at least one class I HLA on the cell surface may be caused by gene editing to modify the class I HLA, B2M, TAP1, TAP2, TAPBP regions on the genome.

[0617] Alternatively, the cells of the present invention may be a cell line established from an individual in which HLA expression has been hereditarily lost.

[0618] In addition, the cells of the present invention may also express a ligand substance for NKG2A on the cell surface. NKG2A (CD159) is a type of inhibitory receptor on the surface of NK cells. As a ligand for NKG2A, HLA-E can be cited.

[0619] The present invention includes a method for producing therapeutic cells in which the risk of immune rejection of a subject is reduced or suppressed, the method comprising expressing on the surface of the therapeutic cells a substance of the present invention having an activity of binding to inhibitory KIR and inhibiting the cytotoxic activity of NK cells.

[0620] 4. CAR-T

[0621] The present invention includes T cells (CAR-T) that express chimeric antigen receptors (CAR: chimeric antigen receptor) targeting desired antigens in addition to the above-mentioned membrane proteins. The CAR-T used in the present invention is not limited to CAR-T targeting tumor-associated antigens. The CAR-T expresses a polypeptide containing a domain that specifically binds to a tumor cell-specific tumor-associated antigen, a transmembrane domain, and an intracellular signaling domain on the surface. The domain that specifically binds to the tumor-associated antigen can use a polypeptide derived from an antibody against the tumor-associated antigen, such as a polypeptide that connects scFv and the Fv region of the immunoglobulin heavy chain and light chain. The CAR of the CAR-T of the present invention is present on the cell membrane, and contains an extracellular domain, a transmembrane domain, and an intracellular signaling domain as an intracellular domain that contains a domain that specifically binds to a tumor cell-specific tumor-associated antigen. The extracellular domain may include an extracellular spacer domain, and the intracellular domain may include a costimulatory domain. The transmembrane domain can use, for example, transmembrane domains of CD8α, CD28, CD3ε, CD3, CD4, etc. The extracellular spacer domain can use spacer sequences such as (G4S)3 and the hinge region of IgG1. The co-stimulatory domain can use CD27, CD28, CD134, CD27, CD2, CD5, CD30, CD40, etc. For example, the intracellular signal transduction domain can use human CD3ζ chain, Fc receptor, FcγRIII, FcεRI, etc. The polynucleotide encoding the CAR of the present invention can be prepared by connecting the polynucleotides encoding the above-mentioned domains. The polynucleotide and the polynucleotide encoding the polypeptide containing the binding site for the inhibitory KIR of the present invention are introduced into T cells, thereby preparing the CAR-T of the present invention.

[0622] The CAR-T of the present invention can be used to treat tumors expressing tumor-associated antigens. The CAR-T cells of the present invention express polypeptides containing binding sites for inhibitory KIR on the cell surface, and in addition to the cell killing activity against target cells, they also have the function of escaping the immune rejection of NK cells. The present invention includes a pharmaceutical composition containing the CAR-T cells of the present invention.

[0623] 5. The substance having the activity of binding to inhibitory KIR and inhibiting the cytotoxic activity of NK cells of the present invention, and the use of the substance having the activity of binding to inhibitory KIR and inhibiting the cytotoxic activity of NK cells expressed on the cell surface

[0624] The present invention includes a substance having the activity of binding to inhibitory KIR and inhibiting the cytotoxic activity of NK cells, and a pharmaceutical composition of cells containing a substance having the activity of binding to inhibitory KIR and inhibiting the cytotoxic activity of NK cells expressed on the cell surface as an active ingredient.

[0625] The pharmaceutical composition can be used as an immunorejection inhibitor when transplanting organs and therapeutic cells. Specifically, it can be used to inhibit rejection during allogeneic transplantation of kidneys and pancreatic islets, prevent and treat GVHD (graft versus host disease), inhibit rejection during allogeneic transplantation of regenerative therapeutic cells, etc. In addition, it can also be used for xenogeneic organ transplantation such as transplantation of organs from genetically modified pigs to humans. The pharmaceutical composition is also called an immunorejection inhibitor or a transplant rejection inhibitor.

[0626] Cells expressing on the cell surface a substance having an activity of binding to inhibitory KIR and inhibiting the cytotoxic activity of NK cells can be used as therapeutic cells for suppressing immune rejection of a subject to which the cells are administered or transplanted. The therapeutic cells can be used as a pharmaceutical composition for transplantation.

[0627] The present invention also includes a method for inhibiting immune rejection of a subject, comprising administering or transplanting a substance of the present invention having the activity of binding to inhibitory KIR and inhibiting the cell killing activity of NK cells to a subject in need of transplantation, and cells expressing on the cell surface the substance having the activity of binding to inhibitory KIR and inhibiting the cell killing activity of NK cells.

[0628] The present invention also includes a method for suppressing immune rejection in a subject, comprising expressing on the surface of therapeutic cells the substance of the present invention having the activity of binding to inhibitory KIR and suppressing the cell-killing activity of NK cells.

[0629] When used for the above purposes, the HLA type of the patient to be treated has an HLA type that becomes a ligand of the inhibitory KIR that is the target. For example, when an agonist substance for inhibitory KIR2DL2 and / or KIR2DL3 or a cell expressing the agonist substance on the cell surface is used, an immunosuppressive effect or an immune rejection escape effect is exerted on a patient who is homozygous for HLA-C1. In addition, when an agonist substance for inhibitory KIR2DL1 or a cell expressing the agonist substance on the cell surface is used, an immunosuppressive effect or an immune rejection escape effect is exerted on a patient who is homozygous for HLA-C2. For example, when an agonist substance against inhibitory KIR2DL2 and / or KIR2DL3 and an agonist substance against inhibitory KIR2DL1 are used in combination, or when cells expressing these two agonist substances in combination on the cell surface are used, an immunosuppressive effect or an immune rejection escape effect will be exerted on patients of all HLA-C genotypes (HLA-C1 homozygous, HLA-C2 homozygous, and HLA-C1 / C2 heterozygous).

[0630] In addition, for HLA-Bw4-negative patients, treatment can be performed corresponding to the above-mentioned HLA-C genotype, while in the case of HLA-Bw4-positive patients, in addition to this, an agonist substance for KIR3DL1 is additionally administered in combination, or cells expressing the combination on the cell surface are administered, thereby exerting an immunosuppressive effect or an immune rejection escape effect.

[0631] The pharmaceutical composition of the present invention may contain a therapeutically effective amount of a substance having an activity that binds to an inhibitory KIR and inhibits the cell killing activity of NK cells, or a substance having an activity that binds to an inhibitory KIR and inhibits the cell killing activity of NK cells, and a cell on which a substance having an activity that binds to an inhibitory KIR and inhibits the cell killing activity of NK cells is expressed on the cell surface, and a pharmaceutically acceptable carrier, diluent, solubilizer, emulsifier, preservative, adjuvant, etc. The method of administration of the pharmaceutical composition of the present invention can be appropriately selected, for example, it can be administered by injection, and local injection, intraperitoneal administration, selective intravenous injection, intravenous injection, subcutaneous injection, organ perfusion fluid injection, etc. can also be used. In addition, the solution for injection can be formulated using a saline solution, a glucose solution, or a mixture of saline and glucose solution, a carrier composed of various buffer solutions, etc. In addition, it can also be formulated in a powder state, and mixed with the above-mentioned liquid carrier to prepare an injection solution when used.

[0632] Regarding other administration methods, they can also be appropriately selected together with the development of the preparation. For example, in the case of oral administration, oral liquids and powders, pills, capsules and tablets can be applied. In the case of oral liquids, as oral liquid preparations such as suspensions and syrups, the following substances can be used for manufacturing: water; sugars such as sucrose, sorbitol, fructose; ethylene glycols such as polyethylene glycol; oils such as sesame oil and soybean oil; preservatives such as alkyl parabens; flavors such as strawberry flavor and mint, etc. Powders, pills, capsules and tablets can be formulated using the following substances: excipients such as lactose, glucose, sucrose, mannitol; disintegrants such as starch and sodium arginine; lubricants such as magnesium stearate and talc; binders such as polyvinyl alcohol, hydroxypropyl cellulose, gelatin; surfactants such as fatty acid esters; plasticizers such as glycerol, etc. Tablets and capsules are the preferred unit administration form in the composition of the present invention because they are easy to administer. When manufacturing tablets and capsules, solid manufacturing carriers are used.

[0633] The amount of the substance that has the activity of binding to inhibitory KIR and inhibiting the cell killing activity of NK cells that is effective for treatment can vary according to the age and condition of the patient, and is ultimately determined by the doctor. For example, the amount at one time is 0.0001 mg to 100 mg per 1 kg of body weight. The prescribed dosage can be once a day for 1 to 180 days, or it can be divided into 2, 3, 4 or more times a day, and administered at appropriate intervals.

[0634] The dosage of the substance having the activity of binding to inhibitory KIR and inhibiting the cytotoxic activity of NK cells, and the substance having the activity of binding to inhibitory KIR and inhibiting the cytotoxic activity of NK cells expressed on the cell surface can be appropriately determined according to the type of transplanted cells, etc.

[0635] 6. Method for evaluating the immune rejection escape activity of the test substance by inhibiting the cytotoxic activity of NK cells against inhibitory KIR

[0636] The present invention includes a method for evaluating the immune rejection escape activity of a test substance, which is the activity of inhibiting the cytotoxic activity of NK cells against inhibitory KIR.

[0637] The test substance is a substance having an activity of binding to any inhibitory KIR and inhibiting the cytotoxic activity of NK cells, and examples thereof include the substances described in the above-mentioned "1."

[0638] The method comprises the following steps.

[0639] (A) a step of contacting NK cells from a subject homozygous for HLA-C type C1 / C1 and target cells from a subject homozygous for HLA-C type C2 / C2 in vitro in the presence of a test substance that binds to KIR, wherein the Bw4 expression type of the donor of the NK cells and the donor of the target cells is the same;

[0640] (B) After step A, a step of measuring the number of surviving target cells.

[0641] The inhibitory KIR may specifically include the following steps.

[0642] (A) a step of contacting NK cells from a subject homozygous for HLA-C type C1 / C1 and target cells from a subject homozygous for HLA-C type C2 / C2 in vitro in the presence of a test substance that binds to KIR2DL2 or KIR2DL3, wherein the expression type of HLA containing the Bw4 motif is the same in the donor of the NK cells and the donor of the target cells;

[0643] (B) After step A, a step of measuring the number of surviving target cells.

[0644] The following steps may also be included.

[0645] (A) a step of contacting NK cells from a subject homozygous for HLA-C type C2 / C2 and target cells from a subject homozygous for HLA-C type C1 / C1 in vitro in the presence of a test substance that binds to KIR2DL1, wherein the expression type of HLA containing the Bw4 motif of the donor of the NK cells and the donor of the target cells is the same;

[0646] (B) After step A, a step of measuring the number of surviving target cells.

[0647] The following steps may also be included.

[0648] (A) a step of contacting NK cells from a subject with HLA-Bw4 type positive / positive homozygous or positive / negative heterozygous and target cells from a subject with HLA-Bw4 type negative / negative homozygous in vitro in the presence of a test substance that binds to KIR3DL1, wherein the donor of the NK cells and the donor of the target cells have the same expression type of HLA-C type;

[0649] (B) After step A, a step of measuring the number of surviving target cells.

[0650] The following steps may also be included.

[0651] (A) a step of contacting NK cells from a subject whose HLA-A type is A3 or A11 positive / positive homozygous or positive / negative heterozygous with target cells from a subject whose HLA-A type is A3 or A11 negative / negative homozygous in vitro in the presence of a test substance that binds to KIR3DL2, wherein the donor of the NK cells and the donor of the target cells have the same expression type of HLA-C type and Bw4 type;

[0652] (B) After step A, a step of measuring the number of surviving target cells.

[0653] As NK cells, for example, NK cells derived from peripheral blood can be used, and as target cells, for example, T cells derived from peripheral blood can be used.

[0654] NK cells recognize their own HLA type (C1, C2, Bw4) and have the property of "missing self" to show cytotoxic activity against target cells. For example, NK cells collected from HLA-C1 / C1 homozygous people will recognize C1 as themselves, so if the target cells do not have C1 type HLA-C, the signal will enter the NK cells through inhibitory KIR, thereby showing cytotoxic activity. On the other hand, if C1 type is present in the target cells, cytotoxic activity will not be shown; in addition, if a substance (agonist binder) that binds to inhibitory KIR2DL2 and / or KIR2DL3 and inhibits the cytotoxic activity of NK cells is expressed as a substitute for C1 type, the cytotoxic activity will be reduced.

[0655] The HLA-C type NK cells used in the above evaluation method are from C1 / C1 homozygous subjects and can be separated from PBMC (peripheral blood mononuclear cells) of people whose HLA-C type is C1 / C1 homozygous using CD56 positivity as an indicator. For example, CD56 microbeads or flow cytometry can be used for separation. The NK cells thus separated have activated KIR2DL2 and KIR2DL3.

[0656] HLA-C type target cells derived from C2 / C2 homozygous subjects can be isolated from PBMC of HLA-C type C2 / C2 homozygous subjects using CD3 positivity as an indicator. Since these target cells do not express C1 type HLA, the above HLA-C type NK cells derived from C1 / C1 homozygous subjects will exhibit cell killing activity.

[0657] When HLA-C type NK cells from C1 / C1 homozygous subjects are used as effector cells and HLA-C type target cells from C2 / C2 homozygous subjects are used as target (target) cells, the target cells are brought into contact with each other. In this case, the target cells are killed by the effector cells. However, if there is a substance that binds to inhibitory KIR2DL2 and / or KIR2DL3 and inhibits the cytotoxic activity of NK cells, the substance will bind to KIR2DL2 and / or KIR2DL3 of NK cells from C1 / C1 homozygous subjects with HLA-C type, thereby inhibiting the cytotoxic activity of NK cells. Therefore, the cytotoxic activity of effector cells will be reduced, and target cells will not be killed. Therefore, by contacting HLA-C type NK cells from C1 / C1 homozygous subjects and HLA-C type target cells from C2 / C2 homozygous subjects in the presence of a test substance that binds to KIR2DL2 and / or KIR2DL3, and then measuring the survival rate of the target cells, the immune rejection escape activity of the test substance that inhibits the cytotoxic activity of NK cells against inhibitory KIR can be evaluated.

[0658] In step B, if the number of surviving target cells is high, it can be determined that the test substance has immune rejection escape activity. The present invention includes a method for screening a substance having immune rejection escape activity, comprising: selecting a test substance having a high number of surviving target cells in step B as a substance having immune rejection escape activity.

[0659] It should be noted that in step (A) of the above method, the test substance is present in the form of a membrane protein expressed on the surface of the target cell as described in "1(2)".

[0660] Specifically, it can be carried out by the methods described in the "Evaluation of cell killing activity" and "Evaluation of agonist activity" in the Examples described later.

[0661] [Example]

[0662] The present invention is specifically described below by the following examples, but the present invention is not limited to these examples.

[0663] method

[0664] The following methods were used for each experimental method.

[0665] [Plasmid vector for virus preparation]

[0666] The preparation of lentivirus for introducing the target gene into the natural killer cell line as effector cells, the K562 cell line as target cells, and human T cells uses the pLVSIN vector from which the gene sequence encoding the green fluorescent protein (ZsGreen1) contained in pLVSIN-EF1αIRES-ZsGreen1 (produced by Takara Bio Co., Ltd., 6191) has been removed. The target gene was introduced into the multiple cloning site of pLVSIN to prepare a plasmid vector for lentivirus preparation.

[0667] [Preparation of lentivirus]

[0668] Lenti-X 293T cell line (manufactured by Takara Bio Co., Ltd., 632180) was cultured in advance to confluence using Dulbecco's Modified Eagle Medium (DMEM) (manufactured by Thermo Fisher Scientific, 10566-016) containing 10 (v / v)% fetal bovine serum (FBS), and on the day of transfection, the cells were detached using trypsin (manufactured by Thermo Fisher Scientific, 12563029) and seeded into T flasks again. After culturing to 70% to 80% confluence, the above-mentioned plasmid vector for lentivirus preparation, lentivirus high titer packaging mixture (produced by Takara Bio, Japan, 6194) and TransIT-293 transfection reagent (produced by Takara Bio, Japan, MIR2704) were mixed in Opti-MEM I reduced serum medium (produced by Thermo Fisher Scientific, 31985070), incubated and added to Lenti-X 293T cell line. The next day, the medium was replaced with DMEM medium containing 10 (v / v)% FBS, cultured for 24 hours, and the supernatant was recovered. If necessary, after the supernatant was recovered, DMEM medium containing 10 (v / v)% FBS was added again and cultured for 24 hours, and the supernatant was recovered again. After filtering the recovered supernatant, 1 / 3 of the Lenti-X concentration reagent (produced by Takara Bio, Japan, 631232) was added, incubated at 4°C for more than 1 hour, and centrifuged to precipitate the virus. The supernatant was removed by aspiration, and the pellet was dissolved in AIM-V medium (manufactured by Thermo Fisher Scientific, 12055083) (hereinafter referred to as "basal medium") containing 5 (v / v)% FBS, and stored frozen at -80°C or -150°C as a lentivirus concentrate.

[0669] [Introduction and preparation of KIR genes into NK92 cells]

[0670] NK92 cells were obtained from ATCC (Cat. No: CRL-2407 TM) to express various KIRs appropriately and used as effector cells for the initial evaluation system. 500 IU / mL (range of 100-2000) of IL-2 and streptomycin / penicillin were added to MyeloCult H5100 medium (produced by STEMCELL Technologies, ST-05150) and cultured at 37°C and 5% CO2.

[0671] In order to establish various KIR-expressing NK92 cell lines described in Table 4, an artificial gene encoding a polypeptide arranged in the order of the target KIR-derived signal peptide (see Table 5), Flag tag (SEQ ID NO: 2), GSGS (SEQ ID NO: 3), and the target receptor peptide (see Table 4) from the N-terminal side was inserted into pLVSINEF1α according to the method described in the above [Preparation of lentivirus] section to prepare lentivirus. In addition, for KIR2DL2*001, without using a Flag tag or a GS linker, an artificial gene encoding a polypeptide arranged in the order of the target KIR-derived signal peptide (see Table 5), the target receptor peptide (SEQ ID NO: 57), the self-cleaving peptide T2A (SEQ ID NO: 20), and the NGFR peptide (SEQ ID NO: 58) was inserted into pLVSIN EF1α according to the method described in the above [Preparation of lentivirus] section to prepare lentivirus.

[0672] It should be noted that each KIR is selected from variants with high expression rates according to the Allele Frequency Net Database. * 003 as KIR2DL2.

[0673] [Table 5]

[0674]

[0675] RetroNectin (T100B, Takara Bio Co., Ltd.) was diluted to 20-100 μg / mL using PBS (Phosphate Buffered Saline) and added to a non-treatment dish. After standing at room temperature for 2 hours or at 4°C overnight, the plate was washed with PBS to prepare a coated plate.

[0676] Use Myelocult H5100 medium to suspend NK92 cells to a concentration of 1-2 × 10 6cells / mL. The lentiviral concentrate and NK cell suspension into which inhibitory KIR and active KIR genes were introduced were mixed using a RetroNectin-coated culture plate and centrifuged at 1000×g at 32°C for 1 hour. Thereafter, culture was performed at 37°C and 5% CO2. After 3 days of culture, the full amount of culture medium was replaced every 1 to 3 days using Myelocult H5100 culture medium. After 1 week of culture, APC-tagged anti-Flag antibody or APC-tagged anti-NGFR antibody, and anti-APC microbeads (produced by Miltenyi Biotec, 130-090-855) were used to select only positive cells. In addition, the expression of KIR introduced using commercially available anti-KIR2DS1 antibody, anti-KIR2DL2 / 3 / 2DS2 antibody, and anti-KIR2DS4 antibody was confirmed. As needed, the recovered cells were suspended in CELLBANKER1 (produced by Takara Bio Co., Ltd., CB011) to a concentration of 0.5 to 1×10 7 cells / mL, and stored frozen at -80°C or -150°C. When using frozen cells for experiments, they were thawed and cultured in NK cell culture medium before use.

[0677] [Flow cytometry and added antibodies]

[0678] In the flow cytometry method for confirming the expression of various cell surface proteins, the antibodies shown in Table 6 were used.

[0679] [Table 6]

[0680]

[0681] The fluorescent-labeled antibody was suspended in autoMACS electrophoresis buffer-MACS separation buffer (manufactured by Miltenyi Biotec, 130-091-221) to a concentration of 1 to 10 μg / mL, and then added to the cells and reacted at 4° C. for 30 minutes.

[0682] The cells after antibody staining were suspended in FACS buffer containing 1-10 μg / mL 7-aminoactinomycin D (7-AAD, produced by WAKO, 016-25241) and measured using a flow cytometer (produced by Miltenyi biotec, MACSQuant Analyzer10, 130-096-343). After extraction as an FCS file, FLOW JO software (produced by FLOWJO LLC, VER.10.7.1) was used to select cell components in the FSC / SSC graph, and the 7-aminoactinomycin D negative / target positive cell group was counted as live cells.

[0683] Example 1: Lirelumab inhibits the cytotoxic activity of NK92 cell line expressing KIR2DL2 against K562 cells

[0684] Through the recognition of HLA by KIR2DL2, an evaluation system for the reduction of cell killing activity of NK cell lines expressing KIR2DL2 was constructed, and then the presence of agonist activity of cell lines expressing KIR2DL2 was evaluated when existing anti-KIR antibodies were added in a soluble form.

[0685] [Gene introduction and preparation into K-562 cells]

[0686] K562 cells were purchased from ATCC (CCL-243 TM ) were obtained and used as target cells for the initial evaluation system. 10 (v / v)% FCS and streptomycin / penicillin were added to RPMI 1640 medium and GlutaMAX (Life Technologies, 61870036) (hereinafter referred to as "RPMI medium") for culture.

[0687] In order to establish a K562 cell line expressing luciferase (Luc-K562 cell line), an artificial gene encoding a polypeptide of firefly luciferase (SEQ ID NO: 17) was first introduced into the K562 cell line via CRISPR / CAS9.

[0688] In order to establish a K562 cell line expressing human HLA-Cw3 ​​(HLA-Cw3-Luc-K562 cell line), an artificial gene encoding a polypeptide to which luciferase (SEQ ID NO: 17), a self-cleaving peptide T2A (SEQ ID NO: 20), a signal peptide derived from human HLA-Cw3 ​​(SEQ ID NO: 21), and a human HLA-Cw3 ​​peptide (SEQ ID NO: 22) were linked in sequence from the N-terminal side was introduced into the K562 cell line via CRISPR / CAS9.

[0689] After cloning of the Luc-K562 cell line, luciferase activity was confirmed and clones were selected. For the HLA-Cw3-K562 cell line, expression was confirmed by anti-HLA-ABC, and after cloning, clones were selected based on luciferase activity and anti-HLA-ABC staining intensity.

[0690] If necessary, the recovered cells were suspended in CELLBANKER1 (manufactured by Takara Bio Co., Ltd., CB011) to a concentration of 0.5 to 1×10 7 cells / mL, and stored frozen at -80°C or -150°C. When frozen cells were used in experiments, they were thawed and cultured in RPMI medium before use.

[0691] [Evaluation of the cytotoxic activity of NK92 cells expressing KIR2DL2 against K562 cells]

[0692] This example uses KIR2DL2 * 001 as KIR2DL2. One day before evaluating the cell killing activity, KIR2DL2-NK92 cells stimulated with 500 IU / mL IL-2 were cultured at 5 × 10 3 Add an equal amount (i.e. 5×10 3 ) Luc-K562 cell line or HLA-Cw3-Luc-K562 cell line was added to separate wells and then cultured. The anti-KIR antibody levelumab (1-7F9, human IgG4) was diluted 9 times from 2μg / mL and added to the mixed wells of KIR2DL2-NK92 cell line and Luc-K562 cell line in 4 stages. In addition, human IgG4 (GeneTex, GTX63305) was treated in the same way at 2μg / mL as a control for levelumab. Basal medium was used for all cultures. After 3 hours, regarding cell killing activity, the luciferase activity in the K562 cell line was expressed as the number of residual target cells when the number of target cells in the wells without KIR2DL2-NK92 cells added was set to 100%.

[0693] The results are shown in Figure 1-1 . The KIR2DL2-NK92 cell line showed cytotoxic activity against the K562 cell line, but the cytotoxic activity against the HLA-Cw3-K562 cell line was weakened. This is because KIR2DL2 that recognizes HLA-Cw3 ​​inputs inhibitory signals into the KIR2DL2-NK92 cell line, thereby inhibiting the cytotoxic activity, thereby constructing an evaluation system that represents the loss of self of KIR2DL2. In the system of KIR2DL2-NK92 cell line against K562 cell line, after adding lirelumab to the culture medium, the cytotoxic activity of KIR2DL2 cell line against K562 cell line was reduced in a concentration-dependent manner, and was inhibited by about 50% at 2μg / mL. It can be seen that lirelumab exhibits agonist activity against inhibitory KIR2DL2 in the cytotoxic activity of KIR2DL2-NK92 cell line against K562 cell line. In addition, the control antibody human IgG4 did not change at 2μg / mL.

[0694] On the other hand, in the evaluation of the cytotoxic activity of the KIR2DL2-NK92 cell line against the HLA-Cw3-K562 cell line, when lirelumab was added at 2 μg / mL, its cytotoxic activity was enhanced. The results are shown in Figure 1-2 In addition, the control antibody human IgG4 showed no change at 2 μg / mL.

[0695] This activity is the same as the related events of lirelumab (PL4: WO2006 / 003179_A2). Lirelumab relieves the inhibitory signal emitted by KIR ligands, enhances the activation of NK cells, and thus inhibits the agonist effect of HLA ligands on inhibitory KIR.

[0696] In summary, in the presence of an agonist of KIR2DL2 called HLA-Cw3, the anti-KIR antibody lirelumab deactivates the ligand and enhances the cytotoxic activity of NK cells. On the other hand, in the absence of a ligand for KIR2DL2, lirelumab functions as a partial agonist that exhibits agonist activity against inhibitory KIR.

[0697] Example 2: Inhibitory KIR-dependent inhibition of the activity of NK cell lines by lirelumab and Pan2D in scFv expression

[0698] When an agonist binder of an inhibitory receptor is used as a secretory protein as a xenotransplantation technique, it may affect the immune system around the target tissue or even in the body. Therefore, the agonist binder needs to be expressed on the surface of the cells to be transplanted in order to render the cells that are to escape immunity immune-tolerant alone, thereby achieving local immune tolerance.

[0699] Soluble lirelumab inhibited the cytotoxic activity of KIR2DL2-NK92 cells against K562 cells, so lirelumab or Pan2D-derived scFv was expressed as a membrane protein on the surface of the K562 cell line to evaluate whether it could inhibit the cytotoxic activity of KIR2DL2-NK92 cells.

[0700] [Introduction and preparation of scFv gene into K-562 cells]

[0701] The sequences of lirelumab and Pan2D (NKVSF1) are cited from WO2006 / 003179A2 (refer to Table 6 for the SEQ ID NO of each variable region).

[0702] In order to establish a K562 cell line expressing levelumab-scFv (levelumab scFv-Luc-K562 cell line) and a cell line expressing Pan2D-scFv (Pan2D-scFv-Luc-K562 cell line), first, a sequence encoding a signal peptide derived from human CD8 (SEQ ID NO: 23), a Flag tag (SEQ ID NO: 2), a linker (GSG), a variable region (VH) in Table 7 below, a GS (G4S) linker (SEQ ID NO: 25), a variable region (VL) in Table 7 below, a hinge region (66 amino acids) sequence derived from CD8 (SEQ ID NO: 24), a cell membrane domain derived from CD8 (SEQ ID NO: 27), an intracellular domain derived from CD8 (SEQ ID NO: 28), a self-cleaving peptide T2A (SEQ ID NO: 20), and a firefly luciferase (SEQ ID NO: 21) were linked in sequence from the N-terminus. The artificial gene of the polypeptide of NO:17) (hereinafter referred to as "membrane-type scFv expression configuration") was inserted into pLVSIN EF1α according to the method described in the above [Preparation of lentivirus] to prepare lentivirus. The schematic diagram of the membrane-type scFv conjugate is shown in Fig. 9 .

[0703] [Table 7]

[0704]

[0705] * The CDR1, CDR2, and CDR3 regions in the table are represented by the range of amino acid numbers in the amino acid sequence of each variable region. The amino acid numbers are determined according to the IMGT definition.

[0706] RetroNectin (T100B, Takara Bio Co., Ltd.) was diluted to 20-100 μg / mL using PBS (Phosphate Buffered Saline) and added to a non-treatment dish. After standing at room temperature for 2 hours or at 4°C overnight, the plate was washed with PBS to prepare a coated plate.

[0707] Use RPMI medium to suspend K562 cells to a concentration of 1-2 × 10 6Cells / mL, the lentiviral concentrate carrying the levelumab scFv-Luc or Pan2D scFv-Luc gene and cells were mixed with RetroNectin-coated culture plates and centrifuged at 1000×g for 1 hour. After 3 days of culture, the full medium was replaced every 1 to 3 days with RPMI medium. After 1 week of culture, APC-tagged anti-Flag antibody and anti-APC microbeads (Miltenyi Biotec, 130-090-855) were used to select only positive cells.

[0708] If necessary, the recovered cells were suspended in CELLBANKER1 (manufactured by Takara Bio Co., Ltd., CB011) to a concentration of 0.5 to 1×10 7 cells / mL, and stored frozen at -80°C or -150°C. When frozen cells were used in experiments, they were thawed and cultured in RPMI medium before use.

[0709] [Evaluation of the cytotoxic activity of levelumab-scFv / Pan2D-scFv-K562 against NK92 cells expressing KIR2DL2]

[0710] This example uses KIR2DL2 * 001 as KIR2DL2. One day before evaluating the cell killing activity, the KIR2DL2-NK92 cell line stimulated with 500 IU / mL IL-2 was added to a 96-well plate, and the Luc-K562 cell line, HLA-Cw3-Luc-K562 cell line, lirelumab-scFv-K562 cell line and Pan2D-scFv-K562 were added to separate wells to make the ratio of NK92 and K562 cells reach 3:1 or 1:1, and then cultured. The basal medium was used for all cultures. After 2 to 5 hours, the activity of luciferase expressed by the K562 cell line was measured, and the luciferase activity in the wells to which KIR2DL2-NK92 cells were added was expressed as the number of residual target cells when the luciferase activity in the wells to which KIR2DL2-NK92 cells were not added was set to 100%. The cell killing activity was expressed as the value obtained by subtracting the luciferase activity in the wells to which KIR2DL2-NK92 cells were added from the luciferase activity in the wells to which KIR2DL2-NK92 cells were not added. The luciferase activity was measured using the ONE-Glo(TM) Luciferase Assay System (manufactured by PROMEGA, E6120).

[0711] In order to determine the KIR-dependent activity and calculate the ratio of the activity dependent on the binder, the activity of the Luc-K-562 strain was set as 1, and the ratio of the cell-killing activity was calculated according to the following formula.

[0712] KIR-dependent cell killing activity ratio = A KIR-NK92 / A NK92

[0713] [A = Cell killing activity in each NK cell % = (luciferase activity of target cells without effector cells added - luciferase activity of target cells with effector cells added) / luciferase activity of target cells without effector cells added × 100]

[0714] The results are shown in Figure 2 The KIR2DL2-NK92 cell line showed cytotoxic activity against the K562 cell line, but the cytotoxic activity against the HLA-Cw3-K562 cell line was weakened. In addition, the cytotoxic activity of lirelumab-scFv-K562 and Pan2D-scFv-K562 was weakened compared with the K562 cell line.

[0715] In summary, when the anti-KIR antibodies lirelumab and Pan2D were expressed as scFv-membrane proteins, the cell killing activity of the KIR2DL2-NK92 cell line was inhibited, showing agonist activity against KIR2DL2.

[0716] Example 3: Lirelumab and Pan2D induce cytotoxic activity of NK cells by activating KIR

[0717] We evaluated whether, in addition to KIR2DL2, other inhibitory and activating KIRs that showed cross-reactivity to lirelumab and Pan2D would also exhibit agonist activity.

[0718] Under the same evaluation conditions as in Example 2, the sensitivity of NK92 cell lines expressing various KIRs to the cell killing activity of levofloxacin or Pan2D-scFv-K562 was evaluated. In order to determine the KIR-dependent activity, the activity ratio was calculated by the following formula. In addition, in order to calculate the activity ratio of the conjugate-dependent activity, the activity of the Luc-K-562 strain was set to 1 and the ratio was calculated. It should be noted that this example uses KIR2DL2 * 001 as KIR2DL2.

[0719] KIR-dependent cell killing activity ratio = A KIR-NK92 / A NK92

[0720] [A = Cell killing activity in each NK cell % = (luciferase activity of target cells without effector cells added - luciferase activity of target cells with effector cells added) / luciferase activity of target cells without effector cells added × 100]

[0721] The results are shown in Figure 3 . The levelumab-scFv-K562 cell line or the Pan2D-scFv-K562 cell line showed an inhibitory effect on the cell killing effect of KIR2DL2-NK92, and showed agonist activity against inhibitory KIR. The result is the same as in Example 2. On the other hand, levelumab-scFv-K562 enhanced the activity of the KIR2DS1-NK92 cell line and the KIR2DS2-NK92 cell line, and thus it was confirmed that it exhibited agonist activity against active KIR2DS1 / 2. In addition, Pan2D-scFv-K562 enhanced the activity of the KIR2DS1-NK92 cell line, the KIR2DS2-NK92 cell line, and the KIR2DS4-NK92 cell line, and thus it was confirmed that it exhibited agonist activity against active KIR2DS1 / 2 / 4.

[0722] The binding specificity of lirelumab and Pan2D is reported in WO2006 / 003179A2. Lirelumab binds to KIR2DS1 and KIR2DS2, and Pan2D binds to KIR2DS1, KIR2DS2, and KIR2DS4. That is, it was confirmed that antibodies against the known inhibitory KIR2DL2 / 3 bind to the corresponding active KIR and enhance the activity of NK cell lines expressing active KIR.

[0723] Example 4: Obtaining KIR2DL2 and / or KIR2DL3 specific binders by phage panning

[0724] Based on the results of Example 3, in order to obtain a novel binder that does not activate active receptors but exhibits agonist activity on inhibitory receptors, an inhibitory KIR-specific binder that does not bind to active KIR was obtained by the following method.

[0725] [Obtaining inhibitory KIR-specific binders by phage panning]

[0726] Phage panning was performed to obtain specific binders that bind to inhibitory KIR2DL2 and / or KIR2DL3 but not to activating KIR2DS1, 2, and 4 using a human antibody phage library.

[0727] In order to implement phage panning, soluble KIRs shown in Table 8 were prepared. For soluble KIR, recombinant proteins were prepared in which a signal peptide derived from IL-2 (SEQ ID NO: 91), each KIR protein extracellular domain described in Table 7, a GGGS linker (SEQ ID NO: 97), an IgG Fc domain (SEQ ID NO: 98), a GAA linker, and an Avi tag (SEQ ID NO: 99) were linked from the N-terminal side. Phage panning used substances that were or were not biotinylated for the soluble KIRs corresponding to each KIR shown in Table 7. In addition, when implementing ELISA, recombinant human KIR2DL3 / CD158b2 Fc chimeric protein (produced by R&D Systems, 2014-KR-050) was used for soluble KIR2DL3.

[0728] [Table 8]

[0729] Recombinant protein name Extracellular domain KIR2DL2 Amino acids 1 to 224 of SEQ ID NO:6 KIR2DS1 Amino acids 1 to 224 of SEQ ID NO:10 KIR2DS2 Amino acids 1 to 224 of SEQ ID NO:12 KIR2DS4 Amino acids 1 to 224 of SEQ ID NO:14

[0730] In this phage panning, positive selection refers to the step of allowing the biotinylated recombinant protein to react with the scFv-expressing phage library solution, and using streptavidin or Neutravidin magnetic beads to concentrate the scFv-expressing phages that bind to the biotinylated recombinant protein. In this phage panning, depletion refers to the step of allowing the biotinylated recombinant protein to react with the scFv-expressing phage library solution, and using streptavidin or Neutravidin magnetic beads to exclude the scFv-expressing phages that bind to the biotinylated recombinant protein. In this phage panning, deselection refers to the operation step in which, during the reaction of the biotinylated recombinant protein and the scFv-expressing phage library solution in the positive selection, non-biotinylated recombinant protein is added, and when the scFv-expressing phage bound to the biotinylated recombinant protein is concentrated using streptavidin or Neutravidin magnetic beads, the scFv-expressing phage that does not bind to the added non-biotinylated recombinant protein but specifically binds to the biotinylated recombinant protein is easily concentrated.

[0731] In each round of phage panning, positive selection for inhibitory KIR, depletion or exclusion of active KIR, or both are performed. After two rounds, a polyclonal phage library enriched for KIR2DL2-specific binders is obtained.

[0732] ELISA was performed using human KIR2DL2 (22-245)-Fc-Avi (the above-mentioned soluble KIR2DL2), and scFv-expressing phages that bind to KIR2DL2 were selected from the polyclonal phage library. The scFv sequence was transferred from the phagemid of the selected scFv-expressing phage to a recombinant protein expression vector for Escherichia coli, introduced into Escherichia coli, and each scFv was prepared from the culture supernatant. For each scFv, ELISA was performed using the soluble KIR or recombinant human KIR2DL3 / CD158b2 Fc chimeric protein (produced by R&D Systems, 2014-KIR-050) described in Table 7 to obtain information on the binding of each scFv to each KIR.

[0733] The variable region of the scFv that binds to KIR2DL2 and / or KIR2DL3 but not to KIR2DS1, KIR2DS2, and KIR2DS4 was introduced into the membrane-type scFv expression configuration described in [Introduction and preparation of scFv gene into K-562 cells] and inserted into pLVSIN EF1α. Lentivirus was prepared according to the method described in [Preparation of lentivirus] using pLVSIN EF1α into which each membrane-type scFv was introduced. K562 cell lines expressing each scFv as a membrane-type scFv were prepared according to the method described in [Introduction and preparation of scFv gene into K-562 cells] using the prepared lentivirus.

[0734] For this 5×10 4 0.1 μM of the Fc-added recombinant proteins shown in Table 7 or recombinant human KIR2DL3 / CD158b2 Fc chimeric protein (manufactured by R&D Systems, 2014-KIR-050) was added to each target cell and reacted at 4°C for 90 minutes.

[0735] The target cells after the reaction were washed with PBS, and APC-tagged anti-human IgG Fc antibody (manufactured by BIOLEGEND, 409306) was added and reacted at 4°C for 30 minutes. Washed twice with PBS, and the binding was confirmed by flow cytometry. The binding to the K562 cell line (the median value of the fluorescence intensity derived from APC) was set to 1, and the ratio relative to this value was calculated.

[0736] The results are shown in Figure 4 .

[0737] In contrast to lirelumab and Pan2D, which exhibited binding to recombinant KIR2DS1, 2, or 4, all 12 binders inhibited binding to recombinant KIR2DS1, 2, or 4, and exhibited binding to recombinant KIR2DL2 and KIR2DL3.

[0738] Example 5: Evaluation of agonist activity of KIR2DL2 and / or KIR2DL3 specific binders (initial evaluation)

[0739] Regarding the specific binders in Example 4, agonist activity was evaluated.

[0740] Regarding these conjugates, genes encoding other polypeptides, in which membrane-type scFv and Lucoferase were linked via the following constructs, were inserted into pLVSINEF1α according to the method described in the section [Preparation of lentivirus] to prepare lentivirus.

[0741] The SEQ ID NOs of the amino acid sequences of the heavy chain variable region and the light chain variable region of each binder are shown in the following Table 8. In order to express a membrane protein having each binder in the extracellular domain in the target cell, an artificial gene encoding a polypeptide in which a self-cleavage peptide T2A (SEQ ID NO: 20) and a firefly luciferase (SEQ ID NO: 17) are sequentially linked to a membrane-type scFv composed of a signal peptide (SEQ ID NO: 23) derived from human CD8, a Flag tag (SEQ ID NO: 2), a linker (GSG), a variable region (VH) in the following Table 8, a GS (G4S) linker (SEQ ID NO: 25), a variable region (VL) in the following Table 8, a hinge region sequence derived from CD8 (SEQ ID NO: 26), a cell membrane domain derived from CD8 (SEQ ID NO: 27), and an intracellular domain derived from CD8 (SEQ ID NO: 28) was inserted into pLVSIN EF1α according to the method described in the above [Preparation of lentivirus] to prepare a lentivirus. In Table 9, "a2DL2 / 3" means binding to 2DL2 and / or 2DL3.

[0742] [Table 9]

[0743]

[0744]

[0745] * The CDR1, CDR2, and CDR3 regions in the table are represented by the range of amino acid numbers in the amino acid sequence of each variable region. The amino acid numbers are determined according to the IMGT definition.

[0746] The sensitivity of various KIR-expressing NK92 cell lines to the cell killing activity of the membrane-type scFv-K562 body of each binder was evaluated under the same evaluation conditions as in Examples 2 and 4. In order to determine the activity dependent on the binder, the activity ratio was calculated by the following formula. In addition, when calculating the activity ratio dependent on the binder, the activity of the Luc-K-562 strain was set to 1 and the ratio was calculated.

[0747] KIR-dependent cell killing activity ratio = A 结合物-scFv-K562 / A K562

[0748] [A = Cell killing activity against each K562 cell % = (luciferase activity of target cells without effector cells added - luciferase activity of target cells with effector cells added) / luciferase activity of target cells without effector cells added × 100]

[0749] The results are shown in Figure 5 All the binders showed agonist activity against KIR2DL2 and KIR2DL3-NK92 cell lines and did not activate KIR2DS1 / 2 / 4-NK92 cell lines as did levofloxacin and Pan2D. In summary, the desired curves, i.e. inhibitory KIR-specific agonist binders, were obtained. These KIRs are KIRs with high frequency expression in the Allele Frequency Network database, so the public can expect their specificity and agonist activity.

[0750] Example 6: Evaluation of agonist activity of KIR2DL2 and / or KIR2DL3 specific binders using primary NK cells

[0751] Currently, the NK92 cell line is used as the effector cell, and inhibitory and active KIRs are introduced. For KIRs expressed on NK cells existing in vivo, it is necessary to examine whether the binders identified in Example 4 exhibit agonist activity, so agonist activity was evaluated using NK cells collected from human peripheral blood mononuclear cells (PBMC).

[0752] NK cells recognize their own HLA type (C1, C2, Bw4) and have the property of "missing self" to show cytotoxic activity against target cells. For example, NK cells collected from HLA-C1 / C1 homozygous people will recognize C1 as themselves, so if the target cells do not have C1 type HLA-C, the signal will enter the NK cells through inhibitory receptors, thereby showing cytotoxic activity. On the other hand, if C1 type is present in the target cells, cytotoxic activity will not be shown; in addition, if an agonist binder that replaces C1 type is expressed, the cytotoxic activity will be reduced.

[0753] The same conclusion applies to HLA-C2 homozygous NK cells and HLA-Bw4-positive NK cells. The inhibitory KIRs that function differently are different. In HLA-C1 homozygous cells, KIR2DL2 and KIR2DL3 function as inhibitory receptors that lose their own self, in HLA-C2 homozygous cells, KIR2DL1 functions as an inhibitory receptor that loses its own self, and in HLA-Bw4-positive cells, KIR3DL1 functions as an inhibitory receptor that loses its own self. Therefore, agonists targeting these KIRs can be evaluated.

[0754] [Preparation of PBMC-derived NK cells]

[0755] As effector cells, two types of HLA-C1 / C1 / Bw4 negative PBMCs (denoted as PBMC-A and PBMC-B) were prepared. From the haplotype of KIR, it was found that PBMC-A had the genes of KIR2DL2, KIR2DL3, KIR2DS1, and KIR2DS2, and PBMC-B had the gene of KIR2DL3.

[0756] Anti-CD16 antibody (manufactured by Biolegend, 302050) was diluted to 5 μg / mL using PBS and added to a non-treatment dish. After standing at room temperature for 2 hours or at 4°C overnight, the culture medium was washed with PBS and suspended human peripheral blood mononuclear cells (PBMC, manufactured by Cellular Technology Limited, CTL-UP1) were added to MyeloCultH5100 culture medium (hereinafter referred to as "NK culture medium") supplemented with 1000U / mL IL-2 and 100ng / mL IL-18 (manufactured by R&D systems, 9124-IL). After 7 to 10 days, the cells were recovered and CD56-positive cells (NK cells) were purified using CD56 microbeads (manufactured by Miltenyi Biotec, 130-050-401). Afterwards, the cells were cultured again with Myelocult supplemented with L-2 / 18.

[0757] [Preparation of T cells expressing target genes]

[0758] As target cells, one type each of HLA-C1 / C1 / Bw4-negative PBMC (denoted as PBMC-A, the same as effector cells) and HLA-C2 / C2 / Bw4-negative PBMC (denoted as PBMC-C) were prepared.

[0759] Anti-CD3 antibody (manufactured by Biolegend, 317347) was diluted to 5-10 μg / mL using PBS (Phosphate Buffered Saline), and RetroNectin (manufactured by Takara Bio, Ltd., T100B) was diluted to 20-100 μg / mL, and added to a non-treatment dish. After standing at room temperature for 2 hours or at 4°C overnight, the plate was washed with PBS to prepare a coated plate (hereinafter referred to as "CD3 Ab / RetroNectin coated plate").

[0760] Lentivirus solutions for expressing membrane-bound scFv of the 10 binders used in Example 5 were prepared. In addition, as a control, an artificial gene containing a polypeptide linked to human CD19 (SEQ ID NO: 53) and a self-cleaving peptide T2A (SEQ ID NO: 20) and a polypeptide of firefly luciferase (SEQ ID NO: 17) was introduced into a lentivirus for preparation.

[0761] The virus concentrate was added to the culture plate and centrifuged at 2000×g for 2 hours to prepare a virus-bound culture plate. Human peripheral blood mononuclear cells (PBMC, Cellular Technology Limited, CTL-UP1) were suspended to 1 to 2×10 using a basal medium (hereinafter referred to as “IL-2 medium”) supplemented with IL-2 (Nipro, 87-890; or Kyowa Pharmaceutical Co., Ltd., 58697900) to a final concentration of 100 U / mL. 6 cells / mL, added to the virus binding culture plate, and centrifuged at 300×g for 3-5 minutes.

[0762] Among them, PBMC-A was infected with human CD19-luciferase, and PBMC-C was infected with human CD19-luciferase or 10 kinds of membrane-type scFv-luciferase lentivirus. After 3 days of culture, the full amount of culture medium was replaced every 1 to 3 days using IL-2 medium. After 1 week of culture, APC-tagged anti-CD19 antibody or APC-tagged anti-Flag antibody and anti-APC microbeads (Miltenyi Biotec, 130-090-855) were used for cell screening to concentrate positive cells.

[0763] [Evaluation of cytotoxic activity using PBMC-derived NK cells]

[0764] 1×10 5 PBMC-NK cells were added to a 96-well plate to evaluate their cell killing activity. 1 / 10 of the PBMC-NK cells (i.e., 1×10 4The target gene-expressing T cells (in the wells without PBMC-NK cells) were added to separate wells and cultured. The basal medium was used for all cultures. After 4 hours, the luciferase activity in the target gene-expressing T cells was expressed as the number of residual target cells when the number of target cells in the wells without PBMC-NK cells was set to 100%.

[0765] The results are shown in Figure 6 .

[0766] First, since the effector cells are C1 / C1 / Bw4 negative, when the target cells are C1 / C1 / Bw4 negative and C2 / C2 / Bw4 negative, they show significant cell-killing activity against C2 / C2 / Bw4 negative T cells. This is "missing self".

[0767] On the other hand, when the KIR2DL2 / 3 specific binding substance was expressed in C2 / C2 / Bw4-negative T cells, the cell killing activity was confirmed to be reduced compared with the case where the C2 / C2 / Bw4-negative T cells expressed CD19-luciferase. Regarding this result, the same trend was confirmed in PBMC-A-derived NK cells and PBMC-B-derived NK cells, which confirmed that the KIR2DL2 / 3 specific binding substance obtained in Example 5 also exhibited agonist activity on primary NK cells.

[0768] Example 7: Binding evaluation of KIR2DL2 and / or KIR2DL3 specific binders

[0769] The binding properties of the conjugates confirmed to have agonist activity in Example 6 to the inhibitory molecules and active molecules on the surface of NK cells were evaluated. As a control, Pan2D was placed.

[0770] For 1×10 4 K562 cells expressing membrane-type a2DL2 / 3-DS4 scFv or membrane-type a2DL2 / 3-DS10scFv prepared in Example 5, or K562 cells expressing membrane-type scFv of Pan2D prepared in Example 3, were added with 0.1 μM of recombinant protein with Fc or His body as a tag, and reacted at 4°C for 90 minutes. Wash with PBS, add APC-tagged anti-human IgG Fc antibody (produced by BIOLEGEND, 409306) or APC-tagged anti-His tag antibody (produced by BIOLEGEND, 362605), and reacted at 4°C for 30 minutes. Wash twice with PBS, and confirm whether it has binding by flow cytometry. The binding to the K562 cell line (the median of the fluorescence intensity derived from APC) is set to 1, and the ratio relative to this value is calculated.

[0771] The recombinant proteins shown in Table 10 were used. The recombinant proteins described as "In house" were the same as those described in Table 7. As Fc-His-Avi, a recombinant KIR protein was prepared in which an IgG Fc domain (SEQ ID NO: 98), a GAA linker, and an Avi tag (SEQ ID NO: 99) were linked from the N-terminal side.

[0772] [Table 10]

[0773]

[0774]

[0775] The results are shown in Figure 7 .

[0776] The binding conditions with Pan2D and KIR2DS1, 2, and 4 were different. The conjugates (a2DL2 / 3-DS4) and (a2DL2 / 3-DS10) not only showed no binding to KIR2DS1, 2, and 4, but also did not show binding to other inhibitory receptors and active receptors.

[0777] The above indicated that the conjugate did not bind to other NK cell receptors and exhibit effects, but instead bound to KIR2DL2 and / or KIR2DL3 and inhibited NK cell activation.

[0778] Example 8: Evaluation of agonist activity of KIR2DL1 binders and KIR3DL1 binders

[0779] It was assessed whether binders showing binding to KIR2DL2 and KIR2DL3 would exhibit agonist activity against NK cell lines expressing each receptor.

[0780] [Gene introduction and preparation into K-562 cells]

[0781] In order to establish a K562 cell line expressing human HLA-C2 (HLA-Cw4-Luc-K562 cell line), first, an artificial gene encoding a polypeptide linked in the order of the signal peptide in Table 11, the Flag tag (SEQ ID NO: 2) and the HLA peptide in Table 10 from the N-terminal side, and then an artificial gene encoding a polypeptide of luciferase (SEQ ID NO: 17) downstream of the IRES (Internal ribosome entry site) sequence was inserted into pLVSIN EF1α according to the method described in the section [Preparation of lentivirus], thereby preparing lentivirus.

[0782] [Table 11]

[0783]

[0784] The anti-3DL1 antibody was cited from WO2018 / 148223A1, and the polypeptide was connected by the following construct and inserted into pLVSIN EF1α according to the method described in [Preparation of lentivirus] to prepare lentivirus.

[0785] The SEQ ID NOs of the amino acid sequences of the heavy chain variable region and the light chain variable region of each binder are shown in Table 12. In order to express a membrane protein having each binder in the extracellular domain in the target cell, an artificial gene encoding a polypeptide in which a self-cleavage peptide T2A (SEQ ID NO: 20) and firefly luciferase (SEQ ID NO: 17) were sequentially linked to the C-terminus of a membrane-type scFv consisting of a signal peptide (SEQ ID NO: 23) derived from human CD8, a Flag tag (SEQ ID NO: 2), a linker (GSG), a variable region (VL) in Table 12, a GS (G4S) linker (SEQ ID NO: 25), a variable region (VH) in Table 12, a hinge region (66 amino acids) sequence derived from CD8 (SEQ ID NO: 24), a cell membrane domain (SEQ ID NO: 27) derived from CD8, and an intracellular domain (SEQ ID NO: 28) derived from CD8 was inserted into pLVSIN EF1α according to the method described in the above [Preparation of lentivirus] to prepare lentivirus.

[0786] [Table 12]

[0787]

[0788] * The CDR1, CDR2, and CDR3 regions in the table are represented by the range of amino acid numbers in the amino acid sequence of each variable region. The amino acid numbers are determined according to the IMGT definition.

[0789] RetroNectin (T100B, Takara Bio Co., Ltd.) was diluted to 20-100 μg / mL using PBS (Phosphate Buffered Saline) and added to a non-treatment dish. After standing at room temperature for 2 hours or at 4°C overnight, the plate was washed with PBS to prepare a coated plate.

[0790] Use PRMI medium to suspend K562 cells to a volume of 1-2 × 10 6Cells / mL. Mix the lentiviral concentrate carrying HLA or membrane-type scFv of the binder and the NK cell suspension using RetroNectin-coated culture plates and centrifuge at 1000×g at 32°C for 1 hour. After that, culture at 37°C and 5% CO2. After 3 days of culture, replace the full medium every 1 to 3 days using PRMI medium. After 1 week of culture, use APC-tagged anti-Flag antibody and anti-APC microbeads (produced by Miltenyi Biotec, 130-090-855) to select only positive cells.

[0791] If necessary, the recovered cells were suspended in CELLBANKER1 (manufactured by Takara Bio Co., Ltd., CB011) to a concentration of 0.5 to 1×10 7 cells / mL, and stored frozen at -80°C or -150°C. When frozen cells were used in experiments, they were thawed and cultured in RPMI medium before use.

[0792] In addition, regarding the anti-2DL1 antibody, Pan2D showing binding property to KIR2DL1 was utilized, and the Pan2D-scFv-K562 cell line of Example 2 was used.

[0793] Under the same evaluation conditions as in Example 2 and Example 4, the sensitivity of various KIR-expressing NK92 cell lines to the cell killing activity of the membrane-bound scFv-K562 of each binder was evaluated, and the activity was calculated by the following formula.

[0794] % of cell killing activity against each K562 cell = (luciferase activity of target cells without effector cells added - luciferase activity of target cells with effector cells added) / luciferase activity of target cells without effector cells added × 100

[0795] The results are shown in Figure 8 .

[0796] HLA-Cw4, as a positive control, showed agonist activity against KIR2DL1-NK92 cell line. In this system, Pan2D showed agonist activity against KIR2DL1. In addition, a3DL1-DS1 showed agonist activity against KIR3DL1.

[0797] In summary, binders that exhibit agonist activity against KIR2DL1, as well as binders that exhibit agonist activity against KIR3DL1 were identified.

[0798] Example 9: Evaluation of the effect of agonist activity of KIR2DL2 and / or KIR2DL3 specific binders on the efficacy of CAR-T cells using primary NK cells

[0799] Using PBMC-derived NK cells as primary NK cells, the CAR-T cells prepared from the same PBMCs as the PBMC-derived NK cells, or the CAR-T cells prepared from PBMCs of different donor origin, were evaluated for their cytotoxic activity against target cells in a state of co-culture with PBMC-derived NK cells.

[0800] In the presence of primary NK cells, CAR-T cells from different donors that do not express HLA that matches the inhibitory KIR of primary NK cells are recognized by primary NK cells as lost self and are attacked.

[0801] In Example 6, it was confirmed that the conjugate identified in Example 4 also exhibited agonist activity on primary NK cells, thereby inhibiting the cell killing activity of primary NK cells. In order to investigate whether inhibiting the cell killing activity of primary NK cells helps maintain the cell killing activity of CAR-T cells, PBMC-derived NK cells were used as primary NK cells, and PBMC-derived NK cells, CAR-T cells from donors different from PBMC-derived NK cells, or CAR-T cells from donors different from PBMC-derived NK cells expressing the conjugate identified in Example 4, and CAR-T cells expressing cell killing activity through CAR were mixed to evaluate the cell killing activity of CAR-T cells against target cells.

[0802] The PBMC-A-derived NK cells prepared according to the method described in [Preparation of PBMC-derived NK cells] were suspended in CELLBANKER1 (manufactured by Takara Bio Co., Ltd., CB011), frozen at -80°C or -150°C, thawed the day before the experiment, and cultured in MyeloCult H5100 medium supplemented with IL-2 and IL-18. The culture medium of PBMC-A-derived NK cells was replaced with MyeloCult H5100 medium without IL-2 and IL-18 the day before the experiment, and cultured.

[0803] [Preparation of T cells expressing CAR and KIR2DL2 / specific binding substances]

[0804] To prepare CAR-expressing T cells, HLA-C2 / C2 / Bw4-negative PBMCs (PBMC-C) were used.

[0805] In order to express anti-CD19 CAR (T-cell clones can be rendered specific for CD19: toward the selective augmentation of the graft-versus-B-lineage leukemia effect. LJ Cooper et al., Blood 2003 Feb 15; 101 (4): 1637-44. CAR) (hereinafter referred to as "CD19CAR"), a CD8-derived signal peptide (SEQ ID NO: 23), CD19CAR VL (SEQ ID NO: 100), a GS (G4S) linker (SEQ ID NO: 25), CD19CAR VH (SEQ ID NO: 101), a CD8-derived hinge region sequence (SEQ ID NO: 26), a CD8-derived cell membrane domain for CAR (SEQ ID NO: 102), an intracellular co-stimulatory molecule (SEQ ID NO: 103), a linker (GSG), a self-cleaving peptide T2A (SEQ ID NO: 20), a blue fluorescent protein (BFP) (SEQ ID NO: 21), and a CD8-derived cell membrane domain (SEQ ID NO: 103). IDNO: 104) was inserted into pLVSIN EF1α, and the anti-CD19CAR-carrying lentiviral concentrate was prepared by the method described in [Preparation of lentivirus]. In addition, in order to express membrane-type a2DL2 / 3-DS10 scFv, the plasmid prepared in Example 4 was used to prepare membrane-type a2DL2 / 3-DS10 scFv-carrying lentiviral concentrate by the method described in [Preparation of lentivirus]. In addition, as a control, an artificial gene encoding a polypeptide configured in the order of NGFR peptide (SEQ ID NO: 58), self-cleavage peptide T2A (SEQID NO: 20) and firefly luciferase (SEQ ID NO: 17) from the N-terminal side (hereinafter referred to as "NGFR-Luc") was used to prepare a lentiviral concentrate carrying NGFR-Luc by the same method.

[0806] PBMC-C was suspended in a medium (hereinafter referred to as "IL-2 medium") in which 5 (v / v)% FCS (10270-106) and streptomycin / penicillin (15240-062) were added to AIM V medium (12055-083) supplemented with IL-2 (87-890, manufactured by Nipro) and mixed with the prepared lentivirus concentrate (four conditions: NGFR-Luc-carried lentivirus alone, membrane-type a2DL2 / 3-DS10 scFv-carried lentivirus alone, CD19CAR-carried lentivirus alone, or a mixture of CD19CAR-carried lentivirus and membrane-type a2DL2 / 3-DS10scFv-carried lentivirus), and added to the CD3Ab / RetroNectin-coated culture plate described in the [Preparation of target gene-expressing T cells]. Centrifuge for 1 hour. The next day, the same virus concentrate as the previous day was added to each well, and the mixture was centrifuged for 1 hour.

[0807] After the full amount of culture medium was replaced with a suitable IL-2 culture medium for cultivation, about the PBMC-C infected with membrane type a2DL2 / 3-DS10 scFv carrying lentivirus, or, CD19CAR carrying lentivirus and membrane type a2DL2 / 3-DS10 scFv carrying lentivirus mixed solution, APC label anti-Flag antibody and anti-APC microbeads (Miltenyi Biotec, 130-090-855) were used to concentrate membrane type a2DL2 / 3-DS10 scFv positive cells. In addition, about the PBMC-C infected with NGFR-Luc carrying lentivirus, anti-LNGFR microbeads (Miltenyi Biotec, 130-099-023) were used to concentrate NGFR positive cells. T cells derived from PBMC-C expressing membrane type a2DL2 / 3-DS10 scFv were obtained by this operation. In addition, in the PBMC-C-derived T cells infected with a mixture of CD19CAR-carrying lentivirus and membrane-type a2DL2 / 3-DS10 scFv-carrying lentivirus, although there were cells expressing both, cells expressing only one of them, and cells expressing neither, a mixed cell group of PBMC-C-derived T cells expressing CD19CAR and membrane-type a2DL2 / 3-DS10 scFv and PBMC-C-derived T cells expressing only membrane-type a2DL2 / 3-DS10 scFv was obtained by concentration. In addition, PBMC-C-derived T cells expressing NGFR-Luc were obtained.

[0808] Regarding PBMC-C-derived T cells expressing NGFR-Luc and PBMC-C-derived T cells expressing membrane-type a2DL2 / 3-DS10 scFv, after concentration, the full amount of culture medium was replaced with an appropriate IL-2 medium for culture, and then suspended in CELLBANKER1 (produced by Takara Bio Co., Ltd., Japan, CB011) and frozen at -80°C or -150°C.

[0809] Regarding PBMC-C infected only with lentivirus carrying CD19CAR, and the mixed cell group of PBMC-C-derived T cells expressing CD19CAR and membrane-type a2DL2 / 3-DS10 scFv obtained by the above concentration and PBMC-C-derived T cells expressing only membrane-type a2DL2 / 3-DS10scFv, the whole amount was suspended in IL-2 medium after culture and added to a CD19-coated plate coated with CD19 human recombinant protein (produced by Origene, TP302922). After culture, it was confirmed that PBMC-C-derived T cells expressing CD19CAR, or PBMC-C-derived T cells expressing CD19CAR and membrane-type a2DL2 / 3-DS10 scFv were concentrated, suspended in CELLBANKER1 (produced by Takara Bio Co., Ltd., Japan, CB011), and frozen at -80°C or -150°C.

[0810] Each PBMC-C-derived T cell was thawed one day before the experiment and cultured in an IL-2 medium.

[0811] [Evaluation of the cytotoxic activity of CAR-T cells against target cells in the presence of PBMC-derived NK cells]

[0812] As a target for T cells expressing CD19CAR, a cell line (hereinafter referred to as "Raji-GFP") in which GFP is introduced into Raji cells that constantly express CD19 using a viral vector was used. A sample in which Raji-GFP cells were mixed with NK cells derived from PBMC-A, or a sample with only Raji-GFP cells was prepared. For each sample, PBMC-C-derived T cells expressing NGFR-Luc, PBMC-C-derived T cells expressing only CD19CAR, PBMC-C-derived T cells expressing only membrane-type a2DL2 / 3-DS10, or PBMC-C-derived T cells expressing CD19CAR and membrane-type a2DL2 / 3-DS10 scFv were added at a ratio of 1:1, 1; 0.5 or 1:0.1 relative to Raji-GFP cells, and cultured with 96-well plates. The culture medium used was MyeloCult H5100 medium, AIM V medium supplemented with 5 (v / v)% FCS, and RPMI 1640 medium GlutaMAX supplemented with 10 (v / v)% FCS (all supplemented with streptomycin / penicillin), and the cell suspensions were mixed to make the volumes of each culture medium equal.

[0813] Four days after the start of culture, the number of cells in each well was determined using the method described in [Flow cytometry and addition of staining antibodies]. The antibody used to detect PBMC-C-derived T cells was APC / Fire 750 anti-human CD3 antibody (manufactured by BIOLEGEND, 300470). In addition, the detection of Raji-GFP cells used GFP expressed by Raji-GFP cells.

[0814] The number of live cells of Raji-GFP cells in the wells to which only Raji-GFP cells were added was set as 100%, the ratio of the number of live cells of Raji cells in each well was plotted on the vertical axis, and the ratio of T cells to Raji-GFP cells in each well at the start of culture was plotted on the horizontal axis. The results are shown in Figure 27-1 .

[0815] In the wells where PBMC-A-derived NK cells were not co-cultured, the cytotoxic activity of PBMC-C-derived T cells expressing only CD19CAR and PBMC-C-derived T cells expressing CD19CAR and membrane-type α2DL2 / 3-DS10 scFv against Raji-GFP cells was the same. On the other hand, in the wells where PBMC-A-derived NK cells were co-cultured, the cytotoxic activity of PBMC-C-derived T cells expressing only CD19CAR against Raji-GFP cells was weakened compared to that of PBMC-C-derived T cells expressing CD19CAR and membrane-type α2DL2 / 3-DS10 scFv against Raji-GFP cells.

[0816] At this time, if Figure 27-2 As shown, in the wells where PBMC-A-derived NK cells were co-cultured, the number of PBMC-C-derived T cells expressing only CD19CAR was reduced compared to the number of PBMC-C-derived T cells expressing CD19CAR and membrane-type a2DL2 / 3-DS10 scFv. The study believes that PBMC-C-derived T cells expressing only CD19CAR were recognized as lost self by PBMC-A-derived NK cells and attacked, so their number was reduced; PBMC-C-derived T cells expressing CD19CAR and membrane-type a2DL2 / 3-DS10scFv showed a2DL2 / 3-DS10-mediated agonist activity against inhibitory KIR against PBMC-A-derived NK cells, escaping the attack of PBMC-A-derived NK cells, so their number did not decrease.

[0817] This indicates that CAR-T cells expressing the binder identified in Example 4 will inhibit the cell killing activity of primary NK cells. In the presence of primary NK cells, the cell killing activity of CAR-T cells against target cells is maintained compared to CAR-T cells that do not express the binder identified in Example 4.

[0818] Example 10: Evaluation of the Effect of CDR Modifications on Agonist Activity of KIR2DL2 and / or KIR2DL3 Specific Binders

[0819] Regarding CDR3 of the variable region (VH) of a2DL2 / 3-DS10, the effect of substituting a specific amino acid residue to alanine on the agonist activity against inhibitory KIR was evaluated.

[0820] In the membrane-type a2DL2 / 3-DS10 scFv, regarding CDR3 of the heavy chain variable region (VH) of a2DL2 / 3-DS10 (amino acids 97 to 112 of SEQID NO: 51), a mutant of the membrane-type a2DL2 / 3-DS10 scFv in which each amino acid residue was substituted with alanine was introduced into the membrane-type scFv expression configuration in the order of VH-VL, and the artificial gene obtained was inserted into pLVSIN EF1α. The names of the mutants and the amino acids substituted with alanine are shown in Table 13 below.

[0821] [Table 13]

[0822] Mutant name VH CDR3 sequences R98A SEQ ID NO:67 V99A SEQ ID NO:68 S100A SEQ ID NO:69 G101A SEQ ID NO:70 T102A SEQ ID NO:71 T103A SEQ ID NO:72 G104A SEQ ID NO:73 G105A SEQ ID NO:74 Y106A SEQ ID NO:75 Y107A SEQ ID NO:76 Y108A SEQ ID NO:77 G109A SEQ ID NO:78 M110A SEQ ID NO:79 D111A SEQ ID NO:80 V112A SEQ ID NO:81

[0823] Using pLVSIN EF1α introduced with each membrane-bound scFv mutant, lentivirus was prepared according to the method described in [Preparation of lentivirus]. Using the prepared lentivirus, K562 cell lines expressing each membrane-bound scFv mutant were prepared according to the method described in [Introduction and preparation of scFv into K-562 cells].

[0824] Using the NK92 cell line, KIR2DL3-NK92 cell line and KIR2DS2-NK92 cell line used in Example 5, the cell killing activity of each NK92 cell line against the K562 cell line expressing each membrane-type scFv mutant was evaluated by the method described in [Evaluation of the cell killing activity of NK92 expressing KIR2DL2 against lirelumab-scFv / Pan2D-scFv-K562].

[0825] The results are shown in Fig.28 . In the mutants R98A, S100A, G101A, T102A, G105A, Y106A, and Y107A, compared with the membrane-type a2DL2 / 3-DS10 WT scFv without the introduction of mutations, it was confirmed that the agonist activity against KIR2DL3 was weakened or eliminated. On the other hand, the mutants other than the above showed the same degree of agonist activity as the membrane-type a2DL2 / 3-DS10 WT scFv, indicating that they are amino acids that are not important for agonist activity. In addition, these mutants did not show agonist activity against KIR2DS2, the active KIR with the highest homology to KIR2DL2 / 3.

[0826] The above results suggest that by modifying the amino acid sequence in the CDR of a binder having specific agonist activity against inhibitory KIR, it may be possible to obtain a binder having the same properties. In addition, it was shown that in a2DL2 / 3-DS10, amino acids R98, S100, G101, T102, G105, Y106, and Y107 of CDR3 contribute to the specific agonist activity against inhibitory KIR.

[0827] Example 11: Evaluation of the KIR2DL2 and / or KIR2DL3-specific agonist activity of CDR grafts grafted to the constant regions of different scFvs

[0828] In order to confirm that the specificity for KIR2DL2 and / or KIR2DL3 is maintained even when only the CDRs of the KIR2DL2 and / or KIR2DL3 specific binders are transplanted, regarding the heavy and light chain CDRs of a2DL2 / 3-DS1, a2DL2 / 3-DS2 or a2DL2 / 3-DS3, K562 cells were expressed as membrane-type scFvs with CDR-transplanted conjugates of the heavy and light chain constant regions transplanted with the CDRs of a2DL2 / 3-DS4 removed (hereinafter referred to as "a2DL2 / 3-DS1-2", "a2DL2 / 3-DS2-2" and "a2DL2 / 3-DS3-2", respectively), and the agonist activity against inhibitory KIR was evaluated.

[0829] a2DL2 / 3-DS1-2, a2DL2 / 3-DS2-2 or a2DL2 / 3-DS3-2 were introduced into membrane-type scFv expression configurations, and the artificial genes obtained were inserted into pLVSIN EF1α. The amino acids of each CDR graft are shown in Table 14 below.

[0830] [Table 14]

[0831] CDR transplant name VH amino acid sequence VL amino acid sequence a2DL2 / 3-DS1-2 SEQ ID NO:82 SEQ ID NO:83 a2DL2 / 3-DS2-2 SEQ ID NO:84 SEQ ID NO:85 a2DL2 / 3-DS3-2 SEQ ID NO:86 SEQ ID NO:87

[0832] Lentivirus was prepared by using pLVSIN EF1α introduced with each CDR-grafted membrane-type scFv according to the method described in [Preparation of Lentivirus]. K562 cell lines expressing each CDR-grafted membrane-type scFv were prepared by using the prepared lentivirus according to the method described in [Introduction and preparation of scFv into K-562 cells]. K562 cells expressing other target genes used the cells used in Example 5.

[0833] Using the NK92 cell line used in Example 5, KIR2DL2-NK92 cell line, KIR2DL3-NK92 cell line, KIR2DS1-NK92 cell line, KIR2DS2-NK92 cell line and KIR2DS4-NK92 cell line, the cell killing activity of each NK92 cell line against the K562 cell line expressing each CDR-grafted membrane-type scFv was evaluated by the method described in [Evaluation of the cell killing activity of NK92 expressing KIR2DL2 against lirelumab-scFv / Pan2D-scFv-K562].

[0834] The results are shown in Fig.29The K562 cell lines expressing each CDR-grafted membrane-type scFv showed agonist activity against KIR2DL2 and KIR2DL3 equivalent to that of the K562 cell lines expressing membrane-type a2DL2 / 3-DS1, membrane-type a2DL2 / 3-DS2, or membrane-type a2DL2 / 3-DS3 without CDR grafting. On the other hand, each CDR-grafted membrane-type scFv did not show agonist activity against KIR2DS1, KIR2DS2, and KIR2DS4 like the scFv of Pan2D.

[0835] The above results show that the CDRs of KIR2DL2 and / or KIR2DL3 specific binders also maintain KIR2DL2 and / or KIR2DL3 specific agonist activity when transplanted into different scFv constant regions. This shows that the sequence of CDRs is important in the KIR2DL2 and / or KIR2DL3 specific agonist activity, and its specificity is maintained even when transplanted into different constant regions.

[0836] Example 12: Evaluation of the additive inhibitory effect on NK cell cytotoxicity when co-expressing KIR2DL2 and / or KIR2DL3 specific binding compounds and HLA-E

[0837] To investigate whether coexpression of membrane-type a2DL2 / 3-DS4 scFv or membrane-type a2DL2 / 3-DS10 scFv and HLA-E would exhibit a stronger inhibitory effect on the cytotoxic activity of NK cells coexpressing KIR2DL2 or KIR2DL3 and NKG2A, the cytotoxic activity of NK92 cells coexpressing KIR2DL2 or KIR2DL3 and NKG2A against K562 cells coexpressing membrane-type a2DL2 / 3-DS4 scFv or membrane-type a2DL2 / 3-DS10 scFv and HLA-E was evaluated.

[0838] In order to prepare a lentivirus for gene introduction into HLA-E, genes encoding polypeptides were linked in the order of a signal peptide derived from human B2M (SEQ ID NO: 18), a peptide derived from an HLA-G signal peptide (SEQ ID NO: 88), a GS (G4S) linker (SEQ ID NO: 25), a human B2M peptide (SEQ ID NO: 19), a GS (G4S) 4 linker (SEQ ID NO: 89), and an HLA-E peptide (SEQ ID NO: 90) from the N-terminal side, and an artificial gene encoding a polypeptide in which a self-cleaving peptide T2A (SEQ ID NO: 20) and a firefly luciferase (SEQ ID NO: 17) were linked to the C-terminal side in this order was inserted into pLVSIN EF1α.

[0839] Lentivirus was prepared using pLVSIN EF1α introduced with HLA-E according to the method described in [Preparation of Lentivirus]. Using the prepared lentivirus, K562 cells expressing membrane-type a2DL2 / 3-DS4 scFv or membrane-type a2DL2 / 3-DS10 scFv prepared in Example 4 were infected with lentivirus according to the method described in [Introduction and preparation of scFv gene into K-562 cells], and K562 cell lines expressing HLA-G signal peptide, HLA-E trimer (hereinafter referred to as "tHLA-E") in which B2M and HLA-E are bound via a linker, and membrane-type a2DL2 / 3-DS4 scFv or membrane-type a2DL2 / 3-DS10 scFv were prepared.

[0840] Since NK92 cells continuously express NKG2A, the NK92 cell line, KIR2DL2-NK92 cell line and KIR2DL3-NK92 cell line used in Example 5 were used to evaluate the cell killing activity of each NK92 cell line against the K562 cell line expressing tHLA-E and membrane-type a2DL2 / 3-DS4 scFv or membrane-type a2DL2 / 3-DS10 scFv by the method described in [Evaluation of the cell killing activity of NK92 expressing KIR2DL2 against lirelumab-scFv / Pan2D-scFv-K562].

[0841] The results are shown in Fig.30 K562 cell lines expressing tHLA-E and membrane-type a2DL2 / 3-DS4 scFv or membrane-type a2DL2 / 3-DS10 scFv showed stronger inhibitory effects on KIR2DL2-NK92 cell lines and KIR2DL3-NK92 cell lines compared with K562 cell lines expressing only tHLA-E or K562 cell lines expressing only membrane-type a2DL2 / 3-DS4 scFv or membrane-type a2DL2 / 3-DS10 scFv.

[0842] The above results indicate that the co-expression of KIR2DL2 and / or KIR2DL3 specific binders and HLA-E has an additive inhibitory effect on the cell-killing activity of NK cells co-expressing KIR2DL2 and / or KIR2DL3 and NKG2A.

[0843] Example 13: Evaluation of the inhibition of NK cell cytotoxicity against iPS cells that do not express class I HLA but express KIR2DL2 and / or KIR2DL3 specific binding agents

[0844] iPS cells that do not express class I HLA are recognized by NK cells as having lost their self-identity and are attacked. In order to evaluate whether iPS cells that do not express class I HLA but express KIR2DL2 and / or KIR2DL3-specific binding compounds inhibit the cytotoxic activity of NK cells, KIR2DL2 and / or KIR2DL3-specific binding compounds were introduced into iPS cells that had been gene-edited to eliminate B2M and thus eliminate the expression of class I HLA. The iPS cells were co-cultured with PBMC-derived NK cells to evaluate the cytotoxic activity of PBMC-derived NK cells against iPS cells.

[0845] In order to prepare iPS cells that do not express class I HLA but express KIR2DL2 and / or KIR2DL3 specific binders, membrane-type a2DL2 / 3-DS10 scFv was introduced into iPS cells in which B2M was deleted using CRISPR / CAS9 (hereinafter referred to as "B2M KO iPS cells"). In addition, as a control, NGFR-Luc was introduced into wild-type iPS cells or B2M KO iPS cells. The membrane-type a2DL2 / 3-DS10 scFv used the plasmid used in Example 4, and the NGFR-Luc used the plasmid used in Example 9, and the lentivirus concentrates were prepared by the method described in the [Preparation of lentivirus].

[0846] Add iMatrix-511silk (produced by Nippi, 892021) and CultureSureR Y-27632 (produced by WAKO, 034-24024) to mTeSR Plus cGMP (produced by VERITAS, 100-0276) medium (hereinafter referred to as "mTeSR medium"). Suspend each iPS cell stripped with Accutase (ICT) (produced by Funakoshi Co., Ltd., AT104) in the above medium. Add the prepared lentivirus concentrate and iPS cell suspension to the culture plate coated with RetroNectin and centrifuge using a centrifuge. Change to mTeSR medium on the second day, and after culture, use APC-labeled anti-Flag antibodies and anti-APC microbeads to select only positive cells.

[0847] The iPS cells used for evaluation of cell killing activity were completely replaced with mTeSR medium containing recombinant human IFN-γ (manufactured by PEPROTECH, 300-02) 3 days, 2 days, and 1 day before the experiment.

[0848] The PBMC-A-derived NK cells prepared according to the method described in [Preparation of PBMC-derived NK cells] were suspended in CELLBANKER1 (manufactured by Takara Bio Co., Ltd., CB011), frozen at -80°C or -150°C, thawed two days before the experiment, and cultured in MyeloCult H5100 medium supplemented with IL-2 and IL-18 (manufactured by R&D systems, 9124-IL). The medium of PBMC-A-derived NK cells was replaced with MyeloCult H5100 medium without IL-2 and IL-18 one day before the experiment, and cultured.

[0849] PBMC-A-derived NK cells and wild-type iPS cells or B2M KO iPS cells or B2M KO iPS expressing membrane-type a2DL2 / 3-DS10 scFv were mixed and cultured in a 96-well plate. The culture medium used MyeloCult H5100 culture medium and mTeSR with CultureSureR Y-27632 added, and the cell suspensions were mixed to make each culture medium reach an equal amount. Two hours after the start of culture, the luciferase activity of each well was measured using the ONE-Glo(TM) luciferase detection system. In addition, the luciferase activity in the wells without the addition of PBMC-A-derived NK cells was set to 100%, and the value after subtracting the luciferase activity in the wells with the addition of PBMC-A-derived NK cells from the luciferase activity in the wells with the addition of PBMC-A-derived NK cells was calculated as the cell killing activity.

[0850] The results are shown in Fig.31 Compared with wild-type iPS cells, B2M KO iPS cells failed to inhibit the cytotoxic activity of PBMC-derived NK cells. On the other hand, B2M KO iPS cells expressing membrane-bound a2DL2 / 3-DS10 scFv showed an inhibitory effect on the cytotoxic activity of PBMC-derived NK cells compared with wild-type iPS cells.

[0851] The above results show that although iPS cells that do not express class I HLA are recognized as lost by NK cells and attacked, the cell killing activity of NK cells is inhibited by expressing KIR2DL2 and / or KIR2DL3 specific binding compounds in iPS cells that do not express class I HLA. In addition, the inhibition of NK cells by KIRs with specific binding compounds of KIR2DL2 and / or KIR2DL3 is effective not only for cell lines that do not express class I HLA without gene editing, such as K562, but also for cells in which the expression of class I HLA is eliminated through gene editing, as shown in the example of iPS cells.

[0852] Example 14: Identification of the epitope of KIR2DL2 by X-ray crystal structure analysis of KIR2DL2 and / or KIR2DL3 specific binders

[0853] In order to identify the epitope of KIR2DL2 and / or KIR2DL3 specific binders to KIR2DL2, the complex of the Fab fragment of KIR2DL2 and / or KIR2DL3 specific binders and recombinant KIR2DL2 was crystallized and subjected to X-ray crystal structure analysis.

[0854] The Fab fragment of the KIR2DL2 and / or KIR2DL3 specific binder used the Fab fragment of a2DL2 / 3-DS10 having the amino acid sequence shown in Table 15. The recombinant KIR2DL2 used a recombinant protein linked in the order of IL-2-derived signal peptide (SEQ ID NO: 91), KIR2DL2 (22-225) (amino acids 1 to 204 of SEQ ID NO: 6), and His tag (SEQ ID NO: 92) from the N-terminal side.

[0855] 340 μL of protein deglycosylation mixture II (produced by NEW ENGLANDBioLabs, P6044S) was added to 9 mg of KIR2DL2 (22-225)-His, and the mixture was reacted at 4°C overnight. The reaction solution was added to Ni Sepharose 6 Fast Flow (produced by Cytiva, 17-5318-03) equilibrated with 50 mM HEPES (pH 8), 500 mM NaCl, 20 mM imidazole, and 5% (v / v) glycerol, and then the column was washed with 50 mM HEPES (pH 8), 500 mM NaCl, 250 mM imidazole, and 5% (v / v) glycerol. Then, the column was eluted with 50 mM HEPES (pH 8), 500 mM NaCl, 500 mM imidazole, and 5% (v / v) glycerol. The recovered deglycosylated KIR2DL2(22-225)-His fraction was purified using HiLoad 16 / 600 Superdex 200 pg (manufactured by Cytiva, 28989336) equilibrated with PBS(-).

[0856] The deglycosylated KIR2DL2(22-225)-His and a2DL2 / 3-DS10 Fab fragments were mixed to a molar ratio of 1.2:1 and incubated at 4°C for 1 hour. The mixture was added to HiLoad16 / 600 Superdex 200pg (Cytiva) equilibrated with 20mM Tris-HCl (pH7.5) and 150mM NaCl to purify the complex of deglycosylated KIR2DL2(22-225)-His and 61_3092_02_F10_Fab.

[0857] The obtained complex of a2DL2 / 3-DS10 Fab and KIR2DL2 was concentrated to 19 mg / mL for crystallization. The crystallization was performed using the vapor diffusion method. A solution of precipitant solution [10% (w / v) polyethylene glycol 3,350 (manufactured by Hampton Research, HR2-527)], 0.2M calcium acetate (manufactured by Hampton Research, HR2-567), and 0.1M imidazole-hydrochloric acid pH 7.5 (manufactured by Nacalai Tesque, 19004-35) was added in equal amounts to 0.1 to 0.2 μL of the protein solution and stored in a sealed container containing 0.05 mL of the precipitant solution, avoiding contact between the two solutions, and standing at 20°C. After 3 days, a single crystal of 0.2 mm×0.1 mm×0.05 mm was obtained.

[0858] The obtained single crystal was immersed in a precipitant solution to which 35% (v / v) polyethylene glycol 3,350 was added, and then frozen in liquid nitrogen. X-ray diffraction data were collected under a nitrogen flow of 95K using BL-17A of the Synchrotron Radiation Science Research Facility of the High Energy Accelerator Research Institute. The diffraction intensity was quantified from the obtained diffraction image using the software XDS (created by Wolfgang Kabsch, Max Planck Institute for Medical Research) to determine the crystal structure factor. Regarding the crystal, the space group in the monoclinic system is C121, and the unit lattice of the crystal is β=92.39°.

[0859] The structural analysis and calculations were performed using the software package Phenix (Python-Based Hierarchical Environment for Integrated Xtallography). Phaser software was used to determine the phase by molecular replacement using the obtained structure factors and the three-dimensional structural coordinates of KIR2DL2 (PDB code: 2DL2) and Fab (PDB code: similar part selected from 1MIM). The crystal contains one complex in the asymmetric unit.

[0860] Use phenix.refine software to refine the structure, and use coot software to correct the model. Repeat this operation. The final R value was 23.4% and the free R value was 27.5% at the resolution. The model consists of a complex, including amino acid residues 1-210 of the VL chain of a2DL2 / 3-DS10 Fab, amino acid residues 1-224 of the H chain, amino acid residues 25-221 of KIR2DL2, 2 N-acetylglucosamines, and 14 water molecules. The model was not constructed because the electron density of the C-terminal 2 residues of the L chain of a2DL2 / 3-DS10 Fab, the C-terminal 6 residues of the H chain and the 8 residues of Ser137-Glu144, and the C-terminal 10 residues including the His tag of KIR2DL2 were unclear.

[0861] Figure 32-1 The ribbon model represents the entire complex. The amino acid residues of KIR2DL2 within are as follows: Glu42, His61, Lys65, Phe66, Lys67, Asp68, Thr69, Leu70, Ser86, Ile87, Gly88, Pro89, Met91, Gln92, Asp93, Leu94, Tyr101, Leu111. The above amino acids are epitopes in KIR2DL2 of a2DL2 / 3-DS10 and are highlighted in the amino acid sequence of KIR2DL2. Comparison with the amino acid sequence of active KIR is shown in Figure 32-2 . The amino acid sequence of each KIR uses the signal peptide and receptor sequence recorded in Table 5. In addition, the amino acid number of each KIR is the combination of the signal peptide and receptor sequence recorded in Table 4, and the numbering starts from 1. That is, the amino acid number of each amino acid in the above-mentioned KIR2DL2 epitope is equivalent to the number of the corresponding amino acid in the amino acid sequence shown in SEQ ID NO:6 plus 21 (the amino acid length of the signal peptide); the corresponding amino acid number of each KIR2DS1 / 2 / 4 is also the same, which is the number of the corresponding amino acid in the amino acid sequence shown in SEQ ID NO:10, SEQ ID NO:12 and SEQ ID NO:14 respectively plus 21.

[0862] In addition, the amino acid sequence that contributes to the selectivity of a2DL2 / 3-DS10 was analyzed using Maestro software released by SCHRODINGER. The analysis results showed that the amino acid corresponding to amino acid Gly88 on KIR2DL2 is amino acid Ser88 in KIR2DS1, but it will sterically hinder amino acid Gly104 on a2DL2 / 3-DS10 VH CDR3, so amino acid Gly104 contributes to the selectivity of KIR2DL2 and KIR2DS1. In addition, the amino acid corresponding to amino acid Pro89 on KIR2DL2 is amino acid Arg89 in KIR2DS1, but it cannot form a hydrogen bond or the formed hydrogen bond is weak with amino acid Tyr106 on a2DL2 / 3-DS10 VH CDR3, indicating that amino acid Tyr106 contributes to the selectivity of KIR2DL2 and KIR2DS1.

[0863] In addition, the amino acid corresponding to amino acid Phe66 on KIR2DL2 is amino acid Tyr66 in KIR2DS2, but it will sterically hinder amino acid Tyr107 on a2DL2 / 3-DS10 VH CDR3, thus indicating that amino acid Tyr107 contributes to the selectivity of KIR2DL2 and KIR2DS2.

[0864] It was also shown that the amino acid corresponding to amino acid Asp68 on KIR2DL2 is amino acid Asn68 in KIR2DS4, but it cannot form a hydrogen bond or forms a weak hydrogen bond with amino acid Tyr107 on a2DL2 / 3-DS10 VH CDR3, so amino acid Tyr107 contributes to the selectivity of KIR2DL2 and KIR2DS4.

[0865] In addition, the above structural analysis shows that the 94th amino acid threonine of the VL chain of a2DL2 / 3-DS10 (the 94th amino acid of SEQ IDNO:52, hereinafter referred to as "DS10-VL-T94") forms a hydrogen bond with the amino acid Asp93 on KIR2DL2, but when it is replaced by the amino acid Val93 in KIR2DS4, it cannot form a hydrogen bond with the NH group of the main chain of DS10-VL-Thr94 or the formed hydrogen bond is weak. Therefore, DS10-VL-Thr94 contributes to the selectivity of a2DL2 / 3-DS10 for KIR2DL2 and KIR2DS4. DS10-VL-T94 was analyzed using the BIOLUMINATE software released by SCHRODINGER. As a result, in the mutant a2DL2 / 3-DS10 in which the amino acid was substituted with the amino acid shown in Table 15, it was observed that the hydrogen bond was formed with Asp93 of KIR2DL2 on the main chain NH, while the hydrogen bond was not formed with Val93 of KIR2DS4.

[0866] It is predicted that substitutions of these amino acids will retain the specificity of binding to inhibitory KIRs but not to active KIRs.

[0867] [Table 15]

[0868] Substituted amino acid Alanine Asparagine Glycine Histidine Isoleucine Serine Valine

[0869] Example 15: Evaluation of the epitope sites of KIR2DL2 and / or KIR2DL3 specific binders by competition of a2DL2 / 3-DS10 with each KIR2DL2 / 3 specific binder in an antigen binding assay

[0870] Fab fragments were prepared for each KIR2DL2 and / or KIR2DL3 specific binder. The Fab fragments and recombinant KIR2DL2 were added to the K562 cell line expressing the membrane-type a2DL2 / 3-DS10 scFv, and the epitope site of each binder was evaluated using as an indicator whether the binding between the K562 cell line expressing the membrane-type a2DL2 / 3-DS10 scFv and the recombinant KIR2DL2 was blocked by competition.

[0871] For the Fab fragment of each KIR2DL2 and / or KIR2DL3 specific binder, a recombinant protein was prepared in which a signal peptide derived from VH (SEQ ID NO: 92), a VH chain variable region listed in Table 15, and a Fab fragment VH chain constant region (SEQ ID NO: 95) were linked in this order from the N-terminus, and a recombinant protein was prepared in which a signal peptide derived from VL (SEQ ID NO: 95), a VL chain variable region listed in Table 16, and a Fab fragment VL chain constant region (SEQ ID NO: 96) were linked in this order from the N-terminus, and the Fab fragment was made to associate. The amino acid sequence of the variable region of each binder is shown in Table 15. In addition, the recombinant KIR2DL2 used in Example 4 was biotinylated with human KIR2DL2 (22-245)-Fc-Avi [hereinafter referred to as "human KIR2DL2 (22-245)-Fc-Biotin"]. In addition, the cell line prepared in Example 4 was used as the K562 cell line expressing the membrane-type a2DL2 / 3-DS10 scFv.

[0872] [Table 16]

[0873] Name of conjugate Variable region a2DL2 / 3-DS1 VH chain (SEQ ID NO:33) a2DL2 / 3-DS1 VL chain (SEQ ID NO:34) a2DL2 / 3-DS2 VH chain (SEQ ID NO:35) a2DL2 / 3-DS2 VL chain (SEQ ID NO:36) a2DL2 / 3-DS3 VH chain (SEQ ID NO:37) a2DL2 / 3-DS3 VL chain (SEQ ID NO:38) a2DL2 / 3-DS4 VH chain (SEQ ID NO:39) a2DL2 / 3-DS4 VL chain (SEQ ID NO:40) a2DL2 / 3-DS5 VH chain (SEQ ID NO:41) a2DL2 / 3-DS5 VL chain (SEQ ID NO:42) a2DL2 / 3-DS6 VH chain (SEQ ID NO:43) a2DL2 / 3-DS6 VL chain (SEQ ID NO:44) a2DL2 / 3-DS7 VH chain (SEQ ID NO:45) a2DL2 / 3-DS7 VL chain (SEQ ID NO:46) a2DL2 / 3-DS9 VH chain (SEQ ID NO:49) a2DL2 / 3-DS9 VL chain (SEQ ID NO:50) a2DL2 / 3-DS10VH chain (SEQ ID NO:51) a2DL2 / 3-DS10VL chain (SEQ ID NO:52) a2DL2 / 3-DS11VH chain (SEQ ID NO:61) a2DL2 / 3-DS11VL chain (SEQ ID NO:62) a2DL2 / 3-DS12VH chain (SEQ ID NO:63) a2DL2 / 3-DS12VL chain (SEQ ID NO:64)

[0874] K562 cell lines or K562 cell lines expressing membrane-type a2DL2 / 3-DS10 scFv were suspended in autoMACS electrophoresis buffer-MACS separation buffer, and the Fab fragments of each conjugate were added to a final concentration of 1.5 μM, 0.5 μM, 0.17 μM, 0.06 μM, 0.02 μM, or 0.01 μM, and the mixture was allowed to stand. Human KIR2DL2(22-245)-Fc-Biotin was added to a final concentration of 0.05 μM, and the mixture was allowed to stand.

[0875] After the reaction, the cells were washed with autoMACS electrophoresis buffer and MACS separation buffer, and APC-tagged anti-human IgG Fc antibody was added and allowed to stand. After the reaction, the cells were washed with autoMACS electrophoresis buffer and MACS separation buffer, and the binding to human KIR2DL2(22-245)-Fc-Biotin was confirmed by flow cytometry using the median value of the fluorescence intensity derived from APC.

[0876] The results are shown in Fig.33. Except for a2DL2 / 3-DS5 and 7, when the Fab fragments were added at a concentration of 1.5 μM, the Fab fragments of KIR2DL2 and / or KIR2DL3 specific binders inhibited the binding of K562 cell lines expressing membrane-bound a2DL2 / 3-DS10 scFv and human KIR2DL2(22-245)-Fc-Biotin by more than 30% compared to control K562 cells. This indicates that a2DL2 / 3-DS10 and most of the KIR2DL2 and / or KIR2DL3 specific binders compete with KIR2DL2.

[0877] The above results show that the epitopes bound by a2DL2 / 3-DS10 and most of the specific binders of KIR2DL2 and / or KIR2DL3 are located near each other. The epitope of a2DL2 / 3-DS10 identified by X-ray crystal structure analysis is very important in showing specificity for KIR2DL2 and / or KIR2DL3 and showing agonist activity.

[0878] Example 16: Obtaining KIR2DL1 or KIR3DL1 specific binders by phage panning

[0879] The above examples show that binders with KIR2DL2 and / or KIR2DL3 specific agonist activity inhibit the cytotoxic activity of NK cells expressing KIR2DL2 and / or KIR2DL3 against cells recognized as lost self. Therefore, in order to obtain the same inhibitory effect on cytotoxic activity of NK cells expressing KIR2DL1 or KIR3DL1, phage panning was performed in the same manner as the method described in Example 4 [Obtaining inhibitory KIR-specific binders by phage panning] for the purpose of obtaining a novel binder that specifically binds to KIR2DL1 or KIR3DL1 and exhibits agonist activity.

[0880] In order to implement phage panning, a recombinant protein was prepared by connecting the signal peptide derived from IL-2 (SEQ ID NO: 91), the extracellular domain of each KIR protein recorded in Table 17, the GGGS linker (SEQ ID NO: 97), the IgG Fc domain (SEQ ID NO: 98), the GAA linker, and the Avi tag (SEQ ID NO: 99) from the N-terminal side as a soluble KIR. The sequence of the extracellular domain of each KIR used by each soluble KIR is shown in Table 17. In addition, the soluble KIR3DS1 uses a recombinant human KIR3DS1 / CD158e2Fc chimeric protein (produced by R&D Systems, 4136-KR-050). Phage panning uses substances that have or have not been biotinylated for each soluble KIR prepared.

[0881] [Table 17]

[0882] Recombinant protein name Extracellular domain KIR2DL1 Amino acids 1 to 224 of SEQ ID NO:4 KIR2DL2 Amino acids 1 to 224 of SEQ ID NO:6 KIR2DL3 Amino acids 1 to 224 of SEQ ID NO:8 KIR2DS1 Amino acids 1 to 224 of SEQ ID NO:10 KIR2DS2 Amino acids 1 to 224 of SEQ ID NO:12 KIR2DS4 Amino acids 1 to 224 of SEQ ID NO:14 KIR3DL1 Amino acids 1 to 319 of SEQ ID NO:16

[0883] In each round of phage panning, positive selection for inhibitory KIR, depletion or exclusion of active KIR, or both are performed. After two rounds, a polyclonal phage library enriched in phages expressing binders that specifically bind to either KIR2DL1 or KIR3DL1 is obtained.

[0884] ELISA was performed using the soluble KIR of the target KIR (KIR2DL1 or KIR3DL1), and the scFv-expressing phage that binds to KIR2DL1 or KIR3DL1 or both was determined from the polyclonal phage library. The sequence of the binder was transferred from the phagemid of the determined scFv-expressing phage to a recombinant protein expression vector for E. coli, introduced into E. coli, and the recombinant protein of each binder was obtained from the culture supernatant. ELISA was performed on each binder using the soluble KIR corresponding to each KIR listed in Table 16 to obtain information on the binding properties of each binder to each KIR.

[0885] The variable regions of the binders that bind to KIR2DL1 or KIR3DL1 but not to KIR2DS1, KIR2DS2, KIR2DS4 and KIR3DS1 were introduced into the membrane-type scFv expression configuration described in the section [Introduction and preparation of scFv gene into K-562 cells] and introduced into pPB[Exp]-EF1A (produced by VectorBuilder).

[0886] The amino acid sequences of the heavy chain variable region and the light chain variable region of the KIR2DL1 specific binder are shown in Table 18. The amino acid sequences of the heavy chain variable region and the light chain variable region of the KIR3DL1 specific binder are shown in Table 19.

[0887] [Table 18]

[0888]

[0889]

[0890] [Table 19]

[0891]

[0892]

[0893] Electroporation was used to introduce genes into K562 cells. When electroporation was performed, pPB[Exp]-EF1A and pRP[Exp]-mCherry-CAG>hyPBase [produced ...

Claims

A substance having an activity of binding to human inhibitory KIR and inhibiting NK cells. 2 . The substance according to claim 1 , which is a polypeptide comprising a binding site for human inhibitory KIR.

3. The substance according to claim 1, characterized in that The binding site of the substance against human inhibitory KIR contains an amino acid sequence from one or two variable regions of a monoclonal antibody or antigen-binding fragment that binds to human inhibitory KIR.

4. The substance according to claim 1, wherein The substance has a higher binding activity to human inhibitory KIR than to the corresponding human active KIR.

5. The substance according to claim 1, wherein The substance has a higher agonist activity against human inhibitory KIR than against the corresponding human active KIR.

6. The substance according to claim 5, wherein The substances do not substantially exhibit agonist activity against the corresponding human active KIR.

7. The substance according to claim 2, wherein The polypeptide is any one of the following (1) to (3): (1) A polypeptide whose binding site for KIR2DL2 and / or KIR2DL3 comprises a C1 epitope motif sequence (amino acid 77 is S and amino acid 80 is N) contained in HLA-C1 type HLA-Cw1, Cw3, Cw7, Cw8, Cw9, Cw10, Cw12, Cw14, Cw16, HLA-B46 or B73, and which lacks or weakens binding to TCR; (2) a polypeptide whose binding site for KIR2DL1 comprises a C2 epitope motif sequence (amino acid 77 is N and amino acid 80 is K) contained in HLA-Cw2, Cw4, Cw5, Cw6, Cw15 or Cw17 belonging to HLA-C2 type, and which lacks or weakens the binding to TCR; (3) A polypeptide whose binding site for KIR3DL1 comprises a Bw4 epitope motif sequence (amino acid 77 is N, amino acid 80 is I or T, and amino acid 83 is R) contained in HLA-B5, B27, B37, B38, B44, B49, B51, B52, B53, B57, B58, B59, B77, A23, A24, A25 or A32 belonging to HLA-Bw4 type, and in which the binding property to TCR is deleted or weakened.

8. The substance according to claim 3, characterized in that The human inhibitory KIR is KIR2DL2 and / or KIR2DL3, and the binding site comprises an amino acid sequence from one or two variable regions of a monoclonal antibody or antigen-binding fragment that binds to KIR2DL2 and / or KIR2DL3.

9. The substance according to claim 8, characterized in that The monoclonal antibody or antigen-binding fragment that binds to KIR2DL2 and / or KIR2DL3 competes with lirelumab, Pan2D, or a scFv consisting of any of the following amino acid sequences (i) to (xii) in binding to KIR2DL2 and / or KIR2DL3: (i) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 33 and the amino acid sequence of the light chain variable region of SEQ ID NO: 34; (ii) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 35 and the amino acid sequence of the light chain variable region of SEQ ID NO: 36; (iii) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 37 and the amino acid sequence of the light chain variable region of SEQ ID NO: 38; (iv) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 39 and the amino acid sequence of the light chain variable region of SEQ ID NO: 40; (v) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 41 and the amino acid sequence of the light chain variable region of SEQ ID NO: 42; (vi) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 43 and the amino acid sequence of the light chain variable region of SEQ ID NO: 44; (vii) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 45 and the amino acid sequence of the light chain variable region of SEQ ID NO: 46; (viii) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 47 and the amino acid sequence of the light chain variable region of SEQ ID NO: 48; (ix) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 49 and the amino acid sequence of the light chain variable region of SEQ ID NO: 50; (x) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 51 and the amino acid sequence of the light chain variable region of SEQ ID NO: 52; (xi) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 61 and the amino acid sequence of the light chain variable region of SEQ ID NO: 62; and, (xii) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 63 and the amino acid sequence of the light chain variable region of SEQ ID NO:

64.

10. The substance according to claim 8, characterized in that The binding site of the substance for KIR2DL2 and / or KIR2DL3 includes a variable region containing amino acids arranged to be close to the following amino acids: 1 to 6 amino acids including Asp at position 47 in the region of amino acids at positions 44 to 49 in the amino acid sequence of KIR2DL2 of SEQ ID NO:6; 2 to 4 amino acids including Gly at position 67 and Pro at position 68 in the region of positions 65 to 68; and 1 to 4 amino acids including Asp at position 72 in the region of positions 70 to 73.

11. The substance according to claim 10, characterized in that In the binding site of the substance for KIR2DL2 and / or KIR2DL3, the amino acid that interacts with Asp at position 47 of SEQ ID NO:6 is Tyr, the amino acid that interacts with Gly at position 67 of SEQ ID NO:6 is Gly, the amino acid that interacts with Pro at position 68 of SEQ ID NO:6 is Tyr, and the amino acid that interacts with Asp at position 72 of SEQ ID NO:6 is Thr, Asn, Gly, His, Ile, Ser, Val or Ala.

12. The substance according to claim 11, characterized in that The amino acids contained in the variable region defined in claim 11 belong to CDR3.

13. The substance according to claim 8, which is a polypeptide comprising any one of the following binding sites (I) to (XII) as a binding site for KIR2DL2 and / or KIR2DL3, and may have one or two amino acid mutations in each CDR: (I) a binding site comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO:33 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO:34; (II) a binding site comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO:35 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO:36; (III) a binding site comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 37 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 38; (IV) a binding site comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO:39 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO:40; (V) a binding site comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO:41 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO:42; (VI) a binding site comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO:43 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO:44; (VII) a binding site comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO:45 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO:46; (VIII) a binding site comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO:47 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO:48; (IX) a binding site comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO:49 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO:50; (X) a binding site comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO:51 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO:52; (XI) a binding site comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO:61 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO:62; and (XII) A binding site comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2 and CDR3 of SEQ ID NO:63 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2 and CDR3 of SEQ ID NO:

64.

14. The substance according to claim 13, wherein The C-terminal side and / or N-terminal side of each CDR may have one or two amino acid mutations.

15. The substance according to claim 13, which is a polypeptide comprising any one of the following binding sites (I) to (XII) as a binding site for KIR2DL2 and / or KIR2DL3: (I) a binding site comprising a combination of a first variable region comprising the amino acid sequence of SEQ ID NO: 33 and a second variable region comprising the amino acid sequence of SEQ ID NO: 34; (II) a binding site comprising a combination of a first variable region comprising the amino acid sequence of SEQ ID NO: 35 and a second variable region comprising the amino acid sequence of SEQ ID NO: 36; (III) a binding site comprising a combination of a first variable region comprising the amino acid sequence of SEQ ID NO: 37 and a second variable region comprising the amino acid sequence of SEQ ID NO: 38; (IV) a binding site comprising a combination of a first variable region comprising the amino acid sequence of SEQ ID NO: 39 and a second variable region comprising the amino acid sequence of SEQ ID NO: 40; (V) a binding site comprising a combination of a first variable region comprising the amino acid sequence of SEQ ID NO:41 and a second variable region comprising the amino acid sequence of SEQ ID NO:42; (VI) a binding site comprising a combination of a first variable region comprising the amino acid sequence of SEQ ID NO: 43 and a second variable region comprising the amino acid sequence of SEQ ID NO: 44; (VII) a binding site comprising a combination of a first variable region comprising the amino acid sequence of SEQ ID NO: 45 and a second variable region comprising the amino acid sequence of SEQ ID NO: 46; (VIII) a binding site comprising a combination of a first variable region comprising the amino acid sequence of SEQ ID NO: 47 and a second variable region comprising the amino acid sequence of SEQ ID NO: 48; (IX) a binding site comprising a combination of a first variable region comprising the amino acid sequence of SEQ ID NO: 49 and a second variable region comprising the amino acid sequence of SEQ ID NO: 50; (X) a binding site comprising a combination of a first variable region comprising the amino acid sequence of SEQ ID NO: 51 and a second variable region comprising the amino acid sequence of SEQ ID NO: 52; (XI) a binding site comprising a combination of a first variable region comprising the amino acid sequence of SEQ ID NO: 61 and a second variable region comprising the amino acid sequence of SEQ ID NO: 62; and (XII) A binding site comprising a combination of a first variable region comprising the amino acid sequence of SEQ ID NO:63 and a second variable region comprising the amino acid sequence of SEQ ID NO:

64.

16. The substance according to claim 8, characterized in that The substance has a higher binding activity against KIR2DL2 and / or KIR2DL3 than against KIR2DS1, KIR2DS2 and / or KIR2DS4.

17. The substance according to claim 8, characterized in that The substance has a higher agonist activity against KIR2DL2 and / or KIR2DL3 than against KIR2DS1, KIR2DS2 and / or KIR2DS4.

18. The substance according to claim 17, characterized in that The substance does not exhibit substantial agonist activity towards KIR2DS1, KIR2DS2 and / or KIR2DS4.

19. The substance according to claim 17, characterized in that The substance does not substantially bind to KIR2DS1, KIR2DS2, and KIR2DS4.

20. The substance according to claim 19, characterized in that The substance comprises an antigen-binding fragment obtained by the following method: Step A: mixing a tagged substance containing a soluble protein of the extracellular domain of KIR2DL2 or KIR2DL3 into a suspension containing a polyclonal display body group, then bringing the suspension into contact with a carrier on which a substance that specifically binds to the tag portion is fixed, and recovering the display body bound to the carrier, thereby obtaining a display body group that displays an antigen binding site that binds to KIR2DL2 or KIR2DL3, wherein the polyclonal display body group includes an antigen binding site that exhibits binding to KIR2DL2 or KIR2DL3 displayed on the surface; Step B: In the suspension containing the display body group obtained in step A, a labeled substance containing a soluble protein of the extracellular domain of KIR2DS1, KIR2DS2 and KIR2DS4 is mixed, and the labeled substance is brought into contact with a carrier on which a substance binding to the tag portion is fixed, and the display body not bound to the carrier is recovered, thereby removing the display body displaying the antigen binding site binding to KIR2DS1, KIR2DS2 and KIR2DS4 from the cell group; Step C: performing gene analysis on the display obtained by the method comprising the above steps to identify the gene sequence encoding the antigen binding site; and Step D: Allowing cells to express the gene containing the identified antigen binding site.

21. The substance according to claim 19, characterized in that The substance comprises an antigen-binding fragment obtained by the following method: Step A: In a suspension containing a polyclonal display body group, a labeled substance containing a soluble protein of the extracellular domain of KIR2DL2 or KIR2DL3 and a soluble protein containing the extracellular domain of KIR2DS1, KIR2DS2 and KIR2DS4 that is not labeled are mixed, and then the suspension is brought into contact with a carrier on which a substance that specifically binds to the label portion is fixed, and the display body bound to the carrier is recovered, thereby obtaining a display body group that displays an antigen binding site that specifically binds to KIR2DL2 or KIR2DL3, wherein the polyclonal display body group includes an antigen binding site that exhibits binding to KIR2DL2 or KIR2DL3 displayed on the surface; Step B: performing gene analysis on the display obtained by the method comprising the above steps to identify the gene sequence encoding the antigen binding site; and Step C: Allowing cells to express the gene containing the identified antigen binding site.

22. The substance according to claim 20 or 21, wherein The combination of the tag and the substance bound to the tag is a combination of biotin-avidin.

23. The substance according to claim 20 or 21, characterized in that The substance is obtained by a method further comprising the step of selecting a substance having agonist activity to KIR2DL2 or KIR2DL3.

24. The substance according to any one of claims 8 to 23, wherein The polypeptide is a monoclonal antibody or an antigen binding fragment.

25. The substance according to claim 24, characterized in that The substance is a single chain antibody or scFv in which the first variable region and the second variable region are connected via a GS linker or a G4S linker.

26. The substance according to any one of claims 8 to 23, wherein The polypeptide is a membrane protein having an extracellular domain comprising a binding site for KIR2DL2 and / or KIR2DL3 and a transmembrane domain.

27. The substance according to claim 3, characterized in that The inhibitory KIR is KIR3DL1, and the binding site contains amino acid sequences from the heavy chain variable region and the light chain variable region of a monoclonal antibody or antigen-binding fragment that binds to KIR3DL1.

28. The substance according to claim 27, characterized in that The monoclonal antibody or antigen-binding fragment that binds to KIR3DL1 competes with a scFv consisting of any one of the following amino acid sequences (xiii) to (xviii) in binding to KIR3DL1: (xiii) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 56 and the amino acid sequence of the light chain variable region of SEQ ID NO: 55; (xiv) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 137 and the amino acid sequence of the light chain variable region of SEQ ID NO: 138; (xv) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 139 and the amino acid sequence of the light chain variable region of SEQ ID NO: 140; (xvi) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 141 and the amino acid sequence of the light chain variable region of SEQ ID NO: 142; (xvii) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 143 and the amino acid sequence of the light chain variable region of SEQ ID NO: 144; and (xviii) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 145 and the amino acid sequence of the light chain variable region of SEQ ID NO:

146.

29. The substance according to claim 27, which is a polypeptide comprising any one of the following binding sites (XIII) to (XVIII) as a binding site for KIR3DL1, and each CDR may have 1 or 2 amino acid mutations: (XIII) a binding site comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO:56 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO:55; (XIV) a binding site comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 137 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 138; (XV) a binding site comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 139 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 140; (XVI) a binding site comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 141 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 142; (XVII) a binding site comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 143 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 144; as well as (XVIII) A binding site comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2 and CDR3 of SEQ ID NO:145 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2 and CDR3 of SEQ ID NO:

146.

30. The substance according to claim 29, wherein The C-terminal side and / or N-terminal side of each CDR may have one or two amino acid mutations.

31. The substance according to claim 27, which is a polypeptide comprising any one of the following binding sites (XIII) to (XVIII) as a binding site for KIR3DL1: (XIII) a binding site comprising a combination of a first variable region comprising the amino acid sequence of SEQ ID NO: 56 and a second variable region comprising the amino acid sequence of SEQ ID NO: 55; (XIIII) a binding site comprising a combination of a first variable region comprising the amino acid sequence of SEQ ID NO: 137 and a second variable region comprising the amino acid sequence of SEQ ID NO: 138; (XV) a binding site comprising a combination of a first variable region comprising the amino acid sequence of SEQ ID NO: 139 and a second variable region comprising the amino acid sequence of SEQ ID NO: 140; (XVI) a binding site comprising a combination of a first variable region comprising the amino acid sequence of SEQ ID NO: 141 and a second variable region comprising the amino acid sequence of SEQ ID NO: 142; (XVII) a binding site comprising a combination of a first variable region comprising the amino acid sequence of SEQ ID NO: 143 and a second variable region comprising the amino acid sequence of SEQ ID NO: 144; and (XVIII) A binding site comprising a combination of a first variable region comprising the amino acid sequence of SEQ ID NO: 145 and a second variable region comprising the amino acid sequence of SEQ ID NO:

146.

32. The substance according to claim 27, characterized in that The binding activity of the substance against KIR3DL1 is higher than the binding activity against KIR3DS1.

33. The substance according to claim 27, characterized in that The substance has a higher agonist activity against KIR3DL1 than against KIR3DS1.

34. The substance according to claim 33, characterized in that The substance does not substantially exhibit agonist activity towards KIR3DS1.

35. The substance according to claim 34, characterized in that The substance does not substantially bind to KIR3DS1.

36. The substance according to claim 35, characterized in that The substance comprises an antigen-binding fragment obtained by a method comprising the steps of: Step A: mixing a tagged substance containing a soluble protein of the extracellular domain of KIR3DL1 in a suspension containing a polyclonal display body group, and then bringing the suspension into contact with a carrier on which a substance that specifically binds to the tag portion is fixed, and recovering the display body bound to the carrier, thereby obtaining a display body group that displays an antigen binding site that binds to KIR3DL1, wherein the polyclonal display body group includes an antigen binding site that exhibits binding to KIR3DL1 and is displayed on the surface; Step B: a labeled substance containing a soluble protein of the extracellular domain of KIR3DS1 is mixed in a suspension containing the display body group obtained in step A, and the labeled substance is brought into contact with a carrier on which a substance binding to the tag portion is fixed, and the display body not bound to the carrier is recovered, thereby removing the display body displaying the antigen binding site bound to KIR3DS1 from the cell group; Step C: performing gene analysis on the display obtained by the method comprising the above steps to identify the gene sequence encoding the antigen binding site; and Step D: Allowing cells to express the gene containing the identified antigen binding site.

37. The substance according to claim 36, characterized in that The substance comprises an antigen-binding fragment obtained by the following method: Step A: In a suspension containing a polyclonal display body group, a labeled substance containing a soluble protein of the extracellular domain of KIR3DL1 and a soluble protein containing an unlabeled extracellular domain of KIR3DS1 are mixed, and then the suspension is brought into contact with a carrier on which a substance that specifically binds to the label portion is fixed, and the display body bound to the carrier is recovered, thereby obtaining a display body group that displays an antigen binding site that specifically binds to KIR3DL1, wherein the polyclonal display body group includes an antigen binding site that exhibits binding to KIR3DL1 and is displayed on the surface; Step B: performing gene analysis on the display obtained by the method comprising the above steps to identify the gene sequence encoding the antigen binding site; and Step C: Allowing cells to express the gene containing the identified antigen binding site.

38. A substance according to claim 36 or 37, wherein The combination of the tag and the substance bound to the tag is a combination of biotin-avidin.

39. The substance according to any one of claims 36 to 38, characterized in that The substance is obtained by a method further comprising the step of selecting a substance having agonist activity on KIR3DL1.

40. The substance according to any one of claims 27 to 39, wherein The polypeptide is a monoclonal antibody or an antigen binding fragment.

41. The substance according to claim 40, characterized in that The substance is a single chain antibody or scFv in which the first variable region and the second variable region are connected via a GS linker or a G4S linker.

42. The substance according to any one of claims 27 to 34, wherein The polypeptide is a membrane protein having an extracellular domain comprising a binding site for KIR3DL1 and a transmembrane domain.

43. The substance according to claim 3, characterized in that The inhibitory KIR is KIR2DL1, and the binding site contains amino acid sequences from the heavy chain variable region and the light chain variable region of a monoclonal antibody or antigen binding fragment that binds to KIR2DL1.

44. The substance according to claim 43, characterized in that The monoclonal antibody or antigen-binding fragment that binds to KIR2DL1 competes with Pan2D in binding to KIR2DL1.

45. The substance according to claim 43, characterized in that The monoclonal antibody or antigen-binding fragment that binds to KIR2DL1 competes with a scFv consisting of any one of the following amino acid sequences (ci) to (cxv) in binding to KIR2DL1: (ci) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 107 and the amino acid sequence of the light chain variable region of SEQ ID NO: 108; (cii) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 109 and the amino acid sequence of the light chain variable region of SEQ ID NO: 110; (ciii) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 111 and the amino acid sequence of the light chain variable region of SEQ ID NO: 112; (civ) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 113 and the amino acid sequence of the light chain variable region of SEQ ID NO: 114; (cv) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 115 and the amino acid sequence of the light chain variable region of SEQ ID NO: 116; (cvi) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 117 and the amino acid sequence of the light chain variable region of SEQ ID NO: 118; (cvii) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 119 and the amino acid sequence of the light chain variable region of SEQ ID NO: 120; (cviii) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 121 and the amino acid sequence of the light chain variable region of SEQ ID NO: 122; (cviiii) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 123 and the amino acid sequence of the light chain variable region of SEQ ID NO: 124; (cx) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 125 and the amino acid sequence of the light chain variable region of SEQ ID NO: 126; (cxi) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 127 and the amino acid sequence of the light chain variable region of SEQ ID NO: 128; (cxii) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 129 and the amino acid sequence of the light chain variable region of SEQ ID NO: 130; (cxiii) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 131 and the amino acid sequence of the light chain variable region of SEQ ID NO: 132; (cxiv) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 133 and the amino acid sequence of the light chain variable region of SEQ ID NO: 134; and (cxv) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 135 and the amino acid sequence of the light chain variable region of SEQ ID NO:

136.

46. ​​The substance according to claim 43, which is a polypeptide comprising any one of the following binding sites (CI) to (CXV) as a binding site for KIR2DL1, and each CDR may have 1 or 2 amino acid mutations: (CI) a binding site comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 107 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 108; (CII) a binding site comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 109 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 110; (CIII) a binding site comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 111 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 112; (CIV) a binding site comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 113 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 114 (preferably a variable region comprising an amino acid sequence of SEQ ID NO: 114); (CV) a binding site comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 115 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 116; (CVI) a binding site comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 117 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 118; (CVII) a binding site comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 119 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 120; (CVIII) a binding site comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 121 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 122; (CVIIII) a binding site comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 123 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 124; (CX) a binding site comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 125 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 126; (CXI) a binding site comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 127 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 128; (CXII) a binding site comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 129 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 130; (CXIII) a binding site comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 131 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 132; (CXIV) a binding site comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 133 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 134; and (CXV) comprises a combined binding site of a first variable region comprising an amino acid sequence derived from CDR1, CDR2 and CDR3 of SEQ ID NO:135 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2 and CDR3 of SEQ ID NO:

136.

47. The substance according to claim 46, wherein The C-terminal side and / or N-terminal side of each CDR may have one or two amino acid mutations.

48. The substance according to claim 46, which is a polypeptide comprising any one of the following binding sites (CI) to (CXV) as a binding site for KIR2DL1: (CI) a binding site comprising a combination of a first variable region comprising the amino acid sequence of SEQ ID NO: 107 and a second variable region comprising the amino acid sequence of SEQ ID NO: 108; (CII) a binding site comprising a combination of a first variable region comprising the amino acid sequence of SEQ ID NO: 109 and a second variable region comprising the amino acid sequence of SEQ ID NO: 110; (CIII) a binding site comprising a combination of a first variable region comprising the amino acid sequence of SEQ ID NO: 111 and a second variable region comprising the amino acid sequence of SEQ ID NO: 112; (CIV) a binding site comprising a combination of a first variable region comprising the amino acid sequence of SEQ ID NO: 113 and a second variable region comprising the amino acid sequence of SEQ ID NO: 114 (preferably a variable region comprising the amino acid sequence of SEQ ID NO: 114); (CV) a binding site comprising a combination of a first variable region comprising the amino acid sequence of SEQ ID NO: 115 and a second variable region comprising the amino acid sequence of SEQ ID NO: 116; (CVI) a binding site comprising a combination of a first variable region comprising the amino acid sequence of SEQ ID NO: 117 and a second variable region comprising the amino acid sequence of SEQ ID NO: 118; (CVII) a binding site comprising a combination of a first variable region comprising the amino acid sequence of SEQ ID NO: 119 and a second variable region comprising the amino acid sequence of SEQ ID NO: 120; (CVIII) a binding site comprising a combination of a first variable region comprising the amino acid sequence of SEQ ID NO: 121 and a second variable region comprising the amino acid sequence of SEQ ID NO: 122; (CVIIII) a binding site comprising a combination of a first variable region comprising the amino acid sequence of SEQ ID NO: 123 and a second variable region comprising the amino acid sequence of SEQ ID NO: 124; (CX) a binding site comprising a combination of a first variable region comprising the amino acid sequence of SEQ ID NO: 125 and a second variable region comprising the amino acid sequence of SEQ ID NO: 126; (CXI) a binding site comprising a combination of a first variable region comprising the amino acid sequence of SEQ ID NO: 127 and a second variable region comprising the amino acid sequence of SEQ ID NO: 128; (CXII) a binding site comprising a combination of a first variable region comprising the amino acid sequence of SEQ ID NO: 129 and a second variable region comprising the amino acid sequence of SEQ ID NO: 130; (CXIII) a binding site comprising a combination of a first variable region comprising the amino acid sequence of SEQ ID NO: 131 and a second variable region comprising the amino acid sequence of SEQ ID NO: 132; (CXIV) a binding site comprising a combination of a first variable region comprising the amino acid sequence of SEQ ID NO: 133 and a second variable region comprising the amino acid sequence of SEQ ID NO: 134; and (CXV) comprises a binding site comprising a first variable region comprising the amino acid sequence of SEQ ID NO:135 and a second variable region comprising the amino acid sequence of SEQ ID NO:

136.

49. The substance according to claim 43, characterized in that The binding activity of the substance to KIR2DL1 is higher than that to KIR2DS1, KIR2DS2 and KIR2DS4.

50. The substance according to claim 43, characterized in that The substance has a higher agonist activity against KIR2DL1 than against KIR2DS1, KIR2DS2 and KIR2DS4.

51. The substance according to claim 50, characterized in that The substance does not substantially exhibit agonist activity towards KIR2DS1, KIR2DS2 and KIR2DS4.

52. The substance according to claim 51, characterized in that The substance does not substantially bind to KIR2DS1, KIR2DS2, and KIR2DS4.

53. The substance according to claim 52, characterized in that The substance comprises an antigen-binding fragment obtained by the following method: Step A: mixing a tagged substance containing a soluble protein of the extracellular domain of KIR2DL1 in a suspension containing a polyclonal display body group, and then bringing the suspension into contact with a carrier on which a substance that specifically binds to the tag portion is fixed, and recovering the display body bound to the carrier, thereby obtaining a display body group that displays an antigen binding site that binds to KIR2DL1, wherein the polyclonal display body group includes an antigen binding site that exhibits binding to KIR2DL1 and is displayed on the surface; Step B: In the suspension containing the display body group obtained in step A, a labeled substance containing a soluble protein of the extracellular domain of KIR2DS1, KIR2DS2 and KIR2DS4 is mixed, and the labeled substance is brought into contact with a carrier on which a substance binding to the tag portion is fixed, and the display body not bound to the carrier is recovered, thereby removing the display body displaying the antigen binding site binding to KIR2DS1, KIR2DS2 and KIR2DS4 from the cell group; Step C: performing gene analysis on the display obtained by the method comprising the above steps to identify the gene sequence encoding the antigen binding site; and Step D: Allowing cells to express the gene containing the identified antigen binding site.

54. The substance according to claim 52, characterized in that The substance comprises an antigen-binding fragment obtained by a method comprising the steps of: Step A: In a suspension containing a polyclonal display body group, a labeled substance containing a soluble protein of the extracellular domain of KIR2DL1 and a soluble protein containing the extracellular domain of KIR2DS1, KIR2DS2 and KIR2DS4 that is not labeled are mixed, and then the suspension is brought into contact with a carrier on which a substance that specifically binds to the tag portion is fixed, and the display body bound to the carrier is recovered, thereby obtaining a display body group that displays an antigen binding site that specifically binds to KIR2DL1, wherein the polyclonal display body group includes an antigen binding site that exhibits binding to KIR2DL1 and is displayed on the surface; Step B: performing gene analysis on the display obtained by the method comprising the above steps to identify the gene sequence encoding the antigen binding site; and Step C: Allowing cells to express the gene containing the identified antigen binding site.

55. A substance according to claim 53 or 54, wherein The combination of the tag and the substance bound to the tag is a combination of biotin-avidin.

56. The substance according to any one of claims 53 to 55, characterized in that The substance is obtained by a method further comprising the step of selecting a substance having agonist activity on KIR3DL1.

57. The substance according to any one of claims 43 to 56, wherein The polypeptide is a monoclonal antibody or an antigen binding fragment.

58. The substance according to claim 57, characterized in that The substance is a single chain antibody or scFv where the first variable region and the second variable region are connected via a GS linker.

59. The substance according to any one of claims 43 to 56, wherein The polypeptide is a membrane protein having an extracellular domain comprising a binding site for KIR2DL1 and a transmembrane domain.

60. A pharmaceutical composition comprising the substance according to any one of claims 1 to 59 as an active ingredient.

61. The pharmaceutical composition according to claim 60, which is an immunorejection inhibitor.

62. A cell, wherein: A substance having an activity of binding to an inhibitory KIR and inhibiting the cytotoxic activity of NK cells according to any one of claims 1 to 59 is expressed on the cell surface.

63. The cell according to claim 62, wherein There are also ligand substances targeting NKG2A expressed on the cell surface.

64. The cell according to claim 63, wherein The ligand for NKG2A is HLA-E.

65. The cell according to claim 62, wherein The expression of at least one HLA class I on the cell surface is absent or reduced.

66. The cell according to claim 65, wherein The disappearance or weakening of the expression of at least one class I HLA on the cell surface is caused by gene editing to modify the class I HLA, B2M, TAP1, TAP2, and TAPBP regions on the genome.

67. The cell according to claim 65, wherein The cells are a cell line established from an individual in which HLA expression has been genetically lost.

68. The cell according to claim 62, wherein There is also ligand substance for NKG2A expressed on the cell surface, and the expression of at least one class I HLA on the cell surface disappears or weakens.

69. The cell according to claim 62, wherein The cells are pluripotent stem cells, T cells, NK cells, peripheral blood mononuclear cells (PBMC), mesenchymal stem cells, cardiomyocytes, chondrocytes, retinal cells, hepatocytes, kidney cells or pancreatic islet cells.

70. The cell according to claim 69, wherein Pluripotent stem cells are iPS cells or ES cells.

71. The cell according to any one of claims 62 to 70, wherein There are also chimeric receptors expressed on the cell surface.

72. The cell according to claim 62, wherein The inhibitory KIR is KIR2DL2 and / or KIR2DL3, and the substance having the activity of binding to KIR2DL2 and / or KIR2DL3 and inhibiting the cytotoxic activity of NK cells is the membrane protein according to claim 26.

73. The cell according to claim 72, characterized in that The membrane protein that binds to KIR2DL2 and / or KIR2DL3 comprises a binding site that competes with lirelumab, Pan2D, or a scFv consisting of any one of the following amino acid sequences (i) to (xii) in binding to KIR2DL2 and / or KIR2DL3: (i) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 33 and the amino acid sequence of the light chain variable region of SEQ ID NO: 34; (ii) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 35 and the amino acid sequence of the light chain variable region of SEQ ID NO: 36; (iii) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 37 and the amino acid sequence of the light chain variable region of SEQ ID NO: 38; (iv) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 39 and the amino acid sequence of the light chain variable region of SEQ ID NO: 40; (v) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 41 and the amino acid sequence of the light chain variable region of SEQ ID NO: 42; (vi) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 43 and the amino acid sequence of the light chain variable region of SEQ ID NO: 44; (vii) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 45 and the amino acid sequence of the light chain variable region of SEQ ID NO: 46; (viii) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 47 and the amino acid sequence of the light chain variable region of SEQ ID NO: 48; (ix) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 49 and the amino acid sequence of the light chain variable region of SEQ ID NO: 50; (x) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 51 and the amino acid sequence of the light chain variable region of SEQ ID NO: 52; (xi) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 61 and the amino acid sequence of the light chain variable region of SEQ ID NO: 62; and, (xii) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 63 and the amino acid sequence of the light chain variable region of SEQ ID NO:

64.

74. The cell according to claim 72, wherein A membrane protein is expressed on the cell surface, the membrane protein comprising any one of the following binding sites (I) to (XII) as a binding site for KIR2DL2 and / or KIR2DL3, and each CDR may have one or two amino acid mutations: (I) a binding site comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO:33 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO:34; (II) a binding site comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO:35 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO:36; (III) a binding site comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 37 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 38; (IV) a binding site comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO:39 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO:40; (V) a binding site comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO:41 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO:42; (VI) a binding site comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO:43 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO:44; (VII) a binding site comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO:45 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO:46; (VIII) a binding site comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO:47 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO:48; (IX) a binding site comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO:49 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO:50; (X) a binding site comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO:51 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO:52; (XI) a binding site comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO:61 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO:62; as well as (XII) A binding site comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2 and CDR3 of SEQ ID NO:63 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2 and CDR3 of SEQ ID NO:

64.

75. The cell according to claim 74, wherein The C-terminal side and / or N-terminal side of each CDR may have one or two amino acid mutations.

76. The cell according to claim 62, wherein The inhibitory KIR is KIR3DL1, and the substance having the activity of binding to KIR3DL1 and inhibiting the cytotoxic activity of NK cells is the membrane protein according to claim 42.

77. The cell according to claim 76, characterized in that The membrane protein that binds to KIR3DL1 comprises a binding site that competes with a scFv consisting of any one of the following amino acid sequences (xiii) to (xviii) in binding to KIR3DL1: (xiii) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 56 and the amino acid sequence of the light chain variable region of SEQ ID NO: 55; (xiv) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 137 and the amino acid sequence of the light chain variable region of SEQ ID NO: 138; (xv) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 139 and the amino acid sequence of the light chain variable region of SEQ ID NO: 140; (xvi) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 141 and the amino acid sequence of the light chain variable region of SEQ ID NO: 142; (xvii) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 143 and the amino acid sequence of the light chain variable region of SEQ ID NO: 144; and (xviii) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 145 and the amino acid sequence of the light chain variable region of SEQ ID NO:

146.

78. The cell according to claim 75, wherein A membrane protein is expressed on the cell surface, the membrane protein comprising any one of the following binding sites (XIII) to (XVIII) as a binding site for KIR3DL1, and each CDR may have one or two amino acid mutations: (XIII) a binding site comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO:56 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO:55; (XIV) a binding site comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 137 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 138 (preferably a variable region comprising an amino acid sequence of SEQ ID NO: 138); (XV) a binding site comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 139 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 140; (XVI) a binding site comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 141 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 142; (XVII) a binding site comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 143 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 144; as well as (XVIII) A binding site comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2 and CDR3 of SEQ ID NO:145 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2 and CDR3 of SEQ ID NO:

146.

79. The cell according to claim 78, wherein The C-terminal side and / or N-terminal side of each CDR may have one or two amino acid mutations.

80. The cell according to claim 62, wherein The inhibitory KIR is KIR2DL1, and the substance having the activity of binding to KIR2DL1 and inhibiting the cytotoxic activity of NK cells is the membrane protein according to claim 59.

81. The cell according to claim 80, characterized in that Membrane proteins that bind to KIR2DL1 contain binding sites that compete with Pan2D in binding to KIR2DL1.

82. The cell according to claim 78, wherein A membrane protein is expressed on the cell surface, and the membrane protein comprises the following (P) binding site as a binding site for KIR2DL1, and each CDR may have one or two amino acid mutations: (P) A binding site comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2 and CDR3 of SEQ ID NO:31 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2 and CDR3 of SEQ ID NO:

32.

83. The cell according to claim 82, wherein The C-terminal side and / or N-terminal side of each CDR may have one or two amino acid mutations.

84. The cell according to claim 80, characterized in that The membrane protein that binds to KIR2DL1 comprises a binding site that competes with a scFv consisting of any one of the following amino acid sequences (ci) to (cxv) in binding to KIR2DL1: (ci) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 107 and the amino acid sequence of the light chain variable region of SEQ ID NO: 108; (cii) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 109 and the amino acid sequence of the light chain variable region of SEQ ID NO: 110; (ciii) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 111 and the amino acid sequence of the light chain variable region of SEQ ID NO: 112; (civ) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 113 and the amino acid sequence of the light chain variable region of SEQ ID NO: 114; (cv) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 115 and the amino acid sequence of the light chain variable region of SEQ ID NO: 116; (cvi) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 117 and the amino acid sequence of the light chain variable region of SEQ ID NO: 118; (cvii) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 119 and the amino acid sequence of the light chain variable region of SEQ ID NO: 120; (cviii) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 121 and the amino acid sequence of the light chain variable region of SEQ ID NO: 122; (cviiii) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 123 and the amino acid sequence of the light chain variable region of SEQ ID NO: 124; (cx) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 125 and the amino acid sequence of the light chain variable region of SEQ ID NO: 126; (cxi) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 127 and the amino acid sequence of the light chain variable region of SEQ ID NO: 128; (cxii) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 129 and the amino acid sequence of the light chain variable region of SEQ ID NO: 130; (cxiii) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 131 and the amino acid sequence of the light chain variable region of SEQ ID NO: 132; (cxiv) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 133 and the amino acid sequence of the light chain variable region of SEQ ID NO: 134; and (cxv) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 135 and the amino acid sequence of the light chain variable region of SEQ ID NO:

136.

85. The cell according to claim 80, wherein A membrane protein is expressed on the cell surface, and the membrane protein comprises any one of the following binding sites (CI) to (CXV) as a binding site for KIR2DL1, and each CDR may have one or two amino acid mutations: (CI) a binding site comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 107 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 108; (CII) a binding site comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 109 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 110; (CIII) a binding site comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 111 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 112; (CIV) a binding site comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 113 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 114 (preferably a variable region comprising an amino acid sequence of SEQ ID NO: 114); (CV) a binding site comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 115 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 116; (CVI) a binding site comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 117 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 118; (CVII) a binding site comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 119 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 120; (CVIII) a binding site comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 121 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 122; (CVIIII) a binding site comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 123 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 124; (CX) a binding site comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 125 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 126; (CXI) a binding site comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 127 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 128; (CXII) a binding site comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 129 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 130; (CXIII) a binding site comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 131 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 132; (CXIV) a binding site comprising a combination of a first variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 133 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 134; and (CXV) comprises a combined binding site of a first variable region comprising an amino acid sequence derived from CDR1, CDR2 and CDR3 of SEQ ID NO:135 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2 and CDR3 of SEQ ID NO:

136.

86. The cell according to claim 85, wherein The C-terminal side and / or N-terminal side of each CDR may have one or two amino acid mutations.

87. A pharmaceutical composition, the active ingredient of which comprises the cell according to any one of claims 62 to 86, wherein the risk of immune rejection in a subject to which the pharmaceutical composition is administered or transplanted is reduced.

88. The pharmaceutical composition of claim 87, which is an immunorejection inhibitor.

89. A method for reducing or inhibiting immune rejection of therapeutic cells by a subject, the method comprising the step of expressing the substance according to any one of claims 1 to 59 on the cell surface of the therapeutic cells.

90. A method for producing therapeutic cells in which the risk of immune rejection in a subject is reduced or suppressed, the method comprising the step of expressing the substance according to any one of claims 1 to 59 on the cell surface of the therapeutic cells.

91. A method for evaluating the immune rejection escape activity caused by the agonist activity of a test substance on inhibitory KIR2DL2 or KIR2DL3, the method comprising the following steps: (A) a step of contacting NK cells from a subject homozygous for HLA-C type C1 / C1 and target cells from a subject homozygous for HLA-C type C2 / C2 in vitro in the presence of a test substance that binds to KIR2DL2 or KIR2DL3, wherein the expression type of HLA containing the Bw4 motif is the same in the donor of the NK cells and the donor of the target cells; (B) After step A, a step of measuring the number of surviving target cells.

92. A method for evaluating the immune rejection escape activity caused by the agonist activity of a test substance on inhibitory KIR2DL1, the method comprising the following steps: (A) a step of contacting NK cells from a subject homozygous for HLA-C type C2 / C2 and target cells from a subject homozygous for HLA-C type C1 / C1 in vitro in the presence of a test substance that binds to KIR2DL1, wherein the expression type of HLA containing the Bw4 motif of the donor of the NK cells and the donor of the target cells is the same; (B) After step A, a step of measuring the number of surviving target cells.

93. A method for evaluating the immune rejection escape activity caused by the agonist activity of a test substance on inhibitory KIR3DL1, the method comprising the following steps: (A) a step of contacting NK cells from a subject with HLA-Bw4 type positive / positive homozygous or positive / negative heterozygous and target cells from a subject with HLA-Bw4 type negative / negative homozygous in vitro in the presence of a test substance that binds to KIR3DL1, wherein the donor of the NK cells and the donor of the target cells have the same expression type of HLA-C type; (B) After step A, a step of measuring the number of surviving target cells.

94. A method for screening a substance having immune rejection escape activity, the method comprising: In the method according to any one of claims 91 to 93, a step is provided in which the test substance having a high number of surviving target cells in step B is selected as a substance having immune rejection escape activity.

95. The method according to any one of claims 91 to 93, characterized in that In step (A), the test substance is present in the form of being expressed on the surface of the target cell as a membrane protein.

96. A method for producing a substance capable of binding to human inhibitory KIR and inhibiting the activity of NK cells, the method comprising the following steps: Step A: mixing a tagged substance of a soluble protein containing an extracellular domain of an inhibitory KIR in a suspension containing a polyclonal display body group, and then bringing the suspension into contact with a carrier on which a substance that specifically binds to the tag portion is fixed, and recovering the display body bound to the carrier, thereby obtaining a display body group that displays an antigen binding site that binds to the KIR, wherein the polyclonal display body group includes an antigen binding site that exhibits binding to the inhibitory KIR and is displayed on the surface; Step B: In a suspension containing the display body group obtained in step A, a labeled substance containing a soluble protein of the extracellular domain of the active KIR corresponding to the inhibitory KIR is mixed, and the labeled substance is brought into contact with a carrier to which a substance bound to the label portion is fixed, and the display body that is not bound to the carrier is recovered, thereby removing the display body that displays the antigen binding site bound to the active KIR from the cell group; Step C: performing gene analysis on the display obtained by the method comprising the above steps to identify the gene sequence encoding the antigen binding site; and Step D: Allowing cells to express the gene containing the identified antigen binding site.

97. A method for producing a substance capable of binding to human inhibitory KIR and inhibiting the activity of NK cells, the method comprising the following steps: Step A: In a suspension containing a polyclonal display body group, a labeled substance containing a soluble protein of the extracellular domain of an inhibitory KIR and a non-labeled soluble protein containing the extracellular domain of an active KIR corresponding to the inhibitory KIR are mixed, and then the suspension is brought into contact with a carrier on which a substance that specifically binds to the label portion is fixed, and the display body bound to the carrier is recovered, thereby obtaining a display body group that displays an antigen binding site that specifically binds to the inhibitory KIR, wherein the polyclonal display body group includes an antigen binding site that exhibits binding to the inhibitory KIR and is displayed on the surface; Step B: performing gene analysis on the display obtained by the method comprising the above steps to identify the gene sequence encoding the antigen binding site; and Step C: Allowing cells to express the gene containing the identified antigen binding site.

98. The method of claim 96 or 97, wherein: The combination of the tag and the substance bound to the tag is a combination of biotin-avidin.

99. The method according to any one of claims 96 to 98, wherein The method further comprises the step of selecting a substance having agonist activity against the inhibitory KIR.

100. The method according to any one of claims 96 to 99, wherein: The inhibitory KIR is KIR2DL2 or KIR2DL3, and the corresponding active KIRs are KIR2DS1, KIR2DS2, and KIR2DS4.

101. The method according to any one of claims 96 to 99, wherein: The inhibitory KIR is KIR3DL1, and the corresponding active KIR is KIR3DS1.

102. The method according to any one of claims 96 to 99, wherein: The inhibitory KIR is KIR2DL1, and the corresponding active KIRs are KIR2DS1, KIR2DS2, and KIR2DS4.

Images