BR112014018539B1 - METHOD OF PREPARING A HYBRIDOMA THAT PRODUCES A MONOCLONAL ANTIBODY, HYBRIDOMA, MONOCLONAL ANTIBODY OR A FRAGMENT CONTAINING AN ANTIGEN-BINDING REGION THEREOF, METHOD OF PREPARING A MONOCLONAL ANTIBODY, EX VIVO METHOD OF DETECTING A PLASMACYTOID DENDRITIC CELL, REAGENT FOR THE DETECTION OF A PLASMACYTOID DENDRITIC CELL, EX VIVO METHOD OF SUPPRESSING AN ACTIVITY OF A PLASMACYTOID DENDRITIC CELL AND AGENT FOR SUPPRESSING AN ACTIVITY OF A PLASMACYTOID DENDRITIC CELL - Google Patents

Abstract

ANTI-PHOSPHOLIPASE D4 ANTIBODY. A monoclonal antibody that binds to a phospholipase D4 (PLD4) protein, or a fragment containing an antigen-binding region thereof.

Description

Technical Field

[0001] The present invention concerns an antibody that binds to phospholipase D4. Hereinafter, “phospholipase D” may be abbreviated as PLD, and “phospholipase D4” and the like may be abbreviated as PLD4 and the like.

Fundamentals of the technique

[0002] Interferon (hereinafter, “interferon” can be abbreviated as IFN) is the most important cytokine in the anti-virus immune response. The cell that produces interferon in human blood (IPC: IPC is an undifferentiated lymphocyte-based dendritic cell positioned as a precursor cell of the dendritic cell (DC). The IPC may also be called a plasmacytoid dendritic cell or plasmacytoid dendritic cell (pDC ).Hereinafter, in the present specification, IPC and pDC are synonymous, and uniformly referred to as a term of pDC in principle below.) expresses CD4 and the major histocompatible complex class II protein. However, particular isolation or characterization of cells has not been carried out so far due to an insufficient number of such cells, rapid apoptosis, and additional lack of lineage marker (system). It was revealed that pDC is a CD4+CD11c-2 type precursor cell of the dendritic cell, and it was discovered that pDC produces IFN more than 200 to 1000 times than other blood cells after stimulation by a microorganism. Consequently, pDC is a conclusive immune system effector cell in an anti-viral/anti-tumor immune response.

[0003] IFNa and IFNß are known as type I IFN having anti-viral activity or anti-tumor activity. On the other hand, it has been revealed that IFNa is associated with autoimmune diseases. For example, abnormal production of IFNa has been reported in patients with autoimmune diseases such as systemic lupus erythematosus and chronic rheumatoid arthritis. Furthermore, a case has been reported where symptoms of autoimmune disease are expressed or aggravated upon administration of a recombinant IFNa2 or IFN. It has also been suggested that autoimmune symptoms are likely to be alleviated by neutralizing IFNa.

[0004] Furthermore, it was also revealed that IFNa induces dendritic cell (DC) differentiation. It was considered that the induction of differentiation of a dendritic cell constitutes an important mechanism in an autoimmune disease since a dendritic cell is an antigen-presenting cell. Indeed, the induction of IFNa dendritic cell differentiation has been suggested to be profoundly associated with the development of systemic lupus erythematosus. As described above, the close relationships of IFNa with autoimmune diseases as well as anti-tumor activity have been highlighted. Furthermore, IFNa is also profoundly associated with the development of psoriasis.

[0005] Only a few pDCs exist in the blood. The ratio of pDC occupying the peripheral blood lymphocyte is considered to be 1% or less. However, pDC has a very high IFN production capacity. The IFN production capacity of pDCs reaches, for example, 3000 pg/ml/104 cells. This means, it can be said that most of the IFNa or IFNß in the blood produced at the time of viral infection is caused by pDC, although the number of cells is small.

[0006] pDC is differentiated into a dendritic cell by viral stimulation, and induces the production of IFN-Y or interleukin (IL)-10 by the T cell. Furthermore, pDC is also differentiated into a dendritic cell by IL-10 stimulation. 3. The differentiated dendritic cell upon IL-3 stimulation induces the production of the Th2 cytokine (IL-4, IL-5, IL-10) by the T cell. As described above, pDC has a property that is differentiated into different dendritic cells depending on the difference in stimulations.

[0007] Consequently, the pDC is a cell that has two sides, that is, one side as a cell that produces IFN, and the other side as a precursor cell of a dendritic cell. Each cell plays an important role in the immune system. This means, pDC is one of the important cells that support the immune system in several aspects.

[0008] In regulating the activity of a humoral factor such as IFN, the administration of an antibody that recognizes the factor is effective. For example, an attempt to treat autoimmune diseases with an antibody against IL-1 or IL-4 has come into practical use. Furthermore, also for IFN similarly, the neutralizing antibody is considered as a therapeutic agent for autoimmune diseases. It can be expected that a similar method will be effective for pDC that produces IFN. However, this method is based on inhibiting the action of a humoral factor after it is produced. If the production of a desired humoral factor can be directly controlled, other essential therapeutic effects can be obtained.

[0009] Antibodies that recognize human pDC have been reported. For example, the anti-BDCA-2 monoclonal antibody is a human pDC-specific monoclonal antibody (Dzionek, A. et al. J. Immunol, 165: 6037-6046, 2000). It has been revealed that the anti-BDCA-2 monoclonal antibody has an action of suppressing the production of IFN from human pDC (J. Exp. Med. l94: 1823-1834, 2001). Additionally, it has also been reported that a monoclonal antibody that recognizes the interferon-producing mouse cell suppresses interferon production (Blood Jun 1 2004; 103/11: 4201-4206. Epub Dec 2003). It has also been reported that the number of dendritic cells decreases in a monoclonal antibody to mouse pDC (J. Immunol. 2003, 171: 6466-6477).

[0010] Similarly, it would be useful if an antibody that could recognize pDC and regulate its activity were provided. For example, the present inventors have already disclosed that an antibody that recognizes Ly49Q specifically binds to mouse pDC. However, the antibody to Ly49Q did not interfere with the activity of mouse pDC (Blood, April 1, 2005, Vol. 105, No. 7, pp. 2787-2792).

[0011] PLD is an enzyme that catalyzes a phosphatidyl choline hydrolysis reaction to produce phosphatidic acid and choline, and causes signaling in various cells. The phosphatidic acid produced is considered to function as a lipid signal molecule.

[0012] PLD1 and PLD2 are conventionally known as two types of mammalian PLDs, and contain Phox homology domain (PX domain), which is capable of binding to phosphatidyl inositide, and pleckstrin homology domain (PH domain) in the region of N terminal of the same. Both domains are involved in membrane targeting of PLD.

[0013] PLD1 and PLD2 also contain two sequences His-x-Lys-x-x-x-x-Asp (HKD motif). This HKD motif is an essential domain in PLD activity.

[0014] It was considered that the phosphatidic acid produced by PLD1 and PLD2 is involved in the reconstitution of the cellular skeleton, exocytosis, phagocytosis, cancerization, cell adhesion, chemotaxis, and the like, and act centrally in the nervous system, the immune system, and similar ones.

[0015] Although human Hu-K4 and mouse SAM9 are officially called PLD3 until now, they are lacking the PX and PH domains, and do not exhibit any PLD activity although they have two HKD motifs. Furthermore, although there are three members of the PLD family, i.e., PLD4, PLD5, and PLD6, these nonclassical PLDs are hardly known.

[0016] The Cerebellar Development Transcriptome Database (CDT-DB) for the gene expression pattern in the developing mouse cerebellum was searched, and as a result of this, PLD4, which was a transcription product that was controlled in developmental time, was identified (see Tao et al., Nat. Methods 2(8), 591-598 (2005)). The basic characteristics of PLD4 have not been reported. It is considered that it should be determined from now on whether PLD4 exhibits enzymatic activity or not, and whether a deglycosylated form of PLD4 has PLD activity or not.

[0017] PLD4 is a 506 amino acid sequence represented by SEQ ID NO: 1 (Tao et al., Nat. Methods 2(8), 591-598 (2005) and Clark et al., Genome Res. 13(10 ), 2265-2270 (2003)). The PLD4 protein has two experimental PDE regions (phosphodiesterase motif), which are made up of two HKD motifs (His-x-Lys-x-x-x-x-Asp amino acid sequence, where x is the other of the amino acids) conserved in the PLD4 protein region. C terminus, and a presumptive phosphorylation site (Thr 472). The protein structure of PLD4 is predicted to be a type II monotropic transmembrane protein. Furthermore, the N-terminal region of the PLD4 protein does not have the PX region and the PH region, which are possessed by PLD1 and PLD2, which are classics of the PLD family (FIGS. 1 and 2).

[0018] On the other hand, although PLD4 belongs to the PLD family due to the fact that it has two HKD motifs, PLD4 lacks the PX domain and PH domain, but on the contrary has a putative transmembrane domain.

[0019] PLD4 mRNA expression, which was characteristically from a low level to a medium level, was found in a cell subpopulation that was preferentially located in the corpus callosum and the periphery of the white matter region including the cerebellar white matter of a mouse 1 week after birth. These cells expressing PLD4 mRNA were identified as Ibal-positive microglia (see Tao et al., Nat. Methods 2(8), 591-598 (2005)).

[0020] The period of 1 week after birth is a period when activation of myelin formation begins in the corpus callosum and cerebellar white matter of a mouse. During this period, PLD4 is highly expressed in amoeboid microglia (activated state) that exist in the white matter. From these facts, a possibility is considered that the PLD4-expressing cell in the white matter is involved in the formation of myelin in this period. Particularly, the accumulation of PLD4 in the phagocytic vesicle becomes evident, and a possibility is suggested that the PLD4-expressing cell is involved in phagocytosis. From amoeboid microglia in the activated state, various cytokines or growth factors are secreted, and phagocytosis is also activated. It is considered that the extra oligodendrocyte (glial cell in the central nervous system, which forms myelin as coiled and attached to the axon) causes apoptosis in the white matter of the brain in the developmental stage. One possibility was considered that the extra oligodendrocyte was degraded and removed from the amoeboid microglia to secrete a signal molecule, thereby preparing the environment for myelin formation in the white matter. It is suggested that PLD4 is involved in these processes including myelin formation.

[0021] PLD4 mRNA expression is universally observed also in non-nervous tissue, but is mainly distributed in the spleen. Strong PLD4 expression is detected at the periphery of the red pulp border zone of the spleen, and splenic PLD4 protein collected from the membrane fraction in the cell is highly N-glycosylated. When PLD4 was expressed in a heterogeneous cell system, they were localized to the endoplasmic reticulum and the Golgi body. Heterologously expressed PLD4 did not show any PLD enzymatic activity (Plos A www.plosone.org, November 2010, Volume 5, Issue 11, e13932).

[0022] From the pattern of time- and location-limited PLD4 expression, it is suggested that PLD4 plays a role in common functions in microglia or the cell in the border region of the spleen at the time of brain development in the early stage after birth.

[0023] PLD4 mRNA expression and distribution of PLD4 in nervous tissue and non-nervous tissue were summarized above. However, the present inventors have discovered that PLD4 mRNA is specifically highly expressed in a resting stage pDC cell (resting pDC) at the level of a cell species described below.

[0024] Mouse anti-human PLD4 polyclonal antibody against the full-length human PLD4 protein is commercially available (purified PLD4 MaxPab mouse polyclonal antibody (B01P), catalog No. H00122618-B01P, manufactured by Abnova Corporation ). However, a monoclonal antibody that binds only to a certain site of PLD4, or a monoclonal antibody that can specifically bind to PLD4, has not been obtained. Citation List Non-Patent Literature (1) Tao et al., Nat. Methods 2(8), 591-598 (2005) (2) Clark et al., Genome Res. 13(10), 2265-2270 ( 2003) (3) Plos A www.plosone.org, November 2010, Volume 5, Issue 11, e13932 (4) Catalog of mouse PLD4 polyclonal antibody against full-length human PLD4 protein (Abnova, catalog No. H00122618 -B01P) (5) Dzionek, A. et al. J. Immunol. 165: 6037-6046, 2000 (6) J. Exp. Med, 194: 1823-1834, 2001 (7) Blood Jun 1, 2004; 103/11: 4201-4206. Epub Dec 2003 (8) J. Immunol. 2003, 171: 6466-6477 (9) Blood, April 1, 2005, Vol. 105, No. 7, pp. 2787-2792 (10) Nat. Methods 2(8), 591-598 (2005)

Summary of the Invention Technical problem

[0025] A problem to be solved by the invention is to provide an antibody that binds to PLD4, and detect, identify, or isolate pDC. Furthermore, a problem to be solved by the invention is regulating pDC activity.

Solution to the Problem

[0026] The present inventors have confirmed through a search for PLD4 that the expression of PLD4 specifically arises in pDC, particularly pDC in the resting stage in addition to pDC in the active stage. Accordingly, the present inventors attempted the preparation of PLD4 antibody and the elucidation of its action.

[0027] In order to obtain an antibody that recognizes a small amount of a protein derived from a living organism, a protein prepared by recombinant gene technology is generally used as an immunogen. The present inventors attempted the expression of PLD4 based on the base sequence information of PLD4 cDNA, and an amino acid sequence (GenBank Accession No. NM_138790.2) encoded by it, which has already been disclosed (Nat. Methods 2(8) , 591-598 (2005)).

[0028] In order to obtain an antibody from a protein, the use of a partial amino acid sequence of a natural protein as an immunogen is often attempted. However, in order for an antibody to recognize a molecule on the cell surface, it is necessary to select a region that constitutes a part recognized by the antibody as an epitope on the cell surface. Consequently, it has been considered that it is unrealistic to use a fragmented amino acid sequence as an immunogen to obtain an antibody specific to PLD4.

[0029] Under such circumstances, the present inventors have revealed that the use of a special immunogen allows obtaining an antibody that binds to pDC. Furthermore, the present inventors confirmed that the antibody thus obtained specifically recognizes human pDC, and furthermore has an action to regulate its activity, and completed the invention. That is to say, the invention concerns an anti-PLD4 antibody, a method of preparation thereof, and use thereof described below. The invention is as follows: (1) A monoclonal antibody that binds to a phospholipase D4 (PLD4) protein, or a fragment containing an antigen-binding region thereof. (2) The monoclonal antibody, or a fragment containing an antigen-binding region thereof as described in (1) mentioned above, which has the sequence SYWMH (SEQ ID NO: 2) as CDR1, the sequence DIYPGSDSTNYNEKFKS (SEQ ID NO: : 3) as CDR2, and the sequence GGWLDAMDY (SEQ ID NO: 4) as CDR3 in the heavy chain variable region. (3) the monoclonal antibody, or a fragment containing an antigen-binding region thereof as described in (1) mentioned above, which has the sequence RASQDISNILN (SEQ ID NO: 5) as CDR1, the sequence YTSRLHS (SEQ ID NO : 6) as CDR2, and sequence QQGNTLPW (SEQ ID NO: 7) as CDR3 in the light chain variable region. (4) the monoclonal antibody, or a fragment containing an antigen-binding region thereof as described in (1) mentioned above, which has the sequence SYWMH as CDR1, the sequence DIYPGSDSTNYNEKFKS as CDR2 and the sequence GGWLDAMDY as CDR3 in the variable region heavy chain, and has the sequence RASQDISNILN as CDR1, the sequence YTSRLHS as CDR2, and the sequence QQGNTLPW as CDR3 in the light chain variable region. (5) the monoclonal antibody (3B4 antibody), or a fragment containing an antigen-binding region thereof as described in (1) mentioned above, which has the sequence TYWMH (SEQ ID NO: 8) as CDR1, the sequence AIYPGNSETSYNQKFKG (SEQ ID NO: 9) as CDR2, and the sequence GYSDFDY (SEQ ID NO: 10) as CDR3 in the heavy chain variable region. (6) the monoclonal antibody, or a fragment containing an antigen-binding region thereof as described in (1) mentioned above, which has the sequence HASQGIRSNIG (SEQ ID NO: 11) as CDR1, the sequence HGTNLED (SEQ ID NO : 12) as CDR2, and the sequence VQYVQFP (SEQ ID NO: 13) as CDR3 in the light chain variable region. (7) the monoclonal antibody, or a fragment containing an antigen-binding region thereof as described in (1) mentioned above, which has the sequence TYWMH as CDR1, the sequence AIYPGNSETSYNQKFKG as CDR2, and the sequence GYSDFDY as CDR3 in the region heavy chain variable region, and has the sequence HASQGIRSNIG as CDR1, the sequence HGTNLED as CDR2, and the sequence VQYVQFP as CDR3 in the light chain variable region. (8) the monoclonal antibody (5B7 antibody), or a fragment containing an antigen-binding region thereof as described in (1) mentioned above, which has the sequence DYNLH (SEQ ID NO: 14) as CDR1, the sequence YIYPYNGNTGYNQKFKR (SEQ ID NO: 15) as CDR2, and the sequence GGIYDDYYDYAIDY (SEQ ID NO: 16) as CDR3 in the heavy chain variable region. (9) the monoclonal antibody, or a fragment containing an antigen-binding region thereof as described in (1) mentioned above, which has the sequence RASENIYSHIA (SEQ ID NO: 17) as CDR1, the sequence GATNLAH (SEQ ID NO : 18) as CDR2, and the sequence QHFWGTP (SEQ ID NO: 19) as CDR3 in the light chain variable region. (10) the monoclonal antibody, or a fragment containing an antigen-binding region thereof as described in (1) mentioned above, which has the sequence DYNLH as CDR1, the sequence YIYPYNGNTGYNQKFKR as CDR2, and the sequence GGIYDDYYDYAIDY as CDR3 in the region heavy chain variable region, and has the sequence RASENIYSHIA as CDR1, the sequence GATNLAH as CDR2, and the sequence QHFWGTP as CDR3 in the light chain variable region. (11) the monoclonal antibody (8C11 antibody), or a fragment containing an antigen-binding region thereof as described in (1) mentioned above, which has the sequence SYILY (SEQ ID NO: 20) as CDR1, the sequence LINPTNSDTIFNEKFKS (SEQ ID NO: 21) as CDR2, and the sequence EGGYGYGPFAY (SEQ ID NO: 22) as CDR3 in the heavy chain variable region. (12) the monoclonal antibody, or a fragment containing an antigen-binding region thereof as described in (1) mentioned above, which has the sequence TSSQTLVHSNGNTILH (SEQ ID NO: 23) as CDR1, the sequence KVSNRFS (SEQ ID NO : 24) as CDR2, and the sequence HSTHVP (SEQ ID NO: 25) as CDR3 in the light chain variable region. (13) the monoclonal antibody, or a fragment containing an antigen-binding region thereof as described in (1) mentioned above, which has the sequence SYILY as CDR1, the sequence LINPTNSDTIFNEKFKS as CDR2, and the sequence EGGYGYGPFAY as CDR3 in the region heavy chain variable region, and has the sequence TSSQTLVHSNGNTILH as CDR1, the sequence KVSNRFS as CDR2, and the sequence HSTHVP as CDR3 in the light chain variable region. (14) the monoclonal antibody (10C3 antibody), or a fragment containing an antigen-binding region thereof as described in (1) mentioned above, which has the sequence SYGMS (SEQ ID NO: 26) as CDR1, the sequence TISSGGSYIYYPESVKG (SEQ ID NO: 27) as CDR2, and the sequence LYGGRRGYGLDY (SEQ ID NO: 28) as CDR3 in the heavy chain variable region. (15) the monoclonal antibody, or a fragment containing an antigen-binding region thereof as described in (1) mentioned above, which has the sequence RSSKSLLHSDGITILY (SEQ ID NO: 29) as CDR1, the sequence QMSNLAS (SEQ ID NO : 30) as CDR2, and the sequence AQNLEL (SEQ ID NO: 31) as CDR3 in the light chain variable region. (16) the monoclonal antibody, or a fragment containing an antigen-binding region thereof as described in (1) mentioned above, which has the sequence SYGMS as CDR1, the sequence TISSGGSYIYYPESVKG as CDR2, and the sequence LYGGRRGYGLDY as CDR3 in the region heavy chain variable region, and has the sequence RSSKSLLHSDGITILY as CDR1, the sequence QMSNLAS as CDR2, and the sequence AQNLEL as CDR3 in the light chain variable region. (17) the monoclonal antibody (13D4 antibody), or a fragment containing an antigen-binding region thereof as described in (1) mentioned above, which has the sequence SHYYWT (SEQ ID NO: 32) as CDR1, the sequence YISYDGSNNYNPSLKN (SEQ ID NO: 33) as CDR2, and the sequence EGPLYYGNPYWYFDV (SEQ ID NO: 34) as CDR3 in the heavy chain variable region. (18) the monoclonal antibody, or a fragment containing an antigen-binding region thereof as described in (1) mentioned above, which has the sequence RASQDIDNILN (SEQ ID NO: 35) as CDR1, the sequence YTSRLHS (SEQ ID NO : 36) as CDR2, and the sequence QQFNTLP (SEQ ID NO: 37) as CDR3 in the light chain variable region. (19) the monoclonal antibody, or a fragment containing an antigen-binding region thereof as described in (1) mentioned above, which has the sequence SHYYWT as CDR1, the sequence YISYDGSNNYNPSLKN as CDR2, and the sequence EGPLYYGNPYWYFDV as CDR3 in the region heavy chain variable region, and has the sequence RASQDIDNILN as CDR1, the sequence YTSRLHS as CDR2, and the sequence QQFNTLP as CDR3 in the light chain variable region. (20) the monoclonal antibody (13H11), or a fragment containing an antigen-binding region thereof as described in (1) mentioned above, which has the sequence SHYYWS (SEQ ID NO: 38) as CDR1, the sequence YISYDGSNNYNPSLKN ( SEQ ID NO: 39) as CDR2, and the sequence EGPLYYGNPYWYFDV (SEQ ID NO: 40) as CDR3 in the heavy chain variable region. (21) the monoclonal antibody, or a fragment containing an antigen-binding region thereof as described in (1) mentioned above, which has the sequence RASQDIDNILN (SEQ ID NO: 41) as CDR1, the sequence YTSRLHS (SEQ ID NO : 42) as CDR2, and the sequence QQFNTLP (SEQ ID NO: 43) as CDR3 in the light chain variable region. (22) the monoclonal antibody, or a fragment containing an antigen-binding region thereof as described in (1) mentioned above, which has the sequence SHYYWS as CDR1, the sequence YISYDGSNNYNPSLKN as CDR2, and the sequence EGPLYYGNPYWYFDV as CDR3 in the region heavy chain variable region, and has the sequence RASQDIDNILN as CDR1, the sequence YTSRLHS as CDR2, and the sequence QQFNTLP as CDR3 in the light chain variable region. (23) A monoclonal antibody, or a fragment containing an antigen-binding region thereof, that is produced by any of the mp5B7, mp7B4, mp13D4, and mp13H11 hybridomas that are deposited under Accession Numbers: NITE BP-1211, NITE BP-1212, NITE BP-1213, and NITE BP-1214. (24) A hybridoma that produces any of the monoclonal antibodies as described in (1) or (2) mentioned above. (25) An mp5B7, mp7B4, mp13D4 or mp13H11 hybridoma which are deposited under Accession Numbers: NITE BP-1211, NITE BP-1212, NITE BP-1213 or NITE BP-1214. (26) A method of preparing an antibody monoclonal, containing a process of cultivating the hybridoma as described in (25) mentioned above, and collecting a monoclonal antibody from the culture. (27) A method of preparing a cell that produces a monoclonal antibody that binds to PLD4, containing the following processes; (1) a method of administering recombinant PLD4-Ig fusion protein encoding an amino acid sequence containing an extracellular PLD4 domain, to an immune animal, and (2) a method of selecting an antibody-producing cell that produces an antibody which binds to PLD4 from cells that produce the immune animal's antibody. (28) The method as described in (27) mentioned above, wherein the cell expressing PLD4 is a cell that retains an extrinsic polynucleotide encoding an amino acid sequence containing the extracellular PLD4 domain in an expressible manner. (29) The method as described in (28) mentioned above, wherein the cell is an animal cell. (30) The method as described in (29) mentioned above, wherein the cell is a human-derived cell. (31) The method as described in (30) mentioned above, wherein the human-derived cell is a HEK-293 T cell. (32) The method as described in any of (27) to (31) mentioned above, containing a process of cloning the incrementally obtained antibody-producing cells. (33) A method of preparing a monoclonal antibody that binds to an extracellular PLD4 domain, containing a method of culturing the antibody-producing cells obtained by the method as described in (29) mentioned above, and collecting a monoclonal antibody from the culture. (34) A monoclonal antibody that recognizes PLD4, or a fragment containing an antigen-binding region thereof, which can be obtained by the following processes; 1) a method of administering a recombinant PLD4-Ig fusion protein encoding an amino acid sequence containing an extracellular PLD4 domain, to an immune animal, 2) a method of selecting an antibody-producing cell that produces an antibody that binds to PLD4 from antibody-producing cells of the immune animal, and 3) a process of culturing the antibody-producing cells selected in process 2), and collecting an antibody that recognizes PLD4 from the culture. (35) An immunogen for preparing an antibody that binds to PLD4, containing (a) an animal cell that retains a polynucleotide encoding an amino acid sequence containing an extracellular PLD4 domain extrinsically in an expressible manner, or a cell membrane fraction the same. (36) The immunogen as described in (35) mentioned above, wherein the animal cell is a human-derived cell. (37) A method of detecting a plasmacytoid dendritic cell, containing a method of bringing a monoclonal antibody that binds to an extracellular PLD4 domain, or a fragment containing an antigen-binding region thereof, into contact with a test cell, and detecting a monoclonal antibody, or a fragment containing an antigen-binding region thereof that binds to the cell. (38) A reagent for the detection of a plasmacytoid dendritic cell, containing a monoclonal antibody that binds to an extracellular PLD4 domain, or a fragment containing an antigen-binding region thereof. (39) A method of suppressing an activity of a plasmacytoid dendritic cell, containing a process of bringing any of the components described below into contact with the plasmacytoid dendritic cell: (a) a monoclonal antibody that binds to PLD4 and suppresses an activity of the plasmacytoid dendritic cell, or a fragment containing an antigen-binding region thereof, and (b) an immunoglobulin into which a complementarity-determining region of the monoclonal antibody (a) is transplanted, or a fragment containing an antigen-binding region of the same. (40) A method of suppressing an activity of a plasmacytoid dendritic cell in a living organism, containing a method of administering any of the components described below to the living organism: (a) a monoclonal antibody that binds to PLD4, and suppresses an activity of the plasmacytoid dendritic cell, or a fragment containing an antigen-binding region thereof, and (b) an immunoglobulin into which a complementarity-determining region of the monoclonal antibody (a) is transplanted, or a fragment containing an antigen-binding region the same. (41) The method as described in (39) or (40) mentioned above, wherein the activity of a plasmacytoid dendritic cell is one of interferon-producing activity and survival of an interferon-producing cell, or both of them. (42) An agent for suppressing a plasmacytoid dendritic cell activity, containing any of the components described below as an active component: (a) a monoclonal antibody that binds to PLD4, and suppresses a plasmacytoid dendritic cell activity, or a fragment containing an antigen-binding region thereof, and (b) an immunoglobulin into which a complementarity-determining region of the monoclonal antibody (a) is transplanted, or a fragment containing an antigen-binding region thereof. (43) The agent for suppressing an activity of an interferon-producing cell as described in (42) mentioned above, wherein the activity of a plasmacytoid dendritic cell is one of interferon-producing activity and survival of an interferon-producing cell, or both of them.

Effects of the Invention

[0030] The invention provides an antibody that specifically recognizes PLD4, immunity that is useful in preparing the antibody, and a method of preparing an anti-PLD4 antibody using the immunogen. PLD4 is a membrane protein that belongs to the PLD4 family. The present inventors have discovered that an antibody that specifically recognizes PLD4 can be readily obtained. The anti-PLD4 antibody that can be obtained by the invention is an antibody having high specificity, which distinguishes human pDC from a cell expressing other PLD families.

[0031] In a preferred aspect, the anti-PLD4 antibody provided by the invention binds to human pDC. Furthermore, the antibody of the invention specifically recognizes human pDC. Accordingly, the antibody of the invention is useful for the detection or isolation of pDC. pDC is a cell that produces more type 1 IFN. Consequently, such detection or isolation is important in diagnosis or research of diseases associated with pDC such as autoimmune diseases.

[0032] Furthermore, in a preferred aspect, the anti-PLD4 antibody provided by the invention has an action to regulate the activity of human pDC. Accordingly, the anti-PLD4 antibody of the invention can be used to suppress pDC activity.

Consequently, if suppression of pDC activity is utilized using the antibody of the invention, therapeutic effects can be expected for a patient of an autoimmune disease in which expression of IFNa has arisen.

[0033] The pDC produces a large amount of IFN in a small number of cells. In IFN neutralization, an antibody is required, which depends on the number of IFN molecules. However, in the invention, the activity of the production cell is directly suppressed. As a result thereof, potent IFN suppression effect can be expected with less amount of the antibody compared to the neutralization of an anti-IFN antibody. Furthermore, in a case where IFN is produced continuously, it is expected that neutralization by the IFN antibody will remain transient suppression. However, in the invention, pDC activity is suppressed, and from this, the suppression effect that produces IFN can be expected over a long time.

Brief Description of the Drawings

[0034] FIG. 1 is a diagram illustrating the amino acid sequence of the human PLD4 protein (C14orfl75) (506 residues). N-terminal residues 31 to 53 are considered to be a transmembrane domain as analyzed using the “SOSUI program (http://bp.nuap.nagoya-u.ac.jp/sosui/sosui_submit.html)”, which is a prognostic system for a transmembrane region. Residues 54 to 506 contain two phosphodiesterase motifs, and this protein is predicted to be a type II transmembrane protein;

[0035] FIG. 2 is a schematic diagram representing a predicted structure of the human PLD4 protein. The structure has two HKD motifs (HxKxxxxD) in the 506 amino acid residues, and threonine residue 472 is possibly a phosphorylation site;

[0036] FIG. 3 is a graph illustrating the homology of the human PLD4 protein with homologous molecular species in a heterogeneous animal. The PLD4 protein was conserved in the process of evolution from a mouse to a human;

[0037] FIG. 4 is a graph illustrating homology to the human PLD4 protein family (paralog);

[0038] FIG. 5 is a graph illustrating human pDC-specific expression of the PLD4 gene in a cell responsible for human immunity. PLD4 gene expression is high in pDC at the resting stage, and expression is low in CD19+ B cells;

[0039] FIG. 6 is a graph illustrating the tissue expression pattern of human PLD4 mRNA. expression is high in spleen and peripheral blood leukocytes;

[0040] FIG. 7 is a schematic diagram illustrating the structure of the recombinant human PLD4-Ig fusion protein. A cDNA fragment corresponding to the human extracellular PLD4 domain (56 to 506 amino acids) was amplified by PCR. This fragment was inserted into the BamHI-EcoRI cloning site of the pcDNA3.1 N-Flag expression vector containing mouse IgK leader segment and mouse IgG2a heavy chain constant Fc region (hinge + CH2 + CH3) at the N terminus. The 293F cell was temporarily transfected with a plasmid, and the culture supernatant was collected;

[0041] FIG. 8 is a FACS analysis diagram illustrating the specificity of binding to human PLD4. The mp11G9.6 and ch11G9.6 antibodies specifically recognized human PLD4, but did not recognize human PLD3-293T or PLD5-293T transfectants;

[0042] FIG. 9 is a FACS analysis diagram illustrating cross-reactivity with cynomolgus monkey PLD4. The mp11G9.6 and ch11G9.6 antibodies were able to recognize cynomolgus monkey PLD4 in the 293T transfectant;

[0043] FIG. 10 is a FACS measurement diagram illustrating staining in the mp11G9.6 antibody of human PBMC. The mp11G9.6 antibody strongly recognized BDCA2 + pDC in human PBMC;

[0044] FIG. 11 is a FACS analysis diagram illustrating staining in the anti-PLD4 antibody purified from the CAL-1 cell;

[0045] FIG. 12 is a FACS analysis diagram illustrating staining in the anti-PLD4 antibody of the human PLD4-CT125 stable cell strain;

[0046] FIG. 13 is a FACS analysis diagram illustrating staining in human PBMC anti-PLD4 antibody. All anti-PLD4 antibodies were able to recognize BDCA2+ pDC in human PBMC;

[0047] FIG. 14 is a diagram representing the multiple alignment, and homology of the proteins of human PLD, cynomolgus monkey PLD4, rhesus monkey PLD4, and mouse PLD4;

[0048] FIG. 15 is a FACS analysis diagram illustrating staining in the anti-PLD4 antibody with transient gene introduction of the Flag-labeled cynomolgus monkey PLD4 expression vector into human PLD4-293 T cell. Cell surface expression of the cynomolgus monkey PLD4 protein was confirmed in the anti-Flag antibody;

[0049] FIG. 16 is a FACS measurement diagram illustrating staining in the cynomolgus monkey PLD4-CT125 cell anti-PLD4 antibody. Among the anti-PLD4 antibodies, seven (3B4, 5B7, 7B4, 13D4, 13H11, 14Cl, and 11G9.6) antibodies can bind to the cynomolgus monkey PLD4-CT125 stable transfectant;

[0050] FIG. 17 is a FACS analysis diagram illustrating staining in the anti-PLD4 antibody, with introduction of Flag-labeled rhesus macaque PLD4 expression vector transient gene into the human PLD4-293 T cell. The cell surface expression of the rhesus monkey PLD4 protein was confirmed in the anti-Flag antibody;

[0051] FIG. 18 is a FACS analysis diagram illustrating staining in the anti-PLD4 antibody of rhesus monkey PBMC. Among the anti-PLD4 antibodies, five (5B7, 7B4, 13D4, 13H11, and 14C1) antibodies specifically bound to the cynomolgus monkey pDC (Lineage-CD123 + HLA-DR+) cell population in rhesus monkey PBMC;

[0052] FIG. 19 is a graph illustrating the molar concentration of dissociation constant (nM unit, Kd value) of anti-PLD4 antibody against human PLD4-CT125 stable cell strain;

[0053] FIG. 20 is a graph illustrating the CDC activity of ten types of anti-PLD4 antibodies. Target cell: human PLD4-CT125 (mouse 2B4 T-cell lymphocyte) stable transfectant antibody concentration: 10 µg/ml effector: 1% immature rabbit complement;

[0054] FIG. 21 is a graph illustrating the CDC activity of anti-PLD4 antibodies (mp11G9.6 antibody and ch11G9.6 antibody). Target cell: human PLD4-CT125 (mouse 2B4 T-cell lymphocyte) stable transfectant antibody concentration: 0.1 µg/ml to 30 µg/ml effector: 1% immature rabbit complement;

[0055] FIG. 22 is a graph illustrating anti-PLD4 chimeric antibody ADCC activity. Target cell: human PLD4-CHO stable transfectant antibody concentration: 10 µg/ml;

[0056] FIG. 23 is a graph illustrating ELISA measurements of inhibition of IFN-a secretion by treatment with chimeric anti-PLD4 antibody (ch11G9.6) in human PBMC isolated from three healthy individuals; It is

[0057] FIG. 24 is a diagram illustrating the loss of human pDC after treatment with chimeric anti-PLD4 antibody.

[0058] FIG. 25 is a result of the ADCC assay with chimeric anti-PLD4 Abs against primary human pDCs.

[0059] FIG. 26 is the production of IFNa from PBMCs by CpG2216 in the presence of chimeric anti-PLD4 Abs.

Mode for Carrying Out the Invention

[0060] The present inventors have discovered that PLD4 is a molecule that is specifically expressed at the mRNA level and protein level in a resting stage plasmacytoid dendritic cell (resting pDC). A method of preparing an antibody that recognizes PLD4 is not established.

[0061] There is a report that mouse PLD4 is a molecule that is expressed in amoeboid microglia (activated state) at the developmental stage in the cerebellum or in the corpus callosum at the early stage after birth. However, the expression of human PLD4 is not known so far. Particularly, the immune system expression, intracellular location, structure, function, and the like of human PLD4 have not been reported so far. It has been confirmed by the invention that human PLD4, which was considered until now to be expressed only in the cytoplasm, is a cell surface marker that is expressed in human plasmacytoid dendritic cell (pDC) as type II transmembrane protein. Consequently, binding of PLD4 antibody to pDC becomes possible, and it has been proven that PLD4 antibody is useful as a molecular target of a therapeutic antibody intended to regulate B cell and pDC cell functions.

[0062] The present inventors confirmed by gene expression analysis that PLD4 is specifically expressed in human pDC. It was considered that if an antibody that can immunologically distinguish PLD4 from other molecules is obtained, it would be useful for pDC research. However, there are many molecules in the PLD family including PLD4, which are very similar in structure to each other. Molecules such as PLD1, PLD2, PLD3, and PLD5, including the PLD that is PLD4, encompass an amino acid sequence having particularly high homology (FIG. 4). Consequently, it has been found that it is difficult to obtain an antibody that distinguishes these molecules from each other using a peptide immunogen that utilizes an amino acid sequence (a partial sequence) that constitutes PLD4 (or extracellular domain). Accordingly, the present inventors have attempted to acquire an antibody against PLD4 using a recombinant PLD4-Ig fusion protein as an immunogen, which encodes an amino acid sequence spanning the extracellular PLD4 domain.

[0063] The present inventors repeated searches in order to acquire an antibody that recognizes PLD4 and revealed that the desired antibody is obtained using a recombinant PLD4-Ig fusion protein as an immunogen, and completed the invention. That is to say, the invention concerns a monoclonal antibody that binds to an extracellular PLD4 domain, or a fragment containing an antigen-binding region thereof.

[0064] In the invention, PLD4 is a natural molecule that is expressed in human pDC, or a molecule immunologically equivalent to PLD4 that is expressed in human pDC. In the invention, binding of an antibody to PLD4 can be confirmed, for example, as described below.

• Confirmation based on reactivity with human cells:

[0065] According to the findings obtained by the present inventors, it is considered that PLD4 can be used as a pDC marker, based on the fact that it exhibits specific expression in human pDC.

[0066] Based on such a PLD4 expression profile, the binding activity with at least the partial subset of pDC is one of the important characteristics of the antibody that binds to PLD4 in the invention. The fact that some cell is pDC can be confirmed by a cell surface marker inherent in each cell family. For example, binding to an intended cell is confirmed by double staining an antibody that binds to a cell surface marker, and an antibody being confirmed for binding activity. That is to say, pDC in the invention encompasses cells that express, for example, BDCA2.

• Confirmation based on reactivity with cells expressing the transformed PLD4 gene:

[0067] The present inventors have confirmed that the immunological characteristics of PLD4 that is expressed in human pDC are reconstituted when the PLD4 gene is expressed under certain conditions. Consequently, reactivity with PLD4 can also be confirmed based on the reactivity of an antibody to a cell into which a gene encoding PLD4 is artificially introduced. That is to say, the invention concerns a monoclonal antibody that binds to a molecule containing an amino acid sequence that constitutes the PLD4 extracellular domain, or a fragment containing an antigen-binding region thereof. Meanwhile, the extracellular domain is constituted by an amino acid sequence corresponding to 54 to 506 of the N terminus in SEQ ID NO 1 (FIG. 1) of the amino acid sequence represented by SEQ ID NO 1.

[0068] For example, in a cell that is transformed with an expression vector that encompasses the DNA encoding PLD4, the immunological characteristics of PLD4 that are expressed in human pDC are maintained. Accordingly, a cell expressing transformed PLD4 is preferable as a cell to confirm binding property of an antibody to the extracellular domain of PLD4 in the invention. When the reactivity of an antibody by a transformed cell is confirmed in the invention, a non-transformed cell is desirably used as a control.

[0069] Next, the antibody that binds to PLD4 in the invention may be an antibody that exhibits cross-property with a cell family that is known to express the PLD family other than PLD4, or may be an antibody that does not exhibit such cross ownership. The antibody that does not exhibit any cross-linking properties is preferred as the antibody that binds to PLD4 in the invention. Specifically, the antibody that binds to PLD4 in the invention is preferably an antibody that cannot be confirmed to bind to a cell family that is known to express the PLD family other than PLD4, under the same conditions as to the conditions where binding to pDC is confirmed.

[0070] That is to say, the monoclonal antibody that binds to an extracellular PLD4 domain in the invention preferably encompasses a monoclonal antibody that has the immunological characteristics described below. a) Binding to human pDC, b) Cannot be confirmed for binding to one or multiple cell species selected from a group consisting of monocyte, macrophage, CD34-positive cell, and dendritic cells derived from these cells, under conditions that allow binding to human pDC.

[0071] Particularly, the monoclonal antibody of the invention is preferably an antibody that cannot be confirmed for binding to monocyte, macrophage, B cell, CD34-positive cell, and dendritic cells derived from these cells, under conditions that allow binding to pDC human.

[0072] Alternatively, the monoclonal antibody that binds to an extracellular PLD4 domain in the invention preferably encompasses a monoclonal antibody that has the immunological characteristics described below. c) Binding to a transformed cell that is transformed with an expression vector that retains DNA encoding PLD4 in an expressible mode, d) Cannot be confirmed for binding to the host cell of c) before being transformed, under conditions that allow attachment to the transformed cell c).

[0073] The fact that the anti-PLD4 monoclonal antibody is not crossed with other PLD family molecules in the invention can be confirmed by using a cell in which each PLD family was obligatory expressed. That is, obligatory expression is accomplished by introducing cDNA encoding the amino acid sequence of each PLD family into an appropriate host cell. The transformed cell obtained is brought into contact with the anti-PLD4 monoclonal antibody to be confirmed for cross-talk. If binding to a cell expressing PLD family molecules other than PLD4 is not shown, it can be confirmed that the antibody can immunologically distinguish PLD4 from other PLD family molecules. For example, it is confirmed in the Examples described below that most of the monoclonal anti-PLD4 antibodies obtained by the invention do not cross with PLD3 and PLD5 having particularly high homology to PLD4, and even PLD1 and PLD2. Accordingly, the monoclonal antibody in the invention is preferably a monoclonal antibody that binds to PLD4, but cannot be detected for binding to PLD3, PLD5, PLD1, or PLD2 under the same conditions. If an antibody that can immunologically distinguish these PLD family molecules from PLD4 is used, the change in PLD4 expression can be specifically detected.

[0074] The binding of a monoclonal antibody to be confirmed regarding binding activity, for each species of a cell, can be confirmed, for example, in the principle of flow cytometry. In order to confirm the reactivity of an antibody by the principle of flow cytometry, it is advantageous to label the antibody with a molecule or group of atoms that produces a detectable signal. In general, a fluorescent label or a luminescent label is used. In order to analyze the binding of a fluorescently labeled antibody to a cell by the principle of flow cytometry, a fluorescence-activated cell sorter (FACS) can be used. By the use of FACS, multiple bindings of an antibody to a cell can be confirmed effectively.

[0075] Specifically, for example, an antibody A that is preliminarily known to be capable of identifying pDC, and an antibody B being analyzed for pDC binding characteristics, are reacted at the same time with a cell family that encompasses pDC. Antibody A and Antibody B are labeled with a fluorescent signal that can distinguish them from each other. If both signals are detected from the same cell family, it can be confirmed that such antibodies bind to the same cell family. That is to say, antibody A and antibody B are found to have the same binding characteristics. If antibody A and antibody B bind to a different cell family, it is clear that their binding characteristics are different from each other.

[0076] Examples of preferred monoclonal antibodies in the invention include, for example, monoclonal antibodies produced by the mp5B7, mp7B4, mp13D4, and mp13H11 hybridomas.

[0077] The mp5B7, mp7B4, mp13D4 and mp13H11 hybridomas have been deposited under Accession Numbers: NITE BP-1211, NITE BP-1212, NITE BP-1213, and NITE BP-1214 at the National Institute of Technology and Evaluation (NITE) Patent Microorganisms Depositary on January 27, 2012. The contents to specify the deposition will be described below. (1) Name and Address of Depositary Authority Name: National Institute of Technology and Evaluation (NITE) Advanced Industrial Science and Technology Patent Microorganisms Depositary Address: 2-5-8 Kazusakamatari, Kisarazu-shi, Chiba-ken, 292-0818 JAPAN ( 2) Filing Date: January 27, 2012 (3) Accession Numbers NITE BP-1211 (mp5B7 hybridoma) NITE BP-1212 (mp7B4 hybridoma) NITE BP-1213 (mp13D4 hybridoma) NITE BP-1214 (mp13H11 hybridoma)

[0078] The monoclonal antibody of the invention may be a fragment containing an antigen-binding region thereof. For example, an antibody fragment encompassing an antigen binding site that is produced by enzymatic digestion of IgG can be used as the antibody of the invention. Specifically, by digestion with papain or pepsin, an antibody fragment such as Fab or F(ab')2 can be obtained. Furthermore, an immunoglobulin fragment encompassing a variable region into which the complementarity determining region (CDR) of some monoclonal antibody is transplanted is encompassed in the antigen-binding region-containing fragment. It is widely known that these antibody fragments can be used as an antibody molecule that has binding affinity for an antigen. Alternatively, an antibody constructed by gene recombination can be used as long as it maintains the activity necessary for antigen binding. Examples of the antibody constructed by gene recombination include, for example, a chimeric antibody, a CDR transplant antibody, a single-chain Fv, a diabody (diabodies), a linear antibody, and a polyspecific antibody formed by antibody fragments. , and the like. A method of obtaining such antibodies based on a monoclonal antibody, or an antibody-producing cell that produces the same, is known.

[0079] The monoclonal antibody of the invention can be obtained by using recombinant PLD4-Ig fusion protein, or a transformed cell that expresses human PLD4 as an immunogen. That is to say, the present invention relates to a method of preparing a cell that produces a monoclonal antibody that binds to an extracellular PLD4 domain, which contains the processes below. (1) Process of administering an extracellular PLD4 domain-containing extrinsic protein to an immune animal, and (2) Process of selecting an antibody-producing cell that produces an antibody that binds to PLD4 from cells that produce antibody of the immune animal.

[0080] The antibody-producing cell thus obtained, or immortalized antibody-producing cell can be cultured, and a desired monoclonal antibody can be collected from the culture. Several methods are known as a method to immortalize the cell that produces the antibody.

[0081] The transformed cell used as an immunogen in the invention can be obtained, for example, by preparing a cell in which an extrinsic polynucleotide (a) encoding the amino acid sequence containing the extracellular PLD4 domain described below is retained in a expressible way.

[0082] The extrinsic polynucleotide in the present invention refers to a polynucleotide that is artificially introduced into a host cell. In a case where a human cell is used as the cell, a human gene is introduced into the human cell. In such a combination, artificially introduced polynucleotide is also referred to as the extrinsic polynucleotide. Consequently, ectopic expression of PLD4 is encompassed under extrinsic polynucleotide expression.

[0083] The extracellular PLD4 domain in the invention refers to an amino acid sequence of 54 to 506 positions that corresponds to the extracellular domain in the amino acid sequence described in SEQ ID NO 1. For example, the amino acid sequences containing each of the Regions in order below the N-terminal side are preferred as the extracellular PLD4 domain-containing amino acid sequence in the invention.

Intracellular region + transmembrane domain + extracellular domain

[0084] Alternatively, an amino acid sequence that is partially lacking in the intracellular region as described below is also encompassed in the amino acid sequence containing the extracellular PLD4 domain in the present invention.

Partial intracellular region + transmembrane domain + extracellular domain

[0085] Furthermore, a structure that is lacking the intracellular region as described below is encompassed in the amino acid sequence containing the extracellular PLD4 domain in the present invention.

Transmembrane domain + extracellular domain

[0086] Regions other than the extracellular domain in the structure mentioned above may be a sequence selected from the amino acid sequence represented by SEQ ID NO 1, or may be a combination with another homologous amino acid sequence. For example, an amino acid sequence that constitutes a signal sequence, a transmembrane domain, and an intracellular region can be used as an amino acid sequence of PLD family molecules other than PLD4. Alternatively, the amino acid sequence of the PLD family from species other than human can also be combined. Furthermore, the amino acid sequence constituting regions other than the extracellular domain may contain mutation within a range where each of the regions' functions can be maintained. Furthermore, other regions can be interposed between each of the regions. For example, between the signal sequence and the extracellular domain, an epitope tag such as FLAG can also be inserted. Particularly, the signal sequence is a region that is individualized for processing in the transport step to the cell membrane surface after protein translation, and is removed. Consequently, an arbitrary amino acid sequence that induces passage of the translated protein across the cell membrane can be used as a signal sequence. More specifically, a PLD4 amino acid sequence (SEQ ID NO 1) is preferred as an amino acid sequence containing the extracellular PLD4 domain.

[0087] Consequently, the polynucleotide (a) mentioned above in the invention can be an arbitrary base sequence that encodes the amino acid sequence that constitutes the structure mentioned above [Intracellular region + transmembrane domain + extracellular domain]. For example, the amino acid sequence of SEQ ID NO 1 is encoded by the cDNA base sequence described in SEQ ID NO 44.

[0088] The recombinant PLD4-Ig fusion protein as an immunogen in the invention can be obtained by introducing an expression vector that retains the aforementioned polynucleotide in an expressible manner, within an appropriate host cell. The cDNA base sequence of the recombinant PLD4-Ig fusion protein is represented by SEQ ID NO 125, and the amino acid sequence is represented by SEQ ID NO 126.

[0089] The host cell in the invention is preferably a mammalian cell. Specifically, a cell derived from a human, a monkey, a mouse, or a rat can be used as the host cell. Particularly, the human-derived cell is preferable as the host cell. For example, the HEK-293 T cell is a human embryo-derived kidney cell strain that can preferably be used as the host cell in the invention. The HEK-293 T cell is available as ATCC CRL-11268. Furthermore, a cell derived from an immune animal can be used as the host cell. If an animal-derived immune cell is used as an immunogen, the immune response against the host cell is small. Therefore, an antibody against the extracellular PLD4 domain, which is extrinsically expressed, can be obtained effectively. Consequently, for example, when a mouse is used as the immune animal, a cell derived from the mouse can also be used as the host cell.

[0090] The above-mentioned polynucleotide can be loaded into a vector that can induce expression in a host cell to transform the cell. A commercially available vector that can induce expression in a mammalian cell can be used. For example, an expression vector such as pCMV-Script(R) Vector, pSG5 Vector (manufactured by Stratagene), pcDNA3.1 (manufactured by Invitrogen), retroviral vector pMXs-IP (manufactured by Cell BioLabs), and the like can be used for the invention.

[0091] The transformed cell thus obtained is administered to an immune animal, with an additional component such as an adjuvant if necessary. As the adjuvant, Freund's complete adjuvant and the like can be used. In a case where a mouse is used as the immune animal, purified recombinant PLD4-Ig fusion protein is administered to the BALB/c mouse. As the adjuvant, Freund's Adjuvants, Complete and Incomplete (manufactured by SIGMA) were used, and administered at 200 µg/mouse for the first time, and 50 µg/mouse for the second time until the fourth time. In general, an immunogen is administered multiple times at intervals until the antibody titer rises. For example, in a case of short-time immunization method, a transformed cell is administered at an interval of 2 to 4 days, more specifically 3 days, and after 2 to 3 times of administration, the antibody-producing cells can be collected. In addition, antibody-producing cells can also be collected after administration 5 to 6 times at an interval of about once a week.

[0092] In order to obtain the monoclonal antibody of the invention, the cell that produces the collected antibody is cloned. For cloning, the cell that produces the antibody is preferably immortalized. For example, a cell fusion method represented by the hybridoma method, or Epstein-Barr virus (EBV) transformation can be used as a method to immortalize the antibody-producing cell.

[0093] A cell that produces antibody produces one type of antibody per cell. Consequently, if a cell population derived from a cell can be established (i.e., cloning), a monoclonal antibody can be obtained. The hybridoma method refers to a method in which an antibody-producing cell is fused with an appropriate cell strain, immortalized, and then cloned. The immortalized antibody-producing cell can be cloned by a method such as limiting dilution method. Many cell strains that are useful in the hybridoma method are known. These cell strains are excellent in the efficiency of immortalizing a lymphocyte-based cell, and have several gene markers that are necessary to select cells that are successful in cell fusion. Furthermore, in a case where acquisition of the antibody-producing cell is intended, a cell strain that is lacking the antibody-producing capacity may also be used.

[0094] For example, mouse myeloma P3x63Ag8.653 (ATCC CRL-1580) or P3x63Ag8U.l (ATCC CRL-1597) is universally used as a useful cell strain for a cell fusion method from a mouse or a mouse. In general, a hybridoma is prepared by fusing homogeneous cells. However, a monoclonal antibody can also be acquired from a heterohybridoma between closely heterogeneous species.

[0095] A specific cell fusion protocol is known. That is, an antibody-producing cell from an immune animal is mixed with an appropriate fusion partner, and individualized for cell fusion. As the cell that produces antibody, a splenic cell, a lymphocyte cell collected from the lymph node, peripheral blood B cell, or the like can be used. As the fusion partner, various cell strains described above can be used. For cell fusion, a polyethylene glycol method or an electrical fusion method is used.

[0096] Next, cells that were successful in cell fusion are selected based on a selection marker possessed by the fusion cell. For example, in a case where the HAT-sensitive cell strain is used in cell fusion, cells that were successful in cell fusion are selected by selecting cells growing in the HAT medium. Furthermore, an antibody produced by the selected cells is confirmed whether it has the intended reactivity.

[0097] Each of the hybridomas is screened based on antibody reactivity. That is to say, a hybridoma that produces an antibody that binds to PLD4 is selected by the method as described above. Preferably, in a case where the selected hybridoma is subcloned, and the production of the desired antibody is finally confirmed, the hybridoma is selected as a hybridoma that produces the monoclonal antibody of the invention.

[0098] Specifically, the desired hybridoma can be selected based on reactivity with a human cell, or reactivity with a transformed cell that expresses the PLD4 gene. An antibody that binds to a cell can be detected at the beginning of an immunoassay. For example, ELISA, in which a cell is used as an antigen, can be used in detecting the intended antibody. Specifically, a hybridoma culture supernatant is brought into contact with a carrier that immobilizes human pDC, or a transformed cell used as an immunogen. In a case where the culture supernatant contains the intended antibody, the antibody is captured by the cell immobilized in the carrier. Then, the solid phase is isolated from the culture supernatant, washed if necessary, and then the antibody captured in the solid phase can be detected. In detecting the antibody, an antibody that recognizes the antibody can be used. For example, an antibody from a mouse can be detected by anti-mouse immunoglobulin antibody. If the antibody that recognizes an antibody is on the label, its detection is easy. As the label, an enzyme, a fluorescent pigment, a luminescent pigment, or the like can be used.

[0099] On the other hand, as the carrier that immobilizes the cell, a particle, or the inner wall of a microtiter plate can be used. The surface of a particle or a container made of plastic can immobilize the cell by physical adsorption. For example, beads or a reaction vessel fabricated from polystyrene can be used as a carrier to immobilize the cell.

[00100] In hybridoma selection, there may be a case where predicted is not the production of an antibody against PLD4, but the production of an antibody against a host cell of a transformed cell used as an immunogen. For example, when a human cell is used as an immunogen and a mouse is used as an immune animal as shown in the Examples, the human cell is recognized as a foreign substance, and an antibody that binds to it is predicted to be produced. In the invention, the acquisition of an antibody that recognizes PLD4 is intended. Consequently, the acquisition of an antibody that recognizes human cell antigens other than PLD4 is not necessary. In order to exclude a hybridoma that produces such an antibody by screening, an unintended antibody may be preliminarily absorbed before confirming the reactivity of the antibody.

[00101] Antibodies that are not intended can be absorbed by an antigen that binds to an antibody predicted to exist. Specifically, for example, an antibody against human cell antigens other than PLD4 may be taken up by a cell for which PLD4 expression cannot be detected. In the invention, the host cell used for the immunogen is preferable as an antigen to absorb unintended antibodies.

[00102] A monoclonal antibody confirmed to have binding activity to an antigen is confirmed for practical influence on pDC activity, if necessary. The influence on pDC can be confirmed, for example, by a method described below.

[00103] The monoclonal antibody of the invention can be collected from a culture that is obtained by cultivating a hybridoma that produces the monoclonal antibody. The hybridoma can be cultured in vitro or in vivo. In in vitro culture, the hybridoma can be grown using a known medium such as RPMI 1640. The immunoglobulins secreted by the hybridoma are accumulated in the culture supernatant. Consequently, the monoclonal antibody of the invention can be obtained by collecting the culture supernatant, and purifying it if necessary. Immunoglobulin purification is easy in a medium that is not added to serum. However, for the purpose of rapid growth of the hybridoma and promotion of antibody production, plus or minus 10% fetal bovine serum can also be added to the medium.

[00104] The hybridoma can also be cultivated in vivo. Specifically, the hybridoma can be cultured in the abdominal cavity by inoculating the hybridoma into the abdominal cavity of a nude mouse. The monoclonal antibody is accumulated in the ascites. Consequently, the ascites is collected, and purified if necessary, to obtain the necessary monoclonal antibody. The obtained monoclonal antibody can be suitably modified or processed depending on the purpose.

[00105] The monoclonal antibody of the invention can be expressed by acquiring the cDNA encoding the antigen-binding region of the antibody from the hybridoma, and inserting it into an appropriate expression vector. A technology for acquiring cDNA encoding a variable region of an antibody and expressing it in an appropriate host cell is known. Furthermore, a method is known in which a variable region containing an antigen-binding region is ligated to a constant region to give a chimeric antibody. For example, a variable region gene can give a chimeric antibody by binding to genes encoding a human IgG1 heavy chain constant region and human IgG1 capla light chain constant region, respectively. Furthermore, it is known that the antigen-binding activity of a monoclonal antibody can be transplanted to another immunoglobulin by incorporating the CDR that constitutes a variable region into a matrix region of another immunoglobulin molecule. Using this, a method is established of transplanting the antigen-binding activity possessed by a heterogeneous immunoglobulin into a human immunoglobulin. For example, there may be a case where a partial amino acid sequence from a template region supporting the CDR is transplanted into a human variable region from a variable region of a mouse antibody. Then, these reconstituted, humanized variable regions of a human antibody can be ligated to a constant region of a human antibody to obtain a humanized antibody.

[00106] Examples of preferable monoclonal antibodies in the invention include, for example, monoclonal antibodies produced by the hybridomas mp5B7, mp7B4, mp13D4, mp13H11, and the like, which have been deposited under Accession Numbers: NITE BP-1211, NITE BP- 1212, NITE BP-1213, and NITE BP-1214, respectively.

[00107] Like the chimeric antibody containing the variable region, or the humanized antibody into which the CDR constituting the variable region is transplanted, an antibody having a constant region derived from IgG or IgM is preferably contained in the antibody of the invention. Particularly, the antibody of the invention is more preferably an antibody that has a combination of: heavy chain CDR1: DYNLH, CDR2: YIYPYNGNTGYNQKFKR, CDR3: GGIYDDYYDYAIDY and light chain CDR1: RASENIYSHIA, CDR2: GATNLAH, CDR3: QHFWGTP, as a CDR sequence which constitutes the variable region of the antibody, an antibody that has a combination of: heavy chain CDR1: SHYYWT, CDR2: YISYDGSNNYNPSLKN, CDR3: EGPLYYGNPYWYFDV and light chain CDR1: RASQDIDNILN, CDR2: YTSRLHS, CDR3: QQFNTLP, as a CDR sequence that constitutes the variable region, or an antibody that has a combination of: heavy chain CDR1: SHYYWS, CDR2: YISYDGSNNYNPSLKN, CDR3: EGPLYYGNPYWYFDV and light chain CDR1: RASQDIDNILN, CDR2: YTSRLHS, CDR3: QQFNTLP, as a sequence of the CDR that constitutes the variable region.

[00108] The present inventors confirmed that the monoclonal antibody against PLD4 has CDC action for a PLD4-expressing cell. Consequently, the antibody that has a constant region derived from IgG or IgM has a cytotoxic action towards the PLD4-expressing cell through the CDC action. Such an antibody is useful for suppressing the cell number of a PLD4-expressing cell such as pDC.

[00109] A chimeric antibody, or a humanized antibody that recognizes PLD4 can be prepared by genetic engineering using a polynucleotide that encodes them.

[00110] An antibody that can specifically recognize PLD4 has not been obtained. An antibody that recognizes PLD4 was first provided by the immunogen of the invention. That is to say, the invention provides an antibody that recognizes PLD4, which can be obtained by the processes described below. (1) Process of administering a protein containing an extracellular PLD4 domain to an immune animal, (2) process of selecting an antibody-producing cell that produces an antibody that binds to PLD4 from antibody-producing cells of the animal immune, and (3) process of culturing the antibody-producing cell selected in (2), and collecting an antibody that recognizes PLD4 from the culture.

[00111] It has been revealed that PLD4 is specifically expressed in human pDC. Specific expression in human pDC was also confirmed by gene expression analysis with SAGE by the present inventors. However, the expression level of PLD4 was all analyzed based on mRNA in a past report. Furthermore, the PLD4 protein is known to be expressed only in the cytoplasm. It has been revealed by the invention that PLD4 is also expressed on the cell surface. Since an antibody that can detect PLD4 was not provided, analysis for the protein expression status was not conventionally performed. The antibody that binds to an extracellular PLD4 domain provided by the invention has performed analysis of the PLD4 protein.

[00112] As confirmed in practice by the present inventors, the monoclonal antibody that binds to an extracellular PLD4 domain based on the invention was specifically detected in human pDC. That is to say, the invention relates to a method of detecting a plasmacytoid dendritic cell, which contains the steps of taking a monoclonal antibody that binds to an extracellular PLD4 domain, or a fragment containing an antigen-binding region thereof into contact with a test cell, and detect a monoclonal antibody that binds to the cell, or a fragment containing an antigen-binding region thereof.

[00113] By detecting PLD4 based on the invention, it can be confirmed whether any cell is pDC or not. That is to say, the invention provides a method of identifying pDC using PLD4 as an index. Alternatively, a cell for which PLD4 is detected can be isolated based on the invention, thereby isolating human pDC. That is to say, the invention provides a method of isolating pDC using PLD4 as an index.

[00114] In the invention, a monoclonal antibody that binds to an extracellular PLD4 domain, or a fragment containing an antigen-binding region thereof can be labeled. For example, the antibody can be easily detected by labeling it with a luminescent pigment or fluorescent pigment. More specifically, antibody labeled with fluorescent pigment is brought into contact with a cell population that possibly contains pDC, and a cell that binds the antibody of the invention can be detected using the fluorescent pigment as an index. Furthermore, if a cell detected with the fluorescent pigment is isolated, pDC can be isolated. A series of steps can be easily implemented in the FACS principle.

[00115] Alternatively, the antibody of the invention can also be linked to a solid phase carrier such as a magnetic particle. The antibody that is bound to the solid phase carrier recognizes PLD4, and pDC is captured on the solid phase carrier. As a result thereof, pDC can be detected or isolated.

[00116] The antibody that is necessary in the pDC detection method based on the invention can be provided as a reagent for pDC detection. That is to say, the invention provides a reagent for detecting pDC containing a monoclonal antibody that binds to an extracellular PLD4 domain, or a fragment containing an antigen-binding region thereof. In the pDC detection reagent of the invention, a positive control or a negative control can be combined in addition to the antibody. For example, a transformed cell expressing extracellular PLD4 domain used as an immunogen, or pDC collected from a human, or the like can be used as a positive control. Usually, only little human pDC can be obtained from peripheral blood. Consequently, a transformed cell is particularly preferable as a positive control in the reagent of the invention. Conversely, as the negative control, an arbitrary cell that does not express PLD4 can be used.

[00117] That is to say, the invention provides a kit for the detection of human pDC containing a monoclonal antibody that binds to an extracellular PLD4 domain, or a fragment containing an antigen-binding region thereof.

[00118] Furthermore, the inventors analyzed the influence of the antibody that binds to an extracellular PLD4 domain on pDC. As a result of the same, it was confirmed that the antibody that binds to an extracellular PLD4 domain suppresses pDC activity. That is to say, the invention concerns a method of suppressing the activity of a cell that produces interferon, which contains a step of bringing any of the components below into contact with the pDC: 1. a monoclonal antibody that binds to PLD4 and suppresses the activity of pDC, or a fragment containing an antigen-binding region thereof, and 2. an immunoglobulin into which a complementarity-determining region of the monoclonal antibody (a) is transplanted, or a fragment containing an antigen-binding region the same.

[00119] Alternatively, the invention relates to a method of suppressing pDC activity in a living organism, which contains a step of administering any of the following components to a living organism: (a) a monoclonal antibody that binds to PLD4 and suppresses the activity of pDC, or a fragment containing an antigen-binding region thereof, (b) an immunoglobulin into which a complementarity-determining region of the monoclonal antibody (a) is transplanted, or a fragment containing an antigen-binding region of the same. antigen thereof, and (c) a polynucleotide encoding the component described in (a) or (b).

[00120] The pDC in the invention refers to a cell that has the capacity to produce IFN, and expresses PLD4 on the cell surface. Hereinafter, unless otherwise particularly stated, the pDC is not only a cell that is a precursor cell of a dendritic cell, but contains a cell that has the ability to produce IFN, and expresses PLD4 on the cell surface. A method of identifying such pDC is known. For example, pDC can be distinguished from other blood cells using various cell surface markers as an index. Specifically, the profile of a human pDC cell surface marker is as described below (Shortman, K. and Liu, YJ. Nature Reviews 2: 151-161, 2002). In recent years, there was also a report that positioned the BDCA-2 positive cell as pDC (Dzione k, A. et al. J. Immunol. 165: 6037-6046, 2000). Cell surface antigen profile of human pDC CD4 positive and CDI23 positive, Lineage (CD3, CD14, CD16, CD19, CD20, and CD56) negative, CD11c negative

[00121] Consequently, a cell that has the expression profile of these known markers, and has the capacity to produce IFN can also be referred to as pDC. Furthermore, a cell that belongs to a cell family having a profile different from the expression pattern of the expression profile of these markers, but having the capacity to produce IFN in a living organism is covered in pDC.

[00122] Furthermore, examples of common characteristics exhibited by human pDC include the characteristics as described below. Morphological Characteristics of the Cell • Similar to a plasma cell • Round cell with a smooth cell surface • Relatively large nucleus Functional Characteristics of the Cell • Production of a large amount of type I IFN in a short time at the time of viral infection • Differentiation into dendritic cell after viral infection

[00123] Suppression of pDC activity in the invention refers to the suppression of at least one of the pDC functions. Examples of pDC functions include IFN production and cell survival. Cell survival can be alluded to as cell number in other words. Consequently, suppression of either or both of these functions is referred to as suppression of pDC activity. It has been revealed that type I IFN produced by pDC is a cause for several diseases. Consequently, suppression of pDC cell number or IFN production is useful as a strategy to treat such diseases.

[00124] For example, the relationship of pathological conditions of an autoimmune disease with IFNa has been highlighted. More IFNa is produced by pDC. Consequently, if its production is suppressed, the pathological conditions caused by IFNa can be alleviated. Meanwhile, in the invention, the suppression of the production of IFN by the pDC refers to the suppression of the production of at least one type of IFN produced by the pDC. The IFN in the invention is preferably type I IFN. Among them, IFNa is important.

[00125] That is to say, the invention concerns a suppressor for the production of IFN, which contains the antibody that binds to an extracellular PLD4 domain as an active component. Alternatively, the invention provides a method of suppressing IFN production, containing a method of administering an antibody that binds to an extracellular PLD4 domain. Furthermore, the invention relates to the use of an antibody that binds to an extracellular PLD4 domain in the preparation of a pharmaceutical composition that suppresses the production of IFN.

[00126] A cell that produces a large amount of IFN in a small number of cells is covered in pDC. For example, a precursor cell of a dendritic cell that is stimulated by a virus or the like produces more than the IFN produced by a living organism. Suppression of the cell number of pDC that produces a large amount of IFN leads to suppression of IFN production as a result. Consequently, suppression of pDC cell number can also alleviate pathological conditions caused by IFNα.

[00127] In a preferred aspect of the invention, it was confirmed that the anti-PLD4 monoclonal antibody binds to a PLD4-expressing cell, and communicates cytotoxicity through the action of Complement-Dependent Cytotoxicity (CDC). The action of CDC is one of the mechanisms of action of an antibody medicine. The anti-PLD4 monoclonal antibody of the invention also has potent cytotoxicity to a PLD4-expressing cell such as pDC through the action of CDC. That is to say, the anti-PLD4 monoclonal antibody in a preferred aspect can be expected to have the effects of suppressing IFN production not only by a mechanism of suppressing IFN production, but also by cytotoxicity to pDC.

[00128] The antibody that recognizes an extracellular PLD4 domain used in the invention can be obtained based on the method as described above. The antibody in the invention can be of any class. Furthermore, a biological species from which the antibody is derived is not limited. Furthermore, a fragment containing an antigen-binding region of the antibody can be used as the antibody. For example, an antibody fragment containing an antigen binding site produced by enzymatic digestion of IgG can be used as the antibody in the invention. Specifically, an antibody fragment such as Fab or F(ab')2 can be obtained by digestion by papain or pepsin. It is widely known that these antibody fragments can be used as an antibody molecule that has binding affinity to an antigen. Alternatively, an antibody constructed by gene recombination can also be used as long as it maintains the necessary antigen-binding activity. Examples of the antibody constructed by gene recombination include a chimeric antibody, a CDR transplant antibody, a single-chain Fv, a diabody (diabodies), a linear antibody, and a polyspecific antibody formed by an antibody fragment, and the similar. A method of obtaining these antibodies based on a monoclonal antibody is known.

[00129] The antibody in the invention can be modified if necessary. According to the invention, the antibody that recognizes an extracellular PLD4 domain has the action of suppressing pDC activity. That is to say, a possibility was considered that the antibody itself has cytotoxicity to pDC. A subclass of antibody that exhibits strong effector action is known. Alternatively, effects of suppressing pDC activity can be further enhanced by modifying the antibody with a cytotoxic agent. Examples of the cytotoxic agent include the agents described below. Toxins: Pseudomonas endotoxin (PE), diphtheria toxin, and ricin Radioactive isotope: Tc99m, Sr89, I131, and Y90 Anti-cancer agent: calicheamicin, mitomycin, and paclitaxel

[00130] Toxins containing a protein can bind to an antibody, a fragment thereof, or the like by a bifunctional reagent. Alternatively, a gene encoding the toxins can also be conjugated to a gene encoding the antibody to give a fusion protein thereof. A method of linking a radioactive isotope to an antibody is also known. For example, a method of labeling an antibody with a radioactive isotope using a chelate agent is known. Furthermore, an anti-cancer agent can bind to an antibody through the use of a sugar chain, a bifunctional reagent, or the like.

[00131] In the invention, antibody artificially modified in structure can also be used as an active component. For example, several modifications are known to improve cytotoxicity or stability of an antibody. Specifically, an immunoglobulin is known, in which the sugar chain of the heavy chain is modified (Shinkawa, T. et al. J. Biol. Chem. 278: 3466-3473. 2003). Modification of the sugar chain increased the Antibody Dependent Cell-Mediated Cytotoxicity (ADCC) activity of immunoglobulin.

[00132] The antibody that binds to an extracellular PLD4 domain suppresses pDC activity when the antibody is brought into contact with the pDC. Accordingly, such an antibody can be used as an agent for suppressing pDC activity, or in a method of suppressing pDC. That is to say, the invention provides an agent for suppressing pDC activity, which contains at least one type of a component selected from a group consisting of (a) to (c) described below as an active component. Alternatively, the invention relates to a method of suppressing pDC activity, which contains a step of administering at least one type of a component selected from a group consisting of (a) to (c) described below. Furthermore, the invention relates to the use of at least one type of a component selected from a group consisting of (a) to (c) described below in the preparation of an agent for regulating pDC activities. (a) Antibody that binds to an extracellular PLD4 domain, or a fragment containing an antigen-binding region thereof, (b) an immunoglobulin into which a complementarity-determining region of the monoclonal antibody (a) is transplanted, or a fragment containing an antigen-binding region thereof.

[00133] As the monoclonal antibody that suppresses pDC activity in the invention, a monoclonal antibody that recognizes an extracellular PLD4 domain can be used. One type or multiple types of monoclonal antibodies can be used in the invention.

[00134] For example, multiple species of monoclonal antibodies that recognize an extracellular PLD4 domain can be mixed, and used in the invention. (c) 134] The fact that the antibody has an action of suppressing the IFN production activity of pDC, can be confirmed in the manner as described below. pDC produces a large amount of IFN upon viral stimulation. The antibody is given to the pDC before, after, or at the same time as viral stimulation, and the ability to produce IFN is compared to a control in which the antibody is not given to the pDC. The ability to produce IFN can be assessed by measuring IFN-a or IFN-ß contained in the supernatant of a pDC culture. As a result of the comparison, if the amount of IFN in the supernatant is significantly decreased by the addition of the antibody, it can be confirmed that the tested antibody has an action to suppress the ability to produce IFN. A method of measuring these IFNs is known. A pDC is a cell that produces the most IFN in a living organism. Consequently, suppression of the IFN production capacity of pDC can regulate the state of IFN production in a living organism.

[00135] The activity of pDC in the invention encompasses the maintenance of pDC cell number. Accordingly, suppression of pDC activity in the invention encompasses suppression of pDC cell number. If pDC cell number is confirmed to be suppressed in the presence of the antibody, the antibody is found to suppress pDC activity. Similarly for the production of IFN, an inactive immunoglobulin that is derived from the same animal species as the species of the antibody to be confirmed for activity, can be used as a control for comparison. The cell number of pDC can be compared quantitatively by cell counting. Cell number can be counted with FACS or a microscope.

[00136] Furthermore, pDC is also assumed to be differentiated into a Th2-inducing cell called Dendritic Cell 2 (DC2) as a result of infection by the virus and the like. If IFN production by pDC upon viral stimulation is suppressed, there is a possibility that differentiation to Th2 could also be suppressed. Consequently, the monoclonal antibody of the invention that suppresses IFN production can also be expected to have therapeutic effects for various allergic diseases.

[00137] In a case where the antibody that recognizes an extracellular PLD4 domain is administered to a host that is different from the biological species from which the antibody is derived, it is desirable to process the antibody in a form that is difficult to recognize as a foreign substance by the host. For example, immunoglobulin can be rendered difficult to recognize as a foreign substance by processing the molecule as described below. A method of processing an immunoglobulin molecule as described below is known. • Fragment containing antigen-binding region, from which the constant region is deleted (Monoclonals Antibodies: Principles and Practice, third edition, Academic Press Limited. l995; Antibody Engineering, A Practical Approach, IRL PRESS, 1996) • Chimeric antibody constituted with an antigen-binding region of a monoclonal antibody and a constant region of the host immunoglobulin (Gene Expression Experiment Manual, Kodansha Ltd. l994 (edited by Isao ISHIDA and Tamie ANDO)) • Antibody substituted in the CDR in which the complementarity-determining region ( CDR) in a host immunoglobulin is replaced with the CDR of a monoclonal antibody (Gene Expression Experiment Manual, Kodansha Ltd. 1994 (edited by Isao ISHIDA and Tamie ANDO))

[00138] Alternatively, the gene for a human immunoglobulin variable region can also be acquired by the phage demonstration method (McCafferty J et al., Nature 348: 552-554, 1990; Kretzschmar T et al., Curr Opin Biotechnol. Dec 2002; 13(6): 598-602). In the phage demonstration method, the gene encoding the human immunoglobulin variable region is incorporated into the phage gene. Using several immunoglobulin genes as a source, a phage library can also be prepared. A phage expresses the variable region as a fusion protein of the protein that constitutes itself. The variable region expressed on the phage surface by the phage maintains antigen-binding activity. Accordingly, selection of a phage that binds an antigen or a cell in which the antigen is expressed, or the like, can screen for a phage in which a variable region having the intended binding activity is expressed from a library of phage. Furthermore, a phage particle thus selected retains a gene encoding the variable region having the intended binding activity. That is to say, a gene encoding a variable region having the desired binding activity can be acquired using the binding activity of the variable region as an index in the phage demonstration method.

[00139] The antibody that recognizes an extracellular PLD4 domain, or an antibody fragment containing at least the antigen-binding region thereof in the agent for suppressing pDC activity, or the method of suppressing pDC activity according to the invention, can be administered as a protein, or a polynucleotide encoding the protein. In administering the polynucleotide, it is desirable to use a vector in which a polynucleotide encoding the desired protein is arranged under the control of an appropriate promoter so as to express the desired protein. In the vector, an enhancer or a terminator may also be arranged. A vector is known that retains genes for a heavy chain and a light chain that constitute an immunoglobulin, and can express the immunoglobulin molecule. The vector that can express an immunoglobulin can be administered by being introduced into a cell. In administration to a living organism, a vector that can infect a cell being administered into a living organism can be administered as such. Alternatively, a vector can also be first introduced into a lymphocyte isolated from a living organism, and returned once again into the living organism (ex vivo).

[00140] The amount of the monoclonal antibody that is administered to a living organism in the agent for suppressing pDC activity, or the method of suppressing pDC activity based on the invention, is usually 0.5 mg to 100 mg, e.g. from 1 mg to 50 mg, preferably from 2 mg to 10 mg per 1 kg of body weight as an immunoglobulin. The interval of administration of the antibody to a living organism can be appropriately regulated such that an effective concentration of the immunoglobulin in the organism can be maintained during the treatment period. Specifically, administration can be carried out, for example, at an interval of 1 to 2 weeks. The route of administration is arbitrary. A person of ordinary skill in the art can adequately select an effective route of administration in treatment. Specifically, examples of the route of administration include oral or non-oral administration. For example, the antibody may be administered systemically or locally by intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, or the like. Examples of a formulation suitable for non-oral administration in the invention include an injection, a suppository, a spray, and the like. Furthermore, in a case where an immunoglobulin is administered to a cell, the immunoglobulin is administered to a culture liquid at usually 1 µg/ml, preferably 10 µg/ml or more, more preferably 50 µg/ml or more. , and even preferably 0.5 mg/ml or more.

[00141] In the agent for suppressing pDC activity or the method of suppressing pDC activity of the invention, the monoclonal antibody can be administered by any method to a living organism. Usually, the monoclonal antibody is mixed with a pharmaceutically acceptable carrier. Along with the monoclonal antibody, additives such as a thickening agent, a stabilizer, a preservative, and a solubilizing agent can be mixed in if necessary. Examples of such carriers or additives include lactose, citric acid, stearic acid, magnesium stearate, sucrose, starch, talc, gelatin, agar, vegetable oil, ethylene glycol, and the like. Terms called “pharmaceutically acceptable” refer to those approved by the government oversight of each country, or those listed in each country's pharmacopoeia or a pharmacopoeia generally known for use in an animal, a mammalian animal, and particularly in human being. The agent for suppressing pDC activity of the invention may also be provided in a lyophilized powder or tablet form in a single dose or multiple doses. The lyophilized powder or tablet may further be combined with sterilized water, physiological saline, or an injection buffer to dissolve the composition to a desired concentration prior to administration.

[00142] Furthermore, in a case where the agent for suppressing pDC activity of the invention is administered as an immunoglobulin expression vector, the heavy chain and the light chain are co-transfected with a separate plasmid, and each of the Plasmids can be administered at 0.1 to 10 mg, for example, 1 to 5 mg per 1 kg of body weight. Additionally, a vector of 1 to 5 µg/106cells is used for introduction into a cell in vitro. Hereinafter, the invention will be specifically explained further based on the Examples.

[00143] Meanwhile, the entire prior art documents cited in the present specification are incorporated into the present specification by reference.

[00144] Although the invention is specifically described further with the Examples below, the invention is in no way limited to such Examples.

Examples Example 1 A. Analysis of PLD4 expression A-1) Analysis using the SAGE library

[00145] Gene expressions in resting pDC, human monocyte, and activated pDCs treated with inactivated herpes virus (HSV-1) were compared by the SAGE® method (Series Analysis of Gene Expression). The analysis method is as described below.

[00146] From human peripheral blood, the monocyte was isolated as a CD14-positive cell and the human pDC cell was isolated as a BDCA-4 positive cell with a BDCA-4+ isolation kit (Miltenyi Biotec company) and MACS system (Miltenyi Biotec company). Furthermore, the human pDC cell was cultured for 12 hours in the presence of inactive HSV-1, to prepare activated pDC (pDC+HSV). Total RNA was extracted from each cell, and the SAGE library was prepared using the I-SAGE® kit (Invitrogen company). Data obtained from about 100,000 labeled base sequences were analyzed with SAGE2000 Analysis Software (Invitrogen company). As a result of the same, the monocyte/pDC/pDC+HSV count value was 0/9/0 gene, that is, PLD4 (Phospholipase D family, member 4, C14orf175; GenBank Accession Number: NM_138790, 2) was discovered, which is a gene known as a gene that exhibits resting pDC cell-specific expression (FIG. 1, Tao et al., Nat. Methods 2(8), 591-598 (2005); Clark et al ., Genome Res. 13(10), 2265-2270 (2003)). PLD4 is a sequence of 506 amino acids (SEQ ID NO: 1) encoded by a base sequence represented by SEQ ID NO: 44. The PLD4 protein has two experimental PDE regions (Phosphodiesterase motif) constituted with two HKD motifs (Phosphodiesterase sequence). amino acid His-x-Lys-x-x-x-x-Asp, the x is the other amino acids) conserved in the C-terminal region, and an experimental phosphorylation site (Thr 472). The protein structure of PLD4 is predicted to be a type II monotropic transmembrane protein. Furthermore, the PLD4 protein lacks the PX region (Phox homology domain) and the PH region (Pleckstrin homology domain) in the N-terminal region, which are possessed by PLD1 and PLD2, which are from the classical PLD family ( FIGS. 1 and 2).

A-2) Analysis of PLD4 mRNA expression in various human cells responsible for immunity by real-time quantitative PCR

[00147] The expression of PLD4 mRNA in a blood cell was specifically reviewed. From human peripheral blood, each cell was isolated and collected by a cell sorter. From each of the isolated and collected cell families, RNA was extracted, and cDNA was synthesized. Using the obtained cDNA as a standard, real-time quantitative PCR was performed, and the expression level of PLD4 mRNA was analyzed.

[00148] For the real-time quantitative PCR reaction, quantitative PCR was performed with ABI PRISM 7000 using the Platinum SYBR Green qPCR Super Mix- UDG kit (Invitrogen company). Sequence Detection System Software (Applied Biosystem company) was used in data analysis. The PCR reaction conditions and the base sequence of the primers used are as follows.

[00149] Advanced primer for PLD4: 5' ATG GAC TGG CGG TCT CTG 3' (SEQ ID NO: 45)

[00150] Reverse primer for PLD4: 5' TGG AAG GTC TTC TCC AGG TC 3' (SEQ ID NO: 46)

[00151] Advanced primer for GAPDH: 5' AGC CAC ATC GCT CAG ACA C 3' (SEQ ID NO: 47)

[00152] Reverse primer for GAPBH: 5' GCC CAA TAC GAC CAA ATC C 3' (SEQ ID NO: 48) 1 cycle at 58 ° C for 2 minutes, 1 cycle at 95 ° C for 10 minutes, 50 cycles of [ 95 °C for 15 seconds, and 60 °C for 60 seconds],

[00153] pDC, pDC (pDC+HSV) stimulated with HSV, B cell (CD19+ cell), activated B cell (CD19+ cell), T cell (CD3+ cell), and activated T cell stimulated with Inomycin and PMA (Forbol 12- myristate 13-acetate) were reviewed. As a result, it was illustrated that PLD4 expression was specifically high in pDC at the resting stage, and low in CDI9+ B cells. The other human blood cDNA fraction used MTC BD® Multi-Tissue cDNA Panels (Cat. No. 636750, Takara Bio company) (FIG. 5).

A-3) analysis of PLD4 mRNA expression in human tissue by real-time quantitative PCR

[00154] Furthermore, expressions in other organs or tissues were reviewed by quantitative PCR using ABI PRISM 7000 (Applied Biosystem company). As the cDNA panel, the MTC BD® multi-tissue cDNA panel (Human I; Cat. No. 636742, Human immune; Cat. No. 636748; all from Takara Bio company) was used. The base sequences of the primers used are represented as follows.

[00155] Advanced primer for PLD4: 5' ATG GAC TGG CGG TCT CTG 3' (SEQ ID NO: 49)

[00156] Reverse primer for PLD4: 5' TGG AAG GTC TTC TCC AGG TC 3' (SEQ ID NO: 50)

[00157] Advanced primer for GAPDH: 5' AGC CAC ATC GCT CAG ACA C 3' (SEQ ID NO: 51)

[00158] Reverse primer for GAPDH: 5' GCC CAA TAC GAC CAA ATC C 3' (SEQ ID NO: 52)

[00159] Quantitative PCR was performed with ABI PRISM 7000 using the Platinum SYBR Green qPCR Super Mix-UDG kit (Invitrogen company). Sequence Detection System Software (Applied Biosystem company) was used in the analysis. The reaction conditions are as described below. Step 1: 1 cycle at 50°C for 2 minutes Step 2: 1 cycle at 95°C for 10 minutes Step 3: 50 cycles at 95°C for 15 seconds and 60°C for 1 minute

[00160] The expressions of the PLD4 genes were compared between each of the tissues by standardizing with the expression level of the GAPDH (glyceraldehyde-3-phosphate dehydrogenase) gene, which is known to be expressed homeostatically. As a result of these, PLD4 mRNA exhibited relatively high expression in the spleen and peripheral blood leukocyte. Furthermore, it was revealed that PLD4 mRNA was widely expressed in other tissues. However, the expression level of PLD4 mRNA was less than 100 times the expression level of the resting pDC cell (FIG. 6).

Preparation of human PLD4 expression vector

[00161] The preparation of a PLD4 gene expression vector was carried out in order to express a human PLD4 protein. From the PLD4 cDNA clone incorporated into a pCR4-TOPO cloning vector (Open Biosystem, Cat. No. MHS4771-99610856), only the PLD4 gene was collected with the EcoRI enzyme, and incorporated into a pcDNA3 expression vector .1 (human PLD4-pcDNA3.1 vector). Using the obtained human PLD4-pcDNA3.1 plasmid as a template, the PLD4 gene was amplified with a primer containing EcoRI, NotI, and Kozak (GCC GCC ACC) sequences (Primer information is as described below). The PCR product was cloned into the EcoRI and NotI sites with pMX-IP retrovirus vector (human PLD4-pMX-IP retrovirus vector). In the PCR reaction, 1 unit of KOD plus DNA polymerase (TOYOBO company) was used, and the reaction conditions were 1 cycle at 94°C for 2 minutes, and then 25 cycles of [94°C for 15 seconds and 68° C for 1 minute and 30 seconds].

[00162] Advanced primer (SEQ ID NO: 53): 5' ttt GAA TTC gcc gcc acc ATG CTG AAG CCT 3' (30-mer)

[00163] Reverse primer (SEQ ID NO: 54): 5' aaa gcg gcc gcT CAG CCC TGC CAA ACG CAG TCC T 3' (34-mer)

[00164] Along with sequence analysis, the HEK (Human Embryonic Kidney)-293 T cell (Hereinafter, 293T cell) was transiently transfected with human PLD4-pMX-IP retroviral vector, and whether human PLD4 was expressed in the 293T cell surface was confirmed with cell staining, and then FACS method.

Example 2 Review of anti-human PLD4 antibody specificity

[00165] The fact that the anti-PLD4 monoclonal antibody does not cross with other molecules of the PLD family can be confirmed using a cell in which each of the PLD family is obligately expressed. Human PLD4 belongs to the PLD family, and there are multiple molecules having high homology. Human PLD4 shows about 41% homology to human PLD3 and about 29.3% homology to human PLD5 (FIG. 4).

1) Preparation of human PLD3 and human PLD5 expression vectors

[00166] For human PLD3 (cDNA SEQ ID NO: 55, amino acid SEQ ID NO: 127) and human PLD5 (cDNA SEQ ID NO: 56, amino acid SEQ ID NO: 123), from the human PLD3 clone (company K.K.DNAFORM, Cat. No. 5189261) incorporated into the pCMV-SPORT6 vector, and human PLD5 clone (company K.K.DNAFORM, Cat. No. 40025860) incorporated into the pCR-Blunt II-TOPO vector, only the PLD3 and PLD5 genes were amplified with PCR, and each of the genes was cloned into the Hind III and EcoRI sites of Flag-labeled pcDNA3.1 (Invitrogen company), to thereby prepare an expression vector (Primer information is as described below). (For human PLD3) Forward primer: huPLD3-SE (Hind III)

[00167] Sequence: 5' ttt AAG CTT gcc gcc acc ATG AAG CCT AAA CTG ATG TAC 3' (39-mer) SEQ ID NO: 57) Reverse primer: huPLD4-1518R (EcoRI)

[00168] Sequence: 5' ttt gaa ttc TCA ctt atc gtc gtc atc ctt gta atc GAG CAG GCG GCA GGC GTT GCC 3' (57-mer) (SEQ ID NO: 58) (For human PLD5) Forward primer: huPLD5 -SE (Hind III).

[00169] Sequence: 5' ttt AAG CTT gcc gcc acc ATG GGA GAG GAT GAG GAT GGA 3' (39-mer) (SEQ ID NO: 59) Reverse primer: huPLD5-1383R (EcoRI)

[00170] Sequence: 5' ttt gaa ttc TCA ctt atc gtc gtc atc ctt gta atc TAC GTT CCG GGG ATC CTT TCC 3' (57-mer) (SEQ ID NO: 60)

[00171] Among the previously mentioned primer sequences, the underlined parts represent base sequences that encode the added FLAG tags, and the italic type represents the Hind III cutting site or EcoRI site of restriction enzymes.

[00172] Along with sequence analysis, the 293T cell was transiently transfected with human PLD3-pcDNA3.1 vector or human PLD5-pcDNA3.1 vector, and whether human PLD4 was expressed on the surface of the 293T cell was confirmed with staining cell, and then FACS method.

[00173] As a result thereof, the antibodies did not react with a cell in which human PLB3 or human PLD5 appeared to be expressed, which suggested that these anti-PLD4 antibodies recognized the human PLD4 molecules specifically (FIG. 8).

Preparation of stable strain of human PLD4 expressing cell

[00174] The prepared human PLD4-pMX-IP vector was cotransfected into 293T cell along with pCL-Eco vector (IMGENEX, Cat. No. 10045P), which is a retrovirus packaging vector, to thus carry out the introduction of the gene . The gene introduction experiment used FuGENE (trademark) HD Transfection Reagent (Roche) as the transfection reagent. After 2 days, the cell culture supernatant, in which the retrovirus containing the human PLD4 gene was secreted, was collected, and it was infected into the cell strain CT125 (mouse T-cell lymphocyte tumor cell series 2B4). . Due to the fact that the retroviral vector pMX-IP contains a gene resistant to puromycin, the culture of infected CT125 cells in the presence of puromycin allows the survival of only cells that express human PLD4, leading to their selection. The selected human PLD4-expressing CT125 cells (hereinafter, human PLD4-CT125) were further selected only for the CT125 cells allowing higher expression of human PLD4 by FACS sorting, and cultured. For confirmation of human PLD4 expression, CT125 cells were stained with commercially available mouse anti-human PLD4 polyclonal antibody (Abnova, Cat #: HOO122618-B01P) adjusted to 5 µg/ml, and FACS analysis was performed . As a result thereof, the stable human PLD4-CT125 cell strain was established, and used in hybridoma FACS screening.

[00175] Construction of cynomolgus monkey and rhesus monkey PLD4 expression vectors, and preparation of the stable strain of human PLD4 expression cell

[00176] For the expressions of PLD4 proteins from cynomolgus monkey and rhesus monkey, cloning of the monkey PLD4 gene and construction of expression vectors were carried out.

1) Cloning of PLD4 gene from cynomolgus monkey and PLD4 from rhesus monkey

[00177] The rhesus monkey PLD4 cDNA sequence is reported in the Genbank database (XM_002805227.1) and the like, but a partial, full-length cDNA thereof is not reported. Furthermore, since the gene sequence of cynomolgus monkey PLD4 is not yet reported, from PBMC (10 ml respectively; SHIN NIPPON BIOMEDICAL LABORATORIES, LTD.) of cynomolgus monkey and rhesus monkey, gene cloning was performed.

[00178] Total RNA was extracted from the monkey's peripheral blood, and from 5 µg of it, cDNA was synthesized using oligo-dT primer and the SuperScript Choice System for cDNA Synthesis kit.

[00179] Using the prepared cDNA as a template, the cynomolgus monkey PLD4 and rhesus monkey PLD4 genes were amplified with the PCR method using the primers of the following base sequences.

[00180] Advanced primer (cynoPLD4-32F): 5' AGA TGC TGA AGC CTC TTC GGA GAG Cg 3' (SEQ ID NO: 61)

[00181] Reverse primer (cynoPLD4-1554R): 5' TCA GCC CTG CCA AAC GCA GTC CTG G3' (SEQ ID NO: 62)

[00182] The amplified cDNA fragments of about 1521 base pairs of cynomolgus monkey PLD4 and 1521 base pairs of rhesus monkey PLD4 were isolated by electrophoresis using 1% agarose gel, collected, and cloned into the plasmid vector pCR4Blunt-TOPO (Invitrogen company) using the Zero Blunt TOPO PCR Cloning kit (Invitrogen company). The base sequences of the obtained gene were analyzed, which are represented by SEQ ID NO: 63 and SEQ ID NO: 124. It was confirmed that the intended cynomolgus monkey PLD4 and rhesus monkey PLD4 genes were able to be cloned.

[00183] The human PLD4 protein represents about 94.4% identity with the cynomolgus monkey PLD4 protein sequence (SEQ ID NO: 129), and about 94% identity with the PLD4 protein sequence of rhesus monkey (SEQ ID NO: 130). FIG. 14 illustrates the homology of human PLD4 protein sequences with cynomolgus monkey PLD4, rhesus monkey PLD4, and mouse PLD4 (cDNA SEQ ID NO: 131, amino acid SEQ ID NO: 132).

2) Preparation of the stable strain of the cynomolgus monkey PLD4 expressing cell

[00184] The stable strain of the cynomolgus monkey PLD4-expressing cell was established by culturing the CT125 cell infected with a retroviral vector in the presence of puromycin, in the same method as the method of preparing the stable strain of the human PLD4-expressing cell using the prepared cynomolgus monkey PLD4-pMX-IP vector.

[00185] To confirm the expression of cynomolgus monkey PLD4, the cells were stained with commercially available mouse anti-human PLD4 polyclonal antibody (Abnova, Cat #: H00122618-B01P) which represents cross-reactivity also with monkey PLD4, and FACS analysis was performed. As a result thereof, the stable cynomolgus monkey PLD4-CT125 cell strain was established, and used in FACS hybridoma screening (FIG. 16).

Preparation of human PLD4-Ig fusion protein

[00186] For use as an immunogen in the preparation of anti-human PLD4 monoclonal antibody, 2142 base pairs of a DNA fragment, in which the extracellular region of the human PLD4 protein (56-506 a.a) and the Fc fragment of Mouse IgG2a (234 a.a containing a portion of the heavy chain hinge, CH2, and CH3) were fused, amplified with the 2-step PCR method, and human PLD4-Ig plasmid expression vectors pcDNA3.1 and PLD4- Human IgG pEE14.4 were constructed (The cDNA and protein sequences are specified in the sequence listing).

[00187] In order to obtain a protein supernatant from the culture, Maxi-prep DNA was transiently transfected into the Freestile 293F cell (hereinafter, 293F cell, catalog No. R790-07; Invitrogen). On day 7 after transfection, the culture liquid from the cotransfected 293F cell was collected in a 50 ml tube, and centrifugation was performed under conditions of 2.070 g at 4 °C for 5 minutes. The supernatant was filtered with a syringe filter having 0.45 µm pore size (catalog No. 431220; CORNING), and the culture supernatants were collected together.

[00188] The collected cell culture supernatant was purified with protein A affinity column of the AKTA-FPLC system, and the recombinant human PLD4-Ig fusion protein protein was purified (FIG. 7).

Example 3 A. Preparation of anti-human PLD4 monoclonal antibody A-1) Immunization

[00189] As an immunogen, the previously mentioned recombinant PLD4-Ig fusion protein was used. The PLD4-Ig fusion protein was administered subcutaneously to the hindquarters of 3 BALB/c mice. Freund's Adjuvants, Complete and Incomplete (SIGMA) were used as an adjuvant, and 200 µg/mouse for the first time and 50 µg/mouse for the second to fourth times were administered.

A-2) Confirmation of antiserum titer

[00190] Blood was collected after the third immunization and the fourth immunization, and the anti-PLD4-Ig titer in the serum was evaluated with ELISA.

[00191] The PLD4-Ig fusion protein was added as a solid phase to a 96-well microtiter plate. The antiserum was diluted stepwise from 1,000-fold to 3-fold, and dilution series were prepared at 729,000-fold. Each of the samples was added to the antigen solid phase plate in 50 µl, and the primary reaction was performed. After washing, the secondary reaction was performed with anti-mouse IgG (K, À) antibody with HRP label, and detected in color (490 nm) with OPD (ortho-phenylenediamine).

A-3) Cell fusion

[00192] Splenic cells were isolated from a mouse for which increased antiserum titer was recognized. The isolated splenic cell and mouse myeloma cell (P3U1) were fused with the PEG method, and the selection and culture of the fusion splenic cell was performed from HAT medium.

FACS screening of hybridoma using CAL-1 cell

[00193] The antibodies producing each clone of the fusion splenic cell obtained from the HAT selection culture were evaluated with FACS. CAL-1 cells the same cell strain as human PDC at 2 x 105 were reacted with 50 µl of culture supernatant from each of the hybridomas described below for 15 minutes at 4 °C. Cells were washed with FACS buffer (1% FBS + PBS) twice, centrifuged, and the supernatant was removed. The reaction was performed for 20 minutes at 4°C using PE-labeled anti-mouse IgG antibody (BD Bioscience: 550589) as a secondary antibody. The culture liquid after 10 days of starting the HAT selection culture was used as the original duplicate for the culture supernatant of each clone. As a result thereof, 3B4, 5B7, 7B4, 8C11, 10C3, 11D10, 13D4, 13H11, 14C1, and 11G9.6 of the hybridoma culture supernatants were well reacted with the CAL-1 cell (FIG. 11).

[00194] Hybridoma FACS screening using human PLD4-CT125 stable cell strain

[00195] The antibodies producing each clone of the fusion splenic cell obtained from the HAT selection culture were evaluated with FACS. Human PLD4-CT125 at 2 x 105 was reacted for 15 minutes at 4°C with 50 µl of culture supernatant from each of the hybridomas described below. Cells were washed with FACS buffer (1% FBS + PBS) twice, centrifuged, and the supernatant was removed. The reaction was performed for 20 min at 4°C using PE-labeled anti-mouse IgG antibody (BD Bioscience: 550589) as a secondary antibody. As a result thereof, 3B4, 5B7, 7B4, 8C11, 10C3, 11D10, 13D4, 13H11, 14C1, and llG9.6 of the hybridoma culture supernatants were well reacted with the human PLD4-CT125 cell (FIG. 12).

A-5) FACS screening using human peripheral blood pDC Isolation of human PBMC

[00196] 20 ml of peripheral blood from a healthy individual was collected, and the peripheral blood mononuclear cell (PBMC) was isolated with specific gravity centrifuge using HISTOPAQUE-1077 (SIGMA company). 1 x 106 PBMCs were dyed for each sample. Cells were washed with FACS buffer, and then added with 25 µl of 5-fold dilution of Fc block reagent (Militenyi company), and reacted at 4 °C for 15 minutes. Cells were washed with FACS buffer, and then added with 50 µl of cell culture supernatant from each hybridoma and mouse IgG2b, K, and reacted at 4°C for 20 minutes. Cells were washed with FACS buffer, and then added with PE-labeled anti-mouse IgG antibody, and reacted at 4°C for 20 minutes. Cells were washed with FACS buffer, and then added with 50 µl of a 10-fold dilution of APC-labeled anti-BDCA2 antibody, and reacted at 4°C for 20 minutes. Cells were washed with FACS buffer, and then resuspended in 300 µl of FACS buffer, and analyzed with FACS Calibur (BD). The mp11G9.6 antibody exhibited binding to pDC (FIG. 10).

[00197] Furthermore, 9 types of PLD4 antibodies of 3B4, 5B7, 7B4, 8C11, 10C3, 11D10, 13D4, 13Hll, and 14C1 exhibited specific binding reaction to the pDC cell population, which was a BDCA2 positive cell ( FIG. 13).

A-6) Cross-reactivity of anti-PLD4 antibody to monkey

[00198] Hybridoma FACS screening using stable cell strain PLD4-CT125 from cynomolgus monkey and transient transfectant cell PLD4-293T from rhesus monkey

[00199] The antibodies producing each clone of the fusion splenic cell obtained from the HAT selection culture were evaluated with FACS. Human PLD4-CT125 at 2 x 105 was reacted with 50 µl of culture supernatant from each of the hybridomas described below for 15 minutes at 4°C. Cells were washed with FACS buffer (1% FBS + PBS) twice, centrifuged, and the supernatant was removed. The reaction was performed for 20 minutes at 4°C using PE-labeled anti-mouse IgG antibody (BD Bioscience: 550589) as a secondary antibody. As a result of the same, 7 types of antibodies of 3B4, 5B7, 7B4, 13D4, 13H11, 14C1, and 11G9.6 antibodies among 10 types of PLD4 antibodies were well reacted with cynomolgus monkey PLD4-CT125 cell and PLD4- 293T of rhesus monkey (FIG. 15, FIG. 16, and FIG. 17).

A-7) Cross-reactivity of anti-PLD4 antibody to monkey PBMC

[00200] Peripheral blood rhesus monkey PBMC (10 ml; SHIN NIPPON BIOMEDICAL LABORATORIES, LTD.), was centrifuged with specific gravity using 96% Ficoll-Paque (registered trademark) PLUS (GE Healthcare company, Cat No. 17 -1440-02). For FACS, 5 x 105 cells were used per sample. Cells were washed with FACS cell buffer, and then added with 10 µl of cynomolgus monkey serum diluted to 10% with FACS buffer, and reacted at 4 °C for 20 minutes. Cells were washed with FACS buffer, and then added with 100 µl of cell culture supernatant from each hybridoma and 10 µg/ml of mouse IgG2a, K or mouse IgG1, K, and reacted at 4°C for 15 minutes. Cells were washed with FACS buffer, and then added with 1 µg/ml APC-labeled anti-mouse IgG antibody, and reacted at 4°C for 20 minutes. Cells were washed with FACS buffer, and then reacted with 25 µl of 10-fold dilution of FITC-labeled anti-Lineage 1 antibody, PE-labeled anti-CD123 antibody, and PcrCP-Cy5.5-labeled anti-HLA-DR antibody at 4 °C for 15 minutes. Cells were washed with FACS buffer, and then resuspended in 300 µl of FACS buffer, and analyzed with FACS calibur. The hybridoma culture supernatant used was specific for PLD4, and 10 types of 3B4, 5B7, 7B4, 8C11, 10C3, 11D10, 13D4, 13H11, 14C1, and 11G9.6, which bind well to CAL-1 cell or pDC human, were selected. As a result thereof, 5 types of cell culture hybridoma supernatant, i.e., 5B7, 7B4, 13D4, 13H11, and 14C1 specifically bind to the cynomolgus monkey pDC cell population (Lineage-CD123+ HLA-DR+) ( FIG. 18).

A-8) Hybridoma cloning using the limiting dilution method 1) Cloning and 2nd screening using the limiting dilution method

[00201] For the cloning of 9 selected types (3B4, 5B7, 7B4, 8C11, 10C3, 11D10, 13D4, 13H11, and 14C1) of hybridomas except the 11G9.6 hybridoma, limiting dilution was performed. The limiting dilution was seeded onto the 2 pieces of the 96-well plate. After 6 days of seeding, the cells were observed under a microscope, and culture supernatants that were monoclone-derived and exhibited good growth in all wells were collected. FACS analysis was performed for the culture supernatant collected as a sample.

[00202] In FACS analysis, the surface antigen of the CAL-1 cell strain was stained using the culture supernatant of each clone followed by PE-labeled anti-mouse IgG antibody (BD Bioscience: 550589) as a secondary antibody . As the culture supernatant of each clone, the culture liquid after 7 days of limiting dilution seeding was used as the original duplication.

2) Cloning and 3rd screening

[00203] Based on the results of FACS analysis from the 2nd screening and the cell status in each reservoir, 1 reservoir was selected from each clone, and limiting dilution was performed once again. Limiting dilution was similarly performed as in 2), and FACS analysis (3a) was performed for the culture supernatant collected as a sample. Based on the FACS analysis data and the like, following 9 types (3B4, 5B7, 7B4, 8C11, 10C3, 11D10, 13D4, 13H11, and 14C1) of the clones were cloned by the limiting dilution method, and the hybridoma producing anti-human PLD4 antibody was established as a stable cell strain.

3) Conversion to single 11G9.6 hybridoma

[00204] The 11G9.6 hybridoma other than the 9 previously mentioned types was collected, and placed in suspension with a classification buffer (1% FBS/PBS) to be 1 x 105 cells/ml. Single-cell sorting was performed using FACS Aria (BD). The data was embedded, and the embedded data was developed for plotting two-dimensional X-axis: FSC and Y-axis: SSC points. In the dot plot, living cells were surrounded with a barrier. The barrier was trimmed to exclude cell doublets in the live cell barrier, the cell population was isolated and collected in a 96-well flat plate at 1 cell/well. Cells after single cell sorting were cultured in HAT medium (RPMI 1640 + 2 mM L-Glutamine, 10 Units/ml Penicillin-Streptomycin, 10 mM HEPES, 1 mM Sodium Pyruvate, 50 µM 2- ME) + HFCS hybridoma growth supplement (Roche company). Then, the CAL-1 cell, human PLD4-CT125 expression stable cell strain, human PBMC, and the monkey PLD4-CT125 expression stable cell strain were stained using the hybridoma cell culture supernatant, and 11G9. 6 single hybridomas were selected.

4) Preparation of frozen cell vial and collection of culture supernatant

[00205] Based on the FACS analysis results described above and the cell status of each reservoir, 1 reservoir was selected from each clone. For the selected reservoir, the expansion culture was performed at the 50 ml scale. The medium was RPMI 1640 containing 10% FCS and penicillin streptomycin. Cells were grown to subconfluent, and frozen and preserved at a cell number of 1 x 106 cells/tube. As the liquid for freezing and preservation, BAMBANKER (NIPPON Genetics, Co. Ltd.) was used. Furthermore, the culture supernatant at this time point was collected and preserved.

Example 4 Antibody purification

[00206] 10 types (3B4, 5B7, 7B4, 8C11, 10C3, 11D10, 13D4, 13H11, 14C1, and 11G9.6) of purified antibodies were obtained from the culture supernatants of the hybridomas by purification using the protein A affinity column (rProtein A Sepharose Fast Flow (catalog No. 17-1279-01, GE Healthcare company). Isotypes were confirmed using the Pierce Rapid ELISA Mouse mAb Isotyping Kit (Thermo Fisher Scientific company). As a result of the same, 3B4 and 14C1 were mouse IgG1, K, 10C3 was mouse IgG2a, K, and the others were mouse IgG2b, K. The measurement of the endotoxin concentration was carried out since if the endotoxin was contained in the antibody, it could have an influence on the results of a property determination test. The kits used were Endospecy ES-50M set, Toxicolor DIA-MP set, and endotoxin standard CSE-L set (SEIKAGAKU BIOBUSINESS CORPORATION company). As a result thereof, the Endotoxin concentration of any purified antibody was 0.3 EU/mg or less of Ab which was the reference value.

Review of purified antibody reactivity

[00207] The binding capacity of the purified antibody was confirmed with a CAL-1 cell, which is the human pDC cell strain. As a result, it could be confirmed that no antibody maintained the ability to bind to human PLD4 on the cell surface (FIG. 11). Furthermore, the antibody also specifically bound to the human peripheral blood pDC (BDCA2+) cell population (FIG. 13).

Calculation of Kd value of purified PLD4 antibody

[00208] Regarding the binding capacity of the purified antibody, a stable cell strain expressing human PLD4-CT125 was reacted to almost 100% positive staining rate at low concentration to high concentration (0.001 µg/ml to 30 µg /ml) of purified PLD4 antibody concentration. The frequency of staining-positive cell and antibody were analyzed in data using Grafic Pad Prism version 5 software, and the molar concentration of the dissociation constant (Kd value) was computed in nM unit. From the fact that the Kd (nM) values of the anti-PLD4 antibodies were all 1 nM or less or 1 nM approximately except the 2 clones of 3B4 and 14C1, the anti-PLD4 antibodies bound very strongly to the PLD4- cell. human CT125 (FIG. 19).

Example 5

[00209] The complement-dependent cytotoxicity activity of the anti-PLD4 antibody to the human PLD4-CT125 expressing cell

[00210] The complement-dependent cytotoxicity activity (hereinafter referred to as CDC activity) of the anti-PLD4 antibody was measured for the stable cell strain CT125 expressing human PLD4 (hereinafter referred to as HuPLD4-CT-125 ) using immature rabbit serum as a source of complement. The activity index was the cell's toxicity calculated from the measured value of Lactase dehydrogenase (LDH) released from the cell. Each cell was dispensed into the 96-well U-bottom plate at 2 x 104 cells/50 µl/well. 1% Baby rabbit complement (CEDARLANE company) was prepared in CDC medium (RPMI 1640 + 0.1% BSA + 10 mM HEPES + 2 mM L-Glutamine + 1% Pen-Strep). Cells were added with 10 µg/ml mouse isotype control antibody (mouse IgG2b, K) and 10 types of the purified mouse anti-PLD4 antibodies (3B4, 5B7, 7B4, 8C11, 10C3, 11D10, 13D4, 13H11, 14C1, and 11G9.6), and reacted for 1 hour. For the assay system, the CytoTox 96 Non-Radioactive Cytotoxicity Assay Kit (Promega company) was used. As a result thereof, for the target cell of HuPLD4-CT-125, 8 types of PLD4 antibodies (5B7, 7B4, 8C11, 10C3, 11D10, 13D4, 13H11, and 11G9.6) exhibited CDC activity of about 33. 5% to 71.1% at 10 µg/ml antibody concentration except 2 types of antibodies PLD4, 3B4 and 14C of which the heavy chain isotype was mouse IgG1 (FIG. 20).

Example 6

[00211] Mouse isotype control antibody of the concentration-dependent, complement-dependent cytotoxicity activity (mouse IgG2b, k) of anti-PLD4 antibody (11G9.6), mouse anti-PLD4 antibody (Ab mp11G9.6) , human isotype control antibody (human IgG1, k), and chimeric anti-PLD4 antibody (Ab ch11G9) were adjusted to 6 total points of antibody concentration from 0.1 µg/ml to 30 µg/ml. As the assay system, the CytoTox 96 Non-Radioactive Cytotoxicity Assay kit (Promega company) was used. As a result thereof, for the HuPLD4-CT-125 target cell, mp11G9.6Ab exhibited about 70% concentration-dependent CDC activity at 3 µg/ml antibody concentration. On the other hand, ch11G9Ab, which was a chimeric antibody, exhibited 10% or less CDC activity even at 30 µg/ml high concentration (FIG. 21).

Example 7 Chimeric antibody preparation

[00212] 10 Types were prepared as a hybridoma that produces mouse anti-PLD4 antibody, and used.

1. Constant region isotype confirmation

[00213] The isotype of the constant region of the mouse anti-PLD4 antibody produced from the hybridoma that produces the anti-PLD4 antibody was confirmed. From the culture supernatant of hybridomas of 10 types (3B4, 5B7, 7B4, 8C11, 10C3, 11D10, 13D4, 13H11, 14C1, and 11G9.6), the isotype was confirmed using the Pierce Rapid ELISA Mouse mAb Isotyping Kit ( company Thermo Fisher Scientific). As a result thereof, 3B4 and 14C1 were mouse IgG1 and mouse Ig kappa, 10C3 was mouse IgG2a and mouse Ig kappa, and the others are mouse IgG2b and mouse Ig kappa.

2. Cloning of cDNA encoding the mouse anti-PLD4 antibody variable region 2-1) Isolation of total RNA

[00214] From the 11G9.6 hybridoma, total RNA was isolated using a commercially available kit, “RNeasy Mini Kit” (Qiagen company, catalog No.: 74106) according to the instruction attached to the kit. The same was prepared from the 5 x 106 cell number of the hybridoma cell strain, and about 79 µg of total RNA was obtained.

2-2) Amplification and fragmentation of the cDNA encoding the mouse heavy chain variable region

[00215] 5 µg of the total RNA isolated in 2-1) was used, and the cDNA encoding the mouse heavy chain variable region was amplified by the 5' RACE PCR method. In amplification, a commercially available kit, “5’ RACE System for Rapid Amplification of cDNA ENDs, Version 2.0” (Invitrogen company, catalog No.: 18374-058) was used. Details are as follows. First, single-stranded cDNA was synthesized by reverse transcription enzyme from total RNA obtained in 2-1). At this time, the antisense primer (GSP1) used was as follows. The GSP1 primers used in cDNA amplification were used differently depending on the isotype of each mouse heavy chain.

[00216] For example, in cloning the heavy chain variable regions of 3B4 and 14C1 hybridomas having mouse IgG1 heavy chains, the following antisense primers are used. GSP1 primer: muIgG1VH-GSP1

[00217] Sequence: 5'-CCA GGA GAG TGG GAG AGG CTC TTC TCA GTA TGG TGG-3' (36-mer) (SEQ ID NO: 64) GSP2 Primer: mu IgG1VH-GSP2

[00218] Sequence: 5'-GGC TCA GGG AAA TAG CCC TTG ACC AGG CAT CC-3' (32-mer) (SEQ ID NO: 65)

[00219] In cloning the 10C3 hybridoma heavy chain variable regions having mouse IgG2a heavy chain, the following antisense primers are used. GSP1 primer: mu IgGHYl-GSPI

[00220] Sequence: 5' TCC AGA GTT CCA GGT CAC TGT CAC 3' (24-mer) (SEQ ID NO: 66) GSP2 Primer: mu IgGHYl-GSP2

[00221] Sequence: 5' AGG GGC CAG TGG ATA GAC AGA TGG 3' (24-mer) (SEQ ID NO: 67)

[00222] In cloning the heavy chain variable regions of hybridomas 5B7, 7B4, 8C11, 11D10, 13D4, 13H11, and 11G9.6 having mouse IgG2b heavy chain, the following antisense primers are used. GSP1 primer: mu IgGH^2B-GSP1

[00223] Sequence: 5' TCC AGA GTT CCA AGT CAC AGT CAC 3' (24-mer) (SEQ ID NO: 68) GSP2 Primer: mu IgGH^2B-GSP2

[00224] Sequence: 5' AGG GGC CAG TGG ATA GAC TGA TGG 3' (24-mer) (SEQ ID NO: 69)

[00225] Furthermore, to the 3' end of the single-stranded cDNA, dC, which is a nucleotide homopolymer, was added using terminal deoxynucleotidyl transferase (TdT). Then, the cDNA was amplified by the PCR method using an anchor primer having a nucleotide polymer complementary to dC (anchor sequence) (SEQ ID NO: 70) at the 3' terminus, and an antisense primer (GSP2). Furthermore, cDNA was amplified by the housed PCR method using the obtained PCR product as a standard and using AUAP primer (SEQ ID NO: 71) and the antisense primer shown in Table 1 (GSP2). Furthermore, this PCR product was purified by the 1.5% low melting point agarose method. Anchor starter for 5’ RACE (SEQ ID NO: 70)

[00226] 5'-GGC CAC GCG TCG ACT AGT ACG GGI IGG GII GGG IIG-3' 36-mer) AUAP Initiator for 5' RACE (SEQ ID NO: 71)

[00227] 5’-GGC CAC GCG TCG ACT AGT AC-3’ 20-mer)

2-3) Amplification and fragmentation of cDNA encoding the variable region of the mouse light chain

[00228] The cDNA encoding the variable region of the mouse light chain was amplified from total RNA in 2-l) isolated, similarly to 2-2). At this time, the GSP1 primers used in cDNA amplification were used differently depending on the isotype of each mouse light chain.

[00229] The following antisense primers are used for light chain cloning since the 10 types of PLD4 antibodies have mouse Ig coat light chain. GSP1 Initiator: muIgG VL capa-GSP1

[00230] Sequence: 5'-CAC TAC TTC CTG TTG AAG CTC TTG ACG ATG G-3' (31-mer) (SEQ ID NO: 72) GSP2 primer: mu IgG VL kappa-GSP2

[00231] Sequence: 5'-GTG AGT GGC CTC ACA GGT ATA GC-3' (23-mer) (SEQ ID NO: 73)

[00232] The PCR product obtained was purified by the 1.5% low melting point agarose method.

2-4) Confirmation of the cDNA base sequence and determination of the CDR region

[00233] The cDNA fragments of the heavy chain variable region obtained in 22), and the light chain variable region obtained in 2-3) were cloned, respectively, with pCR4Blunt-TOPO vector using a commercially available “Zero Blunt TOPO PCR” kit. Cloning Kit” (Invitrogen company, catalog No.: 1325137), according to the instructions attached to the kit, and transformed into a competent Escherichia coli cell to obtain an Escherichia coli transformant. A plasmid was obtained from this transformant, and the plasmid DNA sample was sent for sequence analysis to Operon Biotechnology Co. Ltd (Tokyo), and the cDNA base sequence in the plasmid was confirmed. In sequence analysis, the software “Sequencher DNA sequence assembly and analysis software version 4.2.2 (Gene Codes Corporation)” and the software “GENETYX-MAC Version 11.1.1” (GENETYX CORPORATIOIN)” were used.

[00234] A transcript of a certain sequence was extracted by excluding an inactive RNA transcript, such as matrix change, nonsense mutation, and the like occur at the periphery of a complementarity-determining region (hereinafter referred to as “CDR region” ). Furthermore, for the cDNA base sequence contained in the plasmid, homology with the Immunoglobulins database (IgBLAST, URL: www.ncbi.nlm.nih.gov/Igblast/) was confirmed, and the CDR region sequences in each variable region (CDRs; CDR1, CDR2, and CDR3), the FW region (array working regions), and the variable regions were determined according to the Kabat numbering system analysis method (Kabat et al, 1991 , Sequences of Proteins of Immunological Interest, National Institutes of Health Publication No. 913242, 5th ed., United States Department of Health and Human Services, Bethesda, MD).

[00235] The nucleic acid sequence of the heavy chain variable region of the mouse antibody 11G9.6 obtained is SEQ ID NO: 74, and the amino acid sequence is SEQ ID NO: 75. The amino acid sequences of CDR1, CDR2, and CDR3 in the heavy chain variable region of mouse antibody 11G9.6 are SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, respectively.

[00236] The nucleic acid sequence of the heavy chain variable region of the mouse antibody 3B4 obtained is SEQ ID NO: 76, and the amino acid sequence is SEQ ID NO: 77. The amino acid sequences of CDR1, CDR2, and CDR3 in the heavy chain variable region of the mouse 3B4 antibody are SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10, respectively.

[00237] The nucleic acid sequence of the heavy chain variable region of the mouse antibody 5B7 obtained is SEQ ID NO: 78, and the amino acid sequence is SEQ ID NO: 79. The amino acid sequences of CDR1, CDR2, and CDR3 in the heavy chain variable region of the mouse 5B7 antibody are SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16, respectively.

[00238] The nucleic acid sequence of the heavy chain variable region of the mouse antibody 7B4 obtained is SEQ ID NO: 80, and the amino acid sequence is SEQ ID NO: 81. The amino acid sequences of CDR1, CDR2, and CDR3 in the heavy chain variable region of the mouse 7B4 antibody are SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16, respectively. Antibody 7B4 is an antibody having the same heavy chain and light chain variable region CDR sequences as those of antibody 5B7.

[00239] The nucleic acid sequence of the heavy chain variable region of the mouse antibody 8C11 obtained is SEQ ID NO: 82, and the amino acid sequence is SEQ ID NO: 83. The amino acid sequences of CDR1, CDR2 and CDR3 in the heavy chain variable region of mouse antibody 8C11 are SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 22, respectively.

[00240] The nucleic acid sequence of the heavy chain variable region of the obtained mouse 10C3 antibody is SEQ ID NO: 84, and the amino acid sequence is SEQ ID NO: 85. The amino acid sequences of CDR1, CDR2 and CDR3 in the heavy chain variable region of the mouse 10C3 antibody are SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 28, respectively.

[00241] The nucleic acid sequence of the heavy chain variable region of the mouse antibody 11D10 obtained is SEQ ID NO: 86, and the amino acid sequence is SEQ ID NO: 87. The amino acid sequences of CDR1, CDR2, and CDR3 in the heavy chain variable region of the mouse 11D10 antibody are SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 28, respectively. The 11D10 antibody is an antibody having the same light chain variable region CDR sequence for the heavy chain as the 10C3 antibody. However, the heavy chain isotype (10C3 is the constant region of mouse IgG2a, and 11D10 is the constant region of mouse IgG2b) is different.

[00242] The nucleic acid sequence of the heavy chain variable region of the obtained mouse 13D4 antibody is SEQ ID NO: 88, and the amino acid sequence is SEQ ID NO: 89. The amino acid sequences of CDR1, CDR2, and CDR3 in the heavy chain variable region of mouse 13D4 antibody are SEQ ID NO: 32, SEQ ID NO: 33 and SEQ ID NO: 34, respectively.

[00243] The nucleic acid sequence of the heavy chain variable region of the mouse 13H11 antibody obtained is SEQ ID NO: 90, and the amino acid sequence is SEQ ID NO: 91. The amino acid sequences of CDR1, CDR2, and CDR3 in the heavy chain variable region of the mouse 13H11 antibody are SEQ ID NO: 38, SEQ ID NO: 39, and SEQ ID NO: 40, respectively.

[00244] The nucleic acid sequence of the heavy chain variable region of the obtained mouse 14C1 antibody is SEQ ID NO: 92, and the amino acid sequence is SEQ ID NO: 93. The amino acid sequences of CDR1, CDR2, and CDR3 in the heavy chain variable region of mouse 14C1 antibody are SEQ ID NO: 38, SEQ ID NO: 39, and SEQ ID NO: 40, respectively. The 14C1 antibody is an antibody having the same heavy chain and light chain variable region CDR sequences as those of the 13H11 antibody. However, the heavy chain isotype (13H11 is the constant region of mouse IgG2b, and 14C1 is the constant region of mouse IgG1) is different.

[00245] The nucleic acid sequence of the light chain variable region of mouse antibody 11G9.6 is SEQ ID NO: 94, and the amino acid sequence is SEQ ID NO: 95. The amino acid sequences of CDR1, CDR2 , and CDR3 in the light chain variable region of mouse antibody 11G9.6 are SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, respectively.

[00246] The nucleic acid sequence of the light chain variable region of the mouse 3B4 antibody is SEQ ID NO: 96, and the amino acid sequence is SEQ ID NO: 97. The amino acid sequences of CDR1, CDR2, and CDR3 in the light chain variable region of the mouse 3B4 antibody are SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13, respectively.

[00247] The nucleic acid sequence of the light chain variable region of the mouse 5B7 antibody is SEQ ID NO: 98, and the amino acid sequence is SEQ ID NO: 99. The amino acid sequences of CDR1, CDR2, and CDR3 in the light chain variable region of the mouse 5B7 antibody are SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19, respectively.

[00248] The nucleic acid sequence of the light chain variable region of the mouse 7B4 antibody is SEQ ID NO: 100, and the amino acid sequence is SEQ ID NO: 101. The amino acid sequences of CDR1, CDR2, and CDR3 in the light chain variable region of mouse 7B4 antibody are SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19, respectively.

[00249] The nucleic acid sequence of the light chain variable region of the mouse 8C11 antibody is SEQ ID NO: 102, and the amino acid sequence is SEQ ID NO: 103. The amino acid sequences of CDR1, CDR2, and CDR3 in the light chain variable region of mouse 8C11 antibody are SEQ ID NO: 23, SEQ ID NO: 24, and SEQ ID NO: 25, respectively.

[00250] The nucleic acid sequence of the light chain variable region of the mouse 10C3 antibody is SEQ ID NO: 104, and the amino acid sequence is SEQ ID NO: 105. The amino acid sequences of CDR1, CDR2, and CDR3 in the light chain variable region of the mouse 10C3 antibody are SEQ ID NO: 29, SEQ ID NO: 30, and SEQ ID NO: 31, respectively.

[00251] The nucleic acid sequence of the light chain variable region of the mouse 11D10 antibody is SEQ ID NO: 106, and the amino acid sequence is SEQ ID NO: 107. The amino acid sequences of CDR1, CDR2, and CDR3 in the light chain variable region of the mouse 11D10 antibody are SEQ ID NO: 29, SEQ ID NO: 30, and SEQ ID NO: 31, respectively.

[00252] The nucleic acid sequence of the light chain variable region of the mouse 13D4 antibody is SEQ ID NO: 108, and the amino acid sequence is SEQ ID NO: 109. The amino acid sequences of CDR1, CDR2, and CDR3 in the light chain variable region of the mouse 13D4 antibody are SEQ ID NO: 35, SEQ ID NO: 36, and SEQ ID NO: 37, respectively.

[00253] The nucleic acid sequence of the light chain variable region of the mouse 13H11 antibody is SEQ ID NO: 100, and the amino acid sequence is SEQ ID NO: 111. The amino acid sequences of CDR1, CDR2, and CDR3 in the light chain variable region of the mouse 13H11 antibody are SEQ ID NO: 41, SEQ ID NO: 42, and SEQ ID NO: 43, respectively.

[00254] The nucleic acid sequence of the light chain variable region of the mouse 14C1 antibody is SEQ ID NO: 112, and the amino acid sequence is SEQ ID NO: 113. The amino acid sequences of CDR1, CDR2, and CDR3 in the light chain variable region of the mouse 14C1 antibody are SEQ ID NO: 41, SEQ ID NO: 42, and SEQ ID NO: 43, respectively.

3. Preparation of the 11G9.6 chimeric antibody expression vector 3-1. Cloning of cDNA encoding the human Ig constant region

[00255] From the total RNA of human PBMC, cDNAs of the human IgG1 heavy chain constant region and the human IgG capla light chain constant region were cloned, and cloned with pCR4Blunt-TOPO vector, respectively using a commercially available kit “Zero Blunt TOPO PCR Cloning Kit” (manufactured by the company Invitrogen, catalog No.: 1325137) according to the instruction attached to the kit, and transformed into a competent Escherichia coli cell to obtain an Escherichia coli transformant. A plasmid was obtained from this transformant, and the plasmid DNA sample was sent for sequence analysis to Operon Biotechnology Co. Ltd (Tokyo), and the cDNA base sequence in the plasmid was confirmed.

3-2. Preparation of cDNA encoding the heavy chain of the chimeric PLD4 antibody

[00256] The cDNA encoding the heavy chain of a chimeric PLD4 antibody was prepared by ligation with the pEE6.4 expression vector that has the heavy chain variable region of the mouse 11G9.6 antibody obtained in 2-2, and the region human IgG1 heavy chain constant. The heavy chain variable region of the mouse 11G9.6 antibody was amplified with the PCR method, and a PCR product of length about 450 bases was obtained. At this time, the primer is as shown in Table 1. The obtained PCR product was purified by the 1.5% low melting point agarose method. Table 1

[00257] The heavy chain variable region of the mouse antibody 11G9.6 obtained in 2-2 was individualized for the PCR method to obtain “PCR product encoding the heavy chain variable region of 11G9.6”. The PCR product encoding the 11G9.6 heavy chain variable region was digested with the restriction enzymes Hind III and Apa I, and purified with the 1.5% agarose gel method. This was dissolved in ddH2O, which was collected as a solution of a cDNA fragment encoding the heavy chain variable region.

[00258] The cDNA obtained was amplified with PCR from the plasmid clone pCR4Blunt-TOPO containing the VH region of 11G9.6, using the primers chi11G9VH-SE (Hind III) and chi11G9VL-408R, in which the restriction sites (Hind III and Apa I) preferred for cloning the chimeric 11G9.6 VH coding region into the pEE6.4 vector (manufactured by Lonza Biologics, Slough, UK), and the optimal Kozak sequence (GCCGCCACC) were introduced as the cloning sites of Hind III and Apa I. The Chi11G9VH-pEE6.4 vector contains the constant region of the human IgG1 heavy chain. The VH PCR fragment was inserted into the pEE6.4 vector with an in-frame using Hind III and Apa I. The construct was investigated by cDNA base sequence analysis. For sequence analysis, the plasmid DNA sample was sent to Operon Biotechnology Co. Ltd (Tokyo), and the cDNA base sequence in the plasmid was confirmed.

3.3 Preparation of cDNA encoding chimeric PLD4 antibody light chain

[00259] In order to prepare cDNA encoding the light chain of the chimeric PLD4 antibody, the light chain variable region of the mouse 11G9.6 antibody obtained in 2-3, and the constant region of the human Ig kappa light chain obtained in 3-2 were fused to give a PCR fragment, and the PCR fragment was amplified to a PCR product of about 730 bases in length by a method based on an overlap extension PCR method.

[00260] The PCR product encoding the 11G9.6 light chain variable region was digested with Hind III and EcoRI restriction enzymes, and purified with the 1.5% agarose gel method. This was dissolved in ddH2O, which was collected as a solution of a cDNA fragment encoding the light chain variable region.

[00261] The cDNA encoding VL obtained from chimeric 11G9 was amplified with PCR from the plasmid clone pCR4Blunt-TOPO containing the VL 11G9.6 region, using primers chi11G9VL-SE (Hind) and chi11G9VL-726R (R I), in which the restriction sites (Hind III and EcoR I) preferred for cloning the pEE14.4 vector (manufactured by Lonza Biologics), and optimal Kozak sequence were introduced. The Chi11G9VL-pEE14.4 vector contains the cap light chain constant region. The VL PCR fragment was inserted into the pEE14.4 vector with an in-frame using Hind III and EcoR I. The construct was investigated by cDNA base sequence analysis.

3.4 Construction of chimeric PLD4 antibody dual gene Lonza expression vector (ch11G9VH/VL)

[00262] The chimeric PLD4 antibody Lonza expression vector (chi11G9DG vector) combined into a gene 2 vector was constructed from the chimeric PLD4 antibody heavy chain expression vector (chi11G9VH-pEE6.4), and chain vector light chimeric PLD4 antibody (chi11G9VL-pEE14.4) by standard cloning technology.

4. Transient expression in 293F cell

[00263] 80 µg of Lonza chillG9DG vector DNA, a transient plasmid expression vector, was used.

[00264] 293F cells were combined at 80 ml at 8 x 105 cells/ml in a 250 ml Erlenmeyer flask (catalog No. 431144; manufactured by CORNING) on the day before transfection, and cultured under conditions of 37 ° C and 8% of CO2 concentration for 7 days with agitation.

[00265] After culturing for 7 days, the culture liquid from the transfected 293F cell was collected into a 50 ml tube, and the centrifuge was performed under the conditions of 2,070 g at 4 °C for 5 minutes. The supernatant was filtered with a syringe filter having 0.45 µm pore size (catalog No. 431220; manufactured by CORNING), and the culture supernatant was collected for antibody purification.

5. Purification of anti-PLD4 chimeric antibody

[00266] Using the collected culture supernatant, antibody purification was performed using AKTA-FPLC (manufactured by GE Healthcare Japan) and Unicorn 5.0 software.

[00267] As the column for the purification of chimeric 11G9.6 antibody, HiTrap MabSelect SuRe 1 ml (catalog No. 11-0034-93, Lot No. 10032458; manufactured by GE Healthcare Japan) was used. The column conditions are as follows. Affinity purification was performed using a binding buffer (20 mM sodium phosphate, 0.15 M NaCl, pH 7.4) and an elution buffer (20 mM Sodium citrate, pH 3.4). In order to replace the antibody buffer after purification with PBS, buffer exchange was performed using the Slide-A-Lyzer MINI 10kMWCO dialysis unit.

[00268] The concentration of the purified antibody was calculated by measuring the absorbance at 280 nm, where 1 mg/ml was calculated as 1.38 OD.

[00269] For the purified anti-PLD4 chimeric antibody ch11G9.6Ab, the quality of the protein was analyzed using the Flow Cytometry and SDS-PAGE method. Example 8

[00270] The antibody-dependent cellular cytotoxicity (ADCC activity) of the prepared anti-human PLD4 chimeric antibody (Ab ch11G9.6) was measured. For activity, cell toxicity calculated from the measurement value of lactase dehydrogenase (LDH) released from the cell an index was used. The human peripheral blood mononuclear cell that became an effector cell was purified with specific gravity centrifuge using HISTOPAQUE-1077. As the cell to be a target, human PLD4 gene obligate transformed cell using a CHO-K1 (Chinese hamster ovary cell strain) cell strain (Hereinafter, HuPLD4-CHO) was used (2 x 104/well ). The effector and target cell were mixed in a ratio of 10:1, 20:1, 40:1, and 80:1, and added with 10 mg/ml of ch11G9Ab or isotype control antibody (human IgG1, K) , cultivated at 37 °C for 4 hours, and the effect of antibody cytotoxicity activity was evaluated. As a result thereof, ch11G9Ab of the anti-hPLD4 chimeric antibody exhibited maximum of about 50% or approximately ADCC activity for the HuPLD4-CHO cells that were the target, regardless of the effector cell (FIG. 22). Such results proved that the prepared anti-PLD4 chimeric antibody damaged cells that expressed PLD4 selectively.

[00271] The effects of the anti-PLD4 antibody on pDC were reviewed. Human peripheral blood PBMC was purified, mixed with 10 µg/ml chimeric anti-human PLD4 antibody and cultured for 24 hours. Then, cells were stimulated for 24 hours with CpG2216, which was a Toll-like receptor 9 ligand expressed on pDC, to induce IFNa production. After CpG stimulation, the amount of IFNa produced was tested, and it was confirmed that IFNa production was completely inhibited by the anti-PLD4 chimeric antibody ch11G9Ab treatment (FIG. 23). As for this mechanism, it was found that when cells were harvested 24 hours after ch11G9Ab treatment, and pDC cells were confirmed with triple staining of anti-CD123 antibody, anti-HLA—DR antibody and anti-Lineage 1 antibody, the pDC cell population decreased more than isotype control antibody treatment (human IgG1, K) (FIG. 24). These results indicated that the anti-PLD4 chimeric antibody damaged pDC that specifically expressed PLD4, and as a result thereof, the production of IFNa by stimulation of CpG2216 was inhibited.

[00272] In addition to ch11G9.6Ab, the biological function of chimeric anti-PLD4 Abs such as CH3B4Ab, ch5B7Ab, ch8C11Ab, ch10C3Ab, ch13D4Ab, ch13H11Ab were examined in human primary pDCs. In order to examine the ADCC assay for pDCs, whole human PBMC were cultured with CH3B4Ab, ch5B7Ab, ch8C11Ab, ch10C3Ab, ch13D4Ab, ch13H11Ab, ch11G9.6Ab, or isotype Ab for 14 h. Cells were harvested and stained with BDCA2 and BDCA4 to identify pDCs by flow cytometry. Treatment with the chimeric PLD4 Abs completely depleted pDCs compared to Ab isotype-treated PBMCs (FIG. 25). IFNa production was also measured in cultured PBMCs with the chimeric anti-PLD4 Abs. Whole human PBMC were treated with the CH3B4Ab, ch5B7Ab, ch8C11Ab, ch10C3Ab, ch13D4Ab, ch13H11Ab, ch11G9.6Ab, or isotype Ab. 24 h later, IFNα-inducible CpG2216 was added to the culture and the cells were further cultured for 24 h. Culture supernatants were collected and IFNa production was measured by ELISA. All of the PBMCs treated with chimeric PLD4 Ab completely abolished IFNa production compared to PBMCs treated with isotype Ab (FIG. 26). These results indicated that the chimeric anti-PLD4 Ab abolished pDC function as well as a large amount of IFNa production by depleting pDCs through ADCC activity. 1. The nucleic acid sequence of the heavy chain variable region of the obtained mouse anti-PLD4 antibody 11G9.6 is SEQ ID NO: 74, and the amino acid sequence is SEQ ID NO: 75. The amino acid sequences of CDR1, CDR2, and CDR3 in the heavy chain variable region of mouse antibody 11G9.6 are SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, respectively.

[00273] The nucleic acid sequence of the heavy chain variable region of the mouse anti-PLD4 antibody 11G9.6 (504 base pairs) [upper case: mouse variable region 11G9.6VH, lower case: heavy chain constant region of mouse IgG2b] (SEQ ID NO: 74) ATGAGATCACAG?CTCTATACAG?ACTGAGCACACAGAACCTCACC?GGGATGGAGCTGTATCATCCTC?C?GGT AGCAACAGCTACAGGTGTCCACTCCCAGGTCCAACTGCAGCAGCCTGGGGCTGAACTGGTGAAGCCTGGGACTTCAGTGA AAATGTCCTGCAAGGCTTCTGGCTACACCTTCACCAGCTACTGGATGC ACTGGGTGAAGCAGAGGCCGGGACAAGGCCp GAGTGGATTGGAGATATTTATCCTGGTAGTGATAGTACTAACTACAATGAGAAGTTCAAGAGCAAGGCCACACTGACTGT AGACACATCCTCCAGCACAGCCTACATGCAACTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGTGCAAGAG GAGGGTGGTTGGATGCTATGGACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAgcca aaacaacacccccatca gtctatccactggcccctaagggc

[00274] The amino acid sequence of the mouse 11G9.6 antibody heavy chain variable region (168 a.a.) [Upper case: mouse 11G9.6VH variable region, lower case: mouse IgG2b heavy chain constant region]. The underlined sequence represents a signal sequence, and the double underline represents regions of the CDR (CDR1, CDR2, and CDR3) (SEQ ID NO: 75). MRSOFSIOLLSTQNLTLGWSCIILFLVATATGVHSQVQLQQPGAELVKPGTSVKMSCK ASGYTFTSYWMHWVKORPGOGLEWIGDIYPGSDSTNYNEKFKSKATLTVDTSSSTA YMQLSST.TSEDSAVYYCARGGWLDAMDYWGOGTSVTVSSakttppswplapkg The CDR1 of the 11G9 antibody heavy chain variable region .6 SYWMH (SEQ ID NO: 2) The CDR2 of the antibody heavy chain variable region 11G9.6 DIYPGSDSTNYNEKFKS (SEQ ID NO: 3) The CDR3 of antibody heavy chain variable region 11G9.6 GGWLDAMDY (SEQ ID NO: 4)

[00275] The nucleic acid sequence of the light chain variable region of the mouse anti-PLD4 antibody 11G9.6 obtained is SEQ ID NO: 38, and the amino acid sequence is SEQ ID NO: 39. The amino acid sequences of CDR1, CDR2, and CDR3 in the light chain variable region of mouse antibody 11G9.6 are SEQ ID NO: 40, SEQ ID NO: 41, and SEQ ID NO: 42, respectively.

[00276] The nucleic acid sequence of the light chain variable region of mouse anti-PLD4 antibody 11G9.6 (421 base pairs) [Upper case: mouse 11G9.6VL variable region, lower case: constant chain region light mouse IgK] (SEQ ID NO: 94) ATGATGTCCTCTGCTCAGTTCCTTGGTCTCCTTGTTGCTCTGTTTTCAAGGTACCAGATGTGATATCCAGATGACACAGAC TACATCCTCCCTGTCTGCCTCTCTGGGAGACAGAGTCACCATCAGTTGCAGGGCAAGTCAGGACATTAGCAA?AT?AA ACTGGTATCAGCAGAAACCAGATGGAACTGTTAAACTCCTGATCTACTA CACATCAAGATTACACTCAGGAGTCCCATCA AGGTTCAGTGGCAGTGGGTCTGGAACAGATTATTCTCTCACCATTAGCAACCTGGAGCAAGAAGATATTGCCACTTACTT TTGCCAACAGGGTAAIACGCTTCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAAcgggctgatgctgcaccaa ctgtatccatcaagggcgaat

[00277] The amino acid sequence of the light chain variable region of the mouse 11G9.6 antibody (140 a.a.) [Upper case: mouse 11G9.6VL variable region, lower case: mouse IgK light chain constant region]. The underlined sequence represents a signal sequence, and the double underline represents the CDR regions (CDR1, CDR2, and CDR3) (SEQ ID NO: 95). MMSSAOFLGLLLLCFQGTRCDIQMTOTTSSLSASLGDRVTISCRASODISNYLNWYO OKPnGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTISNLEOEDIATYFCQOGNTLP WTFGGGTKLEIKradaaptvsikge CDR1 of the antibody light chain variable region 11G9.6 RASQDISNILN (SEQ ID NO: 5) region CDR2 antibody light chain variable 11G9.6 YTSRLHS (SEQ ID NO: 6) light chain variable region CDR3 of antibody 11G9.6 QQGNTLPW (SEQ ID NO: 7)

[00278] 2. The nucleic acid sequence of the heavy chain variable region of the obtained mouse anti-PLD4 antibody 3B4 is SEQ ID NO: 76, and the amino acid sequence is SEQ ID NO: 77. The amino acid sequences of CDR1, CDR2, and CDR3 in the heavy chain variable region of the mouse 3B4 antibody are SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10, respectively.

[00279] The nucleic acid sequence of the mouse anti-PLD4 antibody 3B4 heavy chain variable region (437 base pairs) [Upper case: mouse 3B4VH variable region, lower case: IgG1 heavy chain constant region of mouse] ATGGAATGTAACTGGATACTTCCTTTTATTCTGTCGGTAATTTCAGGGGTCTCCTCAGAGGTTCAGCTCCAGCAGTCTGG GACTGTGCTGTCAAGGCCTGGGGCTTCCGTGACGATGTCCTGCAAGGCTTCTGGCGACAGCTTTACCACCTACTGGATGC ACTGGGTAAAACAGAGGCCTGGACAGGGTCTAGAATGGATTGGTGCTATCTATCCTGGAAATAGTGAAACTAGCTACAAC CAGAAGTTCAAGGGCAAGGCCAAACTGACTGCAGTCACATCCGCCAGCACTGCCTATATGGAGTTCACTAGCCTGACAAA TGAGGACTCTGCGGTCTATTACTGTACGGGGGGTTATTCCGACTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCT CCTCAgccaaaacgacacccccatctgtctatccact

[00280] The amino acid sequence of the heavy chain variable region of the mouse 3B4 antibody (145 a.a.) [Upper case: mouse 3B4VH variable region, lower case: mouse IgG1 heavy chain constant region]. The underlined sequence represents a signal sequence, and the double underline represents the CDR regions (CDR1, CDR2, and CDR3). MECNWILPFILSVISGVSSEVOLOOSGTVLSRPGASVTMSCKASGDSFTTYWMHWVK QRPGQGLEWIGAIYPGNSETSYNOKFKGKAKLTAVTSASTAYMEFTSLTNEDSAVYY CTGGYSDFDYWGQGTTLTVSSakttppsvyp 3B4 antibody heavy chain variable region CDR1 TYWMH 3B4 antibody heavy chain variable region CDR2 AIYPGNSETSYNQKFKG CDR3 of antibody heavy chain variable region 3B4 GYSDFDY

[00281] The nucleic acid sequence of the light chain variable region of the mouse anti-PLD4 antibody 3B4 obtained is SEQ ID NO: 96, and the amino acid sequence is SEQ ID NO: 97. The amino acid sequences of CDR1 , CDR2, and CDR3 in the light chain variable region of the mouse 3B4 antibody are SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13, respectively.

[00282] The nucleic acid sequence of the mouse anti-PLD4 antibody 3B4 light chain variable region (459 base pairs) [Upper case: mouse 3B4VL variable region, lower case: mouse IgK light chain constant region] ATGATGGTCCTTGCTCAGTTTCTTGCATTCTTGTTGCT?GG?TCCAGGTGCAGGATGTGACATCCTGATGACCCAATC TCCATCCTCCATGTCTGTATCTCTGGGAGACACAGTCAGCATCACTTGCCATGCAAGTCAGGGCATTAGAAGTAATATAG GGTGGTTGCAGCAGAAACCAGGGAAATCATTTAAGGGCCTGATCTTTCATGGAACCAACTTGGAAGATGGAGTTCCATCA AGGTTCAG TGGCAGAGGATCTGGAGCAGATTATTCTCTCACCATCAACAGCCTGGAATCTGAAGATTTTGCAGACTATTA CTGTGTACAGTATGTTCAGTTTCCTCCAACGTTCGGCTCGGGGACAAAGTTGGAAATAAGAcgggctgatgctgcaccaa ctgtatccatcttcccaccatccagtgagcagttaacatctggaggtgcctcagtcgtg

[00283] The amino acid sequence of the light chain variable region of the mouse 3B4 antibody (153 a.a.) [Upper case: mouse 3B4VL variable region, lower case: mouse IgK light chain constant region]. The underlined sequence represents a signal sequence, and the double underline represents regions of the CDR (CDR1, CDR2, and CDR3). MMVLAQFLAFLLLWFPGAGCDILMTOSPSSMSVSLGDTVSITCHASQGIRSNIGWLO QKPGKSFKGTJFHGTNLEDGVPSRFSGRGSGADYSLTINSLESEDFADYYCVOYVQFP PTFGSGTKLEIRradaaptvsifppsseqltsggasw CDR1 of the 3B4 antibody light chain variable region HASQGIRSNIG CDR2 of the 3B4 antibody light chain variable region antibody 3B4 HGTNLED CDR3 light chain variable region antibody 3B4 VQYVQFP

[00284] 3. The nucleic acid sequence of the heavy chain variable region of the obtained mouse anti-PLD4 antibody 5B7 is SEQ ID NO: 78, and the amino acid sequence is SEQ ID NO: 79. The amino acid sequences of CDR1, CDR2, and CDR3 in the heavy chain variable region of mouse 5B7 antibody are SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16, respectively.

[00285] The nucleic acid sequence of the heavy chain variable region of the mouse anti-PLD4 antibody 5B7 (475 base pairs) [Upper case: mouse 5B7VH variable region, lower case: mouse IgG2b heavy chain constant region] ATGGGATGGAGCTGGATCTTTCTCTTCCTCCTGTCAGGAACTGCAGGCGTCCACTCTGAGGTCCAGCTTCAGCAGTCAGG ACCTGAACTGGTGAAACCTGGGGCCTCAGTGAAGATATCCTGCAAGGCTTCTGGATACACATTCACTGACTACAACTTGC ACTGGGTGAAGCAGAGCCATGGAAAGAGCCTTGAGTGGATTGGATATATTTATCCTTACAATGGTAATACTGGCTACAAC CAGAAGTTCAA GAGGAAGGCCACATTGACTGTAGACAATTCCTCCGGCACAGTCTACATGGÁGCTCCGCAGCCTGACATC TGAGGACTCTGCAGTCTATTACTGTGCAAGAGGAGGGATCTATGATGATTACTACGACTATGCTATCGACTATIGGGGTC AAGGAACCTCAGTCACCGTCTCCTCAgccaaaacaacacccccatcagtctatccactggcccctaagggcgaat

[00286] The amino acid sequence of the heavy chain variable region of the mouse 5B7 antibody (158 a.a.) [Upper case: mouse 5B7VH variable region, lower case: mouse IgG2b heavy chain constant region]. The underlined sequence represents a signal sequence, and the double underline represents regions of the CDR (CDR1, CDR2, and CDR3). MGWSWIFLFLLSGTAGVHSEVOLOQSGPELVKPGASVKISCKASGYTFTDYNLHWV KOSHGKSLEWIGYIYPYNGNTGYNQKFKRKATLTVDNSSGTVYMELRSLTSEDSAV YYCARGGIYDDYYDYAJDYWGOGTSVTVSSakttppsvyplapkge CDR1 of the antibody heavy chain variable region 5B7 DYNLH C DR2 of the heavy chain variable region of the antibody 5B7 YIYPYNGNTGYNQKFKR CDR3 of the heavy chain variable region of the antibody 5B7 GGIYDDYYDYAIDY

[00287] The nucleic acid sequence of the light chain variable region of the mouse anti-PLD4 antibody 5B7 obtained is SEQ ID NO: 98, and the amino acid sequence is SEQ ID NO: 99. The amino acid sequences of CDR1 , CDR2, and CDR3 in the light chain variable region of the mouse 5B7 antibody are SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19, respectively.

[00288] The nucleic acid sequence of the light chain variable region of the mouse anti-PLD4 antibody 5B7 (467 base pairs) [Upper case: mouse 5B7VL variable region, lower case: mouse IgK light chain constant region] ATGAGTGTGCCCACTCAGGTCCTGGGGTTGCTGCTGCTGTGGCTTACAGATGCCAGATGTGACATCCAGATGACTCAGTC TCCAGCCTCCCTATCTGTATCTGTGGGAGAAACTGTCGCCATCACATGTCGAGCAAGTGAGAATATTTACAGTCATATAG CATGGTATCAGCAGAAAGAGGGAAAATCTCCTCAGCGCCTGGTCTATGGTGCAACAAACTTAGCACATGGTGTGCCATCA AGGTTCAGTGG CAGTGGATCAGGCACACAGTATTCCCTCAAGATCAACAGCCTTCAGTCTGAAGATTTTGGGAGTTATTA CTGTCAACATTTTTGGGGTACTCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCÂAAcgggctgatgctgcaccaa ctgtatccatcttcccaccatccagtgagcagttaacatctggaggtgcctcagtcgtgtgcttctt

[00289] The amino acid sequence of the light chain variable region of the mouse 5B7 antibody (155 a.a.) [Upper case: mouse 5B7VL variable region, lower case: mouse Igk light chain constant region]. The underlined sequence represents a signal sequence, and the double underline represents regions of the CDR (CDR1, CDR2, and CDR3). MSVPTOVLGLLLLWLTDARCDIOMTOSPASLSVSVGETVAITCRASENIYSHIAWYO QKEGKSPQRLVYGATNLAHGVPSRFSGSGSGTOYSLKINSLOSEDFGSYYCOHFWGT PWTFGGGTKLEIKradaaptvsifppsseqltsggaswcf CDR1 of the antibody light chain variable region 5B7 RASENIYSHIA CDR2 of the antibody light chain variable region 5B7 GATNLAH CDR3 light chain variable region of 5B7 QHFWGTP antibody

[00290] 4. The nucleic acid sequence of the heavy chain variable region of the obtained mouse anti-PLD4 antibody 7B4 is SEQ ID NO: 80, and the amino acid sequence is SEQ ID NO: 81. The amino acid sequences of CDR1, CDR2, and CDR3 in the heavy chain variable region of mouse 7B4 antibody are SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16, respectively.

[00291] The nucleic acid sequence of the mouse anti-PLD4 antibody 7B4 heavy chain variable region (470 base pairs) [Upper case: mouse 7B4VH variable region, lower case: mouse IgG2b heavy chain constant region] ATCGGATGGAGCTGGAT? TTCAAGAGGAAGGCCACATTGACTGTAGACAATTCCTCCGGCACAGTCTACATGGAGCTCCGCAGCCTGACATC TGAGGACTCTGCAGTCTATTACTGTGCAAGAGGAGGGATCTATGATGATTACTACGACTATGCTATCGACTATTGGGGTC AAGGAACCTCAGTCACCGTCTCCTCAgccaaaacaacacccccatcagtctatccactggcccctaaggg

[00292] The amino acid sequence of the heavy chain variable region of the mouse 7B4 antibody (156 a.a.) [Upper case: mouse 7B4VH variable region, lower case: mouse IgG2b heavy chain constant region]. The underlined sequence represents a signal sequence, and the double underline represents the CDR regions (CDR1, CDR2, and CDR3). MGWSWIFLFLLSGTAGVHSEVOLOOSGPELVKPGASVKISCKASGYTFTDYNLHWV KQSHGKSLEWIGYIYPYNGNTGYNOKFKRKATLTVDNSSGTVYMELRSLTSEDSAV YYCARGGIYDDYYDYAIDYWGOGTSVTVSSakttoDSWDlaDk CDR1 heavy chain variable region of antibody 7B4 DYNLH CDR2 region 7B4 GGIYDDYYDYAIDY Antibody Heavy Chain Variable Region Antibody 7B4 YIYPYNGNTGYNQKFKR CDR3 Heavy Chain Variable

[00293] The nucleic acid sequence of the light chain variable region of the mouse anti-PLD4 antibody 7B4 obtained is SEQ ID NO: 100, and the amino acid sequence is SEQ ID NO: 101. The amino acid sequences of CDR1 , CDR2, and CDR3 in the light chain variable region of the mouse 7B4 antibody are SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19, respectively.

[00294] The nucleic acid sequence of the light chain variable region of the mouse anti-PLD4 antibody 7B4 (454 base pairs) [Upper case: mouse 7B4VL variable region, lower case: mouse Igk light chain constant region ] ATGAGTGTGCCCACTCAGGTCCTGGGGTTGCTGCTGCTGTGGCTTACAGATGCCAGATGTGACATCCAGATGACTCAGTC TCCAGCCTCCCTATCTGTATCTGTGGGAGAAACTGTCGCCATCACATGTCGAGCAAGTGAGAATATTTACAGTCATATAG CATGGTATCAGCAGAAAGAGGGAAAATCTCCTCAGCGCCTGGTCTATGGTGCAACAAACTTAGCACATGGTGTGCCATCA AGGTTCAGT GGCAGTGGATCAGGCACACAGTATTCCCTCAAGATCAACAGCCTTCAGTCTGAAGATTTTGGGAGTTATTA CTGTCAACATTTTTGGGGTACTCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAAcgggctgatgctgcaccaa ctgtatccatcttcccaccatccagtgagcagttaacatctggaggtgcctcag

[00295] The amino acid sequence of the light chain variable region of the mouse 7B4 antibody (151 a.a.) [Upper case: mouse 7B4VL variable region, lower case: mouse Igk light chain constant region]. The underlined sequence represents a signal sequence, and the double underline represents regions of the CDR (CDR1, CDR2, and CDR3). MSVPTOVLGLLLLWLTDARCDIOMTOSPASLSVSVGETVAITCRASENIYSHIAWYO OKEGKSPORLVYGATNLAHGVPSRFSGSGSOTOYSLKINSLQSEDFGSYYCOHFWGT PWTFGGGTKLEIKradaaptvsifppsseqltsggas CDR1 of the light chain variable region of antibody 7B4 RASENIYSHIA CDR2 of the light chain variable region of antibody 7 B4 GATNLAH CDR3 of the light chain variable region of the 7B4 QHFWGTP antibody

[00296] 5. The nucleic acid sequence of the heavy chain variable region of the obtained mouse anti-PLD4 antibody 8C11 is SEQ ID NO: 82, and the amino acid sequence is SEQ ID NO: 83. The amino acid sequences of CDR1, CDR2, and CDR3 in the heavy chain variable region of mouse 8C11 antibody are SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22, respectively.

[00297] The nucleic acid sequence of the heavy chain variable region of the mouse anti-PLD4 antibody 8C11 (462 base pairs) [Upper case: mouse 8C11VH variable region, lower case: mouse IgG2b heavy chain constant region] ATGGGATGGAGCTATATCATCCTCTTTTTGGTAGCAACAGCAACAGGGGTCCACTCCCAGGTCCAACTGCAGCAGTCGGG GGCTGAACTGGTGAAGCCTGGGGCTTCAGTGAAGTTGTCCTGCAAGGCTTCTGGCTACACCTTCACCAGCTACTATTTGT ACTGGGTGAGGCAGAGGCCTGGACAAGGCCTTGAGTGGATTGGACTGATTAATCCTACCAATAGTGATACTATCTTCAAT GAGAAGTT CAAGAGCAAGGCCACACTGACTGTAGACAAATCCTCCAGCACAGCATACATGCAACTCAGCAGCCTGACATC TGAGGACTCTGCGGTCTATTACTGTACACGAGAGGGGGGATATGGTTACGGCCCGTTTGCTTACTGGGGCCAAGGGACTC TGGTCACTGTCTCTGCAgccaaaacaacacccccatcagtctatccactggcccctaagggc

[00298] The amino acid sequence of the heavy chain variable region of the mouse 7B4 antibody (154 a.a.) [Upper case: mouse 8C11VH variable region, lower case: mouse IgG2b heavy chain constant region]. The underlined sequence represents a signal sequence, and the double underline represents regions of the CDR (CDR1, CDR2, and CDR3). MGWSYIILFLVATATGVHSOVOLOOSGAELVKPGASVKLSCKASGYTFTSYYLYWV RORPGOGLEWIGLINPTNSDTIFNEKFKSKATLTVDKSSSTAYMQLSSLTSEDSAVYY CTREGGYGYGPFAYWGOGTLVTVSAakttDDSWDlapkg CDR1 of the heavy chain variable region of the antibody 8C11 SYILY CDR2 of the heavy chain variable region 8C11 EGGYGYGPFAY Antibody Heavy Chain Variable Region Antibody 8C11 LINPTNSDTIFNEKFKS CDR3

[00299] The nucleic acid sequence of the light chain variable region of the mouse anti-PLD4 antibody 8C11 obtained is SEQ ID NO: 102, and the amino acid sequence is SEQ ID NO: 103. The amino acid sequences of CDR1 , CDR2, and CDR3 in the light chain variable region of mouse 8C11 antibody are SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, respectively.

[00300] The nucleic acid sequence of the light chain variable region of the mouse anti-PLD4 antibody 8C11 (457 base pairs) [Upper case: mouse 8C11VL variable region, lower case: mouse Igk light chain constant region ] ATGAAGTTGCCTGTTAGGCTGTTGGTGCTGATGTTCTGGATTCCTGCTTCCAGCAGTGATGTTGTGATGACCCAAACTCC ACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCACATCTAGTCAGACCCTTGTACACAGTAATGGAA ACACCTAT?ACATTGGTACCTGCAGAAGCCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTTTCCAACCGATTTTCT GGGGTCCCA GACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCT GGGAGTTTATTTCTGCTCTCACAGTACACATGTTCCATTCACGTTCGGCTCGGGGACAAAGTTGGAAATAAAAcgggctg atgctgcaccaactgtatccatcttcccaccatccagtgagcagttaacatctggag

[00301] The amino acid sequence of the light chain variable region of the mouse 8C11 antibody (152 a.a.) [Upper case: mouse 8C11VL variable region, lower case: mouse Igk light chain constant region]. The underlined sequence represents a signal sequence, and the double underline represents regions of the CDR (CDR1, CDR2, and CDR3). MKLPVRLLLVLMFWIPASSSDVVMTOTPLSLPVSLGDOASISCTSSOTLVHSNGNTYLH WYLQKPGOSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSHST HVPFTFGSGTKLEIKradaantvsifppssealtsg CDR1 of the light chain variable region of the 8C11 antibody TSSQTLVHSNGNTILH CDR2 of the variable region of 8C11 HSTHVP Antibody Light Chain Variable Region 8C11 KVSNRFS CDR3 Light Chain

[00302] 6. The nucleic acid sequence of the heavy chain variable region of the obtained mouse anti-PLD4 antibody 10C3 is SEQ ID NO: 84, and the amino acid sequence is SEQ ID NO: 85. The amino acid sequences of CDR1, CDR2, and CDR3 in the heavy chain variable region of mouse 10C3 antibody are SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 28, respectively.

[00303] The nucleic acid sequence of the mouse anti-PLD4 antibody 10C3 heavy chain variable region (450 base pairs) [Upper case: mouse 10C3VH variable region, lower case: mouse IgG2a heavy chain constant region ] ATGAACTTCGGGCTCAGCTTGATTTTCCTTGCCCTCATTTTAAAAGGTGTCCAGTGTGAGGTGCAGCTGGTGGAGTCTGG GGGAGACTTAGTGAGGCCTGGAGGGTCCCTGAAACTCTCCTGTGCAGCCTCTGGATTCAGTTTCAGTAGCTATGGCATGT CTTGGTTTCGCCAGACTCCAGACAAGAGGCTGGAGTGGGTCGCAACCATTAGTAGTGGTGGTAGTTACATCTACTATCCA GAAAGTGTGA AGGGGCGAITCACCATCTCCAGAGACAATGCCAGGAACATCCTGTACCTGCAAATGAGCAGTCTGAAGTC TGAGGACACGCCATGTATTATTGTGTAAGACTCTACGGTGGTAGGAGAGGCTATGGTTTGGACTACTGGGGTCAAGGAA CCTCAGTCACCGTCTCCTCAgccaaaacaacagccccatcggtctatcca

[00304] The amino acid sequence of the heavy chain variable region of the mouse 10C3 antibody (150 a.a.) [Upper case: mouse 10C3VH variable region, lower case: mouse IgG2a heavy chain constant region]. The underlined sequence represents a signal sequence, and the double underline represents regions of the CDR (CDR1, CDR2, and CDR3). MNFGLSLIFLALILKGVQCEVOLVESGGDLVRPGGSLKLSCAASGFSFSSYGMSWFRQ TPDKRLEWVATISSGGSYIYYPESVKGRFTISRDNARNILYLOMSSLKSEDTAMYYCV RLYGGRRGYGLDYWGOGTSVTVSSakttansvvn CDR1 of antibody heavy chain variable region 10C3 SYGMS CDR2 of antibody heavy chain variable region 10 10C3 LYGGRRGYGLDY Antibody Heavy Chain Variable Region C3 TISSGGSYIYYPESVKG CDR3

[00305] The nucleic acid sequence of the light chain variable region of the mouse anti-PLD4 antibody 10C3 obtained is SEQ ID NO: 104, and the amino acid sequence is SEQ ID NO: 105. The amino acid sequences of CDR1 , CDR2, and CDR3 in the light chain variable region of the mouse 10C3 antibody are SEQ ID NO: 29, SEQ ID NO: 30, and SEQ ID NO: 31, respectively.

[00306] The nucleic acid sequence of the light chain variable region of the mouse anti-PLD4 antibody 10C3 (423 base pairs) [Upper case: mouse 10C3VL variable region, lower case: mouse Igk light chain constant region] ATGAGGTTCTCTGCTCAGCTTCTGGGGCTGCTTGTGCTCTGGATCCCTGGATCCACTGCGGAAATTGTGATGACGCAGGC TGCATTCTCCAATCCAGTCACTCTTGGAACATCAGCTTCCATCTCCTGCAGGTCTAGTAAGAGTCTCCTACATAGTGATG GCATCACTTATTTGTATTGGTATCTGCAGAAGCCAGGCCAGTCTCCTCAGCTCCTGATTTATCAGATGTCCAACCTTGCC TCAGG AGTCCCAGACAGGTTCAGTAGCAGTGGGTCAGGAACTGATTTCACACTGAGAATCAGCAGAGTGGAGGCTGAGGA TGTGGGTTTATTACTGTGCTCAAAATCTAGAACTTTACACGTTCCGGAGGGGGGACCAAGCTGGAAATAAAAcgggctg atgctgcaccaactgtatccatc

[00307] The amino acid sequence of the light chain variable region of the mouse 10C3 antibody (141 a.a.) [Upper case: mouse 10C3VL variable region, lower case: mouse Igk light chain constant region]. The underlined sequence represents a signal sequence, and the double underline represents regions of the CDR (CDR1, CDR2, and CDR3). MRFSAQLLGLLVLWIPGSTAEIVMTOAAFSNPVTLGTSASISCRSSKSLLHSDGITYLY WYLOKPGOSPOLLIYOMSNLASGVPDRFSSSGSGTDFTLRISRVEAEDVGVYYCAON LELYTFGGGTKLEIKradaaptvsi CDR1 of the light chain variable region of the 10C3 antibody RSSKSLLHSDGITILY CDR2 of the light chain variable region of the 10C3 antibody QMSNLAS CDR3 of the light chain variable region of the 10C3 AQNLEL antibody

[00308] 7. The nucleic acid sequence of the heavy chain variable region of the obtained mouse anti-PLD4 antibody 11D10 is SEQ ID NO: 86, and the amino acid sequence is SEQ ID NO: 87. The amino acid sequences of CDR1, CDR2, and CDR3 in the heavy chain variable region of the mouse 11D10 antibody are SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 28, respectively.

[00309] The nucleic acid sequence of the heavy chain variable region of the mouse anti-PLD4 antibody 11D10 (450 base pairs) [Upper case: mouse 11D10VH variable region, lower case: mouse IgG2b heavy chain constant region] ATGAACTTCGGGCTCAGCTTGATTTTCCTTGCCCTCATTTTAAAAGGTGTCCAGTGTGAGGTGCAGCTGGTGGAGTCTGG GGGAGACTTAGTGAGGCCTGGAGGGTCCCTGAAACTCTCCTGTGCAGCCTCTGGATTCAGTTTCAGTAGCTATGGCATGT CTTGGTTTCGCCAGACTCCAGACAAGAGGCTGGAGTGGGTCGCAACCATTAGTAGTGGTGGTAGTTACATCTACTATCCA GAAAGTGTGAA GGGGCGATTCACCATCTCCAGAGACAATGCCAGGAACATCCTGTACCTGCAAATGAGCAGTCTGAAGTC TGAGGACACGCCATGTATTATTGTGTAAGACTCTACGGTGGTAGGAGAGGCTATGGT?GGACTACTGGGGTCAAGGAA CCTCAGTCACCGTCTCCTCAgccaaaacaacacccccatcagtctatcca

[00310] The amino acid sequence of the heavy chain variable region of the mouse 11D10 antibody (150 a.a.) [Upper case: mouse 11D10VH variable region, lower case: mouse IgG2b heavy chain constant region]. The underlined sequence represents a signal sequence, and the double underline represents regions of the CDR (CDR1, CDR2, and CDR3). MNFGLSLIFLALILKGVQCEVOLVESGGDLVRPGGSLKLSCAASGFSFSSYGMSWFRO TPDKRLEWVATISSGGSYIYYPESVKGRFTISRDNARNILYLOMSSLKSEDTAMYYCV RLYGGRRGYGLDYWGOGTSVTVSS akttppsvyp CDR1 of the antibody heavy chain variable region 11D10 SYGMS CDR2 of the antibody heavy chain variable region 11D10 LYGGRRGYGLDY Antibody Heavy Chain Variable Region 11D10 TISSGGSYIYYPESVKG CDR3

[00311] The nucleic acid sequence of the light chain variable region of the mouse anti-PLD4 antibody 11D10 obtained is SEQ ID NO: 106, and the amino acid sequence is SEQ ID NO: 107. The amino acid sequences of CDR1 , CDR2, and CDR3 in the light chain variable region of the mouse 11D10 antibody are SEQ ID NO: 29, SEQ ID NO: 30, and SEQ ID NO: 31, respectively.

[00312] The nucleic acid sequence of the light chain variable region of the mouse anti-PLD4 antibody 11D10 (423 base pairs) [Upper case: mouse 11D10VL variable region, lower case: mouse Igk light chain constant region ] ATGAGGTTCTCTGCTCAGCTTCTGGGGCTGCTTGTGCTCTGGATCCCTGGATCCACTGCGGAAATTGTGATGACGCAGGC TGCATTCTCCAATCCAGTCACTCTTGGAACATCAGCTTCCATCTCCTGCAGGTCTAGTAAGAGTCTCCTACATAGTGATG GCATCACTTATTTGTATTGGTATCTGCAGAAGCCAGGCCAGTCTCCTCAGCTCCTGATTTATCAGATGTCCAACCTTGCC TCA GGAGTCCCAGACAGGTTCAGTAGCAGTGGGICAGGAACTGATTTCACACTGAGAATCAGCAGAGTGGAGGCTGAGGA TGTGGGTTTATTACTGTGCTCAAAATCTAGAACTTTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAAcgggctg atgctgcaccaactgtatccatc

[00313] The amino acid sequence of the light chain variable region (141 a.a.) of the mouse 11D10 antibody [Upper case: mouse 11D10VL variable region, lower case: mouse Igk light chain constant region]. The underlined sequence represents a signal sequence, and the double underline represents regions of the CDR (CDR1, CDR2, and CDR3). MRFSAOLLGLLVLWIPGSTAEIVMTOAAFSNPVTLGTSASISCRSSKSLLHSDGITYLY WYLQKPGQSPQLLIYOMSNLASGVPDRFSSSGSGTDFTLRISRVEAEDVGVYYCAON LELYTFGGGTKLEIKradaaptvsi CDR1 of antibody light chain variable region 11D10 RSSKSLLHSDGITILY CDR2 of antibody light chain variable region 1 1D10 QMSNLAS CDR3 of 11D10 AQNLEL Antibody Light Chain Variable Region

[00314] 8. The nucleic acid sequence of the heavy chain variable region of the obtained mouse anti-PLD4 antibody 13D4 is SEQ ID NO: 88, and the amino acid sequence is SEQ ID NO: 89. The amino acid sequences of CDR1, CDR2, and CDR3 in the heavy chain variable region of mouse 13D4 antibody are SEQ ID NO: 32, SEQ ID NO: 33, and SEQ ID NO: 34, respectively.

[00315] The nucleic acid sequence of the heavy chain variable region of the mouse anti-PLD4 antibody 13D4 (472 base pairs) [Upper case: mouse 13D4VH variable region, lower case: mouse IgG2b heavy chain constant region] ATGAAAGTGTTGAGTCTGTTGTACCTGTTGACAGCCATTCCTGGTATCCTGTCTGATGTACAGCTTCAGGAGTCAGGACC TGGCCTCGTGAAACCTTCTCAATCTCTGTCTCTCACCTGCTCTGTCACTGGCTACTCCATCACCAGTCATTATTACTGGA CCTGGATCCGGCAGTTTCCAGGAAACAAACTGGAATGGATGGGCTACATAAGCTACGACGGTAGCAATAACTACAACCCA TCTCTCAAAAATCGA ATCTCCATCACTCGTGACACATCTAAGAACCAGTITTTCCTGAAGTTGAATTCTGTGACTACTGA GGACACAGCTACATATAACTGTGCAAGAGAGGGCCGCTCTACTATGGTAACCCCTACTGGTATTTCGATGTCTGGGGCG CAGGGACCAÇGGTCACCGTCTCCTCAgccaaaacaacacccccatcagtctatccactggcccctaagggcg

[00316] The amino acid sequence of the heavy chain variable region of the mouse 13D4 antibody (157 a.a.) [Upper case: mouse 13D4VH variable region, lower case: mouse IgG2b heavy chain constant region]. The underlined sequence represents a signal sequence, and the double underline represents regions of the CDR (CDR1, CDR2, and CDR3). MKVLSLLYLLTAIPGILSDVOLOESGPGLVKPSOSLSLTCSVTGYSITSHYYWTWTRQF PGNKLEWMGYISYDGSNNYNPSLKNRISITRDTSKNOFFLKLNSVTTEDTATYNCAR EGPLYYGNPYWYFDVWGAGTTVTVSSakttDDswDlaükg CDR1 of the heavy chain variable region of the antibody 13D4 SHYYWT CDR2 of the variable region of 13D4 EGPLYYGNPYWYFDV Antibody Heavy Chain Variable Region Antibody 13D4 YISYDGSNNYNPSLKN CDR3 Heavy Chain

[00317] The nucleic acid sequence of the light chain variable region of the mouse anti-PLD4 antibody 13D4 obtained is SEQ ID NO: 108, and the amino acid sequence is SEQ ID NO: 109. The amino acid sequences of CDR1 , CDR2, and CDR3 in the light chain variable region of the mouse 13D4 antibody are SEQ ID NO: 35, SEQ ID NO: 36, and SEQ ID NO: 37, respectively.

[00318] The nucleic acid sequence of the light chain variable region of the mouse anti-PLD4 antibody 13D4 (404 base pairs) [Upper case: mouse 13D4VL variable region, lower case: mouse Igk light chain constant region ] ATGATGTCCTCTGCTCAGTTCCTTGGTCTCCTGTTGCTCTGTTTTCAAGGTACCAGATGTGATATCCAGATGACACAGAC TACATCCTCCCTGTCTGCCTCTCTGGGGGACAGAGTCACCATCAGTTGCAGGGCAAGTCAGGACATTGACAATTATTTAA ACTGGTATCAGCAGAAACCAGATGGAACTGTTAAACTCCTGATCTACTACACATCAAGATTACACTCAGGAGTCCCATCA AGG?CA GTGGCAGTGGGTCTGGMCAGATTATTCTCTCACCATTAGCAACCTGGAGCAAGAAGATG?GCCACTTACTT TTGCCAGCAGTTTAATACGCTTCCTCGGACGTTCGGTGGAGGCACCAAACTGGAAATCAAAcgggctgatgctgcaccaa ctgt

[00319] The amino acid sequence of the light chain variable region of the mouse 13D4 antibody (134 a.a.) [Upper case: mouse 13D4VL variable region, lower case: mouse Igk light chain constant region]. The underlined sequence represents a signal sequence, and the double underline represents regions of the CDR (CDR1, CDR2, and CDR3). MMSSAOFLGLLLLCFOGTRCDIOMTOTTSSLSASLGDRVTISCRASODIDNYLNWYO OKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTISNLEOEDVATYFCOOFNTLPR TFGGGTKLEIKradaapt CDR1 of the light chain variable region of the 13D4 antibody RASQDIDNILN CDR2 of the light chain variable region of the 13D4 antibody YTSRLHS C DR3 of the light chain variable region of the 13D4 QQFNTLP antibody

[00320] 9. The nucleic acid sequence of the heavy chain variable region of the obtained mouse anti-PLD4 antibody 13H11 is SEQ ID NO: 90, and the amino acid sequence is SEQ ID NO: 91. The sequences of amino acid of CDR1, CDR2, and CDR3 in the heavy chain variable region of the mouse 13H11 antibody are SEQ ID NO: 38, SEQ ID NO: 39, and SEQ ID NO: 40, respectively.

[00321] The nucleic acid sequence of the heavy chain variable region of the mouse anti-PLD4 antibody 13H11 (471 base pairs) [Upper case: mouse 13H11VH variable region, lower case: mouse IgG2b heavy chain constant region ] ATGAAAGTGTTGAGTCTGTTGTACCTGTTGACAGCCATTCCTGGTATCCTGTCTGATGTACAGCTTCAGGAGTCAGGACC TGGCCTCGTGAAACCTTCTCAGTCTCTGTCTCTCACCTGCTCTGTCACTGGCTACTCCATCTCCAGTCATTATTACTGGA GTTGGATCCGGCAGTTTCCAGGAAACAGACTGGAATGGATGGGCTACATAAGCTACGACGGTAGCAATAACTACAACCCA TCTCTCAA AAATCGAATCTCCATCACTCGTGACACATCTAAGMCCAGTTTTTCCTGAAGTTGAATTCTGTGACTGA GGACACAGCTACATATAACTGTGCAAGAGAGGGCCCGCTCTACTATGGTAACCCCTACTGGTATTTCGATGTCTGGGCG CAGGGACCACGGTCACCGTCTCCTCAgccaaaacaacacccccatcagtclatccactggcccctaagggc

[00322] The amino acid sequence of the heavy chain variable region of the mouse 13H11 antibody (157 a.a.) [Upper case: mouse 13H11VH variable region, lower case: mouse IgG2b heavy chain constant region]. The underlined sequence represents a signal sequence, and the double underline represents regions of the CDR (CDR1, CDR2, and CDR3). MKVLSLLYLLTAIPGILSDVOLOESGPGLVKPSOSLSLTCSVTGYSISSHYYWSWTRQF PGNRLEWMGYISYDGSNNYNPSLKNRISITRDTSKNOFFIXLNSVTTEDTATYNCARE GPLYYGNPYWYFDVWGAGTTVTVSSakttppsvyplapkg CDR1 of the heavy chain variable region of the antibody 13H11 SHYYWS CDR2 of the variable chain region 13H11 EGPLYYGNPYWYFDV Antibody Heavy Chain Variable Region Antibody 13H11 YISYDGSNNYNPSLKN CDR3 Heavy

[00323] The nucleic acid sequence of the light chain variable region of the mouse anti-PLD4 antibody 13H11 obtained is SEQ ID NO: 110, and the amino acid sequence is SEQ ID NO: 111. The amino acid sequences of CDR1 , CDR2, and CDR3 in the light chain variable region of the mouse 13H11 antibody are SEQ ID NO: 41, SEQ ID NO: 42, and SEQ ID NO: 43, respectively.

[00324] The nucleic acid sequence of the light chain variable region of the mouse anti-PLD4 antibody 13H11 (414 base pairs) [Upper case: mouse 13H11VL variable region, lower case: mouse Igk light chain constant region ] ATGATGTCCTCTGCTCAGTTCCTTGGTCTCCTGTTGCTCTGTTTTCAAGGTACCAGATGTGATATCCAGATGACACAGAC TACATCCTCCCTGTCTGCCTCTCTGGGGGGCAGCGTCACCATCAG?GCAGGGCAAGTCAGGACATTGACAATTA?TAA ACTGGTATCAGCAAAAACCAGATGGAACTGTTAAACTCCTGATCTACTACACATCAAGATTACACTCAGGAGTCCCATCA AGGTTCAG IGGCAGTGGGTCTGGAACAGATTATTCTCTCACCATTAGCAACCTGGAACAAGAAGATATTGCCACTTACTT TTGCCAACAGTTTAATACGCTTCCTCGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAAcgggctgatgctgcaccaa ctgtatccatcttc

[00325] The amino acid sequence of the light chain variable region of the mouse 13H11 antibody (138 a.a.) [Upper case: mouse 13H11VL variable region, lower case: mouse Igk light chain constant region]. The underlined sequence represents a signal sequence, and the double underline represents regions of the CDR (CDR1, CDR2, and CDR3). MMSSAQFLGLLLLCFOGTRCDIOMTOTTSSLSASLGGSVTISCRASQDIDNYLNWYO OKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTISNLEOEDIATYFCOOFNTLPR TFGGGTKLEIKradaaptvsif CDR1 of the light chain variable region of the 13H11 antibody RASQDIDNILN CDR2 of the light chain variable region of the 13H1 antibody 1 YTSRLHS CDR3 of the light chain variable region of the 13H11 QQFNTLP antibody

[00326] 10. The nucleic acid sequence of the heavy chain variable region of the obtained mouse anti-PLD4 antibody 14C1 is SEQ ID NO: 92, and the amino acid sequence is SEQ ID NO: 93. The amino acid sequences of CDR1, CDR2, and CDR3 in the heavy chain variable region of mouse 14C1 antibody are SEQ ID NO: 38, SEQ ID NO: 39, and SEQ ID NO: 40, respectively.

[00327] The nucleic acid sequence of the heavy chain variable region of the mouse anti-PLD4 antibody 14C1 (470 base pairs) [Upper case: mouse 14C1VH variable region, lower case: mouse IgG1 heavy chain constant region ] ATGAAAGTGTTGAGTCTGTTGTACCTGTTGACAGCCATTCCTGGTATCCTGTCTGATGTACAGCTTCAGGAGTCAGGACC TGGCCTCGTGAAACCTTCTCAGTCTCTGTCTCTCACCTGCTCTGTCACTGGCTACTCCATCTCCAGTCATTATTACTGGA GTTGGATCCGGCAGTTTCCAGGAAACAGACTGGAATGGATGGGCTACATAAGCTACGACGGTAGCAATAACTA.CAACCCA TCTCT CAAAAATCGAATCTCCATCACTCGTGACACATCTAAGAACCAGTTTTTCCTGAAGTTGMTTCTGTGACTGA GGACACAGCTACATATAACTGTGCAAGAGAGGGCCCCGCTCTACTATGGTAACCCCTACTGGTATTTCGATGTCTGGGCG CAGGGACCACGGTCACCGTCTCCTCAgccaaaacgacacccccatctgtctatccactggcccctaaggg

[00328] The amino acid sequence of the heavy chain variable region of the mouse 14C1 antibody (156 a.a.) [Upper case: mouse 14C1VH variable region, lower case: mouse IgG1 heavy chain constant region]. The underlined sequence represents a signal sequence, and the double underline represents regions of the CDR (CDR1, CDR2, and CDR3). MKVLSLLYLLTAIPGILSDVOLOESGPGLVKPSOSLSLTCSVTGYSISSHYYWSWIRQF PGNRLEWMGYISYDGSNNYNPSLKNRISITRDTSKNOFFLKLNSVTTEDTATYNCARE GPLYYGNPYWYFDVWGAGTTVTVS S akttppsvyplapk CDR1 of the heavy chain variable region of the antibody 14C1 SHYYWS CDR2 of the variable region of 14C1 EGPLYYGNPYWYFDV Antibody Heavy Chain Variable Region Antibody 14C1 YISYDGSNNYNPSLKN CDR3 Heavy Chain

[00329] The nucleic acid sequence of the light chain variable region of the mouse anti-PLD4 antibody 14C1 obtained is SEQ ID NO: 112, and the amino acid sequence is SEQ ID NO: 113. The amino acid sequences of CDR1 , CDR2, and CDR3 in the light chain variable region of the mouse 14C1 antibody are SEQ ID NO: 41, SEQ ID NO: 42, and SEQ ID NO: 43, respectively.

[00330] The nucleic acid sequence of the light chain variable region of the mouse anti-PLD4 antibody 14C1 (465 base pairs) [Upper case: mouse 14C1VL variable region, lower case: mouse Igk light chain constant region ] ATGATGTCCTCTGCTCAGTTCCTTGGTCTCCTGTTGCTCTGTTTTCAAGGTACCAGATGTGATATCCAGATGACACAGAC TACATCCTCCCTGTCTGCCTCTCTGGGGGGCÂGCGTCACCATCAGTTGCAGGGCAAGTCAGGACATTGACAATTATTTAA ACTGGTATCAGCAAAAACCAGATGGAACTGTTAAACTCCTGATCTACTACACATCAAGATTACACTCAGGAGTCCCATCA AGGTTC AGTGGCAGTGGGTCTGGAACAGATTATTCTCTCACCATTAGCAACCTGGAACAAGAAGATATTGCCACTTACTT TTGCCAACAGmAATACGCTTCCTCGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAAcgggctgatgctgcaccaa ctgtatccatcttcccaccatccagtgagcagttaacatctggaggtgcctcagtcgtgtgcttc

[00331] The amino acid sequence of the light chain variable region of the mouse 14C1 antibody (155 a.a.) [Upper case: mouse 14C1VL variable region, lower case: mouse Igk light chain constant region]. The underlined sequence represents a signal sequence, and the double underline represents regions of the CDR (CDR1, CDR2, and CDR3). MMSSAQFLGLLLLCFQGTRCDIOMTOTTSSLSASLGGSVTISCRASODIDNYLNWYO QKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTISNLEOEDIATYFCOOFNTLPR TFGGGTKLEIKradaaptvsifppsseqltsggaswcf CDR1 of the light chain variable region of the antibody 14C1 RASQDIDNILN CDR2 of the variable region of 14C1 QQFNTLP Antibody Light Chain Variable Region 14C1 YTSRLHS CDR3 Antibody Light Chain

[00332] The base sequences and amino acid sequences of the heavy chain and light chain of the prepared chimeric 11G9 antibody are Sequence Nos. which follow, respectively. Heavy chain SEQ ID NO: 120 (base sequence) SEQ ID NO: 121 (amino acid sequence) Light chain SEQ ID NO: 122 (base sequence) SEQ ID NO: 123 (amino acid sequence)

[00333] 11. The nucleic acid sequence of the chimeric anti-PLD4 antibody 11G9 heavy chain (1401 base pairs) [Upper case: chimeric 11G9VH variable region, lower case: human IgG1 heavy chain constant region] (SEQ ID NO: 120) ATGAAAGTGTTGAGTCTGTTGTACCTGTTGACAGCCATTCCTGGTATCCTGTCTcagGTCCAACTGCAGCAGCCTGGGGC TGAACTGGTGAAGCCTGGGACTTCAGTGAAAATGTCCTGCAAGGCTTCTGGCTACACCTTCACCAGCTACTGGATGCACT GGGTGAAGCAGAGGCCGGGACAAGGCCTTGAGTGGATTGGAGATATTTATCCTGGTAGTGATÀGTACTAACT ACAATGAG AAGTTCAAGAGCAAGGCCACACTGACTGTAGACACATCCTCCAGCACAGCCTACATGCAACTCAGCAGCCTGACATCTGA GGACTCTGCGGTCTATTAGTGTGCAAGAGGAGGGTGGTTGGATGCTATGGACTACTGGGGTCÁAGGAACCTCAGTCACCG TCTCCTCAgctagcaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcaca gcggcc ctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgca caccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggca cccagacctacatctgcaacgtgaatcacaagccca gcaacaccaaggtggacaagaaagttgagcccaaatcttgtgac aaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaa ggacaccctcetgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagt tcaact ggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgt gtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccct cccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggt gtacaccctgcccccatccc gggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgg gagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacag caagctcaccgtggacaagagcaggtggcagcagggg aacgtcttctcatgctccgtgatgcatgaggctctgcacaacc actacacgcagaagagcctctccctgtctccgggtaaatga

[00334] 12. The amino acid sequence of the chimeric anti-PLD4 antibody 11G9 heavy chain (466 a.a.) [Upper case: chimeric 11G9VH variable region, lower case: human IgG1 heavy chain constant region] (SEQ ID NO: 121 ) MKVLSLLYLLTAIPGILSQVQLQQPGAELVKPGTSVKMSCKASGYTFTSYWHWVKQRPGQGLEWIGDIYPGSDSTNYNE KFKSKATLTVDTSSSTAYMQLSSLTSEDSAVYYCARGGMAMDWGQGTSVTVSSastkgpsvfplapsskstsggtaa Igclvkdyfpepvtvswnsgaltsgvh tfpavlqssglyslssvvtvpssslgtQtyicnvnhkpsntkvdkkvepkscd kthtcppcpapellggpsvfIfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyr vvsvltvlhQdwlngkeykckvsnkalpapiektiskakgq prepqvytlppsrdeltkiiQvsltclvkgfypsdiavew esngQpennykt tppvldsdgs f flyskltvdksrwQQgnvf scsvmhealhiihytqksIs 1spgk

[00335] 13. The nucleic acid sequence of the chimeric anti-PLD4 11G9 antibody light chain (705 base pairs) [Upper case: chimeric 11G9VL variable region, lower case: human Ig K light chain constant region] (SEQ ID NO: 122) ATGATGTCCTCTGCTCAGTTCCTTGGTCTCCTGTTGCTCTGTTTTCAAGGTACCAGATGTGATATCCAGATGACACAGAC TACATCCTCCCTGTCTGCCTCTCTGGGAGACAGAGTCACCATCAGTTGCAGGGCAAGTCAGGACATTAGCAATTATTTAA ACTGGTATCAGCAGAAACCAGATGGAACTG?AAACTCCTGATCTACTACACATCAAGATTACACTCAGGAGTC CCATCA AGGTTCAGTGGCAGTGGGTCTGGAACAGATTATTCTCTCACCATTAGCAACCTGGAGCAAGAAGATATTGCCACTTACTT TTGCCAACAGGGTAArACGCTTCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAAcgaactgtggctgcaccat c tgtc t teat c t tccccgccatc tga tgagcagt tgaaatctggaac tgcc tc tg t tgtgtgcctgc tgaat aac 11 ctat cccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcagga cagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcct gcgaagtcacccatca gggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgctag

[00336] 14. The amino acid sequence of the chimeric anti-PLD4 antibody 11G9 light chain (234 a.a.) [Upper case: chimeric 11G9VL variable region, lower case: human IgK light chain constant region] (SEQ ID NO: 123) MMSSAQFLGLLLLCFQGTRCDIQMTQTTSSLSASLGDRVTISCRAQDISNYLNWYQQKPDGTVKLLIY? SRLHSGVPS RFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFGGGTKLEIKrtvaapsvfifppsdeqlksgtasvvcllnnfy preakvqwkvdnalqsgnsqesvteqdskds tyslsstltlskadyekhkvyacevthqglsspvtksfnrgec cDNA and protein sequence in molecular related to PLD4 cDNA of human PLD4 (1521 base pairs) (SEQ ID NO: 44) ATGCTGAAGCCTCTTTGGAAAGCAGCAGTGGCCCCCACATGGCCATGCTCCATGCCGCCCCGCCGCCCGTGGGACAGAGA GGCTGGCACGTTGCAGGTCCTGGGAGCGCTGGCTGTGCTGTGGCTGGGCTCCGTGGCTCTTATCTGCCTCCTGTGGCAAG TGCCCCGTCCTCCCACCTGGGGCCAGGTGCAGCCCAA GGACGTGCCCAGGTCCTGGGAGCATGGCTCCAGCCCAGCTTGG GAGCCCCTGGAAGCAGAGGCCAGGCAGCAGAGGGACTCCTGCCAGCTTGTCCTTGTGGAAAGCATCCCCCAGGACCTGCC ATCTGCAGCCGGCAGCCCCTCTGCCCAGCCTCTGGGCCAGGCCTGGCTGCAGCTGCTGGACACTGCCCAGGAGAGCGTCC ACGTGGC? CATACTACTGGTCCCTCACAGGGCCTGACATCGGGGTCAACGACTCGTCTTCCCAGCTGGGAGAGGCTCTT CTGCAGAAGCTGCAGCAGCTGCTGGGCAGGAACATTTCCCTGGCTGTGGCCACCAGCAGCCCGACACTGGCCAGGACATC CACCGACCTGCAGGTTCTGGCTGCCCGAGGTGCCCATGTACGACAGGTGCCCATGGGGCGGCTCACCAGGGGTGTTTTGC ACTCCAAATTCTGGGTTGTGG ATGGACGGCACATATACATGGGCAGTGCCAACATGGACTGGCGGTCTCTGACGCAGGTG AAGGAGCTTGGCGCTGTCATCTATAACTGCAGCCACCTGGCCCAAGACCTGGAGAAGACCTTCCAGACCTACTGGGTACT GGGGGTGCCCAAGGCTGTCCTCCCCAAAACCTGGCCTCAGAACTTCTCATCTCACTTCAACCGTTTCCAGCCCTTCCACG GCCTCTTTGATGGGGTGCCCACCACTGCCTTCTC TCAGCGTCGCCACCAGCACTCTGTCCCCAGGGCCGCACCCGGGAC CTGGAGGCGCTGCTGGCGGTGATGGGGAGCGCCCAGGAGTTCATCTATGCCTCCGTGATGGAGTATTTCCCCACCACGCG CTTCAGCCACCCCCCGAGGTACTGGCCGGTGCTGGACAACGCGCTGCGGGCGGCAGCCTTCGGCAAGGGCGTGCGCGTGC GCCTGCTGGTCGGCTGCGGACTCAACACGGACCCCACCATGTTC CCCTACCTGCGGTCCCTGCAGGCGCTCAGCAACCCC GCGGCCAACGTCTCTGTGGACGTGAAAGTCTTCATCGTGCCGGTGGGGAACCATTCCAACATCCCATTCAGCAGGGTGAA CCACAGCAAGTTCATGGTCACGGAGAAGGCAGCCTACATAGGCACCTCCAACTGGTCGGAGGATTACTTCAGCAGCACGG CGGGGGTGGGC? GGTGGTCACCCAGAGCCCTGGCGCGCAGCCCGCGGGGGCCACGGTGCAGGAGCAGCTGCGGCAGCTC TTTGAGCGGGACTGGAGTTCGCGCTACGCCGTCGGCCTGGACGGACAGGCTCCGGGCCAGGACTGCGTTTGGCAGGGCTG Human PLD4 Protein (506 amino acids) (SEQ ID NO: 1) MLKPLWKAAVAPTWPCSMPPRRPWDREAGTLQVLGALAVLWLG SVALICLLWQVPRPPTWGQVQPKDVPR SWEHGSSPAWEPLEAEARQQRDSCQLVLVESIPQDLPSAAGSPSAQPLGQAWLQLLDTAQESVHVASYYW SLTGPDIGVNDSSSQLGEALLQKLQQLLGRNISLAVATSSPTLARTSTDLQVLAARGAHVRQVPMGRLTR GVLHSKFWWDGRHIYMGSANMDWRSLTQVK ELGAVIYNCSHLAQDLEKTFQTYWVLGVPKAVLPKTWPQ NFSSHFNRFQPFHGLFDGVPTTAYFSASPPALCPQGRTRDLEALLAVMGSAQEFIYASVMEYFPTTRFSH PPRYWPVLDNALRAAAFGKGVRVRLLVGCGLNTDPTMFPYLRSLQALSNPAANVSVDVKVFIVPVGNHSN IPFSRVNHSKFMVTEKAAYIGTSN WSEDYFSSTAGVGLVVTQSPGAQPAGATVQEQLRQLFERDWSSRYA VGLDGQAPGQPCWQG cynomolgus monkey PLD4 cDNA (1521 base pairs) (SEQ ID NO: 63) ATGCTGAAGCCTCTTCGGAGAGCgGCAGTGACCCCCATGTGGCCGTGCTCCATGCTGCCCCGCCGCCTGTGGGACAGAGA GGCTGGCACGTTGCAGGTCCT GGGAGTGCTGGCTATGCTGTGGCTGGGCTCCATGGCTCTTACCTACCTCCTGTGGCAAG TGCGCCGTCCTCCCACCTGGGGCCAGGTGCAGCCCAAGGACGTGCCCAGGTCCTGGGGGCATGGTTCCAGCCCAGCTCTG GAGCCCCTGGÁAGCGGAGGTCAGGAAGCAGAGGGACTCCTGCCAGCTTGTCCTTGTGGAAAGCATCCCCCAGGACCTGCC ATTTGCAGCCGGCAGCCTCTCCGCC CAGCCTCTGGGCCAGGCCTGGCTGCAGCTGCTGGACACTGCCCAGGAGAGCGTCC ACGTGGCTTCATACTACTGGTCCCTCACAGGGCCCGACATTGGGGTCAACGACTCATCTTCCCAGCTGGGAGAGGCCCTT CTGCAGAAGCTGCAGCAGCTGCTGGGCAGGAACATTTCCTTGGCTGTGGCCACCAGCAGTCCAACACTGGCCAGGAAGTC CACCGACCTGCAGGTCCTGGCTGCCCGAGGTGCCCAGGTACGACGGGTGCCCATGGGGCGGCTCACCAGGGGCGTTTTGC ACTCCAAATTCT GGGTTGTGGATGGACgGCACATATACATGGGCAGTGCcAACATGGACTGGCGGTCCCTGACGCAGGTG AAGGAGCTTGGCGCTGTCATCTATAACTGCAGCCACCTGGCCCAAGACCTGGAGAAGACCTTCCAGACCTACTGGGTGCT GGGGGTGCCCAAGGCTGTCCTCCCCAAAACCTGGCCTCAGAACTTCTCATCTCACATCAACCGTTTCCAGCCCTTCCAGG GCCTCTTTGATGGGGTGCCCACC ACTGCCTACTTCTCAGCATCGCCACCcGCACTCTGTCCCCAGGGCCGCACCCCTGAC CTGGAGGCGCTGTTGGCGGTGATGGGGAGCGCCCAGGAGTTCATCTATGCCTCCGTGATGGAGTATTTCCCTACCACgCG CTTCAGCCACCCCCGCAGGTACTGGCCGGTGCTGGACAACGCGCTGCGGGCGGCAGCCTTCAGCAAGGGTGTGCGCGTGC GCCTGCTGGTCAGCTGCGGACTCAACACGGACC CCACCATGTTCCCCTATCTGCGGTCCCTGCAGGCGCTCAGCAACCCC GCGGCCAACGTCTCTGTGGACGTGAAAGTCTTCATCGTGCCGGTGGGGAATCATTCCAACATCCCGTTCAGCAGGGTGAA CCACAGCAAGTTCATGGTCACGGAGAAGGCAGCCTACATAGGCACCTCCAACTGGTCGGAGGATTACTTCAGCAGCACGA CGGGGGTGGGCCTGGTGGTCACCCAGAGCCCCGGCGCGCAGCCCGC GGGGGCCACGGTACAGGAGCAGCTGCGGCAGCTC TTTGAGCGGGACTGGAGTTCGCGCTACGCCGTCGGCCTGGACGGACAGGCTCCGGGCCAGGACTGCGTTTGGCAGGGCTG The cynomolgus monkey PLD4 protein (506 amino acids) (SEQ ID NO: 129) MLKPLRRAAVTPMWPCSMLPRRLWDREAGTLQVLGVLAMLWLGSMALTYLLWQVRRPPTWGQV QPKDVPRSWGHGSSPAL EPLEAEVRKQRDSCQLVLYESIPQDLPFAAGSLSAQPLGQAWLQLLDTAQESVHVASYYWSLTGPDIGVNPSSSQLGEAL LQKLQQLLGRNISLAVATSSPTLARKSTBLQVLAARGAQVRRVPMGRLTRGVLHSKFWVDGRHIYMGSANMDWRSLTQV KELGAVIYNCSHLAQDLEKTF QTYWVLGVPKAVLPKTWPQNFSSHINRFQPFQGLFDGVPTTAYFSASPPALCPQGRTPD LEALLAVMGSAQEFIYASVMEYFPTTRFSHPRWPVLDNALRAAAFSKGVRVRLLVSCGLNTDPTMFPYLRSLQALSNP AANVSVDVKVFIVPVGNHSNIPFSRVNHSKFMVTEKAAYIGTSNWSEDYFSSTTGYGLVVTQSPGAQ PAGATYQEQLRQL FERDWSSRYAVGLDGQAPGQDCVWQG rhesus monkey PLD4 cDNA (1521 base pairs ) (SEQ ID NO: 124) ATGCTGAAGCCTCTTCGGAGAGCGGCAGTGACCCCCATGTGGCCGTGCTCCATGCTGCCCCGCCGCCTGTGGGACAGAGA GGCTGGCACGTTGCAGGTCCTGGGAGTGCTGGCTATGCTGTGGCTGGGCTCCATGGCTCTTACCTACCTCCTGTGGCAAG TGCGCTGTCCTCCCACCTGGGGCCAGGTGCAGCCCAGGGACGTGCCCAGGTCCTGGGGG CATGGTTCCAGCCTAGCTCTG GAGCCCCTGGAAGCGGAGGTCAGGAAGCAGAGGGACTCCTGCCAGCTTGTCCTTGTGGAAAGCATCCCCCAGGACCTGCC ATTTGCAGCCGGCAGCCTCTCCGCCCAGCCTCTGGGCCAGGCCTGGCTGCAGCTGCTGGACACTGCCCAGGAGAGCGTCC ACGTGGCTTCATACTACTGGTCCCTCACAGGGCCCGACATTGGGGTCAACGACTCATCTTCCCAGCTGG GAGAGGCCCTT CTGCAGAAGCTGCAGCAGCTGCTGGGCAGGAACATTTCCTTGGCTGTGGCCACCAGCAGTCCAACACTGGCCAGGAAGTC CACCGACCTGCAGGTCCTGGCTGCCCGAGGTGCCCAGGTACGACGGGTGCCCATGGGGCGGCTCACCAGGGGCGTTTTGC ACTCCAAATTCTGGGTTGTGGATGGACGGCACATATACATGGGCAGTGCCAACATGGACTGGCGGTCCCTGACGCAGGTG A AGGAGCTTGGCGCTGTCATCTATAACTGCAGCCACCTGGCCCAAGACCTGGAGAAGACCTTCCAGACCTACTGGGTGCT GGGGGTGCCCAAGGCTGTCCTCCCCAAAACCTGGCCTCAGAACTTCTCATCTCACATCAACCGTTTCCAGCCCTTCCAGG GCCTCTTTGATGGGGTGCCCACCACTGCCTACTTCTCAGCATCGCCACCCGCACTCTGTCCCCAGGGCCGCACCCCTGAC CTGGAGGCGCTGTTG GCGGTGATGGGGAGCGCCCAGGAGTTCATCTATGCCTCCGTGATGGAGTATTTCCCTACCACCGCG CTTCAGCCACCCCCGCAGGTACTGGCCGGTGCTGGACAACGCGCTGCGGGCGGCAGCCTTCAGCAAGGGTGTGCGCGTGC GCCTGCTGGTCAGCTGCGGACTCAACACGGACCCCACCATGTTCCCCTATCTGCGGTCCCTGCAGGCGCTCAGCAACCCC GCGGCCAACGTCTCTGTGGACGTGAAA GTCTTCATCGTGCCGGTGGGGAATCATTCCAACATCCCGTTCAGCAGGGTGAA CCACAGCAAGTTCATGGTCACGGAGAAGGCAGCCTACATAGGCACCTCCAACTGGTCGGAGGATTACTTCAGCAGCACGA CGGGGGTGGGCCTGGTGGTCACCCAGAGCCCCGGCGCGCAGCCCGCGGGGGCCACGGTACAGGAGCAGCTGCGGCAGCTC TTTGAGCGGGACTGGAGTTCGCGCTACGCCGTCGGCC TGGACGGACAGGCTCCGGGCCAGGACTGCGTTTGGCAGGGCTG The rhesus monkey PLD4 protein (506 amino acids) (SEQ ID NO: 130) MLKPLRRAAVTPMWPCSMLPRRLTOREAGTLQVLGVLAMLWLGSMAL? LLWQVRCPPWGQVQPRDYPRSWGHGSSLAL EPLEAEVRKQRDSCQLVLVESIPQDLPFAAGSLSAQPLGQAW1QLLDTAQESVIIVASYYWSLTGPDIGVNDSSSQLGEAL LQKLQQLLGRNISLAVATSSPTLARKSTDLQVLAARGAQVRRVPMGRLTRGVLHSKFWVDGRHIYMGSANMDWRSLTQV KELGAVI YNCSHLAQDLEKTFQTYWV1GVPKAVLPKTWPQNFSSHINRFQPFQGLFDGVPTTAYFSASPPALCPQGRTPD LEALLAVMGSAQEFIYASVMEYFPTTRFSHPRWPVLDNALRAAAFSKGVRVRLLVSCGLNTBPTMFPYLRSLQALSNP AANVSVDVKVF1VPVGNHSNIPFSRVNHSKFMVTEKAAYIGTSNWSEDY FSSTTGVGLYVTQSPGAQPAGATVQEQLRQL FERDWSSRYAVGLDGQAPGQDCVWQG Mouse PLD4 cDNA (1512 base pairs) (SEQ ID NO: 131) ATGGACAAGAAGAAAGAGCACCCAGAGATGCGGATACCACTCCAGACAGCAGTGGAGGTCTCTGATTGGCCCTGCTCCAC ATCTCATGATCCACATAGCGGACTTGGCATGGTACT GGGGATGCTAGCTGTACTGGGACTCAGCTCTGTGACTCTCATCT TG? CCTGTGGCAAGGGGCCACTTCTTTCACCAGTCATCGGATGTTCCCTGAGGAAGTGCCCTCCTGGTCCTGGGAGACC CTGAAAGGAGACGCTGAGCAGCAGAATAACTCCTGTCAGCTCATCCTTGTGGAAAGCATCCCCGÀGGACTTGCCATTTGC AGCTGGCAGCCCCACTGCCCAGCCCCTGGCCCAGGCTTGGCTGCAGCTTCTTGACACTGCTCGGGAGAGCGTCCACATTG CCTCGTACTACTGG TCCCTCACTGGACTGGACATTGGAGTCAATGACTCGTCTTCTCGGCAGGGAGAGGCCCTTCTACAG AAG? CCAACAGCTTC? CTCAGGAACATCTCTGTGGTGGTGGCCACCCACAGCCCAACATTGGCCAAGACATCCACTGA CCTCCAGGTCTTGGCTGCCCATGGTGCCCAGATACGACAAGTGCCCATGAAACAGCTTACTGGGGGTGTTCTACACTCCA AATTCTGGGTTGTGGATGGGCGACACGTCTACGTGGGCAGCGCCAACATGGACTGGCGGTCCCTGACTCAGGTGAAGGAA CTTGGTGCAATCATCTACAACTGCAGCAAC CTGGCTCAAGACCTTGAGAAAACATTCCAGACCTACTGGGTGCTAGGGAC TCCCCAAGCTGTTCTCCCTAAAACCTGGCCTCGGAACTTCTCATCCCACATCAACCGCTTCCATCCCTTGCGGGGTCCCT TTGATGGGGTTCCCACCACGGCCTATTTCTCGGCCTCCCTCCCTCCCTCTGCCCGCATGGCCGGACCCGGGATCTGGAC GCAGTGTTGGGAGTGATGGAGGTGCTCGCCAGTTCAT CTATGTCTCGGTGATGGAGTATTTCCCTACCCACGCGCTTCAC CCACCATGCCAGGTACTGGCCCGTGCTGGACAATGCGCTACGGGCAGCGGCCCTCAATAAGGGTGTGCATGTGCGCTTAC TGGTCAGCTGCTGGTTCAACACAGACCCCACCATGTTCGCTTATCTGAGGTCCCTGCAGGCTTTCAGTAACCCCTCGGCT GGCATCTCAGTGGATGTGAAAGTCTTCATCGTGCCTGTGGGAAATC ATTCCAACATCCCGTTCAGCCGCGTGAACCACAG CAAGTTCATGGTCACAGACAAGACAGCCTATGTAGGCACCTCTAACTGGTCAGAAGACTACTTCAGCCACACCGCTGGTG TGGGCCTGATTGTCAGCCAGAAGACCCCCAGAGCCCAGCCAGGCGCAACCACCGTGCAGGAGCAGCTGAGGCAACTCTTT GMCGAGACTGGAGTTCCCACTATGCTATGGACCTAGACAGACAAGTCCCGAGCCAGG ACTGTGTCTGGTAG mouse PLD4 protein (503 amino acids) (SEQ ID NO: 132) MDKKKEHPEMRIPLQTAVEVSDWPCSTSHDPHSGLGMVLGMLAVLGLSSVTLILFLWQGATSFTSHRMFPEEVPSWSWET LKGDAEQQNNSCQLXLVESIPEDLPFAAGSPTAQPLAQAWLQLLDTARESVHIASYYWSLTGLDIGVNDSS SRQGEallq WVLDNALRAAALNKGVHVRLLVSCWFNTDPTMFAYLRSLQAFSNPSA GISVDVKVFIVPVGNHSNIPFSRVNHSKFMVTDKTAYVGTSWSEDYFSHTAGVGLIVSQKTPRAQPGATTVQEQLRQLF ERDWSSHYAMDLDRQVPSQDCVW human PLD3 cDNA sequence (SEQ ID NO: 55) ATGAAGCCTAAACTGATGTACC AGGAGCTGAAGGTGCCTGCAGAGGAGCCCGCCAATGAGCTGCCCATGAATGAGATTGA GGCGTGGAAGGCTGCGGAAAAGAAAGCCCGCTGGGTCCTGCTGGTCCTCATTCTGGCGGTTGTGGGCTTCGGAGCCCTGA TGACTCAGCTGTTTCTATGGGAATACGGCGACTTGCATCTCTTTGGGCCCAACCAGCGCCCAGCCCCCTGCTATGACCCT TGCGAAGCAGTGCTGGTGGAAAGCATTCGTGAGGGCCTGGACTTCCCCAATGCCTCCACGGGGAACCCTTCCACCAGCCA GGCCTGG CTGGCCTGCTCGCCGGTGCGCACAGCAGCCTGGACATCGCCICCTTCTACTGGACCCTCACCAACAATGACA CCCACACGCAGGAGCCCTCTGCCCAGCAGGGTGAGGAGGTCCTCCGGCAGCTGCAGACCCTGGCACCAAAGGGCGTGAAC GTCCGCATCGCTGTGAGCAAGCCCAGCGGGCCCCAGCCACAGGCGGACCTGCAGGCTCTGCTGCAGAGCGGTGCCCAGGT CCGCATGGTGGACAT GCAGAAGCTGACCCATGGCGTCCTGCATACCAAGTTCTGGGTGGTGGACCAGACCCACTTCTACC TGGGCAGTGCCAACATGGACTGGCG? CACTGACCCAGGTCAAGGAGCTGGGCGTGGTCATGTACAACTGCAGCTGCCTG GCTCGAGACCTGACCAAGATCTTTGAGGCCTACTGGTTCCTGGGCCAGGCAGGCAGCTCCATCCCATCAACTTGGCCCCG GTTCTATGACACCCGCTACAACCAAGAGACACCAATGGAGATCTGCCTCAATGGAACCCCTGCTCTGGCCTACCTGGCGA GTGCGCCCCCACCCCTGTGTCCAAGTGGCCGCACT CCAGACCTGAAGGCTCTACTCAACGTGGTGGACAATGCCCGGAGT TTCATCTACGTCGCTGTCATGAACTACCTGCCCACTCTGGAGTTCTCCCACCCTCACAGGTTCTGGCCTGCCATTGACGA TGGGCTGCGGCGGGCCACCTACGAGCGTGGCGTCAAGGTGCGCCTGCTCATCAGCTGCTGGGGACACTCGGAGCCATCCA TGCGGGCCTTCCTGCTCTCTCTGGCTGCCCTGCGTGACAACCATAC CCACTCTGACATCCAGGTGAAACTCTTTGTGGTC CCCGCGGATGAGGCCCAGGCTCGAATCCCATATGCCCGTGTCAACCACAACAAGTACATGGTGACTGAACGCGCCACCTA CATCGGAACCTCCAACTGGTCTGGCAACTACTTCACGGAGACGGCGGGCACCTCGCTGCTGGTGACGCAGAATGGGAGGG GCGGCCTGCGGAGCCAGCTGGAGGCCATTTTCCTGAGGGACTGGGACTCCCCTTACAGCCAT GACC? GACACCTCAGCT GACAGCGTGGGCAACGCCTGCCGCCTGCTCTGA human PLD3 protein (490 amino acids) (SEQ ID NO: 127) MKPKLMYQELKVPAEEPANELPMNBIEAWKAAEKKARWVLLVL1LAVVGFGALMTQLFLWEYGDLHLFGPNQRPAPCYDP CEAVLVESIPEGLDFPNASTGNPSTSQAWLGLLAGAHSSLDIAS FWTLTNNDTHTQEPSAQQGEEVLRQLQTLAPKGVN VRIAVSKPSGPQPQADLQALLQSGAQVRMVDMQKLTHGVLHTKFWWDQTHFYLGSANMDBSLTQVKELGVVMYNCSCL ARDLTKIFEAYWLGQAGSSIPSTWRFYDTRYNQETPMEICLNGTPALAYLASAPPPLCPSGRTPDLKALLNWDNARS FI? AT GGGAGAGGATGAGGATGGACTCTCAGAAAAAAATTGCCAAAATAAATGTCGAATTGCCCTGGTGGAAAATATTCCTGA -AGGCCTTAACTATTCAGAAAATGCACCATTTCACTTATCACTTTTCCAAGGCTGGATGAATTTACTCAACATGGCCAAAA AGTCTGTTGACATAGTGTCTTCCCATTGGGATCTCAACCACACTCATCCATCAGCATGTCAGGGTCAACGTCTTTTTGAA AAGTTGCTCCAGCTGACTTCGCAAAATATT GAAATCAAGCTAGTGAGTGATGTAACAGCTGATTCAAAGGTATTAGAAGC CTTGAAATTAAAGGGAGCCGAGGTGACGTACATGAACATGACCGCTTACAACAAGGGCCGGCTGCAGTCCTCCTTCTGGA TCGTGGACAAACAGCACGTGTATATCGGCAGTGCCGGTTTGGACTGGCAATCCCTGGGACAGATGAAAGAACTCGGTGTC ATCTTCTACAACTGCAGCTGCCTGGTCCTAGATTTACAAAGGATATT TGCTCTATATAGTTCATTAAAATTCAAAAGCAG AGTGCCTCAAACCTGGTCCAAAAGACTCTATGGAGTCTATGACAATGAAAAGAAATTGCAACTTCAGTTGAATGAAACCA AATCTCAAGCATTTGTATCGAATTCTCCAAAACTCTTTTGCCCTAAAAACAGAAGTTTTGACATAGATGCCATCTACAGT GTGATAGATGATGCCAAGCAGTATGTGTACATCGCTGTCATGGACTACCTGCCTATCTCCAGCACAAGCACCA AAAGGAC TTACTGGCCAGACTTGGATGCAAAAATAAGAGAAGCATTAGTTTTACGAAGCGTTAGAGTTCGACTCCTTTTAAGCTTCT GGAAGGAAACTGATCCCC? ACGT? AACT? ATTTCAICTC? AAAGCGAT? GCACTGAAATAGCCAACTGCAGT? G AAAGTTAAATTTTTTGATCTGGAAAGAGAGAATGCTTGTGCTACAAAAGAACAAAAGAATCACACCTTTCCTAGGTTAAA TCGCAACAAGTACATGGTGACAGATGGAGCAGC? ATATTGGAAATTTTGATTGGGTAGGGAATGATTTCACTCAGAATG CTGGCACGGGCCTTGTTATCAACCAGGCAGATGTGAGGAACAACAGAAGCATCATTAAGCAACTTAAAGATGTGTTTGAA AGGGACTGGTATTCACCGTÁTGCCAAAACCTTACAGCCAACCAAACAGCCGAACTGCTCAAGCCTGTTCAAACTCAAACC CCTCTCCAACAAAACTGCCACAGACGACACAGGCGGAAAGGATCCCCGGAAC GTATGA human PLD5 protein (445 amino acids) (SEQ ID NO: 128) NGEDEDGLSEKNCQNKCRIALVENIPEGLHSENAPFHLSLFQGWNLLNMAKKSVDIVSSHTOLNHTHPSACQGQRLFE KLLQLTSQNIEIKLVSDVTADSKVLEALKLKGAEVTYMNMTAYNKGRLQSSFWIVDKQHVYIGSAGLDWSLGQMKELGV IFYNCSCLVLDLQRIFALYSSLKFKSRVPQTWSKRLYGVYDNEKKLQLQLNETKSQAFVSNSPKLFCPKNRSFDIDAIYS VIDDAKQYVYIAVMDYLPISSTSTKRTYWPDLDAKIREALVLRSVRVRLLLSFTOTDPLTFNFISSLKAICTEIANCSL KVKFFDLERENACATKEQKNHTFPRLNRNKYMVTDGAAYIGNFDW GNDFTQNAGTGLVINQADVRNNRSIIKQLKDVFE RDWYSPYAKTLQPTKQPNCSSLFKLKPLSNKTATDDTGGKDPRNV Human PLD4-Ig fusion protein cDNA (2142 base pairs) (SEQ ID NO: 125) ATGGAGTTTCAGACCCAGGTCTTTGTATTCGTGTTGCTCTGGTTGTCTGGTGTTGATGGAgattacaaggatgac gacga taaaGGATCCcccagagggcccacaatcaagccctgtcctccatgcaaatgcccagcacctaacctcttgggtggaccat ccgtcttcatcttccctccaaagatcaaggatgtactcatgatctccctgagccccatagtcacatgtgtggtggtggat gtgagcgaggatgacccagatgtccagatcagctggtttgtgaacaacgtggaagtacacacagctcagacacaaaccca tagagaggattacaacagtactctccgggtggtcagtgccctccccatccagca ccaggactggatgagtggcaaggagt tcaaatgcaaggtcaacaacaaagacctcccagcgcccatcgagagaaccatctcaaaacccaaagggtcagtaagagct ccacaggtatatgtcttgcctccaccagaagaagagatgactaagaaacaggtcactctgacctgcatggtcacagactt catgcctgaagacatttacgtggagtggaccaacaacgg gaaaacagagctaaactacaagaacactgaaccagtcctgg actctgatggttcttact teatgtacagcaagctgagagtggaaaagaactgggtggaaagaaatagctactectgt tcagtggtccacgagggtctgcacaatcaccacacgactaagagcttctcccggactccgggtaaaCGTCCTCCCACCTG GGGCCAGGTGCAGCCCAAGG ACGTGCCCAGGTCCTGGGAGCATGGCTCCAGCCCAGCTTGGGAGCCCCTGGAAGCAGAGG CCAGGCAGCAGAGGGACTCCTGCCAGCTTGTCCTTGTGGAAAGCATCCCCCAGGACCTGCCATCTGCAGCCGGCAGCCCC TCTGCCCAGCCTCTGGGCCAGGCCTGGCTGCAGCTGCTGGACACTGCCCAGGAGAGCGTCCACGTGGCTTCATACTACTG GTCCCTCACAGGGCCTGACATCGGGGT CAACGACTCGTCTTCCCAGCTGGGAGAGGCTCTTCTGCAGAAGCTGCAGCAGC TGCTGGGCAGGAACATTTCCCTGGCTGTGGCCACCAGCAGCCCGACACTGGCCAGGACATCCACCGACCTGCAGGTTCTG GCTGCCCGAGGTGCCCATGTACGACAGGTGCCCATGGGGCGGCTCACCAGGGGTGTTTTGCACTCCAAATTCTGGGTTGT GGATGGACGGCACATATACATGGGCAGTGCCAACATGGACT GGCGGTCTCTGACGCAGGTGAAGGAGCTTGGCGCTGTCA TCTATAACTGCAGCCACCTGGCCCAAGACCTGGAAGACCTTCCAGACCTACTGGGTACTGGGGGTGCCCAAGGCTGTC CTCCCCAAAACCTGGCCTCAGAACTTCTCATCTCACTTCAACCGmCCAGCCCTTCCACGGCCTCTTTGATGGGGTGCC CACCACTGCCTACTTCrCAGCGTCGCCACCAGCACTCTGTCCCCAGGGCC GCACCCGGGACCTGGAGGCGCTGCTGGCGG TGAGGGGAGCGCCCAGGAGTTCATCTATGCCTCCGTGATGGAGTATTTCCCCACCACGCGCTTCAGCCACCCCCCGAGG TACTGGCCGGTGCTGGACAACGCGCTGCGGGCGGCAGCCITCGGCAAGGGCGTGCGCGTGCGCCTGCTGGTCGGCTGCGG ACTCAACACGGACCCCACCATGTTCCCCTACCTGCGGTCCCTGCAGGCGCTCAGCAA CCCCGCGGCCAACGTCTCTGTGG ACGTGAAAGTCTTCATCGTGCCGGTGGGGAACCATTCCAACATCCCATTCAGCAGGGTGAACCACAGCAAGTTCATGGTC ACGGAGAAGGCAGGCTACATAGGCACCTCCAACTG&TCGGAGGAT'TACTTCAGCAGCACGGGGGGTGGGCTTGGTGGT CACCCAGAGCCCTGGCGCGCAGCCCGCGGGGGCCACGGTGCAGGAGCAGCTGCGGCAGCTC TTTGAGCGGGACTGGAGTT CGCGCTACGCCGTCGGCCTGGACGGACAGGCTCCGGGCCAGGACTGCGTTTGGCAGGGCTGA human PLD4-Ig fusion protein (713 amino acids) (SEQ ID NO: 126) MEFQTQVFVFVLLWLSGVDGDYKDDDDKGSPRGPTIKPCPPCKCPAPNLLGPSVFIFPPKIKDVLMISLSPIVTCVVVD V S ED PP D VQISWFVNNVEVHTAQTQTHREDYNSTLRWSALPIQHQDWSGKEFKCKVNNKDLPAPIERTISKPKGSVRA PQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSC SVVHEGLHNHHTTKSFSETPGKRPPTWGQVQPKDVPRS WEHGSSPAWLEAEARQQRDSCQLVLVESIPQDLPSAAGSP SAQPLGQAWLQLLDTAQESVHVASYWSLTGPDIGVNDSSSQLGEALLQKLQQLLGRNISLAVATSSPTLARTSTDLQYL AARGAHVRQYPMGRLTRGVLHSKFWVVDGRHIYMGSANMDWRSLTQVKELGAVIYNCSHLAQDLEKTFQTYWVLGVPK AVLPK? PQNFSSHFNRFQPFHGLFDGYPTTAYFSASPPALCPQGRTRDLEALLAVMGSAQEFIYASVMEYFPTTRFSHPPR YWVLDNALRAAAFGKGVRVRLLVGCGLNTDPTMFPYLRSLQALSNPAANVSVDVKVFIVPVGNHSNIPFSRVNHSKFMV TEKAAYIGTSNWSEDYFSSTAGVGLVVTQSPGAQPAGATVQEQLRQLFER DWSSRYAVGLDGQAPGQDCVWQG Accession Number NITE BP-1211 NITE BP-1212 NITE BP-1213 NITE BP-1214 Sequence List Free Text SEQ ID NO 45: Forward Initiator SEQ ID NO 46: Reverse Initiator SEQ ID NO 47: Forward Initiator SEQ ID NO 48: Reverse starter SEQ ID NO 49: Forward starter SEQ ID NO 50: Reverse starter SEQ ID NO 51: Forward starter SEQ ID NO 52: Reverse starter SEQ ID NO 53: Forward starter SEQ ID NO 54: Reverse starter SEQ ID NO 70: Anchor Initiator SEQ ID NO 70: n is deoxyinosine SEQ ID NO 71: AUAP Initiator SEQ ID NO 72: Initiator SEQ ID NO 73: Initiator SEQ ID NO 114: Initiator SEQ ID NO 115: Initiator SEQ ID NO 116: Initiator SEQ ID NO 117: Starter SEQ ID NO 118: Starter SEQ ID NO 119: Starter

Claims (17)

1. Method of preparing a hybridoma that produces a monoclonal antibody that binds to human PLD4 on a plasmacytoid dendritic cell (pDC), comprising the following processes: 1) a process of administering PLD4-Ig fusion protein recombinant human encoding an amino acid sequence containing an extracellular human PLD4 domain, to an immune non-human animal; 2) a process of producing hybridoma cells that produce antibodies from the immune animal resulting from process 1); and 3) a process of selecting, through the use of a cell expressing human PLD4, a hybridoma that produces an antibody that binds to human PLD4 from the hydridoma cells resulting from process 2). 2. Method according to claim 1, characterized by the fact that the cell expressing human PLD4 is a cell that retains an extrinsic polynucleotide encoding an amino acid sequence containing the extracellular human PLD4 domain in an expressible manner. 3. Method according to claim 2, characterized by the fact that the cell expressing human PLD4 is an animal cell. 4. Method according to claim 3, characterized by the fact that the cell expressing human PLD4 is a human-derived cell. 5. Method according to claim 4, characterized by the fact that the human-derived cell is a HEK-293T or CT125 cell. 6. Method according to claim 1, characterized by the fact that the cell expressing human PLD4 is a cell derived from human pDC. 7. Method according to claim 6, characterized by the fact that the human pDC-derived cell is a CAL-1 cell. 8. Hybridoma that produces a monoclonal antibody that binds to a human PLD4 on a human plasmacytoid dendritic cell (pDC), characterized by the fact that they are called mp5B7, mp7B4, mp13D4 or mp13H11, deposited under Accession Numbers: NITE BP-1211 , NITE BP-1212, NITE BP-1213 or NITE BP-1214. 9. Monoclonal antibody or a fragment containing an antigen-binding region thereof characterized by the fact that it is produced by any of the hybridomas as defined in claim 8, namely, mp5B7, mp7B4, mp13D4 or mp13H11, deposited under Accession Numbers : NITE BP-1211, NITE BP-1212, NITE BP-1213 or NITE BP-1214. 10. Monoclonal antibody or a fragment containing an antigen-binding region thereof that binds to a human PLD4 or a human plasmacytoid dendritic cell (pDC) characterized by the fact that it comprises: (a) the sequence SYWMH (SEQ ID NO: 2 ) as CDR1, the sequence DIYPGSDSTNYNEKFKS (SEQ ID NO: 3) as CDR2 and the sequence GGWLDAMDY (SEQ ID NO: 4) as CDR3 in the heavy chain variable region and the sequence RASQDISNYLN (SEQ ID NO: 5) as CDR1, the sequence YTSRLHS (SEQ ID NO: 6) as CDR2, and the sequence QQGNTLPW (SEQ ID NO: 7) as CDR3 in the light chain variable region; (b) the sequence TYWMH (SEQ ID NO: 8) as CDR1, the sequence AIYPGNSETSYNQKFKG (SEQ ID NO: 9) as CDR2, and the sequence GYSDFDY (SEQ ID NO: 10) as CDR3 in the heavy chain variable region, and the sequence HASQGIRSNIG (SEQ ID NO: 11) as CDR1, the sequence HGTNLED (SEQ ID NO: 12) as CDR2, and the sequence VQYVQFP (SEQ ID NO: 13) as CDR3 in the light chain variable region; (c) the sequence DYNLH (SEQ ID NO: 14) as CDR1, the sequence YIYPYNGNTGYNQKFKR (SEQ ID NO: 15) as CDR2, and the sequence GGIYDDYYDYAIDY (SEQ ID NO: 16) as CDR3 in the heavy chain variable region; and the sequence RASENIYSHIA (SEQ ID NO: 17) as CDR1, the sequence GATNLAH (SEQ ID NO: 18) as CDR2, and the sequence QHFWGTP (SEQ ID NO: 19) as CDR3 in the light chain variable region; (d) the sequence SYYLY (SEQ ID NO: 20) as CDR1, the sequence LINPTNSDTIFNEKFKS (SEQ ID NO: 21) as CDR2, and the sequence EGGYGYGPFAY (SEQ ID NO: 22) as CDR3 in the heavy chain variable region; and the sequence TSSQTLVHSNGNTYLH (SEQ ID NO: 23) as CDR1, the sequence KVSNRFS (SEQ ID NO: 24) as CDR2, and the sequence HSTHVP (SEQ ID NO: 25) as CDR3 in the light chain variable region; (e) the sequence SYGMS (SEQ ID NO: 26) as CDR1, the sequence TISSGGSYIYYPESVKG (SEQ ID NO: 27) as CDR2, and the sequence LYGGRRGYGLDY (SEQ ID NO: 28) as CDR3 in the heavy chain variable region, and the sequence RSSKSLLHSDGITYLY (SEQ ID NO: 29) as CDR1, the sequence QMSNLAS (SEQ ID NO: 30) as CDR2, and the sequence AQNLEL (SEQ ID NO: 31) as CDR3 in the light chain variable region; (f) the sequence SHYYWT (SEQ ID NO: 32) as CDR1, the sequence YISYDGSNNYNPSLKN (SEQ ID NO: 33) as CDR2, and the sequence EGPLYYGNPYWYFDV (SEQ ID NO: 34) as CDR3 in the heavy chain variable region, and the sequence RASQDIDNYLN (SEQ ID NO: 35) as CDR1, the sequence YTSRLHS (SEQ ID NO: 36) as CDR2, and the sequence QQFNTLP (SEQ ID NO: 37) as CDR3 in the light chain variable region; or (g) the sequence SHYYWS (SEQ ID NO: 38) as CDR1, the sequence YISYDGSNNYNPSLKN (SEQ ID NO: 39) as CDR2, and the sequence EGPLYYGNPYWYFDV (SEQ ID NO: 40) as CDR3 in the heavy chain variable region, and the sequence RASQDIDNYLN (SEQ ID NO: 41) as CDR1, the sequence YTSRLHS (SEQ ID NO: 42) as CDR2, and the sequence QQFNTLP (SEQ ID NO: 43) as CDR3 in the light chain variable region. 11. Monoclonal antibody or a fragment thereof, according to any one of claims 9 or 10, characterized by the fact that it is further chimerized or humanized. 12. Method of preparing a monoclonal antibody characterized by the fact that it comprises: cultivating the hybridoma as defined in claim 8; and collect a monoclonal antibody from the culture. 13. Ex vivo method of detecting a plasmacytoid dendritic cell (pDC) characterized by the fact that it comprises: contacting a test cell with a monoclonal antibody or a fragment containing an antigen-binding region thereof, defined in any one of the claims 9 to 11, which binds to an extracellular human PLD4 domain on a human plasmacytoid dendritic cell (pDC); and detecting the monoclonal antibody or fragment that binds to the cell. 14. Reagent for detecting a plasmacytoid dendritic cell (pDC) characterized by the fact that it comprises: a monoclonal antibody or a fragment containing an antigen-binding region thereof, defined in any one of claims 9 to 11. 15. Ex vivo method of suppressing the activity of a plasmacytoid dendritic cell (pDC) characterized by the fact that it comprises: placing any of the components described below in contact with the plasmacytoid dendritic cell, (a) a monoclonal antibody or a fragment containing a region of the same that binds to an extracellular human PLD4 domain on a human plasmacytoid dendritic cell (pDC) and suppresses DC activity, and (b) an immunoglobulin or a fragment containing an antigen-binding region of the same wherein a complementarity-determining region of the monoclonal antibody (a) is transplanted. 16. An agent for suppressing the activity of a plasmacytoid dendritic cell (pDC) characterized in that it comprises: any of the components described below as an active component, (a) a monoclonal antibody or a fragment containing an antigen binding region thereof which binds to an extracellular human PLD4 domain on a human palsmacytoid dendritic cell (pDC), and suppresses DC activity, and (b) an immunoglobulin or a fragment containing an antigen-binding region thereof in which a determinant region of complementarity of the monoclonal antibody (a) is transplanted. 17. An agent for suppressing an activity of a cell that produces interferon, according to claim 16, characterized by the fact that the activity of a plasmacytoid dendritic cell (pDC) is an activity that produces interferon and that survives from a cell that produces interferon, or both of them.