Neutralizing antibody targeting USAG-1 for tooth regeneration therapy

Abstract

The present invention aims to provide a technology for treating odontopathy that utilizes differentiation induction inherent in the tooth organ, rather than using surgical tissue transplantation. [Solution] An antibody or its antigen-binding fragment that specifically binds to and neutralizes USAG-1 is provided, and a pharmaceutical composition containing the antibody or its antigen-binding fragment is provided.

Description

Technical Field

[0001] The present invention relates to a neutralizing antibody targeting USAG-1 for the treatment of anodontia or tooth regeneration.

Background Art

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

[0003] On the other hand, many causative genes of congenital anodontia have been identified, and many are common between humans and mice. For example, RUNX2, MSX1, EDA, WNT10A, PAX9, AXIN2, etc. are known. Among them, congenital anodontia with WNT10A as the causative gene has the largest number of patients. In addition, EDA is the causative gene of anhidrotic ectodermal dysplasia, which is a representative disease of syndromic congenital anodontia. Congenital anodontia is caused by the deficiency or functional suppression of causative genes, which causes tooth development to stop halfway.

Prior Art Documents

Non-Patent Documents

[0004]

Non-Patent Document 1

Non-Patent Document 2

[0005] A new approach to treating congenital anodontia involves promoting differentiation induction to form complete teeth, as described above, in cases where tooth development has stopped midway. Therefore, the present invention aims to provide a technology for treating odontia that utilizes differentiation induction inherent in the tooth organ, rather than relying on surgical tissue transplantation. [Means for solving the problem]

[0006] As a result of diligent research, the inventors have succeeded in developing a neutralizing antibody targeting USAG-1. Furthermore, they have discovered that administration of this antibody can regenerate missing teeth in a congenital anodontia model mouse and can form supernumerary teeth in a congenital anodontia model mouse or wild-type mouse, thus completing the present invention.

[0007] In other words, the present invention is [1]An antibody or antigen-binding fragment that specifically binds to and neutralizes USAG-1, [2] The antibody or antigen fragment described in [1], which specifically binds to USAG-1 and neutralizes the BMP signaling inhibitory activity of USAG-1. [3] An antibody or antigen fragment according to [1] or [2] that specifically binds to USAG-1 and neutralizes the WNT signaling inhibitory activity of USAG-1, [4](a) Three heavy chain complementarity determination regions, each containing an amino acid sequence having at least 90% 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 determination regions, each containing an amino acid sequence having at least 90% sequence identity with the amino acid sequences shown in SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10, (b) Three heavy chain complementarity determination regions, each containing an amino acid sequence having at least 90% sequence identity with 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 determination regions, each containing an amino acid sequence having at least 90% sequence identity with the amino acid sequences shown in SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20, (c) Three heavy chain complementarity determination regions, each containing an amino acid sequence having at least 90% sequence identity with 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 determination regions, each containing an amino acid sequence having at least 90% sequence identity with the amino acid sequences shown in SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 30, (d) Three heavy chain complementarity determination regions, each containing an amino acid sequence having at least 90% sequence identity with the amino acid sequences shown in SEQ ID NOs. 42, 43, and 44, respectively, or three light chain complementarity determination regions, each containing an amino acid sequence having at least 90% sequence identity with the amino acid sequences shown in SEQ ID NOs. 45, 46, and 47, respectively, or (e) Three heavy chain complementarity determination regions, each containing an amino acid sequence having at least 90% sequence identity with 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 determination regions, each containing an amino acid sequence having at least 90% sequence identity with the amino acid sequences shown in SEQ ID NO: 55, SEQ ID NO: 56, and SEQ ID NO: 57, respectively. The antibody or antigen-binding fragment described in any one of items [1] to [3], including [5](f) A heavy chain variable region containing an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in Sequence ID No. 3, or a light chain variable region containing an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in Sequence ID No. 4, (g) A heavy chain variable region containing an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 13, or a light chain variable region containing an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 14. (h) A heavy chain variable region containing an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 23, or a light chain variable region containing an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 24, (i) A heavy chain variable region containing an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 40, or a light chain variable region containing an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 41, or, (j) A heavy chain variable region containing an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 50, or a light chain variable region containing 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 described in any one of items [1] to [4], including [6](k) Three heavy chain complementarity determination regions, each containing an amino acid sequence having at least 90% sequence identity with the amino acid sequences shown in SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, and three light chain complementarity determination regions, each containing an amino acid sequence having at least 90% sequence identity with the amino acid sequences shown in SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10, (l) Three heavy chain complementarity determination regions, each containing an amino acid sequence having at least 90% sequence identity with the amino acid sequences shown in SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17, and three light chain complementarity determination regions, each containing an amino acid sequence having at least 90% sequence identity with the amino acid sequences shown in SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20, (m) Three heavy chain complementarity determination regions, each containing an amino acid sequence having at least 90% sequence identity with the amino acid sequences shown in SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 27, and three light chain complementarity determination regions, each containing an amino acid sequence having at least 90% sequence identity with the amino acid sequences shown in SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 30, (n) Three heavy chain complementarity determination regions, each containing an amino acid sequence having at least 90% sequence identity with the amino acid sequences shown in SEQ ID NOs. 42, 43, and 44, respectively, and three light chain complementarity determination regions, each containing an amino acid sequence having at least 90% sequence identity with the amino acid sequences shown in SEQ ID NOs. 45, 46, and 47, respectively, or (o) Three heavy chain complementarity determination regions, each containing an amino acid sequence having at least 90% sequence identity with the amino acid sequences shown in SEQ ID NOs. 52, 53, and 54, respectively, and three light chain complementarity determination regions, each containing an amino acid sequence having at least 90% sequence identity with the amino acid sequences shown in SEQ ID NOs. 55, 56, and 57, respectively. The antibody or antigen-binding fragment described in any one of items [1] to [3], including [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 containing an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 13, and a light chain variable region containing an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 14, (r) A heavy chain variable region containing an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 23, and a light chain variable region containing an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 24, (s) A heavy chain variable region containing an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 40, and a light chain variable region containing an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 41, or (t) Heavy chain variable region containing an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 50, and light chain variable region containing 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 according to any one of items [1] to [3] and [6], including [8] Regarding binding to USAG-1, an antibody or antigen-binding fragment that competes with any one of the antibodies or antigen-binding fragments described in any one of the items [4] to [7], [9] The antibody or its antigen-binding fragment according to any one of items [1] to [8], wherein the antibody is a humanized antibody or a chimeric antibody, and A pharmaceutical composition for tooth regeneration treatment comprising an antibody or antigen-binding fragment described in any one of items

[10] [1] to [9]. To provide. [Effects of the Invention]

[0008] In this invention, we have succeeded in regenerating teeth in vivo using an antibody preparation. The treatment using the antibody preparation of this invention can be clinically implemented as a regenerative medicine for teeth using conventional oral and maxillofacial surgical approaches such as tooth extraction, orthodontics, and tooth transplantation. [Brief explanation of the drawing]

[0009] [Figure 1] The Wnt inhibitory activity of recombinant human UASG-1 protein derived from Escherichia coli, which was used as an antigen in the examples, is shown. [Figure 2] The BMP inhibitory activity of recombinant human UASG-1 protein derived from Escherichia coli, which was used as an antigen in the examples, is shown. [Figure 3] This shows a newly established USAG-1 KO mouse using CRISPER-CAS9. [Figure 4-1] Shows the results of the primary screening of anti-USAG-1 neutralizing antibodies. [Figure 4-2] Shows the results of the primary screening of anti-USAG-1 neutralizing antibodies. [Figure 5] Shows the results of the purification and concentration of mouse N-terminal PA-tagged USAG-1 (WISE). [Figure 6] Shows the dose-dependent WNT signal inhibitory activity of mouse USAG-1 protein. [Figure 7] Shows the dose-dependent inhibitory activity of mouse USAG-1 protein on BMP signal. [Figure 8] Shows antibodies that neutralize the inhibitory activity of WNT signal by mouse USAG-1 in a dose-dependent manner. [Figure 9] Shows antibodies that neutralize the inhibitory activity of BMP signal by mouse USAG-1 in a dose-dependent manner. [Figure 10] Shows that anti-USAG-1 neutralizing antibodies can induce tooth growth in toothless model mice. [Figure 11] Shows that anti-USAG-1 neutralizing antibodies have an effect equivalent to that of USAG-1KO. [Figure 12] Shows the results of the binding experiment of mouse anti-USAG-1 antibody with mouse / human USAG-1 protein. [Figure 13] Shows immunostaining using mouse anti-USAG-1 antibody against HEK293 cells transiently transfected with human FLAG-tagged USAG-1. [Figure 14] Shows the sequences of the heavy and light chain variable regions of antibody A and antibody B. [Figure 15] Shows the results of the binding experiment of mouse anti-USAG-1 antibody with mouse / human USAG-1 protein. [Figure 16] Shows the competitive binding data of the six obtained antibodies. It is a figure in which the sensorgrams when reacting with the USAG-1 sensor captured by the six antibodies are overlaid for each of the six test antibodies. [Figure 17]The present invention demonstrates the neutralizing activity of the antibody against the WNT signaling and BMP signaling inhibitory activity induced by mouse USAG-1. [Figure 18] This is a dental X-ray image showing the effect of administering USAG-1 neutralizing antibody to dogs with congenital anodontia. [Figure 19] These are μCT images and their 3D reconstructions showing the induction of the third eruptive tooth in the mandibular third premolar region of ferrets by administration of USAG-1 neutralizing antibody. [Figure 20] These are μCT images and their 3D reconstructions showing the induction of the third erupted tooth in the mandibular third premolar region by administration of USAG-1 neutralizing antibody in Sunkus. [Figure 21] The sequences of the heavy chain 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] These are μCT slice images and their 3D reconstructions showing the induction of the third eruptive tooth in the maxillary anterior region of ferrets by administration of USAG-1 neutralizing antibody. [Figure 24] This is a three-dimensional reconstruction image created from μCT data showing the induction of the third eruptive tooth in the mandibular premolar region of ferrets by administration of USAG-1 neutralizing antibody. [Figure 25] The present invention demonstrates the neutralizing activity of the antibody against the WNT signaling and BMP signaling inhibitory activity induced by mouse USAG-1. [Figure 26] The results of a pull-down assay demonstrating the interaction between the mouse anti-USAG-1 antibody and mouse USAG-1 protein complex and the LRP6-E1E2 domain are shown. [Modes for carrying out the invention]

[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. USAG-1-deficient model mice are known to exhibit increased BMP signaling and lead to the formation of supernumerary teeth. The inventors crossed Runx2-deficient mice, a model mouse for congenital anodontia, with USAG-1 gene-deficient mice, a model mouse for supernumerary teeth (teeth present in excess of the normal number), to create double knockout mice. Analysis of these mice revealed that tooth formation was restored. Therefore, it was suggested that inhibiting USAG-1 could treat anodontia.

[0011] In this study, the inventors crossed congenital odontopathy model mice lacking the causative genes Msx1, Eda, and Wnt10a (other than Runx2) with USAG-1 gene-deficient mice, a new supernumerary tooth model mouse created using the CRISPER-CAS9 system, to generate double knockout mice. Analysis of these mice revealed that tooth formation was restored in all odontopathy model mice. Therefore, it was demonstrated that treatment by inhibiting USAG-1 is applicable to patients with congenital odontopathy caused by various gene mutations.

[0012] Therefore, in this invention, antibodies were produced using a human USAG-1 recombinant protein whose activity was confirmed as an antigen, and antibodies that specifically bind to USAG-1 were obtained. These antibodies were confirmed to increase BMP signaling and / or Wnt signaling.

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

[0014] In this specification, neutralization means inhibiting the function of USAG-1. The functions of USAG-1 include, for example, BMP signaling inhibitory activity (also known as "BMP antagonist activity") and Wnt signaling inhibitory activity (also known as "Wnt antagonist activity"). The antibody or antigen-binding fragment of this application inhibits the BMP signaling inhibitory activity and / or Wnt signaling inhibitory activity of USAG-1. Therefore, the antibody or antigen-binding fragment of this application neutralizes either or both of the BMP signaling inhibitory activity and the Wnt signaling inhibitory activity of USAG-1. For example, but not limited to, antibodies or antigen fragments that specifically bind to USAG-1 and neutralize its BMP signaling inhibitory activity but not its Wnt signaling inhibitory activity, or antibodies or antigen fragments that specifically bind to USAG-1 and neutralize its Wnt signaling inhibitory activity but not its BMP signaling inhibitory activity, are also included in the antibodies or antigen-binding fragments of this application. In this specification, "inhibition" includes suppression and reduction.

[0015] The neutralizing activity of an antibody or its antigen-binding fragment can be determined by conventional methods. The activity of USAG-1 to neutralize BMP antagonist activity (also called "BMP antagonist neutralizing activity") can be measured in vitro, for example, by an ALP (alkaline phosphatase) assay or a reporter assay. The ALP assay is performed, for example, by culturing osteoblast progenitor cells, etc., in the presence of BMP with USAG-1 protein and an antibody or its antigen-binding fragment, and measuring the ALP produced when differentiation into osteoblasts is induced. The activity of USAG-1 to neutralize Wnt antagonist activity (also called "Wnt antagonist neutralizing activity") can be measured in vitro, for example, by a reporter assay. A reporter assay is performed, for example, by introducing a vector into cells in which a promoter region responsive to BMP or Wnt is linked to a reporter gene such as luciferase, culturing the cells in the presence of BMP or Wnt with USAG-1 protein and the antibody or its antigen-binding fragment, and measuring the expressed luciferase activity. The BMP antagonist activity neutralized by the anti-USAG-1 antibody and its antigen-binding fragment of this invention may be antagonist activity against any BMP family. For example, the anti-USAG-1 antibody and its antigen-binding fragment of this invention can neutralize antagonist activity against BMP2, BMP4, BMP6, BMP7, etc., but are not limited to these. Furthermore, the Wnt antagonist activity neutralized by the anti-USAG-1 antibody and its antigen-binding fragment of this invention may be antagonist activity against any Wnt family. For example, the anti-USAG-1 antibody and its antigen-binding fragment of this invention can neutralize antagonist activity against Wnt-1, Wnt-3, etc., but are not limited to these.

[0016] Furthermore, in this 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. Specifically, antibody A includes a heavy chain containing the amino acid sequence shown in SEQ ID NO: 1 and a light chain containing the amino acid sequence shown in SEQ ID NO: 2. The heavy chain includes a heavy chain variable region (SEQ ID NO: 3) containing the heavy chain complementarity-determining region containing the amino acid sequences shown in SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7. The light chain includes a light chain variable region (SEQ ID NO: 4) containing the light chain complementarity-determining region shown in SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10. Antibody B comprises a heavy chain containing the amino acid sequence shown in SEQ ID NO: 11 and a light chain containing the amino acid sequence shown in SEQ ID NO: 12. The heavy chain comprises a heavy chain variable region (SEQ ID NO: 13) containing the heavy chain complementarity determination region shown in SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17. The light chain comprises a light chain variable region (SEQ ID NO: 14) containing the light chain complementarity determination region shown in SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20. Antibody C comprises a heavy chain containing the amino acid sequence shown in SEQ ID NO: 21 and a light chain containing the amino acid sequence shown in SEQ ID NO: 22. The heavy chain comprises a heavy chain variable region (SEQ ID NO: 23) containing the heavy chain complementarity determination region shown in SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 27. The light chain comprises a light chain variable region (SEQ ID NO: 24) containing the light chain complementarity determination region shown in SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 30. Antibody D comprises a heavy chain containing the amino acid sequence shown in SEQ ID NO: 38 and a light chain containing the amino acid sequence shown in SEQ ID NO: 39. The heavy chain comprises a heavy chain variable region (SEQ ID NO: 40) containing the heavy chain complementarity determination region shown in SEQ ID NO: 42, SEQ ID NO: 43, and SEQ ID NO: 44. The light chain comprises a light chain variable region (SEQ ID NO: 41) containing the light chain complementarity determination region shown in SEQ ID NO: 45, SEQ ID NO: 46, and SEQ ID NO: 47.Antibody E comprises a heavy chain containing the amino acid sequence shown in SEQ ID NO: 48 and a light chain containing the amino acid sequence shown in SEQ ID NO: 49. The heavy chain comprises a heavy chain variable region (SEQ ID NO: 50) containing the heavy chain complementarity determining region shown in SEQ ID NO: 52, SEQ ID NO: 53, and SEQ ID NO: 54. The light chain comprises a light chain variable region (SEQ ID NO: 51) containing the light chain complementarity determining region shown in SEQ ID NO: 55, SEQ ID NO: 56, and SEQ ID NO: 57. Antibodies A, B, and C have BMP antagonist neutralizing activity in particular. Antibody C has both BMP antagonist neutralizing activity and Wnt antagonist neutralizing activity in particular. Antibodies D and E have Wnt antagonist neutralizing activity in particular.

[0017] Accordingly, in one embodiment of the present invention, antibodies A, B, C, D, and E, as well as their variants, are provided as antibodies or antigen-binding fragments of the present application. As an example of antibody A or its variants, an antibody or antigen-binding fragment is provided that specifically binds to and neutralizes USAG-1, and comprises three heavy chain complementarity-determining regions containing 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 NOs. 5, 6, and 7, respectively, or three light chain complementarity-determining regions containing 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 NOs. 8, 9, and 10, respectively. Preferably, an antibody or antigen-binding fragment that specifically binds to and neutralizes USAG-1 is provided, comprising three heavy chain complementarity-determining regions containing 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 containing amino acid sequences shown in SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10, respectively. More preferably, an antibody or antigen-binding fragment that specifically binds to and neutralizes USAG-1 is provided, 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 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 consisting of 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.More preferably, an antibody or antigen-binding fragment that specifically binds to and neutralizes USAG-1 is provided, comprising three heavy chain complementarity-determining regions consisting of 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 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 its variants, an antibody or its antigen-binding fragment is provided that specifically binds to and neutralizes USAG-1, comprising three heavy chain complementarity-determining regions comprising 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 shown in SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10, respectively, wherein one to several amino acid residues are substituted, deleted, inserted, or added in one or more of the above amino acid sequences. Preferably, an antibody or its antigen-binding fragment is provided that specifically binds to and neutralizes USAG-1, comprising three heavy chain complementarity-determining regions consisting of 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 amino acid sequences shown in SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10, respectively, wherein one to several amino acid residues are substituted, deleted, inserted, or added in one or more of the above amino acid sequences.

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

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

[0021] As a further example of antibody A or its variants, an antibody or antigen-binding fragment is provided which specifically binds to and neutralizes USAG-1, comprising three heavy chain complementarity-determining regions containing 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 NOs. 5, 6, and 7, respectively, and three light chain complementarity-determining regions containing 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 NOs. 8, 9, and 10, respectively. Preferably, an antibody or antigen-binding fragment that specifically binds to and neutralizes USAG-1 is provided, comprising three heavy chain complementarity-determining regions containing amino acid sequences shown in SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, and three light chain complementarity-determining regions containing amino acid sequences shown in SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10, respectively. More preferably, an antibody or antigen-binding fragment that specifically binds to and neutralizes USAG-1 is provided, 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 with 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 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.More preferably, an antibody or antigen-binding fragment that specifically binds to and neutralizes USAG-1 is provided, comprising three heavy chain complementarity-determining regions consisting of amino acid sequences shown in SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, and three light chain complementarity-determining regions consisting of 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 its variants, an antibody or its antigen-binding fragment is provided that specifically binds to and neutralizes USAG-1, comprising three heavy chain complementarity-determining regions comprising 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 amino acid sequences shown in SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10, respectively, wherein one to several amino acid residues are substituted, deleted, inserted, or added in one or more of the above amino acid sequences. Preferably, an antibody or its antigen-binding fragment is provided that specifically binds to and neutralizes USAG-1, comprising three heavy chain complementarity-determining regions comprising 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 amino acid sequences shown in SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10, respectively, wherein one to several amino acid residues are substituted, deleted, inserted, or added in one or more of the above amino acid sequences.

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

[0024] As a further example of antibody A or its variants, an antibody or its antigen-binding fragment that specifically binds to and neutralizes USAG-1 is provided, 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, wherein one to several amino acid residues are substituted, deleted, inserted, or added in one or more of the above amino acid sequences. Preferably, an antibody or its antigen-binding fragment that specifically binds to and neutralizes USAG-1 is provided, 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, wherein one to several amino acid residues are substituted, deleted, inserted, or added in one or more of the above amino acid sequences.

[0025] As an example of antibody B or its variants, an antibody or antigen-binding fragment is provided which specifically binds to and neutralizes USAG-1, and comprises three heavy chain complementarity-determining regions containing 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 NOs. 15, 16, and 17, respectively, or three light chain complementarity-determining regions containing 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 NOs. 18, 19, and 20, respectively. Preferably, an antibody or antigen-binding fragment that specifically binds to and neutralizes USAG-1 is provided, comprising three heavy chain complementarity-determining regions containing amino acid sequences shown in SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17, or three light chain complementarity-determining regions containing amino acid sequences shown in SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20, respectively. More preferably, an antibody or antigen-binding fragment that specifically binds to and neutralizes USAG-1 is provided, 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 with 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 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: 18, SEQ ID NO: 19, and SEQ ID NO: 20, respectively.More preferably, an antibody or antigen-binding fragment that specifically binds to and neutralizes USAG-1 is provided, comprising three heavy chain complementarity-determining regions consisting of amino acid sequences shown in SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17, or three light chain complementarity-determining regions consisting of 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 its variants, an antibody or its antigen-binding fragment is provided that specifically binds to and neutralizes USAG-1, comprising three heavy chain complementarity-determining regions comprising 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 shown in SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20, respectively, wherein one to several amino acid residues are substituted, deleted, inserted, or added in one or more of the above amino acid sequences. Preferably, an antibody or its antigen-binding fragment is provided that specifically binds to and neutralizes USAG-1, comprising three heavy chain complementarity-determining regions consisting of 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 amino acid sequences shown in SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20, respectively, wherein one to several amino acid residues are substituted, deleted, inserted, or added in one or more of the above amino acid sequences.

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

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

[0029] As a further example of antibody B or its variants, an antibody or antigen-binding fragment is provided which specifically binds to and neutralizes USAG-1, comprising three heavy chain complementarity-determining regions containing 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 NOs. 15, 16, and 17, respectively, and three light chain complementarity-determining regions containing 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 NOs. 18, 19, and 20, respectively. Preferably, an antibody or antigen-binding fragment that specifically binds to and neutralizes USAG-1 is provided, comprising three heavy chain complementarity-determining regions containing amino acid sequences shown in SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17, and three light chain complementarity-determining regions containing amino acid sequences shown in SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20, respectively. More preferably, an antibody or antigen-binding fragment that specifically binds to and neutralizes USAG-1 is provided, 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 with 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 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: 18, SEQ ID NO: 19, and SEQ ID NO: 20, respectively.More preferably, an antibody or antigen-binding fragment that specifically binds to and neutralizes USAG-1 is provided, comprising three heavy chain complementarity-determining regions consisting of amino acid sequences shown in SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17, and three light chain complementarity-determining regions consisting of amino acid sequences shown in SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20, respectively.

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

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

[0032] As a further example of antibody B or its variants, an antibody or its antigen-binding fragment that specifically binds to and neutralizes USAG-1 is provided, 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, wherein one to several amino acid residues are substituted, deleted, inserted, or added in one or more of the above amino acid sequences. Preferably, an antibody or its antigen-binding fragment that specifically binds to and neutralizes USAG-1 is provided, 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, wherein one to several amino acid residues are substituted, deleted, inserted, or added in one or more of the above amino acid sequences.

[0033] As an example of antibody C or its variants, an antibody or antigen-binding fragment is provided which specifically binds to and neutralizes USAG-1, and comprises three heavy chain complementarity-determining regions containing 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 NOs. 25, 26, and 27, respectively, or three light chain complementarity-determining regions containing 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 NOs. 28, 29, and 30, respectively. Preferably, an antibody or antigen-binding fragment that specifically binds to and neutralizes USAG-1 is provided, comprising three heavy chain complementarity-determining regions containing amino acid sequences shown in SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 27, or three light chain complementarity-determining regions containing amino acid sequences shown in SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 30, respectively. More preferably, an antibody or antigen-binding fragment that specifically binds to and neutralizes USAG-1 is provided, 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 with 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 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: 28, SEQ ID NO: 29, and SEQ ID NO: 30, respectively.More preferably, an antibody or antigen-binding fragment that specifically binds to and neutralizes USAG-1 is provided, comprising three heavy chain complementarity-determining regions consisting of amino acid sequences shown in SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 27, or three light chain complementarity-determining regions consisting of 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 its variants, an antibody or its antigen-binding fragment is provided that specifically binds to and neutralizes USAG-1, comprising three heavy chain complementarity-determining regions comprising 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 shown in SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 30, respectively, wherein one to several amino acid residues are substituted, deleted, inserted, or added in one or more of the above amino acid sequences. Preferably, an antibody or its antigen-binding fragment is provided that specifically binds to and neutralizes USAG-1, comprising three heavy chain complementarity-determining regions consisting of 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 amino acid sequences shown in SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 30, respectively, wherein one to several amino acid residues are substituted, deleted, inserted, or added in one or more of the above amino acid sequences.

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

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

[0037] As a further example of antibody C or its variants, an antibody or antigen-binding fragment is provided which specifically binds to and neutralizes USAG-1, comprising three heavy chain complementarity-determining regions containing 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 NOs. 25, 26, and 27, respectively, and three light chain complementarity-determining regions containing 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 NOs. 28, 29, and 30, respectively. Preferably, an antibody or antigen-binding fragment that specifically binds to and neutralizes USAG-1 is provided, comprising three heavy chain complementarity-determining regions containing amino acid sequences shown in SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 27, and three light chain complementarity-determining regions containing amino acid sequences shown in SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 30, respectively. More preferably, an antibody or antigen-binding fragment that specifically binds to and neutralizes USAG-1 is provided, 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 with the amino acid sequences shown in SEQ ID NOs. 25, 26, and 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 with the amino acid sequences shown in SEQ ID NOs. 28, 29, and 30, respectively.More preferably, an antibody or antigen-binding fragment that specifically binds to and neutralizes USAG-1 is provided, comprising three heavy chain complementarity-determining regions consisting of amino acid sequences shown in SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 27, and three light chain complementarity-determining regions consisting of amino acid sequences shown in SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 30, respectively.

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

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

[0040] As a further example of antibody C or its variants, an antibody or its antigen-binding fragment that specifically binds to and neutralizes USAG-1 is provided, 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, wherein one to several amino acid residues are substituted, deleted, inserted, or added in one or more of the above amino acid sequences. Preferably, an antibody or its antigen-binding fragment that specifically binds to and neutralizes USAG-1 is provided, 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, wherein one to several amino acid residues are substituted, deleted, inserted, or added in one or more of the above amino acid sequences.

[0041] As an example of antibody D or its variants, an antibody or antigen-binding fragment is provided which specifically binds to and neutralizes USAG-1, and comprises three heavy chain complementarity-determining regions containing 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 NOs. 42, 43, and 44, respectively, or three light chain complementarity-determining regions containing 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 NOs. 45, 46, and 47, respectively. Preferably, an antibody or antigen-binding fragment that specifically binds to and neutralizes USAG-1 is provided, comprising three heavy chain complementarity-determining regions containing amino acid sequences shown in SEQ ID NOs. 42, 43, and 44, respectively, or three light chain complementarity-determining regions containing amino acid sequences shown in SEQ ID NOs. 45, 46, and 47, respectively. More preferably, an antibody or antigen-binding fragment that specifically binds to and neutralizes USAG-1 is provided, 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 with the amino acid sequences shown in SEQ ID NOs. 42, 43, and 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 with the amino acid sequences shown in SEQ ID NOs. 45, 46, and 47, respectively.More preferably, an antibody or antigen-binding fragment that specifically binds to and neutralizes USAG-1 is provided, comprising three heavy chain complementarity-determining regions consisting of amino acid sequences shown in SEQ ID NOs. 42, 43, and 44, respectively, or three light chain complementarity-determining regions consisting of amino acid sequences shown in SEQ ID NOs. 45, 46, and 47, respectively.

[0042] As a further example of antibody D or its variants, an antibody or its antigen-binding fragment is provided that specifically binds to and neutralizes USAG-1, comprising three heavy chain complementarity-determining regions comprising amino acid sequences shown in SEQ ID NOs. 42, 43, and 44, respectively, or three light chain complementarity-determining regions comprising amino acid sequences shown in SEQ ID NOs. 45, 46, and 47, respectively, wherein one to several amino acid residues are substituted, deleted, inserted, or added in one or more of the above amino acid sequences. Preferably, an antibody or its antigen-binding fragment is provided that specifically binds to and neutralizes USAG-1, comprising three heavy chain complementarity-determining regions comprising amino acid sequences shown in SEQ ID NOs. 42, 43, and 44, respectively, or three light chain complementarity-determining regions comprising amino acid sequences shown in SEQ ID NOs. 45, 46, and 47, respectively, wherein one to several amino acid residues are substituted, deleted, inserted, or added in one or more of the above amino acid sequences.

[0043] Further examples of antibody D or its variants are provided, which are antibodies or antigen-binding fragments that specifically bind to and neutralize USAG-1, and which include a heavy chain variable region containing an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 40, or a light chain variable region containing an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 41. Preferably, antibodies or antigen-binding fragments that specifically bind to and neutralize USAG-1 are provided, and which include a heavy chain variable region containing an amino acid sequence shown in SEQ ID NO: 40, or a light chain variable region containing an amino acid sequence shown in SEQ ID NO: 41. More preferably, an antibody or antigen-binding fragment thereof that specifically binds to and neutralizes USAG-1 is provided, 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 with 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 with the amino acid sequence shown in SEQ ID NO: 41.

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

[0045] As a further example of antibody D or its variants, an antibody or antigen-binding fragment is provided which specifically binds to and neutralizes USAG-1, comprising three heavy chain complementarity-determining regions containing 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 NOs. 42, 43, and 44, respectively, and three light chain complementarity-determining regions containing 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 NOs. 45, 46, and 47, respectively. Preferably, an antibody or antigen-binding fragment that specifically binds to and neutralizes USAG-1 is provided, comprising three heavy chain complementarity-determining regions containing amino acid sequences shown in SEQ ID NOs. 42, 43, and 44, respectively, and three light chain complementarity-determining regions containing amino acid sequences shown in SEQ ID NOs. 45, 46, and 47, respectively. More preferably, an antibody or antigen-binding fragment that specifically binds to and neutralizes USAG-1 is provided, 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 with the amino acid sequences shown in SEQ ID NOs. 42, 43, and 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 with the amino acid sequences shown in SEQ ID NOs. 45, 46, and 47, respectively.More preferably, an antibody or antigen-binding fragment that specifically binds to and neutralizes USAG-1 is provided, comprising three heavy chain complementarity-determining regions consisting of amino acid sequences shown in SEQ ID NOs. 42, 43, and 44, respectively, and three light chain complementarity-determining regions consisting of amino acid sequences shown in SEQ ID NOs. 45, 46, and 47, respectively.

[0046] As a further example of antibody D or its variants, an antibody or antigen-binding fragment is provided that specifically binds to and neutralizes USAG-1, comprising three heavy chain complementarity-determining regions comprising amino acid sequences shown in SEQ ID NOs. 42, 43, and 44, respectively, and three light chain complementarity-determining regions comprising amino acid sequences shown in SEQ ID NOs. 45, 46, and 47, respectively, wherein one to several amino acid residues are substituted, deleted, inserted, or added in one or more of the above amino acid sequences. Preferably, an antibody or antigen-binding fragment is provided that specifically binds to and neutralizes USAG-1, comprising three heavy chain complementarity-determining regions comprising amino acid sequences shown in SEQ ID NOs. 42, 43, and 44, respectively, and three light chain complementarity-determining regions comprising amino acid sequences shown in SEQ ID NOs. 45, 46, and 47, respectively, wherein one to several amino acid residues are substituted, deleted, inserted, or added in one or more of the above amino acid sequences.

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

[0048] As a further example of antibody D or its variants, an antibody or its antigen-binding fragment that specifically binds to and neutralizes USAG-1 is provided, 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, wherein one to several amino acid residues are substituted, deleted, inserted, or added in one or more of the above amino acid sequences. Preferably, an antibody or its antigen-binding fragment that specifically binds to and neutralizes USAG-1 is provided, 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, wherein one to several amino acid residues are substituted, deleted, inserted, or added in one or more of the above amino acid sequences.

[0049] As an example of antibody E or its variants, an antibody or antigen-binding fragment is provided which specifically binds to and neutralizes USAG-1, and comprises three heavy chain complementarity-determining regions containing 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 NOs. 52, 53, and 54, respectively, or three light chain complementarity-determining regions containing 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 NOs. 55, 56, and 57, respectively. Preferably, an antibody or antigen-binding fragment that specifically binds to and neutralizes USAG-1 is provided, comprising three heavy chain complementarity-determining regions containing 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 containing amino acid sequences shown in SEQ ID NO: 55, SEQ ID NO: 56, and SEQ ID NO: 57, respectively. More preferably, an antibody or antigen-binding fragment that specifically binds to and neutralizes USAG-1 is provided, 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 with the amino acid sequences shown in SEQ ID NOs. 52, 53, and 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 with the amino acid sequences shown in SEQ ID NOs. 55, 56, and 57, respectively.More preferably, an antibody or antigen-binding fragment that specifically binds to and neutralizes USAG-1 is provided, comprising three heavy chain complementarity-determining regions consisting of 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 amino acid sequences shown in SEQ ID NO: 55, SEQ ID NO: 56, and SEQ ID NO: 57, respectively.

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

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

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

[0053] As a further example of antibody E or its variants, an antibody or antigen-binding fragment is provided which specifically binds to and neutralizes USAG-1, comprising three heavy chain complementarity-determining regions containing 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 NOs. 52, 53, and 54, respectively, and three light chain complementarity-determining regions containing 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 NOs. 55, 56, and 57, respectively. Preferably, an antibody or antigen-binding fragment that specifically binds to and neutralizes USAG-1 is provided, comprising three heavy chain complementarity-determining regions containing amino acid sequences shown in SEQ ID NOs. 52, 53, and 54, respectively, and three light chain complementarity-determining regions containing amino acid sequences shown in SEQ ID NOs. 55, 56, and 57, respectively. More preferably, an antibody or antigen-binding fragment that specifically binds to and neutralizes USAG-1 is provided, 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 with the amino acid sequences shown in SEQ ID NOs. 52, 53, and 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 with the amino acid sequences shown in SEQ ID NOs. 55, 56, and 57, respectively.More preferably, an antibody or antigen-binding fragment that specifically binds to and neutralizes USAG-1 is provided, comprising three heavy chain complementarity-determining regions consisting of amino acid sequences shown in SEQ ID NO: 52, SEQ ID NO: 53, and SEQ ID NO: 54, and three light chain complementarity-determining regions consisting of amino acid sequences shown in SEQ ID NO: 55, SEQ ID NO: 56, and SEQ ID NO: 57, respectively.

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

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

[0056] As a further example of antibody E or its variants, an antibody or its antigen-binding fragment that specifically binds to and neutralizes USAG-1 is provided, 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, wherein one to several amino acid residues are substituted, deleted, inserted, or added in one or more of the above amino acid sequences. Preferably, an antibody or its antigen-binding fragment that specifically binds to and neutralizes USAG-1 is provided, 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, wherein one to several amino acid residues are substituted, deleted, inserted, or added in one or more of the above amino acid sequences.

[0057] In this specification, "several" means approximately 2 to 10 amino acids, and depending on the length of the amino acid sequence, preferably approximately 2 to 7, for example, 3, 4, 5, or 6. Also in this specification, "substitution" may be a conservative substitution or a non-conservative substitution, and 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 substitution of alanine to glycine or serine, substitution of arginine to lysine or histidine, substitution of asparagine to glutamine or histidine, substitution of aspartic acid to glutamic acid or asparagine, substitution of cysteine ​​to serine or alanine, substitution of glutamine to asparagine, substitution of glutamic acid to aspartic acid or glutamine, substitution of glycine to alanine, substitution of histidine to asparagine or glutamine, and substitution of isoleucine to leucine or valine. Examples of substitutions include leucine to isoleucine or valine, lysine to arginine or histidine, methionine to leucine, isoleucine or tyrosine, phenylalanine to tyrosine, methionine or leucine, proline to alanine, serine to threonine, threonine to serine, tryptophan to tyrosine or phenylalanine, tyrosine to tryptophan or phenylalanine, and valine to isoleucine or leucine.

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

[0059] Furthermore, in another aspect of the present invention, an antibody or its antigen-binding fragment is provided which binds to all or part of the same epitope on USAG-1 to which the antibody A, antibody B, antibody C, antibody D, or antibody E, or its variant or antigen-binding fragment, described above, binds.

[0060] Furthermore, the present invention has found that antibody A specifically recognizes and binds to a polypeptide (epitope) containing VNDKTRTQRI (SEQ ID NO: 31) (corresponding to amino acid sequences 134-143 of the human USAG-1 protein) on human USAG-1. Therefore, an antibody or its antigen-binding fragment 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 its antigen-binding fragment of the present application. Furthermore, an antibody or its antigen-binding fragment that competes with antibody A or its variant or its antigen-binding fragment described above 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 its antigen-binding fragment 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. Also, the USAG-1 polypeptide containing the amino acid sequence shown in SEQ ID NO: 31 may be a polypeptide substantially identical to the polypeptide. Examples of such substantially identical polypeptides include polypeptides located at the corresponding positions on the USAG-1 protein of animals other than humans.

[0061] In this specification, “competition” means that an antibody or its antigen-binding fragment competes with a reference antibody (e.g., antibody A, antibody B, antibody C, antibody D, antibody E, or a variant thereof or its antigen-binding fragment) in a binding assay using the USAG-1 protein or polypeptide. For example, if a test antibody or its antigen-binding fragment reduces the binding of the reference antibody to the USAG-1 protein or polypeptide in a binding assay, the test antibody “competes” with the reference antibody. An antibody that competes with a reference antibody reduces the binding of the reference antibody to the 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, but are not limited to, ELISA, flow cytometry, SPR (surface plasmon resonance), BLI (Bio-Layer Interferometry), etc.

[0062] Epitope binning is a technique that classifies two or more antibodies based on their epitopes, for example, using methods such as SPR or BLI. In epitope binning, for example, an antigen protein (target) is added to a biosensor on which a reference antibody is immobilized, causing the reference antibody and target to bind. Then, the biosensor holding the complex of the reference antibody and target is reacted with a test antibody, and the binding and dissociation 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 epitope on the target is already occupied by binding to the reference antibody, and therefore the test antibody cannot bind to the biosensor. Conversely, if the test antibody recognizes a different epitope from 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 reference antibody to the target interferes with the binding of the test antibody to the epitope, and therefore 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 of the present invention binds to USAG-1 or all or part of 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. Furthermore, the antibody may be either a polyclonal antibody or a monoclonal antibody. Also, 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 comprises a human immunoglobulin (recipient antibody) in which residues from the recipient's complementarity-determining region (CDR) are replaced with residues from the CDR of a non-human species (donor antibody) having desired specificity, affinity, and binding ability. Optionally, Fv framework region (FR) residues of the human immunoglobulin may be replaced with corresponding non-human residues. Furthermore, the humanized antibody may contain residues not found in the recipient antibody or donor antibody. Generally, a humanized antibody includes at least one, typically two, variable regions in which all or substantially all of the CDRs are replaced with CDRs of a non-human immunoglobulin, and all or substantially all of the FR region is a human immunoglobulin sequence. Chimeric antibodies include antibodies produced by genetic engineering techniques in which a variable region derived from a donor antibody is ligated to the constant region of a recipient antibody. The production of these antibodies can be carried out by methods known in the field.

[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), and multispecific antibodies formed from antibody fragments. The above antibody fragments can be prepared by methods known in the art.

[0066] Isolated nucleic acids encoding the antibody or its antigen-binding fragment are also included in the present invention.

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

[0068] The pharmaceutical composition of the present application may contain, in addition to the antibody or its antigen-binding fragment, pharmaceutically acceptable carriers, stabilizers, excipients, and other additives. Examples of pharmaceutically acceptable carriers include, but are not limited to, physiological saline, buffers, glycols, glycerol, gelatin, gelatin hydrogel, polylactic acid, collagen sponge, agarose, polyvinyl alcohol, alginic acid, fibrin gel, ethylene-vinyl acetate copolymer, lactic acid-glycolic acid copolymer, and the like. 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 above carriers and additives based on the administration form or route of the pharmaceutical composition. Furthermore, the pharmaceutical composition of the present application can be manufactured by formulating the above antibody or its antigen-binding fragment with the above additives by conventional methods.

[0069] Examples of the forms of the pharmaceutical composition of this application include tablets, powders, capsules, granules, syrups, sustained-release tablets, sustained-release capsules, enteric-coated preparations, inserts, injectable preparations, and the like, with injection preparations being preferred. The pharmaceutical composition of this application is administered by systemic or local administration. The route of administration is not limited and can be appropriately selected by those skilled in the art, but examples include oral, nasal, subcutaneous, intravenous, intramuscular, and intraosseous administration. The pharmaceutical composition of this application may be administered locally, for example, to the tooth formation site.

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

[0071] The target recipients of the pharmaceutical composition of this application include mammals such as humans, dogs, cats, horses, mice, ferrets, squirrels, pigs, monkeys, etc., with humans being preferred.

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

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

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

[0076] Primary screening of 284 wells using ELISA with immunized antigens revealed numerous positive wells (Figures 4-1 and 4-2). A cutoff value of 0.7 was set, and 79 clones were selected. Expansion culture was performed, and ELISA was conducted using immunized antigens and mouse USAG-1 protein derived from a CHO cell expression system produced by Sysmex Corporation. When the absorbance cutoff value was set to 0.025 or higher, approximately half of the wells were positive (Figures 4-1 and 4-2). Antibody subclasses were determined to be IgG1, 2a, 2b, and 2c (Figures 4-1 and 4-2).

[0077] The neutralizing activity of each antibody was assessed in a similar manner to that of the antigen protein. ALP activity, which was elevated by adding 300 ng / ml of BMP7 (R&D Systems) to C2C12 cells, was suppressed by adding 300 ng / ml of rat USAG-1 protein (MyBiosource) derived from a mammalian cell expression system. The neutralization of ALP activity by adding various antibodies was then confirmed. The antibodies were classified into three categories: mild neutralizing activity (*: 60-100% neutralizing activity), moderate neutralizing activity (**: 100-140% neutralizing activity), and highly neutralizing activity (***: 140% or more neutralizing activity). For the BMP reporter assay, the commercially available cell line BMP Responsive Reporter Osteoblast Cell Line (Briter cell) (Kerafast), which incorporates BRE-Luc, was used. In an experimental system where 300 ng / ml of BMP7 was added and the expressed luciferase activity was suppressed by adding 300 ng / ml of rat USAG-1 protein derived from a mammalian cell expression system, the neutralization of luciferase activity suppression was confirmed by adding various antibodies. These were classified into those showing mild neutralization activity (*: 40-60% neutralization activity) and those showing moderate neutralization activity (**: 60% or more neutralization activity). For the WNT reporter assay, HEK293 cells were introduced 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 signaling pathway, the WNT1 gene, and a reporter gene regulated by the HSV-thymidine kinase promoter to obtain an internal standard. These cells were then 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 signaling pathway. The amount of USAG-1 protein added was predetermined. The culture supernatant of antibody-producing hybridomas was added to the culture medium of the screening system in concentrations of 25%, 20%, and 10%, respectively, and the results were evaluated in three experiments.When 25% was added, samples were classified as showing mild neutralizing activity (*: corrected luminescence value of 1.5-2.0) or moderate neutralizing activity (**: corrected luminescence value of 2.0 or higher). When 20% was added, samples were classified as showing mild neutralizing activity (*: corrected luminescence value of 1-1.1) or moderate neutralizing activity (**: corrected luminescence value of 1.1 or higher). When 10% was added, samples were classified as showing mild neutralizing activity (*: corrected luminescence value of 1-1.1) or moderate neutralizing activity (**: corrected luminescence value of 1.1 or higher). The percentage of neutralizing activity was calculated based on the activity under conditions without USAG-1 protein (100%). Furthermore, the neutralizing activity based on luminescence value was calculated with the activity under conditions without antibody added set to 1.

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

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

[0080] Antibody in vitro test 1 After confirming transient expression of recombinant mouse USAG-1(WISE) protein with a PA tag at the N-terminus in Expi293F cells, a stable expression cell line was established. Affinity purification was performed using the PA tag system, yielding 0.2 mg of PA-mUSAG-1(WISE) 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) conditions. N-terminal PA-tagged USAG-1(WISE) protein from mice derived from the mammalian cell Expi293F cell expression system showed dose-dependent inhibitory activity of WNT signaling in the WNT reporter assay (Figure 6) and dose-dependent inhibitory activity of BMP signaling in the BMP ALP assay (Figure 7). Using the mouse USAG-1 protein whose activity was confirmed, the neutralizing activity of five mouse anti-USAG-1 neutralizing antibodies (E12, E16, E37, E48, E57) was confirmed. In the WNT reporter assay, cells into which a vector with a promoter linked to the luciferase gene and a Wnt1 expression vector were introduced were cultured with 1.7 μg of recombinant mouse USAG-1 protein and various antibodies at a ratio of 1 / 1000, 1 / 300, or 1 / 100 of the culture 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 recombinant mouse USAG-1 protein, and various antibodies at 1x (30 ng / ml), 10x (300 ng / ml), and 100x (3 μg / ml) concentrations, and ALP activity was measured.

[0081] As a result, in the WNT reporter assay, the presence of an antibody that neutralizes the WNT signaling inhibitory activity of mouse USAG-1 in a dose-dependent manner was confirmed (Figure 8). In the BMP ALP assay, the presence of an antibody that neutralizes the BMP signaling inhibitory activity of mouse USAG-1 in a dose-dependent manner was confirmed (Figure 9). [Examples]

[0082] In vivo antibody administration study 1 Homozygous EDA-deficient mice, a congenital anodontia model, exhibit a high rate (approximately 90%) of mandibular third molar (M3) absence. Mother mice pregnant with EDA-deficient mice were given a single intraperitoneal dose of mouse anti-USAG-1 neutralizing antibody A (#37). As a result, in 7 out of 8 EDA-deficient mice born, the mandibular third molar (M3) absence was restored (Figure 10). No supernumerary teeth were observed in EDA-deficient mice treated with mouse anti-USAG-1 neutralizing antibody A. Therefore, it was found that antibody A can restore missing teeth. Here, "restoration" refers to the presence of teeth in the area (M3) that is normally missing in EDA-deficient mice (i.e., M3 is not missing) in the newborn EDA-deficient mice. [Examples]

[0083] In vivo antibody administration trial 2 Mother mice pregnant with EDA-deficient mice, a model of congenital anodontia, were given a single intraperitoneal dose of mouse anti-USAG-1 neutralizing antibody B (E57). As a result, in 2 out of 3 EDA-deficient homozygous mice born, the formation of supernumerary teeth in the anterior region or fused teeth in the maxillary molar region was induced (Figure 10). In EDA-deficient heterozygous mice born, supernumerary teeth in the anterior region or fused teeth in the molar region were observed in all 5 cases. Furthermore, when mouse anti-USAG-1 neutralizing antibody B (E57) was administered a single intraperitoneal dose to mother mice pregnant with wild-type mice, supernumerary teeth in the anterior region or fused teeth in the molar region were observed in 11 out of 12 cases (Figure 11). Therefore, 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 trial 3 A mixture of five anti-USAG-1 neutralizing antibodies obtained in Example 1, including neutralizing antibodies A and B, was administered intraperitoneally as a single dose to pregnant mother mice carrying Wnt10a-deficient mice, a model mouse for odontopathy. As a result, supernumerary teeth were formed in the maxillary anterior region (Figure 10). [Examples]

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

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

[0087] Antibody production 2 To produce mouse USAG-1 neutralizing antibodies, rat USAG-1 protein derived from a baculovirus expression system was used as the antigen. Neutralizing antibodies were produced using the iliac lymph node method with 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 a large number of positive wells were observed. A cutoff value of 1.0 was set, and 111 clones were selected. When the immunized antigen was used for expansion culture and sandwich ELISA was performed, positive wells were observed in about half of the samples when the absorbance cutoff values ​​were set to 0.5 or higher (His tag) and 1.4 or higher (Myc tag). The antibody subclasses were measured and found to be IgG1, 2a, 2b, and G3. Furthermore, the neutralizing activity of each obtained antibody was measured using the same method as described in Example 1, and ultimately four types of mouse anti-USAG-1 neutralizing antibodies (B14, B48, B103, B108) were obtained.

[0088] Furthermore, similar to Example 6, a binding assay was performed using mouse / human N-terminal PA-tagged USAG-1 protein to confirm whether the obtained mouse anti-USAG-1 neutralizing antibodies (B14, B48, B103, B108) could recognize the human USAG-1 protein. Each mouse anti-USAG-1 neutralizing antibody (250 μl) was added to 30 μl of Protein A Sepharose, and Protein A / G IgG binding buffer (Pierce TM5 μg of 5 μg of PBS (at room temperature for 1.5 hours) was captured. Then, 0.75 mL of culture supernatant (at room temperature for 2 hours) of human or mouse USAG-1 (WISE) recombinant protein transiently expressed in Expi293F cells with a PA tag at the N-terminus was added and incubated. The cells were washed three times with TBS buffer to remove unbound proteins. All proteins attached to Sepharose were eluted, and bands were confirmed by SDS-PAGE electrophoresis and CBB (Coomassie Brilliant Blue) staining. As a result, although the binding of B103 and B108 was weaker than that of B14 and B48, all tested mouse anti-USAG-1 neutralizing antibodies bound to both mouse USAG-1 and human USAG-1 proteins (Figure 15).

[0089] Of these, the sequence of B14 (referred to as antibody C in this specification) was determined. The full-length heavy chain sequence of antibody C, including the signal sequence, 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 Figure 21. In addition, the sequences of B48 (referred to as antibody D in this specification) and B103 (referred to as antibody E in this specification) were determined. The full-length heavy chain sequences of antibodies D and E, including the signal sequences, are shown in SEQ ID NO: 38 and SEQ ID NO: 48, respectively, and the full-length light chain sequences, including the signal sequences, 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 Figure 22. [Examples]

[0090] Epitope Binning The five mouse USAG-1 neutralizing antibodies selected in Example 1 (E12, E16, E37, E48, E57) and the neutralizing antibodies prepared in Example 7 (B14, B48, B103, B108) were subjected to epitope binning. Figure 16 shows a comparison of the competitive binding data obtained for six of the antibodies. Epitope binning was performed using Octet® Red (Pall ForteBio). In short, nine antibodies were immobilized on a biosensor as capture antibodies, a target containing purified recombinant mouse USAG-1 full-length protein was added and bound, and then the test antibody was reacted to detect the binding signal. This cycle was performed consecutively for nine test antibodies, each containing the same antibody used for capture. Antibodies that do not compete with the capture antibody immobilized on the biosensor for recognition site can bind to the captured USAG-1 protein, resulting in an increased signal. However, competing antibodies show reduced binding, resulting in no increase or a low increase. Based on the obtained signal data, the nine antibodies were grouped according to their epitopes. Specifically, when a test antibody was reacted with the sensors of the nine capture antibodies, antibodies that produced a signal equivalent to or weaker than the signal produced when the same antibody as the capture antibody was added (shown in bold and underlined in Figure 16) were considered to belong to the same group as the capture antibody. As a result, E37 and E48 competed against E12 antibody, E48, E57, and B14 against E16 antibody, E12 against E37 antibody, E16, E57, and B14 against E48 antibody, E16, E48, and B14 against E57 antibody, E16, E48, and B14 against B14 antibody, and B16, E48, and E57 against B14 antibody, and B108 against B103. Based on these results, the nine antibodies were classified into four groups, as shown in Table 1. However, although antibody E48 is basically classified into group 2, it was also close to group 1 because it competed with E12.

[0091] [Table 1] [Examples]

[0092] Epitope Mapping Epitope mapping was performed on antibody E37 (neutralizing antibody A) from Group 1. Simply put, a peptide library of 169 overlapping 15-amino acid peptides was synthesized by shifting the human USAG-1 protein sequence (183 amino acids long), excluding the signal peptide, by one amino acid from the beginning. These 169 peptides were conjugated onto 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 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 134-143 of the full-length USAG-1 amino acid sequence including the signal peptide) was identified as an epitope. [Examples]

[0094] Antibody in vitro test 2 Of the nine antibodies grouped in Example 8, affinity (KD value) was determined for eight of them using the same Octet-based BLI method 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, and 100 nM). Binding and dissociation curves were obtained for each antibody, and these were globally fitted using the analysis program attached to the Octet instrument to calculate the KD value. The results are shown in Table 2.

[0095] [Table 2] [Examples]

[0096] Antibody in vitro trial 3 For antibody B14, which was classified as Group 2 in Example 8, the BMP and Wnt signaling inhibitory neutralizing activity was measured. The experiment was carried out using the same method as in Example 2. Specifically, in the WNT reporter assay, cells into which a vector with a promoter linked to the luciferase gene and a Wnt1 expression vector (1 μg) were introduced were cultured with 1 μg of mouse USAG-1 recombinant protein and antibodies added to the medium at concentrations of 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 various antibodies at concentrations of 30 ng / ml, 150 ng / ml, 300 ng / ml, or 1500 ng / ml, and ALP activity was measured. The results are shown in Figure 17. In a WNT reporter assay, antibody B14 was confirmed to neutralize the inhibitory activity of USAG-1 on WNT signaling (up to 42% neutralization activity). In a BMP ALP assay, antibody B14 was confirmed to neutralize the inhibitory activity of USAG-1 on BMP signaling.

[0097] Furthermore, the EC50 value of antibody B14 against BMP and Wnt signaling inhibition by USAG-1 was calculated by setting the value at 50% inhibition (without antibody addition set to 0, and maximum neutralizing activity set to 100%). The results showed that the EC50 values ​​for BMP and Wnt signaling inhibition were 298 ng / ml and 4.73 μg / ml, respectively. [Examples]

[0098] In vivo antibody administration study 4 (mouse) Six antibodies classified into groups 1 and 2 in Example 8 were administered as a single intraperitoneal dose to EDA homozygous-deficient mice, EDA heterozygous-deficient mice, or mother mice pregnant with wild-type mice. The results are shown in Table 3. All six antibodies were found to induce the formation of supernumerary / fused teeth in EDA-deficient mice, thereby increasing the number of teeth in EDA-deficient mice. In particular, the antibodies in group 1 restored missing teeth in EDA-deficient mice. While the antibodies in group 1 did not induce the formation of supernumerary or fused teeth in wild-type mice, antibodies E57 and B14 from group 2 induced the formation of supernumerary and fused teeth in wild-type mice. Antibody B14 (antibody C), like antibody E57 (antibody B), was found to increase the number of teeth in all EDA-deficient homozygous-deficient mice, EDA-deficient heterozygous mice, and wild-type mice. In this context, "recovery" refers to the presence of teeth in the area (M3) that is normally missing in EDA-deficient mice (i.e., M3 is not missing) in the newly born EDA-deficient mice.

[0099] [Table 3] [Examples]

[0100] In vivo antibody administration trial 5 (dogs) Anti-mouse USAG-1 neutralizing antibody B14 (50 μg / g body weight) was administered as a single intraperitoneal dose to newborn congenital anodontia model dogs. These dogs were TOYO Beagle strains exhibiting congenital anodontia and were obtained from Kitayama Labes Hongo Farm. Some of these dogs lacked the maxillary third premolar, while others lacked the mandibular fourth premolar. Dental X-rays were used to evaluate tooth germ calcification 10 weeks after administration, confirming the recovery of missing teeth (Figure 18). The results are shown in Table 4. Furthermore, blood concentrations of the administered antibody were measured in each individual 3 days, 1, 3, 5, and 7 weeks after administration. The results showed individual differences, but the half-life was 1 week, and the antibody was maintained up to 7 weeks after administration.

[0101] [Table 4]

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

[0103] In vivo antibody administration trial 6 (ferrets) Ferrets, like humans, are diphyodontic, possessing a tooth count similar to the basic mammalian dental formula: 3 incisors, 1 canine, 3 premolars, and 2 molars. 39 ferrets were intraperitoneally administered 39 different USAG-1 neutralizing antibodies (16 μg / g body weight), including those produced in Examples 1 and 7, at 1 and 3 weeks postnatology (one ferret per antibody). 38 ferrets survived at 14 weeks postnatal. Four ferrets administered with four different antibodies, including those from groups 1-4 identified in Example 8, were followed up for 30 weeks postnatal. As a result, several ferrets showed a large number of large teeth in the upper and lower incisors and premolars. Furthermore, in ferrets administered antibody B14, induction of a third phytoe in the lingual third premolar region of the mandible was observed at 14 weeks postnatal (Figure 19). In ferrets administered antibody B103, an extra incisor tooth developed on the lingual (palatal) side of the maxilla at 30 weeks of age, and induction of a third vitreous tooth in the maxillary incisor region was observed (Figure 23). This incisor tooth developed later than the permanent tooth and was morphologically similar to the preceding permanent tooth. This incisor tooth had a short root. Furthermore, in ferrets administered antibody B103, induction of a third vitreous tooth was observed in the left and right premolar regions of the mandible at 30 weeks of age (Figure 24). [Examples]

[0104] Antibody in vivo administration trial 7 (Sunx) Pregnant Sunkus were obtained, and on day 17 of gestation, various USAG-1 neutralizing antibodies (16 μg / g body weight) prepared in Example 1 were administered intraperitoneally. Evaluation was performed by μCT imaging at 7 weeks postnatal. Sunkus administered with USAG-1 neutralizing antibodies did not give birth, but in parent Sunkus aged 4-8 months that were administered with antibodies B (E57) and C (B14), the formation of new teeth was observed around the tooth roots, where ameloemic epithelial stem cells underwent local epithelial-mesenchymal induction. In Sunkus administered with antibody B, induction of a third erupting tooth was observed between the first and second premolars on the mandibular buccal side (Figure 20). Therefore, it was shown that the USAG-1 neutralizing antibody of the present invention induces the formation of a third erupting tooth. [Examples]

[0105] Antibody in vitro testing 4 For antibody B48, which was classified as group 3 in Example 8, the BMP and Wnt signaling inhibitory neutralizing activity was measured. The experiment was carried out using the same method as described in Example 2. Specifically, in the WNT reporter assay, cells into which a vector with a promoter linked to the luciferase gene and a Wnt1 expression vector (1 μg) were introduced were cultured with 1 μg of mouse USAG-1 recombinant protein and antibody at concentrations of 1, 3, 6, 10, or 30 μg / ml relative to the culture 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 0.1 times (3 ng / ml), 10 times (300 ng / ml), or 100 times (3 μg / ml) of antibody, and ALP activity was measured. The results are shown in Figure 25. In a WNT reporter assay, antibody B48 was confirmed to neutralize the WNT signaling inhibitory activity mediated by USAG-1 by almost 100%. [Examples]

[0106] Antibody in vitro test 5 (Pull-down assay using Protein A Sepharose with PA-tagged USAG-1 and E1E2) Wnt transmits signals into cells by binding to the LRP (low-density lipoprotein receptor-related protein) 5 / 6 receptor, which is a coupled receptor for Frizzled. LRP6 has four extracellular domains (E1-E4), of which E1 and E2 are known to be involved in USAG-1 binding. In other words, USAG-1 binds to the E1 and E2 regions of LRP6 and suppresses Wnt signaling. Therefore, we investigated whether the nine mouse anti-USAG-1 neutralizing antibodies (E12, E16, E37, E48, E57, B14, B48, B103, B108) grouped in Example 8 inhibited the binding of USAG-1 to LRP6-E1E2.

[0107] Each sample of Protein A Sepharose (30 μl) was captured with a mouse anti-USAG-1 neutralizing antibody (5 μg in 1 ml of PBS) (room temperature, 1.5 hours). Then, 1 mL of culture supernatant (1 ml) of mouse USAG-1 recombinant protein transiently expressed in Expi293F cells with a PA tag at the N-terminus was added and incubated (room temperature, 2 hours). Next, 1 mL of culture supernatant (1 ml) expressing LRP6-E1E2 (human LRP6 with a His tag fused to amino acid numbers 1-629) was added and incubated (room temperature, 2.5 hours). The samples were washed three times with PBS buffer (1 mL) to remove unbound proteins. All proteins bound to Sepharose were eluted, and bands were confirmed by SDS-PAGE electrophoresis and Oriole gel fluorescence staining. The results are shown in Figure 26. In Figure 26, the right-hand panel shows the following samples: a negative control (no mAb) obtained by performing the same procedure without capturing mouse anti-USAG-1 neutralizing antibody; a positive control (WISE+E1E2 By NZ1) obtained by immunoprecipitating a complex of mouse USAG-1 recombinant protein and LRP6-E1E2 with PA-tagged antibody NZ-1; and a positive control obtained by precipitating LRP6-E1E2 alone with Ni-NTA resin that adsorbs His tags (E1E2 By NiNTA).

[0108] As a result, it was found that LRP6-E1E2 did not bind to the complexes of several antibodies (particularly antibodies B48, B103, and B108 from groups 3 and 4) with USAG-1 (Figure 26). Therefore, it was found that the epitopes of these antibodies are located in regions that overlap with or are structurally close to the LRP6 binding site of USAG-1, and that the binding of these antibodies to USAG-1 inhibits the binding of USAG-1 to LRP6, thereby neutralizing the suppression of Wnt signaling by USAG-1. [Examples]

[0109] In vivo antibody administration study 8 (mouse) In Example 8, three antibodies classified into groups 3 and 4 were administered as a single intraperitoneal dose (16 μg / g body weight) to EDA homozygous deficient congenital anodontia model mice, EDA heterozygous deficient congenital anodontia model mice, or mother mice pregnant with wild-type mice. The presence of supernumerary teeth and fused teeth, as well as the regeneration of missing teeth, were examined in the offspring. The results are shown in Table 5. The three antibodies, in particular, restored missing teeth in EDA-deficient mice. Here, "regeneration" refers to the presence of teeth in the area (M3) that is normally missing in EDA-deficient mice (i.e., M3 is not missing) in the offspring.

[0110] [Table 5] [Examples]

[0111] In vivo antibody administration study 9 (mouse) Five antibodies (E57, B14, B48, B103, B108) were administered as a single intraperitoneal dose to Wnt10a homozygous knockout mice (a congenital anodontia model mouse), Wnt10a heterozygous knockout mice (a congenital anodontia model mouse), or mother mice pregnant with wild-type mice (B103 was administered at 2 μg / g body weight, and the other antibodies at 16 μg / g body weight). The presence of supernumerary teeth and fused teeth, as well as the regeneration of missing teeth, were examined in the offspring. The results are shown in Table 6. Of the antibodies in groups 3 and 4, B48 and B103 induced the formation of supernumerary teeth and fused teeth in homozygous knockout mice.

[0112] [Table 6] [Industrial applicability]

[0113] The antibody or its antigen-binding fragment described herein can be used to treat congenital anodontia and acquired tooth loss. Furthermore, the antibody or its antigen-binding fragment is thought to be effective in the formation of third teeth, which could lead to the development of molecularly targeted drugs for tooth regeneration in the pharmaceutical field, and the establishment of treatment methods for tooth regeneration by inducing the formation of third teeth. [Sequence Listing 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 its antigen-binding fragment that specifically binds to and neutralizes USAG-1.

2. The antibody or antigen fragment 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 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 determination regions, each containing an amino acid sequence having at least 90% 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 determination regions, each containing an amino acid sequence having at least 90% sequence identity with the amino acid sequences shown in SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10, (b) Three heavy chain complementarity determination regions, each containing an amino acid sequence having at least 90% sequence identity with the amino acid sequences shown in SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17, or three light chain complementarity determination regions, each containing an amino acid sequence having at least 90% sequence identity with the amino acid sequences shown in SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20, (c) Three heavy chain complementarity determination regions, each containing an amino acid sequence having at least 90% sequence identity with the amino acid sequences shown in SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 27, or three light chain complementarity determination regions, each containing an amino acid sequence having at least 90% sequence identity with the amino acid sequences shown in SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 30, (d) Three heavy chain complementarity determination regions, each containing an amino acid sequence having at least 90% sequence identity with the amino acid sequences shown in SEQ ID NO: 42, SEQ ID NO: 43, and SEQ ID NO: 44, or three light chain complementarity determination regions, each containing an amino acid sequence having at least 90% sequence identity with the amino acid sequences shown in SEQ ID NO: 45, SEQ ID NO: 46, and SEQ ID NO: 47, or, (e) Three heavy chain complementarity determination regions, each containing an amino acid sequence having at least 90% sequence identity with 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 determination regions, each containing an amino acid sequence having at least 90% sequence identity with the amino acid sequences shown in SEQ ID NO: 55, SEQ ID NO: 56, and SEQ ID NO: 57, respectively. An antibody or antigen-binding fragment according to any one of claims 1 to 3, comprising:

5. (f) A heavy chain variable region containing an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 3, or a light chain variable region containing an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 4, (g) A heavy chain variable region containing an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 13, or a light chain variable region containing an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO:

14. (h) A heavy chain variable region containing an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 23, or a light chain variable region containing an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 24, (i) A heavy chain variable region containing an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 40, or a light chain variable region containing an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 41, or, (j) A heavy chain variable region containing an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 50, or a light chain variable region containing an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 51 An antibody or antigen-binding fragment according to any one of claims 1 to 4, comprising:

6. (k) Three heavy chain complementarity determination regions, each containing an amino acid sequence having at least 90% sequence identity with the amino acid sequences shown in SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, and three light chain complementarity determination regions, each containing an amino acid sequence having at least 90% sequence identity with the amino acid sequences shown in SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10, (l) Three heavy chain complementarity determination regions, each containing an amino acid sequence having at least 90% sequence identity with the amino acid sequences shown in SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17, and three light chain complementarity determination regions, each containing an amino acid sequence having at least 90% sequence identity with the amino acid sequences shown in SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20, (m) Three heavy chain complementarity determination regions, each containing an amino acid sequence having at least 90% sequence identity with the amino acid sequences shown in SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 27, and three light chain complementarity determination regions, each containing an amino acid sequence having at least 90% sequence identity with the amino acid sequences shown in SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 30, (n) Three heavy chain complementarity determination regions, each containing an amino acid sequence having at least 90% sequence identity with the amino acid sequences shown in SEQ ID NO: 42, SEQ ID NO: 43, and SEQ ID NO: 44, and three light chain complementarity determination regions, each containing an amino acid sequence having at least 90% sequence identity with the amino acid sequences shown in SEQ ID NO: 45, SEQ ID NO: 46, and SEQ ID NO: 47, or (o) Three heavy chain complementarity determination regions, each containing an amino acid sequence having at least 90% sequence identity with the amino acid sequences shown in SEQ ID NO: 52, SEQ ID NO: 53, and SEQ ID NO: 54, and three light chain complementarity determination regions, each containing an amino acid sequence having at least 90% sequence identity with the amino acid sequences shown in SEQ ID NO: 55, SEQ ID NO: 56, and SEQ ID NO:

57. An antibody or antigen-binding fragment according to any one of claims 1 to 3, comprising:

7. (p) A heavy chain variable region containing 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 containing 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 containing an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 13, and a light chain variable region containing an amino acid sequence having at least 90% sequence identity with 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 with the amino acid sequence shown in SEQ ID NO: 23, 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: 24, (s) A heavy chain variable region containing an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 40, and a light chain variable region containing an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 41, or (t) Heavy chain variable region including an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 50, and light chain variable region including an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 51 An antibody or antigen-binding fragment according to any one of claims 1 to 3 and 6, comprising:

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

9. The antibody or its antigen-binding fragment 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 treatment comprising an antibody or antigen-binding fragment according to any one of claims 1 to 9.

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