Neutralization antibody having USAG-1 for regeneration treatment of tooth as target molecule

Abstract

To provide a technique regarding a treatment method of anodontia of a tooth which utilizes differentiation induction inherent in an organ of a tooth instead of utilizing surgical tissue transplantation.SOLUTION: An antibody that specifically binds USAG-1 to be neutralized or an antigen-binding fragment thereof, and a pharmaceutical composition containing the antibody or the antigen-binding fragment thereof.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to neutralizing antibodies targeting USAG-1 for the treatment of edentulism or tooth regeneration. [Background technology]

[0002] Most of the causes of edentulism (patients with missing teeth) are acquired causes such as dental caries and periodontal disease, but congenital edentulism has a high incidence rate of 1%. Currently, the only treatments for missing teeth are prosthetic treatments such as dental implants and dentures, and there is no fundamental treatment. Many studies on tooth regeneration using tissue engineering approaches have been reported. Various cells such as stem cells (Non-Patent Document 1) are used as cell sources. In addition, in order to make in vitro created teeth function in the oral cavity, a cell manipulation technique called the "organ primordium method" (Non-Patent Document 2) has been reported, which regenerates the tooth organ primordium, which is the basis of the organ, in a collagen gel. However, all tissue engineering approaches have problems such as the cost of securing a cell source and safety, and have not yet been applied clinically.

[0003] Meanwhile, many causative genes for congenital anodontia have been identified, many of which are common between humans and mice. Examples include RUNX2, MSX1, EDA, WNT10A, PAX9, and AXIN2. Among these, congenital anodontia caused by the WNT10A gene has the largest number of patients. EDA is also the causative gene for anhidrotic ectodermal dysplasia, a representative syndromic congenital anodontia. Congenital anodontia is caused when tooth development stops midway due to a defect or functional inhibition of the causative gene. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Ohazama, J Denr Res, 2004 [Non-Patent Document 2] Nakao, Nat Methods, 2007 Summary of the Invention [Problem to be solved by the invention]

[0005] A new approach to treating congenital edentulism is to induce differentiation from a state in which tooth development has stopped midway as described above, and form complete teeth. Therefore, the present invention aims to provide a technique for treating edentulism that utilizes differentiation induction inherent in the tooth organ, rather than using surgical tissue transplantation. [Means for solving the problem]

[0006] As a result of intensive research, the present inventors have succeeded in developing a neutralizing antibody that targets USAG-1. Furthermore, they have found that administration of the antibody can regenerate missing teeth in a congenital anedentatious model mouse and form supernumerary teeth in a congenital anedentatious model mouse or a wild-type mouse, thereby completing the present invention.

[0007] That is, the present invention provides [1] An antibody or antigen-binding fragment thereof that specifically binds to and neutralizes USAG-1; [2] The antibody or antigen fragment thereof according to [1], which specifically binds to USAG-1 and neutralizes the BMP signaling inhibitory activity of USAG-1. [3] The antibody or antigen fragment thereof according to [1] or [2], which specifically binds to USAG-1 and neutralizes the WNT signaling inhibitory activity of USAG-1. [4] (a) three heavy chain complementarity determining regions comprising amino acid sequences having at least 90% sequence identity to the amino acid sequences shown in SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7, respectively, or three light chain complementarity determining regions comprising amino acid sequences having at least 90% sequence identity to the amino acid sequences shown in SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10, respectively; (b) three heavy chain complementarity determining regions comprising amino acid sequences having at least 90% sequence identity to the amino acid sequences set forth in SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17, respectively, or three light chain complementarity determining regions comprising amino acid sequences having at least 90% sequence identity to the amino acid sequences set forth in SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20, respectively; (c) three heavy chain complementarity determining regions comprising amino acid sequences having at least 90% sequence identity to the amino acid sequences set forth in SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27, respectively, or three light chain complementarity determining regions comprising amino acid sequences having at least 90% sequence identity to the amino acid sequences set forth in SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30, respectively; (d) three heavy chain complementarity determining regions comprising amino acid sequences having at least 90% sequence identity to the amino acid sequences set forth in SEQ ID NO: 42, SEQ ID NO: 43, and SEQ ID NO: 44, respectively; or three light chain complementarity determining regions comprising amino acid sequences having at least 90% sequence identity to the amino acid sequences set forth in SEQ ID NO: 45, SEQ ID NO: 46, and SEQ ID NO: 47, respectively; or (e) three heavy chain complementarity determining regions comprising amino acid sequences having at least 90% sequence identity to the amino acid sequences set forth in SEQ ID NO:52, SEQ ID NO:53, and SEQ ID NO:54, respectively, or three light chain complementarity determining regions comprising amino acid sequences having at least 90% sequence identity to the amino acid sequences set forth in SEQ ID NO:55, SEQ ID NO:56, and SEQ ID NO:57, respectively. The antibody or antigen-binding fragment thereof according to any one of [1] to [3], [5] (f) a heavy chain variable region comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 3, or a light chain variable region comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 4; (g) a heavy chain variable region comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 13, or a light chain variable region comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 14; (h) a heavy chain variable region comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 23, or a light chain variable region comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 24; (i) a heavy chain variable region comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 40, or a light chain variable region comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 41; or, (j) a heavy chain variable region comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 50, or a light chain variable region comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 51. The antibody or antigen-binding fragment thereof according to any one of [1] to [4], [6] (k) three heavy chain complementarity determining regions comprising amino acid sequences having at least 90% sequence identity to the amino acid sequences set forth in SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7, respectively, and three light chain complementarity determining regions comprising amino acid sequences having at least 90% sequence identity to the amino acid sequences set forth in SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10, respectively; (l) three heavy chain complementarity determining regions comprising amino acid sequences having at least 90% sequence identity to the amino acid sequences set forth in SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17, respectively, and three light chain complementarity determining regions comprising amino acid sequences having at least 90% sequence identity to the amino acid sequences set forth in SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20, respectively; (m) three heavy chain complementarity determining regions comprising amino acid sequences having at least 90% sequence identity to the amino acid sequences set forth in SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27, respectively, and three light chain complementarity determining regions comprising amino acid sequences having at least 90% sequence identity to the amino acid sequences set forth in SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30, respectively; (n) three heavy chain complementarity determining regions comprising amino acid sequences having at least 90% sequence identity to the amino acid sequences set forth in SEQ ID NO: 42, SEQ ID NO: 43, and SEQ ID NO: 44, respectively, and three light chain complementarity determining regions comprising amino acid sequences having at least 90% sequence identity to the amino acid sequences set forth in SEQ ID NO: 45, SEQ ID NO: 46, and SEQ ID NO: 47, respectively; or (o) three heavy chain complementarity determining regions comprising amino acid sequences having at least 90% sequence identity to the amino acid sequences set forth in SEQ ID NO:52, SEQ ID NO:53, and SEQ ID NO:54, respectively, and three light chain complementarity determining regions comprising amino acid sequences having at least 90% sequence identity to the amino acid sequences set forth in SEQ ID NO:55, SEQ ID NO:56, and SEQ ID NO:57, respectively. The antibody or antigen-binding fragment thereof according to any one of [1] to [3], [7] (p) a heavy chain variable region comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 3, and a light chain variable region comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 4; (q) a heavy chain variable region comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 13, and a light chain variable region comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 14; (r) a heavy chain variable region comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 23, and a light chain variable region comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 24; (s) a heavy chain variable region comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 40, and a light chain variable region comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 41; or (t) a heavy chain variable region comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 50, and a light chain variable region comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 51. The antibody or antigen-binding fragment thereof according to any one of [1] to [3] and [6], [8] An antibody or an antigen-binding fragment thereof that competes with the antibody or antigen-binding fragment thereof according to any one of [4] to [7] for binding to USAG-1. [9] The antibody or antigen-binding fragment thereof according to any one of [1] to [8], wherein the antibody is a humanized antibody or a chimeric antibody; and

[10] A pharmaceutical composition for tooth regeneration treatment, comprising the antibody or antigen-binding fragment thereof according to any one of [1] to [9]. to provide. Effect of the Invention

[0008] In the present invention, we have succeeded in regenerating teeth in vivo by using an antibody preparation. Treatment with the antibody preparation of the present invention can be clinically deployed as a dental regenerative medicine using general oral and maxillofacial surgical approaches such as conventional tooth extraction, orthodontics, and tooth transplantation. [Brief description of the drawings]

[0009] [Figure 1] 1 shows the Wnt suppressive activity of the E. coli -derived recombinant human UASG-1 protein used as an antigen in the Examples. [Diagram 2] 1 shows the BMP suppressive activity of the E. coli-derived recombinant human UASG-1 protein used as an antigen in the Examples. [Diagram 3] The newly established USAG-1KO mouse is shown using CRISPER-CAS9. [Figure 4-1] 1 shows the results of the primary screening of anti-USAG-1 neutralizing antibodies. [Figure 4-2] 1 shows the results of the primary screening of anti-USAG-1 neutralizing antibodies. [Diagram 5] The results of purification and concentration of mouse N-terminal PA-tagged USAG-1 (WISE) are shown. [Figure 6] 1 shows the dose-dependent WNT signal suppression activity of mouse USAG-1 protein. [Figure 7] 1 shows the dose-dependent inhibitory activity of mouse USAG-1 protein on BMP signaling. [Figure 8] 1 shows an antibody that dose-dependently neutralizes the inhibitory activity of mouse USAG-1 on WNT signaling. [Figure 9] 1 shows an antibody that dose-dependently neutralizes the inhibitory activity of mouse USAG-1 on BMP signaling. [Figure 10] We show that anti-USAG-1 neutralizing antibodies induce tooth growth in an edentulous model mouse. [Figure 11] This shows that anti-USAG-1 neutralizing antibodies have the same effect as USAG-1KO. [Figure 12] 1 shows the results of a binding experiment of mouse anti-USAG-1 antibody with mouse / human USAG-1 protein. [Figure 13] The figure shows immunostaining using mouse anti-USAG-1 antibody on HEK293 cells in which human FLAG-tagged USAG-1 was transiently overexpressed. [Figure 14] The sequences of the heavy and light chain variable regions of Antibody A and Antibody B are shown. [Figure 15] 1 shows the results of a binding experiment of mouse anti-USAG-1 antibody with mouse / human USAG-1 protein. [Figure 16] The competitive binding data obtained for the six antibodies is shown in FIG. 1. Sensorgrams for each of the six test antibodies when reacted with the USAG-1 sensor captured with the six antibodies are overlaid. [Figure 17]1 shows the neutralizing activity of the antibody of the present invention against the inhibitory activity of mouse USAG-1 on WNT signaling and BMP signaling. [Figure 18] 1 shows dental X-rays showing the effect of administration of USAG-1 neutralizing antibody to dogs with congenital edentulism. [Figure 19] These are μCT images and 3D reconstructions showing induction of the third tooth in the mandibular third premolar region in ferrets by administration of USAG-1 neutralizing antibody. [Figure 20] These are μCT images and 3D reconstructions showing induction of the third tooth in the mandibular third premolar region by administration of USAG-1 neutralizing antibody in a musk shrew. [Figure 21] The sequences of the heavy and light chain variable regions of Antibody C are shown. [Figure 22] The sequences of the heavy and light chain variable regions of antibodies D and E are shown. [Figure 23] μCT slice images and 3D reconstructions showing induction of the third maxillary anterior tooth in ferrets by administration of USAG-1 neutralizing antibody. [Figure 24] This is a 3D reconstruction image created based on μCT data showing the induction of the third tooth in the mandibular premolar region in ferrets by administration of USAG-1 neutralizing antibody. [Diagram 25] 1 shows the neutralizing activity of the antibody of the present invention against the inhibitory activity of mouse USAG-1 on WNT signaling and BMP signaling. [Figure 26] 1 shows the results of a pull-down assay demonstrating the interaction of a complex of mouse anti-USAG-1 antibody and mouse USAG-1 protein with the LRP6-E1E2 domain. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] USAG-1 (Uterine Sensitization Associated Gene-1), also known as Sostdc-1, Ectodin, or Wise, is a bone morphogenetic protein (BMP) antagonist and Wnt antagonist. It is known that USAG-1-deficient model mice show increased BMP signaling, which leads to the formation of supernumerary teeth. The inventors crossed Runx2-deficient mice, which are a model of congenital anodontia, with USAG-1-deficient mice, which are a model of supernumerary teeth (teeth present in excess of the normal number of teeth), to generate double knockout mice, and analyzed the mice, finding that tooth formation was restored. Therefore, it was suggested that anodontia could be treated by inhibiting USAG-1.

[0011] In this study, the inventors crossed a congenital edentulism model mouse lacking causative genes Msx1, Eda, and Wnt10a other than Runx2 with a USAG-1 gene-deficient mouse, a supernumerary tooth model mouse newly created using the CRISPER-CAS9 system, to generate double knockout mice, and analyzed the results. They found that tooth formation was restored in all of the edentulism model mice. Therefore, it has become clear that treatment by inhibiting USAG-1 can be applied to patients with congenital edentulism caused by various gene mutations.

[0012] In the present invention, we have produced antibodies using recombinant human USAG-1 protein, the activity of which has been confirmed, as an antigen, and obtained antibodies that specifically bind to USAG-1. These antibodies have been confirmed to increase BMP signaling and / or Wnt signaling.

[0013] Thus, one aspect of the present invention provides an antibody and an antigen-binding fragment thereof that specifically binds to and neutralizes USAG-1, i.e., an anti-USAG-1 neutralizing antibody and an antigen-binding fragment thereof. Unless otherwise specified herein, USAG-1 refers to mammalian USAG-1. Examples of mammalian animals include, but are not limited to, humans, dogs, cats, horses, mice, ferrets, suncus, pigs, monkeys, etc., and preferably humans.

[0014] As used herein, neutralization refers to inhibiting the function of USAG-1. The functions of USAG-1 include, for example, BMP signaling inhibitory activity (also referred to as "BMP antagonist activity") and Wnt signaling inhibitory activity (also referred to as "Wnt antagonist activity"). The antibody or antigen-binding fragment thereof of the present application inhibits the BMP signaling inhibitory activity and / or the Wnt signaling inhibitory activity of USAG-1. Thus, the antibody or antigen-binding fragment thereof of the present application neutralizes either or both of the BMP signaling inhibitory activity of USAG-1 and the Wnt signaling inhibitory activity of USAG-1. For example, but not limited to, the antibody or antigen-binding fragment of the present application includes an antibody or antigen-binding fragment thereof that specifically binds to USAG-1 and neutralizes the BMP signaling inhibitory activity of USAG-1 but does not neutralize the Wnt signaling inhibitory activity of USAG-1, and an antibody or antigen-binding fragment thereof that specifically binds to USAG-1 and neutralizes the Wnt signaling inhibitory activity of USAG-1 but does not neutralize the BMP signaling inhibitory activity of USAG-1. Note that, in this specification, "inhibition" includes suppression and reduction.

[0015] The neutralizing activity of an antibody or an antigen-binding fragment thereof may be determined by a conventional method. The activity of USAG-1 to neutralize the BMP antagonist activity (also referred to as "BMP antagonist neutralizing activity") can be measured in vitro, for example, by ALP (alkaline phosphatase) assay or reporter assay. The ALP assay is performed, for example, by culturing osteoprogenitor cells or the like in the presence of BMP with USAG-1 protein and an antibody or an antigen-binding fragment thereof, and measuring the ALP generated when differentiation into osteoblasts is induced. The activity of USAG-1 to neutralize the Wnt antagonist activity (also referred to as "Wnt antagonist neutralizing activity") can be measured in vitro, for example, by reporter assay. The reporter assay is carried out, for example, by introducing a vector in which a promoter region responsive to BMP or Wnt is linked to a reporter gene such as luciferase into a cell, culturing the cell in the presence of BMP or Wnt by adding USAG-1 protein and an antibody or an antigen-binding fragment thereof, and measuring the expressed luciferase activity. The BMP antagonist activity neutralized by the anti-USAG-1 antibody and its antigen-binding fragment of the present application may be antagonist activity against any of the BMP families. For example, the anti-USAG-1 antibody and its antigen-binding fragment of the present application may neutralize antagonist activity against BMP2, BMP4, BMP6, BMP7, etc., but is not limited thereto. In addition, the Wnt antagonist activity neutralized by the anti-USAG-1 antibody and its antigen-binding fragment of the present application may be antagonist activity against any of the Wnt families. For example, the anti-USAG-1 antibodies and antigen-binding fragments thereof of the present application may neutralize antagonist activity against, but is not limited to, Wnt-1, Wnt-3, and the like.

[0016] Furthermore, in the present invention, the sequences of five of the obtained antibodies, Antibody A, Antibody B, Antibody C, Antibody D, and Antibody E, were determined and analyzed, and the variable region and complementarity determining region of each antibody were also determined. That is, Antibody A comprises a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 1 and a light chain comprising the amino acid sequence shown in SEQ ID NO: 2, the heavy chain comprises a heavy chain variable region (SEQ ID NO: 3) comprising heavy chain complementarity determining regions comprising the amino acid sequences shown in SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, and the light chain comprises a light chain variable region (SEQ ID NO: 4) comprising light chain complementarity determining regions shown in SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10. Antibody B comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:11 and a light chain comprising the amino acid sequence set forth in SEQ ID NO:12, the heavy chain comprising a heavy chain variable region (SEQ ID NO:13) comprising heavy chain complementarity determining regions set forth in SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17, and the light chain comprising a light chain variable region (SEQ ID NO:14) comprising light chain complementarity determining regions set forth in SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20. Antibody C comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:21 and a light chain comprising the amino acid sequence set forth in SEQ ID NO:22, the heavy chain comprising a heavy chain variable region (SEQ ID NO:23) comprising heavy chain complementarity determining regions set forth in SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27, and the light chain comprising a light chain variable region (SEQ ID NO:24) comprising light chain complementarity determining regions set forth in SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30. Antibody D comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:38 and a light chain comprising the amino acid sequence set forth in SEQ ID NO:39, wherein the heavy chain comprises a heavy chain variable region (SEQ ID NO:40) comprising heavy chain complementarity determining regions set forth in SEQ ID NO:42, SEQ ID NO:43, and SEQ ID NO:44, and the light chain comprises a light chain variable region (SEQ ID NO:41) comprising light chain complementarity determining regions set forth in SEQ ID NO:45, SEQ ID NO:46, and SEQ ID NO:47.Antibody E comprises a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 48 and a light chain comprising the amino acid sequence shown in SEQ ID NO: 49, the heavy chain comprising a heavy chain variable region (SEQ ID NO: 50) comprising the heavy chain complementarity determining regions shown in SEQ ID NO: 52, SEQ ID NO: 53, and SEQ ID NO: 54, and the light chain comprising a light chain variable region (SEQ ID NO: 51) comprising the light chain complementarity determining regions shown in SEQ ID NO: 55, SEQ ID NO: 56, and SEQ ID NO: 57. Antibody A, antibody B, and antibody C have particularly BMP antagonist neutralizing activity. Antibody C has particularly both BMP antagonist neutralizing activity and Wnt antagonist neutralizing activity. Antibody D and antibody E have particularly Wnt antagonist neutralizing activity.

[0017] Thus, in one embodiment of the present invention, the antibodies or antigen-binding fragments thereof of the present application include antibody A, antibody B, antibody C, antibody D, and antibody E, and variants thereof. As an example of antibody A or its variant, there is provided an antibody or antigen-binding fragment thereof that specifically binds to and neutralizes USAG-1, and includes three heavy chain complementarity determining regions comprising amino acid sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequences shown in SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7, respectively, or three light chain complementarity determining regions comprising amino acid sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequences shown in SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10, respectively. Preferably, there is provided an antibody or antigen-binding fragment thereof that specifically binds to and neutralizes USAG-1, the antibody or antigen-binding fragment thereof comprising three heavy chain complementarity determining regions comprising the amino acid sequences set forth in SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7, respectively, or three light chain complementarity determining regions comprising the amino acid sequences set forth in SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10, respectively. More preferably, there is provided an antibody or antigen-binding fragment thereof that specifically binds to and neutralizes USAG-1, the antibody or antigen-binding fragment thereof comprising three heavy chain complementarity determining regions consisting of amino acid sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequences set forth in SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7, respectively, or three light chain complementarity determining regions consisting of amino acid sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequences set forth in SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10, respectively.Even more preferably, there is provided an antibody or antigen-binding fragment thereof that specifically binds to and neutralizes USAG-1, the antibody or antigen-binding fragment thereof comprising three heavy chain complementarity determining regions consisting of the amino acid sequences shown in SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7, respectively, or three light chain complementarity determining regions consisting of the amino acid sequences shown in SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10, respectively.

[0018] As a further example of antibody A or a variant thereof, there is provided an antibody or antigen-binding fragment thereof that specifically binds to and neutralizes USAG-1, the antibody or antigen-binding fragment thereof comprising three heavy chain complementarity determining regions comprising the amino acid sequences shown in SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7, respectively, or three light chain complementarity determining regions comprising the amino acid sequences shown in SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10, respectively, and one to several amino acid residues have been substituted, deleted, inserted, or added in one or more of the above amino acid sequences. Preferably, there is provided an antibody or antigen-binding fragment thereof that specifically binds to and neutralizes USAG-1, the antibody or antigen-binding fragment thereof comprising three heavy chain complementarity determining regions consisting of the amino acid sequences shown in SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7, respectively, or three light chain complementarity determining regions consisting of the amino acid sequences shown in SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10, respectively, and one to several amino acid residues have been substituted, deleted, inserted, or added in one or more of the above amino acid sequences.

[0019] As a further example of antibody A or a variant thereof, there is provided an antibody or antigen-binding fragment thereof that specifically binds and neutralizes USAG-1, the antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 3, or a light chain variable region comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 4. Preferably, there is provided an antibody or antigen-binding fragment thereof that specifically binds and neutralizes USAG-1, the antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 3, or a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 4. More preferably, there is provided an antibody or antigen-binding fragment thereof that specifically binds to and neutralizes USAG-1, the antibody or antigen-binding fragment thereof comprising a heavy chain variable region consisting of an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence shown in SEQ ID NO: 3, or a light chain variable region consisting of an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence shown in SEQ ID NO: 4. Still more preferably, there is provided an antibody or antigen-binding fragment thereof that specifically binds to and neutralizes USAG-1, the antibody or antigen-binding fragment thereof comprising a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 3, or a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 4.

[0020] As a further example of antibody A or its variant, there is provided an antibody or antigen-binding fragment thereof that specifically binds to and neutralizes USAG-1, the antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 3 or a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 4, in which one to several amino acid residues have been substituted, deleted, inserted or added in the amino acid sequence. Preferably, there is provided an antibody or antigen-binding fragment thereof that specifically binds to and neutralizes USAG-1, the antibody or antigen-binding fragment thereof comprising a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 3 or a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 4, in which one to several amino acid residues have been substituted, deleted, inserted or added in the amino acid sequence.

[0021] As a further example of antibody A or a variant thereof, there is provided an antibody or antigen-binding fragment thereof that specifically binds and neutralizes USAG-1, the antibody or antigen-binding fragment thereof comprising three heavy chain complementarity determining regions comprising amino acid sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequences set forth in SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7, respectively, and three light chain complementarity determining regions comprising amino acid sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequences set forth in SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10, respectively. Preferably, there is provided an antibody or antigen-binding fragment thereof that specifically binds to and neutralizes USAG-1, the antibody or antigen-binding fragment thereof comprising three heavy chain complementarity determining regions comprising the amino acid sequences set forth in SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7, respectively, and three light chain complementarity determining regions comprising the amino acid sequences set forth in SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10, respectively. More preferably, there is provided an antibody or antigen-binding fragment thereof that specifically binds to and neutralizes USAG-1, the antibody or antigen-binding fragment thereof comprising three heavy chain complementarity determining regions consisting of amino acid sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequences set forth in SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7, respectively, and three light chain complementarity determining regions consisting of amino acid sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequences set forth in SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10, respectively.Even more preferably, there is provided an antibody or antigen-binding fragment thereof that specifically binds to and neutralizes USAG-1, the antibody or antigen-binding fragment thereof comprising three heavy chain complementarity determining regions consisting of the amino acid sequences shown in SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7, respectively, and three light chain complementarity determining regions consisting of the amino acid sequences shown in SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10, respectively.

[0022] As a further example of antibody A or a variant thereof, there is provided an antibody or antigen-binding fragment thereof that specifically binds to and neutralizes USAG-1, the antibody or antigen-binding fragment thereof comprising three heavy chain complementarity determining regions comprising the amino acid sequences shown in SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7, respectively, and three light chain complementarity determining regions comprising the amino acid sequences shown in SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10, respectively, with one to several amino acid residues being substituted, deleted, inserted, or added in one or more of the above amino acid sequences. Preferably, there is provided an antibody or antigen-binding fragment thereof that specifically binds to and neutralizes USAG-1, the antibody or antigen-binding fragment thereof comprising three heavy chain complementarity determining regions consisting of the amino acid sequences shown in SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7, respectively, and three light chain complementarity determining regions consisting of the amino acid sequences shown in SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10, respectively, with one to several amino acid residues being substituted, deleted, inserted, or added in one or more of the above amino acid sequences.

[0023] As a further example of antibody A or a variant thereof, there is provided an antibody or antigen-binding fragment thereof that specifically binds and neutralizes USAG-1, the antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 3, and a light chain variable region comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 4. Preferably, there is provided an antibody or antigen-binding fragment thereof that specifically binds and neutralizes USAG-1, the antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 3, and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 4. More preferably, there is provided an antibody or antigen-binding fragment thereof that specifically binds to and neutralizes USAG-1, the antibody or antigen-binding fragment thereof comprising a heavy chain variable region consisting of an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence shown in SEQ ID NO: 3, and a light chain variable region consisting of an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence shown in SEQ ID NO: 4. Still more preferably, there is provided an antibody or antigen-binding fragment thereof that specifically binds to and neutralizes USAG-1, the antibody or antigen-binding fragment thereof comprising a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 3, and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 4.

[0024] As a further example of antibody A or its variant, there is provided an antibody or antigen-binding fragment thereof that specifically binds to and neutralizes USAG-1, the antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 3 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 4, with one to several amino acid residues being substituted, deleted, inserted or added in one or more of the above amino acid sequences. Preferably, there is provided an antibody or antigen-binding fragment thereof that specifically binds to and neutralizes USAG-1, the antibody or antigen-binding fragment thereof comprising a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 3 and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 4, with one to several amino acid residues being substituted, deleted, inserted or added in one or more of the above amino acid sequences.

[0025] As an example of antibody B or a variant thereof, there is provided an antibody or antigen-binding fragment thereof that specifically binds to and neutralizes USAG-1, the antibody or antigen-binding fragment thereof comprising three heavy chain complementarity determining regions comprising amino acid sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequences shown in SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17, respectively, or three light chain complementarity determining regions comprising amino acid sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequences shown in SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20, respectively. Preferably, there is provided an antibody or antigen-binding fragment thereof that specifically binds to and neutralizes USAG-1, the antibody or antigen-binding fragment thereof comprising three heavy chain complementarity determining regions comprising the amino acid sequences set forth in SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17, respectively, or three light chain complementarity determining regions comprising the amino acid sequences set forth in SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20, respectively. More preferably, there is provided an antibody or antigen-binding fragment thereof that specifically binds to and neutralizes USAG-1, the antibody or antigen-binding fragment thereof comprising three heavy chain complementarity determining regions consisting of amino acid sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequences set forth in SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17, respectively, or three light chain complementarity determining regions consisting of amino acid sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequences set forth in SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20, respectively.Even more preferably, there is provided an antibody or antigen-binding fragment thereof that specifically binds to and neutralizes USAG-1, the antibody or antigen-binding fragment thereof comprising three heavy chain complementarity determining regions consisting of the amino acid sequences shown in SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17, respectively, or three light chain complementarity determining regions consisting of the amino acid sequences shown in SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20, respectively.

[0026] As a further example of antibody B or a variant thereof, there is provided an antibody or antigen-binding fragment thereof that specifically binds to and neutralizes USAG-1, the antibody or antigen-binding fragment thereof comprising three heavy chain complementarity determining regions comprising the amino acid sequences shown in SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17, respectively, or three light chain complementarity determining regions comprising the amino acid sequences shown in SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20, respectively, and one to several amino acid residues have been substituted, deleted, inserted, or added in one or more of the above amino acid sequences. Preferably, there is provided an antibody or antigen-binding fragment thereof that specifically binds to and neutralizes USAG-1, the antibody or antigen-binding fragment thereof comprising three heavy chain complementarity determining regions consisting of the amino acid sequences shown in SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17, respectively, or three light chain complementarity determining regions consisting of the amino acid sequences shown in SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20, respectively, and one to several amino acid residues have been substituted, deleted, inserted, or added in one or more of the above amino acid sequences.

[0027] As a further example of antibody B or a variant thereof, there is provided an antibody or antigen-binding fragment thereof that specifically binds and neutralizes USAG-1, the antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 13, or a light chain variable region comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 14. Preferably, there is provided an antibody or antigen-binding fragment thereof that specifically binds and neutralizes USAG-1, the antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 13, or a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 14. More preferably, there is provided an antibody or antigen-binding fragment thereof that specifically binds to and neutralizes USAG-1, the antibody or antigen-binding fragment thereof comprising a heavy chain variable region consisting of an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence shown in SEQ ID NO: 13, or a light chain variable region consisting of an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence shown in SEQ ID NO: 14. Still more preferably, there is provided an antibody or antigen-binding fragment thereof that specifically binds to and neutralizes USAG-1, the antibody or antigen-binding fragment thereof comprising a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 13, or a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 14.

[0028] As a further example of antibody B or a variant thereof, there is provided an antibody or antigen-binding fragment thereof that specifically binds to and neutralizes USAG-1, the antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 13 or a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 14, in which one to several amino acid residues have been substituted, deleted, inserted or added in the amino acid sequence. Preferably, there is provided an antibody or antigen-binding fragment thereof that specifically binds to and neutralizes USAG-1, the antibody or antigen-binding fragment thereof comprising a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 13 or a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 14, in which one to several amino acid residues have been substituted, deleted, inserted or added in the amino acid sequence.

[0029] As a further example of antibody B or a variant thereof, there is provided an antibody or antigen-binding fragment thereof that specifically binds and neutralizes USAG-1, the antibody or antigen-binding fragment thereof comprising three heavy chain complementarity determining regions comprising amino acid sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequences set forth in SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17, respectively, and three light chain complementarity determining regions comprising amino acid sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequences set forth in SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20, respectively. Preferably, there is provided an antibody or antigen-binding fragment thereof that specifically binds to and neutralizes USAG-1, the antibody or antigen-binding fragment thereof comprising three heavy chain complementarity determining regions comprising the amino acid sequences set forth in SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17, respectively, and three light chain complementarity determining regions comprising the amino acid sequences set forth in SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20, respectively. More preferably, there is provided an antibody or antigen-binding fragment thereof that specifically binds to and neutralizes USAG-1, the antibody or antigen-binding fragment thereof comprising three heavy chain complementarity determining regions consisting of amino acid sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequences set forth in SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17, respectively, and three light chain complementarity determining regions consisting of amino acid sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequences set forth in SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20, respectively.Even more preferably, there is provided an antibody or antigen-binding fragment thereof that specifically binds to and neutralizes USAG-1, the antibody or antigen-binding fragment thereof comprising three heavy chain complementarity determining regions consisting of the amino acid sequences set forth in SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17, respectively, and three light chain complementarity determining regions consisting of the amino acid sequences set forth in SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20, respectively.

[0030] As a further example of antibody B or a variant thereof, there is provided an antibody or antigen-binding fragment thereof that specifically binds to and neutralizes USAG-1, the antibody or antigen-binding fragment thereof comprising three heavy chain complementarity determining regions comprising the amino acid sequences shown in SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17, respectively, and three light chain complementarity determining regions comprising the amino acid sequences shown in SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20, respectively, with one to several amino acid residues being substituted, deleted, inserted, or added in one or more of the above amino acid sequences. Preferably, there is provided an antibody or antigen-binding fragment thereof that specifically binds to and neutralizes USAG-1, the antibody or antigen-binding fragment thereof comprising three heavy chain complementarity determining regions consisting of the amino acid sequences shown in SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17, respectively, and three light chain complementarity determining regions consisting of the amino acid sequences shown in SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20, respectively, with one to several amino acid residues being substituted, deleted, inserted, or added in one or more of the above amino acid sequences.

[0031] As a further example of antibody B or a variant thereof, there is provided an antibody or antigen-binding fragment thereof that specifically binds and neutralizes USAG-1, the antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 13, and a light chain variable region comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 14. Preferably, there is provided an antibody or antigen-binding fragment thereof that specifically binds and neutralizes USAG-1, the antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 13, and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 14. More preferably, there is provided an antibody or antigen-binding fragment thereof that specifically binds to and neutralizes USAG-1, the antibody or antigen-binding fragment thereof comprising a heavy chain variable region consisting of an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 13, and a light chain variable region consisting of an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 14. Still more preferably, there is provided an antibody or antigen-binding fragment thereof that specifically binds to and neutralizes USAG-1, the antibody or antigen-binding fragment thereof comprising a heavy chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 13, and a light chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 14.

[0032] As a further example of antibody B or a variant thereof, there is provided an antibody or antigen-binding fragment thereof that specifically binds to and neutralizes USAG-1, the antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 13 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 14, with one to several amino acid residues being substituted, deleted, inserted or added in one or more of the above amino acid sequences. Preferably, there is provided an antibody or antigen-binding fragment thereof that specifically binds to and neutralizes USAG-1, the antibody or antigen-binding fragment thereof comprising a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 13 and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 14, with one to several amino acid residues being substituted, deleted, inserted or added in one or more of the above amino acid sequences.

[0033] As an example of antibody C or its variant, there is provided an antibody or antigen-binding fragment thereof that specifically binds to and neutralizes USAG-1, the antibody or antigen-binding fragment thereof comprising three heavy chain complementarity determining regions comprising amino acid sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequences shown in SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27, respectively, or three light chain complementarity determining regions comprising amino acid sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequences shown in SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30, respectively. Preferably, there is provided an antibody or antigen-binding fragment thereof that specifically binds to and neutralizes USAG-1, the antibody or antigen-binding fragment thereof comprising three heavy chain complementarity determining regions comprising the amino acid sequences set forth in SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27, respectively, or three light chain complementarity determining regions comprising the amino acid sequences set forth in SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30, respectively. More preferably, there is provided an antibody or antigen-binding fragment thereof that specifically binds to and neutralizes USAG-1, the antibody or antigen-binding fragment thereof comprising three heavy chain complementarity determining regions consisting of amino acid sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequences set forth in SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27, respectively, or three light chain complementarity determining regions consisting of amino acid sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequences set forth in SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30, respectively.Even more preferably, there is provided an antibody or antigen-binding fragment thereof that specifically binds to and neutralizes USAG-1, the antibody or antigen-binding fragment thereof comprising three heavy chain complementarity determining regions consisting of the amino acid sequences shown in SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27, respectively, or three light chain complementarity determining regions consisting of the amino acid sequences shown in SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30, respectively.

[0034] As a further example of antibody C or a variant thereof, there is provided an antibody or antigen-binding fragment thereof that specifically binds to and neutralizes USAG-1, the antibody or antigen-binding fragment thereof comprising three heavy chain complementarity determining regions comprising the amino acid sequences shown in SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27, respectively, or three light chain complementarity determining regions comprising the amino acid sequences shown in SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30, respectively, and one to several amino acid residues have been substituted, deleted, inserted, or added in one or more of the above amino acid sequences. Preferably, there is provided an antibody or antigen-binding fragment thereof that specifically binds to and neutralizes USAG-1, the antibody or antigen-binding fragment thereof comprising three heavy chain complementarity determining regions consisting of the amino acid sequences shown in SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27, respectively, or three light chain complementarity determining regions consisting of the amino acid sequences shown in SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30, respectively, and one to several amino acid residues have been substituted, deleted, inserted, or added in one or more of the above amino acid sequences.

[0035] As a further example of antibody C or a variant thereof, there is provided an antibody or antigen-binding fragment thereof that specifically binds to and neutralizes USAG-1, the antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 23, or a light chain variable region comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 24. Preferably, there is provided an antibody or antigen-binding fragment thereof that specifically binds to and neutralizes USAG-1, the antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 23, or a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 24. More preferably, there is provided an antibody or antigen-binding fragment thereof that specifically binds to and neutralizes USAG-1, the antibody or antigen-binding fragment thereof comprising a heavy chain variable region consisting of an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence shown in SEQ ID NO: 23, or a light chain variable region consisting of an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence shown in SEQ ID NO: 24. Still more preferably, there is provided an antibody or antigen-binding fragment thereof that specifically binds to and neutralizes USAG-1, the antibody or antigen-binding fragment thereof comprising a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 23, or a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 24.

[0036] As a further example of antibody C or a variant thereof, there is provided an antibody or antigen-binding fragment thereof that specifically binds to and neutralizes USAG-1, the antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 23 or a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 24, in which one to several amino acid residues have been substituted, deleted, inserted or added in the amino acid sequence. Preferably, there is provided an antibody or antigen-binding fragment thereof that specifically binds to and neutralizes USAG-1, the antibody or antigen-binding fragment thereof comprising a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 23 or a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 24, in which one to several amino acid residues have been substituted, deleted, inserted or added in the amino acid sequence.

[0037] As a further example of antibody C or a variant thereof, there is provided an antibody or antigen-binding fragment thereof that specifically binds to and neutralizes USAG-1, the antibody or antigen-binding fragment thereof comprising three heavy chain complementarity determining regions comprising amino acid sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequences set forth in SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27, respectively, and three light chain complementarity determining regions comprising amino acid sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequences set forth in SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30, respectively. Preferably, there is provided an antibody or antigen-binding fragment thereof that specifically binds to and neutralizes USAG-1, the antibody or antigen-binding fragment thereof comprising three heavy chain complementarity determining regions comprising the amino acid sequences set forth in SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27, respectively, and three light chain complementarity determining regions comprising the amino acid sequences set forth in SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30, respectively. More preferably, there is provided an antibody or antigen-binding fragment thereof that specifically binds to and neutralizes USAG-1, the antibody or antigen-binding fragment thereof comprising three heavy chain complementarity determining regions consisting of amino acid sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequences set forth in SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27, respectively, and three light chain complementarity determining regions consisting of amino acid sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequences set forth in SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30, respectively.Even more preferably, there is provided an antibody or antigen-binding fragment thereof that specifically binds to and neutralizes USAG-1, the antibody or antigen-binding fragment thereof comprising three heavy chain complementarity determining regions consisting of the amino acid sequences set forth in SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27, respectively, and three light chain complementarity determining regions consisting of the amino acid sequences set forth in SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30, respectively.

[0038] As a further example of antibody C or a variant thereof, there is provided an antibody or antigen-binding fragment thereof that specifically binds to and neutralizes USAG-1, the antibody or antigen-binding fragment thereof comprising three heavy chain complementarity determining regions comprising the amino acid sequences shown in SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27, respectively, and three light chain complementarity determining regions comprising the amino acid sequences shown in SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30, respectively, with one to several amino acid residues being substituted, deleted, inserted, or added in one or more of the above amino acid sequences. Preferably, there is provided an antibody or antigen-binding fragment thereof that specifically binds to and neutralizes USAG-1, the antibody or antigen-binding fragment thereof comprising three heavy chain complementarity determining regions consisting of the amino acid sequences shown in SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27, respectively, and three light chain complementarity determining regions consisting of the amino acid sequences shown in SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30, respectively, with one to several amino acid residues being substituted, deleted, inserted, or added in one or more of the above amino acid sequences.

[0039] As a further example of antibody C or a variant thereof, there is provided an antibody or antigen-binding fragment thereof that specifically binds and neutralizes USAG-1, the antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 23, and a light chain variable region comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 24. Preferably, there is provided an antibody or antigen-binding fragment thereof that specifically binds and neutralizes USAG-1, the antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 23, and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 24. More preferably, there is provided an antibody or antigen-binding fragment thereof that specifically binds to and neutralizes USAG-1, the antibody or antigen-binding fragment thereof comprising a heavy chain variable region consisting of an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 23, and a light chain variable region consisting of an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 24. Still more preferably, there is provided an antibody or antigen-binding fragment thereof that specifically binds to and neutralizes USAG-1, the antibody or antigen-binding fragment thereof comprising a heavy chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 23, and a light chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 24.

[0040] As a further example of antibody C or a variant thereof, there is provided an antibody or antigen-binding fragment thereof that specifically binds to and neutralizes USAG-1, the antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 23 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 24, with one to several amino acid residues being substituted, deleted, inserted or added in one or more of the above amino acid sequences. Preferably, there is provided an antibody or antigen-binding fragment thereof that specifically binds to and neutralizes USAG-1, the antibody or antigen-binding fragment thereof comprising a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 23 and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 24, with one to several amino acid residues being substituted, deleted, inserted or added in one or more of the above amino acid sequences.

[0041] As an example of antibody D or a variant thereof, there is provided an antibody or antigen-binding fragment thereof that specifically binds to and neutralizes USAG-1, the antibody or antigen-binding fragment thereof comprising three heavy chain complementarity determining regions comprising amino acid sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequences set forth in SEQ ID NO:42, SEQ ID NO:43, and SEQ ID NO:44, respectively, or three light chain complementarity determining regions comprising amino acid sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequences set forth in SEQ ID NO:45, SEQ ID NO:46, and SEQ ID NO:47, respectively. Preferably, there is provided an antibody or antigen-binding fragment thereof that specifically binds to and neutralizes USAG-1, the antibody or antigen-binding fragment thereof comprising three heavy chain complementarity determining regions comprising the amino acid sequences set forth in SEQ ID NO:42, SEQ ID NO:43, and SEQ ID NO:44, respectively, or three light chain complementarity determining regions comprising the amino acid sequences set forth in SEQ ID NO:45, SEQ ID NO:46, and SEQ ID NO:47, respectively. More preferably, there is provided an antibody or antigen-binding fragment thereof that specifically binds to and neutralizes USAG-1, the antibody or antigen-binding fragment thereof comprising three heavy chain complementarity determining regions consisting of amino acid sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequences set forth in SEQ ID NO:42, SEQ ID NO:43, and SEQ ID NO:44, respectively, or three light chain complementarity determining regions consisting of amino acid sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequences set forth in SEQ ID NO:45, SEQ ID NO:46, and SEQ ID NO:47, respectively.Even more preferably, there is provided an antibody or antigen-binding fragment thereof that specifically binds to and neutralizes USAG-1, the antibody or antigen-binding fragment thereof comprising three heavy chain complementarity determining regions consisting of the amino acid sequences set forth in SEQ ID NO:42, SEQ ID NO:43, and SEQ ID NO:44, respectively, or three light chain complementarity determining regions consisting of the amino acid sequences set forth in SEQ ID NO:45, SEQ ID NO:46, and SEQ ID NO:47, respectively.

[0042] As a further example of antibody D or its variant, there is provided an antibody or antigen-binding fragment thereof that specifically binds to and neutralizes USAG-1, the antibody or antigen-binding fragment thereof comprising three heavy chain complementarity determining regions comprising the amino acid sequences shown in SEQ ID NO: 42, SEQ ID NO: 43, and SEQ ID NO: 44, respectively, or three light chain complementarity determining regions comprising the amino acid sequences shown in SEQ ID NO: 45, SEQ ID NO: 46, and SEQ ID NO: 47, respectively, and one to several amino acid residues have been substituted, deleted, inserted, or added in one or more of the above amino acid sequences. Preferably, there is provided an antibody or antigen-binding fragment thereof that specifically binds to and neutralizes USAG-1, the antibody or antigen-binding fragment thereof comprising three heavy chain complementarity determining regions consisting of the amino acid sequences shown in SEQ ID NO: 42, SEQ ID NO: 43, and SEQ ID NO: 44, respectively, or three light chain complementarity determining regions consisting of the amino acid sequences shown in SEQ ID NO: 45, SEQ ID NO: 46, and SEQ ID NO: 47, respectively, and one to several amino acid residues have been substituted, deleted, inserted, or added in one or more of the above amino acid sequences.

[0043] As a further example of antibody D or a variant thereof, there is provided an antibody or antigen-binding fragment thereof that specifically binds and neutralizes USAG-1, the antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 40, or a light chain variable region comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 41. Preferably, there is provided an antibody or antigen-binding fragment thereof that specifically binds and neutralizes USAG-1, the antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 40, or a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 41. More preferably, there is provided an antibody or antigen-binding fragment thereof that specifically binds to and neutralizes USAG-1, the antibody or antigen-binding fragment thereof comprising a heavy chain variable region consisting of an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence shown in SEQ ID NO: 40, or a light chain variable region consisting of an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence shown in SEQ ID NO: 41. Still more preferably, there is provided an antibody or antigen-binding fragment thereof that specifically binds to and neutralizes USAG-1, the antibody or antigen-binding fragment thereof comprising a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 40, or a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 41.

[0044] As a further example of antibody D or its variant, there is provided an antibody or antigen-binding fragment thereof that specifically binds to and neutralizes USAG-1, the antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 40 or a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 41, in which one to several amino acid residues have been substituted, deleted, inserted or added in one or more of the amino acid sequences. Preferably, there is provided an antibody or antigen-binding fragment thereof that specifically binds to and neutralizes USAG-1, the antibody or antigen-binding fragment thereof comprising a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 40 or a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 41, in which one to several amino acid residues have been substituted, deleted, inserted or added in one or more of the amino acid sequences.

[0045] As a further example of antibody D or a variant thereof, there is provided an antibody or antigen-binding fragment thereof that specifically binds and neutralizes USAG-1, the antibody or antigen-binding fragment thereof comprising three heavy chain complementarity determining regions comprising amino acid sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequences set forth in SEQ ID NO:42, SEQ ID NO:43, and SEQ ID NO:44, respectively, and three light chain complementarity determining regions comprising amino acid sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequences set forth in SEQ ID NO:45, SEQ ID NO:46, and SEQ ID NO:47, respectively. Preferably, there is provided an antibody or antigen-binding fragment thereof that specifically binds to and neutralizes USAG-1, the antibody or antigen-binding fragment thereof comprising three heavy chain complementarity determining regions comprising the amino acid sequences set forth in SEQ ID NO:42, SEQ ID NO:43, and SEQ ID NO:44, respectively, and three light chain complementarity determining regions comprising the amino acid sequences set forth in SEQ ID NO:45, SEQ ID NO:46, and SEQ ID NO:47, respectively. More preferably, there is provided an antibody or antigen-binding fragment thereof that specifically binds to and neutralizes USAG-1, the antibody or antigen-binding fragment thereof comprising three heavy chain complementarity determining regions consisting of amino acid sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequences set forth in SEQ ID NO:42, SEQ ID NO:43, and SEQ ID NO:44, respectively, and three light chain complementarity determining regions consisting of amino acid sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequences set forth in SEQ ID NO:45, SEQ ID NO:46, and SEQ ID NO:47, respectively.Even more preferably, there is provided an antibody or antigen-binding fragment thereof that specifically binds to and neutralizes USAG-1, the antibody or antigen-binding fragment thereof comprising three heavy chain complementarity determining regions consisting of the amino acid sequences set forth in SEQ ID NO:42, SEQ ID NO:43, and SEQ ID NO:44, respectively, and three light chain complementarity determining regions consisting of the amino acid sequences set forth in SEQ ID NO:45, SEQ ID NO:46, and SEQ ID NO:47, respectively.

[0046] As a further example of antibody D or its variant, there is provided an antibody or antigen-binding fragment thereof that specifically binds to and neutralizes USAG-1, the antibody or antigen-binding fragment thereof comprising three heavy chain complementarity determining regions comprising the amino acid sequences shown in SEQ ID NO: 42, SEQ ID NO: 43, and SEQ ID NO: 44, respectively, and three light chain complementarity determining regions comprising the amino acid sequences shown in SEQ ID NO: 45, SEQ ID NO: 46, and SEQ ID NO: 47, respectively, with one to several amino acid residues being substituted, deleted, inserted, or added in one or more of the amino acid sequences. Preferably, there is provided an antibody or antigen-binding fragment thereof that specifically binds to and neutralizes USAG-1, the antibody or antigen-binding fragment thereof comprising three heavy chain complementarity determining regions consisting of the amino acid sequences shown in SEQ ID NO: 42, SEQ ID NO: 43, and SEQ ID NO: 44, respectively, and three light chain complementarity determining regions consisting of the amino acid sequences shown in SEQ ID NO: 45, SEQ ID NO: 46, and SEQ ID NO: 47, respectively, with one to several amino acid residues being substituted, deleted, inserted, or added in one or more of the amino acid sequences.

[0047] As a further example of antibody D or a variant thereof, there is provided an antibody or antigen-binding fragment thereof that specifically binds and neutralizes USAG-1, the antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 40, and a light chain variable region comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 41. Preferably, there is provided an antibody or antigen-binding fragment thereof that specifically binds and neutralizes USAG-1, the antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 40, and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 41. More preferably, there is provided an antibody or antigen-binding fragment thereof that specifically binds to and neutralizes USAG-1, the antibody or antigen-binding fragment thereof comprising a heavy chain variable region consisting of an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 40, and a light chain variable region consisting of an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 41. Still more preferably, there is provided an antibody or antigen-binding fragment thereof that specifically binds to and neutralizes USAG-1, the antibody or antigen-binding fragment thereof comprising a heavy chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 40, and a light chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 41.

[0048] As a further example of antibody D or a variant thereof, there is provided an antibody or antigen-binding fragment thereof that specifically binds to and neutralizes USAG-1, the antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 40 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 41, with one to several amino acid residues being substituted, deleted, inserted or added in one or more of the above amino acid sequences. Preferably, there is provided an antibody or antigen-binding fragment thereof that specifically binds to and neutralizes USAG-1, the antibody or antigen-binding fragment thereof comprising a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 40 and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 41, with one to several amino acid residues being substituted, deleted, inserted or added in one or more of the above amino acid sequences.

[0049] As an example of antibody E or a variant thereof, there is provided an antibody or antigen-binding fragment thereof that specifically binds to and neutralizes USAG-1, the antibody or antigen-binding fragment thereof comprising three heavy chain complementarity determining regions comprising amino acid sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequences set forth in SEQ ID NO:52, SEQ ID NO:53, and SEQ ID NO:54, respectively, or three light chain complementarity determining regions comprising amino acid sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequences set forth in SEQ ID NO:55, SEQ ID NO:56, and SEQ ID NO:57, respectively. Preferably, there is provided an antibody or antigen-binding fragment thereof that specifically binds to and neutralizes USAG-1, the antibody or antigen-binding fragment thereof comprising three heavy chain complementarity determining regions comprising the amino acid sequences set forth in SEQ ID NO:52, SEQ ID NO:53, and SEQ ID NO:54, respectively, or three light chain complementarity determining regions comprising the amino acid sequences set forth in SEQ ID NO:55, SEQ ID NO:56, and SEQ ID NO:57, respectively. More preferably, there is provided an antibody or antigen-binding fragment thereof that specifically binds to and neutralizes USAG-1, the antibody or antigen-binding fragment thereof comprising three heavy chain complementarity determining regions consisting of amino acid sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequences set forth in SEQ ID NO:52, SEQ ID NO:53, and SEQ ID NO:54, respectively, or three light chain complementarity determining regions consisting of amino acid sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequences set forth in SEQ ID NO:55, SEQ ID NO:56, and SEQ ID NO:57, respectively.Even more preferably, there is provided an antibody or antigen-binding fragment thereof that specifically binds to and neutralizes USAG-1, the antibody or antigen-binding fragment thereof comprising three heavy chain complementarity determining regions consisting of the amino acid sequences set forth in SEQ ID NO:52, SEQ ID NO:53, and SEQ ID NO:54, respectively, or three light chain complementarity determining regions consisting of the amino acid sequences set forth in SEQ ID NO:55, SEQ ID NO:56, and SEQ ID NO:57, respectively.

[0050] As a further example of antibody E or a variant thereof, there is provided an antibody or antigen-binding fragment thereof that specifically binds to and neutralizes USAG-1, the antibody or antigen-binding fragment thereof comprising three heavy chain complementarity determining regions comprising the amino acid sequences shown in SEQ ID NO:52, SEQ ID NO:53, and SEQ ID NO:54, respectively, or three light chain complementarity determining regions comprising the amino acid sequences shown in SEQ ID NO:55, SEQ ID NO:56, and SEQ ID NO:57, respectively, and one to several amino acid residues have been substituted, deleted, inserted, or added in one or more of the above amino acid sequences. Preferably, there is provided an antibody or antigen-binding fragment thereof that specifically binds to and neutralizes USAG-1, the antibody or antigen-binding fragment thereof comprising three heavy chain complementarity determining regions consisting of the amino acid sequences shown in SEQ ID NO:52, SEQ ID NO:53, and SEQ ID NO:54, respectively, or three light chain complementarity determining regions consisting of the amino acid sequences shown in SEQ ID NO:55, SEQ ID NO:56, and SEQ ID NO:57, respectively, and one to several amino acid residues have been substituted, deleted, inserted, or added in one or more of the above amino acid sequences.

[0051] As a further example of antibody E or a variant thereof, there is provided an antibody or antigen-binding fragment thereof that specifically binds and neutralizes USAG-1, the antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 50, or a light chain variable region comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 51. Preferably, there is provided an antibody or antigen-binding fragment thereof that specifically binds and neutralizes USAG-1, the antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 50, or a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 51. More preferably, there is provided an antibody or antigen-binding fragment thereof that specifically binds to and neutralizes USAG-1, the antibody or antigen-binding fragment thereof comprising a heavy chain variable region consisting of an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence shown in SEQ ID NO: 50, or a light chain variable region consisting of an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence shown in SEQ ID NO: 51. Still more preferably, there is provided an antibody or antigen-binding fragment thereof that specifically binds to and neutralizes USAG-1, the antibody or antigen-binding fragment thereof comprising a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 50, or a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 51.

[0052] As a further example of antibody E or a mutant thereof, there is provided an antibody or antigen-binding fragment thereof that specifically binds to and neutralizes USAG-1, the antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 50 or a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 51, in which one to several amino acid residues have been substituted, deleted, inserted or added in the amino acid sequence. Preferably, there is provided an antibody or antigen-binding fragment thereof that specifically binds to and neutralizes USAG-1, the antibody or antigen-binding fragment thereof comprising a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 50 or a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 51, in which one to several amino acid residues have been substituted, deleted, inserted or added in the amino acid sequence.

[0053] As a further example of antibody E or a variant thereof, there is provided an antibody or antigen-binding fragment thereof that specifically binds and neutralizes USAG-1, the antibody or antigen-binding fragment thereof comprising three heavy chain complementarity determining regions comprising amino acid sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequences set forth in SEQ ID NO:52, SEQ ID NO:53, and SEQ ID NO:54, respectively, and three light chain complementarity determining regions comprising amino acid sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequences set forth in SEQ ID NO:55, SEQ ID NO:56, and SEQ ID NO:57, respectively. Preferably, there is provided an antibody or antigen-binding fragment thereof that specifically binds to and neutralizes USAG-1, the antibody or antigen-binding fragment thereof comprising three heavy chain complementarity determining regions comprising the amino acid sequences set forth in SEQ ID NO:52, SEQ ID NO:53, and SEQ ID NO:54, respectively, and three light chain complementarity determining regions comprising the amino acid sequences set forth in SEQ ID NO:55, SEQ ID NO:56, and SEQ ID NO:57, respectively. More preferably, there is provided an antibody or antigen-binding fragment thereof that specifically binds and neutralizes USAG-1, the antibody or antigen-binding fragment thereof comprising three heavy chain complementarity determining regions consisting of amino acid sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequences set forth in SEQ ID NO:52, SEQ ID NO:53, and SEQ ID NO:54, respectively, and three light chain complementarity determining regions consisting of amino acid sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequences set forth in SEQ ID NO:55, SEQ ID NO:56, and SEQ ID NO:57, respectively.Even more preferably, there is provided an antibody or antigen-binding fragment thereof that specifically binds to and neutralizes USAG-1, the antibody or antigen-binding fragment thereof comprising three heavy chain complementarity determining regions consisting of the amino acid sequences set forth in SEQ ID NO:52, SEQ ID NO:53, and SEQ ID NO:54, respectively, and three light chain complementarity determining regions consisting of the amino acid sequences set forth in SEQ ID NO:55, SEQ ID NO:56, and SEQ ID NO:57, respectively.

[0054] As a further example of antibody E or a variant thereof, there is provided an antibody or antigen-binding fragment thereof that specifically binds to and neutralizes USAG-1, the antibody or antigen-binding fragment thereof comprising three heavy chain complementarity determining regions comprising the amino acid sequences shown in SEQ ID NO:52, SEQ ID NO:53, and SEQ ID NO:54, respectively, and three light chain complementarity determining regions comprising the amino acid sequences shown in SEQ ID NO:55, SEQ ID NO:56, and SEQ ID NO:57, respectively, and one to several amino acid residues have been substituted, deleted, inserted, or added in one or more of the above amino acid sequences. Preferably, there is provided an antibody or antigen-binding fragment thereof that specifically binds to and neutralizes USAG-1, the antibody or antigen-binding fragment thereof comprising three heavy chain complementarity determining regions consisting of the amino acid sequences shown in SEQ ID NO:52, SEQ ID NO:53, and SEQ ID NO:54, respectively, and three light chain complementarity determining regions consisting of the amino acid sequences shown in SEQ ID NO:55, SEQ ID NO:56, and SEQ ID NO:57, respectively, and one to several amino acid residues have been substituted, deleted, inserted, or added in one or more of the above amino acid sequences.

[0055] As a further example of antibody E or a variant thereof, there is provided an antibody or antigen-binding fragment thereof that specifically binds and neutralizes USAG-1, the antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 50, and a light chain variable region comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 51. Preferably, there is provided an antibody or antigen-binding fragment thereof that specifically binds and neutralizes USAG-1, the antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 50, and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 51. More preferably, there is provided an antibody or antigen-binding fragment thereof that specifically binds to and neutralizes USAG-1, the antibody or antigen-binding fragment thereof comprising a heavy chain variable region consisting of an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 50, and a light chain variable region consisting of an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 51. Still more preferably, there is provided an antibody or antigen-binding fragment thereof that specifically binds to and neutralizes USAG-1, the antibody or antigen-binding fragment thereof comprising a heavy chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 50, and a light chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 51.

[0056] As a further example of antibody E or a variant thereof, there is provided an antibody or antigen-binding fragment thereof that specifically binds to and neutralizes USAG-1, the antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 50 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 51, with one to several amino acid residues being substituted, deleted, inserted or added in one or more of the above amino acid sequences. Preferably, there is provided an antibody or antigen-binding fragment thereof that specifically binds to and neutralizes USAG-1, the antibody or antigen-binding fragment thereof comprising a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 50 and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 51, with one to several amino acid residues being substituted, deleted, inserted or added in one or more of the above amino acid sequences.

[0057] As used herein, "several" refers to about 2 to 10 amino acids, and although it depends on the length of the amino acid sequence, it is preferably about 2 to 7 amino acids, for example, 3, 4, 5, or 6 amino acids. In addition, as used herein, "substitution" may be a conservative or non-conservative substitution, and is preferably a conservative substitution. A conservative substitution is known to those skilled in the art and refers to a substitution that does not change the biological activity of the resulting molecule. Examples of conservative amino acid substitutions include alanine to glycine or serine, arginine to lysine or histidine, asparagine to glutamine or histidine, aspartic acid to glutamic acid or asparagine, cysteine ​​to serine or alanine, glutamine to asparagine, glutamic acid to aspartic acid or glutamine, glycine to alanine, histidine to asparagine or glutamine, and isoleucine to leucine or valine. , leucine to isoleucine or valine substitution, lysine to arginine or histidine substitution, methionine to leucine, isoleucine or tyrosine substitution, phenylalanine to tyrosine, methionine or leucine substitution, proline to alanine substitution, serine to threonine substitution, threonine to serine substitution, tryptophan to tyrosine or phenylalanine substitution, tyrosine to tryptophan or phenylalanine substitution, and valine to isoleucine or leucine substitution.

[0058] In the present specification, sequence identity may be determined by optimally aligning two sequences and using a conventional method. For example, it may be determined using algorithms known in the art, such as BLAST and FASTA. The antibody or antigen-binding fragment of the present application may have amino acid residues substituted, deleted, inserted or added within the above-mentioned sequence identity range.

[0059] Furthermore, in another aspect of the present invention, the antibody or antigen-binding fragment thereof of the present application is provided as an antibody or antigen-binding fragment thereof that binds to all or a part of the same epitope on USAG-1 to which the above-described antibody A, antibody B, antibody C, antibody D, or antibody E, or a variant or antigen-binding fragment thereof binds. Still further, the present invention provides an antibody or antigen-binding fragment thereof that competes with the above-described antibody A, antibody B, antibody C, antibody D, or antibody E, or a variant or antigen-binding fragment thereof for binding to USAG-1 or to all or a part of an epitope on USAG-1.

[0060] Furthermore, in the present invention, it was found that antibody A specifically recognizes and binds to a polypeptide (epitope) containing VNDKTRTQRI (SEQ ID NO: 31) on human USAG-1 (corresponding to the amino acid sequence of 134th to 143rd amino acids of human USAG-1 protein). Therefore, an antibody or antigen-binding fragment thereof that binds to a USAG-1 polypeptide containing the amino acid sequence shown in SEQ ID NO: 31 is also one embodiment of the antibody or antigen-binding fragment thereof of the present application. Furthermore, an antibody or antigen-binding fragment thereof that competes with the above-described antibody A or its mutant or antigen-binding fragment thereof for binding to a USAG-1 polypeptide containing the amino acid sequence shown in SEQ ID NO: 31 is also one embodiment of the antibody or antigen-binding fragment thereof of the present application. For example, the USAG-1 polypeptide may be a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 31. Furthermore, the USAG-1 polypeptide containing the amino acid sequence shown in SEQ ID NO: 31 may be a polypeptide that is substantially identical to the polypeptide. Examples of the substantially identical polypeptide include a polypeptide that is located at the corresponding position on the USAG-1 protein of an animal other than human.

[0061] As used herein, "competition" means that an antibody or antigen-binding fragment thereof competes with a reference antibody (e.g., antibody A, antibody B, antibody C, antibody D, antibody E, or a variant or antigen-binding fragment thereof) in a binding assay using a USAG-1 protein or polypeptide. For example, a test antibody or antigen-binding fragment thereof "competes" with a reference antibody if the test antibody or antigen-binding fragment thereof reduces the binding of the reference antibody to a USAG-1 protein or polypeptide in a binding assay. An antibody that competes with a reference antibody, for example, reduces the binding of the reference antibody to an antigen protein or polypeptide by at least about 40%, preferably at least about 50%, more preferably at least about 60%, even more preferably at least about 80%, and even more preferably at least 90%. Competitive binding assays can be performed by methods known in the art, including, but not limited to, ELISA, flow cytometry, SPR (surface plasmon resonance) method, BLI (Bio-Layer Interferometry) method, and the like.

[0062] Epitope binning is a technique for classifying two or more antibodies based on their epitopes, for example, using the SPR method or the BLI method. In epitope binning, for example, an antigen protein (target) is added to a biosensor on which a reference antibody is immobilized, and the reference antibody and the target are bound to each other. Then, the biosensor holding the complex of the reference antibody and the target is reacted with a test antibody, and the binding of the test antibody to the biosensor (i.e., binding to the target captured by the reference antibody immobilized on the biosensor) and dissociation are analyzed. If the test antibody shares the same epitope as the reference antibody, the test antibody cannot bind to the biosensor because the epitope on the target is already occupied by binding with the reference antibody. Conversely, if the test antibody recognizes an epitope different from that of the reference antibody, the test antibody can bind to the biosensor. Furthermore, if the test antibody recognizes a region that is structurally close to the epitope of the reference antibody, the binding of the test antibody to the epitope is prevented by the binding of the reference antibody to the target, and the test antibody cannot bind to the biosensor. Thus, epitope binning can be used to determine whether two or more antibody clones compete for binding to a target protein.

[0063] The antibody or antigen-binding fragment thereof of the present application binds to all or a portion of USAG-1 or an epitope on USAG-1 with a KD value of, for example, 1 μM or less, preferably 100 nM or less, more preferably 50 nM or less, even more preferably 30 nM or less, even more preferably 10 nM or less, even more preferably 8 nM or less, and even more preferably 5 nM or less.

[0064] In the present invention, the antibody is preferably an isolated antibody. In the present invention, the antibody may be either a polyclonal antibody or a monoclonal antibody. In the present invention, the antibody may be a human antibody, a humanized antibody, a chimeric antibody, or a multispecific antibody (e.g., a bispecific antibody). A humanized antibody includes a human immunoglobulin (recipient antibody) in which residues from the recipient's complementarity determining region (CDR) are replaced by residues from the CDR of a species other than human (donor antibody) having the desired specificity, affinity, and binding capacity. Optionally, Fv framework region (FR) residues of the human immunoglobulin may be replaced by corresponding non-human residues. Furthermore, a humanized antibody may contain residues that are not found in the recipient antibody or the donor antibody. In general, a humanized antibody comprises at least one, and typically two, variable regions in which all or substantially all of the CDRs are replaced by the CDRs of a non-human immunoglobulin, and all or substantially all of the FR regions are of human immunoglobulin sequences. Chimeric antibodies include antibodies produced by genetic engineering techniques in which the variable regions from a donor antibody are linked to the constant regions of a recipient antibody. The production of such antibodies can be carried out by methods known in the art.

[0065] In the present invention, examples of antigen-binding fragments include, but are not limited to, F(ab')2, Fab', Fab, Fv, rIgG, Fd, linear antibodies, ScFv, Fv-clasp, minibodies, diabodies, triabodies, tetrabodies, single domain antibodies (nanobodies), multispecific antibodies formed from antibody fragments, etc. The antibody fragments can be produced by methods known in the art.

[0066] Also included in the present invention is an isolated nucleic acid encoding an antibody or antigen-binding fragment thereof of the present application.

[0067] The antibody or antigen-binding fragment thereof specifically binds to USAG-1, inhibits the function of USAG-1, and induces tooth formation. Therefore, another aspect of the present invention provides a pharmaceutical composition for tooth regeneration therapy, comprising the antibody or antigen-binding fragment thereof, wherein tooth regeneration includes, for example, regeneration of lost teeth (restoration of missing teeth) and formation of new teeth such as third teeth.

[0068] The pharmaceutical composition of the present application may contain, in addition to the antibody or antigen-binding fragment thereof, a pharma- ceutically acceptable carrier, a stabilizer, an excipient, or other additive. Examples of pharma-ceutically acceptable carriers include, but are not limited to, physiological saline, a buffer, glycol, glycerol, gelatin, gelatin hydrogel, polylactic acid, collagen sponge, agarose, polyvinyl alcohol, alginic acid, fibrin gel, ethylene-vinyl acetate copolymer, and lactic acid-glycolic acid copolymer. Examples of additives include, but are not limited to, carbohydrates such as glucose, sucrose, or dextran, antioxidants such as ascorbic acid or glutathione, chelating agents, and low molecular weight proteins. Those skilled in the art can appropriately select the carrier and additives based on the administration form or administration route of the pharmaceutical composition. The pharmaceutical composition of the present application can be prepared by formulating the antibody or antigen-binding fragment thereof and the additives as appropriate using a conventional method.

[0069] The pharmaceutical composition of the present application may be in the form of, for example, a tablet, a powder, a capsule, a granule, a syrup, a sustained-release tablet, a sustained-release capsule, an enteric-coated drug, an insert, an infusion, an injection, etc., and is preferably an injection. The pharmaceutical composition of the present application is administered by systemic administration or local administration. The route of administration may be appropriately selected by a person skilled in the art, and may include, but is not limited to, oral, nasal, subcutaneous, intravenous, intramuscular, and intraosseous administration. The pharmaceutical composition of the present application may be administered locally, for example, to the site of tooth formation.

[0070] In the present invention, tooth regeneration therapy includes treatment of congenital anodontia and treatment of acquired tooth loss. Congenital anodontia treatable with the pharmaceutical composition of the present application is not particularly limited, and may be congenital anodontia caused by any causative gene. Examples of congenital anodontia treatable with the pharmaceutical composition of the present application include, but are not limited to, congenital anodontia caused by RUNX2, MSX1, EDA, WNT10A, PAX9, or AXIN2, preferably congenital anodontia caused by RUNX2, MSX1, EDA, or WNT10A. In addition, the antibody of the present application induced the formation of supernumerary teeth in wild-type mice. Therefore, the pharmaceutical composition of the present application can induce tooth formation in normal individuals in which the causative gene for anodontia is not deleted and in individuals who have acquired tooth loss.

[0071] The subjects to which the pharmaceutical composition of the present application is administered include mammals, such as humans, dogs, cats, horses, mice, ferrets, suncus, pigs, monkeys, etc., and preferably humans.

[0072] The dosage of the pharmaceutical composition of the present application is not particularly limited. Based on the content of the antibody or antigen-binding fragment thereof in the pharmaceutical composition, the body weight of the subject to be administered, and the like, a person skilled in the art can appropriately determine the desired dosage of the antibody or antigen-binding fragment thereof to be administered. For example, the antibody or antigen-binding fragment thereof is administered in an amount that exhibits a neutralizing activity that increases BMP signaling by at least 30%, preferably at least 60%, and / or a neutralizing activity that increases Wnt signaling by at least 30%, preferably at least 60%, compared to when not administered. The neutralizing activity may be determined based on the activity measured in vitro, for example, by ALP assay or reporter assay.

[0073] A further aspect of the present invention provides a method for tooth regeneration therapy, comprising administering the antibody or antigen-binding fragment thereof of the present application to a subject in need of treatment. The above-mentioned pharmaceutical composition can be used as the antibody or antigen-binding fragment thereof of the present application. The subject in need of treatment is a subject with missing teeth, and includes, for example, the above-mentioned mammals. The administration route and dosage of the antibody or antigen-binding fragment thereof of the present application, and the tooth regeneration therapy are as described above for the pharmaceutical composition of the present application.

[0074] The present invention will be described in more detail below using examples, but the present invention is not limited to these examples. EXAMPLES

[0075] 1. Antibody Preparation To generate neutralizing antibodies against mouse USAG-1, human USAG-1 protein derived from an E. coli expression system (R&D Systems) was used as an antigen. The Wnt inhibitory activity of human USAG-1 protein derived from an E. coli expression system was confirmed in a Wnt reporter assay using HEK293 cells (Figure 1). The BMP inhibitory activity of human USAG-1 protein derived from an E. coli expression system was confirmed in a BMP7-added ALP assay using C2C12 cells (Figure 2). To generate neutralizing antibodies against mouse USAG-1, three strains of supernumerary tooth model mice were newly established using CRISPER-CAS9: USAG-1KO mice (#116, #118, #138) (Figure 3). Neutralizing antibodies were generated by the iliac lymph node method using USAG-1 KO (#116) mice at ITM.

[0076] Primary screening of 284 wells was performed using ELISA with the immunized antigen, and many positive wells were found (Figures 4-1 and 4-2). The cutoff value was set at 0.7, and 79 clones were selected. Expansion culture was performed, and ELISA was performed using the immunized antigen and mouse USAG-1 protein derived from a CHO cell expression system produced by Sysmex Corporation. When the absorbance cutoff value was set at 0.025 or higher, about half of the wells were found to be positive (Figures 4-1 and 4-2). The antibody subclasses were measured, and were found to be IgG1, 2a, 2b, and 2c (Figures 4-1 and 4-2).

[0077] The neutralizing activity of each antibody was confirmed by adding various antibodies to an experimental system in which ALP activity was increased by adding 300ng / ml of BMP7 (R&D systems) to C2C12 cells, as with the antigen protein, and then suppressing the activity by adding 300ng / ml of rat USAG-1 protein (MyBiosource) derived from a mammalian cell expression system. The antibodies were classified into those with mild neutralizing activity (*: 60-100% neutralizing activity), those with moderate neutralizing activity (**: 100-140% neutralizing activity), and those with high neutralizing activity (***: 140% or higher neutralizing activity). For the BMP reporter assay, a commercially available cell line BMP Responsive Reporter Osteoblast Cell Line (Briter cell) (Kerafast) incorporating BRE-Luc was used. In an experimental system in which 300ng / ml of BMP7 was added and the expressed luciferase activity was suppressed by adding 300ng / ml of rat USAG-1 protein derived from a mammalian cell expression system, various antibodies were added to confirm neutralization of the suppression of luciferase activity. The antibodies were classified into those that showed mild neutralization activity (*: 40-60% neutralization activity) and those that showed moderate neutralization activity (**: 60% or more neutralization activity). For the WNT reporter assay, HEK293 cells were transfected with expression plasmids expressing the TOP-Flash reporter gene, which has a DNA sequence that binds to the transcription factor TCF, which is activated downstream of the WNT signal, the WNT1 gene, and a reporter gene controlled by the HSV-thymidine kinase promoter to obtain an internal standard value, and were cultured with mouse USAG-1 protein derived from a mammalian cell expression system at a concentration (EC50) that suppresses 50% of the maximum inhibitory effect of the Wnt signal. The amount of USAG-1 protein added was determined in advance. Culture supernatant of antibody-producing hybridoma was added to the screening medium at 25%, 20%, and 10% respectively, and three experiments were carried out for evaluation.At 25% addition, the samples were classified as follows: mild neutralizing activity (*: corrected luminescence value of 1.5-2.0) and moderate neutralizing activity (**: corrected luminescence value of 2.0 or more); at 20% addition, mild neutralizing activity (*: corrected luminescence value of 1-1.1) and moderate neutralizing activity (**: corrected luminescence value of 1.1 or more); at 10% addition, mild neutralizing activity (*: corrected luminescence value of 1-1.1) and moderate neutralizing activity (**: corrected luminescence value of 1.1 or more). Note that the percentage of neutralizing activity was calculated based on the activity without the addition of USAG-1 protein (100%). The neutralizing activity based on luminescence value was calculated based on the activity without the addition of antibody, which was set at 1.

[0078] Measurements using WNT and BMP reporter assays and BMP7 ALP assays revealed three types of neutralizing antibodies: antibodies that activated either BMP or WNT signaling, and antibodies that simultaneously activated both BMP and WNT signaling. Based on the results of the neutralizing activity measurements, six antibodies were selected (Figures 4-1 and 4-2). The activity of one clone disappeared during expansion and purification. Finally, five mouse anti-USAG-1 neutralizing antibodies (E12, E16, E37, E48, and E57) were obtained.

[0079] Of these, the sequences of E37 (herein referred to as antibody A) and E57 (herein referred to as antibody B) were determined. The full-length heavy chain sequence including the signal sequence of antibody A is shown in SEQ ID NO: 1, and the full-length light chain sequence including the signal sequence is shown in SEQ ID NO: 2. The full-length heavy chain sequence including the signal sequence of antibody B is shown in SEQ ID NO: 11, and the full-length light chain sequence including the signal sequence is shown in SEQ ID NO: 12. Furthermore, the variable regions of antibodies A and B are shown in FIG. 14. EXAMPLES

[0080] 1. In Vitro Testing of Antibodies After confirming the transient expression of recombinant mouse USAG-1 (WISE) protein with an N-terminus PA tag in Expi293F cells, a stable expression line was established. Affinity purification was performed using the PA tag system, and 0.2 mg of PA-mUSAG-1 (WISE) was obtained from 150 mL of culture supernatant (Figure 5). The purified PA-mUSAG-1 (WISE) protein showed a molecular weight of approximately 28 kDa, close to the theoretical value (24 kDa), in electrophoresis under reducing (R) and non-reducing (NR). Mouse N-terminus PA-tagged USAG-1 (WISE) protein derived from the mammalian cell Expi293F cell expression system showed dose-dependent inhibitory activity against WNT signaling in a WNT reporter assay (Figure 6), and dose-dependent inhibitory activity against BMP signaling in a BMP ALP assay (Figure 7). Using the mouse USAG-1 protein, which had been confirmed to have this activity, we confirmed the neutralizing activity of five mouse anti-USAG-1 neutralizing antibodies (E12, E16, E37, E48, and E57). In the WNT reporter assay, cells transfected with a vector in which a promoter was linked to the luciferase gene and a Wnt1 expression vector were cultured with 1.7 μg of mouse USAG-1 recombinant protein and various antibodies at a ratio of 1 / 1000, 1 / 300, or 1 / 100 to the medium, and luciferase activity was measured. In the BMP ALP assay, C2C12 cells were cultured in the presence of 30 ng / ml of BMP7, 30 ng / ml of mouse USAG-1 recombinant protein, and 1-fold (30 ng / ml), 10-fold (300 ng / ml), or 100-fold (3 μg / ml) amounts of various antibodies, and ALP activity was measured.

[0081] As a result, the presence of antibodies that dose-dependently neutralized the inhibitory activity of mouse USAG-1 on WNT signaling was confirmed in the WNT reporter assay (Fig. 8).The presence of antibodies that dose-dependently neutralized the inhibitory activity of mouse USAG-1 on BMP signaling was confirmed in the BMP ALP assay (Fig. 9). EXAMPLES

[0082] In vivo antibody administration test 1 Congenital edentulism model mice, EDA-deficient homozygous mice, show a high rate (approximately 90%) of missing third molars (M3) in the mandible. A single dose of mouse anti-USAG-1 neutralizing antibody A (#37) was administered intraperitoneally to mother mice pregnant with congenital edentulism model mice, EDA-deficient mice. As a result, the missing third molars (M3) in the mandible were restored in 7 out of 8 EDA-deficient mice born (Figure 10). No supernumerary teeth were observed in the congenital edentulism model mice, EDA-deficient mice, that had been administered mouse anti-USAG-1 neutralizing antibody A. Therefore, it was found that antibody A can restore missing teeth. Note that "recovery" here refers to the fact that teeth grow in the site (M3) that is usually missing in EDA-deficient mice (M3 is not missing) in the newborn EDA-deficient mice. EXAMPLES

[0083] In vivo antibody administration test 2 A single intraperitoneal injection of mouse anti-USAG-1 neutralizing antibody B (E57) was administered to mother mice pregnant with EDA-deficient mice, a model mouse for congenital anodontia. As a result, the formation of supernumerary teeth in the anterior region or fused teeth in the upper molar region was induced in two of three EDA-deficient homozygous mice born (Figure 10). In the EDA-deficient heterozygous mice born, supernumerary teeth in the anterior region or fused teeth in the molar region were observed in five of five. Furthermore, a single intraperitoneal injection of mouse anti-USAG-1 neutralizing antibody B (E57) to mother mice pregnant with wild-type mice resulted in supernumerary teeth in the anterior region or fused teeth in the molar region in 11 of 12 mice (Figure 11). Thus, it was found that antibody B can increase the number of teeth in all EDA-deficient homozygous mice, EDA-deficient heterozygous mice, and wild-type mice. EXAMPLES

[0084] In vivo antibody administration study 3 A mixture of the five types of anti-USAG-1 neutralizing antibodies obtained in Example 1, including neutralizing antibodies A and B, was administered intraperitoneally once to a mother mouse pregnant with a Wnt10a-deficient mouse, which is one of the edentulous model mice. As a result, supernumerary teeth were formed in the upper anterior tooth region (FIG. 10). EXAMPLES

[0085] Antibody recognition data for human USAG-1 It was confirmed whether the five kinds of mouse anti-USAG-1 (WISE) neutralizing antibodies (E12, E16, E37, E48, E57) obtained in Example 1 using the human USAG-1 protein derived from the E. coli expression system as an antigen could recognize the human USAG-1 protein. A binding assay was performed using mouse / human N-terminal PA-tagged USAG-1 protein. The five kinds of mouse anti-USAG-1 (WISE) neutralizing antibodies (E12, E16, E37, E48, E57) obtained in Example 1 (5 μg in 1 ml PBS) were captured on Protein A Sepharose (30 μl) (room temperature, 2.5 hours). The culture supernatant (1 mL) of human or mouse USAG-1 (WISE) recombinant protein with a PA tag added to the N-terminus transiently expressed in Expi293F cells and NZ1 Sepharose (30 μl) were added and incubated (room temperature, 2.5 hours). Unbound proteins were removed by washing three times with PBS buffer. All proteins bound to the Sepharose were eluted, and the bands were confirmed by SDS-PAGE electrophoresis. As a result, all five antibodies bound to not only mouse USAG-1 but also human USAG-1 protein (Figure 12).

[0086] Furthermore, human FLAG-tagged USAG-1 cDNA was transiently overexpressed in HEK293 cells, and immunostaining was performed using five mouse anti-USAG-1 neutralizing antibodies (E12, E16, E37, E48, and E57). As a result, clear positive reactions were observed when neutralizing antibodies (E12, E37, and E57) were used. Therefore, it was confirmed that both mouse anti-USAG-1 neutralizing antibody A (E37) and mouse anti-USAG-1 neutralizing antibody B (E57), whose effectiveness was confirmed in vivo, can recognize human USAG-1 protein (Figure 13). EXAMPLES

[0087] 2. Antibody Preparation To prepare neutralizing antibodies against mouse USAG-1, rat USAG-1 protein derived from a baculovirus expression system was used as an antigen. Neutralizing antibodies were prepared by the iliac lymph node method using USAG-1 KO (#116) mice. The rat USAG-1 protein derived from the baculovirus expression system was confirmed to have BMP inhibitory activity and Wnt inhibitory activity by the same method as described in Example 1. Primary screening of the obtained antibody clones was performed by ELISA using the immunized antigen (rat USAG-1 protein derived from a baculovirus expression system) and human USAG-1 protein derived from an E. coli expression system, and many positive wells were observed. The cutoff value was set at 1.0, and 111 clones were selected. After expansion culture, sandwich ELISA was performed using the immunized antigen, and positive wells were observed in about half of the wells when the absorbance cutoff value was set at 0.5 or more (His tag) and 1.4 or more (Myc tag). The antibody subclasses were measured and found to be IgG1, 2a, 2b, and G3. Furthermore, the neutralizing activity of each of the obtained antibodies was measured by the same method as described in Example 1, and finally, four types of mouse anti-USAG-1 neutralizing antibodies (B14, B48, B103, B108) were obtained.

[0088] Furthermore, in order to confirm whether the obtained mouse anti-USAG-1 neutralizing antibodies (B14, B48, B103, and B108) can recognize human USAG-1 protein, a binding assay was performed using mouse / human N-terminal PA-tagged USAG-1 protein in the same manner as in Example 6. Each mouse anti-USAG-1 neutralizing antibody (250 μl, Protein A / G IgG binding buffer (Pierce)) was added to Protein A Sepharose (30 μl). TM) + 5 μg in 250 μl PBS) was captured (room temperature, 1.5 hours). Culture supernatant (0.75 mL) of recombinant human or mouse USAG-1 (WISE) protein with an N-terminus PA tag added to its protein was added transiently in Expi293F cells, and incubated (room temperature, 2 hours). Unbound proteins were removed by washing three times with TBS buffer. All proteins bound to the Sepharose were eluted, and bands were confirmed by SDS-PAGE electrophoresis and CBB (Coomassie Brilliant Blue) staining. As a result, all mouse anti-USAG-1 neutralizing antibodies tested bound to both mouse USAG-1 and human USAG-1 proteins, although the binding of B103 and B108 was weaker than that of B14 and B48 (Figure 15).

[0089] Of these, the sequence of B14 (referred to herein as antibody C) was determined. The full-length heavy chain sequence including the signal sequence of antibody C is shown in SEQ ID NO: 21, and the full-length light chain sequence including the signal sequence is shown in SEQ ID NO: 22. Furthermore, the variable region of antibody C is shown in FIG. 21. Furthermore, the sequences of B48 (referred to herein as antibody D) and B103 (referred to herein as antibody E) were determined. The full-length heavy chain sequences including the signal sequence of antibodies D and E are shown in SEQ ID NO: 38 and SEQ ID NO: 48, respectively, and the full-length light chain sequences including the signal sequence are shown in SEQ ID NO: 39 and SEQ ID NO: 49, respectively. Furthermore, the variable regions of antibodies D and E are shown in FIG. 22. EXAMPLES

[0090] Epitope binning Five types of mouse USAG-1 neutralizing antibodies (E12, E16, E37, E48, E57) selected in Example 1 and the neutralizing antibodies (B14, B48, B103, B108) prepared in Example 7 were subjected to epitope binning. Figure 16 shows a comparison of six types of antibodies among the obtained competitive binding data. Epitope binning was performed using Octet (registered trademark) Red (Pall ForteBio). Briefly, nine types of antibodies were immobilized on a biosensor as capture antibodies, a target containing purified recombinant mouse USAG-1 full length was added and allowed to bind, and then the binding signal was detected by reacting with a test antibody. This cycle was performed continuously for nine test antibodies, including the same antibody used for capture. Antibodies that do not compete with the capture antibody immobilized on the biosensor for their recognition site can bind to the captured USAG-1 protein and the signal increases, but competing antibodies have weaker binding and therefore do not increase this signal or the increase is suppressed to a low level. Based on the signal data obtained, the nine antibodies were grouped according to their epitopes. In other words, when a test antibody was reacted with the sensor of the nine capture antibodies, an antibody that gave a signal equal to or weaker than the signal (indicated by bold underline in FIG. 16) when the same antibody as the capture antibody was added was considered to be in the same group as the capture antibody. As a result, E37 and E48 competed with the E12 antibody, E48, E57, and B14 with the E16 antibody, E12 with the E37 antibody, E16, E57, and B14 with the E48 antibody, E16, E48, and B14 with the E57 antibody, E16, E48, and E57 with the B14 antibody, and B108 with B103. Based on these results, the nine antibodies were classified into four groups. The results are shown in Table 1. However, antibody E48 is basically classified into group 2, but it was close to group 1 because it also competed with E12.

[0091] [Table 1] EXAMPLES

[0092] Epitope mapping Epitope mapping was performed for antibody E37 (neutralizing antibody A) of group 1. Briefly, a peptide library of 169 overlapping 15-amino acid peptides was synthesized, shifting each peptide by one amino acid from the beginning, based on the human USAG-1 protein sequence (183 amino acids long) excluding the signal peptide. The 169 peptides were bound to a cellulose membrane to create a peptide array. Antibody E37 (0.3 g / ml) was added as the primary antibody and incubated. After washing, HRP-conjugated anti-mouse antibody (1 / 25000 dilution) was added as the secondary antibody, and color development was performed using ECL solution.

[0093] As a result, it was found that the antibody E37 specifically binds to six peptides: QEWRCVNDKTRTQRI (SEQ ID NO: 32), EWRCVNDKTRTQRIQ (SEQ ID NO: 33), WRCVNDKTRTQRIQL (SEQ ID NO: 34), RCVNDKTRTQRIQLQ (SEQ ID NO: 35), CVNDKTRTQRIQLQC (SEQ ID NO: 36), and VNDKTRTQRIQLQCQ (SEQ ID NO: 37). Therefore, the amino acid sequence: VNDKTRTQRI (SEQ ID NO: 31) (corresponding to positions 134 to 143 of the full-length USAG-1 amino acid sequence including the signal peptide) was identified as the epitope. EXAMPLES

[0094] 2. In Vitro Testing of Antibodies The affinity (KD value) of eight of the nine antibodies grouped in Example 8 was determined using the BLI method using Octet in the same manner as in Example 8. Specifically, after immobilizing each antibody on a biosensor, purified recombinant mouse USAG-1 protein was added at three different concentrations (10 nM, 30 nM, 100 nM), and binding and dissociation curves were obtained, which were then calculated by global fitting using the analysis program attached to the Octet device. The results are shown in Table 2.

[0095] [Table 2] EXAMPLES

[0096] 3. In Vitro Testing of Antibodies The antibody B14 classified into group 2 in Example 8 was measured for its activity of suppressing and neutralizing BMP and Wnt signaling. The experiment was performed using the same method as that described in Example 2. That is, in the WNT reporter assay, cells transfected with a vector in which a promoter is linked to a luciferase gene and a Wnt1 expression vector (1 μg) were cultured by adding 1 μg of mouse USAG-1 recombinant protein and antibodies to the medium at 1.0, 3.0, 4.0, 6.0, 8.0, 10.0, 12.0, 15.0, or 30.0 μg / ml, and luciferase activity was measured. In the BMP ALP assay, C2C12 cells were cultured in the presence of 30 ng / ml of BMP7, 30 ng / ml of mouse USAG-1 recombinant protein, and 30 ng / ml, 150 ng / ml, 300 ng / ml, or 1500 ng / ml of various antibodies, and ALP activity was measured. The results are shown in FIG. 17. In the WNT reporter assay, antibody B14 was confirmed to neutralize the inhibitory activity of USAG-1 on WNT signaling (maximum neutralization activity of 42%).In the BMP ALP assay, antibody B14 was confirmed to neutralize the inhibitory activity of USAG-1 on BMP signaling.

[0097] Furthermore, the EC50 values ​​of antibody B14 for the inhibition of BMP and Wnt signaling by USAG-1 were calculated as the concentration at which 50% inhibition was achieved, assuming that the concentration without antibody was 0 and the maximum neutralizing activity was 100%. The EC50 values ​​for the inhibition of BMP and Wnt signaling by USAG-1 were 298 ng / ml and 4.73 μg / ml, respectively. EXAMPLES

[0098] In vivo antibody administration test 4 (mouse) The six antibodies classified into groups 1 and 2 in Example 8 were administered once intraperitoneally to mother mice pregnant with congenital anodontia model mice EDA homozygous deficient mice, congenital anodontia model mice EDA heterozygous deficient mice, or wild-type mice. The results are shown in Table 3. It was found that all six antibodies can induce the formation of supernumerary teeth / fused teeth in EDA-deficient mice and increase the number of teeth in EDA-deficient mice. In particular, the antibodies in group 1 restored the missing teeth in EDA-deficient mice. The antibodies in group 1 did not induce the formation of supernumerary teeth and fused teeth in wild-type mice, but the antibodies E57 and B14 of group 2 induced the formation of supernumerary teeth and fused teeth in wild-type mice. It was found that antibody B14 (antibody C), like antibody E57 (antibody B), can increase the number of teeth in all EDA-deficient homozygous mice, EDA-deficient heterozygous mice, and wild-type mice. Here, "recovery" refers to the fact that the newborn EDA-deficient mice have teeth at the site (M3) that is normally missing in EDA-deficient mice (M3 is not missing).

[0099] [Table 3] EXAMPLES

[0100] In vivo antibody administration test 5 (dogs) Anti-mouse USAG-1 neutralizing antibody B14 (50 μg / g body weight) was administered once intraperitoneally to congenital edentulous model dogs immediately after birth. The congenital edentulous model dogs were TOYO beagle strains with congenital edentulousness and were obtained from Kitayama Labes Hongo Farm. The congenital edentulous model dogs included individuals lacking the upper third premolar and individuals lacking the lower fourth premolar. 10 weeks after administration, calcification of the tooth germ was evaluated using dental X-rays, and the recovery of the missing teeth was confirmed (Figure 18). The results are shown in Table 4. In addition, the blood concentration of the administered antibody in each individual was measured 3 days, 1, 3, 5, and 7 weeks after antibody administration. As a result, although there were individual differences, the half-life was 1 week and was maintained up to 7 weeks after administration.

[0101] [Table 4]

[0102] As is clear from Table 4, congenital premolar loss was restored by a single systemic administration of USAG-1 neutralizing antibody B14. The low recovery rate for mandibular premolar loss suggests that the causative gene may be different. EXAMPLES

[0103] In vivo antibody administration test 6 (ferret) Ferrets, like humans, have two teeth, with three incisors, one canine, three premolars, and two molars, which is similar to the basic dental formula of mammals. Thirty-nine ferrets were intraperitoneally administered 39 types of USAG-1 neutralizing antibodies (16 μg / g body weight), including the antibodies prepared in Examples 1 and 7, at 1 and 3 weeks after birth (one for each antibody). Thirty-eight ferrets survived at 14 weeks after birth. Four individuals administered with four types of antibodies, including the antibodies of groups 1 to 4 identified in Example 8, were observed for a long period of time until 30 weeks after birth. As a result, many large teeth were observed in the upper and lower jaw anterior teeth and premolars in several ferrets. Furthermore, in ferrets administered with antibody B14, induction of the third tooth in the third premolar area on the lingual side of the lower jaw was observed at 14 weeks after birth (FIG. 19). In the ferrets administered with antibody B103, at 30 weeks of age, an extra incisor developed on the lingual (palatal) side of the upper jaw, and induction of the third tooth in the upper incisor was observed (Figure 23). The incisor developed later than the permanent teeth and was similar in shape to the preceding permanent tooth. The incisor had a short root. Furthermore, in the ferrets administered with antibody B103, induction of the third tooth in the left and right premolar areas of the lower jaw was observed at 30 weeks of age (Figure 24). EXAMPLES

[0104] In vivo antibody administration test 7 (Suncus murinus) Pregnant suncus were obtained, and on the 17th day of pregnancy, various USAG-1 neutralizing antibodies (16 μg / g body weight) prepared in Example 1 were intraperitoneally administered. At 7 weeks after birth, the suncus was evaluated by μCT imaging. Suncus administered with USAG-1 neutralizing antibodies did not give birth, but the formation of new teeth was observed around the tooth roots of parent suncus aged 4 to 8 months administered with antibody B (E57) and antibody C (B14), which were formed by local epithelial mesenchymal induction of enamel epithelial stem cells. In the suncus administered with antibody B, the induction of the third tooth was observed between the first and second premolars on the buccal side of the mandible (FIG. 20). Therefore, it was shown that the USAG-1 neutralizing antibody of the present invention induces the formation of the third tooth. EXAMPLES

[0105] 4. In Vitro Testing of Antibodies The antibody B48, which was classified into group 3 in Example 8, was measured for its activity of suppressing and neutralizing BMP and Wnt signaling. The experiment was performed using the same method as that described in Example 2. That is, in the WNT reporter assay, cells transfected with a vector in which a promoter is linked to a luciferase gene and a Wnt1 expression vector (1 μg) were cultured by adding 1 μg of mouse USAG-1 recombinant protein and an antibody to the medium at 1, 3, 6, 10, or 30 μg / ml, and the luciferase activity was measured. In the BMP ALP assay, C2C12 cells were cultured in the presence of 30 ng / ml of BMP7, with 30 ng / ml of mouse USAG-1 recombinant protein and 0.1 times (3 ng / ml), 10 times (300 ng / ml), or 100 times (3 μg / ml) of the antibody added, and the ALP activity was measured. The results are shown in FIG. 25. In the WNT reporter assay, it was confirmed that antibody B48 neutralized the inhibitory activity of USAG-1 on WNT signaling by almost 100%. EXAMPLES

[0106] 5. In Vitro Antibody Testing (Protein A Sepharose Pull-Down Assay with PA-Tagged USAG-1 and E1E2) Wnt binds to the LRP (low-density lipoprotein receptor-related protein) 5 / 6 receptor, which is a co-receptor of Frizzled, and transmits a signal into the cell. LRP6 has four extracellular domains (E1 to E4), of which E1E2 is known to be involved in binding to USAG-1. That is, USAG-1 binds to the E1E2 region of LRP6 to suppress Wnt signal transduction. Therefore, we investigated whether the nine types of mouse anti-USAG-1 neutralizing antibodies (E12, E16, E37, E48, E57, B14, B48, B103, and B108) grouped in Example 8 inhibit the binding of USAG-1 to LRP6-E1E2.

[0107] Protein A Sepharose (30 μl) was used to capture each mouse anti-USAG-1 neutralizing antibody (5 μg in 1 ml PBS) (room temperature, 1.5 hours). Culture supernatant (1 mL) of mouse USAG-1 recombinant protein with PA tag added to the N-terminus transiently expressed in Expi293F cells was added and incubated (room temperature, 2 hours). Culture supernatant (1 mL) of LRP6-E1E2 (a region of human LRP6 fused with His tag at amino acid numbers 1 to 629) was added and incubated (room temperature, 2.5 hours). Unbound proteins were removed by washing three times with PBS buffer (1 mL). All proteins bound to the Sepharose were eluted, and bands were confirmed by SDS-PAGE electrophoresis and Oriole gel fluorescent staining. The results are shown in Figure 26. In Figure 26, the right panels show a negative control, a sample (no mAb) that was subjected to the same procedure without capturing mouse anti-USAG-1 neutralizing antibody, a positive control, a complex of mouse USAG-1 recombinant protein and LRP6-E1E2 immunoprecipitated with PA tag antibody NZ-1 (WISE+E1E2 By NZ1), and the expression level of LRP6-E1E2 alone precipitated with Ni-NTA resin that adsorbs the His tag (E1E2 By NiNTA).

[0108] As a result, it was found that LRP6-E1E2 does not bind to the complex of some antibodies (especially antibodies B48, B103, and B108 of groups 3 and 4) with USAG-1 (Figure 26). Therefore, it was found that the epitopes of these antibodies overlap with or are structurally close to the LRP6-binding site of USAG-1, and that the antibodies inhibit the binding of USAG-1 to LRP6 by binding to USAG-1, thereby neutralizing the suppression of Wnt signaling by USAG-1. EXAMPLES

[0109] In vivo antibody administration test 8 (mouse) The three types of antibodies classified into groups 3 and 4 in Example 8 were administered once intraperitoneally to mother mice pregnant with congenital edentulism model mice EDA homozygous deficient mice, congenital edentulism model mice EDA heterozygous deficient mice, or wild-type mice (16 μg / g body weight). The presence of supernumerary teeth and fused teeth, as well as the recovery of missing teeth, were examined in the offspring mice. The results are shown in Table 5. The three types of antibodies in particular restored missing teeth in EDA-deficient mice. Here, "recovery" refers to the fact that teeth grow in the site (M3) that is usually missing in EDA-deficient mice in the newborn EDA-deficient mice (M3 is not missing).

[0110] [Table 5] EXAMPLES

[0111] In vivo antibody administration test 9 (mouse) Five types of antibodies (E57, B14, B48, B103, B108) were administered once intraperitoneally to mother mice pregnant with congenital anodontia model mice, Wnt10a homozygous deficient mice, congenital anodontia model mice, Wnt10a heterozygous deficient mice, or wild-type mice (B103: 2 μg / g body weight, other antibodies: 16 μg / g body weight). The presence of supernumerary teeth and fused teeth, as well as the recovery of missing teeth, were examined in the offspring mice. The results are shown in Table 6. Among the antibodies in groups 3 and 4, B48 and B103 induced the formation of supernumerary teeth and fused teeth in homozygous deficient mice.

[0112] [Table 6] [Industrial Applicability]

[0113] The antibody or antigen-binding fragment thereof of the present application can be used to treat congenital anodontia or acquired tooth loss. The antibody or antigen-binding fragment thereof is also considered to be effective in the formation of the third dentition, which will lead to the development of molecular targeted drugs for tooth regeneration in the pharmaceutical field and the establishment of a treatment method for tooth regeneration by inducing the formation of the third dentition. [Sequence List Free Text]

[0114] SEQ ID NO:32; 15 amino acid peptide for epitope mapping SEQ ID NO:33; 15 amino acid peptide for epitope mapping SEQ ID NO:34; 15 amino acid peptide for epitope mapping SEQ ID NO:35; 15 amino acid peptide for epitope mapping SEQ ID NO:36; 15 amino acid peptide for epitope mapping SEQ ID NO:37; 15 amino acid peptide for epitope mapping

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to and neutralizes USAG-1.

2. The antibody or antigen fragment thereof according to claim 1, which specifically binds to USAG-1 and neutralizes the BMP signaling inhibitory activity of USAG-1.

3. The antibody or antigen fragment thereof according to claim 1 or 2, which specifically binds to USAG-1 and neutralizes the WNT signaling inhibitory activity of USAG-1.

4. (a) three heavy chain complementarity determining regions comprising amino acid sequences having at least 90% sequence identity to the amino acid sequences set forth in SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7, respectively, or three light chain complementarity determining regions comprising amino acid sequences having at least 90% sequence identity to the amino acid sequences set forth in SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10, respectively; (b) three heavy chain complementarity determining regions comprising amino acid sequences having at least 90% sequence identity to the amino acid sequences set forth in SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17, respectively, or three light chain complementarity determining regions comprising amino acid sequences having at least 90% sequence identity to the amino acid sequences set forth in SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20, respectively; (c) three heavy chain complementarity determining regions comprising amino acid sequences having at least 90% sequence identity to the amino acid sequences set forth in SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27, respectively, or three light chain complementarity determining regions comprising amino acid sequences having at least 90% sequence identity to the amino acid sequences set forth in SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30, respectively; (d) three heavy chain complementarity determining regions comprising amino acid sequences having at least 90% sequence identity to the amino acid sequences set forth in SEQ ID NOs: 42, 43, and 44, respectively, or three light chain complementarity determining regions comprising amino acid sequences having at least 90% sequence identity to the amino acid sequences set forth in SEQ ID NOs: 45, 46, and 47, respectively; or, (e) three heavy chain complementarity determining regions comprising amino acid sequences having at least 90% sequence identity to the amino acid sequences set forth in SEQ ID NOs: 52, 53, and 54, respectively, or three light chain complementarity determining regions comprising amino acid sequences having at least 90% sequence identity to the amino acid sequences set forth in SEQ ID NOs: 55, 56, and 57, respectively. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, comprising:

5. (f) a heavy chain variable region comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 3, or a light chain variable region comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 4; (g) a heavy chain variable region comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 13, or a light chain variable region comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 14; (h) a heavy chain variable region comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 23, or a light chain variable region comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 24; (i) a heavy chain variable region comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 40, or a light chain variable region comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 41; or, (j) a heavy chain variable region comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 50, or a light chain variable region comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO:

51. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, comprising:

6. (k) three heavy chain complementarity determining regions comprising amino acid sequences having at least 90% sequence identity to the amino acid sequences set forth in SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7, respectively, and three light chain complementarity determining regions comprising amino acid sequences having at least 90% sequence identity to the amino acid sequences set forth in SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10, respectively; (l) three heavy chain complementarity determining regions comprising amino acid sequences having at least 90% sequence identity to the amino acid sequences set forth in SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17, respectively, and three light chain complementarity determining regions comprising amino acid sequences having at least 90% sequence identity to the amino acid sequences set forth in SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20, respectively; (m) three heavy chain complementarity determining regions comprising amino acid sequences having at least 90% sequence identity to the amino acid sequences set forth in SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27, respectively, and three light chain complementarity determining regions comprising amino acid sequences having at least 90% sequence identity to the amino acid sequences set forth in SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30, respectively; (n) three heavy chain complementarity determining regions comprising amino acid sequences having at least 90% sequence identity to the amino acid sequences set forth in SEQ ID NO:42, SEQ ID NO:43, and SEQ ID NO:44, respectively, and three light chain complementarity determining regions comprising amino acid sequences having at least 90% sequence identity to the amino acid sequences set forth in SEQ ID NO:45, SEQ ID NO:46, and SEQ ID NO:47, respectively; or (o) three heavy chain complementarity determining regions comprising amino acid sequences having at least 90% sequence identity to the amino acid sequences set forth in SEQ ID NOs: 52, 53, and 54, respectively, and three light chain complementarity determining regions comprising amino acid sequences having at least 90% sequence identity to the amino acid sequences set forth in SEQ ID NOs: 55, 56, and 57, respectively. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, comprising:

7. (p) a heavy chain variable region comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 3, and a light chain variable region comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 4; (q) a heavy chain variable region comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 13, and a light chain variable region comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 14; (r) a heavy chain variable region comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 23, and a light chain variable region comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 24; (s) a heavy chain variable region comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 40, and a light chain variable region comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 41; or (T) a heavy chain variable region comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 50, and a light chain variable region comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:

51. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3 and 6, comprising:

8. An antibody or antigen-binding fragment thereof that competes with the antibody or antigen-binding fragment thereof of any one of claims 4 to 7 for binding to USAG-1.

9. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, wherein the antibody is a humanized antibody or a chimeric antibody.

10. A pharmaceutical composition for tooth regeneration therapy, comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9.

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