Anti-GARP antibody and method of use

Abstract

The present invention relates to an antibody capable of binding to GARP, or to a target-binding fragment or derivative thereof that possesses target-binding ability.

Description

[Technical Field]

[0001] [Reference to sequence listings submitted as compliant XML 1.0 format files (.xml)] In accordance with the EFS-Web legal framework and 37 CFR §§1.821-825 (see MPEP §2442.03(a)), EPC Rule 30, and §11 PatV, an electronic sequence listing in XML 1.0 format file compliant with WIPO standard ST.26 is filed concurrently with this application, and the entire contents of the sequence listing are incorporated herein by reference. To avoid any doubt, in the event of any discrepancy between the sequences referred herein and the electronic sequence listing, the sequences herein shall be considered the correct sequences.

[0002] [Field of Invention] The present invention relates to antibodies that bind to GARP and / or GARP / TGFβ, as well as fragments or derivatives thereof, and methods for using them.

[0003] [Integration by reference] All publications, patents, patent applications, and other documents referenced herein are incorporated herein by reference in whole for any purpose to the same extent as when each individual publication, patent, patent application, or other document is individually indicated as being incorporated by reference for any purpose. In the event of any conflict between the teachings of this Specified and any of the references incorporated herein, the teachings of this Specified are intended to prevail. [Background technology]

[0004] The tumor microenvironment (TME) consists of cancer cells, stromal tissue (e.g., blood vessels, fibroblasts, immune cells, and signaling molecules), and the extracellular matrix. The TME is a major determinant of tumor progression, metastasis, and response to treatment. A key aspect of the TME supporting tumor progression, metastasis, and response to treatment is immunosuppression. Many factors may contribute to immunosuppression in the tumor microenvironment, including the loss of tumor cell ligands required for immune cell induction, the upregulation of tumor cell ligands that induce so-called immunosuppressive receptors on immune cells, and the presence of immunosuppressive immune cells, such as a subset of regulatory T cells (Tregs), that actively suppress immune effector cells in the TME. [Overview of the project] [Problems that the invention aims to solve]

[0005] The object of the present invention is to provide a novel therapeutic method that reduces immunosuppression in the tumor environment and / or directly stimulates the death of tumor cells by cytotoxic lymphocytes.

[0006] The objective of this invention is to provide novel and improved therapeutic approaches for treating cancer.

[0007] These and other issues are resolved by embodiments described in the independent claims of this specification. Dependent claims disclose preferred embodiments. [Brief explanation of the drawing]

[0008] [Figure 1A]This figure shows the binding of bifunctional fusion proteins (BFPs), consisting of IgG molecules fused to ULBP2, to GARP, the GARP-TGFβ1 complex, and NKG2D. The table in Figure 1A summarizes the affinity (KD value) measured by molecular binding assays using surface plasmon resonance (SPR), and the binding to cells evaluated by measuring EC50 by flow cytometry. All test compounds were similar to DS-1055a but showed low nanomolar affinity to GARP, in contrast to ABBV-151, which did not show binding to GARP. All compounds showed lower affinity to the GARP-TGFβ1 complex than to GARP, or to antibodies with the VH / VL sequence of DS-1055a (Daiichi Sankyo, Satoh et al. 2021) or ABBV-151 (AbbVie, Tolcher et al. 2022). For convenience, these antibodies are abbreviated as DS-1055a and ABBV-151 in this specification. Binding to the cell expression target, indicated as EC50, was similar for most compounds (between test compounds and compared to DS-1055a and ABBV-151), with the exception of PB003G.23.0104.BFP, which showed slightly lower binding affinity. Binding to NKG2D was confirmed for all BFPs (*N / D - not determined due to very low dissociation rate). [Figure 1B-G] Figures 1B–1G show the results of cell-based assays using HEK293 expressing GARP, GARP-TGFβb1, or NKG2D. All test compounds (PB003G.21.0111.aF(1B), PB003G.21.0091.BFP(1C), PB003G.23.0104.BFP(1D), PB003G.21.0111.BFP(1E), and DS1055a(1F)) showed strong dose-dependent binding to HEK293 T cells expressing the GARP-TGFβ1 complex and to cells expressing GARP. ABBV-151 showed binding only to cells expressing the GARP-TGFβb1 complex (1G). All bifunctional fusion proteins showed binding to NKG2D (1B–1D).

[0009] [Figure 2A] Figure 2 shows a comparison of Fc affinity to FcyRIIIA, and a comparison of ADCC activity between natural (fucosylated) PB003G.21.0111 IgG1 and afucosylated PB003G.21.0111 IgG1 (PB003G.21.0111.aF). The table in Figure 2A shows the affinity of Fc to the human ADCC-inducing receptor FcyRIIIA for two variants (176V and 176F). Afucosylation of PB003G.21.0111 resulted in a 12-fold and 10-fold increase in affinity to FcyRIIIA(176V) and FcyRIIIA(176F), respectively. KD values ​​were measured using surface plasmon resonance (SPR). Increased affinity for FcyRIIIA leads to improved ADDC activity of PB003G.21.0111 IgG1 against GARP-TGFββ1-expressing target cells. [Figure 2B] A comparison of ADCC induced by natural (fucosylated) PB003G.21.0111 IgG1 and afucosylated PB003G.21.0111 IgG1 (PB003G.21.0111.aF) is shown in Figures 2B and 2C. The afucosylated Fc domain of anti-GARP PB003G.21.0111.aF resulted in a significant increase in ADCC induction ability in GARP-expressing Raji cells (2B) and GARP-TGFβ1-expressing Raji cells (2C) compared to the natural (fucosylated) form of PB003G.21.0111 IgG1. ADCC assays were performed using NK cells as effector cells, and cell lysis was measured using flow cytometry. [Figure 2C]A comparison of ADCC induced by natural (fucosylated) PB003G.21.0111 IgG1 and afucosylated PB003G.21.0111 IgG1 (PB003G.21.0111.aF) is shown in Figures 2B and 2C. The afucosylated Fc domain of anti-GARP PB003G.21.0111.aF resulted in a significant increase in ADCC induction ability in GARP-expressing Raji cells (2B) and GARP-TGFβ1-expressing Raji cells (2C) compared to the natural (fucosylated) form of PB003G.21.0111 IgG1. ADCC assays were performed using NK cells as effector cells, and cell lysis was measured using flow cytometry.

[0010] [Figure 3A] This figure shows the ability of PB003G.21.0091.BFP and PB003G.21.0111.aF to block GARP-TGFβ1 complex formation (3A) and inhibit downstream TGFβ1 signaling (3B). (3A) Cells pre-incubated with PB003G.21.0091.BFP or PB003G.21.0111.aF were unable to bind to latent TGFβ1. This indicates that binding of either molecule blocks the ability of GARP to complex with TGFβ1. The assay was performed using flow cytometry. (3B) Incubation of cells carrying the GARP-TGFβ1 complex with PB003G.21.0091.BFP or PB003G.21.0111.aF inhibits downstream TGFβ1 signaling, as measured by the level of phosphorylated SMAD in Western blotting. These results suggest that both molecules block TGFβ1 maturation and its release from the complex with GARP. [Figure 3B]This figure shows the ability of PB003G.21.0091.BFP and PB003G.21.0111.aF to block GARP-TGFβ1 complex formation (3A) and inhibit downstream TGFβ1 signaling (3B). (3A) Cells pre-incubated with PB003G.21.0091.BFP or PB003G.21.0111.aF were unable to bind to latent TGFβ1. This indicates that binding of either molecule blocks the ability of GARP to complex with TGFβ1. The assay was performed using flow cytometry. (3B) Incubation of cells carrying the GARP-TGFβ1 complex with PB003G.21.0091.BFP or PB003G.21.0111.aF inhibits downstream TGFβ1 signaling, as measured by the level of phosphorylated SMAD in Western blotting. These results suggest that both molecules block TGFβ1 maturation and its release from the complex with GARP.

[0011] [Figure 4A] Figure 4 shows the ability of compounds to induce antibody-dependent cell-mediated cytotoxicity (ADCC) in cancer cells and Treg cells. The ADCC assay was performed using NK cells isolated from healthy donors. The table summarizes the calculated semi-effective concentrations (EC50) for ADCC induction by the compounds in cancer cells (Figure 4A). [Figure 4B] Figures 4B–4E show representative results demonstrating dose-dependent NK-mediated death of Raji-GARP cells (4B), Raji-GARP-TGFβ1 cells (4C), L428 cells (4D) naturally expressing the GARP-TGFβ1 complex, and Tregs (4E) induced by PB003G.21.0111.aF, PB003G.21.0111.BFP, PB003G.21.0091.BFP, and control DS1055a. The ADCC-induced EC50 of PB003G.21.0091.BFP against Tregs was estimated to be approximately 1 nM. Analysis was performed using flow cytometry. [Figure 4C]Figures 4B–4E show representative results demonstrating dose-dependent NK-mediated death of Raji-GARP cells (4B), Raji-GARP-TGFβ1 cells (4C), L428 cells (4D) naturally expressing the GARP-TGFβ1 complex, and Tregs (4E) induced by PB003G.21.0111.aF, PB003G.21.0111.BFP, PB003G.21.0091.BFP, and control DS1055a. The ADCC-induced EC50 of PB003G.21.0091.BFP against Tregs was estimated to be approximately 1 nM. Analysis was performed using flow cytometry. [Figure 4D] Figures 4B–4E show representative results demonstrating dose-dependent NK-mediated death of Raji-GARP cells (4B), Raji-GARP-TGFβ1 cells (4C), L428 cells (4D) naturally expressing the GARP-TGFβ1 complex, and Tregs (4E) induced by PB003G.21.0111.aF, PB003G.21.0111.BFP, PB003G.21.0091.BFP, and control DS1055a. The ADCC-induced EC50 of PB003G.21.0091.BFP against Tregs was estimated to be approximately 1 nM. Analysis was performed using flow cytometry. [Figure 4E] Figures 4B–4E show representative results demonstrating dose-dependent NK-mediated death of Raji-GARP cells (4B), Raji-GARP-TGFβ1 cells (4C), L428 cells (4D) naturally expressing the GARP-TGFβ1 complex, and Tregs (4E) induced by PB003G.21.0111.aF, PB003G.21.0111.BFP, PB003G.21.0091.BFP, and control DS1055a. The ADCC-induced EC50 of PB003G.21.0091.BFP against Tregs was estimated to be approximately 1 nM. Analysis was performed using flow cytometry. [Figure 4F]Figures 4F - 4H show NK cell activation when treated with test compound PB003G. NK cell activation was monitored using activation biomarkers - pro-inflammatory cytokines TNFα (4F), INFγ (4G) and degranulation marker CD107a (4H). All compounds resulted in a statistically significant increase in activation markers, indicating that the compounds activate NK cells in the presence of target cells. [Figure 4G] Figures 4F - 4H show NK cell activation when treated with test compound PB003G. NK cell activation was monitored using activation biomarkers - pro-inflammatory cytokines TNFα (4F), INFγ (4G) and degranulation marker CD107a (4H). All compounds resulted in a statistically significant increase in activation markers, indicating that the compounds activate NK cells in the presence of target cells. [Figure 4H] Figures 4F - 4H show NK cell activation when treated with test compound PB003G. NK cell activation was monitored using activation biomarkers - pro-inflammatory cytokines TNFα (4F), INFγ (4G) and degranulation marker CD107a (4H). All compounds resulted in a statistically significant increase in activation markers, indicating that the compounds activate NK cells in the presence of target cells.

[0012] [Figure 5A] Figure 5 shows the anti-tumor effect of PB003G.21.0111.aF in a model bearing human tumors in humanized mice. CD34+ humanized NSG-IL15 mice transplanted with Raji-GARP-TGFβ1 tumors (5A) or HT-29 tumors (5B). Mice were treated with PB003G.21.0111.aF (10 mg / kg i.v.) for 3 weeks, resulting in a statistically significant 39% inhibition of tumor growth compared to the vehicle control in both models. In the Raji-GARP-TGFβ1 tumor model, the number of CD8+ cytotoxic T lymphocytes (CTLs) was estimated in tumor tissue using flow cytometry. An increase in the frequency of CTLs was observed in PB003G.21.0111.aF-treated mice (5C). [Figure 5B] Figure 5 shows the anti-tumor effect of PB003G.21.0111.aF in a model bearing human tumors in humanized mice. CD34+ humanized NSG-IL15 mice transplanted with Raji-GARP-TGFβ1 tumors (5A) or HT-29 tumors (5B). Mice were treated with PB003G.21.0111.aF (10 mg / kg i.v.) for 3 weeks, resulting in a statistically significant 39% inhibition of tumor growth compared to the vehicle control in both models. In the Raji-GARP-TGFβ1 tumor model, the number of CD8+ cytotoxic T cells (CTLs) was estimated in tumor tissues using flow cytometry. An increase in the frequency of CTLs was observed in PB003G.21.0111.aF-treated mice (5C). [Figure 5C] Figure 5 shows the anti-tumor effect of PB003G.21.0111.aF in a model bearing human tumors in humanized mice. CD34+ humanized NSG-IL15 mice transplanted with Raji-GARP-TGFβ1 tumors (5A) or HT-29 tumors (5B). Mice were treated with PB003G.21.0111.aF (10 mg / kg i.v.) for 3 weeks, resulting in a statistically significant 39% inhibition of tumor growth compared to the vehicle control in both models. In the Raji-GARP-TGFβ1 tumor model, the number of CD8+ cytotoxic T cells (CTLs) was estimated in tumor tissues using flow cytometry. An increase in the frequency of CTLs was observed in PB003G.21.0111.aF-treated mice (5C).

[0013] [Figure 6A] Figure 6 shows the safety analysis of PB003G.21.0111.aF treatment in the HT-29 tumor model in mice. Safety was evaluated by monitoring mouse body weight (5A) and graft-versus-host disease (GvHD) symptoms (5B). There was no statistical difference in mice treated with PB003G.21.0111.aF compared to DS-1055a or the vehicle control. [Figure 6B]Figure 6 shows the safety analysis of PB003G.21.0111.aF treatment in a mouse HT-29 tumor model. Safety was assessed by monitoring mouse body weight (5A) and graft-versus-host disease (GvHD) symptoms (5B). There were no statistically significant differences in mice treated with PB003G.21.0111.aF compared to DS-1055a or solvent controls.

[0014] [Figure 7] This figure shows the analysis of binding to platelets. Platelets are the dominant cell population that expresses the GARP-TGFβ1 complex under physiological conditions. Therefore, platelets are capable of binding to anti-GARP antibodies. Platelets were defined as living CD41+CD42b+CD45- cells using flow cytometry. Data represent a twofold increase in normalized MdFI compared to isotype control MdFI. All tested PB003G compounds showed very low levels of binding to platelets, while DS-1055a and ABBV-151 showed significantly higher binding. Bars represent the mean and SD calculated based on data from six independent platelet donors. Statistical analysis was performed using two-way analysis of variance (ANOVA; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001). [Figure 8] This figure shows the schematic structure of a bifunctional fusion protein (BFP), which is an anti-cancer target antibody fused with ULBP2, an immune ligand for the NKG2D receptor, within both arms of Fab. In one embodiment, the anti-cancer antibody is an anti-GARP antibody. In other embodiments, the antibody is preferably in IgG1 form, and ADCC is enhanced when, for example, afucosylated. [Figure 9] This figure compares the ability of two anti-AVB8 antibodies—one in IgG1 (ADWA) form and the other in a bifunctional fusion protein (ADWA_BFP) containing an NKG2D binding domain—to induce antibody-dependent cell-mediated cytotoxicity (ADCC) in cancer cells (Raji-AVB8). The BFP form enhanced the ADCC activity of the AVB8-binding antibody. The ADCC assay was performed using IL-15-stimulated NK cells isolated from healthy donors. [Modes for carrying out the invention]

[0015] According to a first aspect of the present invention, the following heavy chain / light chain variable domain (HCVD / LCVD) pair: Sequence IDs 3 and 4, Sequence IDs 5 and 6, and / or Sequence IDs 7 and 8 The present invention provides an antibody capable of binding to GARP, or a target-binding fragment or derivative thereof that retains target-binding ability.

[0016] Antibodies having the VH / VL sequences of SEQ ID NOs: 3 and 4 are referred to herein as PB003G.21.0091, PB003G.21.0091.BFP, or 091.bfp.

[0017] Antibodies having the VH / VL sequences of SEQ ID NOs. 5 and 6 are referred to herein as PB003G.21.0111, PB003G.21.0111.aF, PB003G.21.0111.BFP, or 0111.aF.

[0018] Antibodies having the VH / VL sequences of SEQ ID NOs. 7 and 8 are referred to herein as PB003G.23.0104 and PB003G.23.104.BFP.

[0019] The afucosylated variants of these antibodies have the tag "aF," while the bifunctional variants have the tag "BFP."

[0020] As used herein, the term "GARP" refers to glycoprotein A repetitions predominantly. GARP (UniProt identifier: Q14392) is encoded by the Lrrc32 gene and plays a crucial role in cell surface docking and latent TGFβ activation.

[0021] The antibody according to the present invention targets the repeat-dominant glycoprotein A (GARP) or the GARP-transforming growth factor beta-1 (TGFβ1) complex, potentially inhibiting the release of active TGFβ1 or directly killing tumor cells or regulatory T cells expressing the target, thereby providing antitumor activity.

[0022] In one embodiment, the antibody or a fragment or derivative thereof is a monoclonal antibody or derived from a monoclonal antibody.

[0023] As used herein, the term “monoclonal antibody (mAb)” refers to an antibody composition having a homogeneous population of antibodies, i.e., a homogeneous population consisting of an entire immunoglobulin, or a fragment or derivative thereof that retains target-binding ability. Particularly preferably, such antibodies are selected from the group consisting of IgG, IgD, IgE, IgA, and / or IgM, or fragments or derivatives thereof that retain target-binding ability.

[0024] As used herein, the term "fragment" refers to a fragment of an antibody that retains its target-binding ability, for example, CDR (Complementarity Determination Area) Hypervariable region, Variable domain (Fv) IgG or IgM heavy chain (consisting of VH, CH1, hinge, CH2, and CH3 regions) IgG or IgM light chain (consisting of VL and CL regions), and / or Fab and / or F(ab)2 This refers to...

[0025] As used herein, the term “derivative” refers to a protein construct that is structurally different from the general concept of an antibody but still has some structural relationship to it (e.g., scFv, Fab, and / or F(ab)2, as well as bispecific, tripspecific, or higher specificity antibody constructs) and further retains target-binding ability. All of these items are described below.

[0026] Other antibody derivatives known to those skilled in the art include diabodies, camelid antibodies, nanobodies, domain antibodies, bivalent homodimers having two chains consisting of scFv, IgA (two IgG structures linked by a J chain and secretory components), shark antibodies, antibodies consisting of a New World primate framework + Old World primate CDR, dimerized constructs containing CH3+VL+VH, and antibody conjugates (e.g., antibodies or fragments or derivatives linked to toxins, cytokines, radioisotopes, or labels). These types are well described in the literature and can be used by those skilled in the art pursuant to this disclosure without further inventive activity.

[0027] Methods for producing hybridoma cells are disclosed in Kohler and Milstein (1975).

[0028] Methods for the preparation and / or selection of chimeric or humanized mAbs are known in the art. For example, U.S. Patent No. 6,331,415 by Genentech describes the preparation of chimeric antibodies, U.S. Patent No. 6,548,640 by the Medical Research Council describes a CDR implantation method, and U.S. Patent No. 5,859,205 by Celltech describes the preparation of humanized antibodies.

[0029] Methods for the preparation and / or selection of fully human mAbs are known in the art. These may require the use of transgenic animals immunized with the respective protein or peptide, or the use of a suitable display method (such as yeast display, phage display, B cell display, or ribosome display) in which antibodies from a library are screened against human iRhom2 in a stationary phase.

[0030] In vitro antibody libraries are disclosed in particular in U.S. Patent No. 6,300064 by MorphoSys and U.S. Patent No. 6,248516 by MRC / Scripps / Stratagene. Phage display methods are disclosed, for example, in U.S. Patent No. 5,223409 by Dyax. Transgenic mammalian platforms are described, for example, in European Patent Application Publication No. 1480515 by TaconicArtemis.

[0031] IgG, IgM, scFv, Fab, and / or F(ab)2 are antibody formats well known to those skilled in the art. Relevant implementation methods are available from their respective textbooks.

[0032] As used herein, the term "Fab" refers to an IgG / IgM fragment containing an antigen-binding region, wherein the fragment consists of one constant domain and one variable domain from each heavy and light chain of the antibody.

[0033] As used herein, the term "F(ab)2" refers to an IgG / IgM fragment consisting of two Fab fragments linked to each other by a disulfide bond.

[0034] As used herein, the term "scFv" refers to a single-chain variable fragment that is a fusion of the variable regions of the heavy and light chains of an immunoglobulin, linked by a short linker, usually serine (S) or glycine (G). This chimeric molecule retains the specificity of the original immunoglobulin despite the removal of the constant region and the introduction of a linker peptide.

[0035] According to one embodiment, an antibody or fragment or derivative thereof has an enhanced ability to induce ADCC compared to naturally occurring antibodies or fragments or derivatives.

[0036] The primary strategies for enhancing IgG's ability to induce ADCC are to modify the Fc portion of the antibody by site-directed mutagenesis, alteration of Fc domain glycosylation, and / or prevention of Fc domain fucosylation, thereby increasing its binding affinity to activated FcγRIIIA. Creating IgG variants with improved binding to activated FcγR through mutagenesis has proven to be an effective strategy for improving the ADCC efficiency of IgG antibodies (Shields et al. 2000, Tang et al. 2007, Zahavi et al. 2018; all of their contents are incorporated herein by reference for feasibility). In addition to Fc residue modification, asymmetric modification of the Fc portion to create heterodimers of different heavy chains has produced more stable antibodies with enhanced ADCC functionality (Liu et al. 2013; their contents are incorporated herein by reference for feasibility).

[0037] According to one embodiment, the antibody or a fragment or derivative thereof comprises an afucosylated Fc domain.

[0038] Afucosylated antibodies are monoclonal antibodies in which the oligosaccharide in the Fc region of the antibody is modified so that it does not contain a fucose sugar unit. When an antibody is in the afucosylated IgG form, its ability to induce antibody-dependent cell-mediated cytotoxicity (ADCC) is increased.

[0039] Technically, afcosylation is, For example, the addition of fucose to existing glycans can be inhibited by overexpressing the enzyme GnTIII (see, e.g., Davies et al. 2001) or by knocking out FUT8 (fucosyltransferase 8, Yamane-Ohnuki et al. 2004). Chemical or enzymatic removal of fucose already attached to existing sugar chains. Modifying fucose synthesis by using heterologous enzymes that deplete the intracellular fucose pool (see also ProBioGen-based GlyMaxx technology, Chung et al., 2012), and / or Use an expression system that does not add fucose residues to the sugar chain (for example, a ciliate-based expression system (see, for example, European Patent Application Publication No. 2542575)). This can be achieved by [method].

[0040] In one embodiment, the antibody according to the present invention was produced by expression in a FUT8-KO CHO (Chinese hamster ovary) cell line containing a knockout of the FUT8 gene. The antibody produced in this manner is fucose-free and therefore afucosylated. In the example shown herein, the afucosylated antibody according to the present invention was produced in this manner. However, it should be noted that similar effects can be expected from the antibody according to the present invention afucosylated by other approaches, such as those described above.

[0041] The publicly available anti-GARP antibody is ABBV-151 from AbbVie (Tolcher et al. 2022). Another commercially available anti-GARP antibody is DS-1055a from Daiichi Sankyo (Satoh et al. 2021).

[0042] The present inventors further demonstrate herein that the antibodies according to the present invention have an improved functional profile compared to DS-1055 and ABBV-151, particularly with respect to effector function (e.g., ADCC) and / or blocking of GARP-TGFβ1 complex formation and TGFβ1 downstream signaling.

[0043] [Table 1]

[0044] Therefore, each of the bifunctional molecules has the same improved functional profile compared to DS-1055 and ABBV-151, particularly with respect to effector function / ADCC and / or blocking of GARP-TGFβ1 complex formation.

[0045] According to another aspect of the present invention, A binding domain that can bind to cancer antigens, A binding domain that can bind to NKG2D and A bifunctional molecule containing the above is provided.

[0046] The term "NKG2D" (UniProt identifier: P26718), as used herein, refers to an activating receptor (transmembrane protein) belonging to the NKG2 family of type C lectin-like receptors. NKG2D is encoded by the KLRK1 (killer cell lectin-like receptor K1) gene located in the NK gene complex (NKC). In humans, NKG2D is expressed by NK cells, γδT cells, and CD8+ αβT cells.

[0047] Generally, the activating receptor NKG2D is unique in its ability to bind to numerous and highly diverse MHC class I-like self-molecules. These ligands are not expressed much in normal cells but can be induced in damaged, transformed, or infected cells, and final NKG2D ligand expression arises from multi-level regulation. While redundant molecular mechanisms may converge on the regulation of all NKG2D ligands, different stimuli may induce specific cellular responses, resulting in the expression of one or fewer ligands. A large amount of evidence indicates that NK cell activation can be caused by different NKG2D ligands, which are often expressed in the same cells. This suggests functional redundancy of these molecules. However, since several evasion mechanisms may reduce the membrane expression of these molecules in both virus-infected and tumor cells, the co-expression of different ligands and / or the presence of alleles of the same ligand ensures NKG2D activation under a variety of stressful and cellular conditions. Notably, NKG2D ligands may differ in their ability to downregulate NKG2D membrane expression in human NK cells. This supports the idea that NKG2D transmits different signals when bound to various ligands. Furthermore, it remains debatable whether NKG2D ligands released by proteolysis, and exosome-associated soluble NKG2D ligands, share the same membrane-bound receptor and ability to induce NKG2D-mediated signaling (Zingoni et al., 2018).

[0048] The use of bifunctional molecules containing a binding domain capable of binding to NKG2D provides options for binding to and activating respective immune cells so that each binding site attacks cancer cells bound to the other binding site of the bifunctional molecule. Therefore, bifunctional molecules containing a binding domain capable of binding to NKG2D offer advantages to well-established bispecific T cell engagers ("biTEs") that contain anti-CD3 binding sites that bind only to T cells and only to CD3-expressing T cells.

[0049] According to that embodiment, a) The binding domain capable of binding to a cancer antigen is an antibody, or a target binding fragment or derivative thereof, and / or b) The binding domains that can bind to NKG2D are the ligand for NKG2D, optionally ULBP2, or its active fragment.

[0050] The term "ULBP2" (UniProt identifier: P26718), as used herein, refers to UL16-binding protein 2, a GPI-anchored cell surface glycoprotein encoded by the ULBP2 gene located on chromosome 6. ULBP2 relates to an MHC class I molecule, but its gene is located outside the MHC locus. The domain structure of ULBP2 is significantly different from that of conventional MHC class I molecules. The domain structure of ULBP2 does not contain an α3 domain and a transmembrane segment. Therefore, ULBP2 consists only of an α1α2 domain linked to the cell membrane by a GPI anchor.

[0051] In short, glycosylphosphatidylinositol (GPI) is a phosphoglyceride that can be added to the C-terminus of a protein during post-translational modification. The resulting GPI-anchored proteins play important roles in a wide variety of biological processes. GPI consists of a phosphatidylinositol group linked to the C-terminal amino acid of a mature protein via a carbohydrate-containing linker (glucosamine and mannose glycosidically bonded to an inositol residue) and an ethanolamine phosphate (EtNP) crosslink. The extracellular fragment of ULBP2 does not have a GPI anchor.

[0052] The term "cancer antigen," as used herein, refers to any molecule (e.g., protein, polypeptide, peptide, lipid, carbohydrate, etc.) that is expressed alone, predominantly, or overexpressed on the surface of tumor cells or cancer cells, or cells within the tumor microenvironment, so that the antigen binds to a tumor or cancer. Cancer antigens may be additionally expressed by normal cells, non-tumor cells, or non-cancerous cells. However, in such cases, the expression of cancer antigens by normal cells, non-tumor cells, or non-cancerous cells is not as robust as that by tumor cells or cancer cells. In this respect, tumor cells or cancer cells may overexpress antigens or express them at significantly higher levels compared to the expression of antigens by normal cells, non-tumor cells, or non-cancerous cells. Furthermore, cancer antigens may be additionally expressed by cells at different developmental or maturing stages. For example, cancer antigens may be additionally expressed by embryonic or fetal cells not typically found in adult hosts. Alternatively, cancer antigens may be additionally expressed by stem cells or precursor cells not typically found in adult hosts.

[0053] According to the embodiment, the binding domain capable of binding to a cancer antigen binds to at least one target selected from the group shown in the table below.

[0054] [Table 2]

[0055] Such bifunctional molecules may also possess an enhanced ability to induce ADCC compared to naturally occurring antibodies, fragments, or derivatives, as discussed elsewhere in this specification. For this purpose, the Fc domain of the antibody that binds to the cancer antigen may be modified as appropriate. ADCC and immune cell engagement via NKG2D binding synergistically complement each other in their cancer cell death effector functions.

[0056] According to the embodiment, ULBP2 contains or consists of the amino acid sequence according to Sequence ID No. 1, and / or The binding domain capable of binding to cancer antigens is the anti-GARP antibody, fragment, or derivative described above.

[0057] Such anti-GARP antibodies, fragments, or derivatives as described above are heavy / light chain variable domain (HCVD / LCVD) pairs as described below: Sequence IDs 3 and 4, Sequence IDs 5 and 6, and / or Sequence IDs 7 and 8 Includes.

[0058] The anti-AVB8 antibody, fragment, or derivative described above contains the heavy chain / light chain variable domain (HCVD / LCVD) pair as defined in SEQ ID NOs. 9 and 10.

[0059] In one embodiment, ULBP2 comprises or consists of the amino acid sequence given by SEQ ID NO: 1. In one embodiment, the active fragment of ULBP2 comprises or consists of the amino acid sequence given by SEQ ID NO: 2.

[0060] In one embodiment of the bifunctional molecule described above, the binding domain capable of binding to NKG2D is either directly fused to the N-terminus of the VH or VL domain of an antibody capable of binding to GARP, or to its target binding fragment or derivative, or fused via a flexible linker.

[0061] Such a linker has, for example, the following amino acid sequence: (GGGGS)4 (SEQ ID NO: 19).

[0062] In one embodiment, the binding domain capable of binding to NKG2D is fused to the N-terminus of the VL domain of an antibody capable of binding to GARP, or to its target binding fragment or derivative.

[0063] In one embodiment of an antibody, its target-binding fragment or derivative, or a bifunctional molecule, the binding domain capable of binding to GARP is in the form of an IgG antibody.

[0064] In one embodiment, the bifunctional molecule comprises a pair of active fragments of ULBP2 fused to the N-terminus of (i) an anti-GARP antibody heavy chain and (ii) an anti-GARP antibody light chain, the pair being Sequence IDs 13 and 14, Sequence IDs 15 and 16, and / or Sequence IDs 17 and 18 It is selected from the group consisting of the following.

[0065] In one embodiment, the bifunctional molecule comprises a pair of active fragments of ULBP2 fused to the N-terminus of (i) an anti-AVB8 antibody heavy chain and (ii) an anti-AVB8 antibody light chain, as described in Sequence IDs 11 and 12.

[0066] According to another aspect of the present invention, (i) Regarding binding to GARP, does it compete with the antibody or its target binding fragment or derivative, or the bifunctional molecule as described above? (ii) Or, an antibody or its target binding fragment or derivative, or a bifunctional molecule that binds to the same GARP epitope as described above, A target-binding molecule is provided.

[0067] In one embodiment, the target-binding molecule is an antibody or a target-binding fragment or derivative thereof, or a bifunctional molecule, as defined in other parts of this specification.

[0068] As used herein, the term “competing for binding” is used with respect to an antibody or its target-binding fragment or derivative, or a target-binding molecule that has the activity to bind to the same substrate to which the bifunctional molecule binds. The efficiency of binding to the target molecule (e.g., kinetic or thermodynamic) may be the same as, greater than, or less than, the efficiency of substrate binding by the antibody or its target-binding fragment or derivative, or the bifunctional molecule. For example, the equilibrium binding constant (Kj) for binding to the substrate may be different. mWhen used herein, the term "Michaelis-Menten constant" refers to the Michaelis-Menten constant for an enzyme and is defined as the specific substrate concentration at which a given enzyme yields half of the maximum rate in an enzyme-catalyzed reaction.

[0069] As used herein, the term “binding to the same epitope” with respect to two or more binding molecules means that the molecules bind to the same segment of amino acid residues determined by a given method. Techniques for determining whether one antibody binds to the same epitope as another antibody include, for example, epitope mapping methods such as X-ray analysis of antigen:antibody complex crystals that exhibit atomic resolution of the epitope, and hydrogen / deuterium exchange mass spectrometry (HDX-MS). Other methods monitor binding to antigen fragments or variant antigens, in which case loss of binding due to modification of amino acid residues in the antigen sequence is often considered an indicator of the epitope component. Furthermore, computer-aided combinatorial methods for epitope mapping may also be used. These methods depend on the ability of the antibody of interest to isolate specific short peptides from combinatorial phage display peptide libraries.

[0070] According to another aspect of the present invention, the use of antibodies or their target-binding fragments or derivatives, or bifunctional molecules or target-binding molecules as described above (for the manufacture of pharmaceuticals) is: Diagnosed with a neoplastic disease, If you have a neoplastic disease, There is a risk of developing neoplastic diseases. It is provided for use in treatments involving humans or animals, or for the prevention of such conditions.

[0071] This language shall encompass both the Swiss-type claim language (in which case parentheses are absent) and the EPC2000 language (in which case parentheses and the content within them are absent), which are accepted in some countries.

[0072] According to another aspect of the present invention, a pharmaceutical composition is provided comprising an antibody or a target-binding fragment or derivative thereof, or a bifunctional molecule, as described above, and one or more pharmaceutically acceptable excipients of any choice.

[0073] According to another aspect of the present invention, a combination is provided comprising (i) an antibody or its target-binding fragment or derivative, or a bifunctional molecule as described above, and (ii) one or more therapeutically active compounds.

[0074] According to another aspect of the present invention, a method for treating or preventing a neoplastic disease is provided, comprising administering to a human or animal in a therapeutically sufficient dose of the antibody or its target-binding fragment or derivative, or a bifunctional molecule, or target-binding molecule, or a pharmaceutical composition, or a combination thereof, as described above. [Examples]

[0075] The present invention is illustrated and described in detail in the drawings and the foregoing description, but such illustrations and descriptions should be considered illustrative or exemplary and not restrictive. The present invention is not limited to the disclosed embodiments. Other modifications of the disclosed embodiments can be understood and achieved by those skilled in the art in carrying out the claimed invention from a study of the drawings, this disclosure and the appended claims. In the claims, the word “comprising” does not exclude other components or steps, and the indefinite article “a” or “an” does not exclude plural. The mere fact that certain means are described in different dependent claims does not imply that combinations of these means cannot be used advantageously. No reference numerals in the claims should be construed as limiting the scope.

[0076] All amino acid sequences disclosed herein are shown in the direction from the N-terminus to the C-terminus.

[0077] Materials and methods Antibody affinity for GARP, GARP-TGFβ1, and NKG2D using surface plasmon resonance (SPR) The affinity of antibodies to GARP and GARP-TGFβ1 was determined using surface plasmon resonance with a BIACORE 8K instrument (Cytiva). Anti-human IgG(Fc) antibodies were immobilized on CM5 chips by direct amine coupling according to the supplier's instructions, capturing selected binding sites in BFP and afucosylated IgG1 forms. Human GARP or GARP-TGFβ1 complexes were injected as analytes into the chip surface in serial dilutions. The sensor surface was regenerated with 10 mM glycine-HCl, pH 2.1 after each binding cycle. Generated data by double reference subtraction (subtracting the reference cell and the blank) was analyzed using BIA evaluation software (Cytiva). Binding (kon) and dissociation (koff) rate constants were evaluated from global fitting based on a 1:1 Langmuir binding model, and the equilibrium dissociation constant was calculated from the formula: KD = koff / kon.

[0078] The affinity of the antibody to NKG2D was determined using surface plasmon resonance with a BIACORE 8K instrument (Cytiva). Anti-human IgG(Fc) antibodies were immobilized on a CM5 chip by direct amine coupling according to the supplier's instructions, capturing selected binding sites in BFP format. Serial dilutions (50-0.78 nM) of human NKG2D / His (Sino Biological) were injected as analytes. The sensor surface was regenerated with 10 mM glycine-HCl, pH 2.1 after each binding cycle. The generated data was analyzed as described above.

[0079] Cellular binding using flow cytometry For cell binding studies, HEK239T cells transfected with GARP, GARP-TGFβ1, NKG2D, or an empty vector (EV) as a control were used. Barcoding was performed to analyze two or more cell lines in a single sample by flow cytometry. Different cell lines were barcoded with fluorescent labeling using the CellTrace® system (Invitrogen). Antibody staining was performed after barcoding. Anti-GARP staining of human LAP (TGF-beta 1) was performed using Alexa Fluor® 647 conjugate antibody (R&D Systems) and human LRRC32 / GARP Alexa Fluor® 647 conjugate antibody (R&D Systems) (incubated on ice for 30 minutes). For staining with the test antibody, serial dilutions were prepared to final concentrations of 40, 20, 10, 5, 0.5, 0.05, 0.005, and 0.0005 μg / mL. Cells were incubated on ice for 30 minutes. For secondary antibody staining, cells were washed and incubated on ice for 30 minutes with allophycocyanin (APC) AffiniPure goat anti-human IgG, Fcγ fragment-specific (Jackson ImmunoResearch) antibody (0.4 μL per sample). After incubation, cells were washed with DPBS + 0.5% FBS, resuspended in 200 μL of DPBS containing SYTOX Blue (Invitrogen) diluted 1:1000 for live / dead staining, and stored on ice until analysis. Samples were analyzed using a CytKick autosampler flow cytometer (Thermo Scientific, USA) and an Attune cytometer with Attune® automated software v.5.1.1. 20,000 events were captured for control samples, and 20,000 events were captured for test samples in the “target cells” gate after discarding Sytox Blue-positive dead cells and doublets. The EC50 value was calculated using a nonlinear regression (curve fitting) function and a log(agonist) vs. response-3 parameter equation.

[0080] Affinity evaluation of antibodies against FcγRIIIA using surface plasmon resonance (SPR) The affinity of PB003G.21.0111 and PB003G.21.0111.aF for FcyRIIIA (176V and 176F) was determined using surface plasmon resonance with a BIACORE 8K instrument (Cytiva). Recombinant biotinylated FcγRIIIA / His-Avi (176V and 176F) were injected onto the surface of a SA chip (Cytiva). Serial dilutions of the antibodies were injected in single-cycle kinetics mode. The sensor surface was regenerated with 10 mM glycine-HCl, pH 3.0 (Cytiva) after each binding cycle. Generated data by dual reference subtraction (subtracting the reference cell and the blank) was analyzed using Biacore Insight evaluation software v4.0 (Cytiva). The binding (kon) and dissociation (koff) rate constants were evaluated based on a 1:1 Langmuir coupling model, and the equilibrium dissociation constant was calculated from the equation: KD = koff / kon. ADCC assay (NK cell-mediated cell death)

[0081] Isolation of effector cells NK cells were isolated from peripheral blood mononuclear cells (PBMCs) obtained from the buffy coat of healthy donors. PBMCs were initially isolated by density centrifugation using SepMate-50 PBMC isolation tubes (Stemcell®). The PBMC layer was transferred to an unused 50 mL conical tube and incubated with erythrocyte lysis buffer (Biolegend, 420302) at room temperature for 10 minutes. The tube was filled with DPBS and centrifuged (350 g, 5 min). After washing, the PBMCs were subjected to immunomagnetic negative selection for NK cell isolation using an NK cell isolation kit (Miltenyi Biotec, 130-092-657) according to the manufacturer's protocol. Isolation purity and NK cell phenotyping were analyzed by flow cytometry (Cytek Northern Lights (NL-00020)). The viability of NK cells was evaluated using the LIVE / DEAD® Fixable Aqua dead cell staining kit (Invitrogen®, L34966, 1:1000). The following markers were tested with antibodies according to the manufacturer's protocol: CD3 (APC-H7 mouse anti-human CD3, BD Biosciences, 560176), CD16 (Alexafluol® 647 mouse anti-human CD16, BD Biosciences, 557710), CD56 (Brilliant Violet 605® anti-human CD56 (NCAM), BioLegend®, 362538), CD69 (BV711 mouse anti-human CD69, BD Biosciences, 563836), CD107a (PE mouse anti-human CD107a, BD Pharmingen®, 555801), and NKG2D (BV421 mouse anti-human CD314 (NKG2D), BD Biosciences, 743558).

[0082] In experiments using Raji-AVB8 cells, NK cells were incubated overnight with IL15 added to the culture medium (5 ng / ml).

[0083] Measurement of ADCC in tumor cells Target cells (Raji-GARP and Raji-GARP-TGFβ1, Raji-AVB8, or L428) were stained with CFSE to isolate them from effector cells by flow cytometry, and their viability was evaluated using the Live / Dead® Fixable Violet dead cell staining kit. PB003G.21.0111 and PB003G.21.0111.aF were added to the cells in serial dilutions. NK cells isolated the previous day were mixed with target cells in a 10:1 ratio (effector vs. target). The cells were incubated for 4 hours, after which cell lysis was detected using the Live / Dead® Fixable Violet dead cell staining kit solution (1:1000 in DPBS, Invitrogen®, L34964), and incubated at 4°C for 20 minutes. Cells were fixed in 4% paraformaldehyde solution (Thermo Scientific (trademark), J19943.K2) for 10 minutes at 4°C. After fixation, the cell pellet was suspended in FACS buffer and subjected to flow cytometry analysis. The percentage of target cell death was plotted against the inverse logarithmic concentration. Data are presented as mean and standard deviation (error bars). Standard one-way ANOVA and Dunnett's multiple comparison test were applied for statistical analysis.

[0084] Measurement of ADCC for Treg Treg cells were isolated from PBMCs of the same donor as NK cells using a human CD4+CD25+ regulatory T cell isolation kit (Miltenyi Biotec, 130-091-301) by immunomagnetic two-step method according to the manufacturer's protocol. Treg cells were seeded at a density of 200,000 cells / well in 96-well plates and mixed with Dynabeads® Human T-Activator CD3 / CD28 for T Cell Expansion and Activation (Gibco, 11161D) in a 1:1 ratio, and cultured in IMDM (Gibco, 31980030) for 2 days. The isolation purity and phenotypic analysis of Treg cells were performed by flow cytometry (Cytek Northern Lights (NL-00020)). The viability of Treg cells was confirmed using the Live / Dead® Fixable Aqua dead cell staining kit (Invitrogen®, L34966, 1:1000). The following markers were tested with antibodies according to the manufacturer's protocol: CD4 (PerCP / Cyanine 5.5 anti-human CD4 antibody, BioLegend, 300529), CD25 (BV605 mouse anti-human CD25, BD Horizon, 562660), CD45 (Brilliant Violet 785 (trademark) anti-human CD45 antibody, BioLegend (registered trademark), 304048), CD127 (BV650 mouse anti-human CD127, BD Horizon, 563225), GARP (PE / Cy7 anti-human GARP, BioLegend, 352508), and FoxP3 (Alexafluol (registered trademark) 647 mouse anti-human FoxP3, BD Pharmingen, 560045).

[0085] DynaBeads® were separated from the cells using a magnet (Miltenyi Biotec) and discarded. Cells were labeled with CellTrace® CFSE Growth Kit (Invitrogen®, C34554) 0.1 μM. Target cells (10,000 cells per well) were seeded into a conical-bottom 96-well plate and incubated with serial dilutions of the test antibody for pre-coating at 37°C, 5% CO2 for 30 minutes. NK cells isolated the previous day were added in a final ratio of effector cells to target cells of 10:1. After overnight incubation, cell lysis levels were estimated using Live / Dead® Fixable Violet Dead Cell Staining Kit (1:1000 in DPBS, Invitrogen®, L34964). Cells were incubated with anti-GARP antibody (BD Pharmingen, 562341). Cells were fixed with 4% paraformaldehyde (ThermoScientific®, J19943.K2) for 10 minutes at 4°C. After fixation, 100 μL of FACS buffer was used for flow cytometry analysis using a Cytek Northern Lights (NL-00020) flow cytometer. The relative percentage of cell lysis was calculated by subtracting the target viability control from the experimental lysis value. Data were expressed on a log10 scale as the relative percentage of lysis relative to the indicated antibody concentration (nM). A graph representing the dose-response curve was fitted using a 4-parameter nonlinear regression equation. Data were analyzed using FlowJo® v10.8.1 software (BD Life Sciences). Percentage of target cell death vs. inverse logarithmic concentration was plotted. Data are expressed as the mean and standard deviation (error bars) from at least two technical replicates. Statistical analysis was not performed due to donor-dependent variability between biological replicates.

[0086] Degranulation assay Target cells were pre-incubated with the test molecule at 50 nM for 30 minutes. PBMCs were then added to an E:T ratio of 1:1. BD GolgiStop®, BD GolgiPlug®, and anti-CD107a antibody were added to the culture for 4 hours. Analysis was performed using the flow cytometry method described below.

[0087] Surface marker and intracellular cytokine staining Degranulating and cytokine-secreting NK cells were analyzed using multiparametric surface markers and intracellular cytokine staining. After co-incubating PBMCs with target cells, all cells were washed and stained with viability markers, anti-CD56 (clone 5.1H11), anti-CD16 (clone 3G8), and anti-CD3 (clone SK7) for 25 minutes at 37°C. Samples were fixed and permeabilized according to the manufacturer's instructions (fixation / permeabilization kit, BD). After washing, cells were further stained with intracellular anti-IFN-γ (clone B27) and anti-TNFα antibody (clone MAb11) for 60 minutes. Data were acquired using a Cytek flow cytometer and analyzed using FlowJo X v.10.0. Results are expressed as mean and standard deviation percentages. The difference in means was calculated using a two-way ANOVA test. Significance is indicated as follows: * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001, **** P ≤ 0.0001.

[0088] GARP-TGFβ1 complex formation blocking assay Raji-GARP cells were incubated with test antibodies and controls in serial dilutions (final concentrations 0.2 ng / mL to 50 μg / mL) and with latent TGFβ1 (15 μg / mL) in complete RPMI medium. After incubation (37°C, 5% CO2, 60 mins) and washing, cells were stained with anti-LAP-AF647 antibody at room temperature for 30 minutes to detect latent TGFβ1. Samples were acquired using an Attune NxT flow cytometer equipped with a CytKick Max autosampler (20,000 events). Data analysis was performed using Attune Cytometric software ver 4.3.002.1. MdFI vs. log concentrations were plotted. Nonlinear curve fitting (inhibitor vs. log agonist, 3 parameters) was applied.

[0089] Western blotting for the evaluation of TGFβ1 downstream signaling: Detection of SMAD2 phosphorylation by SMAD2 phosphorylation Jurkat and Jurkat β8-GARP cells (1 × 10⁻¹⁰ 6Cells (per well) were stimulated with anti-CD3 (OCT3 clone, BioLegend, final concentration 1 μg / mL) and soluble anti-CD28 (BD Biosciences, final concentration 1 μg / mL). Stimulation was performed simultaneously with incubation with test antibodies and controls: PB003G.21.0091.BFP, PB003G.21.0111.aF, DS-1055, ABBV-151, and IgG1 isotype controls. Cell lysates were collected after 24 hours and prepared for Western blotting. 4-20% Mini-Protean TGX gels (BioRad) were used for SDS-PAGE electrophoresis. After transfer, the membranes were incubated overnight with anti-pSMAD2 antibody (1:1000 dilution in 5% BSA / TBST). A secondary antibody solution (anti-rabbit IgG-HRP, R&D Systems, diluted 1:1000 in TBST / 5% BSA) was applied at room temperature for 1 hour. Bands were detected using Immobilon Western Chemiluminescent HRP substrate and ChemiDoc MP (BioRad). After image acquisition, the membrane was washed and incubated with anti-GAPDH antibody for loading control. ImageLAb v. 6.0.1 build 34 was used for concentration analysis.

[0090] In vivo efficacy using a Raji-GARP-TGFβ1 tumor model The antitumor effect, safety, and immune activation of PB003G.21.0111.aF were evaluated at Jackson Laboratories (Bar Harbor, USA) in humanized NSG-hIL15 mice with Raji-GARP-TGFβ1 tumor-carrying CD34+ hematopoietic stem cells (HSCs). 6 The drug was subcutaneously inoculated into the right flank / shoulder of CD34+ HSC humanized NSG-hIL15 mice. The average tumor volume was approximately 150-180 mm². 3When the mice reached [a certain volume], they were randomized by HSC donor and tumor volume. The mice were treated by intravenous injection of solvent, PB003G.21.0111.aF, or DS-1055 at 10 mg / kg body weight (bw) once every two weeks for six times. Tumor volume and mouse body weight were measured two to three times a week. Two-way ANOVA and Bonferroni's multiple comparisons were used for statistical analysis.

[0091] In vivo efficacy using the HT-29 tumor model The antitumor effect of PB003G.21.0111.aF was evaluated in HT29 colon cancer tumor-bearing CD34+ hematopoietic stem cell (HSC) humanized NCG-hIL15 mice at GemPharmatech (Nanjing, China). HT29 cells (5×10 6 ) were subcutaneously inoculated into the right flanks / shoulders of CD34+ HSC humanized NCG-hIL15 mice. When the average tumor volume reached 80 mm 3 , the mice were randomized by HSC donor, humanization level, and tumor volume. The mice were treated by intravenous injection of solvent, PB003G.21.0111.aF, or DS-1055 at 10 mg / kg body weight (bw) once every two weeks for six times. Tumor volume and mouse body weight were measured two to three times a week.

[0092] In vitro binding to platelets using flow cytometry Platelets were isolated from buffy coats obtained from healthy donors by density centrifugation. PBMCs were first isolated by density centrifugation using SepMate™-50 PBMC isolation tubes (StemCell™). The PBMC layer was transferred to an unused 50 mL conical tube and incubated with red blood cell lysis buffer (Biolegend, 420302) for 10 minutes at room temperature. The tube was filled with DPBS and centrifuged (350 g, 5 minutes). After washing and resuspension, the PBMCs were centrifuged (350 g, 5 minutes) and the supernatant was maintained as the platelet-rich fraction. The platelet-rich fraction was then centrifuged at 3200 g for 5 minutes and resuspended in Tyrode's solution.

[0093] 5 × 10 from the platelet-rich fraction of 200 μL of Tyrode's solution 6 Cells were incubated with a biotinylated compound (10 μg / mL) and stained with viability markers (Live / Dead® Fixable Violet-Live / Dead® 405nm stain, ThermoFisher Scientific, catalog #L34955), anti-CD41 (BioLegend, clone HIP8, catalog #303725), anti-CD42b (BioLegend, clone HIP1, catalog #303920), and anti-CD45 (BioLegend, clone HI30, catalog #563716) for 20 minutes at 37°C. After washing, cells were centrifuged at 3000 g for 5 minutes and then stained with FITC streptavidin (BioLegend, catalog #405202) for a further 15 minutes. After washing, cells were centrifuged at 3000 g for 5 minutes and resuspended in 100 μL of final solution. Data was acquired using a Cytek flow cytometer and analyzed using FlowJo X v.10.0. The results are expressed as the mean and standard deviation of the FITC MdFI multiplier change.

[0094] (References) Bouchard A. et al. GARP: A Key Target to Evaluate Tumor Immunosuppressive Microenvironment. Biology (2021) 10, 836. Derynck R., Erine HB Specificity, versatility, and control of TGF-β family signaling. Sci Signal. (2019) 26;12(570). Ollendorff V. et al. The GARP Gene Encodes a New Member of the Family of Leucine-Rich Repeat-Containing Proteins. Cell Growth Differ (1994) 5:213-9. Tolcher A, Roda-Perez D, He K, et al (770) Safety, efficacy, and pharmacokinetic results from a phase I first-in-human study of ABBV-151 with or without anti-PD1 mAb (budigalimab) in patients with locally advanced or metastatic solid tumors Journal for ImmunoTherapy of Cancer 2022;10:doi: 10.1136 / jitc-2022-SITC2022.0770 Satoh K, Kobayashi Y, Fujimaki K, Hayashi S, Ishida S, Sugiyama D, Sato T, Lim K, Miyamoto M, Kozuma S, Kadokura M, Wakita K, Hata M, Hirahara K, Amano M, Watanabe I, Okamoto A, Tuettenberg A, Jonuleit H, Tanemura A, Maruyama S, Agatsuma T, Wada T, Nishikawa H. Novel anti-GARP antibody DS-1055a augments anti-tumor immunity by depleting highly suppressive GARP+ regulatory T cells. Int Immunol. 2021 Jul 23;33(8):435-446. doi: 10.1093 / intimm / dxab027. PMID: 34235533. Zingoni A, Molfetta R, Fionda C, Soriani A, Paolini R, Cippitelli M, Cerboni C, Santoni A. NKG2D and Its Ligands: “One for All, All for One”. Front Immunol. 2018 Mar 12;9:476. doi: 10.3389 / fimmu.2018.00476. PMID: 29662484; PMCID: PMC5890157. Shields, R, Namenuk, A, Hong, Ket al. High resolution mapping of the binding site on human IgG1 for FcγRI, FcγRII, FcγRIII, and FcRn and design of IgG1 variants with improved binding to the FcγR. J Biol Chem 2000; 276: 6591-604. Tang, Y, Lou, J, Alpaugh, Ret al. Regulation of antibody-dependent cellular cytotoxicity by IgG intrinsic and apparent affinity for target antigen. J Immunol 2007; 179: 2815-23. Zahavi D, AlDeghaither D, O’Connell A, Weiner LM. Enhancing antibody-dependent cell-mediated cytotoxicity: a strategy for improving antibody-based immunotherapy. Antib Ther. 2018 Jun 24;1(1):7-12. Liu, Z, Gunasekaran, K, Wang, Wet al. Asymmetrical Fc engineering greatly enhances antibody-dependent cellular cytotoxicity (ADCC) effector function and stability of the modified antibodies. J Biol Chem 2013; 289: 3571-90

[0095] array The following sequences form part of the disclosure of this application. A WIPO standard ST.26 format electronic sequence listing is also provided with this application. To avoid any doubt, if there is any discrepancy between the sequences in the following table and the electronic sequence listing, the sequences in this table shall prevail.

[0096] In some cases, the signal peptide may be incorporated into the reproduced sequence. In such cases, the sequence is disclosed with and without the signal peptide. A readily available tool for identifying the signal peptide in a given protein sequence is SignalP-6.0, provided by Dansk Technical University under https: / / services.healthtech.dtu.dk / service.php?SignalP. Optionally, the signal peptide is underlined where applicable. The same applies to sequences containing the His tag, in which case the sequence is disclosed with and without the His tag.

[0097] [Table 3] TIFF2026520908000005.tif191149TIFF2026520908000006.tif167149TIFF2026520908000007.tif70149

Claims

1. The heavy / light variable domain (HCVD / LCVD) pairs described below: Sequence IDs 3 and 4, Sequence IDs 5 and 6, and / or Sequence IDs 7 and 8 An antibody capable of binding to GARP, or a target-binding fragment or derivative thereof that retains target-binding ability, including the above.

2. The antibody or fragment according to claim 1, which is a monoclonal antibody or derived from a monoclonal antibody.

3. The antibody or fragment or derivative according to claim 1 or 2, wherein the antibody or fragment or derivative has enhanced ability to induce ADCC.

4. The antibody or fragment according to any one of claims 1 to 3, comprising an afucosylated Fc domain.

5. A binding domain that can bind to cancer antigens, A binding domain that can bind to NKG2D and A bifunctional molecule containing [this component].

6. a) The binding domain that can bind to the cancer antigen is an antibody or its target binding fragment or derivative. b) The binding domain that can bind to NKG2D is the ligand for NKG2D, optionally ULBP2, or its active fragment. The bifunctional molecule according to claim 5.

7. The bifunctional molecule according to claim 5 or 6, wherein a binding domain capable of binding to a cancer antigen binds to at least one target selected from the group listed in Table 2.

8. ULBP2 contains or consists of the amino acid sequence according to SEQ ID NO: 1, and / or The binding domain capable of binding to a cancer antigen is the antibody, fragment, or derivative described in claim 1. A bifunctional molecule according to any one of claims 5 to 7.

9. A bifunctional molecule according to any one of claims 5 to 8, wherein the active fragment of ULBP2 contains or comprises the amino acid sequence according to SEQ ID NO:

2.

10. A bifunctional molecule according to any one of claims 5 to 9, wherein the binding domain capable of binding to NKG2D is directly fused to the N-terminus of the VH or VL domain of an antibody capable of binding to GARP, or to its target binding fragment or derivative, or is fused via a flexible linker.

11. A bifunctional molecule according to any one of claims 5 to 9, wherein a binding domain capable of binding to NKG2D is fused to the N-terminus of the VL domain of an antibody capable of binding to GARP, or to its target binding fragment or derivative.

12. An antibody or its target binding fragment or derivative, or a bifunctional molecule, according to any one of claims 1 to 11, wherein the binding domain capable of binding to GARP is in the form of an IgG antibody.

13. (i) comprising a pair of active fragments of ULBP2 fused to the N-terminus of an anti-GARP antibody heavy chain and (ii) an anti-GARP antibody light chain, wherein the pair is Sequence IDs 13 and 14, Sequence IDs 15 and 16, and / or Sequence IDs 17 and 18 A bifunctional molecule according to any one of claims 5 to 11, selected from the group consisting of the following.

14. (i) With respect to binding to GARP, whether it competes with the antibody or its target binding fragment or derivative described in any one of claims 1 to 13, or with the bifunctional molecule, (ii) Or, the antibody or its target binding fragment or derivative, or the bifunctional molecule described in any one of claims 1 to 13, which binds to the same GARP epitope. Target binding molecules.

15. The target-binding molecule according to claim 14, which is an antibody, a target-binding fragment or derivative thereof, or a bifunctional molecule.

16. Diagnosed with a neoplastic disease, If you have a neoplastic disease, There is a risk of developing neoplastic diseases. Use of an antibody or its target-binding fragment or derivative, or a bifunctional molecule or target-binding molecule, according to any one of claims 1 to 15 (for the manufacture of pharmaceuticals), in a treatment of a human or animal subject, or for the prevention of such a condition.

17. A pharmaceutical composition comprising an antibody or a target-binding fragment or derivative thereof according to any one of claims 1 to 13, or a bifunctional molecule, and one or more pharmaceutically acceptable excipients of any choice.

18. (i) an antibody or a target-binding fragment or derivative thereof according to any one of claims 1 to 13, or a bifunctional molecule, and (ii) a combination comprising one or more therapeutically active compounds.

19. A method for treating or preventing a neoplastic disease, comprising administering to a human or animal in a therapeutically sufficient dose of an antibody or its target-binding fragment or derivative, or a bifunctional molecule or target-binding molecule, the pharmaceutical composition according to claim 17, or the combination according to claim 18, as described in any one of claims 1 to 15.

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