Anti-human PD-1 agonist antibodies and pharmaceutical compositions containing them for the treatment or prevention of inflammatory diseases.

Abstract

This invention explores the conditions required for antibodies against human PD-1 to possess agonist activity, identifies optimized agonist antibodies based on these conditions, and uses them as therapeutic agents for human inflammatory diseases. An antibody or a functional fragment thereof is characterized in that it is an agonist antibody against human PD-1 or a functional fragment thereof, binding to the #7 region of human PD-1 represented by sequence number 9.

Description

Technical Field

[0001] This invention relates to a pharmaceutical composition containing a PD-1 (Programmed cell death 1) agonist for the treatment or prevention of inflammatory diseases. Prior Technology

[0002] Inadequate control of the immune response can lead to disease. Insufficient immune responses to pathogens such as bacteria and viruses invading the body result in infections; conversely, harmful immune responses against the body's own tissues lead to autoimmune diseases. To prevent such pathological states and effectively utilize the system, the immune system possesses both mechanisms to activate immune cells and promote immune responses, and mechanisms to suppress and reduce immune responses. Impairment in these endogenous control mechanisms is also a cause of diseases arising from insufficient or excessive immune responses.

[0003] A simple example is immunotherapy for cancer, which has rapidly become a reality in recent years. This new treatment method, different from previous "immunotherapy," utilizes molecules such as CTLA-4 and PD-1, which are also considered endogenous immunosuppressive mechanisms of immune checkpoints. Among similar immunosuppressive mechanisms, their effects are particularly significant. Regarding the course of cancer, the presence of cancer cells that should be expelled can be seen as an inadequate immune response to cancer cells, leading to their proliferation. The fact that cancerous tissue is filled with various immunosuppressive mechanisms is identified as the cause of this inadequacy; that is, cancerous tissue actively utilizes the body's inherent immunosuppressive mechanisms to evade attacks from immune cells. By blocking CTLA-4 and PD-1, representative of this immunosuppressive mechanism, the previously suppressed anti-tumor immunity is reactivated, demonstrating a therapeutic effect. This fact shows that these endogenous immune control mechanisms have a very large influence and are an important target for treating diseases by correcting imbalances in the immune response.

[0004] The presence of an exceptionally strong inflammatory response due to the deficiency of any one of these mechanisms underscores the essential importance of endogenous immunosuppressive mechanisms. For example, mice lacking PD-1 have been shown to exhibit spontaneous onset of various inflammatory diseases (Non-Patent Literature 1: Okazaki et al. Nat. Immunol., 2013). Furthermore, harmful inflammatory responses stemming from excessive immune responses have been observed at a certain rate when cancer is treated with PD-1 inhibitors (Non-Patent Literature 2: Young et al. Cancer Immunol. Res., 2018). These facts suggest that the immunosuppressive mechanisms actively utilized in cancerous tissues are originally physiological feedback mechanisms used to protect normal tissues from excessive inflammatory responses. Importantly, the effects of pharmaceuticals, such as anti-PD-1 antibodies, in cancer treatment suggest that the strength of the immune response can be modulated by controlling PD-1 function. Previous technical documents Non-patent literature

[0005] Non-patent literature 1: Okazaki T, Chikuma S, Iwai Y, Fagarasan S, Honjo T. A rheostat for immune responses: the unique properties of PD-1 and their advantages for clinical application. Nat. Immunol. 14:1212-8 (2013). Non-patent literature 2: Young A, Quandt Z, Bluestone JA. The Balancing Act between Cancer Immunity and Autoimmunity in Response to Immunotherapy. Cancer Immunol. Res. 6:1445-1452 (2018). Summary of the Invention

[0006] [The problem the invention aims to solve]

[0007] As the background above indicates, in cancer treatment where a stronger immune response is needed, agents that inhibit immunosuppressive mechanisms are used. However, in the treatment of inflammatory diseases, it is necessary to suppress unnecessarily hyperactive immune responses; therefore, the opposite approach is required to actively stimulate immunosuppressive mechanisms. PD-1 is expressed in activated T cells and other immune cells, and its mechanism of action is known as follows: during antigen recognition, when ligands such as PD-L1 and PD-L2 are expressed on the surface of the target cells, PD-1 interferes with the activation signals of immune cells through interaction with these ligands. PD-1 itself has the property of inducing immunosuppressive signals; if this property can be artificially stimulated, there is a possibility of treating inflammatory diseases. Anti-PD-1 anti-systems used in cancer treatment induce immune hyperactivity by inhibiting the binding of ligand molecules (resulting in) hyperactivity. However, to achieve active immunosuppression, PD-1 agonists that can induce PD-1 function by binding to PD-1 are promising.

[0008] The purpose of this invention is to explore the conditions required for antibodies against human PD-1 to have agonist activity, to determine the optimal agonist antibody based on these conditions, and to use it as a therapeutic agent for human inflammatory diseases. [Technical means to solve the problem]

[0009] Many inflammatory diseases arise from excessive immune responses, and new, more effective treatments are sought for most of them. The inventors have discovered numerous anti-human PD-1 antibodies that target the immune control molecule PD-1 and induce its immunosuppressive activity. They found that this agonist activity can prevent or treat human inflammatory diseases. Studies of the binding region between these antibodies and human PD-1 revealed that the antibody activity changes depending on this binding region. Furthermore, it was found that to exert the agonist activity of anti-PD-1 antibodies, binding to the Fc receptor is necessary, and that human immunosuppression requires higher Fc receptor affinity. Moreover, it was discovered that by conferring ADCC activity with anti-PD-1 agonist antibodies, activated immune cells expressing PD-1 can be removed, resulting in more ideal immunosuppressive activity.

[0010] This invention was made based on these insights, and its main points are as follows. (1) An antibody or a functional fragment thereof, characterized in that: it is an antibody or a functional fragment thereof that is an agonist antibody against human PD-1, and binds to the #7 region of human PD-1 represented by sequence number 9. (2) The antibody or its functional fragment described in (1) has any of the following (A) to (D): (A) The antibody heavy chain variable region sequence containing CDR1, CDR2 and CDR3 of the amino acid sequence represented by sequence number 17, and the antibody light chain variable region sequence containing CDR1, CDR2 and CDR3 of the amino acid sequence represented by sequence number 34; (B) The antibody heavy chain variable region sequence containing CDR1, CDR2 and CDR3 of the amino acid sequence represented by sequence number 22, and the antibody light chain variable region sequence containing CDR1, CDR2 and CDR3 of the amino acid sequence represented by sequence number 38; (C) The antibody heavy chain variable region sequence containing CDR1, CDR2 and CDR3 of the amino acid sequence represented by sequence number 28, and the antibody light chain variable region sequence containing CDR1, CDR2 and CDR3 of the amino acid sequence represented by sequence number 42; (D) The antibody heavy chain variable region sequence containing CDR1, CDR2 and CDR3 of the amino acid sequence represented by sequence number 33, and the antibody light chain variable region sequence containing CDR1, CDR2 and CDR3 of the amino acid sequence represented by sequence number 46. (3) The antibody or its functional fragment described in (1) has any of the following (A) to (D): (A) An antibody heavy chain variable region comprising complementarity-determining region (CDR) 1 (sequence number 345), CDR 2 (sequence number 346), and CDR 3 (sequence number 347), and an antibody light chain variable region comprising CDR 1 (sequence number 348), CDR 2 (sequence number 349), and CDR 3 (sequence number 350); (B) The antibody heavy chain variable region comprising CDR1 (sequence number 351), CDR2 (sequence number 352), and CDR3 (sequence number 353), and the antibody light chain variable region comprising CDR1 (sequence number 354), CDR2 (sequence number 355), and CDR3 (sequence number 356); (C) The antibody heavy chain variable region comprising CDR1 (sequence number 357), CDR2 (sequence number 358), and CDR3 (sequence number 359), and the antibody light chain variable region comprising CDR1 (sequence number 360), CDR2 (sequence number 361), and CDR3 (sequence number 362); (D) Contains antibody heavy chain variable regions of CDR1 (sequence number 363), CDR2 (sequence number 364), and CDR3 (sequence number 365), and antibody light chain variable regions of CDR1 (sequence number 366), CDR2 (sequence number 367), and CDR3 (sequence number 368). (4) The antibody or its functional fragment as described in (1) to (3), wherein the antibody variable region contains a human antibody architecture or a humanized antibody architecture. (5) The antibody or its functional fragment described in (1) has any of the following (A) to (D): (A) The antibody heavy chain variable region of the amino acid sequence represented by sequence number 20 and the antibody light chain variable region of the amino acid sequence represented by sequence number 37; (B) The antibody heavy chain variable region of the amino acid sequence represented by sequence number 21 and the antibody light chain variable region of the amino acid sequence represented by sequence number 37; (C) The antibody heavy chain variable region of the amino acid sequence represented by sequence number 26 and the antibody light chain variable region of the amino acid sequence represented by sequence number 40; (D) The antibody heavy chain variable region of the amino acid sequence represented by sequence number 30 and the antibody light chain variable region of the amino acid sequence represented by sequence number 44. (6) The antibody or its functional fragment as described in (1) to (5), wherein The constant regions of the antibody heavy chain and the constant regions of the antibody light chain contain amino acid sequences derived from human antibodies. (7) The antibody or its functional fragment as described in (1) to (5), wherein The antibody heavy chain constant region is the antibody heavy chain constant region of human IgG (Immunoglobulin G). (8) The antibody or its functional fragment as described in (7), wherein The aforementioned human IgG antibody heavy chain constant region is the same as the human IgG1 antibody heavy chain constant region. (9) The antibody or its functional fragment as described in (1) to (8), wherein The antibody light chain constant region is the antibody light chain constant region of human Igκ. (10) The antibodies or their functional fragments as described in (1) to (9), The affinity for any one or more of the human FcγRIIA, human FcγRIIB, and human FcγRIIIA receptors is further enhanced. (11) The antibodies or their functional fragments as described in (1) to (9), The affinity for human FcγRIIB has been further enhanced. (12) The antibody or its functional fragment as described in (1) to (9) or (11), The affinity for human FcγRIIIA has been further enhanced. (13) The antibody or its functional fragment as described in (11) or (12) wherein the enhancement of affinity for human FcγRIIB in an Fc receptor binding affinity assay using surface plasma resonance technology is at least 1.5 times, preferably at least 2.0 times, and even more preferably at least 2.5 times, compared with a reference antibody having the Fc region of human IgG1-K322A. (14) The antibody or its functional fragment as described in (11) to (13) wherein the affinity for human FcγRIIB is increased by more than 2 times, preferably more than 5 times, and even more than 20 times, compared with a reference antibody having the Fc region of human IgG1-K322A in a flow cytometry assay of human Fc receptor-expressing cell lines. (15) The antibody or its functional fragment as described in (12) or (13) wherein the enhancement of affinity for human FcγRIIIA in an Fc receptor binding affinity assay using surface plasma resonance technology is at least 1.5 times, preferably at least 2.0 times, even better at least 2.5 times, and most preferably at least 4 times compared to a reference antibody having the Fc region of human IgG1-K322A. (16) The antibody or its functional fragment as described in (12) to (15) wherein the enhancement of affinity for human FcγRIIIA in an Fc receptor binding affinity assay using flow cytometry is at least 1.5 times, preferably at least 2.0 times, even more preferably at least 4.0 times, and most preferably at least 5 times compared to a reference antibody having the Fc region of human IgG1-K322A. (17) The antibody or its functional fragment described in any of (10) to (16) has a modified Fc region of human IgG, thereby enhancing the affinity for the aforementioned receptor by modifying the Fc region of human IgG. (18) The antibody or its functional fragment described in any of (10) to (16) has a modified Fc region of human IgG1 or human IgG4, thereby enhancing the affinity for the aforementioned receptor by modifying the Fc region of human IgG1 or human IgG4. (19) The antibody or its functional fragment as described in (10) to (18) wherein any one of the amino acids at positions 233, 234, 236, 237, 238, 239, 267, 268, 271, 296, 323, 326, 328, 330 and 332 (according to EU numbering) in the amino acid sequence of the Fc region of human IgG1 is modified. (20) The antibody or its functional fragment as described in (19), wherein the modified amino acid is G236D / H268D, S239D / H268D, S239D / H268D / L328Y / I332E, G236D / H268D / K322A, S239D / H268D / K322A, S239D / S267G / H268D / K322A, G236D / H268D / E293A / K322A, S239D / H268D / K322A / L328Y / I332E, S239D / H268D / E Any combination of 293A / K322A, S239D / S267G / H268D / K322A / L328Y, S239D / S267G / H268D / K322A / I332E, S239D / H268D / K322A / L328Y, S239D / H268D / K322A / I332E, S239D / S267G / H268D / K322A / L328Y / I332E, E233D / G237D / P238D / H268D / P271G / A330R, and S267E / L328F. (21) The antibody or its functional fragment as described in (10) to (18) wherein any of the amino acids at positions 236, 239, 268, 328 and 332 (according to EU numbering) of the Fc region of human IgG4 is modified. (22) The antibody or its functional fragment as described in (21), wherein the modified amino acid is any combination of S228P / G236D / Q268D, S228P / S239D / Q268D or S228P / S239D / Q268D / L328Y / I332E. (23) The antibody or its functional fragment described in (10) includes any one of the following (A) to (D): (A) The antibody heavy chain sequence of the amino acid sequence represented by sequence number 245 and the antibody light chain sequence of the amino acid sequence represented by sequence number 247; (B) The antibody heavy chain sequence of the amino acid sequence represented by sequence number 277 and the antibody light chain sequence of the amino acid sequence represented by sequence number 279; (C) The antibody heavy chain sequence of the amino acid sequence represented by sequence number 285 and the antibody light chain sequence of the amino acid sequence represented by sequence number 287; (D) The antibody heavy chain sequence of the amino acid sequence represented by sequence number 289 and the antibody light chain sequence of the amino acid sequence represented by sequence number 291. (24) The antibody or its functional fragment described in any of (12) to (23), wherein the antibody system is "defucosome". (25) A single nucleic acid that encodes an antibody or a functional fragment thereof as described in any of (1) to (24). (26) A carrier comprising nucleic acid as described in (25). (27) A host cell containing nucleic acids as described in (25). (28) A host cell containing a vector as described in (26). (29) A method for producing an antibody or a functional fragment thereof, comprising a stage of culturing host cells as described in (27) or (28). (30) A pharmaceutical composition comprising an antibody or a functional fragment thereof as described in any one of (1) to (24), and a pharmaceutically permissible carrier. [Effects of the Invention]

[0011] According to the present invention, inflammatory diseases can be treated or prevented. This specification contains the contents set forth in the specification and / or drawings of Japanese patent applications Japanese Patent Application No. 2021-81913 and Japanese Patent Application No. 2021-86534, which form the basis of the priority claim in this case. Simple Explanation of the Diagram

[0012] Figure 1 shows an evaluation system for the inhibitory activity of cytokines production in PD-1-stimulated T cells. (A) is a schematic diagram of the evaluation system using DO11.10 T-cell fusion tumors and IIA1.6 B-cell lymphoma cells expressing human PD-1 (hPD-1). DO11.10 T-cell fusion tumors are activated by reacting with the OVA 323-339 peptide presented by MHC class II molecules (IAd) on IIA1.6 cells, but activation is inhibited upon PD-1 stimulation. (B) IL-2 production from DO11.10 T-cell fusion tumors expressing human PD-1 is inhibited upon interaction with PD-L1 on IIA1.6 cells. No inhibition of IL-2 was observed in cells not expressing PD-1 or PD-L1. (C) The inhibitory effect obtained using PD-L1 is relieved when EH12.2H7, a blocking antibody against human PD-1, is added to the evaluation system. This evaluation system can be used to detect PD-1-dependent immunomodulatory activity. Figure 2 is a schematic diagram of the regions of PD-1 used to study the binding properties of human and mouse PD-1 sequences with a novel group of anti-human PD-1 antibodies. (A) Regions represented by numbers 1-8 represent representative regions constituting the surface of the human PD-1 molecule. Regions obtained by replacing one of these regions with the corresponding amino acid sequence of mouse PD-1 were created. When the binding ability of the anti-human PD-1 antibody decreased, this region was defined as the region bound by the antibody. Furthermore, antibody binding studies were also conducted on point variants obtained by only changing the amino acid residues of human PD-1, and on regions obtained by only replacing a portion of mouse PD-1 with the amino acid sequence of human PD-1. (B) Comparison of human PD-1 and mouse PD-1 sequences. The area enclosed by the border line is regions 1-8 in Figure 2A. Figure 3 shows the evaluation of the agonist activity of anti-human PD-1 antibodies. (A) is a schematic diagram of the evaluation system combining DO11.10 T-cell fusion tumors expressing human PD-1 and IIA1.6 B-cell lymphoma cells expressing mouse FcγRIIB but not PD-L1. The inhibitory activity of IL-2 production was used as an indicator for screening anti-human PD-1 antibodies. (B) As part of the evaluation results, approximately 30 immunosuppressive antibodies were obtained, ranging from those with high to low activity. The graph only represents the immunosuppressive antibodies among these anti-human PD-1 antibodies. Figure 4 compares the activities of anti-human PD-1 agonist antibodies. Among the anti-human PD-1 antibodies found to have immunosuppressive activity, those with higher activity were selected, and their IC50 values ​​were determined. Representative antibodies are shown in (A) group #6, 7 (antibody groups binding to regions #6 and #7), (B) group #7 (antibody groups binding only to region #7), and (C) group #6, 8 (antibody groups binding to regions #6 and #8). (D) shows the IC50 values ​​of the antibodies represented by (A)-(C). Novel antibodies prepared by immunizing mice with human PD-1 are self-evident, and commercially available antibodies (J116, MIH4) with proven agonist activity are also mouse IgG. Based on the concentration-dependent IL-2 inhibition curves of these antibodies, the concentration required to achieve 50% inhibition was determined, with the state of no IL-2 production as 0%. Figure 5A shows the correlation between the activity and binding sites of anti-human PD-1 antibodies. (A) Antibodies exhibiting agonist activity show binding characteristics to regions #6 and #8, #6 and #7, or region #7, which is completely different from antibodies exhibiting blocking activity. Regardless of the difference in binding affinity to region #1, all antibodies in groups #6 and #7 showed agonist activity. (B) is a model representing the extracellular region of the human PD-1 molecule. In this model, the human PD-1 molecule is connected to the cell membrane at its upper end. Regions #6, #7, and #8, which are binding sites for anti-human PD-1 agonist antibodies, are located near the cell membrane of human PD-1. On the other hand, the sites associated with the binding of anti-PD-1 blocking antibodies are located far from the cell membrane. It is presumed that the amino acid residue Arg143 is located in the region sandwiched between regions #6 and #7. Figure 5 shows the correlation between the activity and binding sites of CD-line anti-human PD-1 antibodies. (C) is an example of the analysis of binding characteristics. The binding ability of anti-human PD-1 antibodies was evaluated using flow cytometry analysis with fluorescently labeled anti-IgG antibodies to assess binding to forced-expressed PD-1 molecules (wild-type, point variants, and replacements) in cells. The vertical axis represents the fluorescence intensity of GFP (Green fluorescent protein) co-expressed with PD-1, which is proportional to the amount of PD-1 expressed. The horizontal axis represents the amount of bound anti-human PD-1 antibody. From top to bottom, the results are shown using cells expressing wild-type human PD-1, the R143A point variant of human PD-1, and the mouse PD-1-human #7 region replacement (the mouse PD-1 replacement obtained by replacing the #7 region of mouse PD-1 with the amino acid sequence of the #7 region of human PD-1: mouse PD-1 (hu38-48)). HM268, one of the novel anti-human PD-1 antibodies obtained, binds to wild-type human PD-1 and the R143A point variant to approximately the same extent. In contrast, 949, one of the known antibodies, shows a significant decrease in binding to the R143A point variant. (D) Antibodies against human PD-1 are classified based on the different binding regions of human PD-1 and antibodies. Novel anti-human PD-1 antibody groups were obtained that have different binding regions from known antibody groups containing PD-1 blocking antibodies such as nivolumab. These antibody groups are those that do not bind if the #7 region is replaced, or those that do not bind if either the #7 or #6 region is replaced. These are respectively distinguished as antibody groups where the #7 region is important for binding (group #7 / #7 only), and antibody groups where both the #6 and #7 regions are important for binding (groups #6 and #7 / #6,7). The antibody group #7 also includes known antibodies such as 949 and J116. However, the antibody system of this invention maintains its binding ability to point variants of human PD-1 Arg143 such as R143A and R143V, or mouse PD-1-human #7 region replacements (mouse PD-1 (hu38-48)). In contrast, the binding ability of known antibodies to these point variants and replacements is significantly reduced. Furthermore, among the antibodies #6 and #7, some do not bind when the #1 region is replaced (HM292, HM297, HM330, HM624), some show decreased binding when the #1 region is replaced (HM698, HM634, HM643), and some maintain binding even when the #1 region is replaced (HM647, HM654). However, these antibody groups are characterized by their common binding to the #6 and #7 regions, thus distinguishing them from known antibodies. This result indicates that the novel anti-human PD-1 antibody of the present invention has a binding mode different from that of known antibodies. Figure 6 illustrates the induction of allergic asthma in mice and the anti-inflammatory effect of anti-human PD-1 agonist antibodies. (A) shows the induction method of allergic asthma and the timeline of antibody administration. Human PD-1 knock-in mice were sensitized with house dust mite antigen (HDM). One week later, the same antigen was administered intranasally to induce allergic asthma. After seven days of intranasal administration of HDM, bronchoalveolar lavage fluid was collected to investigate the infiltration of inflammatory cells into the alveoli. (BD) With the induction of allergic asthma, the total number of infiltrating inflammatory cells (B) increased, with eosinophils (C) and CD4+ T cells (D) accounting for the majority. However, prophylactic administration of anti-human PD-1 agonist antibodies to this inflammatory model inhibited the infiltration of eosinophils and CD4+ T cells into the alveoli. Furthermore, in order to observe the therapeutic effect, when HM266 was administered only once 3 days after the induction of inflammation in the lung tissue (HM266 (x1)), the infiltration of inflammatory cells into the alveoli was also inhibited. Figure 7 illustrates the anti-inflammatory effect of anti-human PD-1 agonist antibody on allergic asthma and its inhibitory effect on Th2 immune responses. Allergic asthma was induced according to the same timeline as in Figure 6, followed by the recovery of bronchoalveolar lavage fluid and lung tissue. Intracellular staining was used to study the cytokine production of CD4+ T cells that had infiltrated into the alveoli (A) or lung tissue (B). The results showed a decrease in IL-4, IL-5, IL-10, and IL-13 producing cells in the group that received four doses of HM266 (HM266 (x4)). Furthermore, HM266 administration significantly reduced the blood concentration of house dust mite antigen-specific IgE (Immunoglobulin E) (C). The same trend was observed when HM266 was administered only once 3 days after the initiation of lung inflammation (HM266 (x1)), suggesting the usefulness of anti-human PD-1 agonist antibodies in the treatment of allergic diseases. Figure 8 illustrates the induction of experimental encephalomyelitis (EAE) in mice and the anti-inflammatory effect of anti-human PD-1 agonist antibodies. Immunization was derived from myelin lipoprotein (MOG) peptide 35-55 (MOG 35-55 peptide). Spleens and lymph nodes were collected from wild-type mice 13 days after birth to obtain CD4+ T cell fractions. These CD4+ T cells were cultured in the presence of MOG 35-55 peptide and IL-23, while simultaneously overexpressing human PD-1 using a retroviral vector. After 5 days of culture, the cells were recovered and transferred into new wild-type mice to induce EAE. Symptoms appeared approximately one week after cell transfer, with an increased EAE score. However, prophylactic administration of HM266 to this model significantly improved symptoms. Based on these results, it is suggested that anti-human PD-1 agonist antibodies may be useful in the treatment of autoimmune diseases. Figure 9 illustrates the induction of colitis in mice and the anti-inflammatory effect of anti-human PD-1 agonist antibodies. T-cell-dependent colitis was induced in RAG2 knockout mice by transferring CD25-CD4+ T cells derived from human PD-1 knockout mice. (A) Significant weight loss was observed starting 4 weeks after cell transfer, but this weight loss was significantly mitigated by HM266 administration. (BD) In ​​the analysis of colon samples recovered at week 8 post-transfer, compared to the control group, there was a significant reduction in colon length and a marked decrease in the IFN-γ or IL-17 positivity rate of CD4+ T cells infiltrating the lamina propria. Based on these findings, it is suggested that anti-human PD-1 agonist antibodies may be applicable to the treatment of inflammatory bowel disease. Figure 10. Induction of acute graft-versus-host disease (GVHD) and the anti-inflammatory effect of anti-human PD-1 agonist antibody in AB mice. Acute GVHD was induced by transplanting spleen cells from C57BL / 6 human PD-1 knock-in mice (donors) into BDF1 mice (recipients). Induction of GVHD resulted in weight loss and an increase in donor cells in peripheral blood or PD-1 induction on donor T cells. Prophylactic administration of HM266, HM242, HM297, and HM268 to this inflammatory model significantly inhibited weight loss or the increase in donor cells with any of these administrations. Figure 10. Induction of acute GVHD in CDEF mice and the anti-inflammatory effect of anti-human PD-1 agonist antibodies. Acute GVHD was induced by transferring spleen cells from C57BL / 6 human PD-1 knock-in mice (donors) into BDF1 mice (recipients). Prophylactic administration of HM266, HM242, HM297, and HM268 to this inflammatory model, two weeks after induction, significantly suppressed the donor cell percentage (CD) and donor-derived CD8+ cell percentage (EF) in the spleen or liver. Based on these findings, it is suggested that anti-human PD-1 agonist antibodies may be applied in the treatment of inflammatory diseases. Figure 11. Induction of chronic GVHD / lupus-like symptoms and anti-inflammatory effect of anti-human PD-1 agonist antibody in ABCD mice. Chronic GVHD / lupus-like symptoms were induced by removing CD8+ cells from spleen cells derived from C57BL / 6 human PD-1 knock-in mice (donors) and then transferring them into BDF1 mice (recipients). After cell transfer, the number of donor-derived Tfh (follicular helper) cells (A) or recipient-derived germinal center B cells (B), type-converted B cells (C), and plasma cells (D) significantly increased in the recipient spleen. Prophylactic administration of HM266 to this inflammatory model suppressed the increases in all these parameters. "-" indicates recipient mice without donor cell transfer. Figure 11E shows the induction of chronic GVHD / lupus-like symptoms and the anti-inflammatory effect of anti-human PD-1 agonist antibodies in mice. Chronic GVHD / lupus-like symptoms were induced in BDF1 mice by removing CD8+ cells from spleen cells derived from C57BL / 6 human PD-1 knock-in mice (donors) after cell transfer. Following cell transfer, serum anti-dsDNA (double-stranded DNA) antibodies significantly increased. Prophylactic administration of HM266 to this inflammatory model inhibited the increase in serum anti-dsDNA antibodies. Based on these findings, it is suggested that anti-human PD-1 agonist antibodies could be applied to the treatment of inflammatory diseases exhibiting autoantibody production. Figure 12 shows the chimerism and bioactivity-property curve assays of the human Fc region of anti-human PD-1 agonist antibodies. From the obtained anti-human PD-1 agonist antibodies, those with higher activity were selected to prepare chimeric antibodies (human Fc chimeric antibodies) containing the Fab region derived from mice and the Fc region of human IgG1-K322A. (A) Due to the use of the human Fc region, the agonist activity was evaluated using human FcγRIIB expression cell lines. (B) Evaluation of the agonist activity and properties of the human Fc chimeric antibodies. In the column labeled "SDS-PAGE (Sodium dodecyl-sulfate polyacrylamide gel electrophoresis)," 〇 indicates antibodies with no detected abnormal bands, and × indicates antibodies with detected abnormal bands such as heavy chain dimers. Differences were observed in the selection, especially HM242, HM268, and HM297, which showed higher agonist activity and better property curves. Figure 13 shows the humanization and bioactivity-property curve assays of anti-human PD-1 agonist antibodies. The agonist activities of humanized (A) HM242, (B) HM297, and (C) HM268 are shown in Tables 1-3. Furthermore, in Tables 1-3, the homology % (=humanization rate) refers to the homology (%) when comparing the sequence of antibodies other than CDR with the sequence of antibodies derived from human germline cells. Among the antibodies whose agonist activity or stability decreased due to humanization, several antibodies (HM242-C3, HM242-D3, HM297-H5, and HM268-K8) maintained high agonist activity and showed good property curves. Figure 14 illustrates the effect of humanized anti-human PD-1 agonist antibodies in an evaluation system using primary human cultured cells. The study investigated whether humanized anti-human PD-1 agonist antibodies with humanized Fab regions and the Fc region of human IgG1-K322A exhibited T-cell suppression in an evaluation system composed solely of primary human cultured cells. PD-1 expression induction in CD4+ T cells was confirmed using a mixed lymphocyte reaction (MLR) using CD4+ T cells derived from human peripheral blood mononuclear cells (PBMCs) and CD19+ B cells from different donors (data not shown). Upon re-stimulation of these T cells with B cells, the aforementioned humanized anti-human PD-1 agonist antibody or CTLA-4-Ig as a positive control was added. Twelve hours later, the amount of IFN-γ produced in the supernatant was used as an indicator to evaluate the T cell inhibitory activity. The results showed that IFN-γ production was inhibited in the case of CTLA-4-Ig, but no significant inhibition was observed in the case of the aforementioned humanized anti-human PD-1 agonist antibody. In Figure 15, the agonist activity of anti-human PD-1 agonist antibodies depends on Fc receptors, particularly FcγRIIB. The inhibitory activity of humanized anti-human PD-1 agonist antibodies against IL-2 production was investigated using the same evaluation system as in Figures 3 and 12. The IIA1.6 cells used here represented human FcγRIA, FcγRIIA (H131), FcγRIIA (R131), FcγRIIB, FcγRIIIA (F158), and FcγRIIIA (V158) at the same high level. When human FcγRIIB, FcγRIIA(H131), FcγRIIA(R131), FcγRIIIA(F158), and FcγRIIIA(V158) cells were used as expression cells, IL-2 production was inhibited by humanized anti-human PD-1 agonist antibodies. The highest IL-2 production inhibitory activity was observed when FcγRIIB cells were used. However, when FcγRIA cells were used, IL-2 production increased upon antibody addition. These results suggest that humanized anti-human PD-1 agonist antibodies exhibit the highest agonist activity, especially in the presence of FcγRIIB. In Figure 16, the agonist activity of the anti-human PD-1 antibody depends on the expression level of the Fc receptor. In the evaluation system shown in Figure 3, (A) when using IIA1.6 cells that do not express the Fc receptor, the humanized anti-human PD-1 antibody does not show agonist activity. (B, C) On the other hand, when using IIA1.6 cells that express FcγRIIB, the humanized anti-human PD-1 antibody shows agonist activity, but this activity depends on the expression level of FcγRIIB. (D) In ​​cells with high and low expression of FcγRIIB, the expression level of FcγRIIB was measured by FACS (Fluorescence-Activated Cell Sorting), and the results showed a difference of approximately 6-fold in the geometric mean fluorescence intensity (GMFI). Figure 17A shows the increased affinity for human FcγRIIB through Fc region modification. (A) represents the affinity (KD value) of the Fc region modifiers for human FcγRIIB. Compared with IgG1-K322A, the affinity of human IgG1 Fc region modifiers such as X2-K, X3-KS, and X4-K for human FcγRIIB is increased by 2.18 to 9.83 times. Figure 17B shows the enhanced affinity for human FcγRIIB achieved through Fc region modification. (B) shows the GMFI (Gram-to-Fragment Index) of the Fc region modifiers for human FcγRIIB. The affinity of human IgG1 Fc region modifiers such as X2-K, X3-KS, and X4-K for human FcγRIIB is 2.92–84.8 times higher than that of IgG1-K322A, which has the same Fv region. DF indicates defucosomes. Figure 17C shows the increased affinity for human FcγRIIB through Fc region modification. (C) shows the correlation between the affinity (GMFI) of the Fc region modifier for human FcγRIIB and the agonist activity (IC50: ng / ml). Each point represents the antibody described in Figure 17B. When the Fv region is fixed, there is a trend that the higher the affinity of the Fc region for human FcγRIIB, the higher the agonist activity. Figure 17D illustrates the enhanced affinity for human FcγRIIB through Fc region modification. (D) The HM266 Fc region modified chimeric antibody was prepared and its agonist activity was evaluated. When using IIA1.6 cells with low expression of human FcγRIIB, IgG1-K322A or IgG4-S228P, as well as PD1AB-6 or 949, which are known anti-human PD-1 agonist antibodies, showed only weak agonist activity. However, the Fc region modifiers with enhanced affinity for human FcγRIIB showed higher agonist activity. Figure 18 illustrates the immunosuppressive effect of chimeric antibodies against the human PD-1 agonist—human Fc region—using a human cell evaluation system. The MLR system, utilizing CD4+ T cells and CD19+ B cells, evaluated chimeric antibodies containing the HM266 Fab region and various Fc region modifiers, as well as chimeric antibodies against IgG1-K322A and CTLA-4-Ig. Results showed that the chimeric antibody with enhanced affinity for human FcγRIIB via the Fc region of the Fc region modifier significantly improved T cell inhibitory activity compared to the original IgG1-K322A, exhibiting a greater anti-inflammatory effect than CTLA-4-Ig. Figure 19 illustrates the anti-inflammatory effects of humanized anti-human PD-1 agonist antibodies using a human cell evaluation system. Humanized antibodies obtained by combining selected humanized Fab regions (see Figure 13) with the Fc regions of human Fc region modifiers were evaluated using an MLR system with CD4+ T cells and CD19+ B cells. Furthermore, the antibodies shown here are all Fc region modifiers of human IgG1. The results showed that the Fc region modifier humanized antibodies with enhanced FcγRIIB affinity exhibited stronger T-cell inhibitory effects than CTLA-4-Ig, suggesting that these humanized antibodies can exert anti-inflammatory effects through the inhibition of human T-cell function. Figure 20 shows the antibody-dependent cytotoxicity (ADCC) activity of humanized anti-human PD-1 agonist antibodies. Antibodies with the Fc region X2-K showed lower ADCC activity, while antibodies with the X3-KS region had ADCC activity equal to or higher than that of IgG1-K322A. Antibodies with the X4-K region or defucosylated X3-KS (X3-KS-DF) showed higher ADCC activity. Figure 21 illustrates the inhibitory effect of humanized anti-human PD-1 agonist antibodies on antigen-specific T cell activation. Activated and proliferating CD4+ T cells stimulated with tetanus toxin were monitored by observing the decrease in CFSE fluorescence in CD4+ T cells. Data represent the percentage of CD4+ T cells whose CFSE fluorescence intensity decreased due to cell proliferation. In Figure 20, antibodies showing higher ADCC activity exhibited a higher inhibitory effect on proliferation than CTLA-4-Ig. Based on these results, it is suggested that antibodies with higher ADCC activity in addition to agonist activity may have inhibitory effects on a wider range of inflammatory diseases. Figure 22A and B show the inhibitory effect of anti-human PD-1 agonist antibodies on ERK (extracellular regulated protein kinase) phosphorylation. Similar to Figure 3, the effect was evaluated by combining DO11.10 T-cell fusion tumors expressing human PD-1 with IIA1.6 B-cell lymphoma cells expressing mouse FcγRIIB but not PD-L1. HM266 inhibited the increase in phosphorylated ERK positivity rate in DO11.10 cells over time. Based on the above, it is suggested that anti-human PD-1 agonist antibodies inhibit downstream signal transduction of the T-cell receptor. Figure 23 highlights HM266, HM242, HM297, HM268, and J116 from Figure 3B. Compared to J116, HM266, HM242, HM297, and HM268 exhibit higher agonist activity. Figure 24 shows the induction of allergic asthma in mice and the anti-inflammatory effect of antibodies against human PD-1 agonists. (A) shows the induction method of allergic asthma and the timetable of antibody administration. Allergic asthma was induced according to the same timetable as in Figure 6. Regarding (BD), in the in vitro evaluation system of PD-1 agonist activity in Figure 3, by administering J116, which showed weaker activity than HM266, the total number of inflammatory cells infiltrating the tissue (B) was inhibited, especially the infiltration of eosinophils was significantly inhibited (C), but the infiltration of CD4+ T cells was not significantly inhibited (D). Figure 25 illustrates the anti-inflammatory effects and Th2 immune response inhibition of anti-human PD-1 agonist antibodies against allergic asthma. Following the same timeline as in Figure 6, allergic asthma was induced, and bronchoalveolar lavage fluid was recovered. Intracellular staining was used to study cytokine production from CD4+ cells infiltrating the alveoli. The results showed a decrease in IL-5 and IL-13 producing cells in the J116-treated group, but no decrease in IL-4 or IL-10 producing cells obtained by HM266 treatment. These findings suggest the usefulness of anti-human PD-1 agonist antibodies in the treatment of allergic diseases, further indicating a trend of correlation between in vitro agonist activity and the effectiveness of in vivo models. Figure 26 illustrates the induction of colitis in mice and the anti-inflammatory effect of anti-human PD-1 agonist antibodies. T-cell-dependent colitis was induced by transferring CD25-CD4+ T cells derived from human PD-1 knockout mice into RAG2 knockout mice. Regarding (AC), in the analysis of colon samples recovered at week 8 post-transfer, compared to the control group, the total number of cells in the lamina propria (A), the number of CD4+ T cells infiltrating the lamina propria (B), and the prevalence of IFN-γ-positive and IL-17-positive infiltrating CD4+ T cells (C) were significantly decreased. Based on these findings, it is suggested that anti-human PD-1 agonist antibodies may be applicable to the treatment of inflammatory bowel disease. Figure 27A illustrates the induction of acute GVHD in mice and the anti-inflammatory effect of an anti-human PD-1 agonist antibody. Acute GVHD was induced by transferring spleen cells from C57BL / 6 human PD-1 knock-in mice (donors) into BDF1 mice (recipients). (A) shows the expression of human PD-1 in CD4+ or CD8+ T cells from donor cells (H-2Kb+H-2Kd-). In mice induced with GVHD, 61.7% of donor CD4+ T cells and 49.5% of donor CD8+ T cells were positive for human PD-1. Figure 27 shows the induction of acute GVHD in mice (BCD) and the anti-inflammatory effect of antibodies against human PD-1 agonists. (BD) represents the levels of AST (B), ALT (C), and IFN-γ in mouse plasma two weeks after induction. Induction of GVHD resulted in an increase in plasma ALT, AST, and IFN-γ, but this increase was significantly inhibited by prophylactic administration of HM266, HM242, HM297, and HM268. Figure 28. Induction of T cell-dependent antibody response and inhibition of antibody production against human PD-1 agonist antibodies in ABCD mice. In C57BL / 6 human PD-1 knock-in mice, intraperitoneal administration of Alum adjuvant and 4-hydroxy-3-nitrophenylacetyl ovalbumin (NP-OVA) antigen induced a T cell-dependent antibody response. Ten days after antigen administration, the ratio of Tfh cells (A) or germinal center B cells (B), type-transformed B cells (C), and plasma cells (D) in the mouse spleen was suppressed by HM266 administration. In Figure 28E, an increase in blood IL-21 concentration was also observed by antigen administration, but it was inhibited by HM266 administration (E). In Figure 28F, the antibody titers in plasma 7 days after antigen administration were analyzed, and the results showed that low-affinity IgG antibody titers (anti-NP25 IgG) and high-affinity IgG / IgM antibody titers (anti-NP2 IgG or IgM) were inhibited (F). Based on the above, it is suggested that antibody type conversion or affinity maturation can be inhibited by administering anti-human PD-1 agonist antibodies. Figure 29. Induction of chronic GVHD / lupus-like symptoms and anti-inflammatory effect of anti-human PD-1 agonist antibody in ABCD mice. Chronic GVHD / lupus-like symptoms were induced by removing CD8+ cells from spleen cells derived from C57BL / 6 human PD-1 knock-in mice (donors) and then transferring them into BDF1 mice (recipients). Following cell transfer, the number of donor-derived Tfh cells (A), germinal center B cells (B), type-converted B cells (C), and plasma cells (D) significantly increased in the recipient spleen. Prophylactic administration of HM242 or CTLA-4-Ig to this inflammatory model inhibited this increase in cell numbers. "-" indicates recipient mice without donor cell transfer. Figure 29. Induction of chronic GVHD / lupus-like symptoms and anti-inflammatory effects of anti-human PD-1 agonist antibodies in EF mice. Chronic GVHD / lupus-like symptoms were induced in BDF1 mice by removing CD8+ cells from spleen cells derived from C57BL / 6 human PD-1 knock-in mice (donors) after cell transfer. Serum IL-21 concentration (E) and anti-dsDNA antibody titer (F) significantly increased after cell transfer. Prophylactic administration of HM242 or CTLA-4-Ig to this inflammatory model inhibited the increase in serum anti-dsDNA antibodies. Based on these findings, it is suggested that anti-human PD-1 agonist antibodies can be applied to the treatment of inflammatory diseases exhibiting autoantibody production. Figure 30 illustrates the immunosuppressive effect of chimeric antibodies against the human PD-1 agonist—the human Fc region—using a human cell evaluation system. In a co-culture system of CD4+ T cells and THP-1 (a human monocyte cell line), IFN-γ production was used as an indicator to evaluate chimeric antibodies against the Fab region of HM266 and the Fc regions of various Fc region modifiers, as well as chimeric antibodies against the Fc regions of IgG1-K322A or IgG4-S228P. THP-1 cells artificially infused with and highly expressing the human FcγRIIB gene were used for evaluation. The results showed that chimeric antibodies against the Fab region of HM266 and the Fc regions of IgG1-K322A or IgG4-S228P exhibited immunosuppressive activity equal to or higher than that of known antibodies (A). On the other hand, in the evaluation system using THP-1 that expresses intrinsic FcγRIIB without gene transfection, chimeric antibodies of IgG1-K322A or IgG4-S228P and known antibodies did not show immunosuppressive activity, but Fc region modifiers with enhanced affinity for human FcγRIIB showed higher immunosuppressive activity (B). Based on the above, chimeric antibodies with enhanced affinity for human FcγRIIB through Fc region modifiers significantly increased T cell suppressive activity compared to the original IgG1-K322A. Figure 31 shows the ADCC activity of chimeric antibodies against human PD-1 agonists and human Fc regions. Antibodies with the Fc region of type X2 exhibit lower ADCC activity, while antibodies of type X3 or X4 show ADCC activity equal to or higher than that of IgG1-K322A. Fc region modifiers based on the Fc region of IgG4-S228P show increased ADCC activity compared to IgG4-S228P, but not as high as IgG1-K322A or X3 / X4 types. Figure 32 illustrates the inhibitory effect of humanized anti-human PD-1 agonist antibodies on antigen-specific T cell activation. Immunosuppressive activity was evaluated using IFN-γ produced during tetanus toxin stimulation of human PBMCs as an indicator. Figure 21 shows that antibodies with a higher CD4+ cell proliferation inhibitory effect than CTLA-4-Ig also inhibited IFN-γ production; however, antibodies with the Fc region X3-KS-DF inhibited IFN-γ production even at low concentrations. Figure 33 illustrates the inhibitory effect of humanized anti-human PD-1 agonist antibody on the activation of human Tfh cells. Human CD4+ T cells differentiated into Tfh-like cells in the presence of various cytokines were co-cultured with THP-1 cells, and IL-21 production stimulated by CytoStim was used as an indicator for evaluation. The results showed that the humanized anti-human PD-1 agonist antibody inhibited IL-21 production using Tfh-like cells. Figure 34 illustrates the immunosuppressive effect of humanized anti-human PD-1 agonist antibodies on cells derived from patients with autoimmune diseases. Personal patent mulch cells (PBMCs) from patients with rheumatoid arthritis (RA) (A, B) and systemic lupus erythematosus (SLE) (C, D) were cultured in the presence of autoantigens, and the effects were evaluated using CD4+ T cell proliferation (A, C) or IFN-γ production (B, D). The results showed that the humanized anti-human PD-1 agonist antibody exhibited inhibitory activity equal to or greater than CTLA-4-Ig against the activation of cells derived from patients stimulated with autoantigens. Figure 35 shows the increased affinity for human FcγRIIB through Fc region modification. (A) Affinity (KD value) of the Fc region modifier to human FcγRIIB. The affinity of the human IgG1 Fc region modifier to human FcγRIIB is 2.34–8.65 times higher than that of IgG1-K322A. (B) Affinity (GMFI) of the Fc region modifier to human FcγRIIB. The affinity of the human IgG1 Fc region modifier to human FcγRIIB is 26.1–180 times higher than that of IgG1-K322A. Figure 36A shows the increased affinity for human FcγRIIIA through Fc region modification. (A) shows the affinity (KD value) of the Fc region modifiers for human FcγRIIIA (V158). The affinity of human IgG1 Fc region modifiers such as X4-K, X3-KS-DF, X3-K, X3-KSI, X3-KI, and X3-KE for human FcγRIIIA (V158) is increased by 2.92 to 7.55 times compared to IgG1-K322A. Figure 36B shows the enhanced affinity for human FcγRIIIA through Fc region modification. (B) shows the affinity (GMFI) of the Fc region modifiers for human FcγRIIIA (V158). The affinity of human IgG1 Fc region modifiers such as X4-K, X3-KS-DF, X3-K, X3-KSI, X3-KI, and X3-KE for human FcγRIIIA (V158) is increased by 1.92 to 6.98 times compared with IgG1-K322A, which has the same Fv region. Figure 37 illustrates the enhancement of agonist activity through Fc region modification. Evaluation was conducted using IIA1.6 cells with low expression of human FcγRIIB. The results showed that 3.7C6 or antibody 1, known anti-human PD-1 agonist antibodies, exhibited only weaker agonist activity, while the Fc region modifiers with enhanced affinity for human FcγRIIB showed higher agonist activity. Figure 38 illustrates the anti-inflammatory effects of humanized anti-human PD-1 agonist antibodies in an evaluation system using human cells. Humanized antibodies combining the humanized Fab region of HM268-K8 with the Fc region of a human Fc region modifier were evaluated using an MLR system with CD4+ T cells and CD19+ B cells, similar to Figure 19. The results showed that the Fc region-modified humanized antibodies with enhanced FcγRIIB affinity exhibited stronger T-cell inhibitory effects than CTLA-4-Ig, suggesting that these humanized antibodies can exert anti-inflammatory effects through the inhibition of human T-cell function. Figure 39 shows the ADCC activity of humanized anti-human PD-1 agonist antibodies. Antibodies with the Fc region X3-KSL and X3-KL have lower ADCC activity, while antibodies with the Fc region X3-KSI, X3-KI, or X3-KS-DF have higher ADCC activity. Figure 40 illustrates the inhibitory effect of humanized anti-human PD-1 agonist antibodies on antigen-specific T cell activation. Similar to Figure 21, the activation and proliferation of CD4+ T cells stimulated with tetanus toxin were monitored by the reduction of CFSE fluorescence in CD4+ T cells. The data represent the percentage of CD4+ T cells whose CFSE fluorescence intensity decreased due to cell proliferation. Figure 39 shows that antibodies with higher ADCC activity exhibited particularly high inhibitory effects on proliferation. Using cells from a different donor source than those used in Figure 21, CTLA-4-Ig did not show any inhibitory effect on proliferation. Based on these results, it is suggested that antibodies with higher ADCC activity in addition to higher agonist activity have inhibitory effects on a wider range of inflammatory diseases, and can stably exert inhibitory effects even on different donors. Implementation

[0013] The embodiments of the present invention will be described in detail below.

[0014] The present invention provides an antibody or a functional fragment thereof, characterized in that: it is an antibody against human PD-1 agonist (anti-human PD-1 agonist antibody) or a functional fragment thereof, which binds to the #7 region of human PD-1 represented by sequence number 9.

[0015] In this invention, the anti-human PD-1 agonist system refers to an antibody that binds to human PD-1. It can be defined as an antibody that activates the same intracellular signaling system as human PD-L1 in human organisms. In a co-culture system of T cells expressing human PD-1 and antigen-presenting cells expressing human FcγRIIB, the activity of IL-2 produced by T cells in an antigen-stimulation-dependent manner can be used as an indicator for selection (see the examples described below).

[0016] The antibody or its functional fragment of the present invention can bind only to the #7 region of human PD-1 (the region of amino acids 38 to 48 in the amino acid sequence of sequence number 1, sequence number 9), and can also bind to the #6 region of human PD-1 (the region of amino acids 109 to 120 in the amino acid sequence of sequence number 1, sequence number 8), the #1 region (the region of amino acids 129 to 139 in the amino acid sequence of sequence number 1, sequence number 3), etc., preferably only to the #7 region of human PD-1. When the #7 region of human PD-1 (Sequence No. 1) is replaced with the corresponding region of mouse PD-1 (Sequence No. 2), the binding ability of anti-human PD-1 antibodies decreases. This can be defined as the binding of anti-human PD-1 antibodies to the #7 region of human PD-1. In this case, the binding to the #7 region is determined independently of the binding of human PD-1 to other regions. The binding of human PD-1 to regions other than the #7 region is defined similarly. The registry accession number for the amino acid sequence of human PD-1 is NCBI (National Center for Biotechnology Information) accession number: NP_005009.2, and the registry accession number for the amino acid sequence of mouse PD-1 is NCBI accession number: NP_032824.1. Furthermore, the antibody or its functional fragment of the present invention is preferably also bound to mouse PD-1 substitute (mouse PD-1 (hu38-48)) and / or human PD-1 with the 143rd arginine mutated to alanine (R143A point variant). The mouse PD-1 substitute (mouse PD-1 (hu38-48)) is a mouse PD-1 whose #7 region has been replaced with the amino acid sequence of the #7 region of human PD-1.

[0017] Regarding the binding affinity (binding affinity) of the antibody or its functional fragment of the present invention to human PD-1, the equilibrium dissociation constant (KD) should be below 10⁻⁷ M, preferably below 10⁻⁸ M. The equilibrium dissociation constant can be determined by surface plasmon resonance (SPR) method, but the binding affinity of the antibody or its functional fragment of the present invention to human PD-1 can also be determined by flow cytometry analysis based on the concentration dependence of binding to PD-1-expressing cells.

[0018] Examples of the heavy chain CDR1-3 and light chain CDR1-3 sequences of the antibodies of the present invention are shown in Table I below. CDRs can be identified, for example, by algorithms such as Kabat (J Exp Med. 1970; 132: 211-50), Chothia (J Mol Biol. 1987; 196: 901-17), IMGT (Dev Comp Immunol. 2003; 27: 55-77), and Paratome (PLoS Comput Biol. 2012; 8: e1002388), with Kabat's method being preferred. In Table I, HM242, HM266, HM268, and HM297 are mouse anti-human PD-1 antibodies exhibiting PD-1 agonist activity, prepared by immunizing mice with human PD-1. The antibody of the present invention may have a heavy chain variable region comprising heavy chain CDR1-3 and a light chain variable region comprising light chain CDR1-3, as shown in Table I below. In the antibody of the present invention, the heavy chain CDR1-3 and the light chain CDR1-3 may contain sequences in which the CDRs have at least 85%, 85-90%, 90-95%, 95-97%, or more than 97% identity with the sequences shown in Table I below. The identity of the two sequences can be determined using BLAST (Basic Local Alignment Search Tool). [Table I] Sequences of heavy chain CDR1~3 and light chain CDR1~3 of HM242, HM266, HM268 and HM297 The variable region (Fv) of the antibody of this invention may be derived from the Fv of antibodies from animals other than humans (e.g., mice, rabbits, rats, hamsters, guinea pigs, goats, sheep, donkeys, llamas, camels, chickens, ostriches, sharks, etc.), or may be obtained by humanizing the Fv regions of the heavy chain and / or light chain of antibodies from animals other than humans. Humanization can be carried out, for example, by transplanting the CDRs of the VH and VL of antibodies from animals other than humans into the VH and VL structure of human antibodies (Nature, 332, 323-327, 1988). Humanization can be achieved by maintaining the CDR sequence. This can be done by identifying amino acid residues that directly participate in binding with the antigen, amino acid residues that interact with the CDR, and amino acid residues that participate in maintaining the stereostructure of the CDR, and replacing them with amino acid residues of non-humanized antibodies, thereby enhancing antigen binding (MABS, 8(7), 1302-1318, 2016).

[0019] An example of the heavy chain variable region (VH region, positions 1-117, as per EU numbering below) sequence of a mouse-derived antibody demonstrating PD-1 agonist activity and an example of the humanized VH region sequence are shown in Table II below. [Table II] Sequences of the heavy chain variable region (VH region) of HM242, HM266, HM268 and HM297 and sequences of the VH region of humanized HM242, HM266, HM268 and HM297 An example of the light chain variable region (VL region, positions 1-107) sequence of a mouse-derived antibody exhibiting PD-1 agonist activity and an example of the humanized VL region sequence are shown in Table III below. [Table III] Sequences of the variable light chain regions (VL regions) of HM242, HM266, HM268 and HM297 and the VL regions of humanized HM242, HM266, HM268 and HM297 The antibody of the present invention may have the heavy chain variable region and / or light chain variable region shown in Tables II and / or III above. In the antibody of the present invention, the heavy chain variable region and the light chain variable region may respectively contain sequences having at least 90%, 90-95%, 95-99%, or more than 99% identity with the sequences shown in Tables II and III above.

[0020] The antibody of this invention can be one that enhances affinity for the human Fc receptor, preferably the Fcγ receptor, more preferably FcγRII, and even more preferably FcγRIIB. The affinity of the antibody of this invention for the human Fc receptor can be evaluated by flow cytometry assays of binding affinity to human Fc receptor-expressing cell lines, or by Fc receptor binding affinity assays using surface plasma resonance technology with Biacore 8K (Cytiva). The enhancement of the antibody's affinity for the human Fc receptor can be evaluated by any of these methods, but flow cytometry is preferred, and both methods are more preferred. When the affinity of the antibody of the present invention for human FcγRIIB is measured by a human FcγRIIB binding affinity assay using surface plasma resonance technology, it can be expressed as a ratio of the equilibrium dissociation constant (KD) to that of an antibody possessing the Fc region of human IgG1-K322A (reference antibody). The antibody of the present invention has an affinity of at least 1.5 times that of the reference antibody, preferably at least 2 times that of the reference antibody, and more preferably at least 2.5 times that of the reference antibody (see the examples described below). When the affinity of the antibody of the present invention for human FcγRIIB is measured by a binding affinity assay of human FcγRIIB-expressing cell lines using flow cytometry, it can be expressed as a ratio of the GMFI to that of an antibody possessing the Fc region of human IgG1-K322A (reference antibody). Under the measurement conditions where the GMFI is around 40 without the antibody and 300-1500 with the reference antibody, the antibody of the present invention shows more than 2 times the GMFI, more preferably more than 5 times the GMFI, and more preferably more than 20 times the GMFI when a reference antibody with the same Fv region is added.

[0021] The antibody of this invention can be an antibody with enhanced affinity for human FcγRIIIA. When the affinity of the antibody of this invention for human FcγRIIIA is measured using a human FcγRIIIA (V158) binding affinity assay with surface plasma resonance technology, it can be expressed as the ratio of the equilibrium dissociation constant (KD) to that of an antibody possessing the Fc region of human IgG1-K322A (reference antibody). The antibody of this invention has an affinity at least 1.5 times that of the reference antibody, preferably at least 2 times that of the reference antibody, more preferably at least 2.5 times that of the reference antibody, and even more preferably at least 4 times that of the reference antibody (see the examples described below). The affinity of the antibody of the present invention for human FcγRIIIA, when measured by a flow cytometry assay of binding to human FcγRIIIA (V158) expressing cell lines, can be expressed by comparing the GMFI with that of an antibody possessing the Fc region of human IgG1-K322A (reference antibody). Under the assay conditions where the GMFI is approximately 10 without the antibody and 6000-15000 with the reference antibody, the antibody of the present invention shows a GMFI of more than 1.5 times, preferably more than 2 times, more preferably more than 4 times, and even more preferably more than 5 times, compared to the reference antibody with the same Fv region.

[0022] The antibody of this invention can be any antibody possessing the Fc region of a human antibody (e.g., IgG1, IgG4, etc.), and more preferably, it is an Fc region modifier that enhances the affinity of the human antibody for human Fc receptors (human FcγRIIA, human FcγRIIB, human FcγRIIIA, or any one or more of them). By modifying the Fc region (positions 216 to 447) of the antibody, the affinity for human Fc receptors can be enhanced. Furthermore, the affinity for human Fc receptors can also be enhanced by defucosylation of the antibody.

[0023] An example of the amino acid sequence of the Fc region of human IgG1 is shown in sequence number 47. Similarly, an example of the amino acid sequence of the Fc region of human IgG4 is shown in sequence number 64. In the antibody of the present invention, as a method to enhance affinity for the human Fc receptor, for example, one or more variations may be introduced at positions 233, 234, 236, 237, 238, 239, 267, 268, 271, 296, 323, 326, 328, 330, and 332 of the amino acid sequence of the Fc region of human IgG1 represented by sequence number 47, preferably a combination thereof. Furthermore, these variations may also be combined with defucosylation. In cases of combinatorial variation, for example, it could be 236 / 268, 239 / 268, 239 / 268 / 328 / 332, 239 / 267 / 268, 239 / 267 / 268 / 328, 239 / 267 / 268 / 332, 239 / 268 / 328, 239 / 268 / 332, 239 / 267 / 268 / 328 / 332, 233 / 237 / 238 / 268 / 271 / 330, 267 / 3 Any of 28, preferably any of 239 / 268, 239 / 268 / 328 / 332, 239 / 267 / 268, 239 / 267 / 268 / 332, 239 / 268 / 332, 233 / 237 / 238 / 268 / 271 / 330, 267 / 328, more preferably any of 239 / 268 / 328 / 332, 233 / 237 / 238 / 268 / 271 / 330, 267 / 328. A 236-bit G can be mutated into D, E, N, Q, F, H, I, K, L, M, P, R, S, T, V, W, Y, A, etc., preferably into D, E, N, Q, more preferably into D. The 268-bit H can be mutated into D, E, N, Q, A, G, etc., preferably D, E, N, Q, and even more preferably D. The 239-bit S can be mutated into D, E, N, Q, F, T, H, Y, G, L, etc., preferably D, E, N, Q, and even more preferably D. The 328-bit L can be mutated into Y, E, F, H, I, Q, W, D, T, S, M, A, V, N, H, etc., preferably Y, F, D, E, N, Q, and even more preferably Y. The 332-bit I can be mutated into E, D, F, L, R, S, T, D, N, Q, H, Y, A, M, etc., preferably E, T, M, and even more preferably E. The 233-bit E can be mutated into D, etc. The 237-bit G can be mutated into W, F, A, D, E, L, M, Y, etc., preferably into D, E, and even more preferably into D. The 238-bit P can be mutated into D, etc. The 271-bit P can be mutated into G, etc. The 330-bit A can be mutated into K, R, M, etc., preferably into R.The 267-bit letter S can be mutated into E, V, Q, A, L, etc., preferably into E, G, and even more preferably into G. The 326-bit letter K can be mutated into L, Q, N, M, D, S, T, A, etc. The 234-bit letter L can be mutated into D, E, N, Q, T, H, I, V, F, W, Y, etc. The 323-bit letter V can be mutated into I, L, M, etc. The 296-bit letter Y can be mutated into D, etc. []

[0024] When introducing the mutation into the Fc region of human IgG1, it can be combined with other mutations, such as K322A, which is known to inhibit complement-dependent cytotoxicity (CDC) activity by reducing the binding of complement C1q, or E293A, which is known to inhibit ADCC activity by reducing the binding of FcγRIIIA, depending on the purpose.

[0025] Combinations of variants introduced into the Fc region of human IgG1 can be G236D / H268D, S239D / H268D, S239D / H268D / L328Y / I332E, G236D / H268D / K322A, S239D / H268D / K322A, S239D / S267G / H268D / K322A, G236D / H268D / E293A / K322A, and S239D. / H268D / K322A / L328Y / I332E、S239D / H268D / E293A / K322A、S239D / S267G / H268D / K322A / L328Y、S 239D / S267G / H268D / K322A / I332E, S239D / H268D / K322A / L328Y, S239D / H268D / K322A / I332E, S23 Any combination of 9D / S267G / H268D / K322A / L328Y / I332E, E233D / G237D / P238D / H268D / P271G / A330R, and S267E / L328F, preferably S239D / H268D / K322A, S239D / S267G / H268D / K322A, G236D / H268D / E293A / K322A, or S23 Any combination of 9D / H268D / K322A / L328Y / I332E, S239D / H268D / E293A / K322A, S239D / S267G / H268D / K322A / I332E, S239D / H268D / K322A / I332E, E233D / G237D / P238D / H268D / P271G / A330R, and S267E / L328F. When combining Fc region mutations with defucosylation of human antibodies, the combination of Fc region mutations can result in an affinity for human Fc receptors, especially human FcγRIIIA, that is increased by more than 1.5 times compared to antibodies with the same mutations but not defucosylated, preferably by more than 2 times, and even more preferably by more than 3 times. Examples of preferred combinations of mutations and defucosylation include S239D / S267G / H268D / K322A with defucosylation, but this is not a limitation.

[0026] As a method to enhance affinity for the human Fc receptor, for example, the mutation can be introduced into any one or more of the amino acid sequences at positions 236, 239, 268, 328, and 332 of the Fc region of human IgG4 represented by sequence number 64, preferably a combination thereof. Furthermore, these mutations can be combined with defucosylation.

[0027] When the mutation is introduced into the Fc region of human IgG4, it can be combined with other mutations such as S228P, which are known to further enhance antibody stability.

[0028] When combining variations, the combination can be any one of the following combinations: S228P / G236D / Q268D, S228P / S239D / Q268D, or S228P / S239D / Q268D / L328Y / I332E, and preferably any one of the following combinations: S228P / S239D / Q268D or S228P / S239D / Q268D / L328Y / I332E.

[0029] Table IV shows an example of the Fc region sequence of human IgG1 (WT, K322A) and IgG4 (WT, S228P) and the Fc region sequence of the Fc region variant introduced with the mutation. [Table IV] Fc regions of human IgG1 (WT, K322A) and IgG4 (WT, S228P) and Fc regions of Fc region modifiers

[0030] The antibody of this invention can be a monomer with a higher ratio of four chains (two light chains and two heavy chains). The monomer ratio can be determined by SEC (Size Exclusion Chromatography)-HPLC (High-performance liquid chromatography) (see the examples described below). The monomer ratio is preferably 80% or higher, more preferably 90% or higher.

[0031] The antibodies of this invention can be further modified in other ways than those described above, such as amino acid mutation introduction, subtype replacement, glycan modification, etc.

[0032] The ADCC activity of the antibodies of the present invention can be IgG1 or IgG1-K322A or higher when using the same Fv region. Compared with antibodies with lower ADCC activity, antibodies with higher ADCC activity can show a greater T cell inhibitory effect upon antigen stimulation. Regarding ADCC activity, for example, it can be investigated by using genetically modified Jurkat T cells expressing the Fc receptor instead of effector cells, and measuring the amount of luciferase produced by activating the luciferase gene downstream of the NFAT (nuclear factor of activated T cells) response sequence through signaling from the Fc receptor using an illuminometer (see the examples described below).

[0033] The antibody of the present invention preferably comprises a humanized Fab region of the variable region of each of the heavy and light chains of an antibody derived from a non-human animal (e.g., mouse) (exhibiting PD-1 agonist activity), and an Fc region of a modified Fc region of human IgG1 or IgG4. An example of the amino acid sequences of the heavy and light chains of the preferred antibody of the present invention and the nucleic acid sequences encoding them is shown in Table V below. The antibody of the present invention may have the heavy chain sequence and / or the amino acid sequence of the light chain shown in Table V below. In the antibody of the present invention, the heavy and light chains may contain sequences having at least 90%, 90-95%, 95-99%, or more than 99% identity with the amino acid sequences shown in Table V below. The nucleic acid encoding the antibody of the present invention may also be a nucleic acid encoding the amino acid sequences of these heavy and light chains, and may be modified from the nucleic acid sequences shown in Table V below according to the target amino acid sequence. It is common knowledge in the industry that nucleic acid sequences are designed to correspond to target amino acid sequences. Ideally, the nucleic acid sequences (codons) encoding each amino acid should be optimized based on the target amino acid sequence.

[0034] Antibodies of the present invention include, for example, HM242-C3-X3-K, HM242-C3-X3-KE, HM242-C3-X3-KI, HM242-C3-X3-KSI, HM242-C3-X4-K, HM242-D3-X3-K, HM242-D3-X3-KE, HM242-D3-X3-KI, HM242-D3-X3-KS, HM242-D3-X3-KSI, HM242-D3-X4-K, HM268-K8-X3-K, HM268-K8-X3-KE, HM268-K8-X3-KI, HM268-K8- X3-KS, HM268-K8-X3-KSI and HM268-K8-X4-K, preferably HM242-C3-X3-KI, HM242-C3-X3-KSI, HM242-D3-X3-KI, HM242-D3-X3-KS, HM242-D3-X3-K SI, HM268-K8-X3-KI, HM268-K8-X3-KS and HM268-K8-X3-KSI, the best ones are HM242-D3-X3-KS, HM268-K8-X3-KI, HM268-K8-X3-KS and HM268-K8-X3-KSI.

[0035] [Table V](Table V-01)

[0036] [Table V] (Table V continued) (Table V-02)

[0037] [Table V] (Table V continued) (Table V-03)

[0038] [Table V] (Table V continued) (Table V-04)

[0039] [Table V] (Table V continued) (Table V-05)

[0040] [Table V] (Table V continued) (Table V-06)

[0041] [Table V] (Table V continued) (Table V-07)

[0042] [Table V] (Table V continued) (Table V-08)

[0043] [Table V] (Table V continued) (Table V-09)

[0044] [Table V] (Table V continued) (Table V-10)

[0045] [Table V] (Table V continued) (Table V-11)

[0046] [Table V] (Table V continued) (Table V-12)

[0047] [Table V] (Table V continued) (Table V-13)

[0048] [Table V] (Table V continued) (Table V-14)

[0049] [Table V] (Table V continued) (Table V-15)

[0050] [Table V] (Table V continued) (Table V-16)

[0051] [Table V] (Table V continued) (Table V-17)

[0052] [Table V] (Table V continued) (Table V-18)

[0053] [Table V] (Table V continued) (Table V-19)

[0054] [Table V] (Table V continued) (Table V-20)

[0055] [Table V] (Table V continued) (Table V-21)

[0056] [Table V] (Table V continued) (Table V-22)

[0057] [Table V] (Table V continued) (Table V-23)

[0058] [Table V] (Table V continued) (Table V-24)

[0059] [Table V] (Table V continued) (Table V-25)

[0060] [Table V] (Table V continued) (Table V-26)

[0061] [Table V] (Table V continued) (Table V-27)

[0062] [Table V] (Table V continued) (Table V-28)

[0063] [Table V] (Table V continued) (Table V-29)

[0064] [Table V] (Table V continued) (Table V-30)

[0065] [Table V] (Table V continued) (Table V-31)

[0066] [Table V] (Table V continued) (Table V-32)

[0067] [Table V] (Table V continued) (Table V-33)

[0068] [Table V] (Table V continued) (Table V-34)

[0069] [Table V] (Table V continued) (Table V-35)

[0070] In this invention, a functional fragment refers to, for example, a molecule having one or more scFv, Fv, F(ab')2, Fab', or Fab of the antibody of this invention, preferably one that also binds to the Fc receptor. Examples include antibody-drug complexes of the antibody and drug of this invention, polypeptides having scFv of the antibody of this invention and scFv of an anti-Fc receptor antibody, and fusion proteins having scFv of the antibody of this invention and an Fc region, but are not limited to these. When the functional fragment is a fusion protein having an Fc region, such as an scFv of the antibody of this invention and an Fc region, the Fc region is preferably the Fc region of the Fc region modifier described in this specification. The protein improvement and optimization techniques described above can also be applied to the functional fragment.

[0071] The antibodies and their functional fragments of this invention can be fabricated into recombinant antibodies or their functional fragments using genetic engineering techniques. Specifically, the genes encoding the heavy and light chains of the antibodies of this invention, or the DNA (deoxyribonucleic acid) encoding the functional fragments of the antibodies of this invention, are synthesized, inserted into an expression vector (e.g., plasmids, bacteriophages, viruses), and then introduced into host cells (e.g., CHO (Chinese hamster ovary) cells, HEK (Human Embryonic Kidney) cells, etc.). The host cells are then cultured, allowing for the collection of recombinant antibodies or their functional fragments from the culture. When synthesizing the genes encoding the heavy and light chains of the antibodies or the DNA encoding the functional fragments, codon optimization is preferred. The heavy chain gene and light chain gene of the antibody can be inserted into the same expression vector or different expression vectors. The expression vector may, as needed, contain promoters, enhancers, polyadenylation signals, replication origins, selection marker genes, secretion signal sequences, etc.

[0072] The ratio of heavy chain genes to light chain genes in the antibody inserted into the expression vector can be 1:1, but this can be adjusted appropriately to enhance antibody expression levels. To introduce the recombinant expression vector into host cells, known methods such as the calcium phosphate method, the DEAE (diethylaminoethyl)-dextran method, microinjection, lipid transfection, electroporation, transduction, scratch loading, and shotgun assay can be used. If the host cells containing the recombinant expression vector are cultured, recombinant antibodies or their functional fragments will be produced in the culture. Appropriate culture conditions (culture medium, culture time, culture temperature, CO2 concentration, etc.) can be set by the manufacturer. The produced antibodies or their functional fragments can be recovered using known protein separation and purification methods that consider isoelectric point, size, solubility (water, organic solvents, etc.), and affinity for a particular substance (acceptor, coenzyme, etc. in the case of enzymes). To obtain defucosomes of recombinant antibodies or their functional fragments, 2-deoxy-2-fluoro-L-fucose can be added to the culture medium for culturing, or fucosyltransferase 8 (FUT8) deficient cells can be used as host cells, but these methods are not limited to these. The isolation and purification of defucosomes can be carried out by the same methods described above.

[0073] The antibody or its functional fragment of the present invention can exert a stronger T cell inhibitory effect than CTLA-4-Ig in the mixed lymphocyte response (MLR) of human T cells and B cells, and exhibits an anti-inflammatory effect through the inhibition of human T cell function (see the examples described below).

[0074] The antibodies or functional fragments thereof of this invention can be used to treat and / or prevent inflammatory diseases. In this specification, inflammatory diseases refer to diseases caused by excessive inflammation resulting from trauma, chemicals, infection, or the recognition of autologous tissue by immune cells. This includes autoimmune diseases or diseases classified as type I-IV allergies, such as Bessie's disease, systemic lupus erythematosus, lupus nephritis, cutaneous lupus erythematosus, chronic discoid lupus erythematosus, serum sickness, systemic sclerosis (systemic scleroderma, progressive systemic sclerosis), multiple sclerosis, scleroderma, polymyositis, dermatomyositis, periarteritis nodosa (polyarteritis nodosa), arteritis of the large arteries (Gauden's arteritis), malignant rheumatoid arthritis, rheumatoid arthritis, arthritis, juvenile idiopathic arthritis, spondyloarthritis, mixed connective tissue disease, Shoegrange syndrome, adult-onset Still's disease, vasculitis, allergic granulomatous vasculitis, allergic vasculitis, rheumatoid vasculitis, large vessel vasculitis, and ANCA (antineutrophil cytoplasmic) Antibody-associated vasculitis (e.g., microscopic polyangiitis, polyangiitis granulomatosis, and eosinophilic polyangiitis granulomatosis), Cogan's disease, RS3PE (remitting seronegative symmetrical synovitis with pitting edema),The following are considered all types of inflammatory conditions: remission-negative symmetrical synovitis with pitting edema, temporal arteritis, polymyalgia rheumatica, fibromyalgia, antiphospholipid antibody syndrome, eosinophilic bulbar fasciitis, IgG4-related diseases (e.g., primary sclerosing cholangitis, autoimmune pancreatitis), Guillain-Barré syndrome, myasthenia gravis, chronic atrophic gastritis, hepatitis, autoimmune hepatitis, non-alcoholic steatohepatitis, primary biliary cirrhosis, Goupard syndrome, glomerulonephritis, rapidly progressive glomerulonephritis, megaloblastic anemia, hemolytic anemia, autoimmune hemolytic anemia, positive antiglobulin test autoimmune hemolytic anemia, pernicious anemia, autoimmune neutropenia, and idiopathic thrombocytopenic purpura. Bartholomew's disease (Grayish disease (hyperthyroidism)), Hashimoto's disease, autoimmune adrenal insufficiency, primary hypothyroidism, Addison's disease (chronic adrenocortical insufficiency), idiopathic Addison's disease, type 1 diabetes mellitus, slow-progressing type 1 diabetes mellitus (adult latent autoimmune diabetes mellitus), focal scleroderma, psoriasis, psoriatic arthritis, bullous pemphigoid, pemphigus, pemphigoid, herpes gestationis, linear IgA bullous dermatosis, acquired epidermal bullous dermatosis, alopecia areata, leukoplakia, leukoplakia vulgaris, Stevens-Johnson syndrome, fixed drug eruption, neuromyelitis optica, chronic inflammatory demyelinating polyneuritis, multifocal motor neuropathy, sarcoidosis, giant cell arteriosclerosis. Inflammation, Amyotrophic Lateral Sclerosis (ALS), Harada disease, Autoimmune optic neuropathy, Idiopathic azoospermia, Spontaneous abortion, Habitual abortion, Infertility, Infertility due to maternal-fetal intolerance, Inflammatory bowel disease (e.g., ulcerative colitis, Crohn's disease), Celiac disease, Ankylosing spondylitis, Asthma, Severe asthma, Infectious diseases (viral, bacterial, fungal, parasitic), Chronic obstructive pulmonary disease, Urticaria, Chronic urticaria, Immunotransplantation, Familial Mediterranean fever, Eosinophilic globus sinusitis, Eosinophilic gastrointestinal disorders, Dilated cardiomyopathy, Systemic mast cell disease, Inclusion body myositis, Pelvic inflammatory disease, Alzheimer's disease, Peroni disease, Gallbladder disease, Pilopuntia disease, Peritonitis, Postoperative adhesions, Stroke, Lyme disease, Brain Meningoencephalitis, autoimmune uveitis, non-infectious uveitis, allergies, chronic allergic diseases (atopic dermatitis, fever, allergic rhinitis), food allergies, conjunctivitis, anaphylactic shock, contact dermatitis, fibrotic alveolitis, allergic pneumonia, allergic bronchopulmonary aspergillosis, allergic encephalitis, IgA nephropathy, Meniere's disease, cholangitis, pancreatitis, surgical trauma, acute and chronic graft-versus-host disease, transplant rejection, heart disease (myocardial infarction, ischemic diseases such as atherosclerosis), intravascular coagulation syndrome, osteoporosis, osteoarthritis, periodontitis, hypoxia, herpes gestationis, acquired hypogonadism, hypoparathyroidism, cytokine storm, but not limited to these.

[0075] The antibodies or functional fragments thereof of this invention can be used to treat and / or prevent inflammatory diseases, especially those related to T cells or autoantibodies, such as systemic lupus erythematosus, lupus nephritis, multiple sclerosis, systemic sclerosis (systemic scleroderma, progressive systemic sclerosis), polymyositis, dermatomyositis, rheumatoid arthritis, Shoegrange syndrome, allergic granulomatous vasculitis, large vessel vasculitis, ANCA-associated vasculitis (e.g., microscopic polyangiitis, polyangiitis granulomatosis, and eosinophilic polyangiitis). Granulomatous leukoma, antiphospholipid antibody syndrome, IgG4-related diseases (e.g., primary sclerosing cholangitis, autoimmune pancreatitis, etc.), idiopathic thrombocytopenic purpura, Basell's disease (Gray's disease (hyperthyroidism)), Hashimoto's disease, psoriasis, psoriatic arthritis, bullous pemphigoid, pemphigus, bullous pemphigoid, alopecia areata, leukoplakia, common leukoplakia, Stevens-Johnson syndrome, fixed drug eruption, neuromyelitis optica, inflammatory bowel disease (e.g., ulcerative colitis, Crohn's disease), asthma, acute and chronic graft-versus-host disease.

[0076] This invention provides a pharmaceutical composition comprising an antibody or a functional fragment thereof, and a pharmaceutically permissible carrier. The antibody or functional fragment is characterized in that it is a PD-1 agonist antibody (anti-human PD-1 agonist antibody) or a functional fragment thereof, which binds to the #7 region of human PD-1 as indicated by sequence number 9. The pharmaceutical composition of this invention can be used as a medicine.

[0077] The pharmaceutical composition of the present invention can be administered orally or non-orally to the whole body or part of the subject (human or non-human animal).

[0078] The pharmaceutical composition of the present invention comprises an effective amount of anti-human PD-1 agonist antibody or its functional fragment, and can be formulated together with a pharmaceutically permissible carrier by mixing, dissolving, emulsifying, encapsulating, freeze-drying, etc.

[0079] The pharmaceutical compositions of the present invention can be administered by any of the oral or non-oral methods. However, formulations suitable for oral administration include liquids obtained by dissolving an effective amount of anti-human PD-1 agonist antibody or its functional fragment in a diluent such as water or saline; capsules, granules, powders, or tablets containing an effective amount of anti-human PD-1 agonist antibody or its functional fragment in solid or particulate form; suspensions obtained by suspending an effective amount of anti-human PD-1 agonist antibody or its functional fragment in a suitable dispersion medium; and emulsions obtained by dispersing a solution containing an effective amount of anti-human PD-1 agonist antibody or its functional fragment in a suitable dispersion medium and then emulsifying it.

[0080] For non-oral administration, anti-human PD-1 agonist antibodies or their functional fragments can be formulated with pharmaceutically permissible solvents, excipients, binders, stabilizers, dispersants, etc., into dosage forms such as injectable solutions (including freeze-dried formulations), suspensions, emulsions, creams, ointments, inhalers, and suppositories. In injectable formulations, the anti-human PD-1 agonist antibody or its functional fragments can be dissolved in an aqueous solution, preferably Hanks' solution, Ringer's solution, or physiologically compatible buffer solution. Furthermore, the pharmaceuticals of the present invention can be in the form of suspensions, solutions, or emulsions in oily or aqueous media. Alternatively, the anti-human PD-1 agonist antibody or its functional fragments can be manufactured in powder form and prepared into an aqueous solution or suspension using sterilizing water or the like before use. For inhalation administration, anti-human PD-1 agonist antibodies or their functional fragments can be powdered and mixed with suitable bases such as lactose or starch to form a powder mixture. Suppository formulations can be manufactured by mixing anti-human PD-1 agonist antibodies or their functional fragments with conventional suppository bases such as cocoa butter. Furthermore, pharmaceutical compositions can be encapsulated in polymer matrices or the like to formulate sustained-release formulations. Non-oral administration methods include intravenous, intramuscular, subcutaneous, rectal, nasal, oral, and transdermal administration.

[0081] Regarding dosage, for human adults, a single dose of approximately 0.1 to 100 mg / kg (body weight) can be administered, or multiple doses can be administered at intervals of approximately 1 day to 6 months.

[0082] The pharmaceutical composition of this invention can be used alone or in combination with other therapeutic agents such as antihistamines, anti-allergy drugs, vasoconstrictive nasal drops, steroids, low molecular weight immunosuppressants (cyclosporine, tacrolimus, etc.), antibody drugs (anti-IgE antibodies, anti-IL-4 antibodies, anti-IL-5 antibodies, anti-IL-6 antibodies, anti-IL-13 antibodies, anti-IL-4 receptor antibodies, anti-IL-5 receptor antibodies, anti-IL-22 antibodies, anti-IL-25 antibodies, anti-IL-33 antibodies, anti-TNFα antibodies, anti-TSLP antibodies, anti-BAFF antibodies, etc.), and recombinant soluble fusion proteins (CTLA-4-Ig, etc.). A synergistic effect of pharmacodynamics can be expected. [Example]

[0083] The present invention will now be described in more detail through examples. method

[0084] [Commercially available antibodies, commercially available fusion proteins, and known antibodies] [] Commercially available antibodies and recombinant proteins used include anti-human PD-1 antibody (pure line name: EH12.2H7, Biolegend), anti-human PD-1 antibody (pure line name: J116, Invitrogen), anti-human PD-1 antibody (pure line name: MIH4, Invitrogen), control mouse IgG1 (pure line name: MOPC-21, Biolegend), control human IgG1 (pure line name: QA16A12, Biolegend), control human IgG4 (pure line name: QA16A15, Biolegend), and recombinant human CTLA-4-Fc region chimeric protein (CTLA-4-Ig, Cat#: 591908, Biolegend). Regarding known antibodies, 949 (anti-human PD-1 antibody described in WO2011 / 110621, with the Fc region sequence being human IgG4-S228P), PD1AB-6 (PD1AB-6-IgG1-K322A described in WO2017 / 058859), PD1-17 (anti-human PD-1 antibody described in WO2004 / 056875, with the Fc region sequence being IgG1), 3.7C6 (anti-human PD-1 antibody described in WO2020 / 247648, with the Fc region sequence being IgG1), and antibody 1 (anti-human PD-1 antibody described in WO2019 / 168745, with the Fc region sequence being IgG1) were prepared by the following method.

[0085] [Obtaining and Purifying Novel Antibodies] [] Techniques for generating mouse monoclonal antibodies against human PD-1 are known in the art, for example, using the methods described in Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986). A / J and BALB / c mice are used as the immunizing hosts. Immunogens include a plasmonic vector (15-50 μg) expressing the full-length human PD-1 protein (NCBI registry number: NP_005009.2) or a variant thereof, a fusion protein of the recombinant human PD-1 extracellular domain and the human IgG1-Fc region (25 μg), and 293T cells (5 × 10⁷ cells) temporarily expressing human PD-1 or a variant thereof. Any of these are administered intramuscularly, intradermally, intraperitoneally, or intravenously at intervals of 10-50 days. When adjuvants are used, the Sigma adjuvant system (S6322-1VL; Sigma-Aldrich) is used. Three days after final immunization, fusion of spleen cells from the immunized host with P3U1 mouse myeloma cells was performed to prepare fusion tumors. Screening for fusion tumors producing anti-human PD-1 antibodies was conducted as follows: Culture supernatant from each fusion tumor was added to HEK293 cells forcibly expressing human PD-1. After staining with R-phycoerythrin-labeled goat anti-mouse IgG (H+L) F(ab')2 fragment (115-116-146; Jackson ImmunoResearch) as a secondary antibody, the staining was analyzed using flow cytometry. The final selected fusion tumors were colonized using a limiting dilution method, cultured at high density in a Wheaton cell line bioreactor, and then the anti-human PD-1 antibody was purified from the culture supernatant using Ab-Capture ExTra (P-003-10; Protenova).

[0086] [Antibody Sequence Sequencing] [] The specific sequence of the variable region of the anti-human PD-1 antibody was sent to Bizcom Japan Co., Ltd. for analysis using the commissioned analysis service of Fusion Antibodies.

[0087] [Anti-human] [PD-1] [Humanization of Antibodies] [] Humanization is achieved by transplanting the complementarity-determining region (CDR) of mouse antibodies and the residues required to maintain the CDR conformation of mouse antibodies into the human antibody architecture.

[0088] [Characteristics and purification of recombinant antibodies] [] Heavy chain expression vectors were constructed using DNA encoding the heavy chain sequence and an expression vector (pcDNA3.4, Thermo Fisher Scientific). Similarly, light chain expression vectors were constructed using DNA encoding the light chain sequence and an expression vector (pcDNA3.4, Thermo Fisher Scientific). The two expression vectors were mixed in a 1:1 ratio of heavy to light chains, with 0.8 μg of DNA per 1 ml of culture medium, and introduced into CHO cells using ExpiFectamine CHO reagent (Thermo Fisher Scientific). On the second day after transfection, ExpiCHO Feed and ExpiFectamine CHO Enhancer were added, and the cells were cultured at 32°C for 10–12 days. Cells were removed from the culture medium by centrifugation and filtration, and the culture supernatant was recovered. The antibody levels in the culture supernatant were measured using Cedex Bio (Roche Diagnostic). The culture supernatant was applied to a protein A column pre-equilibrated with phosphate-buffered saline (PBS), followed by washing the column with PBS. Antibody lysis was performed using citrate buffer (pH 3.4), with 1 M Tris-HCl (Tris(hydroxymethyl)aminomethane hydrochloride) (pH 9.0) added to 1 ml of lysis buffer at a ratio of 160 μl for neutralization. After concentration by ultrafiltration, purification was achieved using gel filtration chromatography with a Superdex 200 Increase 10 / 300 GL (Cytiva). The defucosylated system was obtained by adding 2-deoxy-2-fluoro-L-fucose (2-DFF) to the culture medium to achieve a final concentration of 10–1000 μM. Equivalent FcγRIIIA affinity was observed within the final concentration range of 10–1000 μM for 2-DFF. Fucosome de-purification was performed using the same method described above.

[0089] [use] [SDS-PAGE] [Analysis of the process] [] The purified antibody was suspended in sample buffer (with and without reducing agent), heated at 98°C for 3 minutes, and then applied to a 4-15% MiniProtein TGX polyacrylamide gel (Bio-Rad) for electrophoresis at a constant voltage of 200 V for 30 minutes. Staining was performed using SYPRO Ruby protein gel staining solution (Thermo Fisher Scientific), and analysis was performed using a LAS500 (Cytiva) analyzer.

[0090] [use] [SEC-HPLC] [Analysis of the process] [] The monomer ratio in the sample was determined by SEC-HPLC. The column used was a TSKgel G3000SWXL, 5 μm, 7.8 mm × 300 mm (TOSOH). The mobile phase consisted of a 1000 ml solution prepared by dissolving 84.4 g of potassium dihydrogen phosphate and 66.2 g of dipotassium hydrogen phosphate in water, which was then diluted 10-fold. The monomer ratio was determined by measuring the peak areas of each component in the sample solution using an automated integration method, and the ratios were calculated using the area percentage method.

[0091] [Evaluation of Thermal Stability Using a Differential Scanning Calorimeter] [] Regarding the thermal stability of anti-human PD-1 antibodies, the modification midpoint temperature (Tm) and modification onset temperature (Tonset), used as predictive indicators, were determined. For 400 μl of anti-human PD-1 antibody sample diluted to 0.1 mg / ml, the heat change at a rate of 200 °C / hr from 25 °C to 100 °C was calculated using a differential scanning calorimeter (Microcal PEAQ-DSC; Malvern Panalytical). PBS was used as the reference for the measurements. The obtained data were corrected for baseline using buffer solutions and analyzed using a non-dimorphic model to calculate the Tm and Tonset for the Fab, CH2, and CH3 regions.

[0092] [Using surface plasma resonance technology] [PD-1] [Binding affinity assay] [] The binding affinity of each antibody to human PD-1 was determined using a Biacore 8K (Cytiva) instrument. Anti-His Tag antibodies were immobilized on eight channels (16 flow cells, 1 channel as 2 flow cells) of a sensor chip immobilized with carboxymethyl dextran on a gold membrane using an Amine Coupling Kit and a His Capture Kit via amine coupling. Immobilization was performed by supplying an activation solution (equal volumes of NHS and EDC mixed) at a flow rate of 10 μl / min for 7 minutes, followed by an immobilization solution at a flow rate of 5 μl / min for 7 minutes, and then by supplying 1 M ethanolamine hydrochloride-NaOH at pH 8.5 at a flow rate of 10 μl / min for 7 minutes. 1×HBS-EP+ buffer was used as the buffer for immobilization. In one of the two flow cells of each immobilized channel, 20 μl of human PD-1 / PDCD1 protein (PD-1) prepared to 1 μg / ml with 1×HBS-EP+ buffer was injected at a flow rate of 10 μl / min before sample injection, and PD-1 was captured on the sensor chip. In the other flow cell, 1×HBS-EP+ buffer was similarly supplied as a control flow cell. Antibodies were prepared to 16 nM with the addition of 1×HBS-EP+ buffer, and then each was diluted 2-fold in seven stages using 1×HBS-EP+ buffer. 30 μl of sample solution was injected at a flow rate of 10 μl / min, and the response was measured. The KD value was calculated by subtracting the response of the control flow cell from the response of the ligand-capturing flow cell, and then using Biacore 8K evaluation software to perform curve fitting.

[0093] [Using surface plasma resonance technology] [Fc] [Receptor binding affinity assay] [] Using a Biacore 8K, the binding affinity of each antibody to the Fc receptor (FcγRIIB / C (CD32b / c) (FcγRIIB) (R&D system) and Fc gamma RIIIA / CD16a, CF (FcγRIIIA(V158)) (R&D system)) was determined. Using an Amine Coupling Kit and His Capture Kit, anti-His Tag antibodies were immobilized on eight channels (16 flow cells, 1 channel as 2 flow cells) of a sensor chip immobilized with carboxymethyl dextran on a gold membrane via amine coupling. Immobilization was performed by supplying an activation solution (equal volumes of NHS and EDC mixed) at a flow rate of 10 μl / min for 7 minutes, followed by an immobilization solution at a flow rate of 5 μl / min for 7 minutes, and then by supplying 1 M ethanolamine hydrochloride-NaOH at pH 8.5 at a flow rate of 10 μl / min for 7 minutes. 1×HBS-EP+ buffer was used as the buffer for immobilization. In one of the two flow cells of each immobilized channel, 20 μl of Fc receptor prepared to 1 μg / ml with 1×HBS-EP+ buffer was injected at a flow rate of 10 μl / min before sample injection, and the Fc receptor was captured on the sensor chip. Similarly, 1×HBS-EP+ buffer was supplied to the other flow cell as a control flow cell. After preparing each antibody to 1 mg / ml with 1×HBS-EP+ buffer, each was diluted 2-fold in 7 stages using 1×HBS-EP+ buffer (4-fold dilution in 7 stages for FcγRIIIA(V158) assay). 15 μl of sample solution was injected at a flow rate of 30 μl / min, and the response was measured. The KD value was calculated by subtracting the response of the control flow cell from the response of the ligand-capturing flow cell, and then using Biacore 8K evaluation software to analyze the equilibrium value.

[0094] [Evaluation of cell line activity] [] The activity of anti-human PD-1 antibodies was evaluated by assessing the effect of the interaction between human PD-1-expressing T cells and antigen-presenting cells on cytokine production. Two T cell lines were prepared: one obtained by knocking out mouse PD-1 using Cas9 (Invitrogen), based on the Lecture Series on Immunogenomics Medicine, Graduate School of Medicine, Kyoto University; and another obtained by forcibly expressing human PD-1. Similarly, two IIA1.6 B cell lines were prepared: one obtained by knocking out mouse PD-L1, based on the Lecture Series on Immunogenomics Medicine, Graduate School of Medicine, Kyoto University; and another obtained by forcibly expressing human PD-L1 or mouse FcγRIIB. These were used as antigen-presenting cells. To evaluate human PD-1 agonist activity, IIA1.6 cells were expressed using mouse or human FcγRIIB; to evaluate antagonist activity, IIA1.6 cells were expressed using human PD-L1. Furthermore, to evaluate Fc receptor dependence, IIA1.6 cells were expressed using various human Fc receptors. Human PD-1 expressed DO11.10 T-cell fusion tumors and IIA1.6 cells were suspended in culture medium (RPMI 1640 medium containing 10% fetal bovine serum). DO11.10 T-cell fusion tumors and IIA1.6 cells were seeded into 96-well round-bottom plates at 5×10⁴ and 1×10⁴ cells / well / 50 μl. Anti-human PD-1 antibody was added to each well at 50 μl to achieve final concentrations of 5, 0.5, 0.05, and 0.005 μg / ml. Subsequently, OVA 323-339 peptide (Eurofins) was added to each well at 50 μl to achieve a final concentration of 2 μg / ml. After 18 hours, the IL-2 concentration in the culture supernatant was measured using a mouse IL-2 DuoSet ELISA (R&D system).

[0095] In the evaluation of human PD-1 agonist activity, cytokine inhibition obtained using human PD-L1-expressing IIA1.6 cells was used as a positive control, and the results obtained using DO11.10 T-cell fusion tumors that do not express human PD-1 were used as a negative control. In the evaluation of human PD-1 antagonist activity, an anti-human PD-1 antibody (pure line name: EH12.2H7) with known antagonist activity was used.

[0096] [Using Flow Cytometry in Humans] [Fc] [Affinity assay of receptor-expressing cell lines] [] In IIA1.6 cells that were forcibly expressed as human FcγRIIB or human FcγRIIIA, 50 μl of various antibodies were added at 5 μg / ml and the cells were incubated at 4°C for 15 minutes. Afterward, the cells were washed, and anti-human IgG Fab antibody labeled with APC was used for staining at 4°C for 15 minutes to allow the antibodies bound to the cells to stain. After washing the cells again, the bound antibodies on the cells were detected by FACS analysis, and the GMFI was calculated as the affinity for FcγRIIB or FcγRIIIA.

[0097] [Anti-human] [PD-1] [Analysis of Antibody Binding Properties] [] Using a vector linked by an IRES (internal ribosome entry site) sequence, the genes of human PD-1 (wild-type) or the human PD-1 replacement with mouse sequences (regions 1-8 in Figure 2A) and fluorescent proteins such as GFP were co-expressed in cell lines (DO11.10 T-cell fusion tumors or HEK293T, etc.). The expression level of PD-1 in this cell line was correlated with the expression level of fluorescent proteins such as GFP. Anti-human PD-1 antibodies were bound to PD-1 / GFP-expressing cells, and the resulting antibodies were detected using secondary antibodies labeled with other fluorescent dyes. Specifically, PD-1-expressing cells were seeded at 1 × 10⁵ cells per well in a V-bottom 96-well plate. For each cell pellet, anti-human PD-1 antibody adjusted to 5 μg / ml was added to 50 μl of the pellet, and the mixture was incubated at 4°C for 15 minutes. After washing, BV421-labeled anti-mouse IgG antibody or BV421-labeled anti-human IgG antibody, adjusted to 5 μg / ml, was added to every 50 μl, and the mixture was incubated at 4°C for 15 minutes. After washing, the antibody was resuspended in 200 μl of buffer, passed through a 70 μm filter, and the fluorescence intensity was measured by flow cytometry. The fluorescence intensity at this point can also be referred to as the binding ability of the anti-human PD-1 antibody to human PD-1 (wild-type or replacement). When the binding ability to a human PD-1 replacement with the corresponding sequence of mouse PD-1 decreases compared to the binding ability to wild-type human PD-1, that region is defined as the region bound by the antibody. The binding characteristics to the R143A point variant or the mouse PD-1 (hu38-48) replacement were also analyzed in the same way.

[0098] [Humans Using Genome Editing] [PD-1] [Making a Knock-in Mouse] [] By microinjection, a donor vector containing gRNA targeting 5'-GCCAGGGGCTCTGGGCATGT-3', the human PD-1 gene, and Cas9 protein (Invitrogen) was introduced into pronuclear zygotes derived from C57BL / 6N mice and then transplanted into the oviducts of foster mother mice. The resulting mice (F0) were screened for indel mice, which were then crossed with wild-type mice to obtain F1 mice. The homozygous F1 mice obtained by further mating confirmed gene-transfected mice were used as human PD-1 knock-in mice for experimental purposes.

[0099] [Utilizing dust mite extract] House dust mite extract [,] [HDM] [Induced allergy model conducted] [PD-1] [Evaluation of Activators] [] In the prophylactic administration experiment, human PD-1 knock-in mice were intraperitoneally administered HDM (D. Pteronyssinus; Greer) with a total protein dose of 400 ng (equivalent to 10 ng of Derp1), and simultaneously, 500 μg of anti-human PD-1 antibody was administered intraperitoneally (day 0). Subsequently, equal amounts of anti-human PD-1 antibody were administered intraperitoneally at days 3, 7, and 10. In the therapeutic administration experiment, anti-human PD-1 antibody was administered only once after day 10. Starting 7 days after intraperitoneal administration of HDM, HDM was administered intranasally under anesthesia with a total protein dose of 25 μg / 25 μl. The mice were administered intranasally for 8 consecutive days. Four hours after the final intranasal administration, blood was collected from the mice, which were then euthanized. Bronchoalveolar lavage fluid and lung tissue were recovered, and monocytes infiltrating the alveoli were isolated from the bronchoalveolar lavage fluid. Regarding the lung tissue, monocytes were isolated by enzyme treatment and density gradient centrifugation. The number of CD4+ T cells and eosinophils (CD11c-SiglecF+) was calculated using FACS analysis. Furthermore, intracellular cytokines from monocytes derived from bronchoalveolar lavage fluid and monocytes derived from the lungs were analyzed using FACS. The serum concentration of HDM-specific IgE was measured using ELISA (enzyme-linked immunosorbent assay) (mouse serum anti-HDM IgE antibody analysis kit; Chondrex).

[0100] [Utilization of Acute Graft-versus-Host Disease] [(GVHD)] [Model implementation] [PD-1] [Evaluation of Activators] [] For BDF1 mice (recipients, H-2Kb+H-2Kd+), cyclophosphamide (Fujifilm and Kodenpak) dissolved in PBS was administered intraperitoneally at a dose of 100 mg / kg (Day -1). One day later, spleen cells from human PD-1 knock-in mice (donors, H-2Kb+H-2Kd-) were recovered, washed with PBS, and resuspended in PBS to a concentration of 5 × 10⁷ cells / 200 μl. 200 μl of this cell suspension was transferred into BDF1 mice via the tail vein (Day 0). From Day 0, anti-human PD-1 agonist antibody was administered intraperitoneally at a dose of 500 μg / mouse every 3–4 days. To evaluate GVHD symptoms, body weight was measured at days 4, 7, 11, and 14, and the rate of weight loss from Day 0 was calculated. Furthermore, blood was collected at 7 and 14 days post-mortem. Plasma was collected by centrifugation, and PBMCs were isolated by density gradient centrifugation. Mice were euthanized at 14 days post-mortem, and spleens and livers were recovered to prepare cell suspensions. The donor cell (H-2Kb+H-2Kd-) percentage and cell number in PBMCs, spleens, or livers were calculated using flow cytometry analysis and cell count measurements. Aspartate transaminase (AST) and alanine transaminase (ALT) levels in plasma were measured using the Transaminase CII-Test Wako (FULIFILM), and IFN-γ concentration was measured using the mouse IFN-γ ELISA MAX Deluxe (BioLegend).

[0101] [use] [EAE] [Model implementation] [PD-1] [Evaluation of Activators] [] For wild-type mice, 100 μg of MOG 35-55 peptide (Eurofins) mixed with complete Freund's adjuvant was administered subcutaneously at two sites on the back, followed by 400 ng of pertussis toxin (Fujifilm and Koichi). One day later, 400 ng of pertussis toxin was administered again. Cells were isolated from the spleen and axillary lymph nodes after 13 days. CD8+ and Gr-1+ cells were removed using AutoMACS, and the remaining cells were seeded at 1.05 × 10⁷ cells per well in 12-well plates and cultured for 5 days in the presence of 20 μg / ml MOG peptide and 10 ng / ml IL-23. Human PD-1 was forcibly expressed using retroviruses on days 1 and 2 of culture. Cells were recovered after 5 days of culture. Activated CD4+ T cells were extracted from 200 μl of lymphocytes at a concentration of 1 × 10⁶ cells. The concentration was adjusted with PBS, and 200 μl of each cell was transferred into wild-type mice (day 0) via the tail vein. From day 0 post-cell transfer, anti-human PD-1 agonist antibody was administered intraperitoneally at a dose of 500 μg / mouse every 3 days. Weight and scores were measured at the time points shown in Figure 8. Scores were recorded as follows: 0: Asymptomatic; 0.5: Tail paralysis; 1.0: Tail paralysis; 1.5: Mild paralysis of a single hind limb; 2.0: Mild paralysis of both hind limbs; 2.5: Paralysis of a single hind limb; 3.0: Paralysis of both hind limbs; 3.5: Paralysis of all four limbs; 4.0: Near death; 5.0: Death.

[0102] [Using a colitis model] [PD-1] [Evaluation of Activators] [] Cells were prepared from the spleen and lymph nodes of human PD-1 knock-in mice, and CD25-CD4+ cells were isolated using a cell sorter. These cells were suspended in PBS at a concentration of 5 × 10⁵ cells / 200 μl, and 200 μl was injected via tail vein into RAG2 knockout mice (day 0). From day 0, anti-human PD-1 agonist antibody was administered intraperitoneally at a dose of 500 μg / mouse every 3-4 days. Body weight was measured twice weekly, and the rate of weight loss from day 0 was calculated. Mice were euthanized at week 8, and the large intestine was recovered and its length measured. CD4+ T cells infiltrating the lamina propria of the large intestine were recovered, stained with intracellular cytokines, and analyzed by flow cytometry.

[0103] [Utilizing chronic] [GVHD / ] [Lupus-like model being implemented] [PD-1] [Evaluation of Activators] [] For BDF1 mice (recipients, H-2Kb+H-2Kd+), cyclophosphamide (Fujifilm and Kodenki) dissolved in PBS was administered intraperitoneally at a dose of 100 mg / kg (Day -1). One day later, spleen cells from human PD-1 knock-in mice (donors, H-2Kb+H-2Kd-) were recovered, and CD8+ cells were removed using AutoMACS. The cells were then washed with PBS to a concentration of 2 × 10⁷ cells / 200 μl. 200 μl of this cell suspension was administered via tail vein to BDF1 mice (Day 0). From Day 0, anti-human PD-1 agonist antibody was administered intraperitoneally at a dose of 500 μg / mouse every 3–4 days. To assess lupus-like symptoms, blood was collected weekly for 11 weeks, and the concentrations of IL-21 or anti-dsDNA antibodies in plasma were measured using ELISA. Mice were euthanized at 7 and 14 days, and their spleens were recovered to prepare cell suspensions. Flow cytometry was used to analyze donor-derived Tfh cells (CXCR5 +ICOS +H-2K d-CD4+), or recipient-derived germinal center B cells (CD95 +GL7 +H-2K d+B220+), type-converted B cells (IgD-IgM-H-2K d+B220+), and plasma cells (CD138 +H-2K d+CD19+).

[0104] [Human] [T] [cell] [B] [Mixed lymphocyte reaction] [(MLR)] [Middle] [PD-1] [Evaluation of Activators] [] This study evaluated the activity of anti-human PD-1 agonist antibodies against T cell responses to allogeneic antigens in MLRs composed of negatively selected human CD4+ T cells (LONZA) and human CD19+ B cells (Precision for Medicine) isolated from healthy human PBMCs. Specifically, CFSE-stained human T cells (2 × 10⁵ cells) and B cells (1 × 10⁵ cells) were mixed and seeded at 200 μl / well in round-bottom 96-well plates. After 6–7 days of culture, the cells were harvested. B cells were negatively selected using CD19 antibody to remove the T cells, and new B cells from the same donor were added to restimulate the T cells. During restimulation, various drugs were added simultaneously to achieve final concentrations of 5, 0.5, 0.05, and 0.005 μg / ml. Twelve hours later, the culture supernatant was recovered, and the IFN-γ concentration was determined using a human IFN-γ ELISA MAX Deluxe (BioLegend).

[0105] [ADCC] [Evaluation of Activity] [] Raji-hPD-1 cells (InvivoGen) were suspended in culture medium at a concentration of 1.1 × 10⁶ cells / ml as target cells, and seeded at 90 μl / well in 96-well round-bottom plates. Simultaneously, 20 μl / well of anti-human PD-1 antibody was added to achieve final concentrations of 1000, 300, 100, 30, 10, 3, 1, 0.3, 0.1, 0.03, and 0.01 ng / ml. After 1 hour of culture, Jurkat-Lucia NFAT-CD16 cells (InvivoGen) were suspended in culture medium at a concentration of 2.2 × 10⁶ cells / ml as effector cells, and 90 μl / well was added. After 6 hours of culture, 50 μl of QUANTI-Luc solution (InvivoGen) was added to 20 μl of the culture supernatant, and the luminescence intensity was measured using a microdisk reader.

[0106] [Human] [PBMC] [In the antigen response] [PD-1] [Evaluation of Activators] [] After thawing frozen healthy human PBMCs (Precision for Medicine), they were suspended in culture medium at a concentration of 1 × 10⁷ cells / 2 ml and seeded at 2 ml / well in 12-well plates. After pre-culturing for 24 hours, the cells were recovered and stained with CFSE. Subsequently, they were suspended in culture medium at a concentration of 3 × 10⁶ cells / ml and seeded at 100 μl / well in 96-well round-bottom plates. Tetanus toxin (Merck) was added at 50 μl / well as an antigen against the cells to achieve a final concentration of 1 μg / ml. Other drugs were added at 50 μl / well to achieve final concentrations of 5, 0.5, 0.05, and 0.005 μg / ml. After 4-5 days, CD4+ T cell proliferation was evaluated by flow cytometry using CFSE as an indicator. The concentration of IFN-γ in the culture supernatant was measured using a human IFN-γ ELISA MAX Deluxe (BioLegend).

[0107] [PD-1] [Action promoter pair] [ERK] [Evaluation of phosphorylation] [] Human PD-1-expressing DO11.10 T-cell fusion tumors and mouse FcγRIIB-expressing IIA1.6 cells were suspended in culture medium (RPMI 1640 medium containing 10% fetal bovine serum). DO11.10 T-cell fusion tumors were seeded at 5 × 10⁴ cells / well and IIA1.6 cells at 1 × 10⁴ cells / well / 50 μl in 96-well round-bottom plates. Control mouse antibody (MOPC-21) or anti-human PD-1 antibody HM266 was added to each well at 50 μl to achieve a final concentration of 5 μg / ml. Subsequently, OVA323-339 peptide (Eurofins) was added to each well at 50 μl to achieve a final concentration of 2 μg / ml. Cells were recovered immediately after OVA addition (0 h) or after incubation at 37°C, 5% CO₂ for 2, 4, and 6 hours. Cells were fixed with 4% paraformaldehyde for 15 minutes, washed, and then treated with 0.1% Triton X-100 for 15 minutes. After washing, the cells were suspended in 50% methanol-PBS and incubated overnight at -20°C. After washing, the cells were stained with Alexa488-anti-phosphorylation-p44 / 42 MAPK (Erk1 / 2) (Thr202 / Tyr204) mAb, PE-anti-DO11.10 TCR mAb, and APC-anti-B220 mAb. The stained cells were analyzed by FACS to calculate the phosphorylated ERK positivity rate in DO11.10 (DO11.10 TCR +B220 -).

[0108] [use] [T] [Cell-dependent antibody response proceeds] [PD-1] [Evaluation of Activators] [] For human PD-1 knock-in mice, a co-precipitate of NP-OVA (total protein 100 μg) and Alum adjuvant was administered intraperitoneally, along with 500 μg of anti-human PD-1 antibody (day 0). Subsequently, on days 3 and 7, an equal amount of anti-human PD-1 antibody was administered intraperitoneally. Blood samples were collected 7 and 10 days after NP-OVA administration, and the antigen-specific antibody titer or IL-21 level in the blood was measured by ELISA. Antibodies (anti-NP2, anti-NP25) binding to 2 or 25 molecules of NP and 1 molecule of bovine serum albumin (BSA) were measured by ELISA and defined as high-affinity and low-affinity antigen-specific antibodies, respectively. Furthermore, euthanasia was performed 10 days after antigen immunization, and the spleen was recovered to prepare a cell suspension. The Tfh cells (CXCR5 +ICOS +CD4 +), germinal center B cells (CD95 +GL7 +B220 +), type-converting B cells (IgD -IgM -B220 +), and plasma cells (CD138 +B220 -) were analyzed by flow cytometry.

[0109] [Revitalizing Humanity] [T] [Cells or people] [Tfh] [like cells and] [THP-1] [In a cell co-culture system] [PD-1] [Evaluation of Activators] [] Activated human T cell lines were prepared as follows: Human CD4+ T cells (LONZA) isolated from healthy human PBMCs were prepared at 1×10⁶ cells / ml and seeded at 1 ml / well in 24-well plates immobilized with anti-CD3 antibody (3 μg / ml), followed by stimulation for 3 days. Human Tfh-like cell lines were prepared as follows: Naïve T cells (CD45RA + CXCR5 - CD11c -) were fractionated from human CD4+ T cells using a cell sorter and prepared at 1×10⁶ cells / ml. 25 μl of washed Dynabeads Human T-activator CD3 / 28 (Thermo Fisher) was added to each ml of these cells, and the cell-bead mixture was seeded at 1 ml / well in 24-well plates. On the second day, cells stimulated by beads were recovered, the beads were removed, and then prepared at a density of 5 × 10⁵ cells / ml. 100 μl / well was seeded into 96-well plates immobilized with anti-CD3 antibody (5 μg / ml). Subsequently, human IL-23 (25 ng / ml), human TGFβ (5 ng / ml), human IL-12 (1 ng / ml), and anti-human CD28 (1 μg / ml) were added to prepare 200 μl / well, and the cells were cultured at 37°C and 5% CO₂ for 48–72 hours. FACS analysis of the cultured cells confirmed that over 50% were Tfh-like cells (CXCR5 + ICOS + PD-1 +). THP-1 cell lines (mother line or human FcγRIIB overexpressing line) were suspended at 1×10⁷ cells / ml in a 500 μg / ml mitomycin C dilution and treated at 37°C for 2 hours, followed by washing three times with culture medium. Activated human T cells or human Tfh-like cells were seeded at 5×10⁴ cells / well and THP-1 cells at 2.5×10⁴ cells / well in 96-well round-bottom plates. CytoStim (Miltenyi Biotec) was added at 0.2 μl / well as a stimulant, along with various other agents, to achieve a final concentration of 5-5000 ng / ml. The plates were then cultured at 37°C and 5% CO₂ for 18 hours. After culture, the supernatant was collected, and cytokine concentrations were measured using human IFN-γ ELISA MAX Deluxe (BioLegend) or human IL-21 Duoset ELISA (R&D).

[0110] [Derived from peripheral blood mononuclear cells of patients with autoimmune diseases] [(PBMC)] [In the context of autoantigen response] [PD-1] [Evaluation of Activators] [] After thawing PBMCs (Precision for Medicine, STEMCELL Technologies) from RA and SLE patients, they were suspended in culture medium at 1×10⁷ cells / 2 ml and seeded at 2 ml / well in 12-well plates. After pre-culturing for 16–24 hours, the cells were recovered, stained with CFSE, and then suspended in culture medium at 3×10⁶ cells / ml and seeded at 100 μl / well in 96-well round-bottom plates. Citrullinated fibrinogen (Cayman) was added to 50 μl / well of RA patient PBMCs, and Sm antigen (AROTEC DIAGNOSTICS) was added to 50 μl / well of SLE patient PBMCs, to achieve final concentrations of 10 μg / ml and 5 μg / ml, respectively. Additionally, each drug was added at 50 μl / well to achieve final concentrations of 5, 0.5, 0.05, and 0.005 μg / ml. Six to seven days later, the proliferation of CD4+ T cells, using CFSE as an indicator, was evaluated by flow cytometry. Furthermore, the concentration of IFN-γ in the culture supernatant was measured using the human IFN-γ ELISA MAX Deluxe (BioLegend).

[0111] Results and Discussion PD-1 antibodies are claimed to inhibit T cell activity through PD-1 agonist activity. (Ref. 1) However, whether these PD-1 antibodies can actually stimulate PD-1 has not been proven. Previous studies have discussed agonist activity based on results from artificial experimental systems, where agonist activity is observed when T cells are stimulated by immobilized anti-CD3ε antibodies, and similarly, immobilized anti-PD-1 antibodies inhibit activation. However, it cannot be said that such artificial conditions accurately reproduce T cell activation in vivo. Whether T cells in vivo can inhibit the activation process through interaction with antigen-presenting cells when administered in a free state of anti-PD-1 antibody is another question.

[0112] To discover anti-human PD-1 agonist antibodies that function under physiological conditions, an ideal system is needed to reproduce the interaction between T cells and antigen-presenting cells, and to evaluate the conditions for antigen-specific T cell activation. Furthermore, an experimental system with easily adaptable conditions is useful for characterizing this action. The established cell line, a combination of DO11.10 T-cell fusion tumor and the IIA1.6 cell line as antigen-presenting cells, provides a useful platform for the activation of antigen-specific T cells and the efficient and reproducible analysis of their mechanism of action (Figure 1). DO11.10 T-cell fusion tumor is a T-cell fusion tumor cell line derived from CD4+ T cells of DO11.10 mice expressing MHC class II (IAd)-restricted T-cell receptors. These MHC class II (IAd)-restricted T-cell receptors recognize peptides derived from ovalbumin (OVA 323-339). DO11.10 T-cell fusion tumors and B-cell lymphoma cell lines, specifically IIA1.6 cells, exhibit an antigenic peptide response to MHC class II (IA d), resulting in the production of IL-2 (Figure 1A). Because this cell combination can induce antigen-specific T-cell activation, this experimental system has advantages in reproducing physiological activation mechanisms that cannot be obtained by experimental systems promoting non-specific T-cell activation. Furthermore, conditions such as gene knockout and forced expression are easily achievable.

[0113] In DO11.10 T-cell fusion tumors expressing human PD-1, IL-2 production was significantly reduced upon antigen stimulation by IIA1.6 cells expressing PD-L1, confirming a PD-1-dependent response (Figure 1B). PD-1 and PD-L1 expressed on the cell membrane are considered to adopt the inherent three-dimensional structure of membrane proteins, a point of advantage over artificial experimental systems utilizing soluble fusion proteins. In fact, the addition of an anti-human PD-1 antibody (EH12.2H7), already identified as a blocking antibody, to this experimental system resulted in a recovery of IL-2 levels reduced by PD-L1, indicating that the experimental system can reliably detect the blocking antibody (Figure 1C). The inventors believe that this experimental system, utilizing cell-to-cell interactions, is suitable for observing combinations of T-cell inhibition via PD-1, characterizing the PD-1 agonist activity and binding properties of anti-human PD-1 antibodies, or exploring the conditions required for agonist activity.

[0114] To discover and characterize biologically functional anti-human PD-1 antibodies, a large number of monoclonal antibodies binding to various regions of human PD-1 were prepared. Fusion tumors producing these antibodies were prepared from mice immunized with human PD-1. The antibody system was classified according to its binding specificity to eight presumed regions on the molecular surface constituting human PD-1 (Figure 2). Analysis of the binding characteristics confirmed that the antibody group contained a large number of antibodies with various binding epitopes.

[0115] Subsequently, the agonist activity of the obtained antibodies was evaluated. The detection of immunosuppressive activity using anti-PD-1 agonist antibodies requires the absence of PD-L1 inhibition. The screening system for agonist antibodies differs from that for blocking antibodies; a combination of DO11.10 T-cell fusion tumors expressing human PD-1 and IIA1.6 cells expressing FcγRIIB lacking PD-L1 was used. The inventors used two systems—one for blocking antibody detection and the other for agonist antibody detection—to evaluate the activity of anti-human PD-1 antibody groups with various binding epitopes and available anti-human PD-1 monoclonal antibodies. As a result, agonist antibodies and blocking antibodies with various activity intensities were obtained (Figures 3-5). It was confirmed that these activities were not observable in the absence of PD-1. No antibodies exhibiting both agonist and blocking activities were found. The results show that HM266, one of the most potent agonist antibodies, can inhibit the phosphorylation of ERK downstream of the T cell receptor in stimulated DO11.10 cells, thus blocking T cell receptor signaling (Figure 22).

[0116] It can be seen that the biological activity of these antibodies is related to their binding sites, and the binding sites of agonist antibodies and blocking antibodies are significantly different (Figure 5). Specifically, agonist antibodies bind to regions #6 (Sequence No. 8), #7 (Sequence No. 9), or #8 (Sequence No. 10) on the surface of the human PD-1 molecule, while blocking antibodies bind to regions #1 (Sequence No. 3), #2 (Sequence No. 4), or #5 (Sequence No. 7). Agonist antibodies binding to region #7 are highly active, including HM266, HM242, HM297, and HM268. This correlation between the binding sites of the human PD-1 molecule and agonist activity has not been reported in the definition of the anti-human PD-1 agonist antibody group.

[0117] The anti-human PD-1 agonist antibody of this invention was selected in an experimental system that activates T cells through the interaction between T cells and antigen-presenting cells. Therefore, it suggests that the immunosuppressive activity resulting from the addition of free anti-human PD-1 antibody is more likely to exert its effect under physiological conditions. Furthermore, the antibody of this invention showed no activity against PD-1-deficient T cells, clearly demonstrating PD-1-dependent immunosuppression. As described in the previous experimental method, when anti-CD3 antibody and anti-PD-1 antibody were co-immobilized for functional evaluation, there was a situation where the amount of anti-CD3 antibody immobilized was reduced due to the influence of the combined anti-PD-1 antibody. In this case, T cell activation was significantly inhibited, due to the reduced stimulation by the anti-CD3 antibody, which is a non-specific phenomenon unrelated to the effect of the anti-PD-1 antibody. In fact, the inventors have also experienced this kind of case. When using this experimental system for PD-1 research, it is necessary to pay close attention to the amount of anti-CD3 antibody immobilization and to use T cells lacking PD-1 to confirm PD-1 specific immunosuppression.

[0118] The anti-human PD-1 agonist antibody of this invention exhibits in vitro immunosuppressive activity targeting activated T cells, thus demonstrating potential effectiveness against a variety of inflammatory diseases. There are reports of activated T cells participating in various organ-specific disorders or type I allergic diseases (Ref. 2), and it is also known that inflammatory responses in these diseases are reduced by neutralizing cytokines derived from activated T cells (Ref. 3). Furthermore, in patients receiving anti-PD-1 blocking antibodies for cancer treatment, symptoms similar to those of diseases with strong T cell involvement have been observed as side effects (Ref. 4). In animal experiments, PD-1 induction has been observed in T cell-dependent inflammation (Ref. 5), and inflammation is enhanced by PD-1 knockout or administration of anti-PD-1 blocking antibodies (Ref. 6), indicating that PD-1 is crucial for controlling activated T cells during disease progression. Based on the above, PD-1-targeted therapeutic interventions have significant potential to become useful treatments that control activated T cells involved in disease progression, showing broad efficacy against various inflammatory diseases. Next, the inventors verified the anti-inflammatory effects of antibodies against human PD-1 agonists in several mouse inflammation models.

[0119] First, using an animal model of asthma induced by inhalation of dust mite antigens, the efficacy against allergic asthma was verified. The anti-human PD-1 agonist antibody HM266 was prophylactically administered to human PD-1 knock-in mice. The results showed that eosinophilic globulin and CD4+ T cell infiltration into the alveoli were inhibited, and the production of IL-4, IL-5, and IL-13 by CD4+ T cells was comprehensively reduced (Figures 6 and 7). With the reduction of Th2 cells, the blood concentration of mite antigen-specific IgE also decreased significantly, indicating that Th2-type immunity was actually suppressed (Figure 7). Furthermore, in a therapeutic environment (treatment regimen), the infiltration of eosinophils and CD4+ T cells producing Th2-type cytokines was significantly inhibited (Figures 6 and 7). These results indicate that the inhibition of T cells by PD-1 agonists can be used as a preventative and therapeutic strategy for allergic inflammation. Similarly, administration of J116, a PD-1 agonist with weaker activity than HM266, significantly inhibited eosinophil infiltration into the alveoli induced by mite antigen stimulation. J116 reduced the number of IL-5 and IL-13 producing cells, but did not significantly reduce CD4+ T cell infiltration or the number of IL-4 producing cells (Figures 24 and 25). Compared with J116, HM266 has higher agonist activity (Figure 23). Therefore, the difference in anti-inflammatory effects reflects its potential as an agonist, and HM266 can be considered an excellent candidate among antibodies that bind to the #7 region of human PD-1.

[0120] The anti-inflammatory effects of anti-human PD-1 agonist antibodies were further evaluated using models of other T-cell-related diseases, including multiple sclerosis (EAE), colitis, acute GVHD, and chronic GVHD / lupus-like disease. These disease models were induced by T-cell translocation, and anti-human PD-1 agonist antibodies were administered concurrently with T-cell translocation. Results showed that in the EAE model, the encephalomyelitis score significantly decreased after HM266 administration (Figure 8). In the colitis model, HM266 reduced weight loss, shortened colon length, and infiltration of cytokine-positive CD4+ cells in the lamina propria of the mucosa (Figures 9 and 26). Furthermore, in the acute GVHD model, anti-human PD-1 agonist antibodies significantly inhibited weight loss, the expansion of donor cells derived from peripheral blood or organs, and the increase of plasma AST, ALT, and IFN-γ, strongly suppressing acute GVHD symptoms. The same anti-inflammatory effects were observed in anti-human PD-1 agonist antibodies such as HM266, HM242, HM268, and HM297 (Figures 10 and 27). Furthermore, anti-human PD-1 agonist antibodies significantly inhibited the induction of autoantibodies and the increase in Tfh cells or antibody-producing B cells associated with their production in a chronic GVHD / lupus-like model (Figures 8-11 and 29). These results suggest that anti-human PD-1 agonist antibodies have an inhibitory effect on T cell-dependent antibody responses.

[0121] Based on the consistent anti-inflammatory effects of anti-human PD-1 agonist antibodies in multiple disease models related to T cells, it is suggested that the antibody of the present invention can be used as a treatment strategy for a wide range of inflammatory diseases. To sustain the effect, continued use of clinically available neutralizing antibodies against cytokines derived from T cells is necessary. However, anti-human PD-1 agonist antibodies, due to their ability to achieve long-term T cell suppression, have the potential to cure these diseases. Specifically, in an acute GVHD model, administration of anti-human PD-1 agonist antibodies significantly inhibited CD8+ T cells; therefore, anti-PD-1 agonist antibodies can be effectively used as a treatment for GVHD or alopecia areata, which are primarily associated with CD8+ T cells. Furthermore, anti-human PD-1 agonist antibodies inhibited CD4+ T cells in asthma, colitis, and EAE models; therefore, anti-PD-1 agonist antibodies can also be used as a treatment for allergic diseases, colitis, multiple sclerosis, and psoriasis, where CD4+ T cells play an important role. Furthermore, by administering PD-1 agonists to lupus-like models, autoantibodies are suppressed. Therefore, PD-1 agonists can be used as therapeutic agents for diseases related to autoantibodies, such as lupus nephritis or rheumatoid arthritis.

[0122] To adapt to human use, the inventors converted anti-human PD-1 agonist antibodies into humanized antibodies. For seven anti-human PD-1 agonist antibodies initially derived from mouse IgG, the Fc region sequence was replaced with the human IgG1-K322A sequence, converting them into chimeric antibodies. The agonist activity of the resulting chimeric antibodies was evaluated using T cell lines overexpressing human PD-1 and antigen-presenting cell lines expressing human FcγRIIB. All chimeric antibodies showed activity equivalent to the mouse antibodies (Figure 12). Of the seven antibodies, three exhibited superior stability in terms of physical properties. Next, the Fv region of these antibodies was converted into a human sequence. For each antibody, the Fv region was modified into 12 or 15 humanized sequences, which were then combined with the constant region of the human IgG1-K322A sequence. The 12 antibodies derived from HM242 maintained high PD-1 agonist activity and exhibited many excellent physical properties. Regarding the 15 antibodies derived from HM297 and 12 antibodies derived from HM268, although they were humanized in the same way as HM242, some of them showed unexpectedly poorer agonist activity or physical property curves compared to their pre-humanization counterparts. Among them, antibodies that could maintain high levels of activity and physical properties were obtained, and four humanized antibodies were ultimately selected (Figure 13, Table 1-3).

[0123] The immunosuppressive activity of these allogeneic antibodies in primary human lymphocytes was evaluated. CD4+ T cells derived from human peripheral blood mononuclear cells were co-cultured with other human B cells to evaluate the effectiveness of the allogeneic reaction in T cell activation. The results showed that CTLA-4-Ig, as a positive candidate, was effective, but none of the four anti-human PD-1 agonist antibodies showed significant inhibitory activity (Figure 14).

[0124] To address this issue, attempts were made to modify humanized anti-human PD-1 agonist antibodies. Among agonist antibodies, some require antibody cross-linking to exert agonist activity, while others utilize Fc receptors as a crucial means of antibody cross-linking. However, there are no examples demonstrating whether the activity of anti-human PD-1 agonist antibodies requires Fc receptor-dependent cross-linking. According to our own verification, in the absence of Fc receptors, anti-human PD-1 agonist antibodies cannot exert agonist activity. Conversely, in the presence of Fc receptors, anti-human PD-1 agonist antibodies exert agonist activity. Among the several human Fc receptors present, FcγRIIB is the most effective in exerting anti-human PD-1 agonist activity (Figures 15 and 16). Furthermore, immunosuppressive activity can also be induced by FcγRIIA (H131) or FcγRIIA (R131), but its intensity is weaker than that of FcγRIIB. The activator activity induced by FcγRIIIA (F158) or FcγRIIIA (V158) is even weaker. Surprisingly, if FcγRIA-expressing antigens are used to present cells for binding to humanized anti-human PD-1 antibodies, cytokine production tends to increase.

[0125] The agonist activity of the humanized anti-human PD-1 agonist antibody increases depending on the level of human FcγRIIB expression on the antigen-presenting cell side (Figure 16). That is, if antigen-presenting cells with high levels of FcγRIIB expression are used, higher immunosuppressive activity is observed; conversely, if FcγRIIB expression is low, only mild immunosuppressive effect is observed. Therefore, it is considered that optimization is needed to enhance the affinity of human FcγRIIB in order for the agonist antibody of the present invention to exert its immunosuppressive effect via the low levels of human FcγRIIB observed in actual human lymphocytes. This problem, or solution, cannot be found in known experimental systems (antibody immobilization) using anti-PD-1 agonist antibodies, and is an insight that ensures the progressiveness of the present invention.

[0126] In response, the inventors considered enhancing the affinity of agonist antibodies for human FcγRIIB. In short, the inventors discovered combinations of FcγRIIB affinity selectively enhanced by amino acid mutations in the Fc region. Chimeric antibodies were prepared by attaching these modified Fc regions to anti-human PD-1 agonist antibodies. The resulting chimeric antibodies exhibited higher agonist activity even under conditions of low FcγRIIB expression, and further demonstrated good immunosuppressive activity in an evaluation system using primary human cells (Figures 17, 18, 30, 35, 37). It was confirmed that these humanized antibodies with Fc modifications exhibited stronger inhibitory effects on human T cell activation than CTLA-4-Ig (Figures 19, 38). These results demonstrate that the Fc-modified anti-human PD-1 agonist antibodies of the present invention can exert a strong immunosuppressive effect through physiologically expressed levels of PD-1 and FcγRIIB in human cells.

[0127] Furthermore, some Fc region modifiers, in addition to enhancing PD-1 agonist activity through increased affinity for FcγRIIB, also exhibit increased affinity for FcγRIIIA, leading to heightened ADCC activity via FcγRIIIA (Figures 20, 31, 36, 39). These antibodies, compared to those with lower FcγRIIIA affinity, showed greater T-cell suppression in an evaluation system using antigen-stimulated human PBMCs (Figures 21, 32, 40). Based on the above, the overall immunosuppressive activity of anti-PD-1 agonist antibodies can be further improved by removing the additional ADCC activity of PD-1-expressing cells.

[0128] Regarding cell surface molecules such as the TNF receptor superfamily, several antibodies are known to exert agonist activity in an FcγRIIB-dependent manner targeting these cell surface molecules (Ref. 7). These agonist antibodies bind to FcγRIIB, causing cross-linking between target molecules, thereby inducing signal transduction via downstream TRAFs (TNF receptor-associated factors) and other pathways (Ref. 8). On the other hand, PD-1 belongs to the immunoglobulin superfamily and possesses an immunosuppressive domain (ITIM / ITSM) within the cell. It recruits dephosphorylating enzymes SHP-1 / 2, causing dephosphorylation of various immune activation signaling molecules, thereby producing immunosuppression (Ref. 9). In other words, the agonist signal transduction via the anti-human PD-1 antibody of this invention, from the perspective of its molecular type or intracellular signal transduction pattern, is completely different from the signal transduction using known agonist antibodies. In fact, there are no reported examples of FcγRIIB-dependent troponin antibodies against PD-1 troponin antibodies belonging to the immunoglobulin superfamily, making it impossible to predict that the anti-human PD-1 troponin antibody of the present invention will have the same properties as the aforementioned known antibodies. Furthermore, it is also impossible to predict that the troponin activity of the anti-human PD-1 antibody will only occur when the antibody binds to a specific region of human PD-1.

[0129] Furthermore, an anti-CD40 antibody, one of the agonist antibodies targeting the aforementioned TNF receptor superfamily, has entered clinical trials, but has not shown significantly greater efficacy than that shown in mouse models (Ref. 10). In that report, it was speculated that Fc modifications enhancing antibody binding to FcγRIIB improve human responses. However, there are no reported cases of FcγRIIB dependence on anti-PD-1 agonist antibodies, and the necessity for higher affinity of anti-human PD-1 agonist antibodies for FcγRIIB is completely unknown. With the inventors' novel idea of ​​enhancing the affinity of anti-human PD-1 agonist antibodies for FcγRIIB, even under conditions of low physiological FcγRIIB expression levels in humans, the antibody of this invention can exert sufficient PD-1 agonist activity, enabling immunosuppressive therapy for inflammatory diseases. Furthermore, there are currently no reports of Fc modifications used to enhance ADCC activity further strengthening the immunosuppressive effect of anti-human PD-1 agonist antibodies. By modifying the Fc to enhance PD-1 agonist activity or enhancing ADCC activity in addition to PD-1 agonist activity, the antibody exhibits a stronger immunosuppressive effect than existing anti-human PD-1 antibodies, demonstrating the progressiveness of the antibody of the present invention.

[0130] [Table 1] Activity and properties of humanized HM242 VH VL sample Productivity (mg / l) Monomer Ratio (%)_Gel Filtration Cell-to-cell contact analysis (ng / ml) SPR(PD-1) KD(M) DSC Tm2 (°C)_Fab Homology with human sequences (%) mice HM242-IgG1-K322A nd 78.3 13.7 <5.105E-11 78.9 91% A 1 HM242-A1 352 88.5 13.1 <3.561E-11 82.5 94% 2 HM242-A2 97 93.4 16.7 <4.080E-11 87.2 95% 3 HM242-A3 252 95.1 18.8 <2.172E-11 90.2 96% B 1 HM242-B1 216 91.4 7.15 <4.482E-11 82.2 95% 2 HM242-B2 91 100.0 8.83 <3.153E-11 85.9 96% 3 HM242-B3 141 100.0 20.8 <3.072E-11 87.1 97% C 1 HM242-C1 485 94.5 19.0 <1.786E-11 82.7 95% 2 HM242-C2 187 73.9 18.1 <2.107E-11 88.3 96% 3 HM242-C3 409 96.0 23.7 <1.861E-11 91.8 97% D 1 HM242-D1 485 95.4 24.8 1.03E-10 82.1 95% 2 HM242-D2 158 86.8 33.3 <6.031E-11 87.7 96% 3 HM242-D3 368 95.7 31.5 <4.924E-11 92.4 97% (nd=Undetermined)

[0131] [Table 2] Activity and properties of humanized HM297 VH VL sample Productivity (mg / l) Monomer Ratio (%)_Gel Filtration Cell-to-cell contact analysis (ng / ml) SPR(PD-1) KD(M) DSC Tm2 (°C)_Fab Homology with human sequences (%) mice HM297-IgG1-K322A nd 95.7 78.3 4.932E-11 78.21 92% E 4 HM297-E4 311 85.70 100 1.293E-10 80.18 94% 5 HM297-E5 614 94.12 78.6 5.022E-08 83.35 94% 6 HM297-E6 508 94.02 65.4 1.259E-08 84.47 95% F 4 HM297-F4 736 87.84 92.3 2.412E-08 80.82 94% 5 HM297-F5 555 85.39 98.9 2.349E-09 85.40 95% 6 HM297-F6 447 84.72 150 4.825E-09 86.45 96% G 4 HM297-G4 449 84.23 55.4 4.507E-10 80.39 94% 5 HM297-G5 276 85.78 102 2.319E-10 82.62 95% 6 HM297-G6 350 79.17 59.7 1.892E-09 82.61 96% H 4 HM297-H4 740 89.90 185 2.206E-10 80.99 95% 5 HM297-H5 728 90.72 96.4 5.630E-10 84.98 95% 6 HM297-H6 757 86.03 141 1.860E-09 85.71 96% I 4 HM297-I4 686 90.07 65.6 4.844E-09 82.61 95% 5 HM297-I5 538 90.11 94.9 2.200E-09 86.13 95% 6 HM297-I6 558 88.57 137 3.479E-09 86.83 96% (nd=Undetermined)

[0132] [Table 3] Activity and properties of humanized HM268 VH VL sample Productivity (mg / l) Monomer Ratio (%)_Gel Filtration Cell-to-cell contact analysis (ng / ml) SPR(PD-1) KD(M) DSC Tm2 (°C)_Fab Homology with human sequences (%) mice HM268-IgG1-K322A nd 94.9 46.5 <1.694E-12 70.4 91% J 7 HM268-J7 332 100.0 70.9 4.816E-12 80.4 95% 8 HM268-J8 639 99.1 63.1 <2.837E-12 82.5 96% 9 HM268-J9 201 97.3 301 2.524E-11 81.4 97% K 7 HM268-K7 473 76.4 43.7 6.906E-12 80.6 95% 8 HM268-K8 905 91.8 39.4 <3.062E-12 86.9 96% 9 HM268-K9 366 65.3 150 1.511E-10 87.5 97% L 7 HM268-L7 369 98.0 103 2.161E-12 82.0 96% 8 HM268-L8 701 99.1 98.2 1.291E-12 87.2 96% 9 HM268-L9 612 87.1 87.4 1.105E-11 87.1 97% M 7 HM268-M7 401 83.7 149 1.199E-11 82.5 96% 8 HM268-M8 521 93.0 75.8 3.588E-12 87.4 97% 9 HM268-M9 295 66.5 1.43E+03 6.143E-11 88.3 97% (nd=Undetermined)

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[0134] This invention can be used to treat and / or prevent inflammatory diseases. [Sequence list non-keyword text]

[0135] Serial Number sequence name 1 Human PD-1 amino acid sequence 2 Mouse PD-1 amino acid sequence 3 Human PD-1 epitope / binding region #1 4 Human PD-1 epitope / binding region #2 5 Human PD-1 epitope / binding region #3 6 Human PD-1 epitope / binding region #4 7 Human PD-1 epitope / binding region #5 8 Human PD-1 epitope / binding region #6 9 Human PD-1 epitope / binding region #7 10 Human PD-1 epitope / binding region #8 11 Human FcgRIA amino acid sequence 12 Human FcgRIIA(H131) amino acid sequence 13 Human FcgRIIA(R131) amino acid sequence 14 Human FcgRIIB amino acid sequence 15 Human FcgRIIIA (F158) amino acid sequence 16 Human FcgRIIIA(V158) amino acid sequence 17 Mouse HM242 VH region amino acid sequence 18 Humanized VH ver.A (HM242) amino acid sequence 19 Humanized VH ver.B (HM242) amino acid sequence 20 Humanized VH ver.C (HM242) amino acid sequence twenty one Humanized VH ver.D (HM242) amino acid sequence twenty two Mouse HM297 VH region amino acid sequence twenty three Humanized VH ver.E (HM297) amino acid sequence twenty four Humanized VH ver.F (HM297) amino acid sequence 25 Humanized VH ver.G (HM297) amino acid sequence 26 Humanized VH ver.H (HM297) amino acid sequence 27 Humanized VH ver.I (HM297) amino acid sequence 28 Mouse HM268 VH region amino acid sequence 29 Humanized VH ver.J (HM268) amino acid sequence 30 Humanized VH ver.K (HM268) amino acid sequence 31 Humanized VH ver.L (HM268) amino acid sequence 32 Humanized VH ver.M (HM268) amino acid sequence 33 Mouse HM266 VH region amino acid sequence 34 Mouse HM242 VL region amino acid sequence 35 Humanized VL ver.1 (HM242) amino acid sequence 36 Humanized VL ver.2 (HM242) amino acid sequence 37 Humanized VL ver.3 (HM242) amino acid sequence 38 Mouse HM297 VL region amino acid sequence 39 Humanized VL ver.4 (HM297) amino acid sequence 40 Humanized VL ver.5 (HM297) amino acid sequence 41 Humanized VL ver.6 (HM297) amino acid sequence 42 Mouse HM268 VL region amino acid sequence 43 Humanized VL ver.7 (HM268) amino acid sequence 44 Humanized VL ver.8 (HM268) amino acid sequence 45 Humanized VL ver.9 (HM268) amino acid sequence 46 Mouse HM266 VL region amino acid sequence 47 Human IgG1 WT Fc region amino acid sequence 48 Human IgG1 Fc_K322A amino acid sequence 49 Human IgG1 Fc_G236D / H268D(X2) amino acid sequence 50 Human IgG1 Fc G236D / H268D / K322A(X2-K) amino acid sequence 51 Human IgG1 Fc G236D / H268D / E293A / K322A(X2-KE) amino acid sequence 52 Human IgG1 Fc_S236D / S267G / H268D / K322A(X2-KS) amino acid sequence 53 Human IgG1 Fc_S239D / H268D(X3) amino acid sequence 54 Human IgG1 Fc_S239D / H268D / K322A(X3-K) amino acid sequence 55 Human IgG1 Fc S239D / H268D / E293A / K322A(X3-KE) amino acid sequence 56 Human IgG1 Fc_S239D / H268D / K322A / I332E(X3-KI) amino acid sequence 57 Human IgG1 Fc_S239D / H268D / L328Y / K322A(X3-KL) amino acid sequence 58 Human IgG1 Fc_S239D / S267G / H268D / K322A(X3-KS) amino acid sequence 59 Human IgG1 Fc_S239D / S267G / H268D / K322A / I332E(X3-KSI) amino acid sequence 60 Human IgG1 Fc_S239D / S267G / H268D / L328Y / K322A(X3-KSL) amino acid sequence 61 Human IgG1 Fc S239D / H268D / L328Y / I332E(X4) amino acid sequence 62 Human IgG1 Fc_S239D / H268D / K322A / L328Y / I332E(X4-K) amino acid sequence 63 Human IgG1 Fc_S239D / S267G / H268D / K322A / L328Y / I332E(X4-KS) amino acid sequence 64 Human IgG4 Fc amino acid sequence 65 Human IgG4 Fc_S228P amino acid sequence 66 Human IgG4 Fc_S228P / G236D / Q268D(X2-P) amino acid sequence 67 Human IgG4 Fc S228P / S239D / Q268D(X3-P) amino acid sequence 68 Human IgG4 Fc S228P / S239D / Q268D / L328Y / I332E(X4-P) amino acid sequence 69 Mouse HM242 heavy chain amino acid sequence 70 Mouse HM242 heavy chain DNA sequence 71 Mouse HM242 light chain amino acid sequence 72 Mouse HM242 light chain DNA sequence 73 Mouse HM266 heavy chain amino acid sequence 74 Mouse HM266 heavy chain DNA sequence 75 Mouse HM266 light chain amino acid sequence 76 Mouse HM266 light chain DNA sequence 77 Mouse HM268 heavy chain amino acid sequence 78 Mouse HM268 heavy chain DNA sequence 79 Mouse HM268 light chain amino acid sequence 80 Mouse HM268 light chain DNA sequence 81 Mouse HM297 heavy chain amino acid sequence 82 Mouse HM297 heavy chain DNA sequence 83 Mouse HM297 light chain amino acid sequence 84 Mouse HM297 light chain DNA sequence 85 Chimeric HM242-IgG1-K322A heavy chain amino acid sequence 86 Chimeric HM242-IgG1-K322A heavy chain DNA sequence 87 Chimeric HM242-IgG1-K322A light chain amino acid sequence 88 Chimeric HM242-IgG1-K322A light chain DNA sequence 89 Chimeric HM266-IgG1-K322A heavy chain amino acid sequence 90 Chimeric HM266-IgG1-K322A heavy chain DNA sequence 91 Chimeric HM266-IgG1-K322A light chain amino acid sequence 92 Chimeric HM266-IgG1-K322A light chain DNA sequence 93 Chimeric HM268-IgG1-K322A heavy chain amino acid sequence 94 Chimeric HM268-IgG1-K322A heavy chain DNA sequence 95 Chimeric HM268-IgG1-K322A light chain amino acid sequence 96 Chimeric HM268-IgG1-K322A light chain DNA sequence 97 Chimeric HM297-IgG1-K322A heavy chain amino acid sequence 98 Chimeric HM297-IgG1-K322A heavy chain DNA sequence 99 Chimeric HM297-IgG1-K322A light chain amino acid sequence 100 Chimeric HM297-IgG1-K322A light chain DNA sequence 101 Chimeric HM624-IgG1-K322A heavy chain amino acid sequence 102 Chimeric HM624-IgG1-K322A heavy chain DNA sequence 103 Chimeric HM624-IgG1-K322A light chain amino acid sequence 104 Chimeric HM624-IgG1-K322A light chain DNA sequence 105 Chimeric HM647-IgG1-K322A heavy chain amino acid sequence 106 Chimeric HM647-IgG1-K322A heavy chain DNA sequence 107 Chimeric HM647-IgG1-K322A light chain amino acid sequence 108 Chimeric HM647-IgG1-K322A light chain DNA sequence 109 Chimeric HM698-IgG1-K322A heavy chain amino acid sequence 110 Chimeric HM698-IgG1-K322A heavy chain DNA sequence 111 Chimeric HM698-IgG1-K322A light chain amino acid sequence 112 Chimeric HM698-IgG1-K322A light chain DNA sequence 113 Chimeric HM266-X2 heavy chain amino acid sequence 114 Chimeric HM266-X2 heavy chain DNA sequence 115 Chimeric HM266-X2 light chain amino acid sequence 116 Chimeric HM266-X2 light chain DNA sequence 117 Chimeric HM266-X2-K heavy chain amino acid sequence 118 Chimeric HM266-X2-K heavy chain DNA sequence 119 Chimeric HM266-X2-K light chain amino acid sequence 120 Chimeric HM266-X2-K light chain DNA sequence 121 Chimeric HM266-X2-KE heavy chain amino acid sequence 122 Chimeric HM266-X2-KE heavy chain DNA sequence 123 Chimeric HM266-X2-KE light chain amino acid sequence 124 Chimeric HM266-X2-KE light chain DNA sequence 125 Chimeric HM266-X2-KS heavy chain amino acid sequence 126 Chimeric HM266-X2-KS heavy chain DNA sequence 127 Chimeric HM266-X2-KS light chain amino acid sequence 128 Chimeric HM266-X2-KS light chain DNA sequence 129 Chimeric HM266-X3 heavy chain amino acid sequence 130 Chimeric HM266-X3 heavy chain DNA sequence 131 Chimeric HM266-X3 light chain amino acid sequence 132 Chimeric HM266-X3 light chain DNA sequence 133 Chimeric HM266-X3-K heavy chain amino acid sequence 134 Chimeric HM266-X3-K heavy chain DNA sequence 135 Chimeric HM266-X3-K light chain amino acid sequence 136 Chimeric HM266-X3-K light chain DNA sequence 137 Chimeric HM266-X3-KE heavy chain amino acid sequence 138 Chimeric HM266-X3-KE heavy chain DNA sequence 139 Chimeric HM266-X3-KE light chain amino acid sequence 140 Chimeric HM266-X3-KE light chain DNA sequence 141 Chimeric HM266-X3-KS heavy chain amino acid sequence 142 Chimeric HM266-X3-KS heavy chain DNA sequence 143 Chimeric HM266-X3-KS light chain amino acid sequence 144 Chimeric HM266-X3-KS light chain DNA sequence 145 Chimeric HM266-X4 heavy chain amino acid sequence 146 Chimeric HM266-X4 heavy chain DNA sequence 147 Chimeric HM266-X4 light chain amino acid sequence 148 Chimeric HM266-X4 light chain DNA sequence 149 Chimeric HM266-X4-K heavy chain amino acid sequence 150 Chimeric HM266-X4-K heavy chain DNA sequence 151 Chimeric HM266-X4-K light chain amino acid sequence 152 Chimeric HM266-X4-K light chain DNA sequence 153 Chimeric HM266-IgG4-S228P heavy chain amino acid sequence 154 Chimeric HM266-IgG4-S228P heavy chain DNA sequence 155 Chimeric HM266-IgG4-S228P light chain amino acid sequence 156 Chimeric HM266-IgG4-S228P light chain DNA sequence 157 Chimeric HM266-X2-P heavy chain amino acid sequence 158 Chimeric HM266-X2-P heavy chain DNA sequence 159 Chimeric HM266-X2-P light chain amino acid sequence 160 Chimeric HM266-X2-P light chain DNA sequence 161 Chimeric HM266-X3-P heavy chain amino acid sequence 162 Chimeric HM266-X3-P heavy chain DNA sequence 163 Chimeric HM266-X3-P light chain amino acid sequence 164 Chimeric HM266-X3-P light chain DNA sequence 165 Chimeric HM266-X4-P heavy chain amino acid sequence 166 Chimeric HM266-X4-P heavy chain DNA sequence 167 Chimeric HM266-X4-P light chain amino acid sequence 168 Chimeric HM266-X4-P light chain DNA sequence 169 Humanized HM242-C3-IgG1-K322A heavy chain amino acid sequence 170 Humanized HM242-C3-IgG1-K322A heavy chain DNA sequence 171 Humanized HM242-C3-IgG1-K322A light chain amino acid sequence 172 Humanized HM242-C3-IgG1-K322A light chain DNA sequence 173 Humanized HM242-D3-IgG1-K322A heavy chain amino acid sequence 174 Humanized HM242-D3-IgG1-K322A heavy chain DNA sequence 175 Humanized HM242-D3-IgG1-K322A light chain amino acid sequence 176 Humanized HM242-D3-IgG1-K322A light chain DNA sequence 177 Humanized HM268-K8-IgG1-K322A heavy chain amino acid sequence 178 Humanized HM268-K8-IgG1-K322A heavy chain DNA sequence 179 Humanized HM268-K8-IgG1-K322A light chain amino acid sequence 180 Humanized HM268-K8-IgG1-K322A light chain DNA sequence 181 Humanized HM297-H5-IgG1-K322A heavy chain amino acid sequence 182 Humanized HM297-H5-IgG1-K322A heavy chain DNA sequence 183 Humanized HM297-H5-IgG1-K322A light chain amino acid sequence 184 Humanized HM297-H5-IgG1-K322A light chain DNA sequence 185 Humanized HM242-C3-X2-K heavy chain amino acid sequence 186 Humanized HM242-C3-X2-K heavy chain DNA sequence 187 Humanized HM242-C3-X2-K light chain amino acid sequence 188 Humanized HM242-C3-X2-K light chain DNA sequence 189 Humanized HM242-C3-X3-K heavy chain amino acid sequence 190 Humanized HM242-C3-X3-K heavy chain DNA sequence 191 Humanized HM242-C3-X3-K light chain amino acid sequence 192 Humanized HM242-C3-X3-K light chain DNA sequence 193 Humanized HM242-C3-X3-KE heavy chain amino acid sequence 194 Humanized HM242-C3-X3-KE heavy chain DNA sequence 195 Humanized HM242-C3-X3-KE light chain amino acid sequence 196 Humanized HM242-C3-X3-KE light chain DNA sequence 197 Humanized HM242-C3-X3-KI heavy chain amino acid sequence 198 Humanized HM242-C3-X3-KI heavy chain DNA sequence 199 Humanized HM242-C3-X3-KI light chain amino acid sequence 200 Humanized HM242-C3-X3-KI light chain DNA sequence 201 Humanized HM242-C3-X3-KL heavy chain amino acid sequence 202 Humanized HM242-C3-X3-KL heavy chain DNA sequence 203 Humanized HM242-C3-X3-KL light chain amino acid sequence 204 Humanized HM242-C3-X3-KL light chain DNA sequence 205 Humanized HM242-C3-X3-KS heavy chain amino acid sequence 206 Humanized HM242-C3-X3-KS heavy chain DNA sequence 207 Humanized HM242-C3-X3-KS light chain amino acid sequence 208 Humanized HM242-C3-X3-KS light chain DNA sequence 209 Humanized HM242-C3-X3-KSI heavy chain amino acid sequence 210 Humanized HM242-C3-X3-KSI heavy chain DNA sequence 211 Humanized HM242-C3-X3-KSI light chain amino acid sequence 212 Humanized HM242-C3-X3-KSI light chain DNA sequence 213 Humanized HM242-C3-X3-KSL heavy chain amino acid sequence 214 Humanized HM242-C3-X3-KSL heavy chain DNA sequence 215 Humanized HM242-C3-X3-KSL light chain amino acid sequence 216 Humanized HM242-C3-X3-KSL light chain DNA sequence 217 Humanized HM242-C3-X4-K heavy chain amino acid sequence 218 Humanized HM242-C3-X4-K heavy chain DNA sequence 219 Humanized HM242-C3-X4-K light chain amino acid sequence 220 Humanized HM242-C3-X4-K light chain DNA sequence 221 Humanized HM242-C3-X4-KS heavy chain amino acid sequence 222 Humanized HM242-C3-X4-KS heavy chain DNA sequence 223 Humanized HM242-C3-X4-KS light chain amino acid sequence 224 Humanized HM242-C3-X4-KS light chain DNA sequence 225 Humanized HM242-D3-X2-K heavy chain amino acid sequence 226 Humanized HM242-D3-X2-K heavy chain DNA sequence 227 Humanized HM242-D3-X2-K light chain amino acid sequence 228 Humanized HM242-D3-X2-K light chain DNA sequence 229 Humanized HM242-D3-X3-K heavy chain amino acid sequence 230 Humanized HM242-D3-X3-K heavy chain DNA sequence 231 Humanized HM242-D3-X3-K light chain amino acid sequence 232 Humanized HM242-D3-X3-K light chain DNA sequence 233 Humanized HM242-D3-X3-KE heavy chain amino acid sequence 234 Humanized HM242-D3-X3-KE heavy chain DNA sequence 235 Humanized HM242-D3-X3-KE light chain amino acid sequence 236 Humanized HM242-D3-X3-KE light chain DNA sequence 237 Humanized HM242-D3-X3-KI heavy chain amino acid sequence 238 Humanized HM242-D3-X3-KI heavy chain DNA sequence 239 Humanized HM242-D3-X3-KI light chain amino acid sequence 240 Humanized HM242-D3-X3-KI light chain DNA sequence 241 Humanized HM242-D3-X3-KL heavy chain amino acid sequence 242 Humanized HM242-D3-X3-KL heavy chain DNA sequence 243 Humanized HM242-D3-X3-KL light chain amino acid sequence 244 Humanized HM242-D3-X3-KL light chain DNA sequence 245 Humanized HM242-D3-X3-KS heavy chain amino acid sequence 246 Humanized HM242-D3-X3-KS heavy chain DNA sequence 247 Humanized HM242-D3-X3-KS light chain amino acid sequence 248 Humanized HM242-D3-X3-KS light chain DNA sequence 249 Humanized HM242-D3-X3-KSI heavy chain amino acid sequence 250 Humanized HM242-D3-X3-KSI heavy chain DNA sequence 251 Humanized HM242-D3-X3-KSI light chain amino acid sequence 252 Humanized HM242-D3-X3-KSI light chain DNA sequence 253 Humanized HM242-D3-X3-KSL heavy chain amino acid sequence 254 Humanized HM242-D3-X3-KSL heavy chain DNA sequence 255 Humanized HM242-D3-X3-KSL light chain amino acid sequence 256 Humanized HM242-D3-X3-KSL light chain DNA sequence 257 Humanized HM242-D3-X4-K heavy chain amino acid sequence 258 Humanized HM242-D3-X4-K heavy chain DNA sequence 259 Humanized HM242-D3-X4-K light chain amino acid sequence 260 Humanized HM242-D3-X4-K light chain DNA sequence 261 Humanized HM242-D3-X4-KS heavy chain amino acid sequence 262 Humanized HM242-D3-X4-KS heavy chain DNA sequence 263 Humanized HM242-D3-X4-KS light chain amino acid sequence 264 Humanized HM242-D3-X4-KS light chain DNA sequence 265 Humanized HM268-K8-X2-K heavy chain amino acid sequence 266 Humanized HM268-K8-X2-K heavy chain DNA sequence 267 Humanized HM268-K8-X2-K light chain amino acid sequence 268 Humanized HM268-K8-X2-K light chain DNA sequence 269 Humanized HM268-K8-X3-K heavy chain amino acid sequence 270 Humanized HM268-K8-X3-K heavy chain DNA sequence 271 Humanized HM268-K8-X3-K light chain amino acid sequence 272 Humanized HM268-K8-X3-K light chain DNA sequence 273 Humanized HM268-K8-X3-KE heavy chain amino acid sequence 274 Humanized HM268-K8-X3-KE heavy chain DNA sequence 275 Humanized HM268-K8-X3-KE light chain amino acid sequence 276 Humanized HM268-K8-X3-KE light chain DNA sequence 277 Humanized HM268-K8-X3-KI heavy chain amino acid sequence 278 Humanized HM268-K8-X3-KI heavy chain DNA sequence 279 Humanized HM268-K8-X3-KI light chain amino acid sequence 280 Humanized HM268-K8-X3-KI light chain DNA sequence 281 Humanized HM268-K8-X3-KL heavy chain amino acid sequence 282 Humanized HM268-K8-X3-KL heavy chain DNA sequence 283 Humanized HM268-K8-X3-KL light chain amino acid sequence 284 Humanized HM268-K8-X3-KL light chain DNA sequence 285 Humanized HM268-K8-X3-KS heavy chain amino acid sequence 286 Humanized HM268-K8-X3-KS heavy chain DNA sequence 287 Humanized HM268-K8-X3-KS light chain amino acid sequence 288 Humanized HM268-K8-X3-KS light chain DNA sequence 289 Humanized HM268-K8-X3-KSI heavy chain amino acid sequence 290 Humanized HM268-K8-X3-KSI heavy chain DNA sequence 291 Humanized HM268-K8-X3-KSI light chain amino acid sequence 292 Humanized HM268-K8-X3-KSI light chain DNA sequence 293 Humanized HM268-K8-X3-KSL heavy chain amino acid sequence 294 Humanized HM268-K8-X3-KSL heavy chain DNA sequence 295 Humanized HM268-K8-X3-KSL light chain amino acid sequence 296 Humanized HM268-K8-X3-KSL light chain DNA sequence 297 Humanized HM268-K8-X4-K heavy chain amino acid sequence 298 Humanized HM268-K8-X4-K heavy chain DNA sequence 299 Humanized HM268-K8-X4-K light chain amino acid sequence 300 Humanized HM268-K8-X4-K light chain DNA sequence 301 Humanized HM268-K8-X4-KS heavy chain amino acid sequence 302 Humanized HM268-K8-X4-KS heavy chain DNA sequence 303 Humanized HM268-K8-X4-KS light chain amino acid sequence 304 Humanized HM268-K8-X4-KS light chain DNA sequence 305 Humanized HM297-H5-X2-K heavy chain amino acid sequence 306 Humanized HM297-H5-X2-K heavy chain DNA sequence 307 Humanized HM297-H5-X2-K light chain amino acid sequence 308 Humanized HM297-H5-X2-K light chain DNA sequence 309 Humanized HM297-H5-X3-KS heavy chain amino acid sequence 310 Humanized HM297-H5-X3-KS heavy chain DNA sequence 311 Humanized HM297-H5-X3-KS light chain amino acid sequence 312 Humanized HM297-H5-X3-KS light chain DNA sequence 313 Humanized HM297-H5-X4-K heavy chain amino acid sequence 314 Humanized HM297-H5-X4-K heavy chain DNA sequence 315 Humanized HM297-H5-X4-K light chain amino acid sequence 316 Humanized HM297-H5-X4-K light chain DNA sequence 317 Humanized HM297-H5-X3-K heavy chain amino acid sequence 318 Humanized HM297-H5-X3-K heavy chain DNA sequence 319 Humanized HM297-H5-X3-K light chain amino acid sequence 320 Humanized HM297-H5-X3-K light chain DNA sequence 321 Humanized HM297-H5-X3-KSL heavy chain amino acid sequence 322 Humanized HM297-H5-X3-KSL heavy chain DNA sequence 323 Humanized HM297-H5-X3-KSL light chain amino acid sequence 324 Humanized HM297-H5-X3-KSL light chain DNA sequence 325 Humanized HM297-H5-X3-KSI heavy chain amino acid sequence 326 Humanized HM297-H5-X3-KSI heavy chain DNA sequence 327 Humanized HM297-H5-X3-KSI light chain amino acid sequence 328 Humanized HM297-H5-X3-KSI light chain DNA sequence 329 Humanized HM297-H5-X3-KL heavy chain amino acid sequence 330 Humanized HM297-H5-X3-KL heavy chain DNA sequence 331 Humanized HM297-H5-X3-KL light chain amino acid sequence 332 Humanized HM297-H5-X3-KL light chain DNA sequence 333 Humanized HM297-H5-X3-KI heavy chain amino acid sequence 334 Humanized HM297-H5-X3-KI heavy chain DNA sequence 335 Humanized HM297-H5-X3-KI light chain amino acid sequence 336 Humanized HM297-H5-X3-KI light chain DNA sequence 337 Humanized HM297-H5-X4-KS heavy chain amino acid sequence 338 Humanized HM297-H5-X4-KS heavy chain DNA sequence 339 Humanized HM297-H5-X4-KS light chain amino acid sequence 340 Humanized HM297-H5-X4-KS light chain DNA sequence 341 Humanized HM297-H5-X3-KE heavy chain amino acid sequence 342 Humanized HM297-H5-X3-KE heavy chain DNA sequence 343 Humanized HM297-H5-X3-KE light chain amino acid sequence 344 Humanized HM297-H5-X3-KE light chain DNA sequence 345 HM242-CDR-H1 amino acid sequence (Kabat) 346 HM242-CDR-H2 amino acid sequence (Kabat) 347 HM242-CDR-H3 amino acid sequence (Kabat) 348 HM242-CDR-L1 amino acid sequence (Kabat) 349 HM242-CDR-L2 amino acid sequence (Kabat) 350 HM242-CDR-L3 amino acid sequence (Kabat) 351 HM266-CDR-H1 amino acid sequence (Kabat) 352 HM266-CDR-H2 amino acid sequence (Kabat) 353 HM266-CDR-H3 amino acid sequence (Kabat) 354 HM266-CDR-L1 amino acid sequence (Kabat) 355 HM266-CDR-L2 amino acid sequence (Kabat) 356 HM266-CDR-L3 amino acid sequence (Kabat) 357 HM268-CDR-H1 amino acid sequence (Kabat) 358 HM268-CDR-H2 amino acid sequence (Kabat) 359 HM268-CDR-H3 amino acid sequence (Kabat) 360 HM268-CDR-L1 amino acid sequence (Kabat) 361 HM268-CDR-L2 amino acid sequence (Kabat) 362 HM268-CDR-L3 amino acid sequence (Kabat) 363 HM297-CDR-H1 amino acid sequence (Kabat) 364 HM297-CDR-H2 amino acid sequence (Kabat) 365 HM297-CDR-H3 amino acid sequence (Kabat) 366 HM297-CDR-L1 amino acid sequence (Kabat) 367 HM297-CDR-L2 amino acid sequence (Kabat) 368 HM297-CDR-L3 amino acid sequence (Kabat) 369 HM242-CDR-H1 amino acid sequence (Chothia) 370 HM242-CDR-H2 amino acid sequence (Chothia) 371 HM242-CDR-H3 amino acid sequence (Chothia) 372 HM242-CDR-L1 amino acid sequence (Chothia) 373 HM242-CDR-L2 amino acid sequence (Chothia) 374 HM242-CDR-L3 amino acid sequence (Chothia) 375 HM266-CDR-H1 amino acid sequence (Chothia) 376 HM266-CDR-H2 amino acid sequence (Chothia) 377 HM266-CDR-H3 amino acid sequence (Chothia) 378 HM266-CDR-L1 amino acid sequence (Chothia) 379 HM266-CDR-L2 amino acid sequence (Chothia) 380 HM266-CDR-L3 amino acid sequence (Chothia) 381 HM268-CDR-H1 amino acid sequence (Chothia) 382 HM268-CDR-H2 amino acid sequence (Chothia) 383 HM268-CDR-H3 amino acid sequence (Chothia) 384 HM268-CDR-L1 amino acid sequence (Chothia) 385 HM268-CDR-L2 amino acid sequence (Chothia) 386 HM268-CDR-L3 amino acid sequence (Chothia) 387 HM297-CDR-H1 amino acid sequence (Chothia) 388 HM297-CDR-H2 amino acid sequence (Chothia) 389 HM297-CDR-H3 amino acid sequence (Chothia) 390 HM297-CDR-L1 amino acid sequence (Chothia) 391 HM297-CDR-L2 amino acid sequence (Chothia) 392 HM297-CDR-L3 amino acid sequence (Chothia) 393 HM242-CDR-H1 amino acid sequence (IMGT) 394 HM242-CDR-H2 amino acid sequence (IMGT) 395 HM242-CDR-H3 amino acid sequence (IMGT) 396 HM242-CDR-L1 amino acid sequence (IMGT) 397 HM242-CDR-L2 amino acid sequence (IMGT) 398 HM242-CDR-L3 amino acid sequence (IMGT) 399 HM266-CDR-H1 amino acid sequence (IMGT) 400 HM266-CDR-H2 amino acid sequence (IMGT) 401 HM266-CDR-H3 amino acid sequence (IMGT) 402 HM266-CDR-L1 amino acid sequence (IMGT) 403 HM266-CDR-L2 amino acid sequence (IMGT) 404 HM266-CDR-L3 amino acid sequence (IMGT) 405 HM268-CDR-H1 amino acid sequence (IMGT) 406 HM268-CDR-H2 amino acid sequence (IMGT) 407 HM268-CDR-H3 amino acid sequence (IMGT) 408 HM268-CDR-L1 amino acid sequence (IMGT) 409 HM268-CDR-L2 amino acid sequence (IMGT) 410 HM268-CDR-L3 amino acid sequence (IMGT) 411 HM297-CDR-H1 amino acid sequence (IMGT) 412 HM297-CDR-H2 amino acid sequence (IMGT) 413 HM297-CDR-H3 amino acid sequence (IMGT) 414 HM297-CDR-L1 amino acid sequence (IMGT) 415 HM297-CDR-L2 amino acid sequence (IMGT) 416 HM297-CDR-L3 amino acid sequence (IMGT) 417 HM242-CDR-H1 amino acid sequence (paratome) 418 HM242-CDR-H2 amino acid sequence (paratome) 419 HM242-CDR-H3 amino acid sequence (paratome) 420 HM242-CDR-L1 amino acid sequence (paratome) 421 HM242-CDR-L2 amino acid sequence (paratome) 422 HM242-CDR-L3 amino acid sequence (paratome) 423 HM266-CDR-H1 amino acid sequence (paratome) 424 HM266-CDR-H2 amino acid sequence (paratome) 425 HM266-CDR-H3 amino acid sequence (paratome) 426 HM266-CDR-L1 amino acid sequence (paratome) 427 HM266-CDR-L2 amino acid sequence (paratome) 428 HM266-CDR-L3 amino acid sequence (paratome) 429 HM268-CDR-H1 amino acid sequence (paratome) 430 HM268-CDR-H2 amino acid sequence (paratome) 431 HM268-CDR-H3 amino acid sequence (paratome) 432 HM268-CDR-L1 amino acid sequence (paratome) 433 HM268-CDR-L2 amino acid sequence (paratome) 434 HM268-CDR-L3 amino acid sequence (paratome) 435 HM297-CDR-H1 amino acid sequence (paratome) 436 HM297-CDR-H2 amino acid sequence (paratome) 437 HM297-CDR-H3 amino acid sequence (paratome) 438 HM297-CDR-L1 amino acid sequence (paratome) 439 HM297-CDR-L2 amino acid sequence (paratome) 440 HM297-CDR-L3 amino acid sequence (paratome)

[0136] <![CDATA[ <110> National Institutes of Biomedical Innovation, Health and Nutrition Public Interest Incorporated Foundation for Biomedical Research and Innovation at Kobe Meiji Seika Pharmaceutical Co., Ltd. <![CDATA[ <120> Anti-human PD-1 agonist antibodies and pharmaceutical compositions containing them for the treatment or prevention of inflammatory diseases. <![CDATA[ <130> FP-314PCT <![CDATA[ <150> JP2021-081913 <![CDATA[ <151> 2021-05-13 <![CDATA[ <150> JP2021-086534 <![CDATA[ <151> 2021-05-21 <![CDATA[ <160> 440 ]]> <![CDATA[ <170> PatentIn version 3.5]]> <![CDATA[ <210> 1]]> <![CDATA[ <211> 288]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Homo sapiens <![CDATA[ <400> 1]]> Met Gln Ile Pro Gln Ala Pro Trp Pro Val Val Trp Ala Val Leu Gln 1 5 10 15 Leu Gly Trp Arg Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp 20 25 30 Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp 35 40 45 Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val 50 55 60 Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala 65 70 75 80 Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg 85 90 95 Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg 100 105 110 Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu 115 120 125 Ala Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val 130 135 140 Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro 145 150 155 160 Arg Pro Ala Gly Gln Phe Gln Thr Leu Val Val Gly Val Val Gly Gly 165 170 175 Leu Leu Gly Ser Leu Val Leu Leu Val Trp Val Leu Ala Val Ile Cys 180 185 190 Ser Arg Ala Ala Arg Gly Thr Ile Gly Ala Arg Arg Thr Gly Gln Pro 195 200 205 Leu Lys Glu Asp Pro Ser Ala Val Pro Val Phe Ser Val Asp Tyr Gly 210 215 220 Glu Leu Asp Phe Gln Trp Arg Glu Lys Thr Pro Glu Pro Pro Val Pro 225 230 235 240 Cys Val Pro Glu Gln Thr Glu Tyr Ala Thr Ile Val Phe Pro Ser Gly 245 250 255 Met Gly Thr Ser Ser Pro Ala Arg Arg Gly Ser Ala Asp Gly Pro Arg 260 265 270 Ser Ala Gln Pro Leu Arg Pro Glu Asp Gly His Cys Ser Trp Pro Leu 275 280 285 <![CDATA[<210> 2]]> <![CDATA[<211> 288]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Mus musculus]]> <![CDATA[<400> 2]]> Met Trp Val Arg Gln Val Pro Trp Ser Phe Thr Trp Ala Val Leu Gln 1 5 10 15 <0x [[ID=5l]]Leu Ser Trp Gln Ser Gly Trp Leu Leu Glu Val Pro Asn Gly Pro Trp 20 25 30 Note: There seems to be an error in the original text where the tag <0x is written with a lowercase 'x' in the middle. I've left it as is in the translation as per the instruction to preserve all tags exactly. If this is a known issue, it might need to be corrected in the original source. Arg Ser Leu Thr Phe Tyr Pro Ala Trp Leu Thr Val Ser Glu Gly Ala 35 40 45 Asn Ala Thr Phe Thr Cys Ser Leu Ser Asn Trp Ser Glu Asp Leu Met 50 55 60 Leu Asn Trp Asn Arg Leu Ser Pro Ser Asn Gln Thr Glu Lys Gln Ala 65 70 75 80 Ala Phe Cys Asn Gly Leu Ser Gln Pro Val Gln Asp Ala Arg Phe Gln 85 90 95 Ile Ile Gln Leu Pro Asn Arg His Asp Phe His Met Asn Ile Leu Asp 100 105 110 Thr Arg Arg Asn Asp Ser Gly Ile Tyr Leu Cys Gly Ala Ile Ser Leu 115 120 125 His Pro Lys Ala Lys Ile Glu Glu Ser Pro Gly Ala Glu Leu Val Val 130 135 140 Thr Glu Arg Ile Leu Glu Thr Ser Thr Arg Tyr Pro Ser Pro Ser Pro 145 150 155 160 Lys Pro Glu Gly Arg Phe Gln Gly Met Val Ile Gly Ile Met Ser Ala 165 170 175 Leu Val Gly Ile Pro Val Leu Leu Leu Leu Ala Trp Ala Leu Ala Val 180 185 190 Phe Cys Ser Thr Ser Met Ser Glu Ala Arg Gly Ala Gly Ser Lys Asp 195 200 205 Asp Thr Leu Lys Glu Glu Pro Ser Ala Ala Pro Val Pro Ser Val Ala 210 215 220 Tyr Glu Glu Leu Asp Phe Gln Gly Arg Glu Lys Thr Pro Glu Leu Pro 225 230 235 240 Thr Ala Cys Val His Thr Glu Tyr Ala Thr Ile Val Phe Thr Glu Gly 245 250 255 Leu Gly Ala Ser Ala Met Gly Arg Arg Gly Ser Ala Asp Gly Leu Gln 260 265 270 Gly Pro Arg Pro Pro Arg His Glu Asp Gly His Cys Ser Trp Pro Leu 275 280 285 <![CDATA[ <210> 3]]> <![CDATA[ <211> 11]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Homo sapiens <![CDATA[ <400> 3]]> Ala Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg 1 5 10 <![CDATA[ <210> 4]]> <![CDATA[ <211> 9]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Homo sapiens <![CDATA[ <400> 4]]> Phe Ser Asn Thr Ser Glu Ser Phe Val 1 5 <![CDATA[ <210> 5]]> <![CDATA[ <211> 11]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Homo sapiens <![CDATA[ <400> 5]]> Asp Ser Pro Asp Arg Pro Trp Asn Pro Pro Thr 1 5 10 <![CDATA[ <210> 6]]> <![CDATA[ <211> 9]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Homo sapiens <![CDATA[ <400> 6]]> Arg Val Thr Gln Leu Pro Asn Gly Arg 1 5 <![CDATA[ <210> 7]]> <![CDATA[<211> 11]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<400> 7]]> Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys 1 5 10 <![CDATA[<210> 8]]> <![CDATA[<211> 12]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<400> 8]]> Ser Val Val Arg Ala Arg Arg Asn Asp Ser Gly Thr 1 5 10 <![CDATA[<210> 9]]> <![CDATA[<211> 11]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<400> 9]]> Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp 1 5 10 <![CDATA[<210> 10]]> <![CDATA[<211> 11]]> <![CDATA[<212> PRT]]> <![CDATA[<213> ]]Homo sapiens <![CDATA[<400> 10]]> Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro 1 5 10 <![CDATA[<210> 11]]> <![CDATA[<211> 374]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<400> 11]]> Met Trp Phe Leu Thr Thr Leu Leu Leu Trp Val Pro Val Asp Gly Gln 1 5 10 15 Val Asp Thr Thr Lys Ala Val Ile Thr Leu Gln Pro Pro Trp Val Ser 20 25 30 Val Phe Gln Glu Glu Thr Val Thr Leu His Cys Glu Val Leu His Leu 35 40 45 Pro Gly Ser Ser Ser Thr Gln Trp Phe Leu Asn Gly Thr Ala Thr Gln 50 55 60 Thr Ser Thr Pro Ser Tyr Arg Ile Thr Ser Ala Ser Val Asn Asp Ser 65 70 75 80 Gly Glu Tyr Arg Cys Gln Arg Gly Leu Ser Gly Arg Ser Asp Pro Ile 85 90 95 Gln Leu Glu Ile His Arg Gly Trp Leu Leu Leu Gln Val Ser Ser Arg 100 105 110 Val Phe Thr Glu Gly Glu Pro Leu Ala Leu Arg Cys His Ala Trp Lys 115 120 125 Asp Lys Leu Val Tyr Asn Val Leu Tyr Tyr Arg Asn Gly Lys Ala Phe 130 135 140 Lys Phe Phe His Trp Asn Ser Asn Leu Thr Ile Leu Lys Thr Asn Ile 145 150 155 160 Ser His Asn Gly Thr Tyr His Cys Ser Gly Met Gly Lys His Arg Tyr 165 170 175 Thr Ser Ala Gly Ile Ser Val Thr Val Lys Glu Leu Phe Pro Ala Pro 180 185 190 Val Leu Asn Ala Ser Val Thr Ser Pro Leu Leu Glu Gly Asn Leu Val 195 200 205 Thr Leu Ser Cys Glu Thr Lys Leu Leu Leu Gln Arg Pro Gly Leu Gln 210 215 220 Leu Tyr Phe Ser Phe Tyr Met Gly Ser Lys Thr Leu Arg Gly Arg Asn 225 230 235 240 Thr Ser Ser Glu Tyr Gln Ile Leu Thr Ala Arg Arg Glu Asp Ser Gly 245 250 255 Leu Tyr Trp Cys Glu Ala Ala Thr Glu Asp Gly Asn Val Leu Lys Arg 260 265 270 Ser Pro Glu Leu Glu Leu Gln Val Leu Gly Leu Gln Leu Pro Thr Pro 275 280 285 Val Trp Phe His Val Leu Phe Tyr Leu Ala Val Gly Ile Met Phe Leu 290 295 300 Val Asn Thr Val Leu Trp Val Thr Ile Arg Lys Glu Leu Lys Arg Lys 305 310 315 320 Lys Lys Trp Asp Leu Glu Ile Ser Leu Asp Ser Gly His Glu Lys Lys 325 330 335 Val Ile Ser Ser Leu Gln Glu Asp Arg His Leu Glu Glu Glu Leu Lys 340 345 350 Cys Gln Glu Gln Lys Glu Glu Gln Leu Gln Glu Gly Val His Arg Lys 355 360 365 <![CDATA[<210> 12]]> <![CDATA[<211> 317]]> Glu Pro Gln Gly Ala Thr <000;2830> 370 [[ID=;1]] <![CDATA[<210> 12]]>[[ID=;7]] <![CDATA[<211> 317]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<400> 12]]> Met Thr Met Glu Thr Gln Met Ser Gln Asn Val Cys Pro Arg Asn Leu 1 5 10 15 It should be noted that there may be some incorrect or unclear parts in the original text, especially in the tags. The above translation is based on the best understanding of the content. If possible, it is recommended to double-check the accuracy of the original text for a more accurate translation. Trp Leu Leu Gln Pro Leu Thr Val Leu Leu Leu Leu Ala Ser Ala Asp 20 25 30 Ser Gln Ala Ala Ala Pro Pro Lys Ala Val Leu Lys Leu Glu Pro Pro 35 40 45 Trp Ile Asn Val Leu Gln Glu Asp Ser Val Thr Leu Thr Cys Gln Gly 50 55 60 Ala Arg Ser Pro Glu Ser Asp Ser Ile Gln Trp Phe His Asn Gly Asn 65 70 75 80 Leu Ile Pro Thr His Thr Gln Pro Ser Tyr Arg Phe Lys Ala Asn Asn 85 90 95 Asn Asp Ser Gly Glu Tyr Thr Cys Gln Thr Gly Gln Thr Ser Leu Ser 100 105 110 Asp Pro Val His Leu Thr Val Leu Ser Glu Trp Leu Val Leu Gln Thr 115 120 125 Pro His Leu Glu Phe Gln Glu Gly Glu Thr Ile Met Leu Arg Cys His 130 135 140 Ser Trp Lys Asp Lys Pro Leu Val Lys Val Thr Phe Phe Gln Asn Gly 145 150 155 160 Lys Ser Gln Lys Phe Ser His Leu Asp Pro Thr Phe Ser Ile Pro Gln 165 170 175 Ala Asn His Ser His Ser Gly Asp Tyr His Cys Thr Gly Asn Ile Gly 180 185 190 Tyr Thr Leu Phe Ser Ser Lys Pro Val Thr Ile Thr Val Gln Val Pro 195 200 205 Ser Met Gly Ser Ser Ser Pro Met Gly Ile Ile Val Ala Val Val Ile 210 215 220 Ala Thr Ala Val Ala Ala Ile Val Ala Ala Val Val Ala Leu Ile Tyr 225 230 235 240 Cys Arg Lys Lys Arg Ile Ser Ala Asn Ser Thr Asp Pro Val Lys Ala 245 250 255 Ala Gln Phe Glu Pro Pro Gly Arg Gln Met Ile Ala Ile Arg Lys Arg 260 265 270 Gln Leu Glu Glu Thr Asn Asn Asp Tyr Glu Thr Ala Asp Gly Gly Tyr 275 280 285 Met Thr Leu Asn Pro Arg Ala Pro Thr Asp Asp Asp Lys Asn Ile Tyr 290 295 300 Leu Thr Leu Pro Pro Asn Asp His Val Asn Ser Asn Asn 305 310 315 <![CDATA[<210> 13]]> <![CDATA[<211> 317]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<400> 13]]> Met Thr Met Glu Thr Gln Met Ser Gln Asn Val Cys Pro Arg Asn Leu 1 5 10 15 Trp Leu Leu Gln Pro Leu Thr Val Leu Leu Leu Leu Ala Ser Ala Asp 20 25 30 Ser Gln Ala Ala Ala Pro Pro Lys Ala Val Leu Lys Leu Glu Pro Pro 35 40 45 Trp Ile Asn Val Leu Gln Glu Asp Ser Val Thr Leu Thr Cys Gln Gly 50 55 60 Ala Arg Ser Pro Glu Ser Asp Ser Ile Gln Trp Phe His Asn Gly Asn 65 70 75 80 Leu Ile Pro Thr His Thr Gln Pro Ser Tyr Arg Phe Lys Ala Asn Asn 85 90 95 Asn Asp Ser Gly Glu Tyr Thr Cys Gln Thr Gly Gln Thr Ser Leu Ser 100 105 110 Asp Pro Val His Leu Thr Val Leu Ser Glu Trp Leu Val Leu Gln Thr 115 120 125 Pro His Leu Glu Phe Gln Glu Gly Glu Thr Ile Met Leu Arg Cys His 130 135 140 Ser Trp Lys Asp Lys Pro Leu Val Lys Val Thr Phe Phe Gln Asn Gly 145 150 155 160 Lys Ser Gln Lys Phe Ser Arg Leu Asp Pro Thr Phe Ser Ile Pro Gln 165 170 175 Ala Asn His Ser His Ser Gly Asp Tyr His Cys Thr Gly Asn Ile Gly 180 185 190 Tyr Thr Leu Phe Ser Ser Lys Pro Val Thr Ile Thr Val Gln Val Pro 195 200 205 Ser Met Gly Ser Ser Ser Pro Met Gly Ile Ile Val Ala Val Val Ile 210 215 220 Ala Thr Ala Val Ala Ala Ile Val Ala Ala Val Val Ala Leu Ile Tyr 225 230 235 240 Cys Arg Lys Lys Arg Ile Ser Ala Asn Ser Thr Asp Pro Val Lys Ala 245 250 255 Ala Gln Phe Glu Pro Pro Gly Arg Gln Met Ile Ala Ile Arg Lys Arg 260 265 270 Gln Leu Glu Glu Thr Asn Asn Asp Tyr Glu Thr Ala Asp Gly Gly Tyr 275 280 285 Met Thr Leu Asn Pro Arg Ala Pro Thr Asp Asp Asp Lys Asn Ile Tyr 290 295 300 Leu Thr Leu Pro Pro Asn Asp His Val Asn Ser Asn Asn 305 310 315 <![CDATA[<210> 14]]> <![CDATA[<211> 310]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<400> 14]]> Met Gly Ile Leu Ser Phe Leu Pro Val Leu Ala Thr Glu Ser Asp Trp 1 5 10 15 Ala Asp Cys Lys Ser Pro Gln Pro Trp Gly His Met Leu Leu Trp Thr 20 25 30 Ala Val Leu Phe Leu Ala Pro Val Ala Gly Thr Pro Ala Ala Pro Pro 35 40 45 Lys Ala Val Leu Lys Leu Glu Pro Gln Trp Ile Asn Val Leu Gln Glu 50 55 60 Asp Ser Val Thr Leu Thr Cys Arg Gly Thr His Ser Pro Glu Ser Asp 65 70 75 80 Ser Ile Gln Trp Phe His Asn Gly Asn Leu Ile Pro Thr His Thr Gln 85 90 95 Pro Ser Tyr Arg Phe Lys Ala Asn Asn Asn Asp Ser Gly Glu Tyr Thr 100 105 110 Cys Gln Thr Gly Gln Thr Ser Leu Ser Asp Pro Val His Leu Thr Val 115 120 125 Leu Ser Glu Trp Leu Val Leu Gln Thr Pro His Leu Glu Phe Gln Glu 130 135 140 Gly Glu Thr Ile Val Leu Arg Cys His Ser Trp Lys Asp Lys Pro Leu 145 150 155 160 Val Lys Val Thr Phe Phe Gln Asn Gly Lys Ser Lys Lys Phe Ser Arg 165 170 175 Ser Asn Pro Asn Phe Ser Ile Pro Gln Ala Asn His Ser His Ser Gly 180 185 190 Asp Tyr His Cys Thr Gly Asn Ile Gly Tyr Thr Leu Tyr Ser Ser Lys 195 200 205 Pro Val Thr Ile Thr Val Gln Ala Pro Ser Ser Ser Pro Met Gly Ile 210 215 220 Ile Val Ala Val Val Thr Gly Ile Ala Val Ala Ala Ile Val Ala Ala 225 230 235 240 Val Val Ala Leu Ile Tyr Cys Arg Lys Lys Arg Ile Ser Ala Leu Pro 245 250 255 Gly Tyr Pro Glu Cys Arg Glu Met Gly Glu Thr Leu Pro Glu Lys Pro 260 265 270 Ala Asn Pro Thr Asn Pro Asp Glu Ala Asp Lys Val Gly Ala Glu Asn 275 280 285 <00033​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​ Gln Pro Gln Thr Phe Glu Gly Val Lys Gly Ala Asp Pro Pro Thr Leu 20 25 30 Pro Pro Gly Ser Phe Leu Pro Gly Pro Val Leu Trp Trp Gly Ser Leu 35 40 45 Ala Arg Leu Gln Thr Glu Lys Ser Asp Glu Val Ser Arg Lys Gly Asn 50 55 60 Trp Trp Val Thr Glu Met Gly Gly Gly Ala Gly Glu Arg Leu Phe Thr 65 70 75 80 Ser Ser Cys Leu Val Gly Leu Val Pro Leu Gly Leu Arg Ile Ser Leu 85 90 95 Val Thr Cys Pro Leu Gln Cys Gly Ile Met Trp Gln Leu Leu Leu Pro 100 105 110 Thr Ala Leu Leu Leu Leu Val Ser Ala Gly Met Arg Thr Glu Asp Leu 115 120 125 Pro Lys Ala Val Val Phe Leu Glu Pro Gln Trp Tyr Arg Val Leu Glu 130 135 140 Lys Asp Ser Val Thr Leu Lys Cys Gln Gly Ala Tyr Ser Pro Glu Asp 145 150 155 160 Asn Ser Thr Gln Trp Phe His Asn Glu Ser Leu Ile Ser Ser Gln Ala 165 170 175 Ser Ser Tyr Phe Ile Asp Ala Ala Thr Val Asp Asp Ser Gly Glu Tyr 180 185 190 Arg Cys Gln Thr Asn Leu Ser Thr Leu Ser Asp Pro Val Gln Leu Glu 195 200 205 Val His Ile Gly Trp Leu Leu Leu Gln Ala Pro Arg Trp Val Phe Lys 210 215 220 Glu Glu Asp Pro Ile His Leu Arg Cys His Ser Trp Lys Asn Thr Ala 225 230 235 240 Leu His Lys Val Thr Tyr Leu Gln Asn Gly Lys Gly Arg Lys Tyr Phe 245 250 255 His His Asn Ser Asp Phe Tyr Ile Pro Lys Ala Thr Leu Lys Asp Ser 260 265 270 Gly Ser Tyr Phe Cys Arg Gly Leu Phe Gly Ser Lys Asn Val Ser Ser 275 280 285 Glu Thr Val Asn Ile Thr Ile Thr Gln Gly Leu Ala Val Ser Thr Ile 290 295 300 Ser Ser Phe Phe Pro Pro Gly Tyr Gln Val Ser Phe Cys Leu Val Met 305 310 315 320 Val Leu Leu Phe Ala Val Asp Thr Gly Leu Tyr Phe Ser Val Lys Thr 325 330 335 Asn Ile Arg Ser Ser Thr Arg Asp Trp Lys Asp His Lys Phe Lys Trp 340 345 350 Arg Lys Asp Pro Gln Asp Lys 355 <![CDATA[<210> 16]]> <![CDATA[<211> 359]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<400> 16]]> Met Ala Glu Gly Thr Leu Trp Gln Ile Leu Cys Val Ser Ser Asp Ala 1 5 10 15 Gln Pro Gln Thr Phe Glu Gly Val Lys Gly Ala Asp Pro Pro Thr Leu <![CDATA[<400> 20]]> 20 25 30 Pro Pro Gly Ser Phe Leu Pro Gly Pro Val Leu Trp Trp Gly Ser Leu 35 40 45 Ala Arg Leu Gln Thr Glu Lys Ser Asp Glu Val Ser Arg Lys Gly Asn 50 55 60 Trp Trp Val Thr Glu Met Gly Gly Gly Ala Gly Glu Arg Leu Phe Thr 65 70 75 80 Ser Ser Cys Leu Val Gly Leu Val Pro Leu Gly Leu Arg Ile Ser Leu 85 90 95 Val Thr Cys Pro Leu Gln Cys Gly Ile Met Trp Gln Leu Leu Leu Pro 100 105 110 Thr Ala Leu Leu Leu Leu Val Ser Ala Gly Met Arg Thr Glu Asp Leu 115 120 125 Pro Lys Ala Val Val Phe Leu Glu Pro Gln Trp Tyr Arg Val Leu Glu 130 135 140 Lys Asp Ser Val Thr Leu Lys Cys Gln Gly Ala Tyr Ser Pro Glu Asp 145 150 155 160 Asn Ser Thr Gln Trp Phe His Asn Glu Ser Leu Ile Ser Ser Gln Ala 165 170 175 Ser Ser Tyr Phe Ile Asp Ala Ala Thr Val Asp Asp Ser Gly Glu Tyr 180 185 190 Arg Cys Gln Thr Asn Leu Ser Thr Leu Ser Asp Pro Val Gln Leu Glu 195 200 205 Val His Ile Gly Trp Leu Leu Leu Gln Ala Pro Arg Trp Val Phe Lys 210 215 220 Glu Glu Asp Pro Ile His Leu Arg Cys His Ser Trp Lys Asn Thr Ala 225 230 235 240 Leu His Lys Val Thr Tyr Leu Gln Asn Gly Lys Gly Arg Lys Tyr Phe 245 250 255 His His Asn Ser Asp Phe Tyr Ile Pro Lys Ala Thr Leu Lys Asp Ser 260 265 270 Gly Ser Tyr Phe Cys Arg Gly Leu Val Gly Ser Lys Asn Val Ser Ser 275 280 285 Glu Thr Val Asn Ile Thr Ile Thr Gln Gly Leu Ala Val Ser Thr Ile 290 295 300 Ser Ser Phe Phe Pro Pro Gly Tyr Gln Val Ser Phe Cys Leu Val Met 305 310 315 320 Val Leu Leu Phe Ala Val Asp Thr Gly Leu Tyr Phe Ser Val Lys Thr 325 330 335 Asn Ile Arg Ser Ser Thr Arg Asp Trp Lys Asp His Lys Phe Lys Trp 340 345 350 Arg Lys Asp Pro Gln Asp Lys 355 <![CDATA[<210> 17]]> <![CDATA[<211> 117]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Mus musculus]]> <![CDATA[<400> 17]]> Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Tyr Ile Gln Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Trp Ile Tyr Pro Gly Asp Gly Thr Thr Lys Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Lys Thr Thr Leu Thr Ile Asp Lys Ser Ser Arg Thr Ala Tyr 65 70 75 80 Met Leu Phe Asn Ser Leu Thr Ser Glu Asp Ser Gly Ile Tyr Phe Cys 85 90 95 Ala Ser Tyr Tyr Gly Ser Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Thr 100 105 110 Leu Thr Val Ser Ser 115 <![CDATA[ <210> 18]]> <![CDATA[ <211> 117]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Humanized VH ver.A (HM242) <![CDATA[ <400> 18]]> Gln Val Gln Leu Val Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Val Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Tyr Ile Gln Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Trp Ile Tyr Pro Gly Asp Gly Thr Thr Lys Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Lys Thr Thr Leu Thr Ile Asp Lys Ser Thr Arg Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ser Tyr Tyr Gly Ser Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <![CDATA[ <210> 19]]> <![CDATA[ <211> 117]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Humanized VH ver.B (HM242) <![CDATA[ <400> 19]]> Gln Val Gln Leu Val Gln Ser Gly Pro Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Val Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Tyr Ile Gln Trp Val Arg Gln Val Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Tyr Pro Gly Asp Gly Thr Thr Lys Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Lys Thr Thr Leu Thr Ile Asp Lys Ser Thr Arg Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ser Tyr Tyr Gly Ser Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <![CDATA[ <210> 20]]> <![CDATA[ <211> 117]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Humanized VH ver.C (HM242) <![CDATA[ <400> 20]]> Gln Val Gln Leu Val Gln Ser Gly Pro Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Val Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Tyr Ile Gln Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Trp Ile Tyr Pro Gly Asp Gly Thr Thr Lys Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Arg Val Thr Met Thr Ile Asp Lys Ser Thr Arg Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ser Tyr Tyr Gly Ser Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <![CDATA[ <210> 21]]> <![CDATA[ <211> 117]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Humanized VH ver.D (HM242) <![CDATA[ <400> 21]]> Gln Val Gln Leu Val Gln Ser Gly Pro Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Val Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Tyr Ile Gln Trp Val Arg Gln Val Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Tyr Pro Gly Asp Gly Thr Thr Lys Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Arg Val Thr Met Thr Ile Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ser Tyr Tyr Gly Ser Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <![CDATA[<210> 22]]> <![CDATA[<211> 116]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Mus musculus]]> <![CDATA[<400> 22]]> Glu Val Leu Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 [[ID=...]] [[ID=2...]] [[ID=2...]] [[ID=2...]] [[ID=2...]] [[ID=2...]] Ser Val Lys Ile Pro Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 <...]] Asn Val Asp Trp Val Arg Gln Ser His Gly Lys Ser Leu Glu Trp Ile 35 40 45 Gly Asp Ile Asp Pro Asn Asn Gly Gly Thr Ile Tyr Asn Gln Lys Phe 50 55 60 It should be noted that some of the "..." in the tags in the original text seem to be incorrect or incomplete notations. I have translated them as accurately as possible based on the existing content. If there are specific requirements or corrections for those tags, please let me know. Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Arg Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 <![CDATA[<210> 23]]> Ala Arg Trp Arg Gly Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val 100 105 110 Thr Val Ser Ser [[ID=3​​​​​​​​​​​​​​​​​​​​​​​​​<![CDATA[<223> Humanized VH ver.E (HM297)]]> <![CDATA[<400> 23]]> Gln Val Gln Leu Val Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Pro Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Asn Val Asp Trp Val Arg Gln Ser His Gly Lys Ser Leu Glu Trp Ile 35 40 45 Gly Asp Ile Asp Pro Asn Asn Gly Gly Thr Ile Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Thr Arg Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Trp Arg Gly Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <![CDATA[ <210> 24]]> <![CDATA[ <211> 116]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Humanized VH ver.F (HM297) <![CDATA[ <400> 24]]> Gln Val Gln Leu Val Gln Ser Gly Pro Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Pro Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Asn Val Asp Trp Val Arg Gln Ser His Gly Lys Ser Leu Glu Trp Ile 35 40 45 Gly Asp Ile Asp Pro Asn Asn Gly Gly Thr Ile Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Arg Val Thr Met Thr Val Asp Lys Ser Thr Arg Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Trp Arg Gly Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <![CDATA[ <210> 25]]> <![CDATA[ <211> 116]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Humanized VH ver.G (HM297) <![CDATA[ <400> 25]]> Gln Val Gln Leu Val Gln Ser Gly Pro Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Pro Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Asn Val Asp Trp Val Arg Gln Val Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Asp Ile Asp Pro Asn Asn Gly Gly Thr Ile Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Thr Arg Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Trp Arg Gly Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <![CDATA[ <210> 26]]> <![CDATA[ <211> 116]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Humanized VH ver.H (HM297) <![CDATA[ <400> 26]]> Gln Val Gln Leu Val Gln Ser Gly Pro Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Pro Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Asn Val Asp Trp Val Arg Gln Val Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Asp Ile Asp Pro Asn Asn Gly Gly Thr Ile Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Arg Val Thr Met Thr Val Asp Lys Ser Thr Arg Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Trp Arg Gly Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <![CDATA[ <210> 27]]> <![CDATA[ <211> 116]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Humanized VH ver.I (HM297) <![CDATA[ <400> 27]]> Gln Val Gln Leu Val Gln Ser Gly Pro Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Val Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Asn Val Asp Trp Val Arg Gln Val Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Asp Ile Asp Pro Asn Asn Gly Gly Thr Ile Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Arg Val Thr Met Thr Val Asp Thr Ser Thr Arg Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Trp Arg Gly Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <![CDATA[<210> 28]]> <![CDATA[<211> 117]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Mus musculus]]> <![CDATA[<400> 28]]> Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Met Pro Cys Lys Thr Ser Gly Tyr Thr Phe Thr Ser Tyr ​​​​​​​​​​35 40 45 Gly Trp Ile Tyr Phe Gly Asp Ala Ser Thr Lys Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Lys Thr Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Leu Leu Ser Ser Leu Thr Ser Glu Asp Ser Gly Ile Tyr Phe Cys 85 90 95 Ala Ser Tyr Tyr Gly Ser Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Thr 100 105 110 Leu Thr Val Ser Ser 115 <![CDATA[ <210> 29]]> <![CDATA[ <211> 117]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Humanized VH ver.J (HM268) <![CDATA[ <400> 29]]> Gln Val Gln Leu Val Gln Ser Gly Pro Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Val Cys Lys Thr Ser Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Tyr Ile Gln Trp Val Arg Gln Val Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Tyr Phe Gly Asp Ala Ser Thr Lys Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Lys Thr Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ser Tyr Tyr Gly Ser Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Thr 100 105 110 Val Thr Val Ser Ser 115 <![CDATA[ <210> 30]]> <![CDATA[ <211> 117]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Humanized VH ver.K (HM268)]]> <![CDATA[<400> 30]]> Gln Val Gln Leu Val Gln Ser Gly Pro Glu Val Lys Lys Pro Gly Ala / / 1 5 10 15 Ser Val Lys Val Val Cys Lys Thr Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Tyr Ile Gln Trp Leu Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Trp Ile Tyr Phe Gly Asp Ala Ser Thr Lys Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Arg Val Thr Met Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ser Tyr Tyr Gly Ser Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Thr 100 105 110 <![CDATA[<210> 31]]> Val Thr Val Ser Ser 115 <![CDATA[<211> 117]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]><![CDATA[<210> 31]]> <![CDATA[<220>]]> <![CDATA[<223> Humanized VH ver.L (HM268)]]> <![CDATA[<400> 31]]> Gln Val Gln Leu Val Gln Ser Gly Pro Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Val Cys Lys Thr Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 <00​​​​​​​​​​​​​​​​​​​​​​​​​ 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ser Tyr Tyr Gly Ser Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Thr 100 105 110 Val Thr Val Ser Ser 115 <![CDATA[ <210> 32]]> <![CDATA[ <211> 117]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Humanized VH ver.M (HM268) <![CDATA[<400> 32]]> Gln Val Gln Leu Val Gln Ser Gly Pro Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Val Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Tyr Ile Gln Trp Val Arg Gln Val Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Tyr Phe Gly Asp Ala Ser Thr Lys Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Arg Val Thr Met Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ser Tyr Tyr Gly Ser Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Thr 100 105 110 Val Thr Val Ser Ser 115 <![CDATA[<210> 33]]> <![CDATA[<211> 117]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Mus musculus]]> <![CDATA[<400> 33]]> Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Tyr Ile Gln Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Trp Ile Tyr Pro Gly Asp Gly Ser Ser Lys Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Lys Thr Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Leu Leu Ser Ser Leu Thr Ser Glu Asp Ser Gly Ile Tyr Phe Cys 85 90 95 Ala Ser Tyr Tyr Gly Ser Ser Phe Asp Tyr Trp Gly Gln Gly Thr Thr 100 105 110 Leu Thr Val Ser Ser 115 <![CDATA[<210> 34]]> <![CDATA[<211> 107]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Mus musculus]]> <![CDATA[<400> 34]]> Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly 1 5 10 15 Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Thr Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Lys Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 <![CDATA[<210> 35]]> Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Thr Asn Leu Glu Gln 65 70 75 80 <00052​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​ <![CDATA[<220>]]> <![CDATA[<223> Humanized VL ver.1 (HM242)]]> <![CDATA[<400> 35]]> Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Thr Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Lys Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Glu Gln 65 70 75 80 Lys Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Ser Thr Leu Pro Phe 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <![CDATA[ <210> 36]]> <![CDATA[ <211> 107]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Humanized VL ver.2 (HM242) <![CDATA[ <400> 36]]> Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Thr Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Lys Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Ser Thr Leu Pro Phe 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <![CDATA[ <210> 37]]> <![CDATA[ <211> 107]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Humanized VL ver.3 (HM242) <![CDATA[ <400> 37]]> Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Lys Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Ser Thr Leu Pro Phe 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <![CDATA[<210> 38]]> <![CDATA[<211> 107]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Mus musculus]]> <![CDATA[<400> 38]]> Asp Val Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly 1 5 10 15 Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Asp Asn Tyr 20 25 30 Leu Lys Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile 35 40 45 Tyr Asp Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Lys Gln 65 70 75 80 Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Tyr Thr Leu Pro Trp 85 90 95 Thr Phe Gly Gly Gly Thr Arg Leu Glu Ile Lys 100 105 <![CDATA[ <210> 39]]> <![CDATA[ <211> 107]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Humanized VL ver.4 (HM297) <![CDATA[ <400> 39]]> Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Asp Asn Tyr 20 25 30 Leu Lys Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile 35 40 45 Tyr Asp Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Lys Gln 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Tyr Thr Leu Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <![CDATA[ <210> 40]]> <![CDATA[ <211> 107]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Humanized VL ver.5 (HM297) <![CDATA[ <400> 40]]> Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Asp Asn Tyr 20 25 30 Leu Lys Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile 35 40 45 Tyr Asp Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Lys Gln 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Tyr Thr Leu Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <![CDATA[ <210> 41]]> <![CDATA[ <211> 107]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Humanized VL ver.6 (HM297) <![CDATA[ <400> 41]]> Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Asp Asn Tyr 20 25 30 Leu Lys Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Asp Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Tyr Thr Leu Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <![CDATA[<210> 42]]> <![CDATA[<211> 107]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Mus musculus]]> <![CDATA[<400> 42]]> Asn Ile Gln Met Thr Gln Asn Thr Ser Ser Leu Ser Ala Thr Leu Gly 1 5 10 15 Val Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile 35 40 45 Tyr Ser Thr Ser Arg Leu His Ser Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln 65 70 75 80 Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Ser Thr Leu Pro Phe 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <![CDATA[ <210> 43]]> <![CDATA[ <211> 107]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Humanized VL ver.7 (HM268) <![CDATA[ <400> 43]]> Asp Ile Gln Met Thr Gln Asn Thr Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile 35 40 45 Tyr Ser Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Glu Gln 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Ser Thr Leu Pro Phe 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <![CDATA[ <210> 44]]> <![CDATA[ <211> 107]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Humanized VL ver.8 (HM268) <![CDATA[ <400> 44]]> Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile 35 40 45 Tyr Ser Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro ​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​<![CDATA[<223> Humanized VL ver.9 (HM268)]]> <![CDATA[<400> 45]]> Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Ser Thr Leu Pro Phe 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <![CDATA[<210> 46]]> <![CDATA[<211> 107]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Mus musculus]]> <![CDATA[<400> 46]]> Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly 1 5 10 15 Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Phe Leu Ile 35 40 45 Tyr Tyr Ser Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln 65 70 75 80 Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Ser Thr Leu Pro Phe 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <![CDATA[<210> 47]]> <![CDATA[<211> 232]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<400> 47]]> Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala 1 5 10 15 [[ID=3l]]Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 20 25 30 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 35 40 45 Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 50 55 60 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 65 70 75 80 Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 85 90 95 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala 100 105 110 Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 115 120 125 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr 130 135 140 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 145 150 155 160 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 165 170 175 Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 180 185 190 Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 195 200 205 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 210 215 220 Ser Leu Ser Leu Ser Pro Gly Lys 225 230 <![CDATA[ <210> 48]]> <![CDATA[ <211> 232]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Human IgG1 Fc_K322A amino acid sequence]]> <![CDATA[ <400> 48]]> Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala 1 5 10 15 Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 20 25 30 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 35 40 45 Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 50 55 60 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 65 70 75 80 Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 85 90 95 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Ala Val Ser Asn Lys Ala 100 105 110 Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 115 120 125 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr 130 135 140 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 145 150 155 160 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 165 170 175 Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 180 185 190 Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 195 200 205 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 210 215 220 Ser Leu Ser Leu Ser Pro Gly Lys 225 230 <![CDATA[ <210> 49]]> <![CDATA[ <211> 232]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Human IgG1 Fc_G236D / H268D(X2) amino acid sequence]]> <![CDATA[ <400> 49]]> Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala 1 5 10 15 Pro Glu Leu Leu Asp Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 20 25 30 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 35 40 45 Val Asp Val Ser Asp Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 50 55 60 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 65 70 75 80 Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 85 90 95 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala 100 105 110 Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 115 120 125 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr 130 135 140 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 145 150 155 160 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 165 170 175 Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 180 185 190 Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 195 200 205 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 210 215 220 Ser Leu Ser Leu Ser Pro Gly Lys 225 230 <![CDATA[ <210> 50]]> <![CDATA[ <211> 232]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Human IgG1 Fc_G236D / H268D / K322A(X2-K) amino acid sequence]]> <![CDATA[ <400> 50]]> Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala 1 5 10 15 Pro Glu Leu Leu Asp Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 20 25 30 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 35 40 45 Val Asp Val Ser Asp Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 50 55 60 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 65 70 75 80 Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 85 90 95 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Ala Val Ser Asn Lys Ala 100 105 110 Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 115 120 125 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr 130 135 140 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 145 150 155 160 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 165 170 175 Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 180 185 190 Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 195 200 205 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 210 215 220 Ser Leu Ser Leu Ser Pro Gly Lys 225 230 <![CDATA[<210> 51]]> <![CDATA[<211> 232]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Synthetic Sequence]]> <![CDATA[<220>]]> <![CDATA[ <223> Human IgG1 Fc_G236D / H268D / E293A / K322A(X2-KE) amino acid sequence]]> <![CDATA[ <400> 51]]> Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala 1 5 10 15 Pro Glu Leu Leu Asp Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 20 25 30 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 35 40 45 Val Asp Val Ser Asp Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 50 55 60 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Ala Glu Gln 65 70 75 80 Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 85 90 95 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Ala Val Ser Asn Lys Ala 100 105 110 Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 115 120 125 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr 130 135 140 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 145 150 155 160 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 165 170 175 Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 180 185 190 Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 195 200 205 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 210 215 220 Ser Leu Ser Leu Ser Pro Gly Lys 225 230 <![CDATA[ <210> 52]]> <![CDATA[ <211> 232]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Human IgG1 Fc_S236D / S267G / H268D / K322A(X2-KS) amino acid sequence]]> <![CDATA[ <400> 52]]> Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala 1 5 10 15 Pro Glu Leu Leu Asp Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 20 25 30 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 35 40 45 Val Asp Val Gly Asp Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 50 55 60 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 65 70 75 80 Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 85 90 95 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Ala Val Ser Asn Lys Ala 100 105 110 Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 115 120 125 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr 130 135 140 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 145 150 155 160 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 165 170 175 Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 180 185 190 Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 195 200 205 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 210 215 220 Ser Leu Ser Leu Ser Pro Gly Lys 225 230 <![CDATA[ <210> 53 <![CDATA[ <211> 232]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Human IgG1 Fc_S239D / H268D(X3) amino acid sequence]]> <![CDATA[ <400> 53]]> Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala 1 5 10 15 Pro Glu Leu Leu Gly Gly Pro Asp Val Phe Leu Phe Pro Pro Lys Pro 20 25 30 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 35 40 45 Val Asp Val Ser Asp Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 50 55 60 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 65 70 75 80 Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 85 90 95 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala 100 105 110 Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 115 120 125 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr 130 135 140 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 145 150 155 160 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 165 170 175 Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 180 185 190 Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 195 200 205 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 210 215 220 Ser Leu Ser Leu Ser Pro Gly Lys 225 230 <![CDATA[ <210> 54]]> <![CDATA[ <211> 232]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Human IgG1 Fc_S239D / H268D / K322A(X3-K) amino acid sequence]]> <![CDATA[ <400> 54]]> Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala 1 5 10 15 Pro Glu Leu Leu Gly Gly Pro Asp Val Phe Leu Phe Pro Pro Lys Pro 20 25 30 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 35 40 45 Val Asp Val Ser Asp Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 50 55 60 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 65 70 75 80 Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 85 90 95 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Ala Val Ser Asn Lys Ala 100 105 110 Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 115 120 125 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr 130 135 140 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 145 150 155 160 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 165 170 175 Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 180 185 190 Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 195 200 205 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 210 215 220 Ser Leu Ser Leu Ser Pro Gly Lys 225 230 <![CDATA[<210> 55]]> <![CDATA[<211> 232]]> <![CDATA[<212> PRT]]> <![CDATA[<2​​​​​ <![CDATA[ <223> Human IgG1 Fc_S239D / H268D / E293A / K322A(X3-KE) amino acid sequence]]> <![CDATA[ <400> 55]]> Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala 1 5 10 15 Pro Glu Leu Leu Gly Gly Pro Asp Val Phe Leu Phe Pro Pro Lys Pro 20 25 30 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 35 40 45 Val Asp Val Ser Asp Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 50 55 60 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Ala Glu Gln 65 70 75 80 Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 85 90 95 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Ala Val Ser Asn Lys Ala 100 105 110 Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 115 120 125 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr 130 135 140 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 145 150 155 160 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 165 170 175 Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 180 185 190 Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 195 200 205 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 210 215 220 Ser Leu Ser Leu Ser Pro Gly Lys 225 230 <![CDATA[ <210> 56]]> <![CDATA[ <211> 232]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Human IgG1 Fc_S239D / H268D / K322A / I332E(X3-KI) amino acid sequence]]> <![CDATA[ <400> 56]]> Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala 1 5 10 15 Pro Glu Leu Leu Gly Gly Pro Asp Val Phe Leu Phe Pro Pro Lys Pro 20 25 30 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 35 40 45 Val Asp Val Ser Asp Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 50 55 60 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 65 70 75 80 Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 85 90 95 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Ala Val Ser Asn Lys Ala 100 105 110 Leu Pro Ala Pro Glu Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 115 120 125 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr 130 135 140 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 145 150 155 160 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 165 170 175 Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 180 185 190 Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 195 200 205 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 210 215 220 Ser Leu Ser Leu Ser Pro Gly Lys 225 230 <![CDATA[ <210> 57]]> <![CDATA[ <211> 232]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Human IgG1 Fc_S239D / H268D / L328Y / K322A(X3-KL) amino acid sequence]]> <![CDATA[ <400> 57]]> Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala 1 5 10 15 Pro Glu Leu Leu Gly Gly Pro Asp Val Phe Leu Phe Pro Pro Lys Pro 20 25 30 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 35 40 45 Val Asp Val Ser Asp Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 50 55 60 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 65 70 75 80 Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 85 90 95 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Ala Val Ser Asn Lys Ala 100 105 110 Tyr Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 115 120 125 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr 130 135 140 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 145 150 155 160 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 165 170 175 Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 180 185 190 Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 195 200 205 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 210 215 220 Ser Leu Ser Leu Ser Pro Gly Lys 225 230 <![CDATA[ <210> 58]]> <![CDATA[ <211> 232]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Human IgG1 Fc_S239D / S267G / H268D / K322A(X3-KS) amino acid sequence]]> <![CDATA[ <400> 58]]> Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala 1 5 10 15 Pro Glu Leu Leu Gly Gly Pro Asp Val Phe Leu Phe Pro Pro Lys Pro 20 25 30 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 35 40 45 Val Asp Val Gly Asp Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 50 55 60 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 65 70 75 80 Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 85 90 95 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Ala Val Ser Asn Lys Ala 100 105 110 Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 115 120 125 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr 130 135 140 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 145 150 155 160 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 165 170 175 Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 180 185 190 Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 195 200 205 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 210 215 220 Ser Leu Ser Leu Ser Pro Gly Lys 225 230 <![CDATA[<210> 59]]> <![CDATA[<211> 232]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Synthetic Sequence]]> <![CDATA[<220>]]>​ <![CDATA[ <223> Human IgG1 Fc_S239D / S267G / H268D / K322A / I332E(X3-K]]>SI) amino acid sequence <![CDATA[ <400> 59]]> Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala 1 5 10 15 Pro Glu Leu Leu Gly Gly Pro Asp Val Phe Leu Phe Pro Pro Lys Pro 20 25 30 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 35 40 45 Val Asp Val Gly Asp Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 50 55 60 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 65 70 75 80 Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 85 90 95 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Ala Val Ser Asn Lys Ala 100 105 110 Leu Pro Ala Pro Glu Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 115 120 125 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr 130 135 140 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 145 150 155 160 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 165 170 175 Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 180 185 190 Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 195 200 205 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 210 215 220 Ser Leu Ser Leu Ser Pro Gly Lys 225 230 <![CDATA[ <210> 60]]> <![CDATA[ <211> 232]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Human IgG1 Fc_S239D / S267G / H268D / L328Y / K322A(X3-KSL) amino acid sequence]]> <![CDATA[ <400> 60]]> Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala 1 5 10 15 Pro Glu Leu Leu Gly Gly Pro Asp Val Phe Leu Phe Pro Pro Lys Pro 20 25 30 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 35 40 45 Val Asp Val Gly Asp Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 50 55 60 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 65 70 75 80 Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 85 90 95 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Ala Val Ser Asn Lys Ala 100 105 110 Tyr Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 115 120 125 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr 130 135 140 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 145 150 155 160 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 165 170 175 Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 180 185 190 Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 195 200 205 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 210 215 220 Ser Leu Ser Leu Ser Pro Gly Lys 225 230 <![CDATA[ <210> 61]]> <![CDATA[ <211> 232]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Human IgG1 Fc_S239D / H268D / L328Y / I332E(X4) amino acid sequence]]> <![CDATA[ <400> 61]]> Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala 1 5 10 15 Pro Glu Leu Leu Gly Gly Pro Asp Val Phe Leu Phe Pro Pro Lys Pro 20 25 30 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 35 40 45 Val Asp Val Ser Asp Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 50 55 60 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 65 70 75 80 Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 85 90 95 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala 100 105 110 Tyr Pro Ala Pro Glu Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 115 120 125 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr 130 135 140 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 145 150 155 160 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 165 170 175 Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 180 185 190 Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 195 200 205 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 210 215 220 Ser Leu Ser Leu Ser Pro Gly Lys 225 230 <![CDATA[ <210> 62]]> <![CDATA[ <211> 232]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Human IgG1 Fc_S239D / H268D / K322A / L328Y / I332E(X4-K) amino acid sequence]]> <![CDATA[ <400> 62]]> Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala 1 5 10 15 Pro Glu Leu Leu Gly Gly Pro Asp Val Phe Leu Phe Pro Pro Lys Pro 20 25 30 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 35 40 45 Val Asp Val Ser Asp Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 50 55 60 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 65 70 75 80 Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 85 90 95 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Ala Val Ser Asn Lys Ala 100 105 110 Tyr Pro Ala Pro Glu Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 115 120 125 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr 130 135 140 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 145 150 155 160 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 165 170 175 Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 180 185 190 Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 195 200 205 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 210 215 220 Ser Leu Ser Leu Ser Pro Gly Lys 225 230 <![CDATA[<210> 63]]> <![CDATA[<211> 232]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Synthetic Sequence]]> <![CDATA[<220>]]> <![CDATA[ <223> Human IgG1 Fc_S239D / S267G / H268D / K322A / L328Y / I332E(X4-KS) amino acid sequence]]> <![CDATA[ <400> 63]]> Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala 1 5 10 15 Pro Glu Leu Leu Gly Gly Pro Asp Val Phe Leu Phe Pro Pro Lys Pro 20 25 30 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 35 40 45 Val Asp Val Gly Asp Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 50 55 60 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 65 70 75 80 Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 85 90 95 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Ala Val Ser Asn Lys Ala 100 105 110 Tyr Pro Ala Pro Glu Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 115 120 125 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr 130 135 140 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 145 150 155 160 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 165 170 175 Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 180 185 190 Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 195 200 205 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 210 215 220 Ser Leu Ser Leu Ser Pro Gly Lys 225 230 <![CDATA[<210> 64]]> <![CDATA[<211> 229]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<400> 64]]> Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro Glu Phe 1 5 10 15 <![CDATA[ ]]>Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 20 25 30 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 35 40 45 Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val 50 55 60 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser 65 70 75 80 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 85 90 95 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser 100 105 110 Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 115 120 125 Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln 130 135 140 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 145 150 155 160 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 165 170 175 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu 180 185 190 Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser 195 200 205 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 210 215 220 Leu Ser Pro Gly Arg 225 <![CDATA[ <210> 65]]> <![CDATA[ <211> 229]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> [H Human IgG4 Fc_S228P Amino Acid Sequence]]> <![CDATA[ <400> 65]]> Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe 1 5 10 15 Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 20 25 30 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 35 40 45 Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val 50 55 60 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser 65 70 75 80 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 85 90 95 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser 100 105 110 Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 115 120 125 Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln 130 135 140 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 145 150 155 160 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 165 170 175 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu 180 185 190 Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser 195 200 205 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 210 215 220 Leu Ser Pro Gly Arg 225 <![CDATA[ <210> 66]]> <![CDATA[ <211> 229]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Human IgG4 Fc_S228P / G236D / Q268D(X2-P) amino acid sequence]]> <![CDATA[ <400> 66]]> Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe 1 5 10 15 Leu Asp Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 20 25 30 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 35 40 45 Ser Asp Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val 50 55 60 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser 65 70 75 80 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 85 90 95 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser 100 105 110 Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 115 120 125 Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln 130 135 140 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 145 150 155 160 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 165 170 175 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu 180 185 190 Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser 195 200 205 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 210 215 220 Leu Ser Pro Gly Arg 225 <![CDATA[ <210> 67]]> <![CDATA[ <211> 229]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Human IgG4 Fc_S2]]>28P / S239D / Q268D(X3-P) amino acid sequence <![CDATA[ <400> 67]]> Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe 1 5 10 15 Leu Gly Gly Pro Asp Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 20 25 30 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 35 40 45 Ser Asp Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val 50 55 60 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser 65 70 75 80 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 85 90 95 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser 100 105 110 Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 115 120 125 Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln 130 135 140 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 145 150 155 160 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 165 170 175 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu 180 185 190 Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser 195 200 205 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 210 215 220 Leu Ser Pro Gly Arg 225 <![CDATA[<210> 68]]> <![CDATA[<211> 229]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Synthetic Sequence]]> <![CDATA[<220>]]> <![CDATA[ <223> including IgG4 Fc_S228P / S239D / Q268D / L328Y / I332E(X4-P) <![CDATA[ <400> 68]]> Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe 1 5 10 15 Leu Gly Gly Pro Asp Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 20 25 30 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 35 40 45 Ser Asp Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val 50 55 60 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser 65 70 75 80 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 85 90 95 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Tyr Pro Ser 100 105 110 Ser Glu Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 115 120 125 Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln 130 135 140 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 145 150 155 160 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 165 170 175 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu 180 185 190 Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser 195 200 205 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 210 215 220 Leu Ser Pro Gly Arg 225 <![CDATA[<210> 69]]> <![CDATA[<211> 447]]> <![CDATA[<212> PRT]]> <![CDATA[<2憨态可掬小家鼠]]> <![CDATA[<400> 69]]> Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Tyr Ile Gln Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 <憨态可掬9345>Gly Trp Ile Tyr Pro Gly Asp Gly Thr Thr Lys Tyr Asn Glu Lys Phe 50 55 60 憨态可掬 It should be noted that the translation of "小家鼠" as "憨态可掬小家鼠" in the provided translation seems incorrect. The correct translation of "小家鼠" is "Mus musculus". The above translation is adjusted based on the correct understanding.Lys Gly Lys Thr Thr Leu Thr Ile Asp Lys Ser Ser Arg Thr Ala Tyr 65 70 75 80 Met Leu Phe Asn Ser Leu Thr Ser Glu Asp Ser Gly Ile Tyr Phe Cys 85 90 95 Ala Ser Tyr Tyr Gly Ser Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Thr 100 105 110 Leu Thr Val Ser Ser Ala Lys Thr Thr Ala Pro Ser Val Tyr Pro Leu 115 120 125 Ala Pro Val Cys Gly Asp Thr Thr Gly Ser Ser Val Thr Leu Gly Cys 130 135 140 Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Leu Thr Trp Asn Ser 145 150 155 160 Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175 Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Thr Ser Ser Thr Trp 180 185 190 Pro Ser Gln Ser Ile Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr 195 200 205 Lys Val Asp Lys Lys Ile Glu Pro Arg Gly Pro Thr Ile Lys Pro Cys 210 215 220 Pro Pro Cys Lys Cys Pro Ala Pro Asn Leu Leu Gly Gly Pro Ser Val 225 230 235 240 Phe Ile Phe Pro Pro Lys Ile Lys Asp Val Leu Met Ile Ser Leu Ser 245 250 255 Pro Ile Val Thr Cys Val Val Val Asp Val Ser Glu Asp Asp Pro Asp 260 265 270 Val Gln Ile Ser Trp Phe Val Asn Asn Val Glu Val His Thr Ala Gln 275 280 285 Thr Gln Thr His Arg Glu Asp Tyr Asn Ser Thr Leu Arg Val Val Ser 290 295 300 Ala Leu Pro Ile Gln His Gln Asp Trp Met Ser Gly Lys Glu Phe Lys 305 310 315 320 Cys Lys Val Asn Asn Lys Asp Leu Pro Ala Pro Ile Glu Arg Thr Ile 325 330 335 Ser Lys Pro Lys Gly Ser Val Arg Ala Pro Gln Val Tyr Val Leu Pro 340 345 350 Pro Pro Glu Glu Glu Met Thr Lys Lys Gln Val Thr Leu Thr Cys Met 355 360 365 Val Thr Asp Phe Met Pro Glu Asp Ile Tyr Val Glu Trp Thr Asn Asn 370 375 380 Gly Lys Thr Glu Leu Asn Tyr Lys Asn Thr Glu Pro Val Leu Asp Ser 385 390 395 400 Asp Gly Ser Tyr Phe Met Tyr Ser Lys Leu Arg Val Glu Lys Lys Asn 405 410 415 Trp Val Glu Arg Asn Ser Tyr Ser Cys Ser Val Val His Glu Gly Leu 420 425 430 His Asn His His Thr Thr Lys Ser Phe Ser Arg Thr Pro Gly Lys 435 440 445 <![CDATA[<210> ]]>70 <![CDATA[<211> 1341]]> <![CDATA[<212> DNA]]> <![CDATA[<213> Mus musculus]]> <![CDATA[<400> 70]]> caggtgcaac tccagcagtc tggtcccgaa ttggtgaaac ctggtgcctc agtcaagatg 60 tcttgcaaag catcagggta tacgtttacc tcctactaca tccagtgggt gaaacagagg 120<00095此文本似乎是一段生物相关的序列信息,包含氨基酸序列和DNA序列等。 ccaggacagg gccttgagtg gattggctgg atttatcccg gagatggcac aaccaagtac 180 aacgagaagt tcaagggcaa aaccacactg actatcgaca agagctccag aactgcctac 240 atgctcttca attccctgac aagcgaagat agtggcatct acttttgtgc cagctactat 300 gggagctatt tcgactattg gggtcaaggg accactctga ccgtatcctc tgcgaaaact 360 actgccccat cagtctatcc ccttgctccc gtctgcggtg ataccacggg ttcctctgtg 420 actcttggtt gtctggttaa agggtatttt cccgaaccag tcaccctcac atggaatagc 480 gggagcctct caagtggagt gcacaccttt cctgccgtcc tgcaatctga cctttacaca 540 ctttcttcaa gcgtgaccgt gacaagtagc acttggccct ctcagtccat tacttgcaat 600 gttgcacatc cagcatcaag tacaaaggtg gataagaaaa tcgaacccag gggcccaacc 660 atcaaaccct gtccgccatg taaatgccct gctccaaatc tcctgggcgg acctagtgtg 720 ttcatctttc ctcccaaaat caaagatgtg ctgatgatca gtctgagccc aatagtgacc 780 tgtgtggtag tggacgtttc cgaggatgac cctgacgtcc agataagctg gtttgtcaac 840 aatgtggaag tgcacacagc ccaaacacag acccatcgcg aggactataa ctccaccctg 900 agggttgtga gcgcactgcc cattcagcac caggactgga tgagtgggaa ggagttcaaa 960 tgcaaggtaa ataacaagga tctgcccgcc ccgattgaac ggactatcag caaaccgaag 1020 gggtccgtta gagcacctca ggtatacgtg ttgccacctc cagaggagga aatgaccaag 1080 aaacaggtta ccctgacatg catggtaacg gacttcatgc ctgaggacat ttacgtggag 1140 tggacgaata acggcaaaac cgagctgaac tacaagaaca ctgagcctgt cctcgattcc 1200 gatggctcct atttcatgta ctctaagctc agagtggaaa agaagaactg ggtggaacgc 1260 aatagctact cttgctcagt ggtccatgag ggcttgcaca atcatcacac tactaagtca 1320 ttctctcgga cacccggaaa g 1341 <![CDATA[<210> 71]]> <![CDATA[<211> 214]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Mus musculus]]> <![CDATA[<400> 71]]> Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly 1 5 10 15 Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Thr Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Lys Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Thr Asn Leu Glu Gln 65 70 75 80 Lys Asp Phe Ala Thr Tyr Phe Cys Gln Gln Ser Ser Thr Leu Pro Phe 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala Asp Ala Ala 100 105 110 Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu Thr Ser Gly 115 120 125 Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile 130 135 140 Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val Leu 145 150 155 160 Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser 165 170 175 Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr 180 185 190 Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys Ser 19� 200 205 Phe Asn Arg Asn Glu Cys 210 <![CDATA[<210> 72]]> <![CDATA[<211> 6۴۲]]> <![CDATA[<212> DNA]]> <![CDATA[<213> Mus musculus]]> <![CDATA[<400> 72]]> gacattcaga tgacccagac aacaagctcc ctttccgcca gtcttggcga tcgggtgacc 60 atcagctgta gagcctcaca agacatctct aactacctga actggtatca gcagaaacct 120 It should be noted that there may be some inaccuracies in the translation of special symbols and specific terms in the original text. It is recommended to check and verify according to the actual situation and relevant professional knowledge. gatggcacag tcactctgct gatctactac accagcaaac tccactctgg tgtgccatct 180 cgcttttcag ggagtggatc tggcactgac tattccctga ccatcaccaa tctcgaacag 240 aaggactttg ccacgtattt ctgccaacag tccagtacct tgcccttcac atttggtgga 300 gggacaaagt tggagataaa gcgagctgat gcagcaccaa ctgtctccat cttccctcct 360 agcagtgaac agctgactag tggaggagcc tctgtggtgt gtttcctcaa caacttctac 420 cccaaagaca tcaatgtcaa gtggaagatc gatgggtcag aacgtcaaaa tggcgtgctg 480 aatagctgga cagatcagga tagcaaggac tccacttact caatgagctc aacactgaca 540 ctgaccaaag acgagtatga gaggcataac agctatacgt gtgaagccac acacaagacc 600 agcacatcac cgattgtgaa gagcttcaat cggaatgagt gc 642 <![CDATA[<210> 73]]> <![CDATA[<211> 447]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Mus musculus]]> <![CDATA[<400> 73]]> Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Tyr Ile Gln Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Trp Ile Tyr Pro Gly Asp Gly Ser Ser Lys Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Lys Thr Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Leu Leu Ser Ser Leu Thr Ser Glu Asp Ser Gly Ile Tyr Phe Cys 85 90 95 Ala Ser Tyr Tyr Gly Ser Ser Phe Asp Tyr Trp Gly Gln Gly Thr Thr 100 105 110 Leu Thr Val Ser Ser Ala Lys Thr Thr Ala Pro Ser Val Tyr Pro Leu 115 120 125 Ala Pro Val Cys Gly Asp Thr Thr Gly Ser Ser Val Thr Leu Gly Cys 130 135 140 Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Leu Thr Trp Asn Ser 145 150 155 160 Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175 Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Thr Ser Ser Thr Trp 180 185 190 Pro Ser Gln Ser Ile Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr 195 200 205 Lys Val Asp Lys Lys Ile Glu Pro Arg Gly Pro Thr Ile Lys Pro Cys 210 215 220 Pro Pro Cys Lys Cys Pro Ala Pro Asn Leu Leu Gly Gly Pro Ser Val 225 230 235 240 Phe Ile Phe Pro Pro Lys Ile Lys Asp Val Leu Met Ile Ser Leu Ser 245 250 255 Pro Ile Val Thr Cys Val Val Val Asp Val Ser Glu Asp Asp Pro Asp 260 265 270 Val Gln Ile Ser Trp Phe Val Asn Asn Val Glu Val His Thr Ala Gln 275 280 285 Thr Gln Thr His Arg Glu Asp Tyr Asn Ser Thr Leu Arg Val Val Ser 290 295 300 Ala Leu Pro Ile Gln His Gln Asp Trp Met Ser Gly Lys Glu Phe Lys 305 310 315 320 Cys Lys Val Asn Asn Lys Asp Leu Pro Ala Pro Ile Glu Arg Thr Ile 325 330 335 Ser Lys Pro Lys Gly Ser Val Arg Ala Pro Gln Val Tyr Val Leu Pro 340 345 350 Pro Pro Glu Glu Glu Met Thr Lys Lys Gln Val Thr Leu Thr Cys Met 355 360 365 Val Thr Asp Phe Met Pro Glu Asp Ile Tyr Val Glu Trp Thr Asn Asn 370 375 380 Gly Lys Thr Glu Leu Asn Tyr Lys Asn Thr Glu Pro Val Leu Asp Ser 385 390 395 400 Asp Gly Ser Tyr Phe Met Tyr Ser Lys Leu Arg Val Glu Lys Lys Asn 405 410 415 Trp Val Glu Arg Asn Ser Tyr Ser Cys Ser Val Val His Glu Gly Leu 420 425 430 His Asn His His Thr Thr Lys Ser Phe Ser Arg Thr Pro Gly Lys [[ID=3​​​​​​​​​​​​​​​​​​​​​​<![CDATA[<400> 74]]> caggtccagc tgcagcaaag tggaccagaa ctggttaaac ccggcgcaag tgtgaagatg 60 agctgtaagg cttcagggta tacatttacc agctactaca tccagtgggt aaagcagcga 120 cctggtcaag gcctggagtg gatagggtgg atttatccgg gagatggatc cagcaaatat 180 aacgagaagt tcaagggcaa gaccacactg acggccgaca aatctagctc aacggcctac 240 atgctgctca gctccttgac ttccgaagat tctgggattt acttctgcgc ttcctactat 300 ggctctagct tcgactattg gggccaaggt accacattga cagtcagctc cgcgaaaact 360 actgccccat cagtctatcc ccttgctccc gtctgcggtg ataccacggg ttcctctgtg 420 actcttggtt gtctggttaa agggtatttt cccgaaccag tcaccctcac atggaatagc 480 gggagcctct caagtggagt gcacaccttt cctgccgtcc tgcaatctga cctttacaca 540 ctttcttcaa gcgtgaccgt gacaagtagc acttggccct ctcagtccat tacttgcaat 600 gttgcacatc cagcatcaag tacaaaggtg gataagaaaa tcgaacccag gggcccaacc 660 atcaaaccct gtccgccatg taaatgccct gctccaaatc tcctgggcgg acctagtgtg 720 ttcatctttc ctcccaaaat caaagatgtg ctgatgatca gtctgagccc aatagtgacc 780 tgtgtggtag tggacgtttc cgaggatgac cctgacgtcc agataagctg gtttgtcaac 840 aatgtggaag tgcacacagc ccaaacacag acccatcgcg aggactataa ctccaccctg 900 agggttgtga gcgcactgcc cattcagcac caggactgga tgagtgggaa ggagttcaaa 960 tgcaaggtaa ataacaagga tctgcccgcc ccgattgaac ggactatcag caaaccgaag 1020 gggtccgtta gagcacctca ggtatacgtg ttgccacctc cagaggagga aatgaccaag 1080 aaacaggtta ccctgacatg catggtaacg gacttcatgc ctgaggacat ttacgtggag 1140 tggacgaata acggcaaaac cgagctgaac tacaagaaca ctgagcctgt cctcgattcc 1200 gatggctcct atttcatgta ctctaagctc agagtggaaa agaagaactg ggtggaacgc 1260 atagctact cttgctcagt ggtccatgag ggcttgcaca atcatcacac tactaagtca 1320 ttctctcgga cacccggaaa g 1341 <![CDATA[ <210> 75]]> <![CDATA[ <211> 214]]> <![CDATA[<212> PRT]]> <![CDATA[<213> House mouse]]> <![CDATA[<400> 75]]> Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly 1 5 10 15 Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Phe Leu Ile 35 40 45 Tyr Tyr Ser Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln 65 70 75 80 Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Ser Thr Leu Pro Phe 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala Asp Ala Ala 100 105 110 Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu Thr Ser Gly 115 120 125 Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile 130 135 140 Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val Leu 145 150 155 160 Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser 165 170 175 Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr 180 185 190 Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys Ser 195 200 205 Phe Asn Arg Asn Glu Cys 210 <00103​​​​​​​​​​​​​​​​​​ gacattcaga tgactcagac cacgtcctcc ttgtccgcct ctttgggaga tcgcgttacc 60 atctcttgca gagcatctca ggatattagc aactacctga actggtatca gcagaaaccc 120 gatgggacgg tgaagttcct catctactat tcctccagac ttcactctgg ggtaccatca 180 cggtttagtg gcagtggtag cggcaccgac tactctctca ccatttccaa ccttgaacag 240 gaggacatag ccacctactt ttgccaacaa gggagcactc tgccctttac atttggcgga 300 ggtaccaaac tggagatcaa acgagctgat gcagcaccaa ctgtctccat cttccctcct 360 agcagtgaac agctgactag tggaggagcc tctgtggtgt gtttcctcaa caacttctac 420 cccaaagaca tcaatgtcaa gtggaagatc gatgggtcag aacgtcaaaa tggcgtgctg 480 aatagctgga cagatcagga tagcaaggac tccacttact caatgagctc aacactgaca 540 ctgaccaaag acgagtatga gaggcataac agctatacgt gtgaagccac acacaagacc 600 agcacatcac cgattgtgaa gagcttcaat cggaatgagt gc 642 <![CDATA[<210> 77]]> <![CDATA[<211> 447]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Mus musculus]]> <![CDATA[<400> 77]]> [[ID=*36]] Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Met Pro Cys Lys Thr Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Tyr Ile Gln Trp Leu Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Trp Ile Tyr Phe Gly Asp Ala Ser Thr Lys Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Lys Thr Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Leu Leu Ser Ser Leu Thr Ser Glu Asp Ser Gly Ile Tyr Phe Cys 85 90 95 Ala Ser Tyr Tyr Gly Ser Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Thr 100 105 110 Leu Thr Val Ser Ser Ala Lys Thr Thr Ala Pro Ser Val Tyr Pro Leu 115 120 125 Ala Pro Val Cys Gly Asp Thr Thr Gly Ser Ser Val Thr Leu Gly Cys 130 135 140 Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Leu Thr Trp Asn Ser 145 150 155 160 Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175 Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Thr Ser Ser Thr Trp 180 185 190 Pro Ser Gln Ser Ile Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr 195 200 205 Lys Val Asp Lys Lys Ile Glu Pro Arg Gly Pro Thr Ile Lys Pro Cys 210 215 220 Pro Pro Cys Lys Cys Pro Ala Pro Asn Leu Leu Gly Gly Pro Ser Val 225 230 235 240 Phe Ile Phe Pro Pro Lys Ile Lys Asp Val Leu Met Ile Ser Leu Ser 245 250 255 Pro Ile Val Thr Cys Val Val Val Asp Val Ser Glu Asp Asp Pro Asp 260 265 270 Val Gln Ile Ser Trp Phe Val Asn Asn Val Glu Val His Thr Ala Gln 275 280 285 Thr Gln Thr His Arg Glu Asp Tyr Asn Ser Thr Leu Arg Val Val Ser 290 295 300 Ala Leu Pro Ile Gln His Gln Asp Trp Met Ser Gly Lys Glu Phe Lys 305 310 315 320 Cys Lys Val Asn Asn Lys Asp Leu Pro Ala Pro Ile Glu Arg Thr Ile 325 330 335 Ser Lys Pro Lys Gly Ser Val Arg Ala Pro Gln Val Tyr Val Leu Pro 340 345 350 Pro Pro Glu Glu Glu Met Thr Lys Lys Gln Val Thr Leu Thr Cys Met 355 360 365 Val Thr Asp Phe Met Pro Glu Asp Ile Tyr Val Glu Trp Thr Asn Asn 370 375 380 Gly Lys Thr Glu Leu Asn Tyr Lys Asn Thr Glu Pro Val Leu Asp Ser 385 390 395 400 Asp Gly Ser Tyr Phe Met Tyr Ser Lys Leu Arg Val Glu Lys Lys Asn 405 410 415 Trp Val Glu Arg Asn Ser Tyr Ser Cys Ser Val Val His Glu Gly Leu 420 425 430 His Asn His His Thr Thr Lys Ser Phe Ser Arg Thr Pro Gly Lys 435 440 445 <![CDATA[<210> 78]]> <![CDATA[<211> 1341]]> <![CDATA[<212> DNA]]> <![CDATA[<213> Mus musculus]]> <![CDATA[<400> 78]]> caggttcagc tgcagcagag tggtcccgaa cttgtgaaac ctggtgcctc tgtgaaaatg 60 ccctgtaaga cctctggata caccttcaca agctactaca tacagtggct gaaacagagg 120 ccaggacaag gcttggaatg gattggctgg atctactttg gggatgccag taccaagtac 180 aacgagaagt tcaaggggaa aaccacactg acagctgaca agtctagcag tacggcctat 240 atgctgctct caagcctcac ttctgaggat tcaggcatct acttttgcgc ctcctattat 300 ggctcctatt tcgactattg gggtcaaggg actaccttga cagtctccag cgcgaaaact 360 actgccccat cagtctatcc ccttgctccc gtctgcggtg ataccacggg ttcctctgtg 420 actcttggtt gtctggttaa agggtatttt cccgaaccag tcaccctcac atggaatagc 480 gggagcctct caagtggagt gcacaccttt cctgccgtcc tgcaatctga cctttacaca 540 ctttcttcaa gcgtgaccgt gacaagtagc acttggccct ctcagtccat tacttgcaat 600 gttgcacatc cagcatcaag tacaaaggtg gataagaaaa tcgaacccag gggcccaacc 660 atcaaaccct gtccgccatg taaatgccct gctccaaatc tcctgggcgg acctagtgtg 720 ttcatctttc ctcccaaaat caaagatgtg ctgatgatca gtctgagccc aatagtgacc 780 tgtgtggtag tggacgtttc cgaggatgac cctgacgtcc agataagctg gtttgtcaac 840 aatgtggaag tgcacacagc ccaaacacag acccatcgcg aggactataa ctccaccctg 900 agggttgtga gcgcactgcc cattcagcac caggactgga tgagtgggaa ggagttcaaa 960 tgcaaggtaa ataacaagga tctgcccgcc ccgattgaac ggactatcag caaaccgaag 1020 gggtccgtta gagcacctca ggtatacgtg ttgccacctc cagaggagga aatgaccaag 1080 aaacaggtta ccctgacatg catggtaacg gacttcatgc ctgaggacat ttacgtggag 1140 tggacgaata acggcaaaac cgagctgaac tacaagaaca ctgagcctgt cctcgattcc 1200 gatggctcct atttcatgta ctctaagctc agagtggaaa agaagaactg ggtggaacgc 1260 aatagctact cttgctcagt ggtccatgag ggcttgcaca atcatcacac tactaagtca 1320 ttctctcgga cacccggaaa g 1341 <![CDATA[<210> 79]]> <![CDATA[<211> 214]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Mus musculus]]> <![CDATA[<400> 79]]> Asn Ile Gln Met Thr Gln Asn Thr Ser Ser Leu Ser Ala Thr Leu Gly 1 5 10 15 Val Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30 [[ID=A7]] Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile A7 35 40 45 It should be noted that there seems to be an "A7" tag in the original text that is not clearly defined in the translation requirements. I have translated it as is while keeping the "A7" in the relevant position. If there are specific instructions regarding this "A7", it may need to be adjusted accordingly.Tyr Ser Thr Ser Arg Leu His Ser Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln 65 70 75 80 Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Ser Thr Leu Pro Phe 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala Asp Ala Ala 100 105 110 Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu Thr Ser Gly 115 120 125 Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile 130 135 140 Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val Leu 145 150 155 160 Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser 165 170 175 Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr 180 185 190 Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys Ser 195 200 205 Phe Asn Arg Asn Glu Cys 210 <![CDATA[<210> 80]]> <![CDATA[<211> 642]]> <![CDATA[<212> DNA]]> <![CDATA[<213> Mus musculus]]> <![CDATA[<400> 80]]> aacattcaga tgacccagaa taccagctcc ttgagtgcca cattgggcgt gagagtgacc 60 atctcatgtc gagcctctca ggacatcagc aattacctga actggtatca gcagaaacct 120 gatgggacag tcaagctgct catctactcc acttctcgcc ttcattccgg gattcccagt 180 cgtttctctg gctctggatc aggcactgac tattccctta ccatcagtaa cctggaacaa 240 gaggacatag ccacgtactt ttgccagcaa ggtagcactc tcccattcac ctttggaggt 300 ggcacaaagc tggagatcaa acgagctgat gcagcaccaa ctgtctccat cttccctcct 360 agcagtgaac agctgactag tggaggagcc tctgtggtgt gtttcctcaa caacttctac 420 cccaaagaca tcaatgtcaa gtggaagatc gatgggtcag aacgtcaaaa tggcgtgctg 480 aatagctgga cagatcagga tagcaaggac tccacttact caatgagctc aacactgaca 540 ctgaccaaag acgagtatga gaggcataac agctatacgt gtgaagccac acacaagacc 600 agcacatcac cgattgtgaa gagcttcaat cggaatgagt gc 642 <![CDATA[<210> 81]]> <![CDATA[<211> 446]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Mus musculus]]> <![CDATA[<400> 81]]> Glu Val Leu Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Pro Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Asn Val Asp Trp Val Arg Gln Ser His Gly Lys Ser Leu Glu Trp Ile 35 40 45 Gly Asp Ile Asp Pro Asn Asn Gly Gly Thr Ile Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Arg Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Trp Arg Gly Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val 100 105 110 Thr Val Ser Ser Ala Lys Thr Thr Ala Pro Ser Val Tyr Pro Leu Ala 115 120 125 Pro Val Cys Gly Asp Thr Thr Gly Ser Ser Val Thr Leu Gly Cys Leu 130 135 140 Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Leu Thr Trp Asn Ser Gly 145 150 155 160 Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Asp 165 170 175 Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Thr Ser Ser Thr Trp Pro 180 185 190 Ser Gln Ser Ile Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys 195 200 205 Val Asp Lys Lys Ile Glu Pro Arg Gly Pro Thr Ile Lys Pro Cys Pro 210 215 220 Pro Cys Lys Cys Pro Ala Pro Asn Leu Leu Gly Gly Pro Ser Val Phe 225 230 235 240 Ile Phe Pro Pro Lys Ile Lys Asp Val Leu Met Ile Ser Leu Ser Pro 245 250 255 Ile Val Thr Cys Val Val Val Asp Val Ser Glu Asp Asp Pro Asp Val 260 265 270 Gln Ile Ser Trp Phe Val Asn Asn Val Glu Val His Thr Ala Gln Thr 275 280 285 Gln Thr His Arg Glu Asp Tyr Asn Ser Thr Leu Arg Val Val Ser Ala 290 295 300 Leu Pro Ile Gln His Gln Asp Trp Met Ser Gly Lys Glu Phe Lys Cys 305 310 315 320 Lys Val Asn Asn Lys Asp Leu Pro Ala Pro Ile Glu Arg Thr Ile Ser 325 330 335 Lys Pro Lys Gly Ser Val Arg Ala Pro Gln Val Tyr Val Leu Pro Pro 340 345 350 Pro Glu Glu Glu Met Thr Lys Lys Gln Val Thr Leu Thr Cys Met Val 355 360 365 Thr Asp Phe Met Pro Glu Asp Ile Tyr Val Glu Trp Thr Asn Asn Gly 370 375 380 Lys Thr Glu Leu Asn Tyr Lys Asn Thr Glu Pro Val Leu Asp Ser Asp 385 390 395 400 Gly Ser Tyr Phe Met Tyr Ser Lys Leu Arg Val Glu Lys Lys Asn Trp 405 410 415 Val Glu Arg Asn Ser Tyr Ser Cys Ser Val Val His Glu Gly Leu His 420 425 430 Asn His His Thr Thr Lys Ser Phe Ser Arg Thr Pro Gly Lys 435 440 445 <![CDATA[<210> 82]]> <![CDATA[<211> 1338]]> <![CDATA[<212> DNA]]> <![CDATA[<213> Mus musculus]]> <![CDATA[<400> 82]]> gaagtgctgt tgcagcagtc aggtcctgaa ctcgtcaaac caggagcatc cgtgaagatt 60 ccctgtaagg cttctgggta taccttcacc gactacaacg tggattgggt tcggcagtct 120 cacggcaaaa gtctggagtg gattggcgac atagatccca ataatggcgg aaccatctac 180 aaccagaagt tcaaggacaa agccaccctg actgtggaca agtctagccg aacagcctac 240 atggagcttc gcagtctgac ttccgaggat actgccgtct actattgcgc acgttggaga 300 ggagccatgg actattgggg ccaagggacc tcagtgacgg ttagctccgc gaaaactact 360 gccccatcag tctatcccct tgctcccgtc tgcggtgata ccacgggttc ctctgtgact 420 cttggttgtc tggttaaagg gtattttccc gaaccagtca ccctcacatg gaatagcggg 480 agcctctcaa gtggagtgca cacctttcct gccgtcctgc aatctgacct ttacacactt 540 tcttcaagcg tgaccgtgac aagtagcact tggccctctc agtccattac ttgcaatgtt 600 gcacatccag catcaagtac aaaggtggat aagaaaatcg aacccagggg cccaaccatc 660 aaaccctgtc cgccatgtaa atgccctgct ccaaatctcc tgggcggacc tagtgtgttc 720 atctttcctc ccaaaatcaa agatgtgctg atgatcagtc tgagcccaat agtgacctgt 780 gtggtagtgg acgtttccga ggatgaccct gacgtccaga tagctggtt tgtcaacaat gtggaagtgc acacagccca aacacagacc catcgcgagg actataactc caccctgagg gttgtgagcg cactgcccat tcagcaccag gactggatga gtggagga gttcaaatgc aaggtaaata acaaggatct gcccgccccg attgaacgga ctatcagcaa accgaagggg tccgttagag cacctcaggt atacgtgttg ccacctccag aggaggaat gaccaagaaa caggttaccc tgacatgcat ggtaacggac ttcatgcctg aggacattta cgtggagtgg 1140 acgaatacg gcaaaaccga gctgaactac aagaacactg agcctgtcct cgattccgat ggctcctatt tcatgtactc taagctcaga gtggaaaaga agaactgggt ggaacgcaat 1260 agctactctt gctcagtggt ccatgagggc ttgcacaatc atcacactac taagtcattc 1320 tctcggacac ccggaaag 1338 <![CDATA[<210> 83]]> <![CDATA[<211> 214]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Mus musculus]]> <![CDATA[<400> 83]]> Asp Val Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly 1 5 10 15 Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Asp Asn Tyr 20 25 30 Leu Lys Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile 35 40 45 Tyr Asp Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Lys Gln 65 70 75 80 Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Tyr Thr Leu Pro Trp 85 90 95 Thr Phe Gly Gly Gly Thr Arg Leu Glu Ile Lys Arg Ala Asp Ala Ala 100 105 110 Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu Thr Ser Gly 115 120 125 Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile 130 135 140 Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val Leu 145 150 155 160 Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser 165 170 175 Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr 180 185 190 Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys Ser 195 200 205 Phe Asn Arg Asn Glu Cys 210 <![CDATA[<210> 84]]> <![CDATA[<211> 642]]> <![CDATA[<212> DNA]]> <![CDATA[<213> Mus musculus]]> <![CDATA[<400> 84]]> gatgtgcaga tgacacagac cacatctagc cttagtgcct cattgggcga tagagtgacc 60 atttcctgtc gtgcctccca agacatcgac aactacctga agtggtatca gcagaaaccc 120 gatggcactg tcaagctgct gatctacgac acatcacggt tgcacagtgg ggtaccttcc 180 cgcttttctg gcagcggatc tggtaccgac tatagcctga ctatctccaa tctgaagcag 240 gaggacattg ccacctactt ctgccagcaa ggctatacgc tcccatggac ttttggaggt 360. ggaactcgac tcgaaatcaa acgagctgat gcagcaccaa ctgtctccat cttccctcct agcagtgaac agctgactag tggaggagcc tctgtggtgt gtttcctcaa caacttctac 420. cccaaagaca tcaatgtcaa gtggaagatc gatgggtcag aacgtcaaaa tggcgtgctg aatagctgga cagatcagga tagcaaggac tccacttact caatgagctc aacactgaca ctgaccaaag acgagtatga gaggcataac agctatacgt gtgaagccac acacaagacc agcacac cgattgtga cgattgt gc 642. to cgattgtga gc <![CDATA[ <210> 85]]> <![CDATA[<211> 447]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Chimeric HM242-IgG1-K322A heavy chain PRT]]> <![CDATA[<400> 85]]> Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Tyr Ile Gln Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 [[ID=,50]] Gly Trp Ile Tyr Pro Gly Asp Gly Thr Thr Lys Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Lys Thr Thr Leu Thr Ile Asp Lys Ser Ser Arg Thr Ala Tyr 65 70 75 80 Met Leu Phe Asn Ser Leu Thr Ser Glu Asp Ser Gly Ile Tyr Phe Cys 85 90 95 Ala Ser Tyr Tyr Gly Ser Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Thr 100 105 110 Leu Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125 Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys 130 135 140 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 145 150 155 160 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180 185 190 Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn 195 200 205 Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His 210 215 220 Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val 225 230 235 240 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250 255 Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu 260 265 270 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 275 280 285 Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 290 295 300 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 305 310 315 320 Cys Ala Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile 325 330 335 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350 Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360 365 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375 380 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 385 390 395 400 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 405 410 415 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420 425 430 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445 <![CDATA[ <210> 86]]> <![CDATA[ <211> 1341]]> <![CDATA[ <212> DNA <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Chimeric HM242-IgG1-K322A heavy-chain DNA <![CDATA[<400> 86]]> caggtgcaac tccagcagtc tggtcccgaa ttggtgaaac ctggtgcctc agtcaagatg 60 tcttgcaaag catcagggta tacgtttacc tcctactaca tccagtgggt gaaacagagg 120 ccaggacagg gccttgagtg gattggctgg atttatcccg gagatggcac aaccaagtac 180 aacgagaagt tcaagggcaa aaccacactg actatcgaca agagctccag aactgcctac 240 atgctcttca attccctgac aagcgaagat agtggcatct acttttgtgc cagctactat 300 gggagctatt tcgactattg gggtcaaggg accactctga ccgtatcctc tgcgtcgacc 360 aagggcccct ccgtgttccc cctggccccc tcctccaagt ccacctccgg cggcaccgcc 420 gccctgggct gcctggtgaa ggactacttc cccgagcccg tgaccgtgtc ctggaactcc 480 ggcgccctga cctccggcgt gcacaccttc cccgccgtgc tccagtcctc cggcctgtac 540 ...

Claims

1. An antibody or a functional fragment thereof, having any one of the following (A) to (D): (A) an antibody heavy chain variable region comprising a complementarity-determining region (CDR) 1 (Sequence No. 345), a CDR 2 (Sequence No. 346), and a CDR 3 (Sequence No. 347), and an antibody light chain variable region comprising a CDR 1 (Sequence No. 348), a CDR 2 (Sequence No. 349), and a CDR 3 (Sequence No. 350); (B) an antibody heavy chain variable region comprising a CDR 1 (Sequence No. 351), a CDR 2 (Sequence No. 352), and a CDR 3 (Sequence No. 353), and an antibody light chain variable region comprising a CDR 1 (Sequence No. 354), a CDR 2 (Sequence No. 355), and a CDR 3 (Sequence No. 356); (C) The antibody heavy chain variable region comprising CDR1 of sequence number 357, CDR2 of sequence number 358, and CDR3 of sequence number 359, and the antibody light chain variable region comprising CDR1 of sequence number 360, CDR2 of sequence number 361, and CDR3 of sequence number 362; (D) The antibody heavy chain variable region comprising CDR1 of sequence number 363, CDR2 of sequence number 364, and CDR3 of sequence number 365, and the antibody light chain variable region comprising CDR1 of sequence number 366, CDR2 of sequence number 367, and CDR3 of sequence number 368.

2. The antibody or a functional fragment thereof as claimed in claim 1, wherein the variable region of the antibody contains a human antibody architecture or a humanized antibody architecture.

3. The antibody or its functional fragment as claimed in claim 1 has any one of the following (A) to (D): (A) the antibody heavy chain variable region of the amino acid sequence represented by sequence number 20 and the antibody light chain variable region of the amino acid sequence represented by sequence number 37; (B) the antibody heavy chain variable region of the amino acid sequence represented by sequence number 21 and the antibody light chain variable region of the amino acid sequence represented by sequence number 37; (C) the antibody heavy chain variable region of the amino acid sequence represented by sequence number 26 and the antibody light chain variable region of the amino acid sequence represented by sequence number 40; (D) the antibody heavy chain variable region of the amino acid sequence represented by sequence number 30 and the antibody light chain variable region of the amino acid sequence represented by sequence number 44.

4. The antibody or its functional fragment as requested in claim 1 or 3, wherein the constant region of the antibody heavy chain and the constant region of the antibody light chain contain an amino acid sequence derived from a human antibody.

5. The antibody or its functional fragment as requested in item 1 or 3, wherein the antibody heavy chain constant region is the antibody heavy chain constant region of human IgG.

6. The antibody or its functional fragment as claimed in claim 5, wherein the constant region of the antibody heavy chain of the aforementioned human IgG is the constant region of the antibody heavy chain of human IgG1.

7. The antibody or its functional fragment as requested in claim 1 or 3, wherein the constant region of the antibody light chain is the constant region of the human Igκ antibody light chain.

8. The antibody or functional fragment thereof, as claimed in claim 1 or 3, further exhibits an affinity for any one or more of the human FcγRIIA, human FcγRIIB, and human FcγRIIIA receptors that is equivalent to or greater than that of a reference antibody having the Fc region of human IgG1-K322A.

9. The antibody or functional fragment thereof, as requested in claim 1 or 3, further exhibits an affinity for human FcγRIIB that is equivalent to or greater than that of a reference antibody having the Fc region of human IgG1-K322A.

10. The antibody or functional fragment thereof, as requested in claim 1 or 3, further exhibits an affinity for human FcγRIIIA equal to or greater than that of a reference antibody having the Fc region of human IgG1-K322A.

11. The antibody or functional fragment thereof as claimed in claim 9, wherein the enhancement of affinity for human FcγRIIB in an Fc receptor binding affinity assay using surface plasma resonance technology is greater than 1.5 times compared to a reference antibody having the Fc region of human IgG1-K322A.

12. The antibody or functional fragment thereof as claimed in claim 9, wherein the enhanced affinity for human FcγRIIB in a flow cytometry assay of human Fc receptor-expressing cell lines is more than twice that of a reference antibody having the Fc region of human IgG1-K322A.

13. The antibody or functional fragment thereof as claimed in claim 10, wherein the affinity for human FcγRIIIA is increased by more than 1.5 times compared with a reference antibody having the Fc region of human IgG1-K322A in an Fc receptor binding affinity assay using surface plasma resonance technology.

14. The antibody or functional fragment thereof, as claimed in claim 10, wherein the enhanced affinity for human FcγRIIIA in an Fc receptor binding affinity assay using flow cytometry is greater than 1.5 times that of a reference antibody having the Fc region of human IgG1-K322A.

15. The antibody or functional fragment thereof of claim 8 has a modified Fc region of human IgG, wherein the affinity for the aforementioned receptor is equal to or greater than that of the antibody before modification due to the modification of the Fc region of human IgG.

16. The antibody or functional fragment thereof of claim 8 has a modified Fc region of human IgG1 or human IgG4, wherein the affinity for the aforementioned receptor is equal to or greater than that of the antibody before modification due to the modification of the Fc region of human IgG1 or human IgG4.

17. The antibody or functional fragment thereof as requested in claim 8, wherein any one of the amino acids at positions 233, 234, 236, 237, 238, 239, 267, 268, 271, 296, 323, 326, 328, 330 and 332 (according to EU designation) in the amino acid sequence of the Fc region of human IgG1 is modified.

18. The antibody or its functional fragment as claimed in claim 17, wherein the modified amino acid is G236D / H268D, S239D / H268D, S239D / H268D / L328Y / I332E, G236D / H268D / K322A, S239D / H268D / K322A, S239D / S267G / H268D / K322A, G236D / H268D / E293A / K322A, S239D / H268D / K322A / L328Y / I332E, S239D / H268D / E2 Any combination of 93A / K322A, S239D / S267G / H268D / K322A / L328Y, S239D / S267G / H268D / K322A / I332E, S239D / H268D / K322A / L328Y, S239D / H268D / K322A / I332E, S239D / S267G / H268D / K322A / L328Y / I332E, E233D / G237D / P238D / H268D / P271G / A330R, and S267E / L328F.

19. The antibody or functional fragment thereof as requested in claim 8, wherein any one of the amino acids at positions 236, 239, 268, 328, and 332 (according to EU designation) in the amino acid sequence of the Fc region of human IgG4 is modified.

20. The antibody or its functional fragment as claimed in claim 19, wherein the modified amino acid is any combination of S228P / G236D / Q268D, S228P / S239D / Q268D or S228P / S239D / Q268D / L328Y / I332E.

21. The antibody or its functional fragment as claimed in claim 8 comprises any one of the following (A) to (D): (A) the antibody heavy chain sequence of the amino acid sequence represented by sequence number 245 and the antibody light chain sequence of the amino acid sequence represented by sequence number 247; (B) the antibody heavy chain sequence of the amino acid sequence represented by sequence number 277 and the antibody light chain sequence of the amino acid sequence represented by sequence number 279; (C) the antibody heavy chain sequence of the amino acid sequence represented by sequence number 285 and the antibody light chain sequence of the amino acid sequence represented by sequence number 287; (D) the antibody heavy chain sequence of the amino acid sequence represented by sequence number 289 and the antibody light chain sequence of the amino acid sequence represented by sequence number 291.

22. The antibody or functional fragment thereof as claimed in claim 10, wherein the antibody system is "defucosomed".

23. A single isolated nucleic acid that encodes an antibody or a functional fragment thereof as described in any one of claims 1 to 22.

24. A vector comprising the nucleic acid as claimed in claim 23.

25. A host cell comprising the nucleic acid as claimed in claim 23.

26. A host cell comprising the vector as claimed in claim 24.

27. A method for producing an antibody or a functional fragment thereof, comprising a stage of culturing host cells as claimed in claim 25 or 26.

28. A pharmaceutical composition comprising an antibody or a functional fragment thereof as claimed in any one of claims 1 to 22, and a pharmaceutically permissible carrier.

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