Anti-ADAMTS13 antibody and its applications

Abstract

A problem addressed by the present invention is to provide a formulation of an antibody or an antibody derivative capable of preventing or treating acquired von Willebrand syndrome (AVWS) that develops in association with diseases that require mechanically assisted circulation.　The present invention solved the problem by providing an antibody or an antibody derivative having specific binding activity for ADAMTS13, a cleaving protease of von Willebrand factor (VWF), which is thought to be the cause of human acquired von Willebrand syndrome (AVWS), and capable of decreasing excessive cleavage of VWF by ADAMTS13.

Description

[Technical Field]

[0001] The present invention relates to an antibody having inhibitory function against the activity of ADAMTS13, a VWF-cleaving protease that has the activity to cleave von Willebrand factor (VWF), and to pharmaceutical applications of such an antibody. [Background technology]

[0002] Von Willebrand factor (VWF) is a high-molecular-weight plasma glycoprotein that plays a role in initial platelet adhesion, platelet aggregation, and stabilization of coagulation factor VIII at the site of vascular injury. It is produced in vascular endothelial cells and megakaryocytes, secreted as a multimer, and cleaved into pieces of appropriate size for hemostasis by ADAMTS13, a VWF cleavage protease. However, excessive cleavage of VWF by ADAMTS13 can lead to bleeding and the development of von Willebrand disease (VWD), a congenital coagulation disorder.

[0003] The inventors of the present invention have produced a monoclonal antibody that has specific affinity for ADAMTS13 for the purpose of treating VMD (Patent Document 1). Using the obtained antibody, they have shown that human ADAMTS13 is localized to satellite cells of the liver (Non-Patent Document 1).

[0004] In recent years, bleeding complications following left ventricular assist device (LVAD) implantation have become a major problem. One important factor has been identified as the loss of high molecular weight multimers of vWF in patients after mechanical circulatory support (Non-Patent Literature 2). This is acquired von Willebrand syndrome (AVWS), which, unlike VMD (Vascular Muscle Disease), is characterized by the absence of a history of bleeding disorders in the past or family. Currently, AVWS is receiving increased attention due to the growing number of diseases requiring mechanical circulatory support.

[0005] However, there is no established method for preventing or treating AVWS, and establishing such a method is considered an urgent task. [Prior art documents] [Patent Documents]

[0006] [Patent Document 1] Patent No. 4533995 [Non-patent literature]

[0007] [Non-Patent Document 1] Uemura M, et al., Blood, 106, 922-924, 2005 [Non-Patent Document 2] Meyer AL, et al. JACC Heart Fail 2141-145, 2014 [Overview of the project] [Problems that the invention aims to solve]

[0008] The present invention aims to provide an antibody or antibody derivative formulation that can prevent or treat AVWS, which occurs in association with diseases requiring mechanical circulatory support. [Means for solving the problem]

[0009] The present invention aims to solve the above problem by providing an antibody or antibody derivative that has specific binding activity to ADAMTS13, a VWF cleavage protease that is thought to be the cause of human AVWS, and that can reduce the excessive cleavage of VWF by ADAMTS13.

[0010] More specifically, this application provides the following embodiments to solve the aforementioned problems: [1]: (1) Heavy chain complementarity determining regions, CDR1(GYSFTGYT, SEQ ID No: 1), CDR2(INPYNGGT, SEQ ID No: 2), and CDR3(ARTSGYLFAY, SEQ ID No: 3), Light chain complementarity-determining regions: CDR1 (EDIYNR, SEQ ID No: 4), CDR2 (GAT, SEQ ID No: 5), and CDR3 (QQYWSSPLT, SEQ ID No: 6); (2) Heavy chain complementarity determination regions, CDR1 (GFSLPRYG, SEQ ID No: 7), CDR2 (IWAGGST, SEQ ID No: 8), and CDR3 (ARAGGSQPFDY, SEQ ID No: 9), and Light chain complementarity-determining regions: CDR1 (RDINTY, SEQ ID No: 10), CDR2 (RAN, SEQ ID No: 11), and CDR3 (LQYDEFPWT, SEQ ID No: 12); (3) Heavy chain complementarity determination regions, CDR1 (GFSLTRYG, SEQ ID No: 13), CDR2 (IWAGGST, SEQ ID No: 14), and CDR3 (ARAGGSSSFDY, SEQ ID No: 15), Light chain complementarity-determining regions: CDR1 (QDINTY, SEQ ID No: 16), CDR2 (RAN, SEQ ID No: 17), and CDR3 (LQYDEFPWT, SEQ ID No: 18); (4) Heavy chain complementarity determination regions, CDR1 (GFSLTGYG, SEQ ID No: 19), CDR2 (IWADGTT, SEQ ID No: 20), and CDR3 (ARAGGSQPFDY, SEQ ID No: 21), Light chain complementarity determination regions: CDR1 (QDINSY, SEQ ID No: 22), CDR2 (RAN, SEQ ID No: 23), and CDR3 (LQYDEFPWT, SEQ ID No: 24); An antibody or antibody derivative thereof that contains a heavy chain / light chain complementarity determining region selected from the group consisting of the following, is capable of binding to ADAMTS13, and has inhibitory activity on cleavage activity of von Willebrand factor (VWF); [2]: Recombinant antibodies or antibody derivatives described in [1]; [3]: The antibody derivative is selected from an antibody variant or a functional fragment thereof, selected from the group consisting of humanized antibodies, chimeric antibodies, single-chain antibodies, polyvalent antibodies, and multispecific antibodies, as described in [1] or [2]; [4]: The amino acid sequence of the heavy chain variable region VH domain of an antibody or antibody derivative is (1-1) The amino acid sequence of SEQ ID No: 25, or the amino acid sequence of SEQ ID No: 25 that includes one or more amino acid substitutions (e.g., conservative substitutions), insertions, or deletions in portions other than CDR1 (SEQ ID No: 1), CDR2 (SEQ ID No: 2), and CDR3 (SEQ ID No: 3), (1-2) The amino acid sequence of SEQ ID No: 27, or the amino acid sequence of SEQ ID No: 27 that includes one or more amino acid substitutions (e.g., conservative substitutions), insertions, or deletions in portions other than CDR1 (SEQ ID No: 1), CDR2 (SEQ ID No: 2), and CDR3 (SEQ ID No: 3), (1-3) The amino acid sequence of SEQ ID No: 29, or the amino acid sequence of SEQ ID No: 29 that includes one or more amino acid substitutions (e.g., conservative substitutions), insertions, or deletions in portions other than CDR1 (SEQ ID No: 1), CDR2 (SEQ ID No: 2), and CDR3 (SEQ ID No: 3), (2) The amino acid sequence of SEQ ID No: 31, or an amino acid sequence comprising one or several amino acid substitutions (e.g., conservative substitutions), insertions, or deletions in the portion of the amino acid sequence of SEQ ID No: 31 other than CDR1 (SEQ ID No: 7), CDR2 (SEQ ID No: 8), and CDR3 (SEQ ID No: 9), (3) The amino acid sequence of SEQ ID No: 33, or an amino acid sequence comprising one or several amino acid substitutions (e.g., conservative substitutions), insertions, or deletions in the portion of the amino acid sequence of SEQ ID No: 33 other than CDR1 (SEQ ID No: 13), CDR2 (SEQ ID No: 14), and CDR3 (SEQ ID No: 15), and (4) The amino acid sequence of SEQ ID No: 35, or an amino acid sequence comprising one or several amino acid substitutions (e.g., conservative substitutions), insertions, or deletions in the portion of the amino acid sequence of SEQ ID No: 35 other than CDR1 (SEQ ID No: 19), CDR2 (SEQ ID No: 20), and CDR3 (SEQ ID No: 21), An antibody or antibody derivative according to any one of [1] to [3], selected from the group consisting of: [5]: The amino acid sequence of the VL domain of the light chain variable region of the antibody or antibody derivative is (1-1) The amino acid sequence of SEQ ID No: 26, or an amino acid sequence comprising one or several amino acid substitutions (e.g., conservative substitutions), insertions, or deletions in the portion of the amino acid sequence of SEQ ID No: 26 other than CDR1 (SEQ ID No: 4), CDR2 (SEQ ID No: 5), and CDR3 (SEQ ID No: 6), (1-2) The amino acid sequence of SEQ ID No: 28, or an amino acid sequence comprising one or several amino acid substitutions (e.g., conservative substitutions), insertions, or deletions in the portion of the amino acid sequence of SEQ ID No: 28 other than CDR1 (SEQ ID No: 4), CDR2 (SEQ ID No: 5), and CDR3 (SEQ ID No: 6), (1-3) The amino acid sequence of SEQ ID No: 30, or an amino acid sequence comprising one or several amino acid substitutions (e.g., conservative substitutions), insertions, or deletions in a portion other than CDR1 (SEQ ID No: 4), CDR2 (SEQ ID No: 5), and CDR3 (SEQ ID No: 6) of the amino acid sequence of SEQ ID No: 30, (2) The amino acid sequence of SEQ ID No: 32, or an amino acid sequence comprising one or several amino acid substitutions (e.g., conservative substitutions), insertions, or deletions in a portion other than CDR1 (SEQ ID No: 10), CDR2 (SEQ ID No: 11), and CDR3 (SEQ ID No: 12) of the amino acid sequence of SEQ ID No: 32, (3) The amino acid sequence of SEQ ID No: 34, or an amino acid sequence comprising one or several amino acid substitutions (e.g., conservative substitutions), insertions, or deletions in a portion other than CDR1 (SEQ ID No: 16), CDR2 (SEQ ID No: 17), and CDR3 (SEQ ID No: 18) of the amino acid sequence of SEQ ID No: 34, and (4) The amino acid sequence of SEQ ID No: 36, or an amino acid sequence comprising one or several amino acid substitutions (e.g., conservative substitutions), insertions, or deletions in a portion other than CDR1 (SEQ ID No: 2), CDR2 (SEQ ID No: 23), and CDR3 (SEQ ID No: 24) of the amino acid sequence of SEQ ID No: 36, An antibody or antibody derivative according to any one of [1] to [4], selected from the group consisting of [6]: The antibody or antibody derivative is (1-1) A heavy chain (the amino acid sequence of SEQ ID No: 37, or an amino acid sequence comprising one or several amino acid substitutions (e.g., conservative substitutions), insertions, or deletions in a portion other than CDR1 (SEQ ID No: 1), CDR2 (SEQ ID No: 2), and CDR3 (SEQ ID No: 3) of the amino acid sequence of SEQ ID No: 37) and Light chain (amino acid sequence of SEQ ID No: 38, or an amino acid sequence of SEQ ID No: 38 that includes one or more amino acid substitutions (e.g., conservative substitutions), insertions, or deletions in portions other than CDR1 (SEQ ID No: 4), CDR2 (SEQ ID No: 5), and CDR3 (SEQ ID No: 6)) Antibodies or antibody derivatives containing the above; (1-2) Heavy chain (amino acid sequence of SEQ ID No: 39, or amino acid sequence of SEQ ID No: 39 that includes one or more amino acid substitutions (e.g., conservative substitutions), insertions, or deletions in parts other than CDR1 (SEQ ID No: 1), CDR2 (SEQ ID No: 2), and CDR3 (SEQ ID No: 3)) and Light chain (amino acid sequence of SEQ ID No: 40, or an amino acid sequence of SEQ ID No: 40 that includes one or more amino acid substitutions (e.g., conservative substitutions), insertions, or deletions in portions other than CDR1 (SEQ ID No: 4), CDR2 (SEQ ID No: 5), and CDR3 (SEQ ID No: 6)); (1-3) Heavy chain (amino acid sequence of SEQ ID No: 41, or amino acid sequence of SEQ ID No: 41 that includes one or more amino acid substitutions (e.g., conservative substitutions), insertions, or deletions in parts other than CDR1 (SEQ ID No: 1), CDR2 (SEQ ID No: 2), and CDR3 (SEQ ID No: 3)) and Light chain (amino acid sequence of SEQ ID No: 42, or an amino acid sequence of SEQ ID No: 42 that includes one or more amino acid substitutions (e.g., conservative substitutions), insertions, or deletions in portions other than CDR1 (SEQ ID No: 4), CDR2 (SEQ ID No: 5), and CDR3 (SEQ ID No: 6)); (2) Heavy chain (amino acid sequence of SEQ ID No: 43, or an amino acid sequence of SEQ ID No: 43 that includes one or more amino acid substitutions (e.g., conservative substitutions), insertions, or deletions in parts other than CDR1 (SEQ ID No: 7), CDR2 (SEQ ID No: 8), and CDR3 (SEQ ID No: 9)) and Light chain (amino acid sequence of SEQ ID No: 44, or an amino acid sequence of SEQ ID No: 44 that includes one or more amino acid substitutions (e.g., conservative substitutions), insertions, or deletions in portions other than CDR1 (SEQ ID No: 10), CDR2 (SEQ ID No: 11), and CDR3 (SEQ ID No: 12)). Antibodies or antibody derivatives containing the above; (3) Heavy chain (amino acid sequence of SEQ ID No: 45, or an amino acid sequence of SEQ ID No: 45 that includes one or more amino acid substitutions (e.g., conservative substitutions), insertions, or deletions in parts other than CDR1 (SEQ ID No: 13), CDR2 (SEQ ID No: 14), and CDR3 (SEQ ID No: 15)) and Antibodies or antibody derivatives containing a light chain (the amino acid sequence of SEQ ID No: 46, or an amino acid sequence of SEQ ID No: 46 that includes one or more amino acid substitutions (e.g., conservative substitutions), insertions, or deletions in portions other than CDR1 (SEQ ID No: 16), CDR2 (SEQ ID No: 17), and CDR3 (SEQ ID No: 18)); (4) Heavy chain (amino acid sequence of SEQ ID No: 47, or an amino acid sequence of SEQ ID No: 47 that includes one or more amino acid substitutions (e.g., conservative substitutions), insertions, or deletions in parts other than CDR1 (SEQ ID No: 19), CDR2 (SEQ ID No: 20), and CDR3 (SEQ ID No: 21)) and Light chain (amino acid sequence of SEQ ID No: 48, or an amino acid sequence of SEQ ID No: 48 that includes one or more amino acid substitutions (e.g., conservative substitutions), insertions, or deletions in portions other than CDR1 (SEQ ID No: 22), CDR2 (SEQ ID No: 23), and CDR3 (SEQ ID No: 24)). Antibodies or antibody derivatives containing the above; An antibody or antibody derivative selected from the group consisting of [1] to [5], as described in any of [1] to [5]; [7]: A pharmaceutical composition for preventing or treating bleeding caused by excessive cleavage of VWF, comprising an antibody or antibody derivative as described in any one of claims 1 to 6; [8]: The pharmaceutical composition according to [7], wherein the bleeding resulting from excessive cleavage of the vein thrombosus is bleeding associated with mechanical circulatory support; [9]: A pharmaceutical composition according to [7] or [8], comprising multiple types of antibodies or antibody derivatives described in any of [1] to [6];

[10] : A pharmaceutical composition according to any one of [7] to [9], by inhibiting the cleavage activity of ADAMTS13 against VWF;

[11] : A pharmaceutical composition according to any one of [7] to

[10] , wherein the bleeding associated with mechanical circulatory support is acquired von Willebrand syndrome (AVWS);

[12] : A pharmaceutical composition according to any one of [7] to

[11] , wherein the mechanical circulatory support is selected from the group consisting of extracorporeal lung (ECMO), implantable ventricular assist device (LVAD), and percutaneous cardiopulmonary support (PCPS);

[13] : A step of contacting a biological sample taken from a subject with an antibody or antibody derivative described in any of [1] to [6] in vitro, A step of detecting and measuring ADAMTS13 in a sample bound to the aforementioned antibody or antibody derivative, A method for detecting and measuring the presence and amount of ADAMTS13 in biological samples, including those containing it;

[14] : A kit for detecting and measuring the presence and amount of ADAMTS13 in a subject, comprising an antibody or antibody derivative described in any of [1] to [6];

[15] : A step of adding an antibody or antibody derivative described in any of [1] to [6] at various concentrations to a biological sample taken from a subject and incubating it, A process for detecting and measuring intact VWF multimers, intact VWF monomers, and VWF cleavage fragments in a sample. A method for detecting and measuring the VWF cleavage activity of ADAMTS13 in biological samples, including those containing ADAMTS13;

[16] : A kit for detecting and measuring intact VWF multimers, intact VWF monomers, and VWF cleavage fragments in a subject body, comprising an antibody or antibody derivative described in any of [1] to [6]. [Effects of the Invention]

[0011] The present invention provides an antibody or antibody derivative that has specific binding activity to ADAMTS13, a VWF cleavage protease, and can reduce the excessive cleavage of VWF by ADAMTS13. Since excessive cleavage of VWF by ADAMTS13 is thought to be the cause of acquired von Willebrand syndrome (AVWS) in humans, the present invention can also provide a pharmaceutical composition for preventing or treating human AVWS, comprising the antibody or antibody derivative of the present invention. [Brief explanation of the drawing]

[0012] [Figure 1] Figure 1 shows the domain structure of ADAMTS13, the target substance of the antibody or antibody derivative of the present invention. The numbers in the figure indicate amino acid numbers. [Figure 2] Figure 2 shows the mechanism of AVWS development. [Figure 3] Figure 3 shows the ADAMTS13 binding activity of anti-ADAMTS13 antibodies (mouse A10 antibody and human chimeric A10c antibody). [Figure 4]Figure 4 shows the inhibitory effect of anti-ADAMTS13 antibody (A10c antibody) on ADAMTS13 activity. [Figure 5] Figure 5 shows the concentration-binding correlation in ADAMTS13 binding activity by anti-ADAMTS13 antibodies (A10h / 8A7 antibody and A10h / 16E8 antibody). [Figure 6] Figure 6 shows the inhibitory effect of anti-ADAMTS13 antibodies (A10h / 8A7 antibody and A10h / 16E8 antibody) on ADAMTS13 activity. [Figure 7] Figure 7 shows the changes in high molecular weight VWF multimer deficiency due to shear stress (Figure 6A) and the inhibition of high molecular weight VWF multimer deficiency by the addition of A10 antibody (Figure 6B). [Modes for carrying out the invention]

[0013] The terms used in this invention are defined as follows: (a) von Willebrand factor (VWF) VWF is a high-molecular-weight plasma glycoprotein that plays a role in initial platelet adhesion, platelet aggregation, and stabilization of coagulation factor VIII at the site of vascular injury. It is produced by vascular endothelial cells and megakaryocytes, secreted as a multimer, and cleaved into pieces of appropriate size for hemostasis by the VWF-cleaving enzyme ADAMTS13. (b) Von Willebrand disease (VWD) is a congenital coagulation disorder characterized by a bleeding tendency due to quantitative and qualitative abnormalities in varicella-walled follicles (VWF). It is classified into six subtypes: type 1, type 2A, type 2B, type 2M, type 2N, and type 3. It is often characterized by bleeding symptoms suggestive of platelet aggregation disorders (e.g., epistaxis, purpura / subcutaneous hematoma, oral mucosal bleeding, menorrhagia). (c) Acquired von Willebrand syndrome (AVWS) is a relatively rare acquired coagulation disorder that presents with a pathological condition similar to congenital von Willebrand disease (VWD) in association with an underlying disease (such as lymphoproliferative disorders, autoimmune diseases, myeloproliferative disorders, cardiovascular diseases, malignant tumors, or hypothyroidism), and is characterized by the absence of a history of bleeding disorders in the past or family.

[0014] This invention demonstrates that the problems of the present invention can be solved by providing an antibody or antibody derivative that has binding affinity to ADAMTS13 and inhibits the cleavage activity of VWF, and includes a heavy-chain / light-chain complementarity determination region having a specific amino acid sequence.

[0015] target antigen ADAMTS13, the target antigen of the antibody or antibody derivative of the present invention, is an enzyme protein (GenBank number AY055376) composed of 1427 amino acids, with a molecular weight of approximately 200 kDa. Its primary structure consists of multiple domains, starting from the N-terminus: a signal peptide (S), a propeptide (P), a metalloproteinase domain (MP), a disintegrin-like domain (D), a thrombospondin-1 motif (T1), a cysteine-rich domain (C), a spacer domain (S), seven repeats of the thrombospondin-1 motif (T2-T8), and finally two CUB domains (CUB1 and CUB2) (Figure 1). The only known physiological substrate of ADAMTS13 is VWF, and the MDTCS domain of ADAMTS13 (a domain consisting of the MP domain, D domain, T1 domain, C domain, and Sp domain) is required for the cleavage of VWF.

[0016] The antibody or antibody derivative of the present invention may target any part of this domain structure. For example, it may target the MDTCS domain itself, which is essential for the VWF cleavage activity described above, by epitoping a partial amino acid sequence of the MDTCS domain and binding to the cleavage function domain of ADAMTS13 to block its function, or it may bind to a part other than the MDTCS domain, so that as a result of binding to that part other than the MDTCS domain, ADAMTS13 cannot bind to VWF in terms of its three-dimensional structure.

[0017] Antibodies or antibody derivatives In one embodiment, the present invention provides an antibody or antibody derivative that binds to ADAMTS13 and inhibits the cleavage activity of VWF, comprising specific heavy-chain / light-chain complementarity-determining regions (CDRs), or variable regions of heavy-chain / light-chain containing these CDRs, or heavy-chain / light-chain containing these CDRs.

[0018] In the present invention, the antibodies or antibody derivatives used are preferably free from pathogens such as viruses that may be present in the blood (and therefore pose a risk of contamination in blood products), such as HBV, hepatitis C virus (HCV), and human immunodeficiency virus (HIV), in order to avoid various problems specific to blood products such as blood-derived antibody preparations (particularly infectivity and immunological problems). Furthermore, they preferably do not contain blood-derived components that may cause immune abnormalities in the administered individual, such as antigenic proteins or antibodies that bind to human proteins present in the blood.

[0019] Antibodies that do not contain blood-derived components can be used, such as antibodies prepared from immortalized cells derived from antibody-producing cells under conditions that do not contain blood-derived components, or recombinant antibodies that can be prepared by recombinantly expressing antibody proteins using DNA that defines the antibody protein obtained from the above-mentioned immortalized cells.

[0020] In other words, in one embodiment, the antibody in the present invention can be obtained by obtaining and immortalizing cell clones that produce antibodies binding to ADAMTS13 from the blood of an individual administered all or part of the target antigen ADAMTS13, thereby obtaining immortalized cells derived from the antibody-producing cells, and then selecting from these immortalized antibody-producing cells those that produce antibodies having the activity to inhibit the VWF cleavage function of ADAMTS13.

[0021] In another embodiment, recombinant antibodies having the activity to bind to ADAMTS13 and inhibit the VWF cleavage function of ADAMTS13 can also be provided using genetic engineering technology. More specifically, the present invention can also provide monoclonal antibodies having the desired activity, obtained as recombinant immunoglobulins (IgG) by obtaining mRNA and creating cDNA from immortalized antibody-producing cells that produce antibodies having the activity to inhibit the VWF cleavage function of ADAMTS13 selected by the method described above, according to well known methods, and expressing them in a mammalian expression system that does not contain blood-derived components using a vector.

[0022] In the present invention, derivatives of these antibodies can also be used. Examples of antibody derivatives that can be used in the present invention include, but are not limited to, antibody modifiers or functional fragments selected from the group consisting of humanized antibodies, chimeric antibodies, single-chain antibodies, polyvalent antibodies, and multispecific antibodies. Among these, examples of functional fragments that can be used include, but are not limited to, F(ab')2.

[0023] These antibody derivatives can be prepared after the antibody has been obtained, according to methods well known in the art, and can be prepared as recombinant antibody derivatives as described above for the antibody of the present invention.

[0024] The antibody or antibody derivative obtained by the method described above is capable of binding to ADAMTS13. However, in the present invention, from among such antibodies or antibody derivatives capable of binding to ADAMTS13, screening is performed based on whether they have the activity to inhibit the VWF cleavage function of ADAMTS13, and the present invention provides an antibody or antibody derivative having said inhibitory activity.

[0025] In this embodiment, the specific heavy-light chain complementarity determining regions (CDRs) contained in the antibody or antibody derivative of the present invention against ADAMTS13 are (1) to (4) below: (1) Heavy chain complementarity determination regions, CDR1 (GYSFTGYT, SEQ ID No: 1), CDR2 (INPYNGGT, SEQ ID No: 2), and CDR3 (ARTSGYLFAY, SEQ ID No: 3), and Light chain complementarity-determining regions: CDR1 (EDIYNR, SEQ ID No: 4), CDR2 (GAT, SEQ ID No: 5), and CDR3 (QQYWSSPLT, SEQ ID No: 6); (2) Heavy chain complementarity determination regions, CDR1 (GFSLPRYG, SEQ ID No: 7), CDR2 (IWAGGST, SEQ ID No: 8), and CDR3 (ARAGGSQPFDY, SEQ ID No: 9), and Light chain complementarity-determining regions: CDR1 (RDINTY, SEQ ID No: 10), CDR2 (RAN, SEQ ID No: 11), and CDR3 (LQYDEFPWT, SEQ ID No: 12); (3) Heavy chain complementarity determination regions, CDR1 (GFSLTRYG, SEQ ID No: 13), CDR2 (IWAGGST, SEQ ID No: 14), and CDR3 (ARAGGSSSFDY, SEQ ID No: 15), Light chain complementarity-determining regions: CDR1 (QDINTY, SEQ ID No: 16), CDR2 (RAN, SEQ ID No: 17), and CDR3 (LQYDEFPWT, SEQ ID No: 18); (4) Heavy chain complementarity determination regions, CDR1 (GFSLTGYG, SEQ ID No: 19), CDR2 (IWADGTT, SEQ ID No: 20), and CDR3 (ARAGGSQPFDY, SEQ ID No: 21), Light chain complementarity determination regions: CDR1 (QDINSY, SEQ ID No: 22), CDR2 (RAN, SEQ ID No: 23), and CDR3 (LQYDEFPWT, SEQ ID No: 24); You can choose from the group consisting of these.

[0026] In this embodiment, the amino acid sequence of a specific heavy chain variable region VH domain contained in the antibody or antibody derivative of the present invention against ADAMTS13 is: (1-1) The amino acid sequence of SEQ ID No: 25, or the amino acid sequence of SEQ ID No: 25 that includes one or more amino acid substitutions (e.g., conservative substitutions), insertions, or deletions in portions other than CDR1 (SEQ ID No: 1), CDR2 (SEQ ID No: 2), and CDR3 (SEQ ID No: 3), (1-2) The amino acid sequence of SEQ ID No: 27, or the amino acid sequence of SEQ ID No: 27 that includes one or more amino acid substitutions (e.g., conservative substitutions), insertions, or deletions in portions other than CDR1 (SEQ ID No: 1), CDR2 (SEQ ID No: 2), and CDR3 (SEQ ID No: 3), (1-3) The amino acid sequence of SEQ ID No: 29, or the amino acid sequence of SEQ ID No: 29 that includes one or more amino acid substitutions (e.g., conservative substitutions), insertions, or deletions in portions other than CDR1 (SEQ ID No: 1), CDR2 (SEQ ID No: 2), and CDR3 (SEQ ID No: 3), (2) The amino acid sequence of SEQ ID No: 31, or the amino acid sequence of SEQ ID No: 31 that includes one or more amino acid substitutions (e.g., conservative substitutions), insertions, or deletions in portions other than CDR1 (SEQ ID No: 7), CDR2 (SEQ ID No: 8), and CDR3 (SEQ ID No: 9), (3) The amino acid sequence of SEQ ID No: 33, or the amino acid sequence of SEQ ID No: 33 that includes one or more amino acid substitutions (e.g., conservative substitutions), insertions, or deletions in parts other than CDR1 (SEQ ID No: 13), CDR2 (SEQ ID No: 14), and CDR3 (SEQ ID No: 15), and (4) The amino acid sequence of SEQ ID No: 35, or the amino acid sequence of SEQ ID No: 35 that includes one or more amino acid substitutions (e.g., conservative substitutions), insertions, or deletions in portions other than CDR1 (SEQ ID No: 19), CDR2 (SEQ ID No: 20), and CDR3 (SEQ ID No: 21), A selection can be made from the group consisting of the following. In this case, the amino acid sequence of the heavy chain constant region CH domain can be that of an existing antibody, for example, the amino acid sequence of the CH domain of any human antibody or an amino acid sequence modified from such an amino acid sequence can be used. Specifically, in the present invention, an amino acid sequence in which each of the CDR1 to CDR3 of the heavy chain constant region CH domain is known to be essential for binding to the target antigen, while the portion other than CDR1 to CDR3 contains one or more amino acid substitutions (e.g., conservative substitutions), insertions, or deletions, can be used as long as it can constitute an antibody or antibody derivative that has binding affinity to ADAMTS13 and inhibits the cleavage activity of von Willebrand factor (VWF).

[0027] In this embodiment, the amino acid sequence of a specific light chain variable region (VL domain) contained in the antibody or antibody derivative of the present invention against ADAMTS13 is (1-1) The amino acid sequence of SEQ ID No: 26, or the amino acid sequence of SEQ ID No: 26 that includes one or more amino acid substitutions (e.g., conservative substitutions), insertions, or deletions in portions other than CDR1 (SEQ ID No: 4), CDR2 (SEQ ID No: 5), and CDR3 (SEQ ID No: 6), (1-2) The amino acid sequence of SEQ ID No: 28, or the amino acid sequence of SEQ ID No: 28 that includes one or more amino acid substitutions (e.g., conservative substitutions), insertions, or deletions in portions other than CDR1 (SEQ ID No: 4), CDR2 (SEQ ID No: 5), and CDR3 (SEQ ID No: 6), (1-3) The amino acid sequence of SEQ ID No: 30, or the amino acid sequence of SEQ ID No: 30 that includes one or more amino acid substitutions (e.g., conservative substitutions), insertions, or deletions in portions other than CDR1 (SEQ ID No: 4), CDR2 (SEQ ID No: 5), and CDR3 (SEQ ID No: 6), (2) The amino acid sequence of SEQ ID No: 32, or the amino acid sequence of SEQ ID No: 32 that includes one or more amino acid substitutions (e.g., conservative substitutions), insertions, or deletions in portions other than CDR1 (SEQ ID No: 10), CDR2 (SEQ ID No: 11), and CDR3 (SEQ ID No: 12), (3) The amino acid sequence of SEQ ID No: 34, or the amino acid sequence of SEQ ID No: 34 that includes one or more amino acid substitutions (e.g., conservative substitutions), insertions, or deletions in parts other than CDR1 (SEQ ID No: 16), CDR2 (SEQ ID No: 17), and CDR3 (SEQ ID No: 18), and (4) The amino acid sequence of SEQ ID No: 36, or the amino acid sequence of SEQ ID No: 36 that includes one or more amino acid substitutions (e.g., conservative substitutions), insertions, or deletions in portions other than CDR1 (SEQ ID No: 22), CDR2 (SEQ ID No: 23), and CDR3 (SEQ ID No: 24), A selection can be made from the group consisting of the following. In this case, the amino acid sequence of the light chain constant region CL domain can be that of an existing antibody, for example, the amino acid sequence of the CL domain of any human antibody, or an amino acid sequence modified from such an amino acid sequence can be used. Specifically, in the present invention, an amino acid sequence in which each of the CDR1 to CDR3 of the light chain constant region CL domain is known to be essential for binding to the target antigen, while the portion other than CDR1 to CDR3 contains one or more amino acid substitutions (e.g., conservative substitutions), insertions, or deletions, can be used as long as it can constitute an antibody or antibody derivative that has binding affinity to ADAMTS13 and inhibits the cleavage activity of von Willebrand factor (VWF).

[0028] In this embodiment, the specific heavy and light chains contained in the antibody or antibody derivative of the present invention against ADAMTS13 are as follows (1-1) to (4): (1-1) Heavy chain (amino acid sequence of SEQ ID No: 37, or amino acid sequence of SEQ ID No: 37 that includes one or more amino acid substitutions (e.g., conservative substitutions), insertions, or deletions in parts other than CDR1 (SEQ ID No: 1), CDR2 (SEQ ID No: 2), and CDR3 (SEQ ID No: 3)) and Light chain (amino acid sequence of SEQ ID No: 38, or an amino acid sequence of SEQ ID No: 38 that includes one or more amino acid substitutions (e.g., conservative substitutions), insertions, or deletions in portions other than CDR1 (SEQ ID No: 4), CDR2 (SEQ ID No: 5), and CDR3 (SEQ ID No: 6)); (1-2) Heavy chain (amino acid sequence of SEQ ID No: 39, or amino acid sequence of SEQ ID No: 39 that includes one or more amino acid substitutions (e.g., conservative substitutions), insertions, or deletions in parts other than CDR1 (SEQ ID No: 1), CDR2 (SEQ ID No: 2), and CDR3 (SEQ ID No: 3)) and Light chain (amino acid sequence of SEQ ID No: 40, or an amino acid sequence of SEQ ID No: 40 that includes one or more amino acid substitutions (e.g., conservative substitutions), insertions, or deletions in portions other than CDR1 (SEQ ID No: 4), CDR2 (SEQ ID No: 5), and CDR3 (SEQ ID No: 6)); (1-3) Heavy chain (amino acid sequence of SEQ ID No: 41, or amino acid sequence of SEQ ID No: 41 that includes one or more amino acid substitutions (e.g., conservative substitutions), insertions, or deletions in parts other than CDR1 (SEQ ID No: 1), CDR2 (SEQ ID No: 2), and CDR3 (SEQ ID No: 3)) and Light chain (amino acid sequence of SEQ ID No: 42, or an amino acid sequence of SEQ ID No: 42 that includes one or more amino acid substitutions (e.g., conservative substitutions), insertions, or deletions in portions other than CDR1 (SEQ ID No: 4), CDR2 (SEQ ID No: 5), and CDR3 (SEQ ID No: 6)); (2) Heavy chain (amino acid sequence of SEQ ID No: 43, or an amino acid sequence of SEQ ID No: 43 that includes one or more amino acid substitutions (e.g., conservative substitutions), insertions, or deletions in parts other than CDR1 (SEQ ID No: 7), CDR2 (SEQ ID No: 8), and CDR3 (SEQ ID No: 9)) and Light chain (amino acid sequence of SEQ ID No: 44, or an amino acid sequence of SEQ ID No: 44 that includes one or more amino acid substitutions (e.g., conservative substitutions), insertions, or deletions in portions other than CDR1 (SEQ ID No: 10), CDR2 (SEQ ID No: 11), and CDR3 (SEQ ID No: 12)); (3) Heavy chain (amino acid sequence of SEQ ID No: 45, or an amino acid sequence of SEQ ID No: 45 that includes one or more amino acid substitutions (e.g., conservative substitutions), insertions, or deletions in parts other than CDR1 (SEQ ID No: 13), CDR2 (SEQ ID No: 14), and CDR3 (SEQ ID No: 15)) and Light chain (amino acid sequence of SEQ ID No: 46, or an amino acid sequence of SEQ ID No: 46 that includes one or more amino acid substitutions (e.g., conservative substitutions), insertions, or deletions in portions other than CDR1 (SEQ ID No: 16), CDR2 (SEQ ID No: 17), and CDR3 (SEQ ID No: 18)); (4) Heavy chain (amino acid sequence of SEQ ID No: 47, or an amino acid sequence of SEQ ID No: 47 that includes one or more amino acid substitutions (e.g., conservative substitutions), insertions, or deletions in parts other than CDR1 (SEQ ID No: 19), CDR2 (SEQ ID No: 20), and CDR3 (SEQ ID No: 21)) and Light chain (amino acid sequence of SEQ ID No: 48, or an amino acid sequence of SEQ ID No: 48 that includes one or more amino acid substitutions (e.g., conservative substitutions), insertions, or deletions in portions other than CDR1 (SEQ ID No: 22), CDR2 (SEQ ID No: 23), and CDR3 (SEQ ID No: 24)); You can choose from the group consisting of these.

[0029] In this case, the heavy chain and light chain can be used in the present invention as long as the amino acid sequences of the heavy chain and light chain, specifically CDR1 to CDR3 of each, are known to be essential for binding to the target antigen, while the other parts of the amino acid sequence may contain one or more amino acid substitutions (e.g., conservative substitutions), insertions, or deletions, as long as they can constitute an antibody or antibody derivative that binds to ADAMTS13 and inhibits the cleavage activity of VWF.

[0030] Inhibition of VWF cleavage activity of ADAMTS13 by antibodies or antibody derivatives VWF is Tyr in its A2 domain 1605 -Met 1606 The A2 domain is the cleavage site for ADAMTS13, but when the multimer exists in plasma, this cleavage site is embedded within the A2 domain, making it structurally inaccessible to ADAMTS13. When a portion of the VWF multimer binds to vascular endothelial cells or platelets, and physical forces such as blood flow are applied, the structure of the A2 domain changes, allowing ADAMTS13 to access the cleavage site of the VWF A2 domain. In such cases, ADAMTS13 functions as a metalloproteinase that specifically cleaves the higher-order structure of the multimer.

[0031] The antibody or antibody derivative of the present invention is characterized by having the effect of inhibiting the VWF cleavage activity of ADAMTS13. Screening to determine whether the antibody or antibody derivative has the effect of inhibiting the VWF cleavage activity of ADAMTS13 can be performed by a method such as confirming that VWF cleavage is inhibited by Western blotting. When screening by Western blotting, a specified amount of monomeric VWF and a specified amount of ADAMTS13 are mixed in vitro, the antibody to be evaluated is added at various concentrations, and the amount of VWF cleaved changes in a concentration-dependent manner of the antibody by Western blotting.

[0032] Pharmaceutical use VWF multimers exhibit higher platelet aggregation ability with increasing molecular weight; therefore, cleavage (reduction of molecular weight) by ADAMTS13 suppresses VWF-dependent platelet aggregation. Consequently, increased ADAMTS13 activity and excessive cleavage of VWF can lead to excessive suppression of platelet aggregation, resulting in a bleeding tendency and potentially causing congenital VWD or acquired AVWS. Conversely, insufficient ADAMTS13 activity can cause excessive platelet aggregation. In a normal living environment, cleavage of VWF multimers by ADAMTS13 occurs at a constant rate, maintaining a balance that prevents bleeding and abnormal coagulation reactions.

[0033] As described above, the antibody or antibody derivative of the present invention is characterized by having the effect of inhibiting the VWF cleavage activity of ADAMTS13, and can therefore intervene in bleeding caused by increased ADAMTS13 activity and excessive cleavage of VWF. Thus, in another embodiment of the present invention, a pharmaceutical composition comprising the antibody or antibody derivative of the present invention can be provided for preventing or treating bleeding caused by excessive cleavage of VWF.

[0034] The pharmaceutical composition in this embodiment may contain one or more types of the antibody or antibody derivative of the present invention as an active ingredient.

[0035] This pharmaceutical composition contains an antibody or antibody derivative of the present invention as an active ingredient, which inhibits the cleavage activity of ADAMTS13 against VWF, thereby enabling it to exhibit pharmaceutical applications aimed at preventing or treating bleeding caused by excessive cleavage of VWF.

[0036] The bleeding caused by excessive cleavage of the vein pulp (VWF), which is the symptom to which the pharmaceutical composition of the present invention is applied, may be of congenital or acquired cause, and is selected from the group consisting of, for example, bleeding associated with mechanical circulatory support and anemia.

[0037] Among the diseases that cause bleeding associated with mechanical circulatory support, which are the symptoms to which the pharmaceutical composition of the present invention is applicable, AVWS can be cited. The mechanism by which AVWS occurs is as shown in Figure 2. It is thought that high shear stress generated in the pump of the device used when mechanical circulatory support is used, such as extracorporeal lung (ECMO), implantable ventricular assist device (LVAD), and percutaneous cardiopulmonary support (PCPS), causes a change in the structure of the VWF multimer, which is thought to trigger excessive degradation of the VWF factor by ADAMTS13.

[0038] Therefore, the mechanical circulatory support that causes AVWS in the present invention is not limited, but mechanical circulatory support when high shear stress occurs in the pump of the device is the target of application of the pharmaceutical composition of the present invention, and such mechanical circulatory support can be selected from the group consisting of extracorporeal membrane oxygenation (ECMO), implantable ventricular assist device (LVAD), and percutaneous cardiopulmonary support (PCPS).

[0039] Other uses of antibodies or antibody derivatives The antibody or antibody derivative of the present invention can also be used for other applications, such as the detection and measurement of the presence and amount of ADAMTS13, and the detection and measurement of the VWF cleavage activity of ADAMTS13.

[0040] For detection and measurement applications of ADAMTS13, A step of contacting a biological sample collected from a subject with the antibody or antibody derivative of the present invention in vitro. A step of detecting and measuring ADAMTS13 in a sample bound to the aforementioned antibody or antibody derivative, By performing this procedure, the presence and amount of ADAMTS13 in biological samples can be detected and measured. In this method, when detecting and measuring ADAMTS13, a detectable label can be directly or indirectly attached to the antibody or antibody derivative of the present invention, and the presence and amount of ADAMTS13 can be detected and measured using known methods such as ELISA and Western blotting.

[0041] In the present invention, a kit for detecting and measuring the presence and amount of ADAMTS13 in a biological sample of a subject, using the method described above, is also provided, which includes the antibody or antibody derivative of the present invention. This kit is characterized in that, among the general kit components used for ELISA, Western blotting, etc., the primary antibody for detection is the antibody or antibody derivative of the present invention.

[0042] In this kit, when detecting the antibody or antibody derivative of the present invention by indirectly attaching a label, the kit may include a labeled secondary antibody for detecting the antibody or antibody derivative of the present invention.

[0043] For detecting and measuring the VWF cleavage activity of ADAMTS13, A step of adding antibodies or antibody derivatives of the present invention at various concentrations to a biological sample collected from a subject, and incubating it for a certain period of time; A process for detecting and measuring intact VWF multimers, intact VWF monomers, and VWF cleavage fragments in a sample. By performing this procedure, the VWF cleavage activity of ADAMTS13 in biological samples can be detected and measured. By adding the antibody or antibody derivative of the present invention, it is possible to determine whether VWF cleavage occurring in the absence of the antibody or antibody derivative is caused by ADAMTS13, and whether excessive VWF cleavage by ADAMTS13 is occurring in the subject's body. In this method, when detecting and measuring intact VWF multimers, intact VWF monomers, and VWF cleavage fragments, known methods such as Western blotting and chromatography can be used to detect and measure intact VWF multimers, intact VWF monomers, and VWF cleavage fragments.

[0044] In the present invention, a kit for detecting and measuring intact VWF multimers, intact VWF monomers, and VWF cleavage fragments, comprising the antibody or antibody derivative of the present invention for detecting and measuring ADAMTS13-mediated VWF cleavage activity in a subject's biological sample using the method described above, can also be provided. This kit is characterized by including the antibody or antibody derivative of the present invention for inhibiting the function of ADAMTS13 in a general kit configuration used for performing Western blotting, chromatography, etc.

[0045] The present invention will be specifically illustrated below with reference to examples. The examples shown below do not limit the present invention in any way. [Examples]

[0046] Example 1: Antibody preparation This example aimed to obtain monoclonal antibodies against ADAMTS13, which are produced in mice as a result of administering ADAMTS13 as an immunogen.

[0047] (1) Antibody production using the hybridoma method Mice (BALB / cAJcl, female, 7 weeks old, CREA Japan) were immunized with 20 μg of recombinant ADAMTS13 full-length antigen (in-house prepared) along with the same amount of Freund's complete adjuvant (Difco) via intraperitoneal administration five times at two-week intervals. One month later, a booster immunization with ADAMTS13 antigen was performed, and blood was collected from the tail vein two weeks later. The increase in ADAMTS13-specific antibody titers in the blood was confirmed by ELISA using a conventional method. Mice with elevated antibody titers were further immunized with ADAMTS13 antigen from the tail vein to complete the immunization, and the mice's spleens were collected three days later.

[0048] Single cell suspensions of splenocytes were prepared from the spleen according to known methods, and 2-amino-6-mercaptopurine (6-thioguanine [2-amino-6-mercaptopurine])-resistant BALB / c mouse-derived myeloma cell line (P3-X63-Ag8·653, hereinafter also referred to as X63 cells) were fused to the parent cell line according to methods commonly used in the art.

[0049] Subsequently, the obtained fusion cells were cultured in a culture medium supplemented with HAT selector solution and cloned. After 2-3 weeks, clones producing the target anti-ADAMTS13 monoclonal antibody were screened using ELISA with a microplate on which ADAMTS13 antigen was adsorbed as a solid phase.

[0050] (2) Screening The culture supernatant of the above hybridoma cells was used to screen for the target antibody by reaction with an ELISA plate immobilized with the ADAMTS13 antigen. In this screening, clones that specifically react only to the ADAMTS13 antigen were screened by removing non-specific reactive clones that reacted with an ELISA plate immobilized with non-specific oligopeptides.

[0051] After selecting hybridoma cell lines that specifically reacted to ELISA plates immobilized with the ADAMTS13 antigen, they were cloned using limiting mechanisms, and anti-monoclonal antibodies and A10 antibodies were obtained from the hybridomas.

[0052] Example 2: Preparation of another antibody This example was conducted with the aim of obtaining a new antibody against ADAMTS13 as a monoclonal antibody, in addition to the antibody obtained in Example 1.

[0053] (1) Antibody production using the hybridoma method Mice (BALB / cA, female, 7 weeks old, Japan SLC) were immunized by injecting 20 μg of a recombinant antigen (MP domain + D domain: hereinafter referred to as "ADAMTS13 MD recombinant antigen," a self-prepared product) containing the metalloproteinase domain (MP) and disintegrin-like domain (D) of ADAMTS13 into the footpad along with TiterMax Gold (TiterMax USA, Inc.). Two weeks later, the antigen was boosted with Freund's incomplete adjuvant (Merck), and three days later, blood was collected from the tail vein. The increase in ADAMTS13 MD-specific antibody titers in the blood was confirmed by ELISA using a conventional method. Popliteal lymph nodes were collected from mice with elevated antibody titers.

[0054] Single-cell suspensions were prepared from popliteal lymph nodes according to known methods, and 2-amino-6-mercaptopurine (6-thioguanine [2-amino-6-mercaptopurine])-resistant mouse-derived myeloma cell line (P3U1 cells) was fused to the parent cell line according to methods commonly used in the art.

[0055] Subsequently, the obtained fusion cells were cultured in a culture medium supplemented with HAT selector solution and cloned. After 2-3 weeks, clones producing the target anti-ADAMTS13 MD monoclonal antibody were screened using ELISA with a microplate on which ADAMTS13 MD antigen was adsorbed as a solid phase.

[0056] (2) Screening The culture supernatant of the above hybridoma cells was used to screen for the target antibody by reaction with an ELISA plate immobilized with ADAMTS13 MD antigen. In this screening, clones that specifically react only to ADAMTS13 MD antigen were screened by removing non-specific reactive clones that reacted with an ELISA plate immobilized with non-specific proteins. As a result, the 1G2 antibody, 12D10 antibody, and 9A7 antibody were obtained.

[0057] Example 3: Human chimerization of A10 antibody In this example, the antibody obtained in Example 1 was subjected to human chimeration. Specifically, human chimeration was performed by substituting the heavy chain constant region (CH) and light chain constant region (CL) of the antibody obtained in Example 1 with the heavy chain constant region (CH) and light chain constant region (CL) of human IgG.

[0058] (1) Obtaining the heavy chain and light chain genes of the A10 antibody From the A10 antibody-producing cells obtained in Example 1, the genes encoding the A10 antibody heavy chain and light chain (heavy chain IgG2b gene, light chain Igk gene) were cloned. Specifically, first, using total RNA extracted from the A10 antibody-producing cells as a template, a reverse transcription reaction was performed using the SMART cDNA Library Construction Kit (TAKARA), which is capable of amplifying 5' full-length cDNA, according to the attached protocol, to produce cDNA.

[0059] Using the cDNA synthesized by the method described above as a template, two PCRs (1st PCR, 2nd PCR) were performed using KOD FX (TOYOBO). For the 1st PCR, 1 μl of cDNA reverse-transcribed from each lymphocyte cell line (LCL) was used as a template, and the following forward primer (Primer 1) and reverse primer (Primer 2) were used as primers for the PCR reaction (reaction conditions: 94°C for 2 minutes → [98°C for 10 seconds, 55°C for 30 seconds, 68°C for 2 minutes] × 35 cycles → 68°C for 3 minutes).

[0060] [Table 1]

[0061] For the second PCR, 0.5 μl of the extension product from the first PCR was used as a template, and the following forward primer (Primer 1) and reverse primer (Primer 2) were used as primers to perform the PCR reaction (reaction conditions: 94°C for 2 minutes → [98°C for 10 seconds, 60°C for 30 seconds, 68°C for 2 minutes] × 35 cycles → 68°C for 3 minutes).

[0062] [Table 2]

[0063] The PCR samples were subjected to the QIAquick Purification Kit (QIAGEN) to remove unreacted primers and enzymes, and purified products were obtained.

[0064] The purified PCR products were restricted enzyme-treated, separated and purified by agarose gel electrophoresis, and then incorporated into vectors. For vector preparation containing heavy chain genes, the pQEFIP vector (a retroviral vector with pQCXIP (TAKARA) as the backbone and the CMV promoter replaced with the human EF1 alpha promoter) was used. For vector preparation containing light chain genes, the pQEFIN vector (a retroviral vector with pQCXIN (TAKARA) as the backbone and the CMV promoter replaced with the human EF1 alpha promoter) was used. The PCR products were incorporated into the vectors using Ligation High (TOYOBO), and competent cells (DH-5alpha: Nippon Gene) were transformed.

[0065] Plasmids were extracted from E. coli cultured in ampicillin-containing LB liquid medium using NucleoSpin Plasmid EasyPure (MACHEREY-NAGEL) according to the attached protocol.

[0066] (2) Chimerization of heavy chains Using a human IgG1 expression plasmid vector (in-house prepared) as a template, fragments containing the human IgG1 constant region were PCR amplified using the following primers (TOYOBO KOD plus neo was used) (reaction conditions: 94°C for 2 minutes → [98°C for 10 seconds, 58°C for 30 seconds, 68°C for 4.5 minutes] × 30 cycles → 68°C for 5 minutes).

[0067] [Table 3]

[0068] Using an A10 antibody IgG2b heavy chain expression plasmid vector as a template, fragments containing the variable region of the A10 antibody heavy chain (IgG2b) were PCR amplified using the following primers (TOYOBO KOD plus neo was used) (reaction conditions: 94°C for 2 minutes → [98°C for 10 seconds, 58°C for 30 seconds, 68°C for 30 seconds] × 30 cycles → 68°C for 3 minutes).

[0069] [Table 4]

[0070] The two PCR products obtained in the above-described process (the constant region of human IgG1 and the variable region of the A10 antibody (IgG2b)) were each treated with the restriction enzyme Dpn I (NEB) at 37°C for 30 minutes, separated by agarose gel electrophoresis, and then DNA was extracted from the gel.

[0071] The two types of DNA obtained were mixed and treated with NEBuilder HiFi DNA Assembly Master Mix (NEB) at 50°C for 15 minutes to conjugate each fragment. This conjugated DNA was transformed into E. coli according to a standard procedure to obtain single colonies, and an A10 chimeric antibody (A10c antibody) heavy chain expression plasmid vector was obtained.

[0072] (3) Chimera formation of light chains Using a human IgG expression plasmid vector (in-house prepared) as a template, fragments containing the human IgG constant region were PCR amplified using the following primers (TOYOBO KOD plus neo was used).

[0073] [Table 5]

[0074] Using an A10 antibody Igk expression plasmid vector as a template, fragments containing the variable region of the A10 antibody light chain (Igk) were PCR amplified using the following primers (TOYOBO KOD plus neo was used).

[0075] [Table 6]

[0076] Each of the two PCR products obtained in the above-described process (human IgG constant region and A10 antibody IgG variable region) was treated with the restriction enzyme Dpn I (NEB) at 37°C for 30 minutes, separated by agarose gel electrophoresis, and then DNA was extracted from the gel.

[0077] The two types of DNA obtained were mixed and treated with NEBuilder HiFi DNA Assembly Master Mix (NEB) at 50°C for 15 minutes to conjugate each fragment. This conjugated DNA was transformed into E. coli according to a standard procedure to obtain single colonies, and an A10 chimeric antibody (A10c antibody) light chain expression plasmid vector was obtained.

[0078] (4) Expression of A10 chimeric antibody (A10c antibody) In this example, the A10 chimeric antibody (A10c antibody) heavy chain expression plasmid vector obtained in (1) above and the A10 chimeric antibody (A10c antibody) light chain expression plasmid vector obtained in (2) above were transiently cotransfected into Expi 293F cells (Thermo Fisher Scientific) using the Expi 293 Expression System (Thermo Fisher Scientific) to induce recombinant antibody expression and secretion.

[0079] 80 μL of ExpiFectamine 293 reagent and 15 μg each of heavy-chain and light-chain plasmids were mixed with 1.5 mL of Opti-MEM I (gibco) and allowed to stand at room temperature for 5 minutes. After standing, the two solutions were combined and allowed to stand at room temperature for 20 minutes, and 4.5–5.5 × 10⁶ cells were cultured in a 125 mL flask (Corning). 6 Cells were added to 30 mL of cells / mL and cultured in a swirling manner (37°C, 8% CO2). After 18-20 hours of culture, 150 μL of Expi Fectamine 293 Transfection Enhancer 1 and 1.5 mL of Expi Fectamine 293 Transfection Enhancer 2 were added, and the cells were cultured in a swirling manner for 5-6 days.

[0080] Recombinant antibodies expressed using an expression system with Expi 293F cells were purified using Protein G affinity chromatography (GE Hitrap protein GHP (1 mL)).

[0081] This example yielded a chimeric antibody based on the A10 antibody obtained in Example 1, and an A10c antibody.

[0082] Example 4: Humanization of antibodies In this example, the antibody obtained in Example 1 was humanized.

[0083] The DNA sequences encoding the amino acid sequence of the heavy chain (hereinafter referred to as the H chain) (SEQ ID NO: 37) and the amino acid sequence of the κ-light chain (hereinafter referred to as the κL chain) (SEQ ID NO: 38) of the anti-mouse ADAMTS13 antibody clone A10 (hereinafter referred to as the mouse A10 antibody) were aligned with mouse antibody germline gene sequences registered in a database. This allowed for the identification of framework regions (FRs) (four types: FR1-FR4), complementarity-determining regions (CDRs) (three types: CDR1-CDR3), and somatic mutation sites in the H and κL chains of the mouse A10 antibody. In the mouse A10 antibody, CDR1-3 of the H chain corresponds to SEQ ID NO: 1-3, and framework regions FR1-4 correspond to the parts of the heavy chain variable region (SEQ ID NO: 25) other than CDR1-3. In the κL chain, CDR1-3 corresponds to SEQ ID NO: 4-6, and framework regions FR1-4 correspond to the parts of the light chain variable region (SEQ ID NO: 26) other than CDR1-3.

[0084] Next, the amino acid sequences of the H chain and κL chain of the mouse A10 antibody (SEQ ID NO: 37 and SEQ ID NO: 38, respectively) were compared with the amino acid sequences of the H chain and κL chain of the known human tetanus antibody clones 8A7 and 16E8 (hereinafter referred to as human 8A7 antibody and human 16E8 antibody, respectively) [Japanese Patent Application No. 2020-089756], and the locations where amino acid residues differ between the mouse A10 antibody and the human antibodies (human 8A7 antibody and human 16E8 antibody) were identified.

[0085] Based on this comparative analysis, we designed a method for transplanting the CDR1-3 portions of the mouse A10 antibody's H chain and κL chain, as well as the amino acids at the somatic mutation site among the amino acid residues near them, onto the H chain and κL chain of known human antibodies (human 8A7 antibody and human 16E8 antibody), using the CDR grafting method, which is a commonly known technique for humanized antibody production.

[0086] After anointing the H chain and κL chain of humanized A10 antibodies (humanized A10h / 8A7 antibody and humanized A10h / 16E8 antibody) with a leader sequence containing the Kozak sequence (ACCATGG), the codon usage frequency of the leader sequence and the variable region (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4) of the humanized antibodies was optimized using a codon optimization tool (https: / / www.vectorbuilder.jp / tool / codon-optimization.html) to optimize antibody molecule expression in human cultured cell lines.

[0087] Using the DNA sequences of humanized A10 antibodies (A10h / 8A7 antibody and A10h / 16E8 antibody) with optimized codon usage frequency, we optimized the codon usage frequency so that the CAI (codon adaptation index, a dimensionless number that varies between 0 and 1, where a higher value indicates better codon usage frequency optimization) was between 0.9 and 1.0.

[0088] The DNA sequences encoding the heavy chain (H chain) and light chain (κL chain) of the obtained humanized A10 antibody were ligated to expression plasmids using the method described in Example 3, and heavy chain expression plasmid vectors and light chain expression plasmid vectors of the humanized A10 antibody (A10h antibody) were obtained.

[0089] The humanized A10 antibody (A10h / 8A7 antibody and A10h / 16E8 antibody) heavy chain expression plasmid vector and the humanized A10 antibody (A10h / 8A7 antibody and A10h / 16E8 antibody) light chain expression plasmid vector obtained in this example were transiently cotransfected into Expi 293F cells (Thermo Fisher Scientific) using the Expi293 Expression System (Thermo Fisher Scientific) to induce recombinant antibody expression and secretion.

[0090] 80 μL of ExpiFectamine 293 reagent and 15 μg each of heavy-chain and light-chain plasmids were mixed with 1.5 mL of Opti-MEM I (gibco) and allowed to stand at room temperature for 5 minutes. After standing, the two solutions were combined and allowed to stand at room temperature for 20 minutes, and 4.5–5.5 × 10⁶ cells were cultured in a 125 mL flask (Corning). 6 Cells were added to 30 mL of cells / mL and cultured in a swirling manner (37°C, 8% CO2). After 18-20 hours of culture, 150 μL of Expi Fectamine 293 Transfection Enhancer 1 and 1.5 mL of Expi Fectamine 293 Transfection Enhancer 2 were added, and the cells were cultured in a swirling manner for 5-6 days.

[0091] Recombinant antibodies expressed using an expression system with Expi 293F cells were purified using Protein G affinity chromatography (GE Hitrap protein GHP (1 mL)).

[0092] In this example, two types of humanized antibodies, A10h / 8A7 antibody (derived from 8A7 antibody) and A10h / 16E8 antibody (derived from 16E8 antibody), were obtained from the A10 antibody obtained in Example 1.

[0093] Example 5: Amino acid sequence analysis of antibodies In this example, amino acid sequence analysis was performed on the antibody produced in Example 1, the human chimeric antibody in Example 2, and the humanized antibody in Example 3.

[0094] (A) Amino acid sequence analysis of A10 antibody derivatives (A10c antibody and A10h / 8A7 antibody) For the amino acid sequence analysis of the A10c antibody, the heavy chain expression plasmid vector and the light chain expression plasmid vector of the A10 chimeric antibody (A10c antibody) obtained in Example 3 were subjected to nucleotide sequence analysis, and the heavy chain amino acid sequence and light chain amino acid sequence were identified based on their respective DNA sequences.

[0095] For the amino acid sequence analysis of the A10h / 8A7 antibody and A10h / 16E8 antibody, the heavy chain expression plasmid vector and the light chain expression plasmid vector of the A10 humanized antibody (A10h / 8A7 antibody and A10h / 16E8 antibody) obtained in Example 4 were subjected to nucleotide sequence analysis, and the heavy chain amino acid sequence and light chain amino acid sequence were identified based on their respective DNA sequences.

[0096] (B) Amino acid sequence analysis of 1G2 antibody, 12D10 antibody, and 9A7 antibody For the amino acid sequence analysis of the 1G2 antibody, 12D10 antibody, and 9A7 antibody obtained in Example 2, the heavy chain IgG1 gene or IgG2b gene and the light chain Igk gene of the antibody genes were isolated from the antibody-producing cells (for 1G2 antibody, heavy chain IgG1 gene and light chain Igk gene; for 12D10 antibody, heavy chain IgG1 gene and light chain Igk gene; and for 9A7 antibody, heavy chain IgG2b gene and light chain Igk gene). Using total RNA extracted from the antibody-producing cells as a template, a reverse transcription reaction was performed according to the attached protocol using the SMART cDNA Library Construction Kit (TAKARA), which is capable of amplifying 5' full-length cDNA, to produce cDNA.

[0097] Using the cDNA synthesized by the method described above as a template, two PCRs (1st PCR, 2nd PCR) were performed using KOD FX (TOYOBO). For the 1st PCR, 1 μl of cDNA reverse-transcribed from each lymphocyte cell line (LCL) was used as a template, and the following forward primer (Primer 1) and reverse primer (Primer 2) were used as primers for the PCR reaction (reaction conditions: 94°C for 2 minutes → [98°C for 10 seconds, 55°C for 30 seconds, 68°C for 2 minutes] × 35 cycles → 68°C for 3 minutes).

[0098] [Table 7]

[0099] For the second PCR, 0.5 μl of the extension product from the first PCR was used as a template, and the following forward primer (Primer 1) and reverse primer (Primer 2) were used as primers to perform the PCR reaction (reaction conditions: 94°C for 2 minutes → [98°C for 10 seconds, 60°C for 30 seconds, 68°C for 2 minutes] × 35 cycles → 68°C for 3 minutes).

[0100] [Table 8]

[0101] The PCR samples were subjected to the QIAquick Purification Kit (QIAGEN) to remove unreacted primers and enzymes, and purified products were obtained.

[0102] The purified PCR products were restricted enzyme-treated, separated and purified by agarose gel electrophoresis, and then incorporated into vectors. For vector preparation containing heavy chain genes, the pQEFIP vector (a retroviral vector with pQCXIP (TAKARA) as the backbone and the CMV promoter replaced with the human EF1 alpha promoter) was used. For vector preparation containing light chain genes, the pQEFIN vector (a retroviral vector with pQCXIN (TAKARA) as the backbone and the CMV promoter replaced with the human EF1 alpha promoter) was used. The PCR products were incorporated into the vectors using Ligation High (TOYOBO), and competent cells (DH-5alpha: Nippon Gene) were transformed.

[0103] Plasmids were extracted from E. coli cultured in ampicillin-containing LB liquid medium using NucleoSpin Plasmid EasyPure (MACHEREY-NAGEL) according to the attached protocol.

[0104] The heavy chain and light chain expression plasmid vectors obtained for each antibody were subjected to nucleotide sequence analysis, and the heavy chain and light chain amino acid sequences of each antibody were identified based on their respective DNA sequences.

[0105] The results of the amino acid sequence analysis were as follows: (1-1) A10c antibody (chimeric A10 antibody) (1-1-1) Heavy chain Full-length heavy chain amino acid sequence: SEQ ID No: 37, Amino acid sequence of the heavy chain variable region (VH domain): SEQ ID No: 25, Of these, when we analyzed the complementarity-determining regions (CDR1, CDR2, CDR3), Heavy chain complementarity determination regions: CDR1 (GYSFTGYT, SEQ ID No: 1), CDR2 (INPYNGGT, SEQ ID No: 2), and CDR3 (ARTSGYLFAY, SEQ ID No: 3), It was confirmed that this was the case; (1-1-2) Light chain Light chain full-length amino acid sequence: SEQ ID No: 38, Amino acid sequence of the light chain variable region (VL) domain: SEQ ID No: 26, Of these, when we analyzed the complementarity-determining regions (CDR1, CDR2, CDR3), Light chain complementarity determination regions: CDR1 (EDIYNR, SEQ ID No: 4), CDR2 (GAT, SEQ ID No: 5), and CDR3 (QQYWSSPLT, SEQ ID No: 6), It was confirmed that this was the case.

[0106] (1-2) A10h / 8A7 antibody (humanized A10 antibody) (1-2-1) Heavy chain Full-length heavy chain amino acid sequence: SEQ ID No: 39, Amino acid sequence of the heavy chain variable region (VH domain): SEQ ID No: 27, Of these, when we analyzed the complementarity-determining regions (CDR1, CDR2, CDR3), Heavy chain complementarity determination regions: CDR1 (GYSFTGYT, SEQ ID No: 1), CDR2 (INPYNGGT, SEQ ID No: 2), and CDR3 (ARTSGYLFAY, SEQ ID No: 3), It was confirmed that this was the case; (1-2-2) Light chain Light chain full-length amino acid sequence: SEQ ID No: 40, Amino acid sequence of the light chain variable region (VL) domain: SEQ ID No: 28, Of these, when we analyzed the complementarity-determining regions (CDR1, CDR2, CDR3), Light chain complementarity determination regions: CDR1 (EDIYNR, SEQ ID No: 4), CDR2 (GAT, SEQ ID No: 5), and CDR3 (QQYWSSPLT, SEQ ID No: 6), It was confirmed that this was the case.

[0107] (1-3) A10h / 16E8 antibody (humanized A10 antibody) (1-3-1) Heavy chain Full-length heavy chain amino acid sequence: SEQ ID No: 41, Amino acid sequence of the heavy chain variable region (VH domain): SEQ ID No: 29, Of these, when we analyzed the complementarity-determining regions (CDR1, CDR2, CDR3), Heavy chain complementarity determination regions: CDR1 (GYSFTGYT, SEQ ID No: 1), CDR2 (INPYNGGT, SEQ ID No: 2), and CDR3 (ARTSGYLFAY, SEQ ID No: 3), It was confirmed that this was the case; (1-3-2) Light chain Light chain full-length amino acid sequence: SEQ ID No: 42, Amino acid sequence of the light chain variable region (VL) domain: SEQ ID No: 30, Of these, when we analyzed the complementarity-determining regions (CDR1, CDR2, CDR3), Light chain complementarity determination regions: CDR1 (EDIYNR, SEQ ID No: 4), CDR2 (GAT, SEQ ID No: 5), and CDR3 (QQYWSSPLT, SEQ ID No: 6), It was confirmed that this was the case.

[0108] (2)1G2 antibody (2-1) Heavy chain Full-length heavy chain amino acid sequence: SEQ ID No: 43, Amino acid sequence of the heavy chain variable region (VH domain): SEQ ID No: 31, Of these, when we analyzed the complementarity-determining regions (CDR1, CDR2, CDR3), Heavy chain complementarity determination regions: CDR1 (GFSLPRYG, SEQ ID No: 7), CDR2 (IWAGGST, SEQ ID No: 8), and CDR3 (ARAGGSQPFDY, SEQ ID No: 9), It was confirmed that this was the case; (2-2) Light chain Light chain full-length amino acid sequence: SEQ ID No: 44, Amino acid sequence of the light chain variable region (VL) domain: SEQ ID No: 32, Of these, when we analyzed the complementarity-determining regions (CDR1, CDR2, CDR3), Light chain complementarity determination regions: CDR1 (RDINTY, SEQ ID No: 10), CDR2 (RAN, SEQ ID No: 11), and CDR3 (LQYDEFPWT, SEQ ID No: 12), It was confirmed that this was the case.

[0109] (3)12D10 antibody (3-1) Heavy chain Full-length heavy chain amino acid sequence: SEQ ID No: 45, Amino acid sequence of the heavy chain variable region (VH domain): SEQ ID No: 33, Of these, when we analyzed the complementarity-determining regions (CDR1, CDR2, CDR3), Heavy chain complementarity determining regions: CDR1 (GFSLTRYG, SEQ ID No: 13), CDR2 (IWAGGST, SEQ ID No: 14), and CDR3 (ARAGGSSSFDY, SEQ ID No: 15), It was confirmed that this was the case; (3-2) Light chain Light chain full-length amino acid sequence: SEQ ID No: 46, Amino acid sequence of the light chain variable region (VL) domain: SEQ ID No: 34, Of these, when we analyzed the complementarity-determining regions (CDR1, CDR2, CDR3), Light chain complementarity determination regions: CDR1 (QDINTY, SEQ ID No: 16), CDR2 (RAN, SEQ ID No: 17), and CDR3 (LQYDEFPWT, SEQ ID No: 18), It was confirmed that this was the case.

[0110] (4)9A7 antibody (4-1) Heavy chain Full-length heavy chain amino acid sequence: SEQ ID No: 47, Amino acid sequence of the heavy chain variable region (VH domain): SEQ ID No: 35, Of these, when we analyzed the complementarity-determining regions (CDR1, CDR2, CDR3), Heavy chain complementarity-determining regions: CDR1 (GFSLTGYG, SEQ ID No: 19), CDR2 (IWADGTT, SEQ ID No: 20), and CDR3 (ARAGGSQPFDY, SEQ ID No: 21), It was confirmed that this was the case; (4-2) Light chain Light chain full-length amino acid sequence: SEQ ID No: 48, Amino acid sequence of the light chain variable region (VL) domain: SEQ ID No: 36, Of these, when we analyzed the complementarity-determining regions (CDR1, CDR2, CDR3), Light chain complementarity determination regions: CDR1 (QDINSY, SEQ ID No: 22), CDR2 (RAN, SEQ ID No: 23), and CDR3 (LQYDEFPWT, SEQ ID No: 24), It was confirmed that this was the case.

[0111] Example 6: Analysis of antibody binding in vitro In this example, the binding affinity of various types of chimeric antibodies, using the heavy and light chains of antibodies produced in Examples 1 and 2, and the heavy and light chains of human chimeric antibodies produced in Example 3, to the target molecule ADAMTS13 was compared and examined, and whether the binding affinity changes depending on the type of chimerization.

[0112] The antibodies used in this example were recombinant A10 antibodies composed of the heavy or light chains of mouse A10 antibody, and recombinant human chimeric antibodies (A10c antibodies) composed of the heavy or light chains of the A10c antibody prepared in Example 3, which were prepared and used.

[0113] For the detection of each antibody (A10c antibody and A10 antibody), the detection antibodies used were anti-human IgG antibody for the A10c antibody and anti-mouse IgG antibody for the A10 antibody, which were used as "secondary antibodies" for detection, respectively.

[0114] Binding was measured by ELISA using wells coated with 50 ng of ADAMTS13 MD recombinant antigen (self-prepared product). Specifically, ADAMTS13 MD was diluted with PBS to a concentration of 1 μg / mL, added to a 96-well ELISA plate (NUNC) at 50 μL / well, and left standing overnight at 4°C. After washing three times (PBS-T (0.1% Tween 20 in PBS), 250 μL / well), 250 μL / well of blocking buffer (2% BSA, 0.05% NaN3 in PBS) was added and left standing at room temperature for 1 hour. After standing, the 96-well plate was washed once (PBS-T, 250 μL / well), and after adjusting the concentrations of each recombinant antibody (A10c antibody and A10 antibody), 50 μL / well was added and left standing at room temperature for 2 hours. Each recombinant antibody was used at concentrations of -3 1×10 -3 μg / mL, 5×10 -2 μg / mL, 1×10 -2 μg / mL, 5×10 -1 μg / mL, 1×10 -1 μg / mL, 1 μg / mL as shown in Figure 3.

[0115] After standing, the 96-well plate was washed three times (PBS-T, 250 μL / well). Secondary antibodies labeled with alkaline phosphatase (goat anti-human IgG-AP, goat anti-mouse IgG-AP (SouthernBiotech)) were diluted 1 / 1000-fold with PBS-T, 50 μL / well, and left to stand at room temperature for 1 hour. After washing the 96-well plate once (PBS-T, 250 μL / well), 100 μL / well of a chromogenic substrate solution (one phosphate substrate (SIGMA) dissolved in 25 mL of 0.1 M glycine buffer) was added. Absorbance at 405 nm and 650 nm was measured using a multimode reader EnSpire (PerkinElmer Enspire).

[0116] As a result, both of the tested antibodies (A10c antibody and A10 antibody) bound to ADAMTS13, and the binding strength of the antibody variants to ADAMTS13 remained unchanged even after chimerization (A10c antibody), showing dose-dependent reactivity to the antigen, similar to the A10 antibody (Figure 3).

[0117] Example 7: Analysis of antibody inhibitory effect in vitro In this example, the inhibitory effect of the A10 antibody produced in Example 1, and the 1G2 antibody, 12D10 antibody, and 9A7 antibody produced in Example 2 on the VWF cleavage activity of the target molecule ADAMTS13 was analyzed.

[0118] To 50 μL of normal human plasma, 20 μL each of the antibodies (A10 antibody obtained in Example 1, and 1G2 antibody, 12D10 antibody, and 9A7 antibody obtained in Example 2) were mixed at concentrations ranging from 0.2 to 200 μg / mL, adjusting the final concentration to 0.01 to 100 μg / mL. After incubation at 37°C for 15 minutes, the activity of ADAMTS13 remaining in the human plasma was measured by ELISA (Kainos Corporation).

[0119] As a result, the A10 antibody showed inhibitory activity against the VWF cleavage activity of ADAMTS13 at an ID50 concentration of approximately 1.55 μg / mL, and exhibited complete (almost 100%) inhibition at concentrations of 50 μg / mL or higher (Figure 4).

[0120] Furthermore, the 1G2 antibody inhibited the VWF cleavage activity of ADAMTS13 with an ID50 concentration of approximately 7.28 μg / mL, the 12D10 antibody inhibited the VWF cleavage activity of ADAMTS13 with an ID50 concentration of approximately 25.4 μg / mL, and the 9A7 antibody inhibited the VWF cleavage activity of ADAMTS13 with an ID50 concentration of approximately 19.5 μg / mL (Figure 4).

[0121] Example 8: Functional analysis of humanized antibodies in vitro In this example, the binding affinity and inhibitory activity of the humanized antibodies (A10h / 8A7 antibody and A10h / 16E8 antibody) produced in Example 4 were compared with the human chimeric antibodies produced in Example 3 to the target molecule ADAMTS13. The study analyzed whether humanization altered the function (binding affinity and inhibitory activity).

[0122] The binding analysis was performed using the same method as in Example 6, except that multiple concentrations of the target molecule ADAMTS13 were set. Goat anti-human IgG-AP was used as the secondary antibody.

[0123] As a result, the tested A10h / 16E8 antibody (humanized A10 antibody) showed almost the same binding affinity to ADAMTS13 as the A10c antibody (human chimeric A10 antibody) whose binding affinity was confirmed in Example 6 (Figure 5). On the other hand, the A10h / 8A7 antibody (humanized A10 antibody) showed decreased binding affinity to ADAMTS13 compared to the A10c antibody (human chimeric A10 antibody), but maintained a binding strength of about 60% of that of the A10c antibody (Figure 5). These results indicate that even when the A10 antibody is humanized, it exhibits dose-dependent reactivity to the ADAMTS13 antigen, similar to the chimeric antibody (A10c antibody).

[0124] Next, the inhibitory activity of each antibody (A10h / 8A7 antibody, A10h / 16E8 antibody, A10 antibody, and A10c antibody) was measured using the same method as in Example 7.

[0125] As a result, the tested A10h / 16E8 antibody (humanized A10 antibody) showed dose-dependent inhibitory activity against ADAMTS13, higher than that of the A10 antibody whose inhibitory activity was confirmed in Example 7 (Figure 6). On the other hand, the A10h / 8A7 antibody (humanized A10 antibody) showed decreased inhibitory activity against ADAMTS13 compared to the A10 antibody, but similarly to the A10h / 16E8 antibody, it showed dose-dependent inhibitory activity against ADAMTS13 (Figure 6). Furthermore, the A10 antibody and A10c antibody (human chimeric antibody of A10 antibody) were shown to have almost equivalent inhibitory activity (Figure 6). These results indicate that even when the A10 antibody is humanized, it exhibits dose-dependent inhibitory activity against the ADAMTS13 antigen, similar to the chimeric antibody (A10c antibody).

[0126] Furthermore, in this embodiment, the ID50 values ​​for the VWF cleavage activity of ADAMTS13 of each antibody were calculated, and it was shown that they had the following ID50 values ​​(Figure 6): A10 antibody=5.97μg / mL; A10c antibody=6.92μg / mL; A10h / 8A7 antibody=13.04μg / mL; A10h / 16E8 antibody = 2.52μg / mL.

[0127] Example 9: Analysis of antibody inhibitory effects in vitro In this embodiment, we investigated the cleavage of VWF multimers generated in blood when high shear stress is applied to the blood, and confirmed how the antibody against ADAMTS13 of the present invention affects the cleavage of VWF multimers.

[0128] First, high shear stress was applied to human plasma, and VWF cleavage by ADAMTS13 was confirmed by VWF multimer analysis. High shear stress was applied by filling two 1-mL syringes or two 2.5-mL syringes (both Terumo) connected by an 18-gauge injection needle (Terumo), and moving the plasma between the syringes once per second for 6 minutes (360 times) (J Thromb Haemost, 2019;17:975-983). In this procedure, the stress was 10⁸ dyne / cm² for plasma volumes of 440 μL, 880 μL, and 1320 μL, respectively. 2 , 216 dyne / cm 2 , and 324 dyne / cm 2 High shear stress occurred. Incidentally, 324 dyne / cm 2 This high shear stress is an extremely high shear stress that is not observed in the body of a healthy person.

[0129] VWF multimer analysis was performed by electrophoresis of plasma on an SDS-1.0% agarose gel and visualization by Western blotting. The detection antibody used was VWF polyclonal antibody (Dako).

[0130] As a result, 108 dyne / cm 2 , 216 dyne / cm 2 324 dyne / cm 2 It was shown that the higher the shear stress, the more the high molecular weight VWF multimer is lost and the stronger the cleavage of VWF becomes (Figure 7A).

[0131] Next, 100 μg / mL of the A10 antibody prepared in Example 1 was added to the plasma, and after 15 minutes, the reaction rate was 324 dyne / cm³ as described above. 2 High shear stress was induced, and the cleavage of the VWF multimer was analyzed using the same method as described above.

[0132] As a result, 324 dyne / cm 2It was observed that even under high shear stress, the addition of anti-ADAMTS13 antibody prevented VWF cleavage (Figure 7B). This suggests that the development of AVWS caused by excessive VWF cleavage by ADAMTS13 due to abnormally high shear stress can be prevented by anti-ADAMTS13 antibody. [Industrial applicability]

[0133] The present invention provides an antibody or antibody derivative that has specific binding activity to ADAMTS13, a VWF cleavage protease, and can reduce the excessive cleavage of VWF by ADAMTS13. Since excessive cleavage of VWF by ADAMTS13 is thought to be the cause of acquired von Willebrand syndrome (AVWS) in humans, the present invention can also provide a pharmaceutical composition for preventing or treating human AVWS, comprising the antibody or antibody derivative of the present invention. [Sequence Listing Free Text]

[0134] Complementarity-determining regions of the heavy chains of A10c antibody, A10h / 8A7 antibody, and A10h / 16E8 antibody: CDR1 (GYSFTGYT, SEQ ID No: 1), CDR2 (INPYNGGT, SEQ ID No: 2), and CDR3 (ARTSGYLFAY, SEQ ID No: 3). Complementarity-determining regions of the light chains of A10c antibody, A10h / 8A7 antibody, and A10h / 16E8 antibody: CDR1 (EDIYNR, SEQ ID No: 4), CDR2 (GAT, SEQ ID No: 5), and CDR3 (QQYWSSPLT, SEQ ID No: 6). Complementarity-determining regions of 1G2 antibody heavy chains: CDR1 (GFSLPRYG, SEQ ID No: 7), CDR2 (IWAGGST, SEQ ID No: 8), and CDR3 (ARAGGSQPFDY, SEQ ID No: 9) Complementarity-determining regions of 1G2 antibody light chains: CDR1 (RDINTY, SEQ ID No: 10), CDR2 (RAN, SEQ ID No: 11), and CDR3 (LQYDEFPWT, SEQ ID No: 12) Complementarity-determining regions of the 12D10 antibody heavy chain: CDR1 (GFSLTRYG, SEQ ID No: 13), CDR2 (IWAGGST, SEQ ID No: 14), and CDR3 (ARAGGSSSFDY, SEQ ID No: 15) Complementarity-determining regions of the 12D10 antibody light chain: CDR1 (QDINTY, SEQ ID No: 16), CDR2 (RAN, SEQ ID No: 17), and CDR3 (LQYDEFPWT, SEQ ID No: 18) Complementarity-determining regions of the 9A7 antibody heavy chain: CDR1 (GFSLTGYG, SEQ ID No: 19), CDR2 (IWADGTT, SEQ ID No: 20), and CDR3 (ARAGGSQPFDY, SEQ ID No: 21) Complementarity-determining regions of the 9A7 antibody light chain: CDR1 (QDINSY, SEQ ID No: 22), CDR2 (RAN, SEQ ID No: 23), and CDR3 (LQYDEFPWT, SEQ ID No: 24) Amino acid sequence of the VH domain in the heavy chain variable region of the A10c antibody: SEQ ID No: 25 Amino acid sequence of the VL domain in the light chain of the A10c antibody: SEQ ID No: 26 Amino acid sequence of the VH domain in the heavy chain variable region of the A10h / 8A7 antibody: SEQ ID No: 27 Amino acid sequence of the VL domain in the light chain of the A10h / 8A7 antibody: SEQ ID No: 28 Amino acid sequence of the VH domain in the heavy chain variable region of the A10h / 16E8 antibody: SEQ ID No: 29 Amino acid sequence of the VL domain in the light chain of the A10h / 16E8 antibody: SEQ ID No: 30 Amino acid sequence of the VH domain in the variable region of the 1G2 antibody heavy chain: SEQ ID No: 31 Amino acid sequence of the VL domain of the 1G2 antibody light chain: SEQ ID No: 32 Amino acid sequence of the VH domain in the heavy chain variable region of the 12D10 antibody: SEQ ID No: 33 Amino acid sequence of the VL domain of the 12D10 antibody light chain: SEQ ID No: 34 Amino acid sequence of the VH domain in the heavy chain variable region of the 9A7 antibody: SEQ ID No: 35 Amino acid sequence of the VL domain of the 9A7 antibody light chain: SEQ ID No: 36 Amino acid sequence of the full-length heavy chain of the A10c antibody: SEQ ID No: 37 Amino acid sequence of the full-length light chain of the A10c antibody: SEQ ID No: 38 Amino acid sequence of the full-length heavy chain of the A10h / 8A7 antibody: SEQ ID No: 39 Amino acid sequence of the full-length light chain of the A10h / 8A7 antibody: SEQ ID No: 40 Amino acid sequence of the full-length heavy chain of the A10h / 16E8 antibody: SEQ ID No: 41 Amino acid sequence of the full-length light chain of the A10h / 16E8 antibody: SEQ ID No: 42 Amino acid sequence of the full-length heavy chain of the 1G2 antibody: SEQ ID No: 43 Amino acid sequence of the full-length light chain of the 1G2 antibody: SEQ ID No: 44 Amino acid sequence of the full-length heavy chain of the 12D10 antibody: SEQ ID No: 45 Amino acid sequence of the full-length light chain of the 12D10 antibody: SEQ ID No: 46 Amino acid sequence of the full-length heavy chain of the 9A7 antibody: SEQ ID No: 47 9A7 antibody light chain full-length amino acid sequence: SEQ ID No: 48 Primer sequences used in Example 5: SEQ ID NO: 49~SEQ ID NO: 64

Claims

1. (1) Heavy chain complementarity determination regions, CDR1 (GYSFTGYT, SEQ ID No: 1), CDR2 (INPYNGGT, SEQ ID No: 2), and CDR3 (ARTSGYLFAY, SEQ ID No: 3), Light chain complementarity-determining regions: CDR1 (EDIYNR, SEQ ID No: 4), CDR2 (GAT, SEQ ID No: 5), and CDR3 (QQYWSSPLT, SEQ ID No: 6); An antibody derivative (hereinafter referred to as "antibody derivative") selected from an antibody variant or functional fragment selected from the group consisting of humanized antibodies, chimeric antibodies, single-chain antibodies, multivalent antibodies, and multispecific antibodies, which have binding affinity to ADAMTS13 and inhibit the cleavage activity of von Willebrand factor (VWF).

2. A recombinant antibody derivative according to claim 1.

3. The amino acid sequence of the VH domain, the heavy chain variable region of antibody derivatives, (1-1) The amino acid sequence of SEQ ID No: 25, or the amino acid sequence of SEQ ID No: 25 that includes one or more amino acid substitutions, insertions, or deletions in portions other than CDR1 (SEQ ID No: 1), CDR2 (SEQ ID No: 2), and CDR3 (SEQ ID No: 3), (1-2) The amino acid sequence of SEQ ID No: 27, or the amino acid sequence of SEQ ID No: 27 that includes substitutions, insertions, or deletions of one or more amino acids in parts other than CDR1 (SEQ ID No: 1), CDR2 (SEQ ID No: 2), and CDR3 (SEQ ID No: 3), and (1-3) The amino acid sequence of SEQ ID No: 29, or the amino acid sequence of SEQ ID No: 29 that includes one or more amino acid substitutions, insertions, or deletions in parts other than CDR1 (SEQ ID No: 1), CDR2 (SEQ ID No: 2), and CDR3 (SEQ ID No: 3), An antibody derivative according to claim 1 or 2, selected from the group consisting of the following.

4. The amino acid sequence of the VL domain of the light chain of antibody derivatives is (1-1) The amino acid sequence of SEQ ID No: 26, or the amino acid sequence of SEQ ID No: 26 that includes one or more amino acid substitutions, insertions, or deletions in portions other than CDR1 (SEQ ID No: 4), CDR2 (SEQ ID No: 5), and CDR3 (SEQ ID No: 6), (1-2) The amino acid sequence of SEQ ID No: 28, or the amino acid sequence of SEQ ID No: 28 that includes one or more amino acid substitutions, insertions, or deletions in parts other than CDR1 (SEQ ID No: 4), CDR2 (SEQ ID No: 5), and CDR3 (SEQ ID No: 6), (1-3) The amino acid sequence of SEQ ID No: 30, or the amino acid sequence of SEQ ID No: 30 that includes one or more amino acid substitutions, insertions, or deletions in portions other than CDR1 (SEQ ID No: 4), CDR2 (SEQ ID No: 5), and CDR3 (SEQ ID No: 6), An antibody derivative according to any one of claims 1 to 3, selected from the group consisting of the following.

5. Antibody derivatives, (1-1) Heavy chain (amino acid sequence of SEQ ID No: 37, or amino acid sequence of SEQ ID No: 37 that includes one or more amino acid substitutions, insertions, or deletions in parts other than CDR1 (SEQ ID No: 1), CDR2 (SEQ ID No: 2), and CDR3 (SEQ ID No: 3)) and Light chain (the amino acid sequence of SEQ ID No: 38, or the amino acid sequence of SEQ ID No: 38 that includes one or more amino acid substitutions, insertions, or deletions in parts other than CDR1 (SEQ ID No: 4), CDR2 (SEQ ID No: 5), and CDR3 (SEQ ID No: 6)) Antibody derivatives containing; (1-2) Heavy chain (amino acid sequence of SEQ ID No: 39, or amino acid sequence of SEQ ID No: 39 that includes one or more amino acid substitutions, insertions, or deletions in parts other than CDR1 (SEQ ID No: 1), CDR2 (SEQ ID No: 2), and CDR3 (SEQ ID No: 3)) and Light chain (the amino acid sequence of SEQ ID No: 40, or the amino acid sequence of SEQ ID No: 40 that includes one or more amino acid substitutions, insertions, or deletions in parts other than CDR1 (SEQ ID No: 4), CDR2 (SEQ ID No: 5), and CDR3 (SEQ ID No: 6)) Antibody derivatives containing; (1-3) Heavy chain (amino acid sequence of SEQ ID No: 41, or amino acid sequence of SEQ ID No: 41 that includes one or more amino acid substitutions, insertions, or deletions in parts other than CDR1 (SEQ ID No: 1), CDR2 (SEQ ID No: 2), and CDR3 (SEQ ID No: 3)) and Light chain (the amino acid sequence of SEQ ID No: 42, or the amino acid sequence of SEQ ID No: 42 that includes one or more amino acid substitutions, insertions, or deletions in parts other than CDR1 (SEQ ID No: 4), CDR2 (SEQ ID No: 5), and CDR3 (SEQ ID No: 6)) Antibody derivatives containing; An antibody derivative according to any one of claims 1 to 4, selected from the group consisting of the following.

6. A pharmaceutical composition for preventing or treating bleeding caused by excessive cleavage of VWF, comprising an antibody derivative according to any one of claims 1 to 5.

7. The pharmaceutical composition according to claim 6, wherein the bleeding resulting from excessive cleavage of the vein whip flap is bleeding associated with mechanically assisted circulation.

8. A pharmaceutical composition according to claim 6 or 7, comprising a plurality of antibody derivatives described in any one of claims 1 to 5.

9. A pharmaceutical composition according to any one of claims 6 to 8, which involves inhibiting the cleavage activity of ADAMTS13 toward VWF.

10. The pharmaceutical composition according to any one of claims 6 to 9, wherein the bleeding associated with mechanical circulatory support is acquired von Willebrand syndrome (AVWS).

11. The pharmaceutical composition according to any one of claims 6 to 10, wherein the mechanical circulatory support is selected from the group consisting of extracorporeal lung (ECMO), implantable ventricular assist device (LVAD), and percutaneous cardiopulmonary support (PCPS).

12. A step of contacting a biological sample collected from a subject with an antibody derivative according to any one of claims 1 to 5 in vitro. A step of detecting and measuring ADAMTS13 in a sample bound to the antibody derivative, A method for detecting and measuring the presence and amount of ADAMTS13 in biological samples, including [specific component].

13. A kit for detecting and measuring the presence and amount of ADAMTS13 in a test subject, comprising the antibody derivative described in any one of claims 1 to 5.

14. A step of adding an antibody derivative according to any one of claims 1 to 5 at various concentrations to a biological sample collected from a subject, and incubating it. A process for detecting and measuring intact VWF multimers, intact VWF monomers, and VWF cleavage fragments in a sample. A method for detecting and measuring the VWF cleavage activity of ADAMTS13 in biological samples, including [specific component].

15. A kit for detecting and measuring intact VWF multimers, intact VWF monomers, and VWF cleavage fragments within a test body, comprising the antibody derivative described in any one of claims 1 to 5.

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