Method for avoiding immune rejection using nkg2a agonist

Abstract

The purpose of the present invention is to provide: a substance that is useful for allogeneic technologies relating to organ transplantation and cell therapy and that has an activity of binding to an inhibitory receptor on the surface of NK cells and suppressing the cytotoxic activity of NK cells; and a method for producing the same　Provided are: a substance that has an activity of binding to an inhibitory receptor on the surface of NK cells and suppressing the cytotoxic activity of NK cells; and a cell in which the substance is expressed on the cell surface.

Description

Method for avoiding immune rejection using NKG2A agonists

[0001] The present invention relates to a substance useful in allogeneic techniques related to organ transplantation and cell therapy, which has the activity of specifically binding to NKG2A and suppressing the cytocidal activity of NK cells, a method for producing the substance, a method for using the substance, etc. Furthermore, the present invention relates to a substance useful in allogeneic techniques related to organ transplantation and cell therapy, which has the activity of binding to inhibitory KIR and suppressing the cytocidal activity of NK cells, a method for producing the substance, a method for using the substance, etc.

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

[0003] Although human pluripotent stem cells have the potential to treat a variety of diseases, a major limitation to their clinical use is the rejection of transplanted cells by recipients due to differences in the major histocompatibility complex (MHC).

[0004] The major MHCs are cell surface multicomponent molecules found in all vertebrates that mediate leukocyte or leukocyte interactions with other cells. The MHC gene family is divided into three groups: Class I, Class II, and Class III. In humans, they are called human leukocyte antigens (HLA). Class I HLA proteins are expressed on all nucleated cells and consist of an HLA Class I heavy chain (or α chain) and β-2 microglobulin (B2M). Class I HLA proteins present peptides to CD8+ cytotoxic T cells. Six Class I HLA α chains have been identified to date, including three classical (HLA-A, HLA-B, and HLA-C) and three non-classical (HLA-E, HLA-F, and HLA-G) α chains. The specificity of peptide binding to the peptide-binding cleft of Class I HLA molecules is determined by the α chain. Recognition of peptides presented by Class I HLA molecules by CD8-positive T cells mediates cell-mediated immunity.

[0005] In transplantation, non-self-derived Class I HLA proteins are recognized as foreign antigens, and recognition of non-self Class I HLA proteins is a major barrier to the use of pluripotent cell transplantation or replacement therapy. Currently, HLA-matched or partially matched cells from stem cell banks or pluripotent stem cell (iPSC) lines derived from individual patients are developed for transplantation. However, developing individually matched cell lines requires significant costs, months of cell culture, highly trained personnel, and extensive validation of the final product, all of which must be approved by regulatory authorities. Furthermore, each cell line may behave somewhat differently in terms of gene expression patterns, culture characteristics, differentiation potential, and genetic mutations, making it extremely difficult to apply this to each individual cell line.

[0006] While personalized stem cell preparations or HLA-diverse stem cell banks could address current transplantation challenges, multiple cell lines must be characterized, differentiated into therapeutic cell products, and approved for human administration. This time-consuming, technically challenging, and costly process is a major factor preventing stem cell-based therapies from entering clinical trials. Therefore, there is a need for more effective, less expensive cell-based therapies that are not hindered by rejection.

[0007] T cells recognize HLA molecules on the cell surface via T cell receptors (TCRs). HLA regulates immune responses and maintains homeostasis by presenting endogenous self-peptides in normal cells and virus-derived non-self-peptides in infected cells.

[0008] NK (Natural Killer) cells have a self-non-self recognition function known as "missing self," which recognizes the self's HLA type and exhibits cytocidal activity against cells that do not express the same type of HLA (Non-Patent Document 1). In the missing self function, receptors such as NKG2 and KIR expressed on the surface of NK cells are responsible for recognizing the self's HLA type. These receptors include inhibitory receptors and activating receptors. In the case of self-cells, the same type of HLA as that of the NK cells binds to the inhibitory receptor on the NK cells, and an inhibitory signal is transmitted into the NK cells, suppressing the cytocidal activity of the NK cells and avoiding elimination by the NK cells.

[0009] Furthermore, with regard to such inhibitory receptors on NK cells, it is known that even the NK cells within a single individual contain NK cell populations that exhibit multiple inhibitory receptor expression types; for example, it has been reported that approximately half of the NK cells contained in human peripheral blood mononuclear cells (PBMCs) and identified as CD56-positive and CD3-negative are NKG2A-positive, while the remaining half are NKG2A-negative, forming an NK cell population that expresses other inhibitory receptors such as KIR and LILB (Non-Patent Document 2).

[0010] NKG2 (CD159) belongs to the C-type lectin superfamily and is composed of seven isoforms: NKG2A, NKG2B, NKG2C, NKG2D, NKG2E, NKG2F, and NKG2H. Of these, NKG2A, NKG2B, NKG2C, NKG2E, and NKG2H form heterodimers with CD94 (KLRD1), a type II transmembrane glycoprotein also belonging to the C-type lectin family, and use HLA-E, which presents peptides derived from Class I HLA, as a ligand. NKG2 is involved in regulating NK cell activation and is classified into activating and inhibitory isoforms. However, because CD94 does not possess a cytoplasmic signaling motif, the determination of activating or inhibitory isoforms is determined by the cytoplasmic motif of NKG2. NKG2C, NKG2E, and NKG2H have charged residues in their transmembrane regions and bind to DAP12, an adaptor protein with an ITAM (Immunoreceptor Tyrosine-based Activation Motif) motif, transducing an active signal. On the other hand, NKG2A and NKG2B have an ITIM (Immunoreceptor Tyrosine-based Inhibitor Motif) motif in the cytoplasm and transduce an inhibitory signal. NKG2D does not form a heterodimer with CD94, and is an NK receptor whose ligands are MICA and MICB, not HLA-E. It binds to DAP10, an adaptor protein with an ITAM motif, transducing an active signal (Non-Patent Document 3).

[0011] KIR (Killer cell immunoglobulin-like receptor) is a single-pass transmembrane protein expressed in NK cells, NKT cells, and a subset of T cells, recognizing HLA-A / B / C as its primary ligand (Non-Patent Document 2). In particular, recognition is based on the host's HLA type. For example, NK cells derived from humans with HLA-C1 / C1 (HLA-C with S at amino acid 77 and N at amino acid 80 is called C1 type) will attack HLA-C2 (HLA-C with N at amino acid 77 and K at amino acid 80 is called C2 type), and vice versa, they exhibit cytocidal activity by the same mechanism (Non-Patent Document 1). This mechanism is known as "missing self." The molecules that control this Missing Self are KIR2DL2 / 3, which function as inhibitory receptors if the host is derived from HLA-C1, and KIR2DL1, which functions as an inhibitory receptor if the host is derived from HLA-C2. Inhibitory receptors have an ITIM (Immunoreceptor Tyrosine-based Inhibitor Motif) in the intracellular compartment, form clusters upon association with HLA, and transmit inhibitory signals inside the cell via adaptor molecules such as SHP-1 (Non-Patent Document 4).

[0012] NK cells are characterized as CD56-positive and CD3-negative cells. NK cells are further broadly classified into CD56bright NK cells and CD56dim NK cells based on differences in their function and phenotype. CD56bright NK cells, which show high responsiveness to cytokine stimulation, play an immunoregulatory role in T cells and other immune responses, migrate to secondary lymphoid organs, the liver, skin, and bone marrow, where they become the predominant NK cell subset. Cytotoxic CD56dim NK cells are known to preferentially receive input from activating and inhibitory receptors and produce the majority of NK cells in peripheral blood. Generally, CD56bright NK cells predominantly express NKG2A, whereas cytotoxic CD56dim NK cells generally have low NKG2A expression and tend to predominantly express NKG2C and activating or inhibitory KIR (Non-Patent Document 5).

[0013] Antibodies against KIR2DL1 / 2 / 3 include lirilumab (BMS-986015, human IgG4), which is currently undergoing clinical trials as an anticancer agent, and the murine antibody Pan2D (NKVFS1, mouse IgG1). Antibodies against KIR3DL1, such as those described in patent document WO 2018 / 148223 A1, have also been reported as potential anticancer agents. Cancer cells have a mechanism for evading cytocidal activity by reducing the expression of activating ligands for immune cell receptors or enhancing the expression of inhibitory ligands. HLA expressed on the surface of cancer cells also acts on inhibitory receptors on NK cells, utilizing the missing-self function to avoid attack by NK cells. It has been reported that the mechanism of action of Lirilumab and Pan2D antibodies is to bind to inhibitory KIR KIR2DL1 / 2 / 3, inhibiting their association with HLA, and promoting the activation of NK cells against cancer (Non-Patent Document 6, Patent Document 1), but there is no description of their agonistic activity against inhibitory KIR.

[0014] To solve the problem of immune cell exclusion due to HLA mismatch between host and donor, Dr. David Russell et al. reported a method in which B2M (beta-2 microglobulin), a part of the protein structure of HLA, is deleted by gene editing, thereby suppressing the expression of Class I HLA on the cell membrane and escaping recognition by CD8-positive T cells. This results in cells lacking HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, and HLA-G on the cell surface. On the other hand, to avoid exclusion by NK cells, HLA-E (naturally a heterotrimer) was forcibly expressed as a single-chain protein on the cell surface of the same cells at the same time as B2M deletion. This resulted in cells expressing only HLA-E, without expressing other HLAs (hereinafter referred to as "universal cell technology"), and it has been reported that this suppressed the cytotoxic activity of NK cells (Patent Document 2). This universal cell technology has been widely used, for example, in the development of allogeneic CAR (chimeric antigen receptor)-T cells (Non-Patent Document 7).

[0015] Antibodies against human NKG2A include monalizumab (IPH2201, Human IgG4, Patent Document 4), which is undergoing clinical trials as an anticancer agent. Additionally, antibodies described in several patent documents (Patent Document 5, US 2015 / 0125464 A1, WO2016 / 041947 A1) have also been reported as candidate anticancer agents. Cancer cells have mechanisms for evading cytocidal activity by reducing the expression of activating ligands for immune cell receptors or enhancing the expression of inhibitory ligands. HLA expressed on the surface of cancer cells also acts on inhibitory receptors on NK cells, utilizing the missing-self function to suppress attacks from NK cells. It has been reported that the mechanism of action of monalizumab and other antibodies against NKG2A as anticancer agents is to bind to NKG2A, inhibit its association with HLA-E, and promote the activation of NK cells against cancer (Patent Documents 4 and 5), but there is no description of their agonistic activity against NKG2A.

[0016] As another technique for evading immune exclusion, it has been reported that cells expressing on their surface an agonist that targets CD45, which is widely expressed on NK cells and T cells, suppressed the cytocidal activity of CD45-expressing immune cells (Patent Document 3).

[0017] International Publication No. WO2006 / 003179 International Publication No. WO2012 / 145384 International Publication No. WO2021 / 113853 International Publication No. WO2016 / 041947 U.S. Publication No. US20200199226

[0018] NK cell receptors: of missing sugar and missing self., P Parham et al., Curr Biol. 2000 Mar 9;10(5):R195-7.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.The CD94 / NKG2 family of receptors: from molecules and cells to clinical relevance. F. Borrego et al., Immunol Res. 2006;35(3):263-78.Negative signaling by inhibitory receptors: the NK cell paradigm. E O Long et al., Immunol Rev. 2008 Aug;224:70-84.A subpopulation of human peripheral blood NK cells that lacks inhibitory reseptors fwor self-MHC is developmentally immature.Sarah Cooley,et al.Blood.2007 Jul 15.110(2): 578-586.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. Endowing universal CAR T-sell with immune-evasive properties using TALEN-gene editing. Sumin Jo, et al. Nat Commun. 2022 Nov 30.13, 3453. ;

[0019] The universal cell technology disclosed in Patent Document 2 has the following problem. Not all NK cells possessed by a human individual express NKG2A. For example, among NK cells contained in human peripheral blood mononuclear cells (PBMCs) and identified as CD56-positive and CD3-negative, CD56bright NK cells tend to predominantly express NKG2A, while CD56dim NK cells tend to have low NKG2A expression and express other NK receptors such as NKG2C and KIR. In other words, HLA-E-transfected cells incorporating universal cell technology may be attacked by NKG2C-positive NK cells that express low or no NKG2A, such as CD56dim NK cells with high cytotoxic activity, because HLA-E binds to NKG2C and activates the cytotoxic activity of the NK cells.

[0020] Non-Patent Document 7 shows that when CAR-T cells equipped with universal technology are co-cultured with NK cells obtained from a donor with a higher proportion of NKG2C-positive NK cells than NKG2A-positive NK cells, the CAR-T cells equipped with universal technology are attacked by the NKG2C-positive NK cells, resulting in a significant decrease in cell viability. These results suggest that the activation of host NK cells via NKG2C of HLA-E in universal technology is a non-negligible issue in immune evasion of allogeneic cell technology.

[0021] Regarding the technology disclosed in Patent Document 2, because CD45 is expressed on T cells, there is a risk that loading a CD45 agonist onto CAR-T cells, for example, may suppress their own function and activity. Furthermore, since CD45, a target molecule expressed on a wide range of immune cells, is targeted, there is a risk of side effects and the inability to avoid treatment cells by rejection if they become cancerous. For these reasons, there is a need for a technology to avoid immune rejection that uses an agonist substance that targets a target that has the minimum necessary expression distribution, rather than a wide range, and is directly linked to the properties of immune cells.

[0022] The expression distribution of NKG2A and KIR on the surface of NK cells is inversely correlated (Non-Patent Document 2), and NK cells with low NKG2A expression have high KIR expression levels. This suggests that, in universal cell technology, HLA-E-introduced cells are effective in cells with high NKG2A expression, but are not effective in NK cells with low or no NKG2A expression and inhibitory KIR expression. On the other hand, since the ligands for inhibitory KIR are HLA-A, HLA-B, and HLA-C, direct introduction of these is incompatible with avoiding TCR-mediated rejection of recipient T cells, and therefore a substance with HLA-like agonist activity is required.

[0023] Monalizumab, a known antibody against NKG2A, and other antibodies against NKG2A as anticancer drugs have been reported to inhibit the inhibitory signal induced by natural ligand binding to NKG2A. However, it was unclear whether these antibodies exhibited agonistic activity against NKG2A. It was also unclear whether these antibodies did not exhibit agonistic activity against NKG2C.

[0024] Lirilumab (BMS-986015, human IgG4), known as an antibody against KIR, and the mouse antibody Pan2D (NKVFS1, mouse IgG1) have been reported to inhibit the inhibitory signal induced by natural ligand binding to inhibitory KIR, but their effects in the absence of ligand were unknown.

[0025] The present inventors have discovered that by expressing scFvs (single chain Fvs) created from the gene sequences of multiple anti-NKG2A antibodies on the cell surface as membrane proteins, they can exhibit agonistic activity against NKG2A, similar to cells expressing HLA-E on the cell surface, and thereby avoid attack from NKG2A-positive NK cells. Furthermore, they have discovered that, while HLA-E also has binding affinity to NKG2C, one of the scFvs is an NKG2A-specific scFv that exhibits high binding affinity to NKG2A but no binding affinity to NKG2C.

[0026] When cells expressing HLA-E or NKG2A-specific scFv on the cell surface were co-cultured with NKG2C-positive NK cells, the HLA-E-expressing cells activated the NKG2C-positive NK cells, whereas the NKG2A-specific scFv-expressing cells did not activate the NKG2C-positive NK cells, demonstrating that the scFv has NKG2A-specific agonistic activity.

[0027] Furthermore, the present inventors have found that when cells co-expressing an scFv having specific agonistic activity against NKG2A and an scFv that binds to an inhibitory KIR and exhibits agonistic activity are co-cultured with NKG2A-positive NK cells or NKG2C-positive and inhibitory KIR-positive NK cells, the cytocidal activity of the NKG2A-positive NK cells and the NKG2C-positive and inhibitory KIR-positive NK cells is suppressed.

[0028] Furthermore, with regard to the invention of inhibitory KIR agonists, we have discovered that lirilumab exhibits agonistic activity against inhibitory KIR on NK cells in the absence of a ligand. We have also discovered that cells expressing scFv containing the gene sequence on the cell membrane surface can avoid attack by NK cells. We have also discovered that Pan2D (NKVSF1), which binds to KIR, also exhibits agonistic activity against inhibitory KIR both in soluble form and on the cell surface. However, we have confirmed that lirilumab and Pan2D bind not only to inhibitory KIR but also to activating KIR, and enhance NK cell activity through their agonistic action against activating KIR (KIR2DS1, 2, 4, etc.).

[0029] Based on this, the inventors decided to obtain an agonist binder that has selectivity for activating KIR, binds only to inhibitory KIR, and further binds to inhibitory KIR. As a result, by phage panning using a human antibody phage library, they identified 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. Further investigation led to the completion of the present invention.

[0030] That is, the present invention provides the following: [1] A substance that binds to a human inhibitory KIR and inhibits the cytocidal activity of NK cells. [2] The substance of [1], which is a polypeptide containing a binding site for a human inhibitory KIR. [3] The substance of [2], wherein the binding site for a human inhibitory KIR comprises amino acid sequences derived from the heavy chain variable region and light chain variable region of a monoclonal antibody or antigen-binding fragment that binds to a human inhibitory KIR. [4] The substance of [1], which has a higher binding activity for a human inhibitory KIR than for the corresponding human activating KIR. [5] The substance of [1], which has a higher agonistic activity for a human inhibitory KIR than for the corresponding human activating KIR. [6] The substance of [1], which exhibits substantially no agonistic activity for the corresponding human activating KIR. [7] The substance of [2], wherein the polypeptide is any one of the following (1) to (3): (1) a polypeptide whose binding site to KIR2DL2 and / or KIR2DL3 contains a C1 epitope motif sequence (in which 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, HLA-B46, or B73 belonging to the HLA-C1 type, and whose binding ability to TCR is deleted or weakened; (2) (3) A polypeptide whose binding site to KIR2DL1 comprises a C2 epitope motif sequence (in which the 77th amino acid is N, and the 80th amino acid is K) contained in HLA-Cw2, Cw4, Cw5, Cw6, Cw15, or Cw17 belonging to the HLA-C2 type, and whose TCR-binding ability is deleted or weakened; (4) A polypeptide whose binding site to KIR3DL1 comprises a Bw4 epitope motif sequence (in which the 77th amino acid is N, the 80th amino acid is I or T, and the 83rd amino acid 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 the HLA-Bw4 type, and whose TCR-binding ability is deleted or weakened.[8] The substance according to [3], characterized in that the human inhibitory KIR is KIR2DL2 and / or KIR2DL3, and the binding site comprises an amino acid sequence derived from the heavy chain variable region and light chain variable region of a monoclonal antibody or antigen-binding fragment that binds to KIR2DL2 and / or KIR2DL3. [9] The substance of [8], wherein the monoclonal antibody or antigen-binding fragment that binds to KIR2DL2 and / or KIR2DL3 competes with Lirilumab, Pan2D, or an scFv consisting of any of the following amino acid sequences (i) to (xii) for binding to KIR2DL2 and / or KIR2DL3: (i) an amino acid sequence comprising a combination of the amino acid sequence of a heavy chain variable region of SEQ ID NO: 33 and the amino acid sequence of a light chain variable region of SEQ ID NO: 34; (ii) an amino acid sequence comprising a combination of the amino acid sequence of a heavy chain variable region of SEQ ID NO: 35 and the amino acid sequence of a light chain variable region of SEQ ID NO: 36; (iii) an amino acid sequence comprising a combination of the amino acid sequence of a heavy chain variable region of SEQ ID NO: 37 and the amino acid sequence of a light chain variable region of SEQ ID NO: 38; (iv) an amino acid sequence comprising a combination of the amino acid sequence of a heavy chain variable region of SEQ ID NO: 39 and the amino acid sequence of a light chain variable region of SEQ ID NO: 40; (v) (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 a heavy chain variable region of SEQ ID NO: 63 and the amino acid sequence of a light chain variable region of SEQ ID NO: 64. [9-2] The substance of [9], characterized in that it competes with an scFv consisting of an amino acid sequence comprising a combination of the amino acid sequence of a heavy chain variable region of SEQ ID NO: 51 and the amino acid sequence of a light chain variable region of SEQ ID NO: 52.

[10] The substance of [8], characterized in that the binding site for KIR2DL2 and / or KIR2DL3 comprises a variable region including amino acids positioned adjacent to 1 to 6 amino acids including Asp at position 47 in the region of amino acids 44 to 49, 2 to 4 amino acids including Gly at position 67 and Pro at position 68 in the region of amino acids 65 to 68, and 1 to 4 amino acids including Asp at position 72 in the region of amino acids 70 to 73 in the amino acid sequence of KIR2DL2, in a model structural analysis based on the X-ray crystal structure of the binding complex with KIR2DL2.

[11] The substance of

[10] , wherein, in the binding site to KIR2DL2 and / or KIR2DL3, the amino acid calculated by the structural analysis to interact with Asp at position 47 of SEQ ID NO: 6 is Tyr, the amino acid calculated by the structural analysis to interact with Gly at position 67 of SEQ ID NO: 6 is Gly, the amino acid calculated by the structural analysis to interact with Pro at position 68 of SEQ ID NO: 6 is Tyr, and the amino acid calculated by the structural analysis to interact with Asp at position 72 of SEQ ID NO: 6 is Thr, Asn, Gly, His, Ile, Ser, Val, or Ala.

[12] The substance of

[11] , wherein the amino acid contained in the variable region specified in

[11] belongs to CDR3.

[13] The substance of [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 which may have one or two amino acid mutations in each CDR: (I) a binding site comprising a combination of a first variable region comprising amino acid sequences derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 33 and a second variable region comprising amino acid sequences 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) (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: 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. [13-2] The substance of

[13] , which is a polypeptide comprising 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.

[14] The substance of

[13] , which may have one or two amino acid mutations on the C- and / or N-terminal side of each CDR. [14-2] The substance according to

[13] or

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

[15] The substance of

[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. [15-2] The substance of

[15] , which is a polypeptide comprising 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.

[16] The substance of [8], which has higher binding activity to KIR2DL2 and / or KIR2DL3 than to KIR2DS1, KIR2DS2 and / or KIR2DS4.

[17] The substance of [8], which has higher agonist activity to KIR2DL2 and / or KIR2DL3 than to KIR2DS1, KIR2DS2 and / or KIR2DS4.

[18] The substance of

[17] , which exhibits substantially no agonist activity to KIR2DS1, KIR2DS2 and / or KIR2DS4.

[19] The substance according to

[17] , which does not substantially bind to KIR2DS1, KIR2DS2 and KIR2DS4.

[20] The substance according to

[19] , characterized in that it contains an antigen-binding fragment obtained by the following method: Step A: mixing a labeled solubilized protein containing the extracellular domain of KIR2DL2 or KIR2DL3 with a suspension containing a polyclonal population of presenters including presenters that present on their surface an antigen-binding site that binds to KIR2DL2 or KIR2DL3, and then contacting the suspension with a carrier on which a substance that specifically binds to the labeled portion is immobilized, and recovering the presenters bound to the carrier, thereby obtaining a population of presenters that present an antigen-binding site that binds to KIR2DL2 or KIR2DL3; Step B: A step of mixing labeled solubilized proteins containing the extracellular domains of KIR2DS1, KIR2DS2, and KIR2DS4 into a suspension containing the presenter population obtained in Step A, bringing the suspension into contact with a carrier on which a substance that binds to the label is immobilized, and recovering presenters that do not bind to the carrier, thereby removing presenters that present antigen-binding sites that bind to KIR2DS1, KIR2DS2, and KIR2DS4 from the cell population; Step C: A step of performing genetic analysis of the presenters obtained by a method including the above steps and identifying the gene sequence that encodes the antigen-binding site; and Step D: A step of expressing a gene containing the identified antigen-binding site in a cell.

[21] The substance of

[19] , characterized in that it contains an antigen-binding fragment obtained by the following method: Step A: mixing a labeled solubilized protein containing the extracellular domain of KIR2DL2 or KIR2DL3 and unlabeled solubilized proteins containing the extracellular domains of KIR2DS1, KIR2DS2, and KIR2DS4 with a suspension containing a polyclonal population of presenters, including presenters that present on their surface an antigen-binding site that binds to KIR2DL2 or KIR2DL3, and then contacting the suspension with a carrier on which a substance that specifically binds to the labeled portion has been immobilized, and recovering the presenters bound to the carrier, thereby obtaining a population of presenters that present an antigen-binding site that specifically binds to KIR2DL2 or KIR2DL3; Step B: performing genetic analysis of the presenters obtained by the method comprising the above steps, and identifying the gene sequence encoding the antigen-binding site; and Step C: expressing the gene containing the identified antigen-binding site in cells.

[22] The substance of

[20] or

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

[23] The substance of

[20] or

[21] , wherein the substance is obtained by a method further comprising a step of selecting a substance that has agonistic activity against KIR2DL2 or KIR2DL3.

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

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

[25] The substance of

[24] , wherein the first variable region and the second variable region are linked via a GS linker or a G4S linker, and the polypeptide is a single-chain antibody or scFv.

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

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

[27] The substance according to [3], wherein the inhibitory KIR is KIR3DL1, and the binding site comprises amino acid sequences derived 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 of

[27] , wherein the monoclonal antibody or antigen-binding fragment that binds to KIR3DL1 competes for binding to KIR3DL1 with an scFv consisting of any of the following amino acid sequences (xiii) to (xviii): (xiii) an amino acid sequence comprising a combination of the amino acid sequence of a heavy chain variable region of SEQ ID NO: 56 and the amino acid sequence of a light chain variable region of SEQ ID NO: 55; (xiv) an amino acid sequence comprising a combination of the amino acid sequence of a heavy chain variable region of SEQ ID NO: 137 and the amino acid sequence of a light chain variable region of SEQ ID NO: 138; (xv) an amino acid sequence comprising a combination of the amino acid sequence of a heavy chain variable region of SEQ ID NO: 139 and the amino acid sequence of a light chain variable region of SEQ ID NO: 140; (xvi) an amino acid sequence comprising a combination of the amino acid sequence of a heavy chain variable region of SEQ ID NO: 141 and the amino acid sequence of a light chain variable region of SEQ ID NO: 142; (xvii) (xviii) an amino acid sequence comprising a combination of the amino acid sequence of a heavy chain variable region of SEQ ID NO: 145 and the amino acid sequence of a light chain variable region of SEQ ID NO: 146.

[29] The substance of

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

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

[31] The substance of

[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;(XIV) 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

[27] , characterized in that its binding activity to KIR3DL1 is higher than its binding activity to KIR3DS1.

[33] The substance according to

[27] , which has higher agonist activity against KIR3DL1 than against KIR3DS1.

[34] The substance according to

[33] , which exhibits substantially no agonist activity against KIR3DS1.

[35] The substance according to

[34] , which does not substantially bind to KIR3DS1.

[36] The substance according to

[35] , characterized in that it comprises an antigen-binding fragment obtained by a method comprising the following steps: Step A: mixing a labeled solubilized protein comprising the extracellular domain of KIR3DL1 with a suspension containing a polyclonal presenter population including presenters that present an antigen-binding site that binds to KIR3DL1 on their surface, then contacting the suspension with a carrier on which a substance that specifically binds to the label is immobilized, and recovering presenters that bind to the carrier, thereby obtaining a presenter population that presents an antigen-binding site that binds to KIR3DL1; Step B: mixing a labeled solubilized protein comprising the extracellular domain of KIR3DS1 with a suspension containing the presenter population obtained in Step A, then contacting the suspension with a carrier on which a substance that binds to the label is immobilized, and recovering presenters that do not bind to the carrier, thereby removing presenters that present an antigen-binding site that binds to KIR3DS1 from the cell population;

[0023] Step C: performing genetic analysis of the presenter obtained by the method comprising the steps to identify a gene sequence encoding the antigen-binding site, and Step D: expressing the gene comprising the identified antigen-binding site in cells.

[37] The substance of

[36] , characterized by comprising an antigen-binding fragment obtained by the following method: Step A: mixing a labeled solubilized protein comprising the extracellular domain of KIR3DL1 and an unlabeled solubilized protein comprising the extracellular domain of KIR3DS1 with a suspension containing a polyclonal population of presenters including presenters that present an antigen-binding site that binds to KIR3DL1 on their surface, and then contacting the suspension with a carrier on which a substance that specifically binds to the labeled moiety is immobilized, and recovering the presenters bound to the carrier, thereby obtaining a population of presenters that present an antigen-binding site that specifically binds to KIR3DL1, Step B: performing genetic analysis of the presenter obtained by the method comprising the steps to identify a gene sequence encoding the antigen-binding site, and Step C: expressing the gene comprising the identified antigen-binding site in cells.

[38] The substance of

[36] or

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

[39] The substance of any of

[36] to

[38] , wherein the substance is obtained by a method further comprising a step of selecting a substance having agonistic activity against KIR3DL1.

[40] The substance of any of

[27] to

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

[41] The substance of

[40] , wherein the polypeptide is a single-chain antibody or scFv, wherein the first variable region and the second variable region are linked via a GS linker or a G4S linker.

[42] The substance of any of

[27] to

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

[43] The substance according to [3], wherein the inhibitory KIR is KIR2DL1, and the binding site comprises amino acid sequences derived 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

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

[45] The substance of

[43] , wherein the monoclonal antibody or antigen-binding fragment that binds to KIR2DL1 competes for binding to KIR2DL1 with an scFv consisting of any of the following amino acid sequences (ci) to (cxv): (ci) an amino acid sequence comprising a combination of the amino acid sequence of a heavy chain variable region of SEQ ID NO: 107 and the amino acid sequence of a light chain variable region of SEQ ID NO: 108; (cii) an amino acid sequence comprising a combination of the amino acid sequence of a heavy chain variable region of SEQ ID NO: 109 and the amino acid sequence of a light chain variable region of SEQ ID NO: 110; (ciii) an amino acid sequence comprising a combination of the amino acid sequence of a heavy chain variable region of SEQ ID NO: 111 and the amino acid sequence of a light chain variable region of SEQ ID NO: 112; (civ) an amino acid sequence comprising a combination of the amino acid sequence of a heavy chain variable region of SEQ ID NO: 113 and the amino acid sequence of a light chain variable region of SEQ ID NO: 114; (cv) an amino acid sequence comprising a combination of the amino acid sequence of a heavy chain variable region of SEQ ID NO: 115 and the amino acid sequence of a light chain variable region of SEQ ID NO: 116; (cvi) an amino acid sequence comprising a combination of the amino acid sequence of a heavy chain variable region of SEQ ID NO: 117 and the amino acid sequence of a light chain variable region of SEQ ID NO: 118; (cvii) an amino acid sequence comprising a combination of the amino acid sequence of a heavy chain variable region of SEQ ID NO: 119 and the amino acid sequence of a light chain variable region of SEQ ID NO: 120; (cviii) an amino acid sequence comprising a combination of the amino acid sequence of a heavy chain variable region of SEQ ID NO: 121 and the amino acid sequence of a light chain variable region of SEQ ID NO: 122; (cix) an amino acid sequence comprising a combination of the amino acid sequence of a heavy chain variable region of SEQ ID NO: 123 and the amino acid sequence of a light chain variable region of SEQ ID NO: 124; (cx) an amino acid sequence comprising a combination of the amino acid sequence of a heavy chain variable region of SEQ ID NO: 125 and the amino acid sequence of a light chain variable region of SEQ ID NO: 126; (cxi) an amino acid sequence comprising a combination of the amino acid sequence of a heavy chain variable region of SEQ ID NO: 127 and the amino acid sequence of a 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 a heavy chain variable region of SEQ ID NO: 131 and the amino acid sequence of a light chain variable region of SEQ ID NO: 132; (cxiv) an amino acid sequence comprising a combination of the amino acid sequence of a heavy chain variable region of SEQ ID NO: 133 and the amino acid sequence of a light chain variable region of SEQ ID NO: 134; and (cxv) an amino acid sequence comprising a combination of the amino acid sequence of a heavy chain variable region of SEQ ID NO: 135 and the amino acid sequence of a light chain variable region of SEQ ID NO: 136.

[46] The substance of

[43] , which is a polypeptide comprising any one of the following binding sites (CI) to (CXV) as a binding site for KIR2DL1, and which may have one or two amino acid mutations in each CDR; (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 the 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; (CIX) 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) (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: 125 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2 and CDR3 of SEQ ID NO: 126; (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: 127 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2 and CDR3 of SEQ ID NO: 128; (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: 129 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2 and CDR3 of SEQ ID NO: 130; (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: 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) 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: 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 of

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

[48] The substance of

[46] , which is a polypeptide comprising any 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) (CVI) 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; (CVII) 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; (CIX) 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) (CXII) 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) A binding site comprising a combination of 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 of

[43] , which has higher binding activity to KIR2DL1 than to KIR2DS1.

[50] The substance of

[43] , which has higher agonistic activity to KIR2DL1 than to KIR2DS1.

[51] The substance of

[50] , which exhibits substantially no agonistic activity to KIR2DS1.

[52] The substance according to

[51] , which does not substantially bind to KIR2DS1, KIR2DS2 and KIR2DS4.

[53] The substance of

[52] , characterized in that it contains an antigen-binding fragment obtained by the following method: Step A: mixing a labeled solubilized protein containing the extracellular domain of KIR2DL1 with a suspension containing a polyclonal presenter population including presenters that present an antigen-binding site that binds to KIR2DL1 on their surface, then contacting the suspension with a carrier on which a substance that specifically binds to the label is immobilized, and recovering presenters that bind to the carrier, thereby obtaining a presenter population that presents an antigen-binding site that binds to KIR2DL1; Step B: mixing a labeled solubilized protein containing the extracellular domains of KIR2DS1, KIR2DS2, and KIR2DS4 with a suspension containing the presenter population obtained in Step A, then contacting the suspension with a carrier on which a substance that binds to the label is immobilized, and recovering presenters that do not bind to the carrier, thereby removing presenters that present antigen-binding sites that bind to KIR2DS1, KIR2DS2, and KIR2DS4 from the cell population; Step C: performing genetic analysis of the display body obtained by the method comprising the steps above to identify the gene sequence encoding the antigen-binding site; and Step D: expressing the identified gene comprising the antigen-binding site in a cell.

[54] The substance of

[52] , characterized in that it contains an antigen-binding fragment obtained by a method comprising the following steps: Step A: mixing a labeled solubilized protein containing the extracellular domain of KIR2DL1 and unlabeled solubilized proteins containing the extracellular domains of KIR2DS1, KIR2DS2, and KIR2DS4 with a suspension containing a polyclonal population of presenters, including presenters that present an antigen-binding site that binds to KIR2DL1 on their surface, and then contacting the suspension with a carrier on which a substance that specifically binds to the labeled portion has been immobilized, and recovering the presenters bound to the carrier, thereby obtaining a population of presenters that present an antigen-binding site that specifically binds to KIR2DL1; Step B: performing genetic analysis of the presenters obtained by the method comprising the steps and identifying the gene sequence encoding the antigen-binding site; and Step C: expressing the gene containing the identified antigen-binding site in cells.

[55] The substance of

[53] or

[54] , wherein the combination of the label and the substance binding to the label is a biotin-avidin combination.

[56] The substance of any of

[53] to

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

[57] The substance of any of

[43] to

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

[58] The substance of

[57] , wherein the polypeptide is a single-chain antibody or scFv, characterized in that the first variable region and the second variable region are linked via a GS linker.

[59] The substance of any of

[43] to

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

[60] A pharmaceutical composition comprising as an active ingredient any of the substances of [1] to

[59] .

[61] The pharmaceutical composition of

[60] , wherein the pharmaceutical composition is an immunorejection suppressant.

[62] A cell expressing on its cell surface a substance that binds to any of the inhibitory KIRs [1] to

[59] and inhibits the cytocidal activity of NK cells.

[63] The cell of

[62] , further expressing on its cell surface a ligand substance for NKG2A.

[64] The cell of

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

[65] The cell of

[62] , further having lost or attenuated expression of at least one Class I HLA on the cell surface.

[66] The cell of

[65] , wherein the lost or attenuated expression of at least one Class I HLA on the cell surface is due to at least one modification (preferably a B2M modification, more preferably a B2M deletion) selected from the group consisting of Class I HLA, B2M, TAP1, TAP2, and TAPBP regions in the genome, achieved by gene editing.

[67] The cell of

[65] , which is a cell line established from an individual with genetically lost HLA expression.

[68] The cell of

[62] , further having a ligand substance for NKG2A expressed on the cell surface and having lost or attenuated expression of at least one Class I HLA on the cell surface.

[69] The cell of

[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 liver cell, a kidney cell, or a pancreatic cell.

[70] The cell of

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

[71] The cell of any of

[62] to

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

[72] The cell of

[62] , wherein the inhibitory KIR is KIR2DL2 and / or KIR2DL3, and the substance that binds to KIR2DL2 and / or KIR2DL3 and has the activity of inhibiting the cytotoxic activity of NK cells is the membrane protein of

[26] .

[73] The cell according to

[72] , wherein the membrane protein that binds to KIR2DL2 and / or KIR2DL3 comprises a binding site that competes with Lirilumab, Pan2D, or an 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 a heavy chain variable region of SEQ ID NO: 33 and the amino acid sequence of a light chain variable region of SEQ ID NO: 34; (ii) an amino acid sequence comprising a combination of the amino acid sequence of a heavy chain variable region of SEQ ID NO: 35 and the amino acid sequence of a light chain variable region of SEQ ID NO: 36; (iii) an amino acid sequence comprising a combination of the amino acid sequence of a heavy chain variable region of SEQ ID NO: 37 and the amino acid sequence of a 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 of

[72] , which expresses on its cell surface a membrane protein comprising any one of the following binding sites (I) to (XII) as a binding site for KIR2DL2 and / or KIR2DL3, and which 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) (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: 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.

[75] The cell of

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

[76] The cell of

[62] , wherein the inhibitory KIR is KIR3DL1, and the substance that binds to KIR3DL1 and has the activity of suppressing the cytotoxic activity of NK cells is the membrane protein of

[42] .

[77] The cell of

[76] , wherein the membrane-type protein that binds to KIR3DL1 comprises a binding site that competes with an scFv consisting of any of the following amino acid sequences (xiii) to (xviii) for binding to KIR3DL1; (xiii) an amino acid sequence comprising a combination of the amino acid sequence of a heavy chain variable region of SEQ ID NO: 56 and the amino acid sequence of a light chain variable region of SEQ ID NO: 55; (xiv) an amino acid sequence comprising a combination of the amino acid sequence of a heavy chain variable region of SEQ ID NO: 137 and the amino acid sequence of a light chain variable region of SEQ ID NO: 138; (xv) an amino acid sequence comprising a combination of the amino acid sequence of a heavy chain variable region of SEQ ID NO: 139 and the amino acid sequence of a light chain variable region of SEQ ID NO: 140; (xvi) an amino acid sequence comprising a combination of the amino acid sequence of a heavy chain variable region of SEQ ID NO: 141 and the amino acid sequence of a light chain variable region of SEQ ID NO: 142; (xvii) an amino acid sequence comprising a combination of the amino acid sequence of a heavy chain variable region of SEQ ID NO: 143 and the amino acid sequence of a light chain variable region of SEQ ID NO: 144; (xviii) An amino acid sequence comprising a combination of the amino acid sequence of a heavy chain variable region of SEQ ID NO: 145 and the amino acid sequence of a light chain variable region of SEQ ID NO: 146.

[78] The cell of

[75] , which expresses on its cell surface a membrane protein comprising any one of the following binding sites (XIII) to (XVIII) as a binding site for KIR3DL1, which may have one or two amino acid mutations in each CDR; (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 the 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; and (XVIII) A binding site comprising a combination of a first variable region comprising amino acid sequences derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 145 and a second variable region comprising amino acid sequences derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 146.

[79] The cell of

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

[80] The cell of

[62] , wherein the inhibitory KIR is KIR2DL1, and the substance that binds to KIR2DL1 and has activity of inhibiting the cytotoxic activity of NK cells is the membrane protein of

[59] .

[81] The cell of

[80] , wherein the membrane protein that binds to KIR2DL1 comprises a binding site that competes with Pan2D for binding to KIR2DL1.

[82] The cell of

[78] , which expresses on its cell surface a membrane protein comprising the following binding site (P) as a binding site for KIR2DL1, and which may have one or two amino acid mutations in each CDR: (P) A binding site comprising a combination of a first variable region comprising amino acid sequences derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 31 and a second variable region comprising amino acid sequences derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 32.

[83] The cell of

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

[84] The cell of

[80] , wherein the membrane protein that binds to KIR2DL1 comprises a binding site that competes with an scFv consisting of any of the following amino acid sequences (ci) to (cxv) for binding to KIR2DL1: (ci) an amino acid sequence comprising a combination of the amino acid sequence of a heavy chain variable region of SEQ ID NO: 107 and the amino acid sequence of a light chain variable region of SEQ ID NO: 108; (cii) an amino acid sequence comprising a combination of the amino acid sequence of a heavy chain variable region of SEQ ID NO: 109 and the amino acid sequence of a light chain variable region of SEQ ID NO: 110; (ciii) an amino acid sequence comprising a combination of the amino acid sequence of a heavy chain variable region of SEQ ID NO: 111 and the amino acid sequence of a light chain variable region of SEQ ID NO: 112; (civ) an amino acid sequence comprising a combination of the amino acid sequence of a heavy chain variable region of SEQ ID NO: 113 and the amino acid sequence of a light chain variable region of SEQ ID NO: 114; (cv) an amino acid sequence comprising a combination of the amino acid sequence of a heavy chain variable region of SEQ ID NO: 115 and the amino acid sequence of a light chain variable region of SEQ ID NO: 116; (cvi) an amino acid sequence comprising a combination of the amino acid sequence of a heavy chain variable region of SEQ ID NO: 117 and the amino acid sequence of a light chain variable region of SEQ ID NO: 118; (cvii) an amino acid sequence comprising a combination of the amino acid sequence of a heavy chain variable region of SEQ ID NO: 119 and the amino acid sequence of a light chain variable region of SEQ ID NO: 120; (cviii) an amino acid sequence comprising a combination of the amino acid sequence of a heavy chain variable region of SEQ ID NO: 121 and the amino acid sequence of a light chain variable region of SEQ ID NO: 122; (cix) an amino acid sequence comprising a combination of the amino acid sequence of a heavy chain variable region of SEQ ID NO: 123 and the amino acid sequence of a light chain variable region of SEQ ID NO: 124; (cx) an amino acid sequence comprising a combination of the amino acid sequence of a heavy chain variable region of SEQ ID NO: 125 and the amino acid sequence of a light chain variable region of SEQ ID NO: 126; (cxi) an amino acid sequence comprising a combination of the amino acid sequence of a heavy chain variable region of SEQ ID NO: 127 and the amino acid sequence of a light chain variable region of SEQ ID NO: 128; (cxii) (cxiii) an amino acid sequence comprising a combination of the amino acid sequence of a heavy chain variable region of SEQ ID NO: 129 and the amino acid sequence of a light chain variable region of SEQ ID NO: 130; (cxiii) an amino acid sequence comprising a combination of the amino acid sequence of a heavy chain variable region of SEQ ID NO: 131 and the amino acid sequence of a 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 of

[80] , which expresses on its cell surface a membrane protein comprising any one of the following binding sites (CI) to (CXV) as a binding site for KIR2DL1, and which may have one or two amino acid mutations in each CDR; (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) (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: 111 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2 and CDR3 of SEQ ID NO: 112; (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: 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 the 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; (CIX) 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) (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: 125 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2 and CDR3 of SEQ ID NO: 126; (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: 127 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2 and CDR3 of SEQ ID NO: 128; (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: 129 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2 and CDR3 of SEQ ID NO: 130; (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: 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) 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: 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 of

[85] , which may have one or two amino acid mutations at the C- and / or N-terminus of each CDR.

[87] A pharmaceutical composition comprising any of the cells of

[62] to

[86] as an active ingredient, which reduces the risk of immune rejection in a recipient of administration or transplantation.

[88] The pharmaceutical composition of

[87] , which is an immune rejection suppressant.

[89] A method for reducing or suppressing immune rejection of therapeutic cells by a recipient, which comprises the step of expressing any of the substances of [1] to

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

[90] A method for producing therapeutic cells, which reduces or suppresses the risk of immune rejection by a recipient, which comprises the step of expressing any of the substances of [1] to

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

[91] A method for evaluating the immune rejection avoidance activity of a test substance due to its agonistic activity against inhibitory KIR2DL2 or KIR2DL3, comprising the following steps: (A) contacting in vitro NK cells derived from a subject whose HLA-C type is C1 / C1 homozygous with target cells derived from a subject whose HLA-C type is C2 / C2 homozygous in the presence of a test substance that binds to KIR2DL2 or KIR2DL3 (wherein the NK cell donor and the target cell donor have the same Bw4 motif-containing HLA phenotype); and (B) measuring the number of surviving target cells after step A.

[92] A method for evaluating the immune rejection avoidance activity of a test substance due to its agonistic activity against inhibitory KIR2DL1, comprising the following steps: (A) contacting in vitro NK cells derived from a subject whose HLA-C type is C2 / C2 homozygous with target cells derived from a subject whose HLA-C type is C1 / C1 homozygous in the presence of a test substance that binds to KIR2DL1 (wherein the NK cell donor and the target cell donor have the same Bw4 motif-containing HLA phenotype); and (B) measuring the number of surviving target cells after step A.

[93] A method for evaluating the immune rejection avoidance activity of a test substance due to its agonistic activity against inhibitory KIR3DL1, comprising the following steps: (A) contacting in vitro, in the presence of a test substance that binds to KIR3DL1, NK cells derived from a subject whose HLA-Bw4 type is positive / homopositive or heteropositive / negative, with target cells derived from a subject whose HLA-Bw4 type is negative / homonegative (wherein the HLA-C type phenotype of the NK cell donor and that of the target cell donor are identical);(B) A step of measuring the number of surviving target cells after step A.

[94] A method for screening for a substance having immune rejection evasion activity, comprising a step of selecting, as a substance having immune rejection evasion activity, a test substance that results in a high number of surviving target cells in step B in any of the methods

[91] to

[93] .

[95] Any of the methods

[91] to

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

[96] A method for producing a substance that binds to a human inhibitory KIR and has the activity of inhibiting NK cells, comprising the following steps: Step A: mixing a labeled solubilized protein comprising the extracellular domain of the inhibitory KIR with a suspension containing a polyclonal presenter population including presenters that present on their surface an antigen-binding site that binds to the inhibitory KIR, then contacting the suspension with a carrier on which a substance that specifically binds to the label is immobilized, and recovering presenters that bind to the carrier, thereby obtaining a presenter population that presents an antigen-binding site that binds to the KIR; Step B: mixing a labeled solubilized protein comprising the extracellular domain of the activating KIR corresponding to the inhibitory KIR with a suspension containing the presenter population obtained in Step A, contacting the suspension with a carrier on which a substance that binds to the label is immobilized, and recovering presenters that do not bind to the carrier, thereby removing presenters that present an antigen-binding site that binds to the activating KIR from the cell population; Step C: performing genetic analysis of the presenters obtained by a method comprising the above steps, and identifying the gene sequence that encodes the antigen-binding site;

[97] A method for producing a substance that binds to human inhibitory KIR and has the activity of inhibiting NK cells, comprising the following steps: Step A: mixing a labeled solubilized protein comprising the extracellular domain of an inhibitory KIR and an unlabeled solubilized protein comprising the extracellular domain of an activating KIR corresponding to the inhibitory form with a suspension containing a polyclonal presenter population including presenters that present on their surface an antigen-binding site that binds to the inhibitory KIR, and then contacting the suspension with a carrier on which a substance that specifically binds to the labeled portion is immobilized, and recovering the presenters bound to the carrier, thereby obtaining a presenter population that presents an antigen-binding site that specifically binds to the inhibitory KIR;Step B: performing genetic analysis of the presenter obtained by the method comprising the steps to identify a gene sequence encoding the antigen-binding site, and Step C: expressing the identified gene containing the antigen-binding site in a cell.

[98] The method of

[96] or

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

[99] Any of the methods of

[96] to

[98] , further comprising a step of selecting a substance that has agonist activity against the inhibitory KIR.

[100] Any of the methods of

[96] to

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

[101] Any of the methods of

[96] to

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

[102] The method according to any one of

[96] to

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

[0031] The present invention further provides the following: <1> A polypeptide that is a membrane protein having an extracellular domain containing an NKG2A-binding site and a transmembrane domain, the polypeptide (1) having activity of binding to NKG2A on the surface of an NK cell and suppressing the NK cell, and (2) having substantially no agonist activity for NKG2C. <2> The polypeptide according to <1>, characterized in that the binding site has a higher binding activity to NKG2A than to NKG2C. <3> The polypeptide according to <1>, characterized in that the binding site does not substantially bind to NKG2C. <4> The polypeptide according to any of <1> to <3>, characterized in that the binding site comprises an amino acid sequence derived from one or two variable regions of a monoclonal antibody or antigen-binding fragment that binds to NKG2A. <5> The polypeptide of <4>, wherein the binding site comprises an amino acid sequence derived from one or two variable regions contained in an antigen-binding fragment obtained by the following method: Step A: mixing a labeled solubilized protein comprising the extracellular domain of NKG2A with a suspension containing a polyclonal presenter population including presenters that present an antigen-binding site that binds to NKG2A on their surface, and then contacting the suspension with a carrier on which a substance that specifically binds to the label moiety is immobilized, and recovering presenters that bind to the carrier, thereby obtaining a presenter population that presents an antigen-binding site that binds to NKG2A; Step B: mixing a labeled solubilized protein comprising the extracellular domain of NKG2C with the suspension containing the presenter population obtained in Step A, contacting the suspension with a carrier on which a substance that binds to the label moiety is immobilized, and recovering presenters that do not bind to the carrier, thereby removing presenters that present an antigen-binding site that binds to NKG2C from the cell population; Step C: performing genetic analysis of the presenters obtained by the method comprising the above steps, and identifying the gene sequence that encodes the antigen-binding site; and Step D: A step of expressing a gene containing the identified antigen-binding site in a cell.<6> The polypeptide of <4>, wherein the binding site comprises an amino acid sequence derived from one or two variable regions of an antigen-binding fragment obtained by the following method: Step A: mixing a labeled solubilized protein comprising the extracellular domain of NKG2A and an unlabeled solubilized protein comprising the extracellular domain of NKG2C with a suspension containing a polyclonal population of presenters, the suspension including presenters that present an antigen-binding site that binds to NKG2A on their surface, and then contacting the suspension with a carrier on which a substance that specifically binds to the label is immobilized, and recovering the presenters bound to the carrier, thereby obtaining a population of presenters that present an antigen-binding site that specifically binds to NKG2A; Step B: performing genetic analysis of the presenters obtained by the method comprising the above steps to identify a gene sequence encoding the antigen-binding site; and Step C: expressing the identified gene comprising the antigen-binding site in a cell. <7> The polypeptide of <5> or <6>, wherein the combination of the label and the substance that binds to the label is a combination of biotin and avidin. <8> The polypeptide of <5> or <6>, characterized in that it is obtained by a method further comprising a step of selecting an antigen-binding fragment having agonistic activity against NKG2A. <9> The polypeptide of any of <1> to <8>, wherein the binding site is a single-chain antibody or scFv, characterized in that the first variable region and the second variable region are linked via a GS linker or a G4S linker. <10> The polypeptide of <1>, characterized in that the binding site competes for binding to NKG2A with an scFv consisting of the following amino acid sequence: (Ni) an amino acid sequence comprising a combination of the amino acid sequence of a heavy chain variable region of SEQ ID NO: 163 and the amino acid sequence of a light chain variable region of SEQ ID NO: 164. <11> The polypeptide of <1>, which comprises the following binding site as the binding site, and which may have one or two amino acid mutations in each CDR: (N-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: 163 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 164.<12> The polypeptide of <11>, which may have one or two amino acid mutations at the C- and / or N-terminus of each CDR. <13> The polypeptide of <11>, which is a polypeptide comprising the following binding site as the binding site: (N-I) A binding site comprising a combination of a first variable region comprising the amino acid sequence of SEQ ID NO: 163 and a second variable region comprising the amino acid sequence of SEQ ID NO: 164. <14> A polynucleotide encoding any one of the polypeptides <1> to <13>. <15> A cell expressing the polypeptide of any one of <1> to <13> on its cell surface. <16> The cell of <15>, further expressing on its cell surface a membrane-type protein exhibiting specific agonistic activity against an inhibitory KIR. Here, the membrane-type protein exhibiting specific agonistic activity against an inhibitory KIR is preferably the membrane-type protein against KIR2DL2 / 3 described in

[26] , the membrane-type protein against KIR3DL1 described in

[42] , or the membrane-type protein against KIR2DL1 described in

[59] . <17> The cell of <16>, wherein the inhibitory KIR is at least one of KIR2DL2 / 3, KIR3DL1, and KIR2DL1 (preferably KIR2DL2 / 3). <18> The cell of any one of <15> to <17>, further comprising: loss or attenuation of expression of at least one Class I HLA on the cell surface. <19> The cell of <18>, wherein loss or attenuation of expression of at least one Class I HLA on the cell surface is due to gene editing-induced alteration of at least one region selected from the group consisting of Class I HLA, B2M, TAP1, TAP2, and TAPBP in the genome (preferably alteration of B2M, more preferably loss of B2M). More preferred such cells are those that satisfy all of the following conditions: The polypeptide according to any one of the above <1> to <13> is expressed on the cell surface, <ii> A membrane-type protein exhibiting specific agonist activity against an inhibitory KIR (preferably at least one selected from the group consisting of the membrane-type protein for KIR2DL2 / 3 described in

[26] , the membrane-type protein for KIR3DL1 described in

[42] , and the membrane-type protein for KIR2DL1 described in

[59] ) is expressed on the cell surface, and <iii> Expression of at least one (preferably all) Class I HLA on the cell surface has been eliminated or attenuated by modification (preferably deletion) of the B2M gene on the genome by gene editing. <20> The cell of <18>, which is a cell line established from an individual in which HLA expression has been genetically eliminated. <21> The cell of any one of <15> to <20>, 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 liver cell, a kidney cell, or a pancreatic cell. <22> The cell of <21>, wherein the pluripotent stem cell is an iPS cell or an ES cell. <23> The cell of any one of <15> to <22>, wherein a chimeric receptor is further expressed on the cell surface. <24> A pharmaceutical composition comprising as an active ingredient any one of the cells of <15> to <23>, thereby reducing the risk of immune rejection in a subject to administration or transplantation. <25> A method for reducing or suppressing immune rejection of therapeutic cells by a subject to be treated, comprising a step of expressing any one of the polypeptides of <1> to <13> on the cell surface of the therapeutic cells.Preferably, the method is similar to the above-mentioned method, and further comprises the step of expressing a membrane-type protein exhibiting specific agonist activity against inhibitory KIR (preferably at least one selected from the group consisting of the membrane-type protein against KIR2DL2 / 3 described in

[26] , the membrane-type protein against KIR3DL1 described in

[42] , and the membrane-type protein against KIR2DL1 described in

[59] ) on the cell surface of the therapeutic cells, and / or the step of modifying at least one gene selected from the Class I HLA, B2M, TAP1, TAP2, and TAPBP regions in the genome (preferably modifying the B2M gene, more preferably deleting the B2M gene) to eliminate or attenuate the expression of at least one (preferably all) Class I HLA on the cell surface by gene editing. <26> A method for producing therapeutic cells with a reduced or suppressed risk of immune rejection by the subject to be treated, comprising the step of expressing any one of the polypeptides described in <1> to <13> on the cell surface of the therapeutic cells. Preferably, the method further comprises the steps of: expressing a membrane-type protein that exhibits specific agonist activity against inhibitory KIR (preferably at least one selected from the group consisting of the membrane-type protein against KIR2DL2 / 3 described in

[26] , the membrane-type protein against KIR3DL1 described in

[42] , and the membrane-type protein against KIR2DL1 described in

[59] ) on the cell surface of the therapeutic cells; and / or modifying at least one gene selected from Class I HLA, B2M, TAP1, TAP2, and TAPBP regions on the genome (preferably modifying the B2M gene, more preferably deleting the B2M gene) to eliminate or attenuate the expression of at least one (preferably all) Class I HLA on the cell surface by gene editing.<27> A method for producing the cells of <15>, comprising the following steps: Step A: mixing a labeled solubilized protein comprising the extracellular domain of NKG2A with a suspension containing a polyclonal presenter population including presenters that present an antigen-binding site that binds to NKG2A on their surface, and then contacting the suspension with a carrier on which a substance that specifically binds to the label is immobilized, and recovering presenters that bind to the carrier, thereby obtaining a presenter population that presents the antigen-binding site that binds to NKG2A; Step B: mixing a labeled solubilized protein comprising the extracellular domain of NKG2C with a suspension containing the presenter population obtained in Step A, contacting the suspension with a carrier on which a substance that binds to the label is immobilized, and recovering presenters that do not bind to the carrier, thereby removing presenters that present the antigen-binding site that binds to NKG2C from the cell population; Step C: performing genetic analysis of the presenters obtained by a method comprising the steps to identify a gene sequence that encodes the antigen-binding site; and <28> A method for producing the cell of <15>, comprising the following steps: step A: mixing a labeled solubilized protein comprising the extracellular domain of NKG2C and an unlabeled solubilized protein comprising the extracellular domain of NKG2C with a suspension containing a polyclonal population of presenters, the suspension including presenters that present an antigen-binding site that binds to NKG2A on their surface, and then contacting the suspension with a carrier on which a substance that specifically binds to the labeled portion is immobilized, and recovering the presenters bound to the carrier, thereby obtaining a population of presenters that present an antigen-binding site that specifically binds to NKG2A; step B: performing genetic analysis of the presenters obtained by a method comprising the above steps, and identifying a gene sequence that encodes the antigen-binding site; and step C: expressing a gene encoding the polypeptide of <1> that comprises the identified antigen-binding site in the extracellular domain in a cell. <29> The method according to <27> or <28>, wherein the combination of the label and the substance that binds to the label is a combination of biotin and avidin. <30> The method according to any one of <27> to <29>, further comprising a step of selecting cells having agonistic activity against NKG2A.This specification includes the disclosure of Japanese Patent Application No. 2024-070975, from which the present application claims priority.

[0032] Substances that bind to NKG2A and have the activity of inhibiting the cytocidal activity of NK cells, or cells that express such substances, can suppress the rejection reaction during organ or therapeutic cell transplantation by inhibiting the cytocidal activity of NK cells. Therefore, substances that bind to NKG2A and have the activity of inhibiting the cytocidal activity of NK cells, or cells that express such substances, can be used as a new allogeneic technology that will greatly change the versatility of organ transplantation and cell therapy.

[0033] Furthermore, substances that bind to inhibitory KIR and have the activity of suppressing the cytocidal activity of NK cells, or cells that express such substances, can suppress the rejection reaction during organ or therapeutic cell transplantation by suppressing the cytocidal activity of NK cells. Therefore, substances that bind to inhibitory KIR and have the activity of suppressing the cytocidal activity of NK cells, or cells that express such substances, can be used as a new allogeneic technology that will greatly change the versatility of organ transplantation and cell therapy.

[0034]

[0033] FIG. 1 shows the results of evaluating the cytocidal activity of KIR2DL2-expressing NK92 against K562, and is a diagram showing the agonist activity of lirilumab against inhibitory KIR2DL2.

[0034] FIG. 1 shows the results of evaluating the cytocidal activity of KIR2DL2-expressing NK92 against HLA-Cw3-K562, and is a diagram showing the inhibitory activity of lirilumab against an agonist for inhibitory KIR2DL2.

[0035] FIG. 1 shows that when anti-KIR antibodies lirilumab and Pan2D are expressed as scFv-membrane proteins, their cytocidal activity against KIR2DL2-NK92 cell lines is attenuated.

[0036] FIG. 1 shows that binders that exhibit binding affinity to activating KIR enhance the activity of NK cell lines expressing activating KIR. 6A and 6B show the results of evaluating the agonistic activity of KIR2DL2 / 3-specific binders using primary NK cells. FIG. 6A shows the results using NK cells carrying KIR2DL2, KIR2DL3, KIR2DS1, and KIR2DS2 genes, and FIG. 6B shows the results using NK cells carrying KIR2DL3 genes. These figures show that the newly obtained binders bind to KIR2DL2 or 3 and inhibit NK cell activation. 1 shows the results of evaluating whether binders (scFv) that exhibit binding to KIR2DL1 and KIR3DL1 change the cytocidal activity of NK cell lines that express each receptor. FIG. 1 shows an alignment of the full-length amino acid sequences of KIR2DL2*001 (SEQ ID NO: 6, extracellular regions 1-224), KIR2DL2*003 (SEQ ID NO: 57, extracellular regions 1-224), KIR2DL3*001 (SEQ ID NO: 8, extracellular regions 1-224), KIR2DS1*002 (SEQ ID NO: 10, extracellular regions 1-224), KIR2DS2*001 (SEQ ID NO: 12, extracellular regions 1-224), and KIR2DS4*001 (SEQ ID NO: 14, extracellular regions 1-224) relative to KIR2DL1*003 (SEQ ID NO: 4, extracellular regions 1-222).FIG. 1 shows an alignment of the full-length amino acid sequences of KIR2DL2*003 (SEQ ID NO: 57, extracellular regions 1-224), KIR2DL1*003 (SEQ ID NO: 4, extracellular regions 1-224), KIR2DL3*001 (SEQ ID NO: 8, extracellular regions 1-224), KIR2DS1*002 (SEQ ID NO: 10, extracellular regions 1-224), KIR2DS2*001 (SEQ ID NO: 12, extracellular regions 1-224), and KIR2DS4*001 (SEQ ID NO: 14, extracellular regions 1-224) based on KIR2DL2*001 (SEQ ID NO: 6, extracellular regions 1-224).

[0023] Figure 1 shows the alignment of the full-length amino acid sequences of KIR3DL2*002 (SEQ ID NO: 59, extracellular regions 1 to 319) and KIR3DS1*013 (SEQ ID NO: 60, extracellular regions 1 to 319) with KIR3DL1*015 (SEQ ID NO: 16, extracellular regions 1 to 319) as the reference.

[0024] Figure 1 shows the amino acid sequences 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 plasma membrane domain (SEQ ID NO: 27), and a CD8-derived intracellular domain (SEQ ID NO: 28), as well as the amino acid sequences of Lirilumab (1-7F9) VH (SEQ ID NO: 29) and Lirilumab (1-7F9) VL (SEQ ID NO: 30). The underlined sequences show the sequences of CDR1 to CDR3.

[0023] Figure 1 shows the amino acid sequences of Pan2D VH (SEQ ID NO: 31) and Pan2D VL (SEQ ID NO: 32), and the amino acid sequences of a2DL2 / 3-DS1 VH (SEQ ID NO: 33) and a2DL2 / 3-DS1 VL (SEQ ID NO: 34). The underlined sequences indicate the sequences of CDR1 to CDR3.

[0024] Figure 1 shows the amino acid sequences of a2DL2 / 3-DS2 VH (SEQ ID NO: 35) and a2DL2 / 3-DS2 VL (SEQ ID NO: 36), and the amino acid sequences of a2DL2 / 3-DS3 VH (SEQ ID NO: 37) and a2DL2 / 3-DS3 VL (SEQ ID NO: 38). The underlined sequences indicate the sequences of CDR1 to CDR3.

[0023] Figure 1 shows the amino acid sequences of a2DL2 / 3-DS4 VH (SEQ ID NO: 39) and a2DL2 / 3-DS4 VL (SEQ ID NO: 40), as well as the amino acid sequences of a2DL2 / 3-DS5 VH (SEQ ID NO: 41) and a2DL2 / 3-DS5 VL (SEQ ID NO: 42). The underlined sequences indicate the sequences of CDR1 to CDR3.

[0024] Figure 1 shows the amino acid sequences of a2DL2 / 3-DS6 VH (SEQ ID NO: 43) and a2DL2 / 3-DS6 VL (SEQ ID NO: 44), as well as the amino acid sequences of a2DL2 / 3-DS7 VH (SEQ ID NO: 45) and a2DL2 / 3-DS7 VL (SEQ ID NO: 46). The underlined sequences indicate the sequences of CDR1 to CDR3.

[0023] Figure 1 shows the amino acid sequences of a2DL2 / 3-DS8 VH (SEQ ID NO: 47) and a2DL2 / 3-DS8 VL (SEQ ID NO: 48), as well as the amino acid sequences of a2DL2 / 3-DS9 VH (SEQ ID NO: 49) and a2DL2 / 3-DS9 VL (SEQ ID NO: 50). The underlined sequences indicate the sequences of CDR1 to CDR3.

[0024] Figure 1 shows the amino acid sequences of a2DL2 / 3-DS10 VH (SEQ ID NO: 51) and a2DL2 / 3-DS10 VL (SEQ ID NO: 52), as well as the amino acid sequences of a3DL1-DS1 VL (SEQ ID NO: 55) and a3DL1-DS1 VH (SEQ ID NO: 56). The underlined sequences indicate the sequences of CDR1 to CDR3.

[0046] Figure 20 shows that expression of a KIR2DL2 / 3-specific binder by CAR-T cells suppresses the cytocidal activity of NK cells against CAR-T cells, while maintaining the cytocidal activity of CAR-T cells against target cells. Figure 20A shows the cytocidal activity of CAR-T cells in wells to which NK cells were not added, and Figure 20B shows the cytocidal activity of CAR-T cells in wells to which NK cells were added. Figure 20B shows the number of PBMC-C-derived T cells in wells co-cultured with PBMC-A-derived NK cells. Figure 20C shows the agonistic activity against inhibitory KIR of a mutant in which every amino acid in the CDR3 of the variable region (VH) of a2DL2 / 3-DS10, a KIR2DL2 / 3-specific binder, is substituted with alanine.1 is a diagram showing the agonistic activity against inhibitory KIR of a CDR-graft in which the CDR of a KIR2DL2 / 3-specific binder is grafted onto the constant region of a different scFv. 2 is a diagram showing the additive inhibitory effect of cells co-expressing a KIR2DL2 / 3-specific binder and HLA-E on the cytocidal activity of NK cells expressing an inhibitory KIR and NKG2A. 3 is a diagram showing the inhibitory effect of iPS cells expressing a KIR2DL2 / 3-specific binder without expressing Class I HLA on the cytocidal activity of NK cells. 4 is a diagram showing a binding model between the Fab fragment of a2DL2 / 3-DS10, a KIR2DL2 / 3-specific binder identified by X-ray crystal structure analysis, and KIR2DL2. 1 is a diagram showing the mapping of the epitope site on KIR2DL2 of a2DL2 / 3-DS10, a KIR2DL2 / 3-specific binder identified by X-ray crystal structure analysis.

[0034] FIG. 1 shows the results of examining the competition between a2DL2 / 3-DS10 and each KIR2DL2 / 3-specific binder in an antigen binding test for KIR2DL2 / 3-specific binders.

[0035] FIG. 1 shows the binding affinity of KIR2DL1-specific binders or KIR3DL1-specific binders newly obtained by phage panning to inhibitory and activating KIRs.

[0036] FIG. 1 shows the agonistic activity of KIR2DL1-specific binders newly obtained by phage panning to inhibitory KIRs.

[0037] FIG. 1 shows the agonistic activity of KIR3DL1-specific binders newly obtained by phage panning to inhibitory KIRs.

[0023] FIG. 1 shows the amino acid sequences of a2DL2 / 3-DS11 VH (SEQ ID NO: 61) and a2DL2 / 3-DS11 VL (SEQ ID NO: 62), as well as the amino acid sequences of a2DL2 / 3-DS12 VH (SEQ ID NO: 63) and a2DL2 / 3-DS12 VL (SEQ ID NO: 64). The underlined sequences represent the sequences of CDR1 to CDR3.7 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), as well as 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).

[0023] FIG. 1 shows the amino acid sequences of a2DL2 / 3-DS1-2 VH (SEQ ID NO: 82) and a2DL2 / 3-DS1-2 VL (SEQ ID NO: 83), as well as the amino acid sequences of a2DL2 / 3-DS2-2 VH (SEQ ID NO: 84) and a2DL2 / 3-DS2-2 VL (SEQ ID NO: 85). The underlined sequences represent the sequences of CDR1 to CDR3. FIG. 1 shows 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) (the underlined sequences indicate the sequences of CDR1 to 3), as well as the amino acid sequence of an HLA-G signal peptide-derived peptide (SEQ ID NO: 88), the amino acid sequence of a G4S linker (SEQ ID NO: 89), the amino acid sequence of HLA-E (SEQ ID NO: 90), the amino acid sequence of an IL-2 signal peptide (SEQ ID NO: 91), the amino acid sequence of a His tag (SEQ ID NO: 92), and the amino acid sequence of a VH signal peptide (SEQ ID NO: 93).

[0023] Figure 1 shows 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), the amino acid sequence of the Fab fragment L-chain constant region (SEQ ID NO:96), 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), the amino acid sequence of the Avi tag (SEQ ID NO:99), the amino acid sequence of the CA19CAR VL (SEQ ID NO:100), and the amino acid sequence of the CA19CAR VH (SEQ ID NO:101).

[0024] Figure 1 shows the amino acid sequences of the CD8-derived plasma membrane domain and intracellular domain (SEQ ID NO:102), the amino acid sequence of an intracellular costimulatory molecule (SEQ ID NO:103), the amino acid sequence of a blue fluorescent protein (SEQ ID NO:104), the amino acid sequence of an IL-21 fragment (SEQ ID NO:105), and the amino acid sequence of the CD8 hinge (62 aa) (SEQ ID NO:106).

[0023] Figure 1 shows the amino acid sequences of a2DL1-DS1 VH (SEQ ID NO: 107) and a2DL1-DS1 VL (SEQ ID NO: 108), as well as the amino acid sequences of a2DL1-DS2 VH (SEQ ID NO: 109) and a2DL1-DS2 VL (SEQ ID NO: 110). The underlined sequences indicate the sequences of CDR1 to CDR3.

[0024] Figure 1 shows the amino acid sequences of a2DL1-DS3 VH (SEQ ID NO: 111) and a2DL1-DS1 VL (SEQ ID NO: 112), as well as the amino acid sequences of a2DL1-DS4 VH (SEQ ID NO: 113) and a2DL1-DS4 VL (SEQ ID NO: 114). The underlined sequences indicate the sequences of CDR1 to CDR3.

[0033] Figure 1 shows the amino acid sequences of a2DL1-DS5 VH (SEQ ID NO: 115) and a2DL1-DS5 VL (SEQ ID NO: 116), as well as the amino acid sequences of a2DL1-DS6 VH (SEQ ID NO: 117) and a2DL1-DS6 VL (SEQ ID NO: 118). The underlined sequences indicate the sequences of CDR1 to CDR3.

[0034] Figure 1 shows the amino acid sequences of a2DL1-DS7 VH (SEQ ID NO: 119) and a2DL1-DS7 VL (SEQ ID NO: 120), as well as the amino acid sequences of a2DL1-DS8 VH (SEQ ID NO: 121) and a2DL1-DS8 VL (SEQ ID NO: 122). The underlined sequences indicate the sequences of CDR1 to CDR3.

[0023] Figure 1 shows the amino acid sequences of a2DL1-DS9 VH (SEQ ID NO: 123) and a2DL1-DS9 VL (SEQ ID NO: 124), as well as the amino acid sequences of a2DL1-DS10 VH (SEQ ID NO: 125) and a2DL1-DS10 VL (SEQ ID NO: 126). The underlined sequences represent the sequences of CDR1 to CDR3.

[0024] Figure 1 shows the amino acid sequences of a2DL1-DS11 VH (SEQ ID NO: 127) and a2DL1-DS11 VL (SEQ ID NO: 128), as well as the amino acid sequences of a2DL1-DS12 VH (SEQ ID NO: 129) and a2DL1-DS12 VL (SEQ ID NO: 130). The underlined sequences represent the sequences of CDR1 to CDR3.

[0033] Figure 1 shows the amino acid sequences of a2DL1-DS13 VH (SEQ ID NO: 131) and a2DL1-DS13 VL (SEQ ID NO: 132), and the amino acid sequences of a2DL1-DS14 VH (SEQ ID NO: 133) and a2DL1-DS14 VL (SEQ ID NO: 134). The underlined sequences indicate the sequences of CDR1 to CDR3.

[0034] Figure 1 shows the amino acid sequences of a2DL1-DS15 VH (SEQ ID NO: 135) and a2DL1-DS15 VL (SEQ ID NO: 136), and the amino acid sequences of a3DL1-DS2 VH (SEQ ID NO: 137) and a3DL1-DS2 VL (SEQ ID NO: 138). The underlined sequences indicate the sequences of CDR1 to CDR3.

[0033] Figure 1 shows the amino acid sequence of a3DL1-DS3 VH (SEQ ID NO: 139) and a3DL1-DS3 VL (SEQ ID NO: 140), as well as the amino acid sequence of a3DL1-DS4 VH (SEQ ID NO: 141) and a3DL1-DS4 VL (SEQ ID NO: 142). The underlined sequences indicate the sequences of CDR1 to 3.

[0034] Figure 1 shows the amino acid sequence of a3DL1-DS5 VH (SEQ ID NO: 143) and a3DL1-DS5 VL (SEQ ID NO: 144), as well as the amino acid sequence of a3DL1-DS6 VH (SEQ ID NO: 145) and a3DL1-DS6 VL (SEQ ID NO: 146).

[0035] Figure 1 shows the results of cytocidal activity evaluation of membrane-type anti-NKG2A-scFv-expressing K562 cell line using NK92 cells.This is a figure showing the expression of cell surface markers of NK92 cells by flow cytometry, indicating that it is positive for NKG2A and negative for NKG2C. It is a figure showing the amino acid sequence of aNKG2A-DS11 VH (SEQ ID NO: 163) and the amino acid sequence of aNKG2A-DS11 VL (SEQ ID NO: 164). The underlined sequences indicate the sequences of CDR1-3. Among the membrane-type anti-NKG2A-scFvs showing agonist activity against NKG2A, aNKG2A-DS11 showed binding to the NKG2A-CD94 recombinant protein, while it did not show binding to the NKG2C-CD94 recombinant protein. This is a figure showing the expression of cell surface markers of each PBMC-derived NK cell used in Example 21 by flow cytometry. It is a figure showing the killing activity of Class I HLA-negative iPS cells expressing tHLA-E or membrane-type aNKG2A-DS11 of each PBMC-derived NK cell. In Figure 52-2A, it shows the killing activity of PBMC-derived NK cells in which NKG2A-positive NK cells are dominant over NKG2C-positive NK cells, and in Figure 52-2B, it shows the killing activity of PBMC-derived NK cells in which NKG2C-positive NK cells are dominant over NKG2A-positive NK cells. This is a figure (Part 1) showing the expression of cell surface markers of each PBMC-derived NK cell used in Example 22 by flow cytometry. This is a figure (Part 2) showing the expression of cell surface markers of each PBMC-derived NK cell used in Example 22 by flow cytometry. It is a figure showing the killing activity of Class I HLA-negative iPS cells co-expressing tHLA-E and membrane-type aNKG2A-DS11 of each PBMC-derived NK cell. In Figure 53-3A, it shows the killing activity of PBMC-derived NK cells in which NKG2A-positive NK cells are dominant over NKG2C-positive NK cells, and in Figure 53-3B, it shows the killing activity of PBMC-derived NK cells in which NKG2C-positive NK cells are dominant over NKG2A-positive NK cells, respectively.

[0035] The present invention is described in detail below. 1. Substances Having Activity of Binding to Inhibitory Receptors on the Surface of NK Cells and Inhibiting the Cytocidal Activity of NK Cells The present invention provides substances having activity of binding to inhibitory receptors on the surface of NK cells and inhibiting the cytocidal activity of NK cells. Such substances are sometimes referred to as substances having agonistic activity for inhibitory receptors on the surface of NK cells. That is, the activity of binding to inhibitory receptors on the surface of NK cells and inhibiting the cytocidal activity of NK cells is sometimes referred to as agonistic activity for inhibitory receptors on the surface of NK cells. Furthermore, in the present invention, a substance that binds to a receptor on the surface of NK cells is called a binder, and a substance that binds to a receptor on the surface of NK cells and has the effect of activating a signal from the receptor to the NK cell is called an agonist binder for the receptor. That is, an agonist binder for inhibitory receptors on the surface of NK cells exhibits the effect of inhibiting NK cell activity, and an agonist binder for activating receptors on the surface of NK cells exhibits the effect of activating NK cells.

[0036] Inhibitory receptors on the surface of NK cells include NKG2A and inhibitory KIR.

[0037] NKG2 is expressed on the surface of NK cells and some T cells, and is involved in the NK cell's function of recognizing self and non-self, known as "missing self." NKG2 includes inhibitory NKG2A and NKG2B, and activating NKG2C, NKG2D, NKG2E, NKG2F, and NKG2H. HLA-E and HLA-G have been reported as ligands for NKG2A, and the ligand HLA-E also binds to NKG2C.

[0038] KIRs are expressed on the surface of NK cells and are involved in the NK cell's function of recognizing self and non-self, known as "missing self." KIRs include inhibitory KIRs and activating KIRs, each of which has multiple subtypes. Signals from inhibitory KIRs suppress the cytocidal activity of NK cells. In other words, when NK cells in a steady state recognize a surface antigen on a target cell, they become activated unless a signal is received from an inhibitory receptor, and acquire the ability to damage or kill the recognized cell (hereinafter referred to as "NK cell cytocidal activity"). In other words, when a target cell expresses the same type of HLA as the NK cell, the cell's HLA binds to the inhibitory KIR on the NK cell, transmitting an inhibitory signal into the NK cell and suppressing the cytocidal activity of the NK cell. As a result, cells expressing the same type of HLA as the NK cell can avoid elimination by the NK cell.

[0039] On the other hand, activating KIR recognizes HLA presenting virus-derived peptides on cells expressing the same type of HLA as NK cells, and the peptides carried by them, and an activation signal is transmitted into the NK cells, activating the cytocidal activity of the NK cells. Unlike inhibitory KIR, activating KIR functions to recognize and eliminate viral peptides presented on HLA.

[0040] In the present invention, "binding" is also referred to as "recognition." In the present invention, "recognition," i.e., "binding," means binding that is not nonspecific adsorption. The dissociation constant (hereinafter referred to as "KD") can be used as a criterion for determining whether or not recognition has occurred, i.e., whether or not binding has occurred. The KD value of a suitable antibody of the present invention for an antigen protein is 1 x 10 －５ M or less, 5×10 －６ M or less, 2×10 －６ M or less, 1×10 －６M or less. In the present invention, the binding can be measured or determined by a biomolecular interaction analysis system such as SPR or BLI, or by ELISA or RIA. The binding between a substance expressed on the cell surface and a recombinant protein of inhibitory KIR can be measured by flow cytometry or the like.

[0041] A substance that binds to an inhibitory receptor on the surface of an NK cell and has the activity of inhibiting the cytocidal activity of an NK cell is a substance that binds to an inhibitory receptor on the surface of an NK cell, activates the inhibitory receptor on the surface of the NK cell, transmits an inhibitory signal into the NK cell, and inhibits the cytocidal activity of the NK cell.

[0042] Inhibitory NKG2s include NKG2A and NKG2B. Active NKG2s include NKG2C. Ligands for NKG2A include HLA-E and HLA-G, and the ligand HLA-E also binds to NKG2C.

[0043] The substance of the present invention that binds to NKG2A and has the activity of suppressing the cytocidal activity of NK cells is a substance that binds to NKG2A and has the activity of suppressing the cytocidal activity of NK cells that express the NKG2A to which it binds. That is, it is a substance that binds to NKG2A and has the activity of suppressing the cytocidal activity of NK cells. The substance binds to NKG2A and exerts agonist activity.

[0044] The substances of the present invention that bind to NKG2A and have the activity of inhibiting the cytocidal activity of NK cells have low activity of activating the cytocidal activity of NK cells. For example, the activity of the substances of the present invention that bind to NKG2A and have the activity of inhibiting the cytocidal activity of NK cells is higher than the activity of the corresponding substance that binds to the corresponding NKG2C and activates the cytocidal activity of NK cells. The substances of the present invention that bind to NKG2A and have the activity of inhibiting the cytocidal activity of NK cells preferably do not bind to NKG2C. That is, they do not substantially exhibit agonist activity against NKG2C.

[0045] Inhibitory KIRs include those belonging to the KIR2DL subclass and the KIR3DL subclass. Those belonging to the KIR2DL subclass include KIR2DL1, KIR2DL2, KIR2DL3, and KIR2DL5, while those belonging to the KIR3DL subclass include KIR3DL1, KIR3DL2, and KIR3DL3. Furthermore, activating KIR subtypes include KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5, and KIR3DS1. Furthermore, KIR2DL4 exists as a subtype that has both inhibitory and activating properties. The ligands (HLA) that bind to these inhibitory KIRs and activating KIRs are shown in Table 1.

[0046]

[0047] There are various variants of the inhibitory and activating KIRs of the present invention, but in the examples of the present invention, variants having amino acid sequences with high expression frequencies were selected and used. Therefore, the effects of the present invention are not limited to individual variants, but are applicable to KIRs belonging to each subtype, and to cells, hosts, etc. having an HLA type corresponding to the KIR. For KIR2DL1, KIR2DL1*003 (SEQ ID NO: 4, IPD Accession No.: KIR00003), for KIR2DL2, KIR2DL2*001 (SEQ ID NO: 6, IPD Accession No.: KIR00010) and KIR2DL2*003 (SEQ ID NO: 57, IPD Accession No.: KIR00012), for KIR2DL3, KIR2DL3*001 (SEQ ID NO: 8, IPD Accession No.: KIR00014), and for KIR2DS1, KIR2DL1*003 (SEQ ID NO: 4, IPD Accession No.: KIR00003), for KIR2DL2, KIR2DL2*001 (SEQ ID NO: 6, IPD Accession No.: KIR00010), and for KIR2DL2*003 (SEQ ID NO: 57, IPD Accession No.: KIR00012), for KIR2DL3, KIR2DL3*001 (SEQ ID NO: 8, IPD Accession No.: KIR00014), and for KIR2DS1, KIR2DL1*003 (SEQ ID NO: 57, IPD Accession No.: KIR00012), for KIR2DL3, KIR2DL3*001 (SEQ ID NO: 8, IPD Accession No.: KIR00014), for KIR2DS1, KIR2DL1*003 (SEQ ID NO: 5 For KIR2DS1, KIR2DS1*002 (SEQ ID NO: 10, IPD accession number: KIR00034) was used; for KIR2DS2, KIR2DS2*001 (SEQ ID NO: 12, IPD accession number: KIR00037); for KIR2DS4, KIR2DS4*001 (SEQ ID NO: 14, IPD accession number: KIR00045); and for KIR3DL1, KIR3DL1*015 (SEQ ID NO: 16, IPD accession number: KIR00102) was used.

[0048] As shown in Figure 11, the amino acid sequences of KIR2DL2*003 and KIR2DL3*001 are highly identical to each other (in particular, the amino acid sequence identity of the extracellular domains is very high), and are also highly identical to the amino acid sequences of the active KIRs KIR2DS1*002, KIR2DS2*001, and KIR2DS4*001.

[0049] The substance of the present invention that binds to an inhibitory KIR and has the activity of suppressing the cytocidal activity of NK cells is a substance that binds to at least one of the subclasses of inhibitory KIR described above and has the activity of suppressing the cytocidal activity of NK cells that express the KIR to which it binds. The inhibitory KIR to which it binds is not particularly limited, but is preferably a substance that binds to KIR2DL1, KIR2DL2, KIR2DL3, and / or KIR3DL1, more preferably KIR2DL1, KIR2DL2, and / or KIR2DL3, and has the activity of suppressing the cytocidal activity of NK cells. The substance binds to an inhibitory KIR and exhibits agonist activity.

[0050] KIR2DL1 recognizes C2 epitopes (Cw2, Cw4, Cw5, Cw6, Cw15, and Cw17) of HLA-C type, which is a Class I MHC molecule.

[0051] KIR2DL2 and KIR2DL3 recognize C1 epitopes (Cw1, Cw3, Cw7, Cw8, Cw9, Cw10, Cw12, Cw14, and Cw16) of HLA-C type, which are Class I MHC molecules, and C1 epitopes (B46 and B73) of HLA-B type.

[0052] KIR3DL1 recognizes the Bw4 epitope (A23, A24, A25, A32, B5, B27, B37, B38, B44, B49, B51, B52, B53, B57, B58, B59, B77) of HLA-B class I MHC molecules. KIR3DL2 recognizes A3 and A11 of HLA-A class I MHC molecules.

[0053] Substances that bind to the inhibitory KIR of the present invention and have the activity of inhibiting the cytocidal activity of NK cells have low activity of activating the cytocidal activity of NK cells. For example, the activity of a substance that binds to the inhibitory KIR of the present invention and has the activity of inhibiting the cytocidal activity of NK cells is higher than the activity of the substance that binds to the corresponding activating KIR and activates the cytocidal activity of NK cells. Here, the corresponding activating KIR for an inhibitory KIR is KIR2DS1, KIR2DS2, KIR2DS3, and KIR2DS4, and particularly important are KIR2DS1, KIR2DS2, and KIR2DS4. Furthermore, the activating KIR for KIR3DL1 is KIR3DS1. The substance of the present invention that binds to the inhibitory KIR and inhibits the cytocidal activity of NK cells preferably does not bind to the activating KIR. That is, it does not substantially exhibit agonist activity against the activating KIR. Therefore, the substance that binds to the inhibitory KIR and inhibits the cytocidal activity of NK cells is preferably a substance that has few identical sequences or binds to partial sequences that have different accessibility when the protein assumes a three-dimensional structure in the alignment of the amino acid sequences of KIR2DL2, KIR2DL3, KIR2DS1, KIR2DS2, and KIR2DS4 shown in Figure 10.

[0054] The NK cells whose activation is inhibited by the substance of the present invention refer to NK cells expressing an inhibitory receptor on the cell surface that is the target. When the inhibitory receptor is NKG2A, a blood sample from a subject containing an NKG2A-positive NK cell population, or an NK cell population isolated from the blood, can be used as such NK cells. When the inhibitory receptor is an inhibitory KIR, a blood sample from a subject having an HLA type corresponding to the inhibitory KIR, or an NK cell population isolated from the blood, can be used. Alternatively, NK cells obtained by genetically engineering a commercially available NK cell line to express an inhibitory receptor on the surface of the target NK cells may also be used.

[0055] The agonistic activity of the substances of the present invention against inhibitory receptors on the surface of NK cells can be evaluated by measuring the cytocidal activity of the following effector cells against the following target cells in the presence and absence of a test substance, and calculating the percentage of inhibition of cytocidal activity in the presence of the test substance. NK cells expressing the inhibitory receptor on the surface of the target NK cells are used as effector cells. Cells not expressing HLA corresponding to the inhibitory receptor on the surface of the target NK cells are used as target cells. In measuring this agonistic activity, the test substance may be added to the culture medium in a soluble form, or may be expressed on the surface of the target cells in the form of a membrane protein, as described below.

[0056] For example, in the case of NKG2A, to measure the agonistic activity of a substance that binds to NKG2A, NK cells that express NKG2A are used as effector cells, and cells that do not express Class I HLA (e.g., cells lacking the B2M gene) or cells that barely express Class I HLA are used as target cells. Furthermore, by further evaluating the target cells using NK cells that express NKG2C as effector cells, the agonistic activity of a test substance against NKG2C can be evaluated, and substances having agonistic activity specific to NKG2A can be selected.

[0057] For example, in the case of inhibitory KIR, to measure the agonist activity of a substance that binds to KIR2DL2 / 3, NK cells derived from HLA-C1 homotype or HLA-C1 / C2 heterotype and expressing KIR2DL2 and / or KIR2DL3 are used as effector cells, and cells that do not express HLA-C1 type (e.g., cells collected from a subject with a C2 / C2 homotype) are used as target cells. It is preferable that both the effector cells and target cells are of the same Bw4 type (both negative homotypes or both positive homotypes).

[0058] For example, to measure the agonist activity of a substance that binds to KIR2DL1, NK cells derived from HLA-C2 homotype or HLA-C1 / C2 heterotype and expressing KIR2DL1 are used as effector cells, and cells that do not express HLA-C2 type (e.g., cells collected from a subject with a C1 / C1 homotype) are used as target cells. It is preferable that both the effector cells and the target cells are of the same BW4 type (both negative homotypes or both positive homotypes).

[0059] Furthermore, for example, to measure the agonist activity of a substance that binds to KIR3DL1, NK cells derived from the HLA-Bw4 type and expressing KIR3DL1 are used as effector cells, and cells that do not express the HLA-Bw4 type (e.g., cells collected from a subject with a Bw4-positive / Bw4-negative homozygous HLA type) are used as target cells. It is preferable that the HLA-C types of both the effector cells and the target cells are the same (both C1 / C1 homozygous, both C2 / C2 homozygous, or both C1 / C2 heterozygous).

[0060] For example, to measure the agonist activity of a substance that binds to KIR3DL2, NK cells derived from HLA-A03 or A11 types and expressing KIR3DL2 are used as effector cells, and cells that do not express HLA-A03 or A11 types are used as target cells. Preferably, the effector and target cells are matched in terms of HLA-C type (both C1 / C1 homozygous, both C2 / C2 homozygous, or both C1 / C2 heterozygous) and both BW4 type (both negative homozygous, both positive homozygous, or both positive / negative heterozygous).

[0061] Substances that bind to inhibitory receptors on the surface of NK cells and inhibit the cytocidal activity of NK cells include substances that have a binding site for inhibitory receptors on the surface of NK cells, such as polypeptides that have a binding site for inhibitory KIR and bind to inhibitory receptors on the surface of NK cells.

[0062] Examples of polypeptides that have a binding site for an inhibitory receptor on the surface of NK cells and bind to the inhibitory receptor on the surface of NK cells include the following polypeptides (A) and (C):

[0063] (A) Antibodies or antigen-binding fragments against inhibitory receptors Examples of antibodies against NKG2A include monalizumab, a monoclonal antibody into which a CDR or variable region contained in the scFv (aNKG2A-DS11) described below has been grafted, and the like. Furthermore, examples of antibodies against inhibitory KIR include lirilumab, Pan2D, and a monoclonal antibody into which a CDR or variable region contained in scFv (i) to (x) described below has been grafted. The present invention is not limited to these antibodies, and includes all antibodies that have the activity of binding to an inhibitory receptor expressed on the surface of NK cells and inhibiting the cytocidal activity of NK cells.

[0064] Antibodies and antigen-binding fragments that bind to inhibitory receptors on the surface of NK cells and inhibit the cytocidal activity of NK cells (hereinafter also referred to as "antibodies of the present invention") may be either monoclonal or polyclonal antibodies. The isotype of the monoclonal antibodies of the present invention is not particularly limited, and examples include IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgA (e.g., IgM, IgA1, IgA2), IgD, and Ig. The isotype and subclass of monoclonal antibodies can be determined, for example, by the Ouchterlony method, ELISA, or RIA. Examples of monoclonal antibodies of the present invention include antibodies derived from non-human animals (non-human animal antibodies), human antibodies, chimeric antibodies (also referred to as "chimeric antibodies"), and humanized antibodies. Preferably, human antibodies can be used. The scope of the antibodies of the present invention also includes antibody mutants; for example, the scope of human antibodies also includes human mutant antibodies.

[0065] Non-human animal antibodies include antibodies derived from vertebrates such as mammals and birds. Mammal-derived antibodies include rodent-derived antibodies such as mouse antibodies and rat antibodies, and single-chain antibodies having only one variable region derived from camels, alpacas, llamas, etc. Bird-derived antibodies include chicken antibodies, etc.

[0066] Examples of chimeric antibodies include, but are not limited to, antibodies formed by combining a variable region derived from a non-human animal antibody with a constant region of a human antibody (human immunoglobulin).

[0067] Examples of humanized antibodies include, but are not limited to, antibodies in which the CDRs in the variable regions of a non-human animal antibody have been transplanted onto a human antibody (the variable regions of human immunoglobulins), antibodies in which not only the CDRs but also part of the framework region sequence of a non-human animal antibody has been transplanted onto a human antibody, and antibodies in which one or more amino acids derived from any of these non-human animal antibodies have been replaced with human amino acids.

[0068] Examples of CDR-grafted antibodies include, but are not limited to, antibodies or antigen-binding fragments in which the CDRs in the variable region of a given antibody or antigen-binding fragment have been grafted onto the variable region of another immunoglobulin; antibodies in which not only the CDRs but also part of the framework region sequence of the original antibody or antigen-binding fragment have been grafted onto the variable region of the antibody; and antibodies in which one or more amino acids from either of these antibodies or antigen-binding fragments have been replaced with other amino acids.

[0069] In the above-described CDR grafting, when an amino acid in a CDR is substituted with another amino acid, 1 to 6 amino acids selected from 1, 2, or 3 amino acids on the N-terminal and / or C-terminal side of the CDR region may be substituted, more preferably 1 to 4 amino acids selected from 2 amino acids on the N-terminal and / or C-terminal side, and even more preferably 1 or 2 amino acids selected from 1 amino acid on the N-terminal and / or C-terminal side.

[0070] Antibodies can be produced by various known methods. Known methods include methods using hybridomas and cell-based immunization, and they can also be produced by genetic recombination techniques. Methods for obtaining human antibodies selected from a human antibody phage library are also known. For example, a phage display method can be used in which the variable regions of human antibodies are expressed on the surface of phages as scFvs and phages that bind to the antigen are selected. The DNA sequence encoding the variable regions of human antibodies that bind to the antigen can be determined by analyzing the genes of phages selected by binding to the antigen. Once the DNA sequence of an antigen-binding scFv is determined, an expression vector containing that sequence can be constructed and introduced into an appropriate host for expression, thereby enabling the production of a human antibody (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 consisting only of heavy chains are antibodies possessed by camels, alpacas, llamas, etc., and can be obtained by immunizing these animals or using phage libraries derived from these animals (see, for example, 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.).

[0071] In the present invention, the term "antigen-binding fragment" refers to a peptide that is composed of one or two variable regions and has antigen-binding activity. Examples of "antigen-binding fragments" include scFv, Fab, and F(ab'). ２Examples of antigen-binding fragments include, but are not limited to, Fab', Fv, and single-domain antibodies (sdAbs). Such antigen-binding fragments may be obtained by treating full-length antibody protein molecules with enzymes such as papain and pepsin, as well as 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 produced by genetic engineering techniques, the heavy chain variable region and light chain variable region (in the case of a single-chain antibody, only the heavy chain variable region may be present) contained in a monoclonal antibody or antigen-binding fragment that binds to a target antigen, or the CDR1, CDR2, and CDR3 contained therein may be grafted onto the framework sequence of the desired antigen-binding fragment. Antigen-binding fragments derived from single-chain antibodies consisting only of heavy chains can be produced by using displayer libraries such as phage or yeast that contain genes encoding the variable regions of single-chain antibodies derived from camels, alpacas, llamas, etc. that bind to the antigen of interest, and incorporating them into an expression format suitable for a single variable region, in a manner similar to that used for scFv and membrane-type scFv, according to publicly known methods or methods disclosed herein (see, for example, 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.). Furthermore, when grafting a variable region or CDR, examples include, but are not limited to, substitution of one or more amino acids in the sequence of the original antibody or the like with amino acids of the desired antigen-binding fragment.When the antigen-binding fragment of the present invention is a single chain such as an scFv, it is known that binding may be maintained even if the heavy chain and light chain are swapped when grafting the variable region or CDR from the original antibody or antigen-binding fragment to the desired antigen-binding fragment, and the present invention includes those in which the heavy chain variable region and light chain variable region are swapped in this manner (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. 2021 Mar; 37(2): 33102).

[0072] Among these, scFv is preferred. scFv is an antigen-binding fragment obtained by linking a heavy chain variable region and a light chain variable region with a polypeptide linker (Pluckthun A. The Pharmacology of Monoclonal Antibodies 113, edited by Rosenburg and Moore, 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. Known artificial linker sequences are amino acid sequences rich in glycine and serine, and examples thereof include a GS linker consisting of three amino acids GSG or four amino acids GSGS (SEQ ID NO: 3), and a linker sequence represented by the formula (G4S)n. In the formula, n is 1 to 10, preferably n=3 (GGGGGSGGGGSGGGGS: SEQ ID NO: 25). A linker domain is also called a peptide linker, and known peptide linkers can be used as the extracellular linker domain. The sequence represented by the above formula (G4S)n is called a G4S linker.

[0073] An scFv that binds to a target antigen can be obtained by the phage display method (Nature Biotechnology (2005), 23, (9), pp. 1105-1116), in which a library of variable regions is expressed on the surface of a phage as single-chain antibodies (scFv), and phages that bind to the antigen are selected. By analyzing the genes of phages selected by binding to the antigen, the DNA sequence encoding the variable regions that bind to the antigen can be determined. Once the sequences of the heavy and light chain variable regions are known, the sequences of the CDRs contained in the variable regions can also be identified. An expression vector having a sequence in which these CDR or variable region sequences are incorporated into the sequence format of a desired antibody or antigen-binding fragment is prepared, and the vector is introduced into an appropriate host for expression, thereby enabling the production of an antibody or antigen-binding fragment having the desired antigen-binding ability (WO92 / 01047, WO92 / 20791, WO93 / 06213, WO93 / 11236, WO93 / 19172, WO95 / 01438, WO95 / 15388, Annu. Rev. Immunol (1994) 12, pp. 433-455, Nature Biotechnology (2005) 23(9), pp. 1105-1116).

[0074] CDRs can be modified to the extent that the agonist activity of the polypeptide of the present invention is not altered. That is, one specific amino acid residue in the CDR of the polypeptide of the present invention can be substituted with alanine, and then it can be confirmed whether the agonist activity against inhibitory KIR is maintained. Amino acids that maintain the agonist activity of the polypeptide against inhibitory KIR even after substitution with alanine can be determined to be unimportant for agonist activity, and such amino acids in the CDR can be substituted with other amino acids, and the polypeptide with the substituted amino acids will have the same reactivity as the polypeptide before the amino acid substitution. When substituting amino acids in the CDR with other amino acids, it is preferable to substitute 1 to 6 amino acids selected from 1, 2, or 3 amino acids on the N-terminus and / or C-terminus of the CDR region, more preferably 1 to 4 amino acids selected from 2 amino acids on the N-terminus and / or C-terminus, and even more preferably 1 or 2 amino acids selected from 1 amino acid on the N-terminus and / or C-terminus.

[0075] The scFv against NKG2A of the present invention includes scFvs consisting of the amino acid sequence of (Ni) below. Here, the heavy chain variable region and light chain variable region can be linked, for example, by a GS linker, a G4S linker, etc. (Ni) An amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 163 and the amino acid sequence of the light chain variable region of SEQ ID NO: 164.

[0076] Examples of the scFv against inhibitory KIR2DL2 or KIR2DL3 of the present invention include scFvs consisting of any of the following amino acid sequences (i) to (xii), where the heavy chain variable region and the light chain variable region can be linked by, for example, a GS linker, a G4S linker, or the like. (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) (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.

[0077] Furthermore, examples of the scFv against inhibitory KIR3DL1 of the present invention include scFvs consisting of any of the amino acid sequences (xiii) to (xviii) below, where the heavy chain variable region and light chain variable region can be linked by, for example, a GS linker, a G4S linker, or the like. (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.

[0078] Examples of scFv against inhibitory KIR2DL1 of the present invention include scFvs consisting of any of the amino acid sequences (ci) to (cxv) below, where the heavy chain variable region and light chain variable region can be linked by, for example, a GS linker, a G4S linker, or the like.(ci) an amino acid sequence comprising a combination of the amino acid sequence of a heavy chain variable region of SEQ ID NO: 107 and the amino acid sequence of a light chain variable region of SEQ ID NO: 108; (cii) an amino acid sequence comprising a combination of the amino acid sequence of a heavy chain variable region of SEQ ID NO: 109 and the amino acid sequence of a light chain variable region of SEQ ID NO: 110; (ciii) an amino acid sequence comprising a combination of the amino acid sequence of a heavy chain variable region of SEQ ID NO: 111 and the amino acid sequence of a light chain variable region of SEQ ID NO: 112; (civ) an amino acid sequence comprising a combination of the amino acid sequence of a heavy chain variable region of SEQ ID NO: 113 and the amino acid sequence of a light chain variable region of SEQ ID NO: 114; (cv) an amino acid sequence comprising a combination of the amino acid sequence of a heavy chain variable region of SEQ ID NO: 115 and the amino acid sequence of a light chain variable region of SEQ ID NO: 116; (cvi) an amino acid sequence comprising a combination of the amino acid sequence of a heavy chain variable region of SEQ ID NO: 117 and the amino acid sequence of a light chain variable region of SEQ ID NO: 118; (cvii) (cviii) an amino acid sequence comprising a combination of the amino acid sequence of a heavy chain variable region of SEQ ID NO: 121 and the amino acid sequence of a light chain variable region of SEQ ID NO: 122; (cix) an amino acid sequence comprising a combination of the amino acid sequence of a heavy chain variable region of SEQ ID NO: 123 and the amino acid sequence of a light chain variable region of SEQ ID NO: 124; (cx) an amino acid sequence comprising a combination of the amino acid sequence of a heavy chain variable region of SEQ ID NO: 125 and the amino acid sequence of a light chain variable region of SEQ ID NO: 126; (cxi) an amino acid sequence comprising a combination of the amino acid sequence of a heavy chain variable region of SEQ ID NO: 127 and the amino acid sequence of a light chain variable region of SEQ ID NO: 128; (cxii) an amino acid sequence comprising a combination of the amino acid sequence of a heavy chain variable region of SEQ ID NO: 129 and the amino acid sequence of a light chain variable region of SEQ ID NO: 130; (cxiii) (cxiv) an amino acid sequence comprising a combination of the amino acid sequence of a heavy chain variable region of SEQ ID NO: 133 and the amino acid sequence of a light chain variable region of SEQ ID NO: 134; and (cxv) an amino acid sequence comprising a combination of the amino acid sequence of a heavy chain variable region of SEQ ID NO: 135 and the amino acid sequence of a light chain variable region of SEQ ID NO: 136.

[0079] Furthermore, the polypeptide having a binding site for an inhibitory receptor on the surface of NK cells and binding to the inhibitory receptor on the surface of NK cells includes a polypeptide having a binding site consisting of an amino acid sequence derived from an antibody or scFv that recognizes and binds to the inhibitory receptor on the surface of NK cells. Such polypeptides can take various forms, and preferred examples include a monoclonal antibody, an antigen-binding fragment such as scFv, or a membrane-type protein that has the amino acid sequence of the antigen-binding fragment on the outside of the cell.

[0080] The amino acid sequence derived from an antibody that recognizes and binds to an inhibitory receptor on the surface of NK cells or an scFv includes the amino acid sequence of a site in an antibody that recognizes and binds to an inhibitory receptor on the surface of NK cells or an scFv variable region that binds to an inhibitory receptor on the surface of NK cells, and further includes an amino acid sequence in which one or several amino acids have been substituted, deleted, or added (addition includes insertion) (hereinafter collectively referred to as "mutation") in the amino acid sequence of an antibody that recognizes and binds to an inhibitory receptor on the surface of NK cells or an amino acid sequence of a site in an scFv that binds to an inhibitory receptor on the surface of NK cells, and which has the activity of binding to an inhibitory receptor on the surface of NK cells and inhibiting the cytocidal activity of NK cells. Here, "several" in "one or several" refers to 2 to 10, preferably 7 or less, more preferably 5 or less, and even more preferably 4, 3, 2, or 1.

[0081] Specific examples of binding sites to inhibitory receptors on the surface of NK cells include sites containing amino acid sequences derived from CDR1, CDR2, and CDR3 contained in the light chain variable region and heavy chain variable region of a monoclonal antibody or scFv that binds to an inhibitory receptor on the surface of NK cells, amino acid sequences derived from variable regions containing the respective CDR1, CDR2, and CDR3, or amino acid sequences derived from the light chain variable region and heavy chain variable region. The amino acid sequence derived from an antibody or scFv that binds to an inhibitory receptor on the surface of NK cells has the same meaning as above.

[0082] For example, a polypeptide that has a binding site for NKG2A and binds to NKG2A has the combination of heavy chain variable region and light chain variable region shown in Table 21, aNKG2A-DS11.

[0083] One embodiment of the polypeptide of the present invention that has a binding site for NKG2A and binds to NKG2A includes, for example, a polypeptide comprising the amino acid sequence of the following (N-I) (preferably a monoclonal antibody, an antigen-binding fragment, an scFv consisting of said amino acid sequence, or a membrane-type protein comprising said scFv in its extracellular domain): (N-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: 163 (preferably a variable region comprising the amino acid sequence of SEQ ID NO: 163) and a second variable region comprising an amino acid sequence derived from CDR1, CDR2, and CDR3 of SEQ ID NO: 164 (preferably a variable region comprising the amino acid sequence of SEQ ID NO: 164);

[0084] For example, a polypeptide that has a binding site for inhibitory KIR2DL2 and / or KIR2DL3 and binds to inhibitory KIR2DL2 and / or KIR2DL3 has a combination of heavy chain variable region and light chain variable region shown in Table 2. In Table 2, "a2DL2 / 3" indicates that the binder binds to inhibitory KIR2DL2 and / or KIR2DL3.

[0085] *The CDR1, CDR2, and CDR3 regions in the table are indicated by the range of amino acid numbers in the amino acid sequence of each variable region. The amino acid numbers are specified according to the IMGT definition.

[0086] Furthermore, polypeptides that have a binding site for inhibitory KIR2DL1 and bind to inhibitory KIR2DL1 have the combination of heavy chain variable regions and light chain variable regions shown in Table 3. In Table 3, "a2DL1" indicates that the binder binds to inhibitory KIR2DL1.

[0087]

[0088] Furthermore, polypeptides that have a binding site for inhibitory KIR3DL1 and bind to inhibitory KIR3DL1 have the combination of heavy chain variable regions and light chain variable regions shown in Table 4. In Table 4, "a3DL1" indicates that the binder binds to inhibitory KIR3DL1.

[0089] *The CDR1, CDR2, and CDR3 regions in the table are indicated by the range of amino acid numbers in the amino acid sequence of each variable region. The amino acid numbers are specified according to the IMGT definition.

[0090] One embodiment of the polypeptide of the present invention that has a binding site for inhibitory KIR2DL2 or KIR2DL3 and binds to inhibitory KIR is, for example, a polypeptide comprising any of the following amino acid sequences (I) to (XII) (preferably a monoclonal antibody, an antigen-binding fragment, an scFv consisting of the amino acid sequence, or a membrane-type protein comprising the scFv in its extracellular domain). (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); (III) (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: 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); (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);(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); (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 the 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 the amino acid sequence of SEQ ID NO: 44); (VII) (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); (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: 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); 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 the 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 the amino acid sequence of SEQ ID NO: 50);(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); (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 (preferably a variable region comprising the amino acid sequence 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 (preferably a 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 an amino acid sequence derived from CDR1, CDR2 and CDR3 of SEQ ID NO: 63 (preferably a variable region comprising the amino acid sequence 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 (preferably a variable region comprising the amino acid sequence of SEQ ID NO: 64).

[0091] Furthermore, one embodiment of the polypeptide of the present invention that has a binding site for inhibitory KIR3DL1 and binds to inhibitory KIR3DL1 includes, for example, a polypeptide comprising any of the amino acid sequences of (XIII) to (XVIII) below (preferably, a monoclonal antibody, an antigen-binding fragment, an scFv consisting of the amino acid sequence, or a membrane protein comprising the scFv in its extracellular domain).(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); (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); (XV) (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: 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); (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: 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); (XVII) (XVIII) 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 the 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 the amino acid sequence of SEQ ID NO: 144); and (XVIII) 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: 145 (preferably a variable region comprising the amino acid sequence 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 (preferably a variable region comprising the amino acid sequence of SEQ ID NO: 146).

[0092] Furthermore, one embodiment of the polypeptide of the present invention that has a binding site for inhibitory KIR2DL1 and binds to inhibitory KIR2DL1 includes, for example, a polypeptide comprising the amino acid sequence of the following (P) (preferably, a monoclonal antibody, an antigen-binding fragment, an scFv consisting of said amino acid sequence, or a membrane-type protein comprising said scFv in its extracellular domain): (P) 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: 31 (preferably, a variable region comprising the amino acid sequence 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 (preferably, a variable region comprising the amino acid sequence of SEQ ID NO: 32).

[0093] Furthermore, one embodiment of the polypeptide of the present invention has a binding site for inhibitory KIR2DL1 and binds to inhibitory KIR2DL1, and includes, for example, a polypeptide comprising any of the amino acid sequences (CI) to (CXV) below (preferably, a monoclonal antibody, an antigen-binding fragment, an scFv consisting of the amino acid sequence, or a membrane protein comprising the scFv in its extracellular domain). (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); (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); (CIII) (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: 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); (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 the 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 the amino acid sequence of SEQ ID NO: 114);(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); (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); (CVII) (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: 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); (CIX) 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); (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: 123 (preferably a variable region comprising the 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 the amino acid sequence of SEQ ID NO: 124); (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);(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); (CXII) 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: 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); (CXIII) (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: 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); (CXV) 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 the 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 the amino acid sequence of SEQ ID NO: 134); and (CXV) 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: 135 (preferably a variable region comprising the 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 the amino acid sequence of SEQ ID NO: 136).

[0094] 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 abolish antigen reactivity. Such mutations include, for example, substitution of one or two amino acids in the amino acid sequence of the CDR, preferably substitution of one or two amino acids at the C- and / or N-terminus of the amino acid sequence of the CDR.

[0095] Here, in the polypeptides (N-I) (polypeptides having a binding site for NKG2A and binding to NKG2A) and the polypeptides (I) to (XIII), (P), and (CI) to (CXV) (polypeptides having a binding site for inhibitory KIR and binding to inhibitory KIR), the structure other than the variable region can be appropriately selected depending on the form of the selected polypeptide (monoclonal antibody, antigen-binding fragment, etc.). When the polypeptide is a single chain, the first variable region may be arranged in the following order from the N-terminus: first variable region-linker sequence-second variable region, or the second variable region-linker sequence-first variable region. Furthermore, when the polypeptide is in the form of a divided heavy chain and light chain, the first variable region may be arranged in the heavy chain and the second variable region in the light chain, or the first variable region may be arranged in the light chain and the second variable region in the heavy chain.

[0096] In one embodiment, the present invention provides a substance having agonistic activity specific to an inhibitory receptor on the surface of an NK cell. "An agonistic activity specific to an inhibitory receptor on the surface of an NK cell" refers to a substance that exhibits agonistic activity against an inhibitory receptor on the surface of an NK cell, and whose agonistic activity against an activating receptor on the surface of an NK cell corresponding to the inhibitory receptor on the surface of the NK cell is 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, 20% or less, 15% or less, 10% or less, or 5% or less of the agonistic activity against the inhibitory receptor on the surface of the NK cell, and most preferably, exhibits substantially no agonistic activity against the activating receptor on the surface of an NK cell. Here, "an activating receptor on the surface of an NK cell corresponding to an inhibitory receptor on the surface of an NK cell" refers to an activating receptor on the surface of an NK cell, the amino acid sequence of whose extracellular domain is highly homologous to the amino acid sequence of the extracellular domain of the inhibitory receptor on the surface of the target NK cell. For example, an active receptor for NKG2A includes NKG2C. Furthermore, for example, an active KIR corresponding to KIR2DL2 / 3 includes KIR2DS1, KIR2DS2, or KIR2DS4. Furthermore, an active KIR corresponding to KIR2DL1 is KIR2DS1, an active KIR corresponding to KIR2DL2 / 3 is KIR2DS2, and an active KIR corresponding to KIR3DL1 and KIR3DL2 is KIR3DS1.

[0097] Such antibodies can be obtained, for example, by contacting an antibody or antigen-binding fragment thereof, which binds to an inhibitory receptor on the surface of NK cells and exhibits agonistic activity, obtained by the above-described method, with NK cells expressing the corresponding activating KIR, measuring the cytocidal activity of the NK cells against target cells, and selecting antibodies or antigen-binding fragments exhibiting a predetermined activity or less. In this agonistic activity, the antibody or antigen-binding fragment may be added as a soluble protein, or may be expressed on the surface of the target cells in the form of a membrane protein, as described below.

[0098] Furthermore, in obtaining such antibodies, antibodies that bind to inhibitory receptors on the surface of NK cells but have low binding affinity to activating receptors on the surface of NK cells can be obtained, for example, by the phage display method (Nature Biotechnology (2005), 23, (9), pp. 1105-1116), in which the variable regions of an antibody are expressed on the surface of a phage as single-chain fragments (scFv), and phages that bind to the antigen are selected. A method for obtaining binders that specifically bind to inhibitory receptors on the surface of NK cells using the phage display method involves, for example, recovering and concentrating phages that express scFv that bind to inhibitory receptors on the surface of NK cells from a human antibody phage library (positive selection), and then removing phages that express scFv that bind to activating receptors on the surface of NK cells from the resulting phage mixture (depletion), thereby selecting phages that express scFv that bind to inhibitory receptors on the surface of NK cells but do not bind to activating receptors on the surface of NK cells. Here, the inhibitory or activating receptor on the surface of NK cells can be a recombinant protein / peptide consisting of the amino acid sequence of the extracellular region of each molecule, including a partial sequence important for the agonist activity of the inhibitory receptor on the surface of NK cells. The phage recovery / concentration or removal process can employ various methods used to select substances with binding or affinity. For example, two molecules (e.g., a combination of biotin and avidin) that exhibit strong binding affinity can be bound to an NKG2 fragment or a KIR fragment (labeled NKG2 or KIR), and the other can be bound to a carrier (e.g., magnetic beads). After mixing the phage solution with labeled NKG2 or KIR, the mixture is brought into contact with the carrier, and phages that bind to labeled NKG2 or KIR are recovered and concentrated on the carrier. In positive selection, inhibitory receptors on the surface of NK cells are labeled, and phages that are concentrated and recovered on the carrier are obtained. Conversely, in depletion, activating receptors on the surface of NK cells are labeled, and phages in solution that do not bind to the carrier are collected, thereby obtaining phages that do not bind to activating receptors on the surface of NK cells.

[0099] Alternatively, in the positive selection step, an excess amount of unlabeled activating receptor fragments on the surface of NK cells can be added to recover and concentrate (deselect) phages with high binding specificity to labeled NKG2 or KIR. By repeating these methods multiple times or performing them in combination, the rate at which scFvs with high binding specificity to inhibitory receptors on the surface of NK cells can be obtained increases.

[0100] Specifically, an antibody or the like that binds to an inhibitory receptor on the surface of NK cells but does not bind to an activating receptor on the surface of NK cells can be produced by a method comprising the steps shown in (1) or (2) below, and the present invention provides such a production method, a substance produced by the production method, etc. Here, the term "display body" refers to a gene carrier for cells that has the function of displaying a protein or peptide encoded by a gene incorporated by a vector on the surface of a lipid membrane, and examples thereof include phages, yeast, and cells. When phages are used as display bodies, known techniques such as the phage display method or phage panning method described above can be used. In addition, the method of using yeast as a display body is known as the yeast display method (for example, Lukas Roth, et al., Methods Mol Biol, 2020, Vol. 207, pp. 173-189, doi: 10.1007 / 978-1-4939-9853-1_10, etc.).

[0101] (1) A method for producing a substance that binds to an inhibitory receptor on the surface of human NK cells and has the activity of inhibiting NK cells, comprising the following steps: Step A: mixing a labeled solubilized protein containing the extracellular domain of an inhibitory receptor on the surface of NK cells with a suspension containing a polyclonal presenter population including presenters that present on their surface an antigen-binding site that binds to the inhibitory receptor on the surface of the NK cells, then contacting the suspension with a carrier on which a substance that specifically binds to the label moiety is immobilized, and recovering presenters that bind to the carrier, thereby obtaining a presenter population that presents an antigen-binding site that binds to the inhibitory receptor on the surface of the NK cells; Step B: mixing a labeled solubilized protein containing the extracellular domain of an activating receptor on the surface of NK cells that corresponds to the inhibitory receptor on the surface of the NK cells with a suspension containing the presenter population obtained in Step A, then contacting the suspension with a carrier on which a substance that binds to the label moiety is immobilized, and recovering presenters that do not bind to the carrier, thereby removing presenters that present an antigen-binding site that binds to the activating receptor on the surface of the NK cells from the cell population; Step C: performing genetic analysis of the presenter obtained by the method comprising the steps above to identify the gene sequence encoding the antigen-binding site; and Step D: allowing a cell to express a gene in which the gene encoding the identified antigen-binding site has been incorporated into an expression format for an antibody or antigen-binding fragment.

[0102] (2) A method for producing a substance that binds to an inhibitory receptor on the surface of human NK cells and has the activity of inhibiting NK cells, comprising the following steps: Step A: mixing a labeled solubilized protein containing the extracellular domain of an inhibitory receptor on the surface of NK cells and an unlabeled solubilized protein containing the extracellular domain of an inhibitory receptor on the surface of NK cells corresponding to the inhibitory form with a suspension containing a polyclonal presenter population including presenters that present on their surface an antigen-binding site that binds to the inhibitory receptor on the surface of NK cells, and then contacting the cell solution with a carrier on which a substance that specifically binds to the labeled portion is immobilized, and recovering the presenters bound to the carrier, thereby obtaining a presenter population that presents an antigen-binding site that specifically binds to the inhibitory receptor on the surface of the NK cells; Step B: performing genetic analysis of the presenter obtained by a method comprising the above steps and identifying the gene sequence encoding the antigen-binding site; and Step C: expressing a gene in a cell, in which the gene encoding the identified antigen-binding site has been incorporated into an expression format for an antibody or antigen-binding fragment.

[0103] The steps A-B of the method (1) and the step A of the method (2) may each be repeated multiple times, or both may be performed in combination.

[0104] In the method (1) or (2), the combination of the label and the substance that binds to the label can be any of various known combinations of substances, such as a combination of biotin and avidin.

[0105] The method may further comprise a step of selecting a substance having agonistic activity against the inhibitory KIR from the antibody or antigen-binding fragment (soluble or membrane protein) expressed by method (1) or method (2) according to the method described herein.

[0106] In the above method, the inhibitory receptor on the surface of the NK cell may be NKG2A, and the corresponding activating receptor may be NKG2C. When the inhibitory receptor on the surface of the NK cell is an inhibitory KIR, the inhibitory KIR may be KIR2DL2 or KIR2DL3, and the corresponding activating KIR may be KIR2DS1, KIR2DS2, or KIR2DS4. Alternatively, the inhibitory KIR may be KIR3DL1, and the corresponding activating KIR may be KIR3DS1. Furthermore, the inhibitory KIR may be KIR2DL1, and the corresponding activating KIR may be KIR2DS1, KIR2DS2, or KIR2DS4.

[0107] A polyclonal population of presenters that presents antigen-binding sites that bind to inhibitory receptors on the surface of NK cells can be obtained or produced by known methods. For example, a phage library that displays diverse variable regions, which is used in phage display methods to obtain antibodies, or a yeast library used in yeast display methods, can be used. In another embodiment, an animal is immunized with an antigen protein containing the extracellular domain of an inhibitory receptor on the surface of NK cells, and cells carrying antigen-specific antibody genes, such as B cells, are collected from the spleen cells of the animal that produce antibodies or antigen-binding fragments against the antigen. Gene sequences encoding the variable regions of the antibody genes of the cells are amplified and inserted into a vector that displays the variable regions in a predetermined format on the surface of a lipid membrane, thereby producing a presenter library. Such techniques are described, for example, in David R. Maass et al. J Immunol Methods, (2007 Jul 31); 324(1-2):13-25 and Lukas Roth et al. , Methods Mol Biol., (2020); 2070: 173-189, etc. When the display body is a phage, a phagemid vector is used as the vector.

[0108] In method (1) or method (2), various cells such as animal cells and Escherichia coli can be used as cells for expressing the antibody or antigen-binding fragment, depending on the purpose. When expressing the antibody or antigen-binding fragment as a membrane-type protein, target cells used in agonist activity measurement tests or transplant cells for cell transplantation (examples include, but are not limited to, blood cells, T cells, and pluripotent stem cells) can be used.

[0109] Examples of substances having agonistic activity specific to NKG2A include the above-mentioned (NI) polypeptide, scFv, and membrane proteins containing the scFv in the extracellular domain.

[0110] Examples of substances having agonistic activity specific to inhibitory KIR2DL2 / 3 include the polypeptides of any of (I) to (XII) above, scFv, membrane-type proteins containing the scFv in their extracellular domains, etc. Furthermore, examples of substances having agonistic activity specific to inhibitory KIR3DL1 include the polypeptides of (XIII) above, scFv, membrane-type proteins containing the scFv in their extracellular domains, scFv, membrane-type proteins containing the scFv in their extracellular domains, etc.

[0111] Examples of substances having agonistic activity specific to inhibitory KIR2DL1 include any of the above polypeptides (CI) to (CXV), scFv, and membrane proteins containing the scFv in the extracellular domain.

[0112] Furthermore, in one embodiment, the present invention provides a polypeptide comprising, as a binding site for an inhibitory receptor on the surface of NK cells, 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 (e.g., a competitive-binding antibody) that competes with the monoclonal antibody or antigen-binding fragment that binds to the inhibitory receptor on the surface of NK cells for binding to the inhibitory receptor on the surface of NK cells. Such a competitive-binding antibody recognizes and binds to the same epitope as the monoclonal antibody or scFv, or an epitope that overlaps with or is located in the vicinity of the epitope, and is therefore likely to exhibit the same agonistic activity as the monoclonal antibody or antigen-binding fragment for the inhibitory receptor on the surface of NK cells. Therefore, by obtaining multiple antibodies or antigen-binding fragments that competitively bind to antibodies or antigen-binding fragments whose agonistic activity has been confirmed in the Examples herein from antibodies against inhibitory receptors on the surface of NK cells, and then selecting from among these antibodies or antigen-binding fragments that exhibit the desired agonistic activity (particularly, agonistic activity specific to inhibitory receptors on the surface of NK cells), it is possible to obtain polypeptides that have the desired agonistic activity against inhibitory receptors on the surface of NK cells.

[0113] For example, a polypeptide of the present invention containing a binding site for NKG2A contains binding sites 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 consisting of the amino acid sequence of (Ni) above in binding to NKG2A.

[0114] For example, the polypeptide of the present invention containing a binding site for inhibitory KIR2DL2 and / or KIR2DL3 contains 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 scFv, Lirilumab, or Pan2D, consisting of any of the amino acid sequences (i) to (xii) above, in binding to inhibitory KIR2DL2 and / or KIR2DL3.

[0115] For example, a polypeptide containing a binding site for KIR2DL1 contains binding sites derived from the heavy chain variable region and light chain variable region of a monoclonal antibody or antigen-binding fragment that competes with Pan2D or the above (P) in binding to KIR2DL1.

[0116] Furthermore, for example, a polypeptide containing a binding site for KIR2DL1 contains 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 an scFv consisting of any of the amino acid sequences (ci) to (cxv) above for binding to KIR2DL1.

[0117] Furthermore, for example, the polypeptide of the present invention containing a binding site for inhibitory KIR3DL1 contains binding sites 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 consisting of any of the amino acid sequences (xiii) to (xviii) above in binding to inhibitory KIR3DL1.

[0118] The epitope of an NK cell surface inhibitory receptor recognized by a polypeptide of the present invention containing a binding site for an NK cell surface inhibitory receptor can be analyzed by X-ray crystal structure analysis of a complex between the polypeptide containing a binding site for an NK cell surface inhibitory receptor and the NK cell surface inhibitory receptor, i.e., model structural analysis based on the X-ray crystal structure. Epitope analysis by X-ray crystal structure analysis can be performed, for example, by crystallizing a complex between a polypeptide of the present invention containing a binding site for an NK cell surface inhibitory receptor and the NK cell surface inhibitory receptor and performing X-ray structural analysis. In this case, a Fab fragment can be used as the polypeptide containing a binding site for an NK cell surface inhibitory receptor. For example, the analysis can be performed by obtaining a ribbon model and identifying amino acid residues located near the Fab fragment, for example, within 4 Å, and determining that these amino acid residues form the epitope. The X-ray structural analysis can be used to calculate that specific amino acids in the binding site interact with other specific amino acids.

[0119] For example, the amino acids constituting the epitope of inhibitory KIR2DL2 in binding to a2DL2 / 3-DS10 (VH: SEQ ID NO: 51, VL: SEQ ID NO: 52) are Glu42 (amino acid number 21 of SEQ ID NO: 6), His61 (amino acid number 40 of SEQ ID NO: 6), Lys65 (amino acid number 44 of SEQ ID NO: 6), Phe66 (amino acid number 45 of SEQ ID NO: 6), Lys67 (amino acid number 46 of SEQ ID NO: 6), Asp68 (amino acid number 47 of SEQ ID NO: 6), Thr69 (amino acid number 48 of SEQ ID NO: 6), Leu70 (amino acid number 49 of SEQ ID NO: 6), and Glu41 (amino acid number 50 of SEQ ID NO: 6). 6), Ser86 (amino acid number 65 of SEQ ID NO:6), Ile87 (amino acid number 66 of SEQ ID NO:6), Gly88 (amino acid number 67 of SEQ ID NO:6), Pro89 (amino acid number 68 of SEQ ID NO:6), Met91 (amino acid number 70 of SEQ ID NO:6), Gln92 (amino acid number 71 of SEQ ID NO:6), Asp93 (amino acid number 72 of SEQ ID NO:6), Leu94 (amino acid number 73 of SEQ ID NO:6), Tyr101 (amino acid number 80 of SEQ ID NO:6), and Leu111 (amino acid number 90 of SEQ ID NO:6). Here, 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, interaction with these amino acids and amino acids nearby is important for a KIR2DL2 / 3-specific binder.

[0120] Analysis of the interaction between a2DL2 / 3-DS10 and KIR2DL2 confirmed that Tyr at position 107 of the VH chain of a2DL2 / 3-DS10 (amino acid number 107 of SEQ ID NO: 51; hereinafter referred to as "DS10-VH-Y107") forms a hydrogen bond with Asp68 on KIR2DL2 and is positioned adjacent to Phe66. On the other hand, analysis of the interaction between a2DL2 / 3-DS10 and the corresponding activated KIR showed that DS10-VH-Y107 either weakly or cannot form a hydrogen bond with Asn68 (47 in SEQ ID NO: 14) of KIR2DS4 corresponding to Asp68, and that steric hindrance occurs with Tyr66 (Tyr at position 45 in SEQ ID NO: 12) of KIR2DS2 corresponding to Phe66, preventing them from adopting a proximal configuration, suggesting that these factors contribute to the property of a2DL2 / 3-DS10 not binding to KIR2DS2 and KIR2DS4. That is, when the amino acid corresponding to Phe66 in the receptor is Tyr, steric hindrance occurs in the amino acids in the variable region adjacent to Phe66 and Asp68 of KIR2DL2, and these amino acids are capable of forming hydrogen bonds with Asp but have weak or no hydrogen-bonding ability with Asn, preferably Tyr.

[0121] In an analysis of the interaction between a2DL2 / 3-DS10 and KIR2DL2, it was confirmed that Tyr at position 106 of the VH chain of a2DL2 / 3-DS10 (Tyr at amino acid number 106 of SEQ ID NO: 51; hereinafter "DS10-VH-Y106") forms a hydrogen bond with the main chain carbonyl group of Pro89 on KIR2DL2. On the other hand, in an analysis of the interaction between a2DL2 / 3-DS10 and the corresponding activated KIR, it was suggested that DS10-VH-Y106 is unable to form or only weakly forms a hydrogen bond with the main chain carbonyl group of Arg89 (Arg at position 68 of SEQ ID NO: 10) of KIR2DS1, which corresponds to Pro89, suggesting that this contributes to the property of a2DL2 / 3-DS10 not binding to KIR2DS1. That is, the amino acid in the variable region adjacent to Pro89 on KIR2DL2 is an amino acid that can form a hydrogen bond with Pro and has weak or no ability to form a hydrogen bond with Arg, and is preferably Tyr.

[0122] In an analysis of the interaction between a2DL2 / 3-DS10 and KIR2DL2, it was confirmed that the main chain carbonyl group of Gly at position 104 of the VH chain of a2DL2 / 3-DS10 (Gly at amino acid number 104 of SEQ ID NO: 51, hereinafter referred to as "DS10-VH-G104") forms a hydrogen bond with the main chain imino group of Gly88 on KIR2DL2. On the other hand, in an analysis of the interaction between a2DL2 / 3-DS10 and the corresponding activated KIR, it was suggested that DS10-VH-G104 cannot form or weakens a hydrogen bond between the main chains of Ser88 of KIR2DS1 (Ser at position 67 of SEQ ID NO: 10) corresponding to Gly88 due to steric hindrance, suggesting that this contributes to the property of a2DL2 / 3-DS10 not binding to KIR2DS1. That is, the amino acid in the variable region adjacent to Gly88 on KIR2DL2 is an amino acid that can form a hydrogen bond with the imino group in the main chain of Gly and has weak or no ability to form a hydrogen bond with the imino group in the main chain of Ser, and is preferably Gly.

[0123] In an analysis of the interaction between a2DL2 / 3-DS10 and KIR2DL2, it was confirmed that Thr at position 94 of the VL chain of a2DL2 / 3-DS10 (Thr at amino acid number 94 of SEQ ID NO: 52; hereinafter referred to as "DS10-VL-T94") forms a hydrogen bond with the side chain of Asp93 on KIR2DL2 via its main chain imino group. On the other hand, in an analysis of the interaction between a2DL2 / 3-DS10 and the corresponding activated KIR, it was suggested that DS10-VL-T94 is unable to form or only weakly forms a hydrogen bond with Val93 of KIR2DS4 (Val at position 72 of SEQ ID NO: 14) corresponding to Asp93, suggesting that this contributes to the inability of a2DL2 / 3-DS10 to bind to KIR2DS4. Furthermore, it was suggested that this specificity of 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 adjacent to Asp93 on KIR2DL2 is capable of forming a hydrogen bond with the side chain of Asp and is an amino acid that has weak or no ability to form a hydrogen bond with Val, and is preferably Thr, Asn, Gly, His, Ile, Ser, Val, or Ala, and more preferably Thr.

[0124] One embodiment 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 1 to 6 (preferably 3 to 6, more preferably 4 to 6, and even more preferably 5 or 6) amino acids in the region of amino acids 44 to 49, including the amino acid at position 47, 2 to 4 (preferably 2, 3, or 4) amino acids in the region of amino acids 65 to 68, including the amino acids at positions 67 and 68, and 1 to 4 (preferably 2, 3, or 4) amino acids in the region of amino acids 70 to 73, including the amino acid at position 72, in the amino acid sequence of KIR2DL2 of SEQ ID NO: 6. Preferred examples of such substances are binders comprising a variable region that interacts with the 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); the 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 the 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.

[0125] One embodiment of the KIR2DL2-specific agonist of the present invention is an antibody or antigen-binding fragment in which the amino acid in the variable region that interacts with Asp at position 47 of SEQ ID NO: 6 of KIR2DL2 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. Preferably, in addition to the above amino acids, such an antibody or antigen-binding fragment has Tyr as the amino acid adjacent to Phe at position 45 of SEQ ID NO: 6, and more preferably, all of the above amino acids are contained in CDR3 of the variable region. Such an antibody or antigen-binding fragment is preferably characterized by having a first variable region having a CDR3 comprising the amino acid sequence of 97 to 112 of SEQ ID NO: 51, and a second variable region having a CDR3 comprising the amino acid sequence of 89 to 97 of SEQ ID NO: 52, in which Thr at position 94 may be substituted with Asn, Gly, His, Ile, Ser, Val, or Ala; more preferably, an antibody or antigen-binding fragment is characterized by having the first variable region comprising the amino acid sequence of SEQ ID NO: 51, and having the second variable region having an amino acid sequence of SEQ ID NO: 52, in which Thr at position 94 may be substituted with Asn, Gly, His, Ile, Ser, Val, or Ala.

[0126] Furthermore, in antigen binding tests, epitopes can be analyzed by conducting competition tests in which polypeptides containing binding sites for inhibitory receptors on the surface of NK cells compete with each other. For example, a K562 cell line expressing an scFv of a polypeptide having a binding site for an inhibitory receptor on the surface of NK cells and a Fab fragment of a different polypeptide can be mixed with recombinant NKG2A or KIR, and the binding to recombinant NKG2A or KIR can be allowed to compete between the K562 cell line expressing the scFv and the Fab fragment. The epitope site of each binder can be evaluated using as an indicator whether binding between the K562 cell line expressing the scFv and recombinant NKG2A or KIR is inhibited by competition. In the present invention, the terms recombinant, recombinant, and soluble are used to refer to the same thing.

[0127] Furthermore, the polypeptide of the present invention containing a binding site for an inhibitory receptor on the surface of NK cells includes other polypeptides that recognize the epitope recognized by the polypeptide containing a binding site for an inhibitory receptor on the surface of NK cells analyzed by the above method. The other polypeptides compete with the polypeptide of the present invention containing a binding site for an inhibitory receptor on the surface of NK cells for binding to the inhibitory receptor on the surface of NK cells and have inhibitory selectivity.

[0128] (B) The membrane-type protein of the present invention has an extracellular domain containing a binding site for an inhibitory receptor on the surface of NK cells and a transmembrane domain, and therefore, when expressed in a cell, a polypeptide that has a binding site for an inhibitory receptor on the surface of NK cells and recognizes and binds to the inhibitory receptor on the surface of NK cells can be present on the cell membrane surface. The membrane-type protein of the present invention may further contain an intracellular domain.

[0129] The polypeptide has a binding site for an inhibitory receptor on the surface of an NK cell, and binds to the inhibitory receptor on the surface of an NK cell, and is linked to the transmembrane domain by an extracellular linker domain.

[0130] Here, a domain refers to a region present in a polypeptide that folds into a specific structure independently of other regions. In membrane proteins, a polypeptide that has a binding site for an inhibitory receptor on the surface of NK cells and binds to the inhibitory receptor on the surface of NK cells can be referred to as a binding domain. When the binding domain includes a first variable region and a second variable region derived from the heavy chain variable region and light chain variable region of the monoclonal antibody or antigen-binding fragment described in (1) above, the two variable regions can employ a linker sequence that can be employed in the scFv described above. Furthermore, the first variable region may be arranged in the following order from the N-terminus of the polypeptide: first variable region-linker sequence-second variable region, or second variable region-linker sequence-first variable region.

[0131] The transmembrane domain is a domain that has affinity for the lipid bilayer that constitutes the cell membrane, and examples thereof include, but are not limited to, transmembrane domains of HLA, CD28, CD3, CD8, CD19, CD4, or 4-1BB. Transmembrane domains derived from various natural membrane proteins and artificially designed transmembrane sequences can also be used. A preferred example is the CD8α-derived transmembrane domain (SEQ ID NO: 27).

[0132] Examples of extracellular linkers include, but are not limited to, full-length or partial extracellular domains of HLA, CD28, CD8, CD19, CD3, CD4, or 4-1BB, and include linker sequences and hinge sequences derived from various natural proteins (e.g., immunoglobulin hinge sequences), artificially designed flexible linker sequences, etc. Preferred are the 45-amino acid long hinge sequence derived from CD8α (SEQ ID NO: 26) and the 66-amino acid long hinge sequence derived from CD8α (SEQ ID NO: 24).

[0133] The intracellular domain is necessary for the stability of the scFv, and a portion of the intracellular domain (preferably 10 amino acids or less, 9 amino acids or less, 8 amino acids or less, 7 amino acids or less, 6 amino acids or less, 5 amino acids or less, 4 amino acids or less, 3 amino acids or less, 2 amino acids or less, or 1 amino acid) that is not involved in intracellular signaling, such as HLA, CD28, CD3, CD8, CD19, CD4, or 4-1BB, can be used. However, when it is necessary to promote activation or suppression of intracellular signals by binding to KIR, a portion of the intracellular domain involved in intracellular signaling can also be used.

[0134] Examples of membrane proteins of the present invention include the following.

[0135] Examples of membrane proteins that exhibit agonistic activity against NKG2A include membrane proteins having an extracellular domain containing an NKG2A-binding site (specifically, an scFv containing the first and second variable regions described in (NI) above) and a transmembrane domain.

[0136] Examples of membrane-type proteins that exhibit agonistic activity against KIR2DL2 or KIR2DL3 include membrane-type proteins having an extracellular domain and a transmembrane domain that include a binding site for KIR2DL2 and / or KIR2DL3 (specifically, an scFv comprising the first and second variable regions described in any of (I) to (XII) above).

[0137] Examples of membrane-type proteins that exhibit agonistic activity against KIR3DL1 include membrane-type proteins having an extracellular domain containing a binding site for KIR3DL1 (specifically, an scFv containing the first and second variable regions described in any of (XIII) to (XVIII) above) and a transmembrane domain.

[0138] Examples of membrane-type proteins that exhibit agonistic activity against KIR2DL1 include membrane-type proteins having an extracellular domain and a transmembrane domain that include a binding site for KIR2DL1 (specifically, an scFv including the first and second variable regions described in any of (CI) to (CXV) above).

[0139] (C) A polypeptide whose binding site for an inhibitory receptor on the surface of an NK cell includes a sequence derived from the binding site of an HLA molecule, which is a natural ligand of the inhibitory receptor on the surface of the NK cell, with the inhibitory receptor on the surface of the NK cell, and whose binding affinity to a TCR (T cell receptor) is deleted or weakened.

[0140] For example, when the inhibitory KIR is KIR2DL2 or KIR2DL3, a polypeptide can be used which contains a C1 epitope motif sequence (in which 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, B73, etc., which belong to the HLA-C1 type, and which contains a domain that is not recognized by TCR by adding a mutation to or deleting the region recognized by TCR of the HLA molecule.

[0141] For example, when the inhibitory KIR is KIR2DL1, a polypeptide can be used that contains a C2 epitope motif sequence (in which the 77th amino acid is N and the 80th amino acid is K) contained in HLA-Cw2, Cw4, Cw5, Cw6, Cw15, Cw17, etc., which belong to the HLA-C2 type, and that contains a domain that is not recognized by TCR by adding a mutation to or deleting the region recognized by TCR of the HLA molecule.

[0142] For example, when the inhibitory KIR is KIR3DL1, a polypeptide can be used that contains a Bw4 epitope motif sequence (in which the 77th amino acid is N, the 80th amino acid is I or T, and the 83rd amino acid is R) found in HLA-B5, B27, B37, B38, B44, B49, B51, B52, B53, B57, B58, B59, B77, A23, A24, A25, A32, etc., which belong to the HLA-Bw4 type, and that contains a domain that is not recognized by TCR by adding a mutation to or deleting the region of the HLA molecule that is recognized by TCR. Such a polypeptide may be in the form of a soluble ligand containing an amino acid sequence that includes the epitope motif sequence, or in the form of a membrane protein that contains the epitope motif sequence in its extracellular domain.

[0143] For example, when the inhibitory KIR is KIR3DL2, a polypeptide can be used that includes a free heavy chain of HLA-A3, A11, or B27, or a domain that is not recognized by TCR by adding a mutation to or deleting the region of the HLA molecule that is recognized by TCR. Such a polypeptide may be in the form of a soluble ligand or a membrane protein.

[0144] Further examples of substances having agonistic activity against inhibitory receptors on the surface of NK cells include low-molecular-weight compounds that bind to inhibitory receptors on the surface of NK cells and exhibit agonistic activity against the inhibitory receptors on the surface of NK cells. Such compounds can be obtained, for example, by screening from a compound library using the above-mentioned method for evaluating agonistic activity against inhibitory receptors on the surface of NK cells. Furthermore, compounds selected by this method can be further derivatived based on their structures, and their binding activity to the target inhibitory receptor on the surface of NK cells and their agonistic activity can be measured, structure-activity relationships can be analyzed, and further derivative development can be repeated to obtain compounds with better binding affinity and / or agonistic activity.

[0145] 2. Method for producing a substance that binds to an inhibitory receptor on the surface of NK cells and has the activity of inhibiting the cytocidal activity of NK cells The polypeptide of the present invention that has a binding site for an inhibitory receptor on the surface of NK cells and binds to the inhibitory receptor on the surface of NK cells can be prepared in the form of a secreted protein (soluble protein) or a membrane protein by genetic recombination, genome editing, in vitro translation, chemical synthesis, peptide synthesis, or the like.

[0146] For example, the polypeptide of the present invention can be produced by introducing a polynucleotide comprising a nucleotide sequence encoding the amino acid sequence of the polypeptide into an expression system (such as a cell, phage, yeast, microorganism, or in vitro expression system) in a form that allows expression in the target expression system for the polypeptide, functionally linked to necessary factors, and recovering the polypeptide from the expression system after expression, or recovering cells on whose surface the polypeptide is expressed.

[0147] Here, "functionally linked" refers to elements linked so as to perform their functions. When the expression system is a cell, yeast, microorganism, or the like, these nucleotides are inserted into an expression vector, host cells are transformed with the vector, the host cells are cultured to produce the polypeptide, and the polypeptide or cells expressing the polypeptide on their surface are recovered from the culture. The vector may also contain nucleotides encoding a signal peptide that promotes secretion of the polypeptide from the host cells. In this case, the nucleotides encoding the signal peptide and the nucleotides encoding the polypeptide are linked in-frame. After the polypeptide is produced, the signal peptide is removed, allowing the polypeptide to be obtained as a mature protein. For example, the CD8 signal peptide can be used as a signal peptide.

[0148] The expression vector is not particularly limited as long as it can be replicated in a host such as an animal cell, bacteria, or yeast, and examples thereof include known plasmids and phages. Examples of vectors used to construct an expression vector include pcDNA™ (ThermoFisher SCIENTIFIC), Flexi™ Vector (Promega), pUC19, pUEX2 (Amersham), pGEX-4T, pKK233-2 (Pharmacia), and pMAM-neo (Clontech). Prokaryotic cells such as Escherichia coli and Bacillus subtilis, as well as eukaryotic cells such as yeast and animal cells, can be used as host cells, but eukaryotic cells are preferred. For example, HEK293 cells, a human embryonic kidney cell line, or Chinese hamster ovary (CHO) cells may be used as animal cells. The expression vector can be introduced into host cells by known methods to transform the host cells. Examples of such methods include electroporation, calcium phosphate precipitation, and DEAE-dextran transfection. The produced antibodies can be purified using separation and purification methods commonly used for proteins. For example, affinity chromatography, other chromatographies, filters, ultrafiltration, salting out, dialysis, and the like may be appropriately selected and combined.

[0149] 3. Cells expressing a membrane-type protein of the present invention on their surface and a method for producing the same Cells expressing on their surface a substance that binds to an inhibitory receptor on the surface of an NK cell and inhibits the cytocidal activity of an NK cell are preferably cells expressing a polypeptide that has a binding site for an inhibitory receptor on the surface of an NK cell and binds to the inhibitory receptor on the surface of an NK cell, as described in 1. (B) above, and a membrane-type protein having a transmembrane domain. The transmembrane domain of the membrane-type protein is present in a state that it spans the cell membrane, and a polypeptide that has a binding site for an inhibitory receptor on the surface of an NK cell and binds to an inhibitory KIR is present on the cell surface.

[0150] Examples of such cells include cells expressing on their surface a membrane protein exhibiting agonistic activity against NKG2A (a membrane protein having an extracellular domain and a transmembrane domain containing a binding site for NKG2A (specifically, an scFv containing the first and second variable regions described in (N-I) above)) and cells expressing on their surface a membrane protein exhibiting agonistic activity against KIR2DL2 or KIR2DL3 (a membrane protein having an extracellular domain and a transmembrane domain containing a binding site for KIR2DL2 and / or KIR2DL3 (specifically, an scFv containing the first and second variable regions described in any of (I) to (XII) above)). Examples of such a cell include a cell in which a membrane-type protein exhibiting agonistic activity against KIR3DL1 (a membrane-type protein having an extracellular domain and a transmembrane domain that includes a binding site for KIR3DL1 (specifically, an scFv including the first and second variable regions described in any of (XIII) to (XVIII) above)) is expressed on the cell surface, and a cell in which a membrane-type protein exhibiting agonistic activity against KIR2DL1 (a membrane-type protein having an extracellular domain and a transmembrane domain that includes a binding site for KIR2DL1 (specifically, an scFv including the first and second variable regions described in any of (CI) to (CXV) above)) is expressed on the cell surface.

[0151] These cells may express multiple types of membrane-type proteins against different inhibitory receptors on their cell surface. Cells expressing multiple types of membrane-type proteins exert agonistic activity against the inhibitory receptors on NK cells targeted by each membrane-type protein, thereby avoiding attack from heterogeneous NK cell populations that express NK cell receptors (KIR, NKG2, etc.) that distinguish between self and non-self Class I MHC in diverse expression patterns. In particular, cells co-expressing a membrane-type protein that exhibits specific agonistic activity against NKG2A and a membrane-type protein that exhibits specific agonistic activity against inhibitory KIR can avoid attack from NKG2A-positive NK cells, NKG2C-positive NK cells, inhibitory KIR-positive NK cells, and the corresponding activating KIR-positive NK cells.

[0152] The cells have a binding site for an inhibitory receptor on the surface of NK cells, and a polynucleotide encoding a polypeptide that binds to an inhibitory receptor on the surface of NK cells may be introduced into the cells and expressed.

[0153] For a polypeptide having a binding site for an inhibitory receptor on the surface of NK cells and binding to the inhibitory receptor on the surface of NK cells, and a membrane-type protein having a transmembrane domain, polynucleotides encoding the amino acid sequences of each domain may be linked and further functionally linked to elements such as a promoter, an enhancer, a polyadenylation signal, etc. Furthermore, the extracellular linker domain may be present between the polypeptide that binds to the inhibitory receptor on the surface of NK cells and the transmembrane domain by linking nucleotide sequences encoding the amino acid sequences of each domain, inserting the nucleotide sequences into a vector, and expressing the vector in a host cell.

[0154] Specific examples of such polynucleotides include: a polynucleotide comprising a nucleotide sequence encoding the amino acid sequence of a membrane-type protein exhibiting agonistic activity against NKG2A (a membrane-type protein having an extracellular domain and a transmembrane domain, which includes a binding site for NKG2A (specifically, an scFv comprising the first and second variable regions described in (N-I) above)); a polynucleotide comprising a nucleotide sequence encoding the amino acid sequence of a membrane-type protein exhibiting agonistic activity against KIR2DL2 or KIR2DL3 (a membrane-type protein having an extracellular domain and a transmembrane domain, which includes a binding site for KIR2DL2 and / or KIR2DL3 (specifically, an scFv comprising the first and second variable regions described in any of (I) to (XII) above)); a membrane-type protein exhibiting agonistic activity against KIR3DL1 (a membrane-type protein having a binding site for KIR3DL1 (specifically, an scFv comprising the first and second variable regions described in any of (XIII) to (XVIII) above)); Examples of such polynucleotides include a polynucleotide comprising a nucleotide sequence encoding the amino acid sequence of a membrane-type protein having an extracellular domain and a transmembrane domain, the membrane-type protein exhibiting agonistic activity against KIR2DL1 (a membrane-type protein having an extracellular domain and a transmembrane domain, the membrane-type protein comprising a binding site for KIR2DL1 (specifically, a membrane-type protein having an extracellular domain and a transmembrane domain, the membrane-type protein comprising an scFv comprising the first and second variable regions described in any of (CI) to (CXV) above)). A single polynucleotide may encode multiple membrane-type proteins for different inhibitory receptors. In this case, by linking the nucleotide sequences encoding each membrane-type protein with a nucleotide sequence encoding a self-cleaving peptide, the membrane-type proteins can be cleaved sequentially and expressed on the cell surface as individual membrane-type proteins. When the protein is expressed on the cell surface as a membrane-type protein, a polypeptide having a secretory signal peptide (e.g., a signal peptide derived from CD8) linked to the N-terminus of the extracellular domain may be used as the membrane-type protein, and a polypeptide containing a nucleotide sequence encoding the amino acid sequence of a membrane-type protein having such a signal peptide at the N-terminus may be used as the polynucleotide.

[0155] The method for introducing a polynucleotide may be any commonly used method, and is not particularly limited. When the polynucleotide is introduced using a vector, examples of the vector that can be used include, but are not limited to, a lentivirus vector, a retrovirus vector, a foamy virus vector, and an adeno-associated virus vector.

[0156] When expressing the membrane-type protein, an expression control element such as an enhancer or promoter is linked to the nucleotide encoding the membrane-type protein, introduced into an expression vector, and cells are transformed with the expression vector to express the membrane-type protein in the cells. When introducing the nucleotide, it is desirable to link an appropriate promoter upstream of the nucleotide to be introduced in a functional manner. The promoter is not limited, but examples that can be used include viral promoters such as lentivirus, retrovirus, adenovirus, SV40 virus, and cytomegalovirus, as well as promoters derived from mammalian cells. The method for producing the polypeptide described in 2. above can be used to produce cells that express the membrane-type protein of the present invention.

[0157] When the nucleotide encoding the membrane-type protein is introduced into cells, the expression vector may contain a selection marker gene, which may be a known marker gene such as a thymidine kinase gene.

[0158] The present invention also encompasses DNA encoding the above membrane-type protein and RNA that is a transcription product of the DNA.

[0159] Genome editing technology may also be employed for the expression of the membrane-type protein of the present invention. Genome editing technology includes, but is not limited to, various methods such as CRISPR-Cas, Zinc-Finger, and TaLEN. For example, the method described in WO2018 / 018534 is a method for producing genetically modified cells using genome editing technology, which can efficiently perform genome editing not only on normal cells but also on pluripotent stem cells such as iPS cells.

[0160] Cells expressing the above-mentioned membrane-type proteins include cells for allogeneic transplantation for transplantation or cell therapy. These cells are called therapeutic cells. Cells expressing the above-mentioned membrane-type proteins express on their surface a polypeptide that exhibits agonistic activity at an inhibitory receptor on the surface of NK cells. When this polypeptide binds to the inhibitory receptor on the surface of NK cells, it transmits an inhibitory signal into the NK cells and suppresses the cytocidal activity of the NK cells. As a result, immune rejection during transplantation can be suppressed, making the cells less susceptible to immune rejection and reducing the risk of immune rejection.

[0161] Pluripotent stem cells, particularly ES cells and iPS cells, are used as cells for allogeneic transplantation or cell therapy. These pluripotent stem cells can be used to express the membrane-type protein of the present invention or for useful gene editing, as described above. Other examples include mesenchymal stem cells and T cells, NK cells, and macrophages derived from PBMCs (peripheral blood mononuclear cells) as primary cells. Cells collected from these biological tissues can be modified, for example, to knock out HLA or to express NKG2A agonists and / or KIR agonists, thereby suppressing NK activity. Furthermore, mesenchymal cells can be modified to express and secrete useful therapeutic factors, such as cancer treatment factors, and then transplanted. T cells and NK cells can be modified to have anti-cancer activity, such as CAR-T / NK, and used as therapeutic cells. Furthermore, examples of cells for allogeneic transplantation for transplantation or cell therapy include cardiomyocytes, chondrocytes, retinal cells, liver cells, kidney cells, pancreatic cells, etc. These cells may be cells collected from a donor, or may be various cells produced by artificially inducing differentiation from pluripotent stem cells. Regardless of the type of cell, if a membrane protein capable of binding to an inhibitory receptor on the surface of an NK cell is expressed on the cell surface, it can bind to the inhibitory receptor on the surface of the NK cell, transmit an inhibitory signal into the NK cell, and suppress the cytocidal activity of the NK cell. Furthermore, if the membrane protein has agonist activity specific to the inhibitory receptor on the surface of the NK cell (i.e., it does not exhibit agonist activity at the corresponding activating receptor), it can avoid attack from NK cells expressing the corresponding activating receptor.

[0162] The cells of the present invention may further have at least one Class I HLA expression on the cell surface that has been eliminated or attenuated. It is preferable that the expression of all Class I HLAs has been eliminated or attenuated. The elimination or attenuation of the expression of at least one Class I HLA on the cell surface may be due to modification of at least one of the Class I HLA, B2M, TAP1, TAP2, and TAPBP regions on the genome by gene editing. Among these, modification of B2M (preferably deletion of B2M) is preferred. Methods for eliminating or attenuating Class I HLA expression on the cell surface can be appropriately adopted, for example, with reference to methods used in Universal Cell technology (see Patent Document 2, etc.).

[0163] The cells of the present invention may also be a cell line established from an individual in which HLA expression has been genetically lost.

[0164] Furthermore, the cells of the present invention may further express both a membrane protein that binds to an inhibitory KIR and a membrane protein that binds to NKG2A on the cell surface. NKG2A (CD159) is a type of inhibitory receptor on the surface of NK cells. HLA-E is an example of a ligand for NKG2A.

[0165] The membrane protein that binds to NKG2A and is expressed in the cells is preferably a membrane protein that has, in its extracellular domain, an scFv that exhibits agonistic activity against NKG2A but does not exhibit agonistic activity against NKG2C.

[0166] The present invention encompasses a method for producing therapeutic cells with a reduced or suppressed risk of immune rejection in a subject, comprising expressing on the surface of the therapeutic cells a substance that has the activity of binding to an inhibitory receptor on the surface of the NK cells of the present invention and suppressing the cytocidal activity of the NK cells.

[0167] 4. CAR-T The present invention includes T cells (CAR-T) that further express a chimeric antigen receptor (CAR) that targets a desired antigen in addition to the above-mentioned membrane-type protein. The CAR-T used in the present invention is not limited to a CAR-T that targets a tumor-associated antigen. The CAR-T expresses on its surface a polypeptide comprising a domain that specifically binds to a tumor-associated antigen specific to tumor cells, a transmembrane domain, and an intracellular signaling domain. The domain that specifically binds to a tumor-associated antigen can be a polypeptide derived from an antibody against the tumor-associated antigen, such as an scFv or a polypeptide linking the Fv regions of an immunoglobulin heavy chain and light chain. The CAR of the CAR-T of the present invention is present on the cell membrane and comprises an extracellular domain comprising a domain that specifically binds to a tumor-associated antigen specific to tumor cells, a transmembrane domain, and an intracellular domain that contains an intracellular signaling domain. The extracellular domain may include an extracellular spacer domain, and the intracellular domain may include a costimulatory domain. Examples of transmembrane domains that can be used include transmembrane domains such as CD8α, CD28, CD3ε, CD3, and CD4. Examples of extracellular spacer domains that can be used include spacer sequences such as (G4S)3 and the hinge region of IgG1. Examples of costimulatory domains that can be used include CD27, CD28, CD134, CD27, CD2, CD5, CD30, and CD40. Examples of intracellular signaling domains that can be used include human CD3ζ chain, Fc receptor, FcγRIII, and FcεRI. A polynucleotide encoding the CAR of the present invention can be prepared by linking polynucleotides encoding each of the above domains. A CAR-T of the present invention can be prepared by introducing this polynucleotide and a polynucleotide encoding a polypeptide containing a binding site for an inhibitory KIR of the present invention into T cells.

[0168] The CAR-T cells of the present invention can be used to treat tumors expressing tumor-associated antigens. The CAR-T cells of the present invention express on their cell surface a polypeptide containing a binding site for an inhibitory receptor on the surface of NK cells, and have the ability to avoid immune rejection by NK cells in addition to cytocidal activity against target cells. The present invention encompasses pharmaceutical compositions comprising the CAR-T cells of the present invention.

[0169] 5. Use of the Substance of the Present Invention That Binds to an Inhibitory Receptor on the Surface of NK Cells and Inhibits the Cytocidal Activity of NK Cells, and Cells Expressing on Their Surface a Substance That Binds to an Inhibitory Receptor on the Surface of NK Cells and Inhibits the Cytocidal Activity of NK Cells The present invention encompasses pharmaceutical compositions comprising, as active ingredients, the Substance of the Present Invention that Binds to an Inhibitory Receptor on the Surface of NK Cells and Inhibits the Cytocidal Activity of NK Cells, and cells expressing on their surface a Substance That Binds to an Inhibitory Receptor on the Surface of NK Cells and Inhibits the Cytocidal Activity of NK Cells. Examples of such cells include cells expressing on their surface a polypeptide that is a membrane-type protein having an extracellular domain and a transmembrane domain that includes an NKG2A-binding site, the polypeptide (1) having activity to inhibit NK cells by binding to NKG2A on the surface of NK cells, and (2) substantially no agonist activity against NKG2C, and further expressing a membrane-type protein that exhibits specific agonist activity against an inhibitory KIR, and further having lost or attenuated expression of at least one Class I HLA on the cell surface.

[0170] The pharmaceutical composition can be used as an agent for suppressing immune rejection during organ or therapeutic cell transplantation. Specifically, it can be used to suppress rejection during allogeneic transplantation of kidneys, pancreatic islets, etc., to prevent or treat GVHD (graft-versus-host disease), and to suppress rejection during allogeneic transplantation of regenerative therapy cells. It can also be used for xenogeneic organ transplantation, such as transplantation of organs derived from genetically modified pigs into humans. The pharmaceutical composition is also referred to as an immune rejection suppressant or transplant rejection suppressant.

[0171] Cells expressing on their surface a substance that binds to an inhibitory receptor on the surface of NK cells and inhibits the cytocidal activity of NK cells can be used as therapeutic cells that suppress immune rejection in a subject to which the cells are administered or transplanted, and these therapeutic cells can be used as a pharmaceutical composition for transplantation.

[0172] Furthermore, the present invention encompasses a method for suppressing immune rejection in a subject to be treated, comprising administering or transplanting to a subject in need of a transplant a substance of the present invention that binds to an inhibitory receptor on the surface of an NK cell and has the activity of suppressing the cytocidal activity of an NK cell, or cells on the cell surface of which a substance that binds to an inhibitory receptor on the surface of an NK cell and has the activity of suppressing the cytocidal activity of an NK cell is expressed.

[0173] Furthermore, the present invention encompasses a method for suppressing immune rejection in a subject to be treated, comprising expressing on the surface of therapeutic cells a substance that binds to an inhibitory receptor on the surface of NK cells of the present invention and inhibits the cytocidal activity of NK cells. Examples of such methods include expressing on the cell surface a polypeptide that is a membrane-type protein having an extracellular domain containing an NKG2A-binding site and a transmembrane domain, the polypeptide (1) having activity to bind to NKG2A on the surface of NK cells and inhibit the NK cells, and (2) having substantially no agonist activity against NKG2C, further expressing a membrane-type protein that exhibits specific agonist activity against an inhibitory KIR, and further genetically modifying the cells so that expression of at least one Class I HLA on the cell surface is eliminated or attenuated. This method is also a method for producing cells that will suppress immune rejection in a subject to be treated when transplanted. Furthermore, this method is also a method for suppressing immune rejection in a subject when the therapeutic cells are administered to the subject, comprising expressing on the surface of the therapeutic cells a substance that has activity of binding to an inhibitory receptor on the surface of the NK cells of the present invention and suppressing the cytocidal activity of the NK cells.

[0174] When used for the above purposes, the patient to be treated has an HLA type that serves as a ligand for an inhibitory receptor on the surface of the target NK cell. For example, when an agonist substance for inhibitory KIR2DL2 and / or KIR2DL3 or cells expressing such an agonist substance on their cell surface is used, the substance exerts an immunosuppressive effect or an immune rejection evasion effect on patients who are HLA-C1 homozygous. Furthermore, when an agonist substance for inhibitory KIR2DL1 or cells expressing such an agonist substance on their cell surface is used, the substance exerts an immunosuppressive effect or an immune rejection evasion effect on patients who are HLA-C2 homozygous. When an agonist substance for inhibitory KIR2DL2 and / or KIR2DL3 and an agonist substance for inhibitory KIR2DL1 are used in combination, or when cells expressing these two types of agonist substances in combination on the cell surface are used, an immunosuppressive effect or an immune rejection avoidance effect is exerted on patients of all HLA-C genotypes (HLA-C1 homotype, HLA-C2 homotype, and HLA-C1 / C2 heterotype).

[0175] Furthermore, for HLA-Bw4-negative patients, treatment corresponding to the above-mentioned HLA-C genotype can be performed, but for HLA-Bw4-positive patients, an agonist substance for KIR3DL1 can be additionally administered in combination with the above, or cells expressing the combination on the cell surface can be used to exert an immunosuppressive effect or an immune rejection avoidance effect.

[0176] Furthermore, when an agonist substance for NKG2A or cells expressing the agonist substance on the cell surface are used, an immunosuppressive effect or immune rejection avoidance effect is exerted on patients having NKG2A-positive NK cells. Furthermore, when an agonist substance for inhibitory KIR2DL2 and / or KIR2DL3 and an agonist substance for NKG2A are used in combination, or when cells expressing a combination of these two types of agonist substances on the cell surface are used, an additive immunosuppressive effect or immune rejection avoidance effect is exerted on HLA-C1 homozygous patients compared to when an agonist substance for inhibitory KIR2DL2 and / or KIR2DL3 is used alone. Furthermore, when an agonist substance for inhibitory KIR2DL1 is used in combination with an agonist substance for NKG2A, or when cells expressing a combination of these two types of agonist substances on the cell surface are used, an additive immunosuppressive effect or immune rejection avoidance effect is exerted in HLA-C2 homozygous patients compared to when an agonist substance for inhibitory KIR2DL1 is used alone. Compared with the use of a combination of an agonist substance for inhibitory KIR2DL2 and / or KIR2DL3, an agonist substance for inhibitory KIR2DL1, and an agonist substance for NKG2A, or the use of cells expressing a combination of these three types of agonist substances on the cell surface, the use of a combination of an agonist substance for inhibitory KIR2DL2 and / or KIR2DL3 and an agonist substance for inhibitory KIR2DL1, or the use of cells expressing a combination of these two types of agonist substances on the cell surface, an additive immunosuppressive effect or immune rejection avoidance effect is exerted on patients of all HLA-C genotypes (HLA-C1 homotype, HLA-C2 homotype, and HLA-C1 / C2 heterotype).

[0177] Furthermore, in the case of HLA-Bw4-positive patients, an immunosuppressive effect or immune rejection avoidance effect can be exerted by administering an agonist substance for inhibitory KIR2DL2 and / or KIR2DL3, an agonist substance for inhibitory KIR2DL1, and an agonist substance for KIR3DL1 in combination, or by using cells in which a combination of these substances is expressed on the cell surface. However, an additive immunosuppressive effect or immune rejection avoidance effect can be exerted by administering an agonist substance for NKG2A in combination with these three types of agonist substances, or by using cells in which a combination of these substances is expressed on the cell surface.

[0178] The pharmaceutical composition of the present invention may contain a therapeutically effective amount of a substance that binds to an inhibitory receptor on the surface of NK cells and inhibits the cytocidal activity of NK cells, or a substance that binds to an inhibitory receptor on the surface of NK cells and inhibits the cytocidal activity of NK cells, or cells on whose surface a substance that binds to an inhibitory receptor on the surface of NK cells and inhibits the cytocidal activity of NK cells is expressed, as well as pharmaceutically acceptable carriers, diluents, solubilizers, emulsifiers, preservatives, adjuvants, and the like. The pharmaceutical composition of the present invention may be administered by injection, for example, via local injection, intraperitoneal administration, selective intravenous injection, intravenous injection, subcutaneous injection, or organ perfusion injection. Injectable solutions may also be formulated using carriers such as salt solutions, glucose solutions, mixtures of salt solutions and glucose solutions, and various buffer solutions. Alternatively, the composition may be formulated in powder form, which can be mixed with the liquid carrier at the time of use to prepare an injectable solution.

[0179] Other administration methods can also be appropriately selected based on the development of the formulation. For example, oral administration can be performed in the form of oral liquids, powders, pills, capsules, tablets, etc. Oral liquid preparations such as suspensions and syrups can be prepared using water, sugars such as sucrose, sorbitol, and fructose, glycols such as polyethylene glycol, oils such as sesame oil and soybean oil, preservatives such as alkyl parahydroxybenzoates, and flavors such as strawberry flavor and peppermint. Powders, pills, capsules, and tablets can be formulated using excipients such as lactose, glucose, sucrose, and mannitol, disintegrants such as starch and sodium alginate, lubricants such as magnesium stearate and talc, binders such as polyvinyl alcohol, hydroxypropyl cellulose, and gelatin, surfactants such as fatty acid esters, and plasticizers such as glycerin. Tablets and capsules are preferred unit dosage forms for the composition of the present invention because of their ease of administration. When tablets or capsules are made, solid manufacturing carriers are used.

[0180] The effective therapeutic amount of a substance that binds to an inhibitory receptor on the surface of NK cells and inhibits the cytocidal activity of NK cells varies depending on the age and condition of the patient and is ultimately determined by a physician. For example, it is 0.0001 mg to 100 mg per kg of body weight per dose. The prescribed dose may be administered once every 1 to 180 days, or may be administered in divided doses two, three, four, or more times per day at appropriate intervals.

[0181] The dosage of the substance that binds to an inhibitory receptor on the surface of NK cells and has the activity of inhibiting the cytocidal activity of NK cells, and the dosage of the cells on whose surface a substance that binds to an inhibitory receptor on the surface of NK cells and has the activity of inhibiting the cytocidal activity of NK cells is expressed can be determined appropriately depending on the type of cells to be transplanted, etc.

[0182] 5. Method for Evaluating the Immune Rejection Avoidance Activity of a Test Substance The present invention encompasses a method for evaluating the activity of a test substance to suppress the cytocidal activity of NK cells mediated by inhibitory receptors on the surface of NK cells, i.e., the immune rejection avoidance activity.

[0183] The test substance is a substance that binds to any inhibitory receptor on the surface of NK cells and has the activity of inhibiting the cytocidal activity of NK cells, and examples thereof include the substances described in 1 above.

[0184] The method comprises the following steps: (A) contacting ex vivo NK cells derived from a subject homozygous for HLA-C type C1 / C1 with target cells derived from a subject homozygous for HLA-C type C2 / C2 in the presence of a test substance that binds to KIR (wherein the Bw4 phenotype of the donor of the NK cells is the same as that of the donor of the target cells), and (B) measuring the number of surviving target cells after step A.

[0185] Specifically, the inhibitory KIR may comprise the following steps: (A) contacting ex vivo NK cells derived from a subject whose HLA-C type is C1 / C1 homozygous with target cells derived from a subject whose HLA-C type is C2 / C2 homozygous in the presence of a test substance that binds to KIR2DL2 or KIR2DL3 (wherein the NK cell donor and the target cell donor have the same phenotype of Bw4 motif-containing HLA); and (B) measuring the number of surviving target cells after step A.

[0186] The method may further comprise the following steps: (A) contacting ex vivo NK cells derived from a subject whose HLA-C type is C2 / C2 homozygous with target cells derived from a subject whose HLA-C type is C1 / C1 homozygous in the presence of a test substance that binds to KIR2DL1 (wherein the phenotype of Bw4 motif-containing HLA is the same as that of the donor of the NK cells and the donor of the target cells), and (B) measuring the number of surviving target cells after step A.

[0187] The method may further comprise the following steps: (A) contacting in vitro NK cells derived from a subject whose HLA-Bw4 type is positive / homopositive or heteropositive / negative with target cells derived from a subject whose HLA-Bw4 type is negative / homonegative in the presence of a test substance that binds to KIR3DL1 (wherein the HLA-C phenotype of the NK cell donor and that of the target cell donor are identical); and (B) measuring the number of surviving target cells after step A.

[0188] The method may further comprise the following steps: (A) contacting in vitro, in the presence of a test substance that binds to KIR3DL2, NK cells derived from a subject whose HLA-A type is A3 or A11 positive / homopositive or heteropositive / negative, with target cells derived from a subject whose HLA-A type is A3 or A11 negative / homonegative (wherein the HLA-C and Bw4 phenotypes of the NK cell donor and the target cell donor are identical); and (B) measuring the number of surviving target cells after step A.

[0189] As the NK cells, for example, peripheral blood-derived NK cells can be used, and as the target cells, for example, peripheral blood-derived T cells can be used.

[0190] NK cells have the property of "missing-self," in that they recognize their own HLA types (C1, C2, Bw4) and exhibit cytocidal activity against target cells. For example, NK cells collected from an HLA-C1 / C1 homozygous individual recognize C1 as self, and therefore, unless the target cells possess C1-type HLA-C, signals do not enter the NK cells via inhibitory KIR, resulting in cytocidal activity. On the other hand, if the target cells possess C1 type, they do not exhibit cytocidal activity, and if a substance (agonist binder) that binds to inhibitory KIR2DL2 and / or KIR2DL3, which are substitutes for C1 type, and has the activity of inhibiting the cytocidal activity of NK cells, is expressed, the cytocidal activity can be reduced.

[0191] The NK cells derived from a subject with a homozygous HLA-C type C1 / C1 used in the above evaluation method can be isolated from PBMCs (peripheral blood mononuclear cells) of a homozygous HLA-C type C1 / C1 human using CD56 positivity as an indicator. For example, they can be isolated using CD56 microbeads or flow cytometry. NK cells isolated in this manner have activated KIR2DL2 and KIR2DL3.

[0192] Target cells derived from a subject whose HLA-C type is C2 / C2 homozygous may be T cells, which can be isolated from PBMCs of a human whose HLA-C type is C2 / C2 homozygous using CD3 positivity as an indicator. Because the target cells do not express C1 type HLA, NK cells derived from the subject whose HLA-C type is C1 / C1 homozygous exhibit cytocidal activity.

[0193] When NK cells derived from a subject whose HLA-C type is C1 / C1 homozygous are contacted as effector cells with target cells derived from a subject whose HLA-C type is C2 / C2 homozygous, the target cells are killed by the effector cells. However, if a substance that binds to inhibitory KIR2DL2 and / or KIR2DL3 and has the activity of inhibiting the cytocidal activity of NK cells is present, the substance will bind to KIR2DL2 and / or KIR2DL3 on NK cells derived from a subject whose HLA-C type is C1 / C1 homozygous, inhibiting the cytocidal activity of the NK cells, thereby reducing the cytocidal activity of the effector cells and preventing the target cells from being killed. Therefore, by contacting NK cells derived from a subject whose HLA-C type is C1 / C1 homozygous with target cells derived from a subject whose HLA-C type is C2 / C2 homozygous 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 avoidance activity of the test substance, which is the activity of suppressing the cytocidal activity of NK cells against inhibitory KIR, can be evaluated.

[0194] The test substance can be determined to have immune rejection evasion activity when the number of surviving target cells is high in step B. The present invention encompasses a method for screening for a substance having immune rejection evasion activity, which comprises a step of selecting a test substance that has a high number of surviving target cells in step B as a substance having immune rejection evasion activity.

[0195] In step (A) of the above method, the test substance may be present on the surface of the target cell in the form of expressed membrane protein 1(2).

[0196] Specifically, this can be carried out by the methods described in the "Cytocidal Activity Evaluation" and "Agonist Activity Evaluation" sections of the Examples below.

[0197] The present invention will be specifically explained by the following examples, but the present invention is not limited to these examples.

[0198] Methods The following methods were used for each experiment. [Plasmid vector for virus production] To produce lentivirus used to introduce a target gene into a natural killer cell line as effector cells, a K562 cell line as target cells, and human T cells, a pLVSIN vector was used, in which the gene sequence encoding green fluorescent protein (ZsGreen1) contained in pLVSIN-EF1α IRES-ZsGreen1 (Takara Bio, 6191) had been removed. The target gene was introduced into the multicloning site of pLVSIN to prepare a plasmid vector for lentivirus production.

[0199] [Preparation of lentivirus] Lenti-X 293T cell line (Takara Bio, 632180) was cultured to confluence in Dulbecco's Modified Eagle Medium (DMEM) (Thermo Fisher Scientific, 10566-016) containing 10 (v / v)% fetal bovine serum (FBS). On the day of transfection, the cells were detached using trypsin (Thermo Fisher Scientific, 12563029) and reseeded in a T-flask. After culturing until the cells reached 70-80% confluence, the above-mentioned lentivirus production plasmid vector, Lentiviral High Titer Packaging Mix (Takara Bio, 6194), and TransIT-293 Transfection Reagent (Takara Bio, MIR2704) were mixed with Opti-MEM I Reduced Serum Media (Thermo Fisher Scientific, 31985070). After incubation, the mixture was added to the Lenti-X 293T cell line. The next day, the medium was replaced with DMEM medium containing 10 (v / v)% FBS, and the cells were cultured for 24 hours, after which the supernatant was collected. If necessary, after collecting the supernatant, 10 (v / v)% FBS-containing DMEM medium was added again and cultured for 24 hours, and the supernatant was collected again. The collected supernatant was filtered, and then 1 / 3 of the volume of Lenti-X concentrator (Takara Bio, 631232) was added. The mixture was incubated at 4°C for at least 1 hour and centrifuged to precipitate the virus. The supernatant was aspirated, and the pellet was dissolved in 5 (v / v)% FBS-containing AIM-V medium (Thermo Fisher Scientific, 12055083) (hereinafter referred to as "basal medium") and frozen and stored at -80°C or -150°C as a lentivirus concentrate.

[0200] [KIR Gene Introduction into NK92 Cells and Preparation] NK92 cells were obtained from ATCC (Cat. No.: CRL-2407 TMThe cells were cultured in MyeloCult H5100 medium (ST-05150, manufactured by STEMCELL Technologies) supplemented with 500 IU / mL (100-2000) of IL-2 and streptmycin / penicillin at 37°C and 5% CO ２ The cells were cultured under the conditions.

[0201] To establish the various KIR-expressing NK92 cell lines listed in Table 4, an artificial gene encoding a polypeptide arranged in the following order from the N-terminus: a signal peptide derived from the target KIR (see Table 5), a Flag tag (SEQ ID NO: 2), GSGS (SEQ ID NO: 3), and a target receptor peptide (see Table 4) was inserted into pLVSIN EF1α according to the method described in the above section "Preparation of lentivirus," to produce a lentivirus. For KIR2DL2*001, an artificial gene encoding a polypeptide arranged in the following order: a signal peptide derived from the target KIR (see Table 5), a target receptor peptide (SEQ ID NO: 57), a self-cleaving peptide T2A (SEQ ID NO: 20), and a NGFR peptide (SEQ ID NO: 58) was inserted into pLVSIN EF1α according to the method described in the above section "Preparation of lentivirus," to produce a lentivirus.

[0202] Each KIR was selected from variants with high expression rates based on the Allele Frequency Net Database. Unless otherwise specified in the Examples of this specification, KIR2DL2 is referred to as KIR2DL2. ＊ 003 was used.

[0203]

[0204] Retronectin (T100B, manufactured by Takara Bio) was diluted with PBS (phosphate buffered saline) to 20-100 μg / mL and added to a non-treatment dish. After leaving the dish at room temperature for 2 hours or overnight at 4°C, the dish was washed with PBS to prepare a coated plate.

[0205] NK92 cells were cultured at 1-2 × 10 in Myelocult H5100 medium. ６ The lentivirus concentrate incorporating the inhibitory KIR and activating KIR genes and the NK cell suspension were mixed in a RetroNectin-coated plate and centrifuged at 1000 × g at 32°C for 1 hour. Then, the plate was incubated at 37°C, 5% CO ２ The cells were cultured under these conditions. From day 3 of culture, the entire medium was replaced every 1 to 3 days using Myelocult H5100 medium. After 1 week of culture, only positive cells were selected using an APC-labeled anti-Flag antibody or an APC-labeled anti-NGFR antibody, and anti-APC Microbeads (Miltenyi: 130-090-855). Furthermore, the expression of the introduced KIR was confirmed using commercially available anti-KIR2DS1 antibody, anti-KIR2DL2 / 3 / 2DS2 antibody, and anti-KIR2DS4 antibody. If necessary, the collected cells were transferred to CELLBANKER1 (Takara Bio, CB011) at a concentration of 0.5 to 1 x 10 ７ The cells were suspended at 1000 cells / mL and stored frozen at −80° C. or −150° C. When using frozen cells in an experiment, they were thawed and cultured in NK cell culture medium before use.

[0206] [Flow cytometry and added antibodies] The antibodies shown in Table 6 were used in the flow cytometry method for confirming the expression of various cell surface proteins.

[0207]

[0208] The above fluorescently labeled antibody was suspended in autoMACS Running Buffer-MACS Separation Buffer (Miltenyi Biotec, 130-091-221) to a concentration of 1 to 10 μg / mL, then added to the cells and allowed to react at 4° C. for 30 minutes.

[0209] The antibody-stained cells were suspended in FACS buffer containing 1-10 μg / mL of 7-aminoactinomycin D (7-AAD; WAKO, 016-25241) and analyzed using a flow cytometer (Miltenyi Biotec, MACSQuant Analyzer 10, 130-096-343). After extraction as an FCS file, cell fractions were selected on an FSC / SSC plot using FLOW JO software (FLOWJO LLC, VER. 10.7.1), and 7-aminoactinomycin D-negative / target-positive cell groups were counted as live cells.

[0210] Example 1: Lirilumab inhibits the cytocidal activity of K562 by NK92-expressing KIR2DL2 cell line An evaluation system was constructed in which the cytocidal activity of KIR2DL2-expressing NK cell line is attenuated by HLA recognition by KIR2DL2, and further, an evaluation was conducted to determine whether agonist activity of KIR2DL2-expressing cell line is present when an existing anti-KIR antibody is added in a soluble form.

[0211] [Gene introduction into K-562 cells and preparation] K562 cells were obtained from ATCC (CCL-243 ＴＭ These cells were obtained from the University of Tokyo and used as target cells for the initial evaluation system. RPMI 1640 Medium, GlutaMAX (Life Technologies, 61870036) (hereinafter referred to as "RPMI medium") was supplemented with 10 (v / v)% FCS and streptmycin / penicillin, and cultured.

[0212] To establish a luciferase-expressing K562 cell line (Luc-K562 cell line), an artificial gene encoding the Firefly luciferase (SEQ ID NO: 17) polypeptide was first introduced into the K562 cell line by CRISPR / CAS9.

[0213] To establish a human HLA-Cw3-expressing K562 cell line (HLA-Cw3-Luc-K562 cell line), an artificial gene encoding a polypeptide in which luciferase (SEQ ID NO: 17), self-cleaving peptide T2A (SEQ ID NO: 20), human HLA-Cw3-derived signal peptide (SEQ ID NO: 21), and human HLA-Cw3 ​​peptide (SEQ ID NO: 22) are linked in this order from the N-terminus was introduced into the K562 cell line using CRISPR / CAS9.

[0214] After cloning the Luc-K562 cell line, the luciferase activity was confirmed and clones were selected.For the HLA-Cw3-K562 cell line, expression was confirmed with anti-HLA-ABC, and after cloning, clones were selected based on luciferase activity and anti-HLA-ABC staining intensity.

[0215] If necessary, the collected cells were cultured in CELLBANKER1 (Takara Bio, CB011) at a concentration of 0.5 to 1 × 10 ７ The cells were suspended at 1000 cells / mL and stored frozen at −80° C. or −150° C. When using frozen cells in an experiment, they were thawed and cultured in RPMI medium before use.

[0216] [Evaluation of the cytotoxic activity of KIR2DL2-expressing NK92 against K562] In this example, KIR2DL2 was used as KIR2DL2. ＊ The KIR2DL2-NK92 cell line was stimulated with 500 IU / mL of IL-2 on the day before the evaluation of cytotoxic activity, and 5 × 10 cells were placed in a 96-well plate. ３ Add an equal amount (i.e., 5 x 10 ３The Luc-K562 cell line or HLA-Cw3-Luc-K562 cell line (100 cells / ml) was added to separate wells and further cultured. The anti-KIR antibody Lirilumab (1-7F9, human IgG4) was added separately to the mixed wells of KIR2DL2-NK92 cell line and Luc-K562 cells at four 9-fold dilutions starting from 2 μg / mL. As a Lirilumab control, human IgG4 (GeneTex, GTX63305) was treated at 2 μg / mL in the same manner. All cultures were performed in basal medium. After 3 hours, the cytotoxic activity of luciferase in the K562 cell line was expressed as the number of remaining target cells, assuming that the number of target cells in the wells to which KIR2DL2-NK92 cells were not added was 100%.

[0217] The results are shown in Figure 1-1. The KIR2DL2-NK92 cell line exhibited cytocidal activity against the K562 cell line, while its cytocidal activity was attenuated against the HLA-Cw3-K562 cell line. This is thought to be because KIR2DL2, which recognized HLA-Cw3, suppressed the cytocidal activity by introducing an inhibitory signal into the KIR2DL2-NK92 cell line, and an evaluation system demonstrating the missing self of KIR2DL2 was constructed. When lirilumab was added to the culture medium in this system of the KIR2DL2-NK92 cell line against the K562 cell line, the cytocidal activity of the KIR2DL2 cell line against the K562 cell line was concentration-dependently attenuated, with approximately 50% inhibition at 2 μg / mL. These results demonstrate that Lirilumab exhibits agonistic activity against inhibitory KIR2DL2 in the cytotoxicity of the KIR2DL2-NK92 cell line against K562 cells. There was no change with the control antibody, human IgG4, at 2 μg / mL.

[0218] On the other hand, when lirilumab was added at 2 μg / mL in an evaluation of the cytocidal activity of the KIR2DL2-NK92 cell line against the HLA-Cw3-K562 cell line, its cytocidal activity was enhanced. The results are shown in Figure 1-2. There was no change when the control antibody, human IgG4, was added at 2 μg / mL.

[0219] This is equivalent to the event related to lirilumab (PL4: WO2006 / 003179_A2), and it is presumed that lirilumab removes the inhibitory signal induced by the KIR ligand, enhancing the activation of NK cells, and this activity inhibits the agonistic action of HLA ligands against inhibitory KIR.

[0220] These results suggest that in the presence of a KIR2DL2 agonist such as HLA-Cw3, the anti-KIR antibody lirilumab neutralizes the ligand's activity and enhances the cytotoxic activity of NK cells. However, in the absence of a KIR2DL2 ligand, lirilumab functions as a partial agonist, exhibiting agonistic activity against inhibitory KIR.

[0221] Example 2: Inhibition of inhibitory KIR-dependent NK cell line activity by expression of scFv of Lirilumab and Pan2D When an agonist binder of an inhibitory receptor is made to act as a secreted protein as an allogeneic technology, there is a possibility that it may affect the immune system around the target tissue and even within the body. Therefore, we aimed to achieve local immune tolerance by expressing the agonist binder on the surface of the transplanted cells so that only the cells desired to evade the immune system would be immune tolerant.

[0222] Since solubilized lirilumab inhibited the cytocidal activity of KIR2DL2-NK92 cells against K562 cells, we evaluated whether lirilumab or Pan2D-derived scFv could be expressed as a membrane protein on the surface of the K562 cell line and inhibit the cytocidal activity of KIR2DL2-NK92 cells.

[0223] [ScFv gene transfection into K-562 cells and preparation] The sequences of Lirilumab and Pan2D (NKVSF1) were cited from WO2006 / 003179 A2 (see Table 6 for the sequence numbers of each variable region).

[0224] To establish the Lirilumab-scFv-expressing K562 cell line (Lirilumab-scFv-Luc-K562 cell line) and the Pan2D-scFv-expressing K562 cell line (Pan2D-scFv-Luc-K562 cell line), the following sequences were first synthesized from the N-terminus: a human CD8-derived signal peptide (SEQ ID NO: 23), a Flag tag (SEQ ID NO: 2), a linker (GSG), a variable region (VH) shown in Table 7 below, a GS (GS) linker (SEQ ID NO: 25), a variable region (VL) shown in Table 7 below, a CD8-derived hinge region (66 amino acids) sequence (SEQ ID NO: 24), a CD8-derived plasma membrane domain (SEQ ID NO: 27), a CD8-derived intracellular domain (SEQ ID NO: 28), and the self-cleaving peptide T2A (SEQ ID NO: 20) and Firefly The artificial gene encoding a polypeptide in which the nucleotide sequence of nucleotides 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 12

[0225] *The CDR1, CDR2, and CDR3 regions in the table are indicated by the range of amino acid numbers in the amino acid sequence of each variable region. The amino acid numbers are specified according to the IMGT definition.

[0226] Retronectin (T100B, manufactured by Takara Bio) was diluted with PBS (phosphate buffered saline) to 20-100 μg / mL and added to a non-treatment dish. After leaving the dish at room temperature for 2 hours or overnight at 4°C, the dish was washed with PBS to prepare a coated plate.

[0227] 1-2 x 10 using PRMI medium ６K562 cell lines were suspended to a concentration of 1000 cells / mL, and the lentivirus concentrate carrying the Lirilumab scFv-Luc or Pan2D scFv-Luc gene and the cells were mixed in a RetroNectin-coated plate and centrifuged at 1,000 x g for 1 hour. After 3 days of culture, the medium was completely replaced with RPMI medium every 1 to 3 days, and after 1 week of culture, only positive cells were selected using an APC-labeled anti-Flag antibody and anti-APC Microbeads (Miltenyi Biotec, 130-090-855).

[0228] If necessary, the collected cells were cultured in CELLBANKER1 (Takara Bio, CB011) at a concentration of 0.5 to 1 × 10 ７ The cells were suspended at 1000 cells / mL and stored frozen at −80° C. or −150° C. When using frozen cells in an experiment, they were thawed and cultured in RPMI medium before use.

[0229] [Evaluation of the cytotoxic activity of Lirilumab-scFv / Pan2D-scFv-K562 by KIR2DL2-expressing NK92] In this example, KIR2DL2 was used as KIR2DL2. ＊001 was used. The day before the evaluation of cytocidal activity, the KIR2DL2-NK92 cell line, stimulated with 500 IU / mL of IL-2, was added to a 96-well plate, and the Luc-K562 cell line, HLA-Cw3-Luc-K562 cell line, Lirilumab-scFv-K562 cell line, and Pan2D-scFv-K562 were added to separate wells at a ratio of NK92 to K562 cells of 3:1 or 1:1, and further cultured. Basal medium was used for all cultures. After 2 to 5 hours, the luciferase activity expressed by the K562 cell line was measured, and the luciferase activity in the wells containing KIR2DL2-NK92 cells was expressed as the number of remaining target cells, with the luciferase activity in the wells containing KIR2DL2-NK92 cells taken as 100%. Furthermore, the value obtained by subtracting the luciferase activity in the wells containing KIR2DL2-NK92 cells from the luciferase activity in the wells containing KIR2DL2-NK92 cells was expressed as the cell killing activity. A ONE-Glo™ Luciferase Assay System (PROMEGA, E6120) was used to measure luciferase activity. To determine the KIR-dependent activity and to calculate the binder-dependent activity ratio, the activity of the Luc-K562 strain was set to 1, and the cytocidal activity ratio was calculated using the following formula: KIR-dependent cytocidal activity ratio = A ＫＩＲ―ＮＫ９２ / A ＮＫ９２ [A = % cytotoxic activity of 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 x 100]

[0230] The results are shown in Figure 2. The KIR2DL2-NK92 cell line exhibited cytocidal activity against the K562 cell line, whereas its cytocidal activity against the HLA-Cw3-K562 cell line was attenuated. Furthermore, the cytocidal activity of Lirilumab-scFv-K562 and Pan2D-scFv-K562 was attenuated compared to the K562 cell line.

[0231] From the above, when anti-KIR antibodies Lirilumab and Pan2D were expressed as scFv-membrane proteins, they suppressed the cytocidal activity of the KIR2DL2-NK92 cell line, and exhibited agonistic activity against KIR2DL2.

[0232] Example 3: Lirilumab and Pan2D induce cytocidal activity of NK cells via activating KIRs We evaluated whether lirilumab and Pan2D exhibit agonistic activity not only against KIR2DL2 but also against other inhibitory KIRs and activating KIRs with which they show cross-reactivity.

[0233] The cytotoxic activity sensitivity of Lirilumab or Pan2D-scFv-K562 was evaluated using various KIR-expressing NK92 cell lines under the same evaluation conditions as in Example 2. To determine KIR-dependent activity, the activity ratio was calculated using the following formula. Furthermore, to calculate the binder-dependent activity ratio, the activity of the Luc-K562 line was set to 1, and the ratio was calculated. In this example, KIR2DL2 was used as KIR2DL2. ＊ 001 was used. KIR-dependent cell killing activity ratio = A ＫＩＲ―ＮＫ９２ / A ＮＫ９２ [A = % cytotoxic activity of 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 x 100]

[0234] The results are shown in Figure 3. The Lirilumab-scFv-K562 cell line and Pan2D-scFv-K562 cell line exhibited an inhibitory effect on the cytotoxicity of KIR2DL2-NK92, demonstrating agonistic activity against inhibitory KIR. This is the same result as in Example 2. On the other hand, Lirilumab-scFv-K562 enhanced the activity of the KIR2DS1-NK92 cell line and KIR2DS2-NK92 cell line, confirming that it exhibited agonistic activity against active KIR2DS1 / 2. Furthermore, Pan2D-scFv-K562 enhanced the activity of the KIR2DS1-NK92 cell line, the KIR2DS2-NK92 cell line, and the KIR2DS4-NK92 cell line, confirming that it exhibited agonistic activity against the active forms of KIR2DS1 / 2 / 4.

[0235] The binding specificities of Lirilumab and Pan2D are reported in WO2006 / 003179 A2, and it has been reported that Lirilumab exhibits binding affinity to KIR2DS1 and KIR2DS2, and Pan2D exhibits binding affinity to KIR2DS1, KIR2DS2, and KIR2DS4. That is, it has been confirmed that an antibody against the known inhibitory KIR2DL2 / 3 exhibits binding affinity to the corresponding activating KIR and enhances the activity of NK cell lines expressing activating KIR.

[0236] Example 4: Obtaining KIR2DL2- and / or KIR2DL3-specific binders by phage panning Based on the results of Example 3, aiming to obtain a novel binder that does not activate activating receptors and exhibits agonist activity to inhibitory receptors, an inhibitory KIR-specific binder that does not exhibit binding affinity to activating KIRs was obtained by the following method.

[0237] [Identification of inhibitory KIR-specific binders by phage panning] Phage panning was carried out using a human antibody phage library to obtain specific binders that bind to inhibitory KIR2DL2 and / or KIR2DL3 but do not bind to activating KIR2DS1, 2, and 4.

[0238] To perform phage panning, soluble KIRs shown in Table 8 were prepared. Recombinant proteins of soluble KIRs were prepared by linking, from the N-terminus, an IL-2-derived signal peptide (SEQ ID NO: 91), an extracellular domain of each KIR protein shown 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). For phage panning, soluble KIRs corresponding to each KIR shown in Table 7 were used, with or without biotinylation treatment. Furthermore, when performing ELISA, Recombinant Human KIR2DL3 / CD158b2 Fc Chimera Protein (R&D Systems, 2014-KR-050) was used as soluble KIR2DL3.

[0239]

[0240] In this phage panning, positive selection refers to the process of reacting a biotinylated recombinant protein with an scFv-expressing phage library solution and enriching scFv-expressing phages that bind to the biotinylated recombinant protein using streptavidin or neutravidin beads. In this phage panning, depletion refers to the process of reacting a biotinylated recombinant protein with an scFv-expressing phage library solution and eliminating scFv-expressing phages that bind to the biotinylated recombinant protein using streptavidin or neutravidin beads. In this phage panning, deselection refers to a process in which, by adding a non-biotinylated recombinant protein during the reaction process between a biotinylated recombinant protein and an scFv-expressing phage library solution in positive selection, when scFv-expressing phages that bind to the biotinylated recombinant protein are concentrated using Streptavidin or Neutravidin Beads, scFv-expressing phages that do not bind to the added non-biotinylated recombinant protein but specifically bind to the biotinylated recombinant protein are more likely to be concentrated.

[0241] In each round of phage panning, positive selection against inhibitory KIRs and depletion or deselection against activating KIRs were performed. After two rounds, a polyclonal phage library enriched for KIR2DL2-specific binders was obtained.

[0242] ELISA was performed using human KIR2DL2 (22-245)-Fc-Avi (the soluble KIR2DL2 described above), and scFv-expressing phages that bind to KIR2DL2 were selected from a polyclonal phage library. The scFv sequence from the phagemid of the selected scFv-expressing phage was transferred to a recombinant protein expression vector for E. coli, which was then introduced into E. coli, and each scFv was prepared from the culture supernatant. ELISA was performed for each scFv using the soluble KIR or Recombinant Human KIR2DL3 / CD158b2 Fc Chimera Protein (R&D Systems, 2014-KIR-050) listed in Table 7, and information on the binding ability of each scFv to each KIR was obtained.

[0243] Among these, the variable regions of scFvs that bind to KIR2DL2 and / or KIR2DL3 but not KIR2DS1, KIR2DS2, or KIR2DS4 were introduced into the membrane-type scFv expression format described in the section "ScFv Gene Transduction and Preparation into K-562 Cells" and inserted into pLVSIN EF1α. Using pLVSIN EF1α containing each membrane-type scFv, lentiviruses were produced according to the method described in the section "Lentivirus Preparation." Using the produced lentiviruses, K562 cell lines expressing each scFv as membrane-type scFv were generated according to the method described in the section "ScFv Gene Transduction and Preparation into K-562 Cells."

[0244] 5x10 of these target cells ４ To each well, 0.1 μM of the Fc-added recombinant protein shown in Table 7 or Recombinant Human KIR2DL3 / CD158b2 Fc Chimera Protein (R&D Systems, 2014-KIR-050) was added and reacted at 4° C. for 90 minutes.

[0245] After the reaction, the target cells were washed with PBS, and an APC-labeled anti-human IgG Fc antibody (BIOLEGEND, 409306) was added and reacted at 4°C for 30 minutes. The cells were washed twice with PBS, and the presence or absence of binding was confirmed using flow cytometry. The binding to the K562 cell line (median APC-derived fluorescence intensity) was set to 1, and the ratio relative to this was calculated.

[0246] The results are shown in Figure 4. Lirilumab and Pan2D showed binding to recombinant KIR2DS1, 2, or 4, whereas all 12 binders suppressed binding to recombinant KIR2DS1, 2, or 4 and showed binding to recombinant KIR2DL2 and KIR2DL3.

[0247] Example 5: Evaluation of agonist activity of KIR2DL2- and / or KIR2DL3-specific binders (initial evaluation) The agonist activity was evaluated for the binders whose specificity was ensured in Example 4. Note that for these binders, the following constructs, in which a membrane-type scFv and lucoferase are linked to encode other polypeptides, were inserted into pLVSIN EF1α according to the method described in the section [Preparation of lentivirus] to produce lentiviruses.

[0248] The SEQ ID NOs for the amino acid sequences of the heavy chain variable region and light chain variable region of each binder are shown in Table 8 below. To express in target cells membrane-type proteins having each binder in the extracellular domain, a gene encoding a polypeptide in which the self-cleaving peptide T2A (SEQ ID NO: 20) and Firefly luciferase (SEQ ID NO: 17) were linked in this order to a membrane-type scFv consisting of, from the N-terminus, a human CD8-derived signal peptide (SEQ ID NO: 23), a Flag tag (SEQ ID NO: 2), a linker (GSG), a variable region (VH) shown in Table 8 below, a GS (GS) linker (SEQ ID NO: 25), a variable region (VL) shown in Table 8 below, a CD8-derived hinge region sequence (SEQ ID NO: 26), a CD8-derived plasma membrane domain (SEQ ID NO: 27), and a CD8-derived intracellular domain (SEQ ID NO: 28) was inserted into pLVSIN EF1α according to the method described in the above section [Preparation of lentivirus] to prepare a lentivirus. In Table 9, "a2DL2 / 3" indicates binding to 2DL2 and / or 2DL3.

[0249] *The CDR1, CDR2, and CDR3 regions in the table are indicated by the range of amino acid numbers in the amino acid sequence of each variable region. The amino acid numbers are specified according to the IMGT definition.

[0250] The cytocidal activity sensitivity of each binder membrane-type scFv-K562 was evaluated using various KIR-expressing NK92 cell lines under the same evaluation conditions as in Examples 2 and 4. To determine binder-dependent activity, the activity ratio was calculated using the following formula. Furthermore, when calculating the binder-dependent activity ratio, the activity of the Luc-K562 line was set to 1, and the ratio was calculated. KIR-dependent cytocidal activity ratio = A バインダー－ｓｃＦｖ－Ｋ５６２ / A Ｋ５６２ [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 x 100]

[0251] The results are shown in Figure 5. All binders exhibited agonistic activity against the KIR2DL2 and KIR2DL3-NK92 cell lines, and did not activate the KIR2DS1 / 2 / 4-NK92 cell line, as did lirilumab and Pan2D. From the above, agonistic binders specific to inhibitory KIRs, which are the desired profile, were obtained. Since these KIRs are KIRs that are frequently expressed on the Allele Frequency Net Database, their specificity and agonistic activity can be expected in many individuals.

[0252] Example 6: Evaluation of agonist activity of KIR2DL2- and / or KIR2DL3-specific binders using primary NK cells

[0066] Until now, the NK92 cell line has been used as an effector, and inhibitory and activating KIRs have been introduced. To examine whether the binders identified in Example 4 also exhibit agonist activity against KIRs expressed on NK cells in vivo, agonist activity was evaluated using NK cells collected from human peripheral blood mononuclear cells (PBMC).

[0253] NK cells have the property of "missing-self," in which they recognize their own HLA type (C1, C2, Bw4) and exhibit cytocidal activity against target cells. For example, NK cells collected from an HLA-C1 / C1 homozygous individual recognize C1 as self, and therefore, unless the target cells possess HLA-C of the C1 type, an inhibitory signal does not enter the NK cells via an inhibitory receptor, resulting in cytocidal activity. On the other hand, if the target cells possess the C1 type, they will not exhibit cytocidal activity, and if an agonist binder that substitutes for the C1 type is expressed, cytocidal activity can be reduced.

[0254] The same can be applied to HLA-C2 homozygous NK cells and HLA-Bw4 positive NK cells. The inhibitory KIRs that function in each group are different: KIR2DL2 and KIR2DL3 in HLA-C1 homozygous cells, KIR2DL1 in HLA-C2 homozygous cells, and KIR3DL1 in HLA-Bw4 positive cells function as missing self inhibitory receptors, so agonists for these KIRs can be evaluated.

[0255] [Preparation of PBMC-derived NK cells] Two types of HLA-C1 / C1 / Bw4-negative PBMCs were prepared as effector cells (PBMC-A and PBMC-B). Based on the KIR haplotype, PBMC-A was found to have the KIR2DL2, KIR2DL3, KIR2DS1, and KIR2DS2 genes, while PBMC-B had the KIR2DL3 gene.

[0256] Anti-CD16 antibody (Biolegend, 302050) was diluted with PBS to 5 μg / mL and added to a non-treatment dish. After leaving the dish to stand for 2 hours at room temperature or overnight at 4°C, the dish was washed with PBS, and human peripheral blood mononuclear cells (PBMCs; Cellular Technology Limited, CTL-UP1) suspended in MyeloCult H5100 medium (hereinafter referred to as "NK medium") containing IL-2 and IL-18 (R&D systems, 9124-IL) at 1000 U / mL and 100 ng / mL, respectively, were added. After 7 to 10 days, the cells were collected and CD56-positive cells (NK cells) were purified using CD56 MicroBeads (Miltenyi Biotec, 130-050-401). Thereafter, the cells were again cultured in Myelocult containing IL-2 / 18.

[0257] [Preparation of target gene-expressing T cells] As target cells, one type each of HLA-C1 / C1 / Bw4-negative PBMCs, designated PBMC-A (the same as the effector), and HLA-C2 / C2 / Bw4-negative PBMCs (referred to as PBMC-C) were prepared.

[0258] Anti-CD3 antibody (Biolegend, 317347) and RetroNectin (Takara Bio, T100B) were diluted with PBS (phosphate buffered saline) to 5-10 μg / mL and 20-100 μg / mL, respectively, and added to a non-treatment dish. After allowing to stand at room temperature for 2 hours or overnight at 4°C, the dish was washed with PBS to prepare a coated plate (hereafter referred to as a "CD3 Ab / RetroNectin coated plate").

[0259] A lentivirus solution was prepared for expressing membrane-type scFv of the 10 binders used in Example 5. As a control, an artificial gene containing a polypeptide in which a human CD19 polypeptide (SEQ ID NO: 53) was linked to the self-cleaving peptide T2A (SEQ ID NO: 20) and Firefly luciferase (SEQ ID NO: 17) was introduced into a lentivirus to produce an artificial gene.

[0260] The virus concentrate was added to the plate and centrifuged at 2000 × g for 2 hours to prepare a virus-binding plate. Human peripheral blood mononuclear cells (PBMCs; CTL-UP1, Cellular Technology Limited) were cultured at a concentration of 1 to 2 × 10 in a basal medium (hereinafter referred to as "IL-2 medium") supplemented with IL-2 (Nipro, 87-890 or Kyowa Pharmaceutical, 58697900) to a final concentration of 100 U / mL. ６ The cells were suspended to a concentration of 100 cells / mL, added to a virus binding plate, and centrifuged at 300×g for 3 to 5 minutes.

[0261] PBMC-A were infected with human CD19-luciferase, and PBMC-C were infected with lentivirus containing human CD19-luciferase or membrane-bound scFv-luciferase of 10 types of binders. After 3 days of culture, the entire medium was replaced every 1 to 3 days using IL-2 medium. After 1 week of culture, cells were selected using an APC-labeled anti-CD19 antibody, an APC-labeled anti-Flag antibody, and anti-APC Microbeads (Miltenyi, 130-090-855) to enrich for positive cells.

[0262] [Evaluation of Cytotoxic Activity Using PBMC-Derived NK Cells] PBMC-derived NK cells to be evaluated for cytotoxic activity were placed in a 96-well plate at 1 × 10 ５ Add 1 / 10 of the amount (i.e., 1 x 10 ４ (100%) of target gene-expressing T cells were added to separate wells and further cultured. Basal medium was used for all cultures. After 4 hours, the cytotoxic activity of the target gene-expressing T cells was evaluated by expressing the luciferase activity as the number of remaining target cells, relative to the number of target cells in wells without PBMC-derived NK cells, which was defined as 100%.

[0263] The results are shown in Figure 6. First, because the effector cells were C1 / C1 / Bw4 negative, when the target cells were C1 / C1 / Bw4 negative or C2 / C2 / Bw4 negative, they showed clear cytotoxic activity against C2 / C2 / Bw4 negative T cells. This is thought to be a missing self.

[0264] On the other hand, when the KIR2DL2 / 3-specific binder was expressed in C2 / C2 / Bw4-negative T cells, the cytocidal activity was attenuated compared to when CD19-luciferase was expressed in C2 / C2 / Bw4-negative T cells. Similar trends were observed in PBMC-A-derived NK cells and PBMC-B-derived NK cells, confirming that the KIR2DL2 / 3-specific binder obtained in Example 5 also exhibits agonistic activity against primary NK cells.

[0265] Example 7: Evaluation of binding ability of KIR2DL2- and / or KIR2DL3-specific binders The binders confirmed to have agonist activity in Example 6 were evaluated for their binding ability to inhibitory and activating molecules on the surface of NK cells. Pan2D was used as a control.

[0266] 1x10 K562 cells expressing the membrane-type a(anti)2DL2 / 3-DS4 scFv or membrane-type a2DL2 / 3-DS10 scFv prepared in Example 5, or 1x10 K562 cells expressing the membrane-type scFv of Pan2D prepared in Example 3 ４ To each well, 0.1 μM recombinant protein tagged with Fc or His was added and incubated at 4°C for 90 minutes. After washing with PBS, APC-labeled anti-human IgG Fc antibody (BIOLEGEND, 409306) or APC-labeled anti-His tag antibody (BIOLEGEND, 362605) was added and incubated at 4°C for 30 minutes. After washing twice with PBS, binding was confirmed using flow cytometry. The binding to the K562 cell line (median APC-derived fluorescence intensity) was set to 1, and the ratio relative to this was calculated.

[0267] The recombinant proteins used were those shown in Table 10. The recombinant proteins marked "In house" were the same as those listed in Table 7. A recombinant KIR protein was produced as Fc-His-Avi, 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-terminus.

[0268]

[0269] The results are shown in Figure 7. While Pan2D showed binding to KIR2DS1, 2, and 4, the binder (a2DL2 / 3-DS4) and binder (a2DL2 / 3-DS10) not only showed no binding to KIR2DS1, 2, or 4, but also showed no binding to other inhibitory or activating receptors.

[0270] From the above, it was suggested that the binder does not exert its action by binding to other NK cell receptors, but rather binds to KIR2DL2 and / or KIR2DL3 to suppress NK cell activation.

[0271] Example 8: Evaluation of agonist activity of KIR2DL1 binders and KIR3DL1 binders Whether binders that exhibit binding affinity to KIR2DL1 and KIR3DL1 exhibit agonist activity against NK cell lines expressing each receptor was evaluated.

[0272] [Gene introduction into K-562 cells and preparation] To establish a human HLA-C2-expressing K562 cell line (HLA-Cw4-Luc-K562 cell line), first, an artificial gene encoding a polypeptide in which a signal peptide in Table 11, a Flag tag (SEQ ID NO: 2), and an HLA peptide in Table 10 were linked in this order from the N-terminus, and an artificial gene encoding a luciferase polypeptide (SEQ ID NO: 17) downstream of an IRES (internal ribosome entry site) sequence was inserted into pLVSIN EF1α according to the method described in the section [Preparation of lentivirus] to prepare a lentivirus.

[0273]

[0274] The sequence of the anti-3DL1 antibody was cited from WO2018 / 148223 A1, and the polypeptides were linked using the following construct. This was inserted into pLVSIN EF1α according to the method described in the section "Preparation of lentivirus," to produce a lentivirus.

[0275] The SEQ ID NOs for the amino acid sequences of the heavy chain variable region and light chain variable region of each binder are shown in Table 12. To express in target cells membrane-type proteins having each binder in the extracellular domain, a gene encoding a polypeptide in which the self-cleaving peptide T2A (SEQ ID NO: 20) and Firefly luciferase (SEQ ID NO: 17) were linked in this order to the C-terminus of a membrane-type scFv consisting of, from the N-terminus, a human CD8-derived signal peptide (SEQ ID NO: 23), a Flag tag (SEQ ID NO: 2), a linker (GSG), a variable region (VL) in Table 12, a GS (GS) linker (SEQ ID NO: 25), a variable region (VH) in Table 12, a CD8-derived hinge region (66 amino acids) sequence (SEQ ID NO: 24), a CD8-derived membrane domain (SEQ ID NO: 27), and a CD8-derived intracellular domain (SEQ ID NO: 28) was inserted into pLVSIN EF1α according to the method described in the above section [Preparation of lentivirus] to prepare a lentivirus.

[0276] *The CDR1, CDR2, and CDR3 regions in the table are indicated by the range of amino acid numbers in the amino acid sequence of each variable region. The amino acid numbers are specified according to the IMGT definition.

[0277] Retronectin (T100B, manufactured by Takara Bio) was diluted with PBS (phosphate buffered saline) to 20-100 μg / mL and added to a non-treatment dish. After leaving the dish at room temperature for 2 hours or overnight at 4°C, the dish was washed with PBS to prepare a coated plate.

[0278] K562 cells were cultured in PRMI medium at 1-2 x 10 ６The lentivirus concentrate carrying HLA or binder membrane-type scFv and the NK cell suspension were mixed in a RetroNectin-coated plate and centrifuged at 1000 × g at 32°C for 1 hour. Then, the plate was incubated at 37°C, 5% CO ２ After 3 days of culture, the entire medium was replaced with PRMI medium every 1 to 3 days, and after 1 week of culture, only positive cells were selected using an APC-labeled anti-Flag antibody and anti-APC Microbeads (Miltenyi: 130-090-855).

[0279] If necessary, the collected cells were cultured in CELLBANKER1 (Takara Bio, CB011) at a concentration of 0.5 to 1 × 10 ７ The cells were suspended at 1000 cells / mL and stored frozen at −80° C. or −150° C. When using frozen cells in an experiment, they were thawed and cultured in RPMI medium before use.

[0280] As for the anti-2DL1 antibody, Pan2D, which exhibits binding to KIR2DL1, was used, and the Pan2D-scFv-K562 cell line of Example 2 was used.

[0281] The cytocidal activity sensitivity of each binder membrane-type scFv-K562 was evaluated using various KIR-expressing NK92 cell lines under the same evaluation conditions as in Examples 2 and 4, and the activity was calculated using the following formula: % cytocidal activity against each K562 = (luciferase activity of target cells in the absence of effector cells - luciferase activity of target cells in the presence of effector cells) / luciferase activity of target cells in the absence of effector cells x 100. The results are shown in Figure 8.

[0282] The positive control, HLA-Cw4, exhibited agonistic activity against the KIR2DL1-NK92 cell line. In this system, Pan2D exhibited agonistic activity against KIR2DL1, and a3DL1-DS1 exhibited agonistic activity against KIR3DL1.

[0283] From the above, binders exhibiting agonistic activity against KIR2DL1 and binders exhibiting agonistic activity against KIR3DL1 were identified.

[0284] Example 9 Evaluation of the Effect of Agonistic Activity of KIR2DL2- and / or KIR2DL3-Specific Binder on the Efficacy of CAR-T Cells Using Primary NK Cells PBMC-derived NK cells were used as primary NK cells, and CAR-T cells prepared from the same PBMCs as the PBMC-derived NK cells, or CAR-T cells prepared from PBMCs derived from a different donor, were co-cultured with the PBMC-derived NK cells to evaluate the cytocidal activity of the CAR-T cells against target cells.

[0285] In the presence of primary NK cells, CAR-T cells derived from a different donor that does not express HLA that matches the inhibitory KIR of the primary NK cells are recognized by the primary NK cells as missing-self and become targets of attack.

[0286] In Example 6, it was confirmed that the binders identified in Example 4 also exhibit agonist activity against primary NK cells and suppress the cytocidal activity of primary NK cells. To investigate whether suppressing the cytocidal activity of primary NK cells contributes to maintaining the cytocidal activity of CAR-T cells, PBMC-derived NK cells were used as primary NK cells, and three types of cells were mixed: PBMC-derived NK cells, CAR-T cells derived from a donor different from the PBMC-derived NK cells, or CAR-T cells derived from a donor different from the PBMC-derived NK cells expressing the binder identified in Example 4, and CAR-T cells exhibiting cytocidal activity via CAR, and the cytocidal activity of the CAR-T cells against target cells was evaluated.

[0287] PBMC-A-derived NK cells were prepared according to the method described in the section "Preparation of PBMC-derived NK cells," suspended in CELLBANKER1 (Takara Bio, CB011), and stored frozen at -80°C or -150°C. These cells were thawed two days before the test and cultured in MyeloCult H5100 medium supplemented with IL-2 and IL-18. The day before the test, the medium for PBMC-A-derived NK cells was replaced with MyeloCult H5100 medium without IL-2 or IL-18, and the cells were cultured.

[0288] [Preparation of T cells expressing CAR and KIR2DL2 / specific binder] HLA-C2 / C2 / Bw4-negative PBMCs (PBMC-C) were used to generate T cells expressing CAR.

[0289] 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. L J Cooper et al., Blood 2003 Feb 15; 101(4): 1637-44.) (hereinafter referred to as "CD19CAR"), the vector was constructed by combining, from the N-terminus, a CD8-derived signal peptide (SEQ ID NO: 23), CD19CAR VL (SEQ ID NO: 100), a GS (GS) linker (SEQ ID NO: 25), CD19CAR VL (SEQ ID NO: 100), and CD19CAR VL (SEQ ID NO: 100). An artificial gene encoding VH (SEQ ID NO: 101), CD8-derived hinge region sequence (SEQ ID NO: 26), CD8-derived cell membrane domain for CAR (SEQ ID NO: 102), intracellular costimulatory molecule (SEQ ID NO: 103), linker (GSG), self-cleaving peptide T2A (SEQ ID NO: 20), and blue fluorescent protein (BFP) (SEQ ID NO: 104) was inserted into pLVSIN EF1α, and a lentivirus concentrate carrying anti-CD19 CAR was prepared by the method described in the section "Preparation of lentivirus." Furthermore, to express membrane-type a2DL2 / 3-DS10 scFv, a lentivirus concentrate carrying membrane-type a2DL2 / 3-DS10 scFv was prepared using the plasmid prepared in Example 4 by the method described in the section "Preparation of lentivirus." As a control, a gene encoding a polypeptide in which the NGFR peptide (SEQ ID NO: 58), the self-cleaving peptide T2A (SEQ ID NO: 20), and Firefly luciferase (SEQ ID NO: 17) were arranged in this order from the N-terminus (hereinafter referred to as "NGFR-Luc") was used as an artificial gene, and a NGFR-Luc-loaded lentivirus concentrate was prepared by a similar method.

[0290] PBMC-C were suspended in AIM V Medium (Thermo Fisher Scientific, 12055-083) supplemented with IL-2 (Nipro, 87-890), 5 (v / v)% FCS (Thermo Fisher Scientific, 10270-106) and Streptmycin / Penicillin (Thermo Fisher Scientific, 15240-062) (hereinafter referred to as "IL-2 medium"), and the resulting lentivirus concentrate (NGFR-Luc-loaded lentivirus monolayer a2DL2 / 3-DS10) was used. The virus was mixed with four conditions (scFv-carrying lentivirus alone, CD19CAR-carrying lentivirus alone, or a mixture of CD19CAR-carrying lentivirus and membrane-type a2DL2 / 3-DS10 scFv-carrying lentivirus) and added to a CD3 Ab / RetroNectin-coated plate described in the section "Preparation of target gene-expressing T cells." Centrifuged for 1 hour using a centrifuge. The following day, the same virus concentrate as the previous day was added to each well, and the plate was centrifuged for 1 hour using a centrifuge.

[0291] After appropriate medium replacement with IL-2 medium and culturing, PBMC-C infected with membrane-type a2DL2 / 3-DS10 scFv-carrying lentivirus or a mixture of CD19CAR-carrying lentivirus and membrane-type a2DL2 / 3-DS10 scFv-carrying lentivirus were enriched for membrane-type a2DL2 / 3-DS10 scFv-positive cells using an APC-labeled anti-Flag antibody and anti-APC Microbeads (Miltenyi Biotec, 130-090-855). Also, PBMC-C infected with NGFR-Luc-carrying lentivirus were enriched for NGFR-positive cells using anti-LNGFR Microbeads (Miltenyi Biotec, 130-099-023). This procedure yielded PBMC-C-derived T cells expressing membrane-type a2DL2 / 3-DS10 scFv. Furthermore, PBMC-C-derived T cells infected with a mixture of CAR-carrying lentivirus and membrane-type a2DL2 / 3-DS10 scFv-carrying lentivirus contained a mixture of cells expressing both, cells expressing only one of them, and cells expressing neither. However, concentration yielded a mixed cell population 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. Furthermore, PBMC-C-derived T cells expressing NGFR-Luc were obtained.

[0292] PBMC-C-derived T cells expressing NGFR-Luc and PBMC-C-derived T cells expressing membrane-type a2DL2 / 3-DS10 scFv were concentrated and then cultured under appropriate IL-2 medium replacement. The cells were then suspended in CELLBANKER1 (Takara Bio, CB011) and cryopreserved at −80°C or −150°C.

[0293] After culturing PBMC-C infected with only the CD19CAR-carrying lentivirus, and a mixed cell group of PBMC-C-derived T cells expressing CD19CAR and membrane-type a2DL2 / 3-DS10 scFv obtained by the above-mentioned enrichment, and PBMC-C-derived T cells expressing only membrane-type a2DL2 / 3-DS10 scFv, the entire amount was suspended in IL-2 medium and added to a CD19-coated plate coated with CD19 Human Recombinant Protein (manufactured by Origene, TP302922). After the 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 enriched, and the cells were suspended in CELLBANKER1 (manufactured by Takara Bio, CB011) and cryopreserved at −80°C or −150°C.

[0294] Each PBMC-C-derived T cell was thawed the day before the test and cultured in IL-2 medium.

[0295] [Evaluation of cytocidal activity of CAR-T cells against target cells in the presence of PBMC-derived NK cells] A cell line (hereinafter referred to as "Raji-GFP") in which GFP was introduced into Raji cells that constitutively express CD19 using a viral vector was used as a target for T cells expressing CD19CAR. A sample in which Raji-GFP cells were mixed with PBMC-A-derived NK cells or a sample containing 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 to Raji-GFP cells at a ratio of 1:1, 1:0.5, or 1:0.1, and the cells were cultured in a 96-well plate. The cell suspension was mixed using equal volumes of MyeloCult H5100 medium, AIM V medium supplemented with 5 (v / v)% FCS, RPMI 1640 medium supplemented with 10 (v / v)% FCS, and GlutaMAX (all containing streptmycin / penicillin).

[0296] Four days after the start of culture, the number of cells in each well was measured using the method described in the section "Flow cytometry and added staining antibodies." APC / Fire 750 anti-human CD3 antibody (BIOLEGEND, 300470) was used to detect PBMC-C-derived T cells. Raji-GFP cells were detected using GFP, which is expressed by Raji-GFP cells.

[0297] The number of viable Raji-GFP cells in the well to which only Raji-GFP cells were added was set to 100%, and the percentage of viable Raji cells in each well was plotted on the vertical axis, while the percentage 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 20.

[0298] In wells not co-cultured with PBMC-A-derived NK cells, the cytotoxic activity of PBMC-C-derived T cells expressing only CD19CAR and that of PBMC-C-derived T cells expressing CD19CAR and membrane-type a2DL2 / 3-DS10 scFv against Raji-GFP cells were comparable. On the other hand, in wells co-cultured with PBMC-A-derived NK cells, the cytotoxic activity of PBMC-C-derived T cells expressing only CD19CAR against Raji-GFP cells was weakened compared to the cytotoxic activity of PBMC-C-derived T cells expressing CD19CAR and membrane-type a2DL2 / 3-DS10 scFv again...

Claims

1. A polypeptide that is a membrane protein having an extracellular domain containing a binding site for NKG2A and a transmembrane domain, which (1) has the activity of binding to NKG2A on the surface of NK cells and suppressing the NK cells, and (2) has substantially no agonist activity against NKG2C.

2. The polypeptide according to claim 1, wherein the binding site has a higher activity of binding to NKG2A than to NKG2C.

3. The polypeptide of claim 1, wherein the binding site does not substantially bind to NKG2C.

4. A polypeptide according to any one of claims 1 to 3, characterized in that the binding site comprises an amino acid sequence derived from one or two variable regions of a monoclonal antibody or antigen-binding fragment that binds to NKG2A.

5. The polypeptide according to claim 4, characterized in that the binding site comprises an amino acid sequence derived from one or two variable regions contained in an antigen-binding fragment obtained by the following method: Step A: a step of mixing a labeled solubilized protein comprising the extracellular domain of NKG2A with a suspension containing a polyclonal presenter population including presenters that present an antigen-binding site that binds to NKG2A on their surface, and then contacting the suspension with a carrier on which a substance that specifically binds to the label moiety is immobilized, and recovering presenters that bind to the carrier, thereby obtaining a presenter population that presents an antigen-binding site that binds to NKG2A; Step B: a step of mixing a labeled solubilized protein comprising the extracellular domain of NKG2C with the suspension containing the presenter population obtained in Step A, contacting the suspension with a carrier on which a substance that binds to the label moiety is immobilized, and recovering presenters that do not bind to the carrier, thereby removing presenters that present an antigen-binding site that binds to NKG2C from the cell population; Step C: a step of performing genetic analysis of the presenters obtained by the method comprising the above steps, and identifying the gene sequence that encodes the antigen-binding site; Step D: A step of expressing a gene containing the identified antigen-binding site in a cell.

6. The polypeptide according to claim 4, characterized in that the binding site comprises an amino acid sequence derived from one or two variable regions of an antigen-binding fragment obtained by the following method: Step A: mixing a labeled solubilized protein comprising the extracellular domain of NKG2A and an unlabeled solubilized protein comprising the extracellular domain of NKG2C with a suspension containing a polyclonal population of presenters, including presenters that present on their surface an antigen-binding site that binds to NKG2A, and then contacting the suspension with a carrier on which a substance that specifically binds to the labeled portion has been immobilized, and recovering the presenters bound to the carrier, thereby obtaining a population of presenters that present an antigen-binding site that specifically binds to NKG2A; Step B: performing genetic analysis of the presenters obtained by a method comprising the above steps, and identifying the gene sequence encoding the antigen-binding site; and Step C: expressing the gene comprising the identified antigen-binding site in cells.

7. The polypeptide according to claim 5 or 6, wherein the combination of the label and the substance that binds to the label is a biotin-avidin combination.

8. The polypeptide according to claim 5 or 6, characterized in that it is obtained by a method further comprising the step of selecting an antigen-binding fragment having agonistic activity against NKG2A.

9. A polypeptide according to any one of claims 1 to 8, wherein the binding site is a single chain antibody or scFv, characterized in that the first variable region and the second variable region are linked via a GS linker or a G4S linker.

10. The polypeptide of claim 1, wherein the binding site competes with an scFv consisting of the following amino acid sequence for binding to NKG2A: (Ni) an amino acid sequence comprising a combination of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 163 and the amino acid sequence of the light chain variable region of SEQ ID NO:

164.

11. A polypeptide according to claim 1, which comprises the following binding site as the binding site, and each CDR may have one or two amino acid mutations: (N-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: 163 and a second variable region comprising an amino acid sequence derived from CDR1, CDR2 and CDR3 of SEQ ID NO:

164.

12. The polypeptide of claim 11, which may have one or two amino acid mutations on the C- and / or N-terminal side of each CDR.

13. The polypeptide according to claim 11, which is a polypeptide comprising the following binding site as the binding site: (NI) A binding site comprising a combination of a first variable region comprising the amino acid sequence of SEQ ID NO: 163 and a second variable region comprising the amino acid sequence of SEQ ID NO:

164.

14. A polynucleotide encoding any one of the polypeptides of claims 1 to 13.

15. A cell expressing the polypeptide according to any one of claims 1 to 13 on its cell surface.

16. The cell according to claim 15, further expressing on the cell surface a membrane protein that exhibits specific agonist activity against inhibitory KIR.

17. The cell according to claim 16, wherein the inhibitory KIR is at least one of KIR2DL2 / 3, KIR3DL1 and KIR2DL1.

18. The cell according to any one of claims 15 to 17, further comprising a cell surface that lacks or attenuates the expression of at least one Class I HLA.

19. The cell of claim 18, wherein the loss or attenuation of expression of at least one Class I HLA on the cell surface is due to modification of at least one selected from the group consisting of Class I HLA, B2M, TAP1, TAP2, and TAPBP regions on the genome by gene editing.

20. The cells of claim 18, which are a cell line established from an individual who has genetically lost HLA expression.

21. The cell of any one of claims 15 to 20, 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 liver cell, a kidney cell, or a pancreatic cell.

22. The cell according to claim 21, wherein the pluripotent stem cell is an iPS cell or an ES cell.

23. The cell according to any one of claims 15 to 22, further expressing a chimeric receptor on the cell surface.

24. A pharmaceutical composition comprising the cells according to any one of claims 15 to 23 as an active ingredient, which reduces the risk of immune rejection in a subject to administration or transplantation.

25. A method for reducing or suppressing immune rejection of therapeutic cells by a subject, comprising the step of expressing the polypeptide described in any one of claims 1 to 13 on the cell surface of the therapeutic cells.

26. A method for producing therapeutic cells with reduced or suppressed risk of immune rejection by a subject, comprising the step of expressing the polypeptide described in any one of claims 1 to 13 on the cell surface of the therapeutic cells.

27. A method for producing the cells of claim 15, comprising the following steps: Step A: mixing a labeled solubilized protein comprising the extracellular domain of NKG2A with a suspension containing a polyclonal population of presenters comprising presenters that present an antigen-binding site that binds to NKG2A on their surface, then contacting the suspension with a carrier on which a substance that specifically binds to the label is immobilized, and recovering presenters that bind to the carrier, thereby obtaining a population of presenters that present an antigen-binding site that binds to NKG2A; Step B: mixing a labeled solubilized protein comprising the extracellular domain of NKG2C with a suspension containing the presenter population obtained in Step A, then contacting the suspension with a carrier on which a substance that binds to the label is immobilized, and recovering presenters that do not bind to the carrier, thereby removing presenters that present an antigen-binding site that binds to NKG2C from the cell population; Step C: performing genetic analysis of the presenters obtained by a method comprising the above steps, and identifying the gene sequence that encodes the antigen-binding site; and Step D: A step of expressing in a cell a gene encoding the polypeptide of claim 1, which contains the identified antigen-binding site in its extracellular domain.

28. A method for producing the cells of claim 15, comprising the following steps: Step A: mixing a labeled solubilized protein comprising the extracellular domain of NKG2C and an unlabeled solubilized protein comprising the extracellular domain of NKG2C with a suspension containing a polyclonal population of presenters, including presenters that present an antigen-binding site that binds to NKG2A on their surface, and then contacting the suspension with a carrier on which a substance that specifically binds to the labeled portion has been immobilized, and recovering the presenters bound to the carrier, thereby obtaining a population of presenters that present an antigen-binding site that specifically binds to NKG2A; Step B: performing genetic analysis of the presenters obtained by a method comprising the above steps, and identifying the gene sequence encoding the antigen-binding site; and Step C: expressing in the cells a gene encoding the polypeptide of claim 1 that contains the identified antigen-binding site in its extracellular domain.

29. The method according to claim 27 or 28, wherein the combination of the label and the substance that binds to the label is a biotin-avidin combination.

30. The method according to any one of claims 27 to 29, further comprising the step of selecting cells having agonistic activity against NKG2A.

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