Antibodies and antigen-binding peptides as factor XIA inhibitors, and their use

Abstract

The present invention provides novel antigen-binding peptides, such as antibodies or antibody fragments, that specifically bind to selective FXIa inhibitors and / or dual inhibitors of FXIa and plasma kallikrein.The present invention further relates to a method of reducing the antithrombotic effect of FXIa inhibitors by administering to a subject a pharma- ceutical effective amount of the antigen-binding peptides disclosed herein.In addition, the present invention provides detection reagents and methods for detecting FXIa inhibitor levels in biological samples. TIFF2024505390000129.tif113125

Description

Technical Field

[0001] (Cross - reference to Related Applications) This application claims priority based on U.S. Provisional Application No. 63 / 135,016 filed on January 8, 2021, U.S. Provisional Application No. 63 / 148,767 filed on February 12, 2021, U.S. Provisional Application No. 63 / 152,595 filed on February 23, 2021, and U.S. Provisional Application No. 63 / 153,045 filed on February 24, 2021, and each of them is hereby incorporated by reference in its entirety into this specification.

[0002] (Reference to Sequence Listing) This application includes a sequence listing with the file name "055920 - 553P01US_Sequence_Lising.txt" (about 316,130 bytes, created on December 23, 2020, and electronically submitted in ASCII format), and the entire sequence listing is hereby incorporated by reference.

Background Art

[0003] Thromboembolic diseases remain a leading cause of death in developed countries despite the availability of anticoagulants (e.g., dabigatran, apixaban, rivaroxaban, warfarin (COUMADIN®), heparin, low molecular weight heparin (LMWH), and synthetic pentasaccharides) and platelet agents (e.g., aspirin and clopidogrel (PLAVIX®))). The discovery and development of safe and effective oral anticoagulants for the prevention and treatment of a wide range of thromboembolic diseases remains crucial. One approach involves reducing thrombin production by inhibiting factor XIa (FXIa) coagulation. FXIa is a plasma serine protease involved in blood coagulation control, and in vivo, tissue factor (TF) binding to factor VII (FVII) promotes the production of factor VIIa (FVIIa). The resulting TF:FVIIa complex activates factor IX (FIX) and factor X (FX), leading to the production of factor Xa (FXa). The produced FXa catalyzes the conversion of prothrombin to a small amount of thrombin, and this pathway is subsequently inhibited by tissue factor pathway inhibitor (TFPI). The catalytic amount of thrombin further promotes coagulation by activating factors V, VIII, and XI. The rapid increase in thrombin converts fibrinogen into fibrin, a polymer that forms the structural framework of the thrombus, and activates platelets, the key cellular component of coagulation. In short, because FVIIa plays a crucial role in this series of reactions in blood coagulation, it is an ideal target in antithrombotic therapy.

[0004] Plasma prekallikrein is an enzyme precursor of trypsin-like serine protease and is present in plasma at a concentration of 35-50 μg / mL. Its structure is similar to factor XI (FXI), and overall, the amino acid sequence of plasma kallikrein is 58% identical to that of FXI. Plasma kallikrein is thought to play an important role in numerous inflammatory diseases. The main inhibitor of plasma kallikrein is serpin C1 esterase inhibitor. Patients with hereditary angioedema (HAE), who lack the gene for C1 esterase inhibitor, experience intermittent swelling of the face, hands, throat, gastrointestinal tract, and genitals. Blisters formed due to sudden onset contain high levels of plasma kallikrein. Plasma kallikrein hydrolyzes high molecular weight kininogen, releasing bradykinin, which increases vascular permeability. Treatment with the large protein plasma kallikrein inhibitor has been shown to effectively treat HAE by inhibiting the release of bradykinin, which increases vascular permeability.

[0005] The plasma kallikrein-kinin system is abnormally high in patients with advanced diabetic macular edema. Recently, it has been reported that plasma kallikrein is involved in retinal vascular dysfunction in diabetic rats. Furthermore, administration of the plasma kallikrein inhibitor ASP-440 improved both retinal vascular permeability and retinal blood flow abnormalities in diabetic rats. Therefore, plasma kallikrein inhibitors should be useful as therapeutic agents to reduce retinal vascular permeability associated with diabetic retinopathy and diabetic macular edema. Other complications of diabetes associated with plasma kallikrein, such as cerebral hemorrhage, nephropathy, cardiomyopathy, and neuropathy, may also be targets for plasma kallikrein inhibitors. To date, no small-molecule synthetic plasma kallikrein inhibitors have been approved for medical use. As reported with ecalantide, large-protein plasma kallikrein inhibitors carry a risk of anaphylactic reactions.

[0006] Novel and effective selective FXIa inhibitors, or dual inhibitors of FXIa and plasma kallikrein, for the treatment of thromboembolic and / or inflammatory diseases are disclosed in WO2016053455A1, incorporated herein by reference. These selective FXIa inhibitors or FXIa / plasma kallikrein dual inhibitors (e.g., compounds provided in the present invention) have been developed based on their excellent antithrombotic effects with little to no risk of bleeding. However, bleeding can rarely occur in clinical trials in which such FXIa inhibitors are administered to patients. In humans, bleeding due to factor XI deficiency can occur, particularly in trauma to highly fibrinolytic tissues (e.g., the pharyngeal orifice and urinary tract).

[0007] While hemostatic methods exist, such as coagulation factor preparations and recombinant activated factor VII, and these drugs are primarily approved for use in hemophilia patients, they may also be used in bleeding patients treated with thrombin or FXa inhibitors when specific antagonists are unavailable. However, these methods carry the risk of thrombus formation. Therefore, there is an urgent need to develop compounds that can immediately restore the antithrombotic effect of the aforementioned selective FXIa inhibitors or FXIa / plasma kallikrein dual inhibitors (e.g., the compounds of this disclosure) without the risk of thrombus formation, for patients experiencing severe bleeding or requiring emergency surgical intervention.

[0008] (Summary of the present invention) This invention discloses novel antibodies or antigen-binding peptides that specifically bind to selective FXIa inhibitors and / or FXIa / plasma kallikrein dual inhibitors. Furthermore, this invention discloses a method for reducing the antithrombotic effect of FXIa inhibitors by administering a pharmaceutically effective amount of the antibody or antigen-binding peptide disclosed herein to a target. Furthermore, this invention discloses detection reagents and methods for detecting FXIa inhibition levels in biological samples.

[0009] Specific Embodiment 1: An isolated antigen-binding peptide comprising at least one heavy chain variable region (VH) and at least one light chain variable region (VL), wherein at least one VH is: (a) VH complementarity determination region 1 (VH-CDR1) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 12; (b) VH-CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 13-22; or (c) VH-CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs. 23-28 It includes at least one of the following, and at least one VL is: (d) VL-CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs. 29-37; (e) VL-CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs. 38-43; or (f) VL-CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 44-51 It includes at least one of the following.

[0010] Specific Embodiment 2: below: (a) at least one heavy chain variable region having an amino acid sequence selected from the group consisting of SEQ ID NOs. 52 to 83; and (b) At least one light chain variable region having an amino acid sequence selected from the group consisting of SEQ ID NOs: 84-99 Isolated antigen-binding peptides containing [the specified substance].

[0011] Specific Embodiment 3: An isolated antigen-binding peptide comprising at least one heavy chain variable region (VH) and at least one light chain variable region (VL), wherein VH comprises three complementarity-determining regions (CDRs): VH-CDR1, VH-CDR2, and VH-CDR3, and VL comprises three CDRs: VL-CDR1, VL-CDR2, and VL-CDR3. Furthermore, the amino acid sequences of VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3 each have sequences selected from the group consisting of the following. (a) Sequence numbers for each: 1, 13, 23, 29, 38, and 44; (b) Sequence numbers for each: 1, 14, 23, 29, 38, and 45; (c) Sequence numbers for each: 1, 13, 24, 30, 38, and 45; (d) Sequence numbers for each: 1, 13, 24, 29, 39, and 45; (e) Sequence numbers for each: 1, 14, 25, 29, 38, and 46; (f) Sequence numbers for each: 2, 13, 26, 31, 40, and 47; (g) Sequence numbers for each: 3, 15, 24, 32, 40, and 47; (h) Sequence numbers for each: 4, 16, 24, 29, 38, and 46; (i) Sequence numbers for each: 5, 15, 24, 29, 38, and 46; (j) Sequence numbers for each: 1, 14, 24, 29, 38, and 46; (k) Sequence numbers for each: 6, 13, 24, 31, 40, and 47; (l) Sequence numbers for each: 3, 15, 24, 32, 41, and 48; (m) Sequence numbers for each: 1, 14, 24, 33, 38, and 49; (n) Sequence numbers for each: 1, 14, 26, 29, 38, and 46; (o) Sequence numbers for each: 7, 17, 26, 29, 38, and 46; (p) Sequence numbers: 8, 17, 24, 34, 38, and 46; (q) Sequence numbers for each: 1, 17, 26, 29, 38, and 46; (r) Sequence numbers for each: 1, 17, 26, 35, 38, and 46; (s) Sequence numbers for each: 1, 17, 24, 33, 38, and 49; (t) Sequence numbers: 9, 14, 26, 29, 38, and 46; (u) Sequence numbers: 9, 14, 26, 35, 38, and 46; (v) Sequence numbers for each: 9, 17, 24, 29, 38, and 46; (w) Sequence numbers: 9, 17, 24, 35, 38, and 46; (x) Sequence numbers for each: 9, 17, 24, 34, 38, and 46; (y) Sequence numbers for each: 9, 14, 24, 29, 38, and 46; (z) Sequence numbers: 9, 18, 26, 35, 38, and 46; (aa) Sequence numbers for each: 8, 14, 24, 29, 38, and 46; (bb) Sequence numbers: 8, 17, 26, 29, 38, and 46; (cc) Sequence numbers for each: 9, 19, 26, 29, 38, and 46; (dd) Sequence numbers for each: 9, 17, 26, 34, 38, and 46; (ee) Sequence numbers: 10, 20, 27, 36, 42, and 50; (ff) Sequence numbers: 11, 21, 28, 37, 43, and 51; (gg) Sequence numbers for each: 12, 22, 26, 33, 38, and 46; (hh) Sequence numbers for each: 12, 17, 26, 33, 38, and 46; (ii) Each sequence number: 9, 17, 26, 33, 38, and 46; and (jj) A variant of (a)-(ii) in which any amino acid sequence contains one, two, or three conserved substitutions.

[0012] Specific Embodiment 4: An isolated antigen-binding peptide of Embodiment 3, wherein at least one VH region and at least one VL region each have an amino acid sequence selected from the group consisting of the following. (a) Sequence ID 52 and Sequence ID 84, respectively; (b) Sequence ID 53 and Sequence ID 85, respectively; (c) Sequence IDs 54 and 86, respectively; (d) Sequence IDs 54 and 87, respectively; (e) Sequence IDs 55 and 88, respectively; (f) Sequence ID 56 and Sequence ID 89, respectively; (g) Sequence IDs 57 and 90, respectively; (h) Sequence IDs 58 and 88, respectively; (i) Sequence ID 59 and Sequence ID 88, respectively; (j) Sequence IDs 60 and 91, respectively; (k) Sequence IDs 61 and 89, respectively; (l) Sequence ID 57 and Sequence ID 92, respectively; (m) Sequence IDs 60 and 93, respectively; (n) Sequence ID 60 and Sequence ID 88, respectively; (o) Sequence IDs 62 and 88, respectively; (p) Sequence IDs 63 and 88, respectively; (q) Sequence ID 64 and Sequence ID 88, respectively; (r) Sequence IDs 65 and 94, respectively; (s) Sequence ID 66 and Sequence ID 88, respectively; (t) Sequence ID 66 and Sequence ID 95, respectively; (u) Sequence IDs 67 and 88, respectively; (v) Sequence IDs 68 and 93, respectively; (w) Sequence IDs 69 and 88, respectively; (x) Sequence ID 69 and Sequence ID 95, respectively; (y) Sequence IDs 70 and 88, respectively; (z) Sequence IDs 70 and 95, respectively; (aa) Sequence IDs 71 and 88, respectively; (bb) Sequence IDs 71 and 94, respectively; (cc) Sequence IDs 72 and 88, respectively; (dd) Sequence IDs 73 and 95, respectively; (ee) Sequence IDs 74 and 88, respectively; (ff) Sequence IDs 75 and 88, respectively; (gg) Sequence IDs 76 and 88, respectively; (hh) Sequence IDs 77 and 94, respectively; (ii) Sequence IDs 78 and 96, respectively; (jj) Sequence IDs 79 and 97, respectively; (kk) Sequence IDs 80 and 98 respectively; (ll) Sequence IDs 81 and 99, respectively; (mm) Sequence IDs 81 and 98, respectively; (nn) Sequence IDs 82 and 99, respectively; (oo) Sequence ID 83 and Sequence ID 98 respectively; and Variants of (a)~(oo) that include (pp) 1, 2, 3, or 4 conservative substitutions.

[0013] Specific Embodiment 5: An isolated antigen-binding peptide from any of the above embodiments, comprising two heavy chain variable regions, each paired with a light chain variable region.

[0014] Specific Embodiment 6: The isolated antigen-binding peptide of Embodiment 5, further comprising a polypeptide linker having a sequence selected from SEQ ID NOs. 196-199.

[0015] Specific Embodiment 7: An isolated antigen-binding peptide of any one of the embodiments described above, wherein formula (I): [ka] [In the formula, R 1 C 1-4 It is alkyl; R 2 These are independently selected from F, Cl, CF3, CHF2, CH2F, and CH3; R 3 These are independently selected from CF3, CHF2, CH2F, and CH3; R 4 is H; and R 5 [It is independently selected from F and Cl.] An antigen-binding peptide that specifically binds to the compound indicated by, or to its stereoisomer or tautomer.

[0016] Specific Embodiment 8: The isolated antigen-binding peptide of Embodiment 7, wherein the compound is of formula (II). [ka]

[0017] Specific Embodiment 9: The isolated antigen-binding peptide in any one of the embodiments described above is an antibody.

[0018] Specific Embodiment 10: An isolated antigen-binding peptide according to any one of the embodiments described above, wherein the antigen-binding peptide is Fab, Fab', F(ab')2, Fd, single-chain Fv or scFv, disulfide-bonded Fv, V-NAR domain, IgNar, intrabody, IgGACH2, minibody, F(ab')3, tetrabody, triabody, bispecific antibody, single-domain antibody, DVD-Ig, Fcab, mAb 2, (scFv)2, scFv-Fc, or tandem Fab.

[0019] Specific Embodiment 11: The following: (a) Sequence ID 100 and Sequence ID 160, respectively; (b) Sequence ID 101 and Sequence ID 160, respectively; (c) Sequence IDs 102 and 161, respectively; (d) Sequence IDs 103 and 161, respectively; (e) Sequence ID 104 and Sequence ID 162, respectively; (f) Sequence IDs 105 and 162, respectively; (g) Sequence IDs 104 and 163, respectively; (h) Sequence IDs 105 and 163, respectively; (i) Sequence ID 106 and Sequence ID 164, respectively; (j) Sequence ID 107 and Sequence ID 164, respectively; (k) Sequence ID 108 and Sequence ID 165, respectively; (l) Sequence ID 109 and Sequence ID 165, respectively; (m) Sequence IDs 110 and 166, respectively; (n) Sequence ID 111 and Sequence ID 166, respectively; (o) Sequence IDs 112 and 164, respectively; (p) Sequence IDs 113 and 164, respectively; (q) Sequence IDs 114 and 164, respectively; (r) Sequence IDs 115 and 164, respectively; (s) Sequence IDs 116 and 167, respectively; (t) Sequence IDs 117 and 167, respectively; (u) Sequence ID 118 and Sequence ID 165, respectively; (v) Sequence IDs 119 and 165, respectively; (w) Sequence IDs 110 and 168, respectively; (x) Sequence ID 111 and Sequence ID 168, respectively; (y) Sequence IDs 116 and 169, respectively; (z) Sequence IDs 117 and 169, respectively; (aa) Sequence ID 116 and Sequence ID 164, respectively; (bb) Sequence IDs 117 and 164, respectively; (cc) Sequence IDs 120 and 164, respectively; (dd) Sequence IDs 121 and 164, respectively; (ee) Sequence IDs 122 and 164, respectively; (ff) Sequence IDs 123 and 164, respectively; (gg) Sequence IDs 124 and 164, respectively; (hh) Sequence IDs 125 and 164, respectively; (ii) Sequence IDs 126 and 170, respectively; (jj) Sequence IDs 127 and 170, respectively; (kk) Sequence IDs 128 and 164, respectively; (ll) Sequence IDs 129 and 164, respectively; (mm) Sequence IDs 128 and 171, respectively; (nn) Sequence IDs 129 and 171, respectively; (oo) Sequence IDs 130 and 164, respectively; (pp) Sequence IDs 131 and 164, respectively; (qq) Sequence IDs 132 and 169, respectively; (rr) Sequence IDs 133 and 169, respectively; (ss) Sequence IDs 134 and 164, respectively; (tt) Sequence IDs 135 and 164, respectively; (uu) Sequence IDs 134 and 171, respectively; (vv) Sequence IDs 135 and 171, respectively; (ww) Sequence IDs 136 and 164, respectively; (xx) Sequence IDs 137 and 164, respectively; (yy) Sequence IDs 136 and 171, respectively; (zz) Sequence IDs 137 and 171, respectively; (aaa) Sequence ID 138 and Sequence ID 164, respectively; (bbb) Sequence IDs 139 and 164, respectively; (ccc) Sequence ID 138 and Sequence ID 170, respectively; (ddd) Sequence ID 139 and Sequence ID 170, respectively; (eee) Sequence IDs 140 and 164, respectively; (fff) Sequence IDs 141 and 164, respectively; (ggg) Sequence IDs 142 and 171, respectively; (hhh) Sequence IDs 143 and 171, respectively; (iii) Sequence ID 144 and Sequence ID 164, respectively; (jjj) Sequence IDs 145 and 164, respectively; (kkk) Sequence IDs 146 and 164, respectively; (lll) Sequence IDs 147 and 164, respectively; (mmm) Sequence IDs 148 and 164, respectively; (nnn) Sequence IDs 149 and 164, respectively; (ooo) Sequence ID 150 and Sequence ID 170, respectively; (ppp) Sequence IDs 151 and 170, respectively; (qqq) Sequence IDs 152 and 172, respectively; (rrr) Sequence IDs 153 and 172, respectively; (sss) Sequence IDs 154 and 173 respectively; (ttt) Sequence IDs 155 and 173, respectively; (uuu) Sequence IDs 156 and 174, respectively; (vvv) Sequence IDs 157 and 174, respectively; (www) Sequence ID 158 and Sequence ID 175, respectively; (xxx) Sequence IDs 159 and 175, respectively; (yyy) Sequence ID 158 and Sequence ID 174 respectively; and (zzz) Sequence IDs 159 and 174 respectively An isolated antigen-binding peptide having a sequence selected from the group consisting of the above embodiments, wherein the isolated antigen-binding peptide is of formula (II) [ka] It specifically binds to the compound.

[0020] Specific Embodiment 12: The following: (a) Sequence ID 176 and Sequence ID 160, respectively; (b) Sequence ID 177 and Sequence ID 160, respectively; (c) Sequence ID 178 and Sequence ID 160, respectively; (d) Sequence IDs 179 and 160, respectively; (e) Sequence ID 180 and Sequence ID 164, respectively; (f) Sequence IDs 181 and 164, respectively; (g) Sequence IDs 182 and 164, respectively; (h) Sequence IDs 183 and 164, respectively; (i) Sequence ID 184 and Sequence ID 163, respectively; (j) Sequence ID 185 and Sequence ID 163, respectively; (k) Sequence ID 186 and Sequence ID 163, respectively; (l) Sequence IDs 187 and 163, respectively; (m) Sequence IDs 184 and 162, respectively; (n) Sequence ID 185 and Sequence ID 162, respectively; (o) Sequence IDs 186 and 162, respectively; (p) Sequence IDs 187 and 162, respectively; (q) Sequence ID 188 and Sequence ID 165, respectively; (r) Sequence IDs 189 and 165, respectively; (s) Sequence ID 190 and Sequence ID 165, respectively; (t) Sequence IDs 191 and 165, respectively; (u) Sequence IDs 192 and 161, respectively; (v) Sequence IDs 193 and 161, respectively; (w) Sequence ID 194 and Sequence ID 161 respectively; and (x) Sequence IDs 195 and 161 respectively An isolated antigen-binding peptide having a sequence selected from the group consisting of the above embodiments, wherein the isolated antigen-binding peptide specifically binds to the compound of formula (II). [ka]

[0021] Specific Embodiment 13: An isolated antigen-binding peptide of Embodiment 11 having the sequences of SEQ ID NO: 106 and SEQ ID NO: 164, respectively, wherein the isolated antigen-binding peptide specifically binds to a compound of formula (II). [ka]

[0022] Specific Embodiment 14: An isolated Fab fragment antibody having the sequences of SEQ ID NO: 106 and SEQ ID NO: 164, wherein the isolated Fab fragment antibody specifically binds to a compound of formula (II). [ka]

[0023] Specific Embodiment 15: An isolated antigen-binding peptide of Embodiment 12 having the sequences of SEQ ID NO: 180 and SEQ ID NO: 164, respectively, wherein the isolated antigen-binding peptide specifically binds to a compound of formula (II). [ka]

[0024] Specific Embodiment 16: An isolated tandem Fab fragment antibody having the sequences of SEQ ID NO: 180 and SEQ ID NO: 164, wherein the isolated tandem Fab fragment antibody specifically binds to a compound of formula (II). [ka]

[0025] Specific Embodiment 17: An isolated antigen-binding peptide of Embodiment 12 having the sequences of SEQ ID NO: 181 and SEQ ID NO: 164, respectively, wherein the isolated antigen-binding peptide specifically binds to a compound of formula (II). [ka]

[0026] Specific Embodiment 18: An isolated tandem Fab fragment antibody having the sequences of SEQ ID NO: 181 and SEQ ID NO: 164, wherein the isolated tandem Fab fragment antibody specifically binds to a compound of formula (II). [ka]

[0027] Specific Embodiment 19: An isolated antigen-binding peptide of Embodiment 12 having the sequences of SEQ ID NO: 182 and SEQ ID NO: 164, respectively, wherein the isolated antigen-binding peptide specifically binds to a compound of formula (II). [ka]

[0028] Specific Embodiment 20: An isolated tandem Fab fragment antibody having the sequences of SEQ ID NO: 182 and SEQ ID NO: 164, wherein the isolated tandem Fab fragment antibody specifically binds to a compound of formula (II). [ka]

[0029] Specific Embodiment 21: An isolated antigen-binding peptide of Embodiment 12 having the sequences of SEQ ID NO: 183 and SEQ ID NO: 164, respectively, wherein the isolated antigen-binding peptide specifically binds to a compound of formula (II). [ka]

[0030] Specific Embodiment 22: An isolated tandem Fab fragment antibody having the sequences of SEQ ID NO: 183 and SEQ ID NO: 164, wherein the isolated tandem Fab fragment antibody specifically binds to a compound of formula (II). [ka]

[0031] Specific Embodiment 23: An isolated antigen-binding peptide of Embodiment 12 having the sequences of SEQ ID NO: 176 and SEQ ID NO: 160, respectively, wherein the isolated antigen-binding peptide specifically binds to a compound of formula (II). [ka]

[0032] Specific Embodiment 24: An isolated tandem Fab fragment antibody having the sequences of SEQ ID NO: 176 and SEQ ID NO: 160, wherein the isolated tandem Fab fragment antibody specifically binds to a compound of formula (II). [ka]

[0033] Specific Embodiment 25: An isolated antigen-binding peptide of Embodiment 12 having the sequences of SEQ ID NO: 177 and SEQ ID NO: 160, respectively, wherein the isolated antigen-binding peptide specifically binds to a compound of formula (II). [ka]

[0034] Specific Embodiment 26: An isolated tandem Fab fragment antibody having the sequences of SEQ ID NO: 177 and SEQ ID NO: 160, wherein the isolated tandem Fab fragment antibody specifically binds to a compound of formula (II). [ka]

[0035] Specific Embodiment 27: An isolated antigen-binding peptide of Embodiment 12 having the sequences of SEQ ID NO: 184 and SEQ ID NO: 162, respectively, wherein the isolated antigen-binding peptide specifically binds to a compound of formula (II). [ka]

[0036] Specific Embodiment 28: An isolated tandem Fab fragment antibody having the sequences of SEQ ID NO: 184 and SEQ ID NO: 162, wherein the isolated tandem Fab fragment antibody specifically binds to the compound of formula (II). [ka]

[0037] Specific Embodiment 29: An isolated antigen-binding peptide of Embodiment 12 having the sequences of SEQ ID NO: 184 and SEQ ID NO: 163, respectively, wherein the isolated antigen-binding peptide specifically binds to a compound of formula (II). [ka]

[0038] Specific Embodiment 30: An isolated tandem Fab fragment antibody having the sequences of SEQ ID NO: 184 and SEQ ID NO: 163, wherein the isolated tandem Fab fragment antibody specifically binds to a compound of formula (II). [ka]

[0039] Specific Embodiment 31: An isolated antigen-binding peptide of Embodiment 12 having the sequences of SEQ ID NO: 188 and SEQ ID NO: 165, respectively, wherein the isolated antigen-binding peptide specifically binds to a compound of formula (II). [ka]

[0040] Specific Embodiment 32: An isolated tandem Fab fragment antibody having the sequences of SEQ ID NO: 188 and SEQ ID NO: 165, wherein the isolated tandem Fab fragment antibody specifically binds to the compound of formula (II). [ka]

[0041] Specific Embodiment 33: An isolated antigen-binding peptide of Embodiment 12 having the sequences of SEQ ID NO: 192 and SEQ ID NO: 161, respectively, wherein the isolated antigen-binding peptide specifically binds to a compound of formula (II). [ka]

[0042] Specific Embodiment 34: An isolated tandem Fab fragment antibody having the sequences of SEQ ID NO: 192 and SEQ ID NO: 161, wherein the isolated tandem Fab fragment antibody specifically binds to the compound of formula (II). [ka]

[0043] Specific Embodiment 35: An isolated polynucleotide having a nucleic acid sequence encoding an antigen-binding peptide or a Fab fragment antibody or a tandem Fab fragment antibody, as per any one of Embodiments 1 to 34.

[0044] Specific Embodiment 36: An isolated vector containing the polynucleotide of Embodiment 35.

[0045] Specific Embodiment 37: Isolated host cells containing the vector of Embodiment 36.

[0046] Specific Embodiment 38: A method for producing an antigen-binding peptide, a Fab fragment antibody, or a tandem Fab fragment antibody, characterized by (a) culturing the host cells of Embodiment 37 under conditions that promote protein production, such that the host cells produce an antigen-binding peptide, a Fab fragment antibody, or a tandem Fab fragment antibody, and (b) isolating the antigen-binding peptide, Fab fragment antibody, or tandem Fab fragment antibody from the culture medium in (a).

[0047] Specific Embodiment 39: A detection reagent comprising an isolated antigen-binding peptide or isolated Fab fragment antibody or isolated tandem Fab fragment antibody as described in any one of Embodiments 1 to 34, and a detectable label.

[0048] Specific Embodiment 40: The detection reagent of Embodiment 39, wherein an isolated antigen-binding peptide or an isolated Fab fragment antibody or an isolated tandem Fab fragment antibody is conjugated to a detectable label.

[0049] Specific Embodiment 41: A method for reducing the antithrombotic effect of a compound of formula (I) or a stereoisomer or tautomer thereof, which comprises administering to a subject in need an isolated antigen-binding peptide or an isolated Fab fragment antibody or an isolated tandem Fab fragment antibody according to any one of Embodiments 1 to 34 in a pharmaceutically effective amount.

Chemical formula

[0050] Specific Embodiment 42: The method of Embodiment 41, wherein the compound of formula (I) is of formula (II).

Chemical formula

[0051] Specific Embodiment 43: The method of Embodiment 41 or 42, wherein the pharmaceutically effective amount of the isolated antigen-binding peptide or the isolated Fab fragment antibody or the isolated tandem Fab fragment antibody means that the antigen-binding peptide or the Fab fragment antibody or the tandem Fab fragment antibody is at least about 1:1 molar ratio to the dosage of the compound of formula (I) or (II), or at least about 1:1 molar ratio to the abundance of the compound of formula (I) or (II) in the subject.

[0052] Specific Embodiment 44: A method according to any one of Embodiments 41 to 43, wherein the antigen-binding peptide or isolated Fab fragment antibody or isolated tandem Fab fragment antibody isolated herein is administered simultaneously with or after the administration of the compound of formula (I) or (II).

[0053] Specific Embodiment 45: A method according to any one of Embodiments 41 to 44, wherein the antigen-binding peptide or isolated Fab fragment antibody or isolated tandem Fab fragment antibody isolated herein is administered intravenously, intramuscularly, or subcutaneously.

[0054] Specific Embodiment 46: The method according to any one of Embodiments 41 to 45, wherein the subject is a human.

[0055] Specific Embodiment 47: A method for detecting the level of a compound of formula (I) or its stereoisomers, tautomers, or pharmaceutically acceptable salts in a biological sample. [ka] Here, R 1 C 1-4 It is alkyl; R 2 These are independently selected from F, Cl, CF3, CHF2, CH2F, and CH3; R 3 These are independently selected from CF3, CHF2, CH2F, and CH3; R 4 is H; and R 5 These are independently selected from F and Cl. The above method is as follows: (a) Contacting a biological sample with an isolated antigen-binding peptide or isolated Fab fragment antibody or isolated tandem Fab fragment antibody as described in any one of Embodiments 1 to 34; and (b) Detect the amount of a complex to which the compound and the isolated antigen-binding peptide or isolated Fab fragment antibody or isolated tandem Fab fragment antibody are bound. It is characterized by the following.

[0056] Specific Embodiment 48: The method according to Embodiment 47, wherein the compound of formula (I) is of formula (II). [ka]

[0057] Specific Embodiment 49: The method according to Embodiment 47 or 48, wherein an isolated antigen-binding peptide or an isolated Fab fragment antibody or an isolated tandem Fab fragment antibody is labeled.

[0058] Specific embodiment 50: The method according to any one of embodiments 47 to 49, wherein detection is performed by an immunoassay.

[0059] Specific Embodiment 51: The method according to any one of Embodiments 47 to 50, wherein the biological sample includes urine, feces, saliva, whole blood, plasma, organ tissue, hair, skin, cells, or cell culture.

[0060] Specific Embodiment 52: A method for conjugating a compound of formula (I) or its stereoisomer or tautomer to a subject who is taking a therapeutically effective amount of the compound of formula (I) or its stereoisomer or tautomer, characterized in that the subject is administered a pharmaceutically effective amount of an isolated antigen-binding peptide or isolated Fab fragment antibody or isolated tandem Fab fragment antibody as described in any one of claims 1 to 34. [ka] Here, R 1 C 1-4 It is alkyl; R 2 These are independently selected from F, Cl, CF3, CHF2, CH2F, and CH3; R 3 These are independently selected from CF3, CHF2, CH2F, and CH3; R 4 is H; and R 5 These are independently selected from F and Cl.

[0061] Specific Embodiment 53: The method according to Embodiment 52, wherein the compound of formula (I) is of formula (II). [ka]

[0062] Specific Embodiment 54: The method according to either Embodiment 52 or 53, wherein a pharmaceutically effective amount of isolated antigen-binding peptide or isolated Fab fragment antibody or isolated tandem Fab fragment antibody comprises the antigen-binding peptide or Fab fragment antibody or tandem Fab fragment antibody in a molar ratio of at least about 1:1 with respect to the dose of the compound of formula (I) or (II), or in a molar ratio of at least about 1:1 with respect to the amount of the compound of formula (I) or (II) in the subject.

[0063] Specific Embodiment 55: A method according to any one of Embodiments 52 to 54, wherein the antigen-binding peptide or isolated Fab fragment antibody or isolated tandem Fab fragment antibody isolated herein is administered simultaneously with or after the administration of the compound of formula (I) or (II).

[0064] Specific Embodiment 56: A method according to any one of Embodiments 52 to 55, wherein the antigen-binding peptide or isolated Fab fragment antibody or isolated tandem Fab fragment antibody isolated herein is administered intravenously, intramuscularly, or subcutaneously.

[0065] Specific Embodiment 57: The method according to any one of Embodiments 52 to 56, wherein the subject is a human. [Brief explanation of the drawing]

[0066] [Figure 1] Figures 1A and 1B show a comparison of the 26D5 mAb sequence with the nearest human germline V and J gene sequences. Figure 1A shows a comparison of the 26D5 VH sequence with the germline sequences of IGHV3-53 and IGHJ4. Kabat numbering and Kabat definitions of HCDR2 and HCDR3 are shown. Kabat and AbM definitions of HCDR1 are shown. Figure 1A shows sequence numbers 205, 83, and 220 in the order they were expressed. Figure 1B shows a comparison of the 26D5 VK sequence with the germline sequences of IGKV1-12 and IGKJ4. Kabat numbering and Kabat definitions of CDR are shown. Figure 1B shows sequence numbers 206, 98, and 207 in the order they were expressed.

[0067] [Figure 2] Figure 2 shows the randomized positions of 26D5-GV-Q mAbs by mutation scanning. The Kabat numbering indicates each randomized position by mutation scanning. Kabat definitions of LCDR1-3 and HCDR2-3 (where the last six amino acids are missing from HCDR2) are shown; the AbM definition of HCDR1 is also shown. Figure 2 shows, from top to bottom, sequence numbers 33, 46, 208-209, and 26, respectively.

[0068] [Figure 3] Figures 3A-E show heatmap scans indicating the locations where mutations occur at the binding site of the 26D5-GV-Q mAb antibody (favorable = likely, neutral = neutral, unfavorable = unlikely). Figure 3A shows LCDR1 (SEQ ID NO: 33), Figure 3B shows LCDR3 (SEQ ID NO: 46), Figure 3C shows HCDR1 (SEQ ID NO: 208), Figure 3D shows HCDR2 (SEQ ID NO: 209), and Figure 3E shows HCDR3 (SEQ ID NO: 26).

[0069] [Figure 4]Figures 4A and 4B show the alignment of the progenitor amino acid sequences of the affinity-matured 26D5-GV-Q antibody. Kabat was used for CDR definition and numbering. Figure 4A shows the alignment of the heavy chain variable region (sequence numbers from top to bottom are 83, 82, 74, 60, 67, 62, 75, 66, 64, 69, 71, 60, 68, 77, 66, 76, 69, 70, 71, 73, 72, 65, 70, and 63). Figure 4B shows the alignment of the light chain variable region (sequence numbers from top to bottom are 98, 99, 88, 88, 88, 88, 88, 88, 88, 93, 93, 94, 95, 88, 95, 88, 94, 95, 88, 94, 95, and 88).

[0070] [Figure 5] Figure 5 shows the randomized locations of 26D5-295-B08 mAb cells in the mutation scan. The Kabat numbering indicates each randomized location in the mutation scan. Figure 5 shows sequence numbers 210 to 215 from top to bottom.

[0071] [Figure 6] Figures 6A-F show heatmap scans representing the locations of mutations in the CDR of the antibody binding site and the adjacent framework region of the 26D5-295-B08 mAb (see Figure 3A for details). Figure 6A shows LCDR1 (SEQ ID NO: 210), Figure 6B shows LCDR2 (SEQ ID NO: 211), Figure 6C shows LCDR3 (SEQ ID NO: 212), Figure 6D shows HCDR1 (SEQ ID NO: 213), Figure 6E shows HCDR2 (SEQ ID NO: 214), and Figure 6F shows HCDR3 (SEQ ID NO: 215).

[0072] [Figure 6] Figures 7A and 7B show the randomized amino acid positions of 26D5-295-B08 mAb to create a library of the complex (Figure 7A: Library of a portion of the complex (sequence numbers 216-219 from top to bottom, respectively); Figure 7B: Doped library of the complex (sequence number 215)).

[0073] [Figure 8] Figures 8A and 8B show the alignment of the progenitor amino acid sequences of 26D5-295-B08 mAb after affinity maturation. Kabat was used for CDR definition and numbering. Figure 8A shows the alignment of the heavy chain variable region (sequence numbers from top to bottom are 83, 82, 60, 54, 52, 54, 60, 53, 61, 56, 59, 57, 57, 58, and 55). Figure 8B shows the alignment of the light chain variable region (sequence numbers from top to bottom are 98, 99, 88, 86, 84, 87, 91, 85, 89, 89, 88, 92, 90, 88, and 88).

[0074] [Figure 9] Figure 9 shows the results of a chromogenic enzyme assay to determine the amount of antibody required to restore the activity of compound A by more than 50%. The concentrations of factor XIa substrate and compound A were kept constant, and the activity of factor XIa enzyme (y-axis) was plotted as a function of the concentration of a representative affinity-mature mAb (x-axis). The upper graph shows data obtained using the parent 26D5 mAb IgG1f (sequence numbers 83 and 98; also referred to herein as P1-072224) (indicated as "26D5 mAb" in the figure). The lower graph shows data obtained using the 26D5-296-G07 mAb IgG1f (sequence numbers 65 and 94; also referred to herein as P1-073056) (indicated as "26D5-296-G07 mAb" in the figure).

[0075] [Figure 10] Figure 10 shows surface plasmon resonance (SPR) graphs representing the binding affinity of the parent compound 26D5 mAb (P1-072224) to compound A at various concentrations (indicated as "26D5" in the figure).

[0076] [Figure 11] Figure 11 shows graphs of surface plasmon resonance (SPR) representing the binding affinity of mAb 26D5-GV-Q (top), mAb 26D5-295-B08 (middle), and Fab fragment antibody 26D5-295-B08 (bottom) to compound 2, measured at various mAb / Fab concentrations.

[0077] [Figure 12] Figure 12 shows graphs of surface plasmon resonance (SPR) representing the binding affinity of antibody tandem Fab (TanFab) fragment 26D5-75616-348-F10-TanFab (top) and antibody tandem Fab (TanFab) fragment 26D5-75214-343-F06-TanFab (bottom) to compound 5, measured at various TanFab concentrations.

[0078] [Figure 13] Figure 13 shows the time-resolved fluorescence-to-fluorescence resonance energy transitions (TR-FRETs) obtained from competing data for compound 5 in the following forms: (A) 26D5-GVR-Q-FT-Fab-LONG, (B) 26D5-295-B08-Fab-LONG, and (C) 26D5-75747-348-D07-Fab-LONG.

[0079] [Figure 14] Figure 14 shows the time-resolved fluorescence-to-fluorescence resonance energy transitions (TR-FRETs) obtained from the competitive data of the described antibody and Fab fragment antibody against compound 5.

[0080] [Figure 15] Figure 15 shows the DSC thermogram of each Fab fragment antibody.

[0081] [Figure 16] Figure 16 shows the structure of the Fab fragment antibody 26D5-GVR-Q-FTFab bound to compound A, as determined by crystal structure analysis.

[0082] [Figure 17] Figure 17 shows the structure of the Fab fragment antibody 26D5-75616-348-F10-Fab-SHORT bound to compound A, as determined by crystal structure analysis.

[0083] [Figure 18] Figure 18 shows the restoration of the anticoagulant effect of compound A by the neutralizing Fab fragment antibody 26D5-75616-348-F10-Fab-SHORT. Human plasma clotting time (aPTT) was plotted as a function of the plasma concentration of compound A and the plasma concentration of the neutralizing Fab fragment antibody 26D5-75616-348-F10-Fab-SHORT.

[0084] [Figure 19] Figure 19 shows that human plasma clotting time (aPTT) is a function of the plasma concentration of unbound compound A, regardless of the presence or absence of the Fab fragment antibody 26D5-75616-348-F10-Fab-SHORT. Human plasma clotting time (aPTT) is plotted as a function of the plasma concentration of compound A in the absence of the neutralizing Fab fragment antibody 26D5-75616-348-F10-Fab-SHORT (black circles) and in the presence of the antibody (white circles).

[0085] [Figure 20] Figure 20 shows the restoration of the anticoagulant effect of compound A by the neutralizing Fab fragment antibody 26D5-75616-348-F10-Fab-SHORT. Rabbits were intravenously administered compound A (1 mg / kg), and 20 minutes later, Fab fragment antibody 26D5-75616-348-F10-Fab-SHORT (160 mg / kg) was administered intravenously. Plasma clotting time (aPTT) was measured immediately before administration of compound A and approximately 24 hours later as reference.

[0086] [Figure 21] Figure 21 shows the pharmacokinetics (plasma concentrations in rabbits) of Fab fragment antibody 26D5-75616-348-F10-Fab-SHORT, compound A, and free compound A after intravenous administration of compound A (1 mg / kg) and intravenous administration of Fab fragment antibody 26D5-75616-348-F10-Fab-SHORT (160 mg / kg) 20 minutes later.

[0087] [Figure 22] Figure 22 shows the pharmacokinetics (plasma concentrations in rats) of the Fab fragment antibody 26D5-75616-348-F10-Fab-SHORT and the tandem Fab fragment antibody 26D5-75616-348-F10-TanFab after intravenous administration of 10 mg / kg over 10 minutes by drip infusion. TANDEM FAB(○) indicates data for the tandem Fab fragment antibody 26D5-75616-348-F10-TanFab. FAB(△) indicates data for the Fab fragment antibody 26D5-75616-348-F10-Fab-SHORT.

[0088] [Figure 23] Figure 23 shows the pharmacokinetics (plasma concentrations in rabbits) of tandem Fab fragment antibody 26D5-75616-348-F10-TanFab, compound A, and free compound A after intravenous administration of compound A (0.4 mg / kg) by drip infusion over 10 minutes, followed 20 minutes later by intravenous administration of tandem Fab fragment antibody 26D5-75616-348-F10-TanFab by drip infusion over 10 minutes. TANDEM Fab (○) indicates data for tandem Fab fragment antibody 26D5-75616-348-F10-TanFab.

[0089] [Figure 24] Figure 24 shows the restoration of the anticoagulant effect of compound A by the neutralizing tandem Fab fragment antibody 26D5-75616-348-F10-TanFab. Human plasma clotting time (aPTT) is plotted as a function of the plasma concentration of compound A and the plasma concentration of the neutralizing tandem Fab fragment antibody 26D5-75616-348-F10-TanFab. "Tandem Fab" refers to the tandem Fab fragment antibody 26D5-75616-348-F10-TanFab. [Modes for carrying out the invention]

[0090] The present invention provides novel antibodies or antigen-binding peptides that bind to selective FXIa inhibitors and / or FXIa / plasma kallikrein biinhibitors. As used herein, FXIa inhibitors are compounds represented by formula (I) that can inhibit the activity or function of FXIa. Accordingly, in some embodiments, the antigen-binding peptides disclosed herein (e.g., antibodies or fragment antibodies, but not limited to the following) specifically bind to the compound represented by formula (I) or its stereoisomers or tautomers. In some embodiments, R of formula (I) 1 is C 1-4 It is alkyl; R of formula (I) 2 R is independently selected from F, Cl, CF3, CHF2, CH2F, CH3; R in formula (I) 3 R is independently selected from CF3, CHF2, CH2F, and CH3; R in equation (I) 4 is H; and R of equation (I) 5 is independently selected from F and Cl. In certain embodiments, the antigen-binding peptides disclosed herein (e.g., antibodies or fragment antibodies, but not limited to those described below) specifically bind to the compound represented by formula (II). The terms used herein, the compound of formula (I) or (II), include all compounds having formula (I) or (II), or their stereoisomers or tautomers. [ka]

[0091] In certain embodiments, the antigen that binds to the antigen-binding peptide of the present invention is the compound of formula (II) (also referred to herein as compound A and known as milvexian). Milvexian is a highly affinity and highly selective, directly acting, reversible small molecule therapeutic agent that binds to and inhibits human active coagulation factor XI (FXIa). The chemical name of milvexian is (5R,9S)-9-(4-(5-chloro-2-(4-chloro-1H-1,2,3-triazole-1-yl)phenyl)-6-oxopyrimidine-1(6H)-yl)-21-(difluoromethyl)-5-methyl-21H-3-aza-1(4,2)-pyridina-2(5,4)-pyrazolacyclononaphan-4-one. Milvexian and methods for producing milvexian are described in U.S. Patent No. 9,453,018, all of which are incorporated by reference.

[0092] As used herein, FXIa refers to an intrinsic serine protease involved in the control of blood coagulation. The structure and physiological function of FXIa are generally well known to those skilled in the art, and it is mainly synthesized in hepatocytes and circulates in the form of its enzyme precursor, FXI. FXI is then physiologically activated by FXIa and thrombin. See Mohammed B. et al. Thromb Res., 161:94-105 (2018), incorporated by reference.

[0093] As used herein, the term "antigen-binding peptide" refers to a protein or polypeptide molecule that recognizes and specifically binds to a target molecule (i.e., an antigen). Examples of target molecules include, but are not limited to, small molecules, proteins, polypeptides, peptides, carbohydrates, polynucleotides, lipids, or any part or combination thereof.

[0094] In some embodiments, the antigen-binding peptide of the present invention is an antibody, or, but is not limited to, (Fab), Fab', F(ab')2, Fd, single-chain Fv or scFv, disulfide-bonded Fv, V-NAR domain, IgNar, intrabody, IgGACH2, minibody, F(ab')3, tetrabody, triabody, bispecific antibody, single-domain antibody, DVD-Ig, Fcab, mAb 2 These are fragment antibodies such as (scFv)2, scFv-Fc, or tandem Fab.

[0095] In some embodiments, the antigen-binding peptides of the present invention (e.g., antibodies or Fab fragments) may be isolated. As used herein, “isolated” means that a nucleic acid, peptide, or protein has been isolated from, for example, the original environment in which a cell or organism produced them, or from a naturally occurring liquid. In the case of a peptide or protein having a novel non-natural amino acid sequence, an “isolated” peptide or protein means that the protein or peptide has been isolated from the modified cell that produces it. A peptide or protein is considered isolated for the purposes of the present invention even if the peptide or protein is a component of a mixture or composition such as a pharmaceutical formulation, as long as it is not in the cell or original environment in which the peptide or protein is produced.

[0096] In one specific embodiment of the present invention, Fab is used as an antigen-binding peptide. The terms "Fab" or "Fab fragment antibody" as used herein are well known terms and refer to the antigen-binding region of a full-length antibody. In some embodiments, the Fab fragment antibody consists of at least a full-length light chain and the N-terminal portion of a heavy chain. As used herein, the full-length light chain includes at least a light chain constant region (CL) and a light chain variable region (VL); and the N-terminal portion of the heavy chain includes at least the CH1 domain of the heavy chain constant region and the heavy chain variable region (VH).

[0097] In one specific embodiment of the present invention, the tandem Fab is used as an antigen-binding peptide. The tandem Fab disclosed herein comprises at least one N-terminal portion of a heavy chain (VH-CH1) and at least one full-length light chain (VL-CL). In some embodiments, the tandem Fab disclosed herein comprises two or more N-terminal portions of heavy chains linked via a linker (e.g., VH-CH1-linker-CH1-VH or VH-CH1-linker-VH-CH1), each paired with a full-length light chain (VL-CL). In some embodiments, the tandem Fab disclosed herein comprises two N-terminal portions of heavy chains linked via a linker (e.g., VH-CH1-linker-CH1-VH or VH-CH1-linker-VH-CH1), each paired with a full-length light chain (VL-CL). In some embodiments, the linker is a polypeptide linker. Examples of the tandem Fab are disclosed in Table 4 of this application. In this specification, the terms "Tandem Fab," "Tandem Fab fragment antibody," and "TanFab fragment antibody" are used synonymously.

[0098] The "variable region" of an antibody is a well-known technical term that contributes to the antibody's specificity and refers to the end of the light or heavy chain to which the antigen binds. The terms "heavy chain variable region," "variable heavy chain," and "VH" are used synonymously and refer to the end of the heavy chain to which the antigen binds, contributing to the antibody's specificity. Similarly, the terms "light chain variable region," "variable light chain," and "VL" are used synonymously and refer to the end of the light chain to which the antigen binds, contributing to the antibody's specificity.

[0099] The variable regions of the heavy and light chains generally consist of four framework regions (FRs) linked by three complementarity-determining regions (CDRs), also known as hypervariable regions. The CDRs of each chain are close together by the FRs and, together with the CDRs of other chains, contribute to the formation of the antibody's antigen-binding domain. Methods for determining CDRs are generally known to those skilled in the art. For example, there are at least two methods for determining CDRs: (1) a method based on the diversity of interspecies sequences; and (2) a method based on the crystal structure analysis of the antigen-antibody complex. Furthermore, those skilled in the art may use a combination of these two methods to determine CDRs. The CDRs of each chain are numbered CDR1, CDR2, and CDR3 from the amino terminus to the carboxyl terminus.

[0100] The "constant region" of an antibody is a term well known to those skilled in the art, referring to the portion of the antibody whose amino acid sequence remains relatively constant even among different antibody molecules. Generally, the heavy chain constant region consists of three distinct regions called CH1, CH2, and CH3, numbered from the amino terminus (N-terminus) to the carboxyl terminus (C-terminus). A typical light chain has only one constant region, called CL. The constant region of an antibody determines its respective effector function. Those skilled in the art will readily understand the terminology and structural characteristics of the constant region of an antibody.

[0101] In some embodiments, the antigen-binding peptide includes any modified polypeptide molecule having at least one antigen recognition site, provided that the modified polypeptide molecule exhibits the desired antigen-binding activity. The antigen-binding peptide disclosed herein may or may not be bound to other molecules (e.g., toxins, radioisotopes, fluorescent labels, etc.).

[0102] As used herein, the term "antibody" is a well-known technical term referring to an immunoglobulin molecule that recognizes and specifically binds to a target molecule via at least one antigen recognition site in at least one portion of the variable region of the immunoglobulin molecule. The structure of antibodies is generally known to those skilled in the art and often consists of at least two full-length heavy chains. With the exception of well-known examples such as camelid antibodies, the vast majority of antibodies consist of at least two full-length heavy chains and at least two full-length light chains. Antibodies as used herein include polyclonal antibodies, monoclonal antibodies (also referred to herein as "mAb"), multispecific antibodies (e.g., bispecific antibodies made from at least two antibodies), chimeric antibodies, humanized antibodies, human antibodies, and non-human antibodies. The term "antibody" as used herein refers to one of the five main classes of immunoglobulins (IgA, IgD, IgE, IgG, and IgM, or their subclasses (isotypes, e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2)), based on the type of constant region of the heavy chain, which is designated as α, δ, ε, γ, and μ, respectively. It is well known that different classes of immunoglobulins have different subunit structures and three-dimensional structures.

[0103] The Kabat numbering system is generally used to refer to residues in the heavy chain variable region or light chain variable region (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain) (Kabat et al., Sequences of Proteins of Immunological Interest, 5 th See Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991). The residues in the CDR that interact with the antigen can also be determined by crystal structure analysis of the antigen-antibody complex.

[0104] The terms “polypeptide,” “peptide,” and “protein” as used herein are synonymous and refer to polymers of amino acids of any length. Amino acid polymers may be linear or branched and may contain modified amino acids. They may also be blocked by non-amino acids. The term also includes naturally or artificially modified amino acid polymers. In some embodiments, amino acid polymers are modified by disulfide bond formation, glycosylation, lipid addition, acetylation, phosphorylation, or any other treatment or modification. In some embodiments, amino acid polymers are modified by binding with a labeling component. This definition also includes peptides having one or more analogues of amino acids and non-natural amino acids known to those skilled in the art.

[0105] "Specific binding" (or "specific binding") is a term well known to those skilled in the art and generally means that the antigen-binding site of an antigen-binding peptide (e.g., an antibody or fragment antibody, but not limited to the following) specifically recognizes the antigen via its antigen-binding domain, and that the binding has at least some complementarity between the antigen-binding domain and the antigen. As described in this definition, antigen-binding peptides (e.g., an antibody or fragment antibody, but not limited to the following) are said to "specifically bind" to the epitope of an antigen via their antigen-binding domain and bind more easily than any unrelated antigen.

[0106] In some embodiments, the antibody or fragment antibody of the present invention comprises a combination of VH and VL CDR sequences as listed in Table 1. In some embodiments, the antibody or fragment antibody of the present invention disclosed herein comprises VH and VL having CDRs that specifically bind to the FXIa inhibitor of the present disclosure (for example, an FXIa inhibitor of formula (I)) and undergo up to four (i.e., 0, 1, 2, 3, or 4) conservative amino acid substitutions independently of the corresponding CDRs listed in Table 1. Table 1: Amino acid sequences of variable heavy chain (VH) and light chain (VL) CDRs [Table 1] [Table 2] [Table 3] [Table 4] [Table 5]

[0107] In some embodiments, the antibody or fragment antibody of the present invention includes a combination of VH and VL amino acid sequences listed in Table 2. In some embodiments, the antibody or fragment antibody of the present invention includes a combination of a partial heavy chain amino acid sequence and a full-length light chain amino acid sequence listed in Table 3. In some embodiments, the antibody or fragment antibody of the present invention includes a combination of a tandem partial heavy chain amino acid sequence and a full-length light chain amino acid sequence listed in Table 4.

[0108] In some embodiments, the antibody or fragment antibody of the present invention comprises one or more independent variable light chains or variable heavy chains as described herein. In some embodiments, the antibody or fragment antibody of the present invention comprises both variable light chains and variable heavy chains as described herein. In some embodiments, the antibody or fragment antibody of the present invention comprises one variable heavy chain paired with one variable light chain as described herein. In some embodiments, the antibody or fragment antibody of the present invention comprises one or more variable heavy chains, each paired with one variable light chain as described herein. In some embodiments, the antibody or fragment antibody of the present invention comprises two variable heavy chains, each paired with one variable light chain as described herein.

[0109] The present invention also includes antibodies or fragment antibodies having VH sequences and VL sequences that are at least about 80%, 85%, 89%, 90%, 95%, or 99% identical to the VH sequences and VL sequences disclosed in Table 2 of this specification. Table 2: Variable heavy and light chain amino acid sequences (the underlined parts are CDRs, and the non-underlined parts are framework regions.) [Table 6] [Table 7] [Table 8] [Table 9] [Table 10] [Table 11]

[0110] The present invention also includes antibodies or fragment antibodies having partial heavy chain amino acid sequences and full-length light chain amino acid sequences that are at least about 80%, 85%, 89%, 90%, 95%, or 99% identical to the partial heavy chain amino acid sequences and full-length light chain amino acid sequences disclosed in Table 3. The present invention also includes antibodies or fragment antibodies (e.g., Fab fragment antibodies) that include or are composed of any combination of one of the N-terminal portions of the heavy chains in Table 3 and one of the full-length light chains in Table 3. Furthermore, the present invention includes antibodies or fragment antibodies (e.g., Fab fragment antibodies) that include or are composed of any pair of the N-terminal portions of the heavy chains and full-length light chains shown in Table 3. The present invention also includes antibodies or fragment antibodies (e.g., Fab fragment antibodies) that have or are composed of the sequences of SEQ ID NO: 106 and SEQ ID NO: 164. The present invention also includes antibodies or fragment antibodies (e.g., Fab fragment antibodies) that have the sequences of SEQ ID NO: 106 and SEQ ID NO: 164. The present invention also includes antibodies or fragment antibodies (e.g., Fab fragment antibodies) that essentially have the sequences of SEQ ID NO: 106 and SEQ ID NO: 164. The present invention also includes antibodies or fragment antibodies (e.g., Fab fragment antibodies) composed of the sequences of SEQ ID NO: 106 and SEQ ID NO: 164. Table 3: Fab sequences (non-bold portions are variable regions of the CDR (underlined) and framework region (ununderlined); bold amino acids are constant regions of the Fab regions of the heavy and light chains, respectively) [Table 12] [Table 13] [Table 14] [Table 15] [Table 16] Table 17 Table 18 Table 19 Table 20 Table 21 Table 22 Table 23 Table 24 Table 25 Table 26 Table 27 Table 28 Table 29 Table 30 Table 31

[0111] The present invention also includes antibodies or fragment antibodies having tandem partial heavy chain amino acid sequences and full-length light chain amino acid sequences that are at least about 80%, 85%, 89%, 90%, 95%, or 99% identical to the partial heavy chain amino acid sequences and full-length light chain amino acid sequences disclosed in Table 4. Table 4: Tandem Fab sequence (The non-bold portion is the variable region of the CDR (underlined) and framework region (ununderlined); the bold amino acids are the constant regions of the heavy and light chains of the Fab region, respectively; the double-lined portion is the linker sequence) [Table 32] [Table 33] [Table 34] [Table 35] [Table 36] [Table 37] [Table 38] [Table 39] [Table 40] [Table 41] [Table 42] [Table 43]

[0112] The term "identity" itself has a recognized meaning in the relevant technical field and can be calculated using published techniques (see, for example, COMPUTATIONAL MOLECULAR BIOLOGY, Lesk, AM, ed., Oxford University Press, New York, (1988); BIOCOMPUTING: INFORMATICS AND GENOME PROJECTS, Smith, DW, ed., Academic Press, New York, (1993); COMPUTER ANALYSIS OF SEQUENCE DATA, PART I, Griffin, AM, and Griffin, HG, eds., Humana Press, New Jersey, (1994); SEQUENCE ANALYSIS IN MOLECULAR BIOLOGY, von Heinje, G., Academic Press, (1987); and SEQUENCE ANALYSIS PRIMER, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, (1991)). Numerous methods exist for determining the identity between two polynucleotide sequences or two polypeptide sequences, and the term "identity" is well known to those skilled in the art (Carillo, H., and Lipton, D., SIAM J. Applied Math. 48:1073 (1988)). Methods commonly used to determine the identity or similarity between two sequences include, but are not limited to, those disclosed in “Guide to Huge Computers” edited by Martin J. Bishop (Academic Press, San Diego, (1994)) or in SIAM J. Applied Math. 48:1073 (1988) by Carillo, H. and Lipton, D.Methods for arranging polynucleotides or polypeptides consist of computer programs including the GCG program package (Devereux, J., et al., Nucleic Acids Research 12(1):387 (1984)), BLASTP, BLASTN, FASTA (Atschul, SF et al., J. Mol. Biol. 215:403 (1990)), and the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, Wis. 53711 (using Smith-Waterman's local homology algorithm (Advances in Applied Mathematics 2:482 489 (1981))).

[0113] For example, the statement that a polynucleotide is at least 95% "identical" to a nucleotide reference sequence means that the nucleotide sequence of the polynucleotide is identical to the reference sequence, except that the polynucleotide sequence may contain up to five mutations per 100 nucleotides compared to the nucleotide reference sequence. For example, to obtain a polynucleotide having a nucleotide sequence that is at least 95% identical to the nucleotide reference sequence, up to 5% of the nucleotides in the reference sequence may be deleted or replaced with other nucleotides. Alternatively, up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence.

[0114] In practice, whether any particular nucleic acid molecule is at least 80%, 85%, 89%, 90%, 95%, or 99% identical to the nucleotide sequence of the present invention can be determined using a known computer program. A method for determining the best overall match between a query sequence and a reference sequence, also known as global alignment, can be determined using the FASTDB computer program (Comp. App. Biosci. 6:237-245 (1990)), based on the algorithm of Brutlag et al. In conventional nucleotide sequence alignment, both the query sequence and the reference sequence are DNA sequences. However, comparison with RNA sequences is also possible by converting U to T. The result of global alignment is expressed as identity (%). In one embodiment of the present invention, the parameters used in FASTDB alignment of DNA sequences to calculate identity (%) are Matrix=Unitary, k-tuple=4, Mismatch Penalty=1, Joining Penalty=30, Randomization Group Length=0, Cutoff Score=1, Gap Penalty=5, Gap Size Penalty=0.05, and Window Size=500 (or the shorter of the lengths of the target nucleotide sequences).

[0115] If the reference sequence is shorter than the query sequence due to a deletion at the 5' or 3' end, rather than an internal sequence deletion, manual correction is essential to obtain results. This is because the FASTDB program does not take into account deletions at the 5' and 3' ends of the reference sequence when calculating identity (%). For reference sequences truncated at the 5' or 3' end relative to the query sequence, the identity (%) is corrected by calculating the number of bases at the 5' and 3' ends of the reference sequence that do not match / cannot be aligned with the query sequence as a percentage of the total bases of the query sequence. Then, this percentage (%) is subtracted from the identity (%) calculated by a FASTDB program using specific parameters to obtain the final identity (%) score. This corrected score is used for the purposes of this invention. Only the bases other than those at the 5' and 3' ends of the reference sequence that do not match / cannot be aligned with the query sequence, as displayed by FASTDB alignment, are calculated to manually adjust the identity (%) score.

[0116] For example, a 90-base reference sequence is aligned with a 100-base query sequence to determine identity (%). If a deletion occurs at the 5' end of the reference sequence, the FASTDB alignment will not show a match / match in the first 10 bases of the 5' end. Since these 10 unpaired bases represent 10% of the sequence (number of unmatched bases at the 5' and 3' ends / total number of bases in the query sequence), 10% is subtracted from the identity (%) score calculated by the FASTDB program. If the remaining 90 bases are a perfect match, the final identity (%) will be 90%. Another example of comparing a 100-base query sequence with a 90-base reference sequence is when the deletion is an internal sequence deletion; in this case, there are no unmatched / unalignable bases in the reference sequence at the 5' or 3' end. In this case, the identity (%) calculated by FASTDB is not manually corrected. Again, only the unmatched / unalignable bases at the 5' and 3' ends of the reference sequence are manually corrected.

[0117] For example, a polypeptide having an amino acid sequence that is at least 95% "identical" to the amino acid query sequence of the present invention means that the amino acid sequence of the polypeptide is identical to the query sequence, except that the target polypeptide sequence may contain up to five amino acid changes for every 100 amino acids of the query sequence. In other words, to obtain a polypeptide having an amino acid sequence that is at least 95% identical to the amino acid query sequence, up to 5% of the amino acid residues in the reference sequence may be inserted, deleted, or substituted with other amino acids. These changes to the reference sequence may occur at the amino terminus, carboxyl terminus, or anywhere between these terms of the reference amino acid sequence, individually or interspersed across one or more contiguous segments.

[0118] In practice, whether any particular polypeptide is at least 80%, 85%, 89%, 90%, 95%, or 99% identical to, for example, any of the amino acid sequences shown in Tables 1-4 can conventionally be determined using known computer programs. A preferred method for determining the best overall match between a query sequence (the sequence of the present invention) and a reference sequence, also known as global alignment, can be determined using the FASTDB computer program described above. In sequence alignment, both the query sequence and the reference sequence are amino acid sequences. The result of the global alignment is expressed as identity (%). In one embodiment of the present invention, the parameters used in FASTDB alignment of amino acid sequences to calculate identity (%) are Matrix=PAM 0, k-tuple=2, Mismatch Penalty=1, Joining Penalty=20, Randomization Group Length=0, Cutoff Score=1, Window Size=sequence length, Gap Penalty=5, Gap Size Penalty=0.05, Window Size=500 (or the shorter of the lengths of the amino acid sequences in question).

[0119] If the reference sequence is shorter than the query sequence due to an N-terminus or C-terminus deletion rather than an internal sequence deletion, manual correction is essential to obtain results. This is because the FASTDB program does not take into account N- and C-terminus deletions of the reference sequence when calculating identity (%). For reference sequences truncated at the N-terminus or C-terminus, identity (%) is corrected by calculating the number of N- and C-terminus residues of the reference sequence that do not match / cannot be aligned with the corresponding query sequence residues as a percentage of the total bases of the query sequence. Whether the residues match / align is determined by the results of FASTDB's sequence alignment. This percentage is then subtracted from the identity (%) calculated by the FASTDB program using specific parameters to obtain the final identity (%) score. This final identity (%) score is used for the purposes of this invention. Only the N- and C-terminus bases of the reference sequence that do not match / cannot be aligned with the query sequence are considered for manual adjustment of the identity (%) score. In other words, the residues in the query sequence are located in the reference sequence other than the furthest N-terminal and C-terminal residues.

[0120] For example, a reference sequence of 90 amino acid residues is aligned with a query sequence of 100 residues to determine identity (%). If a deletion occurs at the N-terminus of the reference sequence, the FASTDB alignment will not show a match / match for the first 10 residues of the N-terminus. Since these 10 unpaired residues represent 10% of the sequence (number of unmatched residues at the N-terminus and C-terminus / total number of residues in the query sequence), 10% is subtracted from the identity (%) score calculated by the FASTDB program. If the remaining 90 residues match perfectly, the final identity (%) will be 90%. Another example of comparing a 100-residue query sequence with a 90-residue reference sequence is when the deletion is an internal sequence deletion; in this case, there are no unmatched / unalignable residues in the reference sequence at the N-terminus or C-terminus. In this case, the identity (%) calculated by FASTDB is not manually corrected. Again, only the N- and C-terminal residues of the reference sequence that do not match / unalign with the query sequence are manually corrected.

[0121] Within the scope of the disclosed identity (%), the present invention also relates to substitutional variants of the disclosed polypeptides of the present invention. Substitutional variants include polypeptides in which one or more amino acid residues are deleted and substituted by other residues. In some embodiments, the identity (%) disclosed above relates to the entire sequence of a particular identified sequence, where amino acid residues that do not undergo substitution and amino acid residues that are not subject to substitution constitute the CDR of the sequence, and amino acid residues that are subject to substitution constitute the framework region of the sequence. For example, in a particular embodiment, if the antigen-binding peptide of the present invention (e.g., an antibody or fragment antibody, but not limited to the following) contains at least one VH having an amino acid sequence that is at least about 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence of SEQ ID NO: 64, then the CDR region of the VH is constant, and the framework region may vary. However, the overall identity (%) with SEQ ID NO: 64 is within the scope of the embodiments. Mutations are substitutions of a conserved nature, but in some embodiments of the present invention, non-conservative substitutions are also included. Conservative substitutions in the present invention can be defined as shown in Tables 5-7 below. Amino acids are classified according to their physical properties and their contribution to the secondary and tertiary structures of proteins. Conservative substitutions are recognized by those skilled in the art as the substitution of one amino acid with another amino acid having similar properties. Examples of conservative substitutions are given below. [Table 44] Alternatively, amino acid conservation may be grouped as shown below, as described in Lehninger (1975) Biochemistry, Second Edition; Worth Publishers, pp. 71-77. [Table 45]

[0122] Another example of a conservative substitution is shown below. [Table 46]

[0123] In some embodiments of the antibody or fragment antibody of the present invention, the CH1 domain includes a partial heavy chain constant region having the amino acid sequence ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC (SEQ ID NO: 202). The present invention also includes an antibody or fragment antibody having a CH1 domain with an amino acid sequence that is at least about 80%, 85%, 89%, 90%, 95%, or 99% identical to the CH1 domain of SEQ ID NO: 202. In some embodiments of the antibody or fragment antibody of the present invention, the CH1 domain includes a partial heavy chain constant region having the amino acid sequence ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTH (SEQ ID NO: 203). Furthermore, the present invention also includes antibodies or fragment antibodies having a CH1 domain whose amino acid sequence is at least about 80%, 85%, 89%, 90%, 95%, or 99% identical to the CH1 domain of SEQ ID NO: 203. In some embodiments of the antibodies or fragment antibodies of the present invention, the CL domain includes a light chain constant region having the amino acid sequence RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 204). Furthermore, the present invention also includes antibodies or fragment antibodies having a CL domain whose amino acid sequence is at least about 80%, 85%, 89%, 90%, 95%, or 99% identical to the CH1 domain of SEQ ID NO: 204.

[0124] In some embodiments, the antibody or fragment antibody of the present invention comprises one or more heavy chain N-terminal portions and full-length light chains as described herein. In some embodiments, the antigen-binding peptide (for example, an antibody or fragment antibody, but not limited to the following) comprises both the heavy chain N-terminal portion and the full-length light chain sequence as described herein. In some embodiments, the antibody or fragment antibody of the present invention comprises one heavy chain N-terminal portion paired with one full-length light chain as described herein. In some embodiments, the antibody or fragment antibody of the present invention comprises one or more heavy chain N-terminal portions, each paired with one full-length light chain as described herein. In some embodiments, the antibody or fragment antibody of the present invention comprises two heavy chain N-terminal portions, each paired with one full-length light chain as described herein. In certain embodiments, the two heavy chain N-terminal portions are linked via a linker.

[0125] Table 4 shows the heavy and light chain sequences of examples of the tandem Fab of the present invention. In some embodiments, the heavy chain of the tandem Fab contains the N-terminal portions of the heavy chains of one or two antibodies, which are linked via a linker, and the light chain of the tandem Fab contains the full-length light chain (VL-CL) of the antibody. In some embodiments, the heavy chain of the tandem Fab may be represented as "VH-CH1-linker-CH1-VH" or "VH-CH1-linker-VH-CH1".

[0126] The linker included in the present invention may be any suitable molecule of various structures. In certain embodiments, the linker is a polypeptide linker. Polypeptide linkers may be of various lengths. In some embodiments, the linker is a polypeptide having about 20 or fewer amino acids. Examples of polypeptide linker sequences are disclosed in the double-underlined portions of Tables 8 and 4. [Table 47] [Table 48]

[0127] In some embodiments, the present invention provides an antigen-binding peptide having the amino acid sequence of SEQ ID NO: 201.

[0128] In some embodiments, the present invention provides an antigen-binding peptide (for example, an antibody or fragment antibody, but not limited to the following) comprising at least one VH and at least one VL. In some embodiments, the at least one VH comprises a VH complementarity-determining region 1 (VH-CDR1) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 12; a VH-CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 22; or a VH-CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 23 to 28. In some embodiments, the at least one VL comprises at least one of the following: a VL-CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 29 to 37; a VL-CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 38 to 43; or a VL-CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 44 to 51. In some embodiments, the antigen-binding peptides of the present invention (e.g., antibodies or fragment antibodies, but not limited to those listed below) include VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3 having 1, 2, 3, or 4 conservative substitutions.

[0129] In some embodiments, the antigen-binding peptide (e.g., an antibody or fragment antibody, but not limited to the following) includes at least one VH having an amino acid sequence that is at least about 80%, 85%, 90%, 95%, or 99% identical to a sequence selected from the group consisting of SEQ ID NOs. 52 to 83; and at least one VL having an amino acid sequence that is at least about 80%, 85%, 90%, 95%, or 99% identical to a sequence selected from the group consisting of SEQ ID NOs. 84 to 99.

[0130] In some embodiments, the variant sequences disclosed in Table 2 of this specification also include at least one VH region and at least one VL region having 1, 2, 3, or 4 conservative substitutions.

[0131] In some embodiments, the tandem Fab of the present invention has sequences that are at least about 80%, 85%, 90%, 95%, and 99% identical to the sequences in Table 4.

[0132] Furthermore, the present invention includes a polynucleotide having a nucleic acid sequence that partially or completely encodes an antigen-binding peptide disclosed herein (for example, an antibody or fragment antibody, but not limited to the following).

[0133] In some embodiments, the polynucleotide has a nucleic acid sequence encoding one of the CDR sequences listed in Table 1. In some embodiments, the polynucleotide has a nucleic acid sequence encoding one of the VH or VL sequences listed in Table 2. In some embodiments, the polynucleotide has a nucleic acid sequence encoding one of the N-terminal portions of either the heavy chain or the full-length light chain listed in Table 3. In some embodiments, the polynucleotide has a nucleic acid sequence encoding one of the tandem Fab heavy chain and light chain sequences listed in Table 4.

[0134] The present invention also includes polynucleotides having a sequence identical to any polynucleotide of the present disclosure by at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%. Furthermore, the present invention also provides polynucleotide variants encoding fragments, analogs, and derivatives of antigen-binding peptides of the present disclosure (e.g., antibodies or fragment antibodies, but not limited to the following). Polynucleotide variants may include mutations in the coding region, non-coding region, or both. In some embodiments, polynucleotide variants include mutations that result in deexpressive mutations, insertions, or deletions, but do not alter the properties or activity of the encoded polypeptide. In some embodiments, polynucleotide variants are obtained by deexpressive mutations due to genetic code degeneracy. Polynucleotide variants may be obtained for a variety of reasons, such as optimizing codon expression for a particular host.

[0135] In certain embodiments, the polynucleotide of the present invention has a coding sequence of a mature polypeptide linked in the same reading frame as a polynucleotide encoding a polypeptide that assists in the expression and secretion of polypeptides from a host cell, for example. In some embodiments, the mature polypeptide is an antigen-binding peptide of the present disclosure (e.g., an antibody or fragment antibody, but not limited to the following). In certain embodiments, the polynucleotide has a sequence encoding a signal polypeptide sequence that functions as a secretion sequence for controlling the transport of polypeptides from cells. The polypeptide having the signal sequence is a precursor protein, and the signal sequence can be degraded by the host cell to form a mature polypeptide. The polynucleotide may also encode a precursor protein obtained by adding a 5' amino acid residue to the mature protein. The mature polypeptide having the pro sequence is a precursor protein and is an inactive protein. Once the pro sequence is degraded, an active mature protein remains. In certain embodiments, the polynucleotide has a coding sequence of a mature polypeptide linked in the same reading frame as a marker sequence that can, for example, purify the encoded polypeptide.

[0136] In some embodiments, the present invention provides vectors comprising any one of the polynucleotides disclosed herein. As used herein, the term “vector” means a transportable construct capable of expressing, as appropriate, one or more target polynucleotides, proteins, or sequences in a host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or naked RNA expression vectors, plasmids, cosmid vectors or phage vectors, DNA or RNA expression vectors associated with cationic condensers, liposome-encapsulated DNA or RNA expression vectors, and certain eukaryotic cells (e.g., producing cells).

[0137] Furthermore, the present invention provides host cells containing the vectors disclosed herein. In some embodiments, the host cells are isolated cells. In some embodiments, the isolated host cells produce the antigen-binding peptides disclosed herein (e.g., antibodies or fragment antibodies, but not limited to those disclosed herein). Suitable host cells include prokaryotes, yeasts, insects, or higher eukaryotic cells. Prokaryotes include Gram-negative or Gram-positive bacteria (e.g., Escherichia coli or Bacillus). Higher eukaryotic cells include the established mammalian cell lines described below. Cell-free translation systems may also be used. Suitable cloning vectors and expression vectors for use as hosts for bacterial, fungal, yeast, and mammalian cells are generally known to those skilled in the art. Various mammalian or insect cell culture systems are also utilized to express recombinant proteins. Recombinant proteins are expressed in mammalian cells because such recombinant proteins are generally correctly folded, appropriately modified, and fully functional. Examples of suitable mammalian host cell lines include the monkey kidney cell lines COS-7, L cells, C127, 3T3, Chinese hamster ovary (CHO), HeLa, and BHK cell lines. Furthermore, baculoviruses that produce heterologous proteins in insect cells are generally known to those skilled in the art.

[0138] The antigen-binding peptides of the present invention (e.g., antibodies or fragment antibodies, but not limited to those listed below) obtained from a transmuted host can be purified by any suitable method. Standard methods include chromatography (e.g., ion-exchange chromatography, affinity chromatography, and size exclusion column chromatography), centrifugation, solubility differential, or any other standard technique used for protein purification. Affinity tags (e.g., hexahistidine (SEQ ID NO: 221), maltose-binding domain, influenza coat sequence, and glutathione-S-transferase) may be conjugated to the protein to facilitate purification by passing it through a suitable affinity column. The isolated proteins can be physically identified using techniques such as proteolysis, nuclear magnetic resonance, mass spectrometry, and X-ray crystallography. Methods for purifying antibodies and other proteins are generally known to those skilled in the art.

[0139] In certain embodiments, the present invention provides a method for producing antigen-binding peptides of the present disclosure (e.g., antibodies or fragment antibodies, but not limited to those described below). In the examples, the method is characterized by (a) culturing the host cells disclosed above under culture conditions that promote the production of proteins such as antigen-binding peptides (e.g., antibodies or fragment antibodies, but not limited to those described below); and (b) isolating the antigen-binding peptides (e.g., antibodies or fragment antibodies, but not limited to those described below) from the cultured cells. The antigen-binding peptides of the present invention (e.g., antibodies or fragment antibodies, but not limited to those described below) can be obtained using methods for producing antigen-binding peptides that are generally known to those skilled in the art.

[0140] In some embodiments, the antigen-binding peptides disclosed herein (e.g., antibodies or fragment antibodies, but not limited to these) are used as detection reagents. In some embodiments, the antigen-binding peptides (e.g., antibodies or fragment antibodies, but not limited to these) are labeled for detection. As used herein, “label” means a detectable compound that binds directly or indirectly to an antigen-binding peptide (e.g., antibodies or fragment antibodies, but not limited to these). The label may be detectable by itself (e.g., radioisotope labeling or fluorescent labeling), or, in the case of enzymatic labeling, it may catalyze a chemical change in a detectable substrate. In certain embodiments, the label is selected from the group consisting of immunofluorescence, chemiluminescence, phosphorescence, enzymatic labeling, isotope labeling, avidin / biotin, gold colloid, colored particles, and magnetic particles.

[0141] In some embodiments, an antigen-binding peptide (e.g., an antibody or fragment antibody, but not limited to the following) forms a complex with a compound of formula (I) or (II) in vitro or in vivo. In some embodiments, the complex is an immune complex. In general, detection of immune complex formation is well known to those skilled in the art and can be detected by various methods. In some embodiments, it is detected by immunological assays.

[0142] As used herein, an immunological assay refers to any assay that utilizes the specificity of antibody-antigen binding in vitro or in vivo. In some embodiments, the assay may be used to confirm the presence or absence of a target molecule in a biological sample. In some embodiments, the assay may be used to measure the amount or level of the target molecule. In some embodiments, the target molecule is an immunocomplex of an antigen-binding peptide (e.g., an antibody or fragment antibody, but not limited to the following) conjugated to a compound of formula (I) or (II) in vitro or in vivo. In some embodiments, the target molecule is the compound of formula (I) or (II) itself. In some embodiments, immunological assays include, but are not limited to, radioimmunoassay, immunohistochemical staining, chemiluminescence immunoassay (CLIA), enzyme immunoassay (EIA) or enzyme-linked immunosorbent assay (ELISA), Western blotting, counting immunoassay, flow cytometry, fluorescence immunoassay, and fluorescence-activated cell sorting (FACS).

[0143] The biological sample used herein may be any sample obtained from a patient. In some embodiments, the biological sample is urine, feces, saliva, whole blood, plasma, organ tissue, hair, skin, cells, or cell culture. In some embodiments, the biological sample is a liquid. In some embodiments, the biological sample may be fixed with a fixative. For example, aldehyde fixatives (e.g., formalin (formaldehyde) and glutaraldehyde) are commonly used.

[0144] Furthermore, the present invention provides a method for suppressing the antithrombotic effects of an FXIa inhibitor, or a dual inhibitor of FXIa and plasma kallikrein, in a subject where necessary. In some embodiments, the present invention relates to a method for suppressing the antithrombotic effects of an FXIa inhibitor. In some embodiments, the FXIa inhibitor is a compound of formula (I) or (II). In some embodiments, the method is characterized by administering a pharmaceutically effective amount of the antigen-binding peptide disclosed herein (e.g., an antibody or fragment antibody, but not limited to the following) to a subject. In some embodiments, the antigen-binding peptide (e.g., an antibody or fragment antibody, but not limited to the following) binds with high affinity to the compound of formula (I) or (II), and can suppress its antithrombotic effect in vitro or in vivo. In some embodiments, the binding of the antigen-binding peptide (e.g., an antibody or fragment antibody, but not limited to the following) to the compound of formula (I) or (II) can neutralize its antithrombotic effect in vitro or in vivo. In some embodiments, the antigen-binding peptide binds to an FXIa inhibitor (e.g., a compound of formula (I) or (II)), thereby preventing the FXIa inhibitor from binding to FXIa.

[0145] The term "subject" refers to any animal, including but not limited to humans and non-human primates. In some embodiments, the subject is the recipient of a particular treatment. In some embodiments, the subject is a human. In certain embodiments, the subject is a human patient in need of the treatment disclosed herein. The terms "subject" and "patient" are used synonymously herein.

[0146] For example, the terms “treatment” or “to treat” are used synonymously and refer to a treatment method that cures, delays, suppresses, reduces, recovers from or eliminates the effects of a disease, and / or halts the progression of a disease. As used herein, the term “treatment” is used to mean receiving at least one of the antigen-binding peptides of the present invention (e.g., antibodies or fragment antibodies, but not limited to those described below). The terms “prevent” or “avoid risk” are used to mean a preventive method that prevents the onset of a disease in question and / or delays its progression, or reduces the risk of developing abnormal symptoms compared to those who do not receive treatment. In other words, those who require treatment include those who already have the condition (e.g., thrombosis), those who are susceptible to developing the condition, and those who should be prevented from developing the condition.

[0147] The present invention also provides pharmaceutical compositions comprising the antigen-binding peptide of the present disclosure (e.g., antibodies or fragment antibodies, but not limited to those listed below). In some embodiments, the pharmaceutical compositions of the present invention comprise therapeutic and / or prophylactic compositions. In some embodiments, the pharmaceutical compositions comprise a therapeutically effective dose of the antigen-binding peptide (e.g., antibodies or fragment antibodies, but not limited to those listed below), and a pharmaceutically acceptable carrier or excipient. Such pharmaceutically acceptable excipients are generally known to those skilled in the art. Common excipients include, but are not limited to, preservatives, solubilizers, stabilizers, wetting agents, emulsifiers, disintegrants, fluidizers, lubricants, adsorbents, vehicles, sweeteners, flavorings, colorants, odorants, salts (the substance of the present invention itself may be used in the form of a pharmaceutically acceptable salt), buffers, coatings, and antioxidants. Examples of excipients include, but are not limited to, physiological saline, buffered physiological saline, dextrose, water, glycerol, ethanol, sucrose, sorbitol, and any combination thereof. In some embodiments, the pharmaceutically acceptable excipient is an inactive component. However, it is understood that some pharmaceutically acceptable excipients may affect the manufacture, quality, safety, or efficacy of the pharmaceutical composition. In some embodiments, the pharmaceutical composition may include a therapeutic activator in addition to the antigen-binding peptide of the present invention (e.g., an antibody or fragment antibody, but not limited to the following).

[0148] The pharmaceutical compositions of the present invention may be stored in a lyophilized state, contained in dose- or multi-dose containers (e.g., sealed ampoules and vials), requiring only the addition of a sterile liquid carrier for injection (e.g., water or saline solution) immediately before use. Injectable solutions and suspensions may be prepared immediately from sterile powders, granules, and tablets.

[0149] The pharmaceutical composition may be administered by a simple and appropriate method depending on its intended use. In some embodiments, the pharmaceutical composition may be administered via parenteral routes. In some embodiments, parenteral administration may be intravenous, intraperitoneal, intramuscular, intratumoral, subcutaneous, intranasal, or intradermal.

[0150] Pharmaceutical compositions for transdermal administration can be formulated as individual patches to remain closely adhered to the recipient's epidermis for an extended period. For example, as commonly described in Pharmaceutical Research, 3(6):318 (1986), the active ingredient may be released from the patch by ion electrophoresis.

[0151] Pharmaceutical compositions for nasal administration with a solid carrier contain coarse powder with a particle size of, for example, 20 to 500 μm, which are administered by inhalation, i.e., by rapid nasal inhalation from a powder container held close to the nose. Compositions with a liquid carrier suitable for administration as a nasal spray or nasal dropper contain an aqueous solution or oily solution of the active ingredient.

[0152] Pharmaceutical compositions for parenteral administration may include water-soluble and water-insoluble sterile injection solutions, which may include water-soluble and water-insoluble suspensions containing antioxidants, buffers, bacteriostatic agents, and solutes for making a preparation substantially isotonic with the blood of the target recipient; and suspending agents and thickeners. Excipients that may be used in injection solutions include, for example, water, alcohol, polyols, glycerin, and vegetable oils.

[0153] In this specification, the terms “effective dose,” “therapeutic dose,” and “pharmaceutically effective dose” are used synonymously and refer to a dose sufficient to obtain a physiological effect. In some embodiments, the pharmaceutically effective dose of the antigen-binding peptide disclosed herein (e.g., an antibody or fragment antibody, but not limited to the following) refers to the amount of antigen-binding peptide (e.g., an antibody or fragment antibody, but not limited to the following) that is effective in reducing or neutralizing the antithrombotic effect of the compound disclosed herein in the subject concerned. In some embodiments, administering a certain pharmaceutically effective dose of antigen-binding peptide (e.g., an antibody or fragment antibody, but not limited to the following) to a subject experiencing severe bleeding can immediately restore the antithrombotic effect of the compound of formula (I) or (II). In some embodiments, administration of a certain pharmaceutically effective dose of antigen-binding peptide (e.g., an antibody or fragment antibody, but not limited to the following) has a limited duration of action. In certain embodiments, a single dose of a pharmaceutically effective dose of antigen-binding peptide (e.g., an antibody or fragment antibody, but not limited to the following) has a sufficient duration of action to restore the antithrombotic effect of the compound of formula (I) or (II). On the other hand, a single dose of a pharmaceutically effective amount of antigen-binding peptide (e.g., an antibody or fragment antibody, but not limited to the following) has a sufficiently short duration of action to allow for the resumption of antithrombotic therapy immediately after the single dose of the pharmaceutically effective amount, minimizing the time during which the risk of thromboembolism is increased.

[0154] In some embodiments, the pharmaceutically effective dose may be determined by a predetermined empirical method for the purposes described above. For example, in some embodiments, the dose of the compound of formula (II) is approximately 25 mg (once daily) to approximately 375 mg (twice daily). In some embodiments, the pharmaceutically effective dose of antigen-binding peptide (e.g., antibody or fragment antibody, but not limited to the following) is approximately 25 mg once daily. In some embodiments, the pharmaceutically effective dose of antigen-binding peptide (e.g., antibody or fragment antibody, but not limited to the following) is approximately 50 mg once daily. In some embodiments, the pharmaceutically effective dose of antigen-binding peptide (e.g., antibody or fragment antibody, but not limited to the following) is approximately 75 mg once daily. In some embodiments, the pharmaceutically effective dose of antigen-binding peptide (e.g., antibody or fragment antibody, but not limited to the following) is approximately 100 mg once daily. In some embodiments, the pharmaceutically effective dose of antigen-binding peptide (e.g., antibody or fragment antibody, but not limited to the following) is approximately 125 mg once daily. In some embodiments, the pharmaceutically effective amount of antigen-binding peptide (e.g., an antibody or fragment antibody, but not limited to the following) is approximately 150 mg once daily. In some embodiments, the pharmaceutically effective amount of antigen-binding peptide (e.g., an antibody or fragment antibody, but not limited to the following) is approximately 175 mg once daily. In some embodiments, the pharmaceutically effective amount of antigen-binding peptide (e.g., an antibody or fragment antibody, but not limited to the following) is approximately 200 mg once daily. In some embodiments, the pharmaceutically effective amount of antigen-binding peptide (e.g., an antibody or fragment antibody, but not limited to the following) is approximately 375 mg once daily.

[0155] In some embodiments, a pharmaceutically effective amount of antigen-binding peptide (e.g., an antibody or fragment antibody, but not limited to the following) is approximately 25 mg twice daily. In some embodiments, a pharmaceutically effective amount of antigen-binding peptide (e.g., an antibody or fragment antibody, but not limited to the following) is approximately 50 mg twice daily. In some embodiments, a pharmaceutically effective amount of antigen-binding peptide (e.g., an antibody or fragment antibody, but not limited to the following) is approximately 75 mg twice daily. In some embodiments, a pharmaceutically effective amount of antigen-binding peptide (e.g., an antibody or fragment antibody, but not limited to the following) is approximately 100 mg twice daily. In some embodiments, a pharmaceutically effective amount of antigen-binding peptide (e.g., an antibody or fragment antibody, but not limited to the following) is approximately 125 mg twice daily. In some embodiments, a pharmaceutically effective amount of antigen-binding peptide (e.g., an antibody or fragment antibody, but not limited to the following) is approximately 150 mg twice daily. In some embodiments, a pharmaceutically effective amount of antigen-binding peptide (e.g., an antibody or fragment antibody, but not limited to the following) is approximately 175 mg twice daily. In some embodiments, the pharmaceutically effective amount of antigen-binding peptide (e.g., an antibody or fragment antibody, but not limited to the following) is approximately 200 mg twice daily. In some embodiments, the pharmaceutically effective amount of antigen-binding peptide (e.g., an antibody or fragment antibody, but not limited to the following) is approximately 375 mg twice daily.

[0156] In some embodiments of the present invention, the pharmaceutically effective amount of antigen-binding peptide (e.g., an antibody or fragment antibody, but not limited to the following) is determined primarily in relation to the amount of compound of formula (I) or (II) administered previously. In some embodiments, the pharmaceutically effective amount includes antigen-binding peptide (e.g., an antibody or fragment antibody, but not limited to the following) in a molar ratio of at least about 1:1 with respect to the amount of compound of formula (I) or (II) administered previously to the subject. In some embodiments, the pharmaceutically effective amount includes antigen-binding peptide (e.g., an antibody or fragment antibody, but not limited to the following) in a molar ratio of at least about 2:1 to about 10:1 with respect to the amount of compound of formula (I) or (II) administered to the subject. In some embodiments, the pharmaceutically effective amount includes an antigen-binding peptide (e.g., an antibody or fragment antibody, but not limited to the following) in a molar ratio of at least 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, 55:1, 60:1, 65:1, 70:1, 75:1, 80:1, 85:1, 90:1, 95:1, or 100:1 with respect to the amount of compound of formula (I) or (II) previously administered to the subject.

[0157] In some embodiments, the pharmaceutically effective amount includes an antigen-binding peptide (e.g., an antibody or fragment antibody, but not limited to the following) in a molar ratio of at least about 1:1 to the amount of compound of formula (I) or (II) in the subject. In some embodiments, the pharmaceutically effective amount includes an antigen-binding peptide (e.g., an antibody or fragment antibody, but not limited to the following) in a molar ratio of at least about 2:1 to about 10:1 to the amount of compound of formula (I) or (II) in the subject. In some embodiments, the pharmaceutically effective amount includes an amount of antigen-binding peptide (e.g., an antibody or fragment antibody, but not limited to the following) in a molar ratio of at least about 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, 55:1, 60:1, 65:1, 70:1, 75:1, 80:1, 85:1, 90:1, 95:1, or 100:1 to the amount of compound of formula (I) or (II) in the subject.

[0158] In some embodiments, the pharmaceutically effective amount of antigen-binding peptide (e.g., an antibody or fragment antibody, but not limited to the following) is calculated by mass ratio. For example, the molecular weight (MW) of the antigen-binding peptide (e.g., an antibody or fragment antibody, but not limited to the following) may be about 75 times the MW of the compound of formula (II). In this example, for about 100 mg of the compound of formula (II), the equivalent molar of antigen-binding peptide (e.g., an antibody or fragment antibody, but not limited to the following) is about 7.5 g. Since the molar mass of the substance is described herein, those skilled in the art can easily calculate the mass ratio of the antigen-binding peptide (e.g., an antibody or fragment antibody, but not limited to the following) to the compound of formula (I) or (II).

[0159] In some embodiments, the dosage of the antigen-binding peptide (e.g., an antibody or fragment antibody, but not limited to the following) is determined in clinical trials. Prior to two trials, computational models and simulations were conducted incorporating (1) information on human pharmacokinetics and human pharmacological effects (obtained from Phase 1 trials), (2) binding rates, and (3) predicted human PK parameters.

[0160] The antigen-binding peptide of the present invention (e.g., an antibody or fragment antibody, but not limited to the following) may be administered simultaneously with or after the administration of the compound of formula (I) or (II). In some embodiments, the antigen-binding peptide (e.g., an antibody or fragment antibody, but not limited to the following) is administered simultaneously with the administration of the compound of formula (I) or (II). In some embodiments, the antigen-binding peptide (e.g., an antibody or fragment antibody, but not limited to the following) is administered immediately after the administration of the compound of formula (I) or (II). In the examples, the antigen-binding peptide (e.g., an antibody or fragment antibody, but not limited to the following) is administered about 30 minutes after the start of administration of the compound of formula (I) or (II). In some embodiments, the antigen-binding peptide (e.g., an antibody or fragment antibody, but not limited to the following) is administered about 20 minutes after the completion of administration of the compound of formula (I) or (II). However, the antigen-binding peptide (e.g., an antibody or fragment antibody, but not limited to the following) and the compound of formula (I) or (II) may be administered simultaneously or sequentially in any order, as deemed appropriate by those skilled in the art.

[0161] The antigen-binding peptide of the present invention (e.g., an antibody or fragment antibody, but not limited to the following) may be administered by any route deemed appropriate by those skilled in the art. In some embodiments, the antigen-binding peptide (e.g., an antibody or fragment antibody, but not limited to the following) is administered intravenously, intramuscularly, or subcutaneously. In some embodiments, the antigen-binding peptide (e.g., an antibody or fragment antibody, but not limited to the following) is administered once daily. In some embodiments, the antigen-binding peptide (e.g., an antibody or fragment antibody, but not limited to the following) is administered once or more daily. In some embodiments, the antigen-binding peptide (e.g., an antibody or fragment antibody, but not limited to the following) is administered over approximately 10 minutes. In some embodiments, the antigen-binding peptide (e.g., an antibody or fragment antibody, but not limited to the following) is administered over a period of less than approximately 10 minutes. In some embodiments, the antigen-binding peptide (e.g., an antibody or fragment antibody, but not limited to the following) is administered over a period of approximately 10 minutes or more.

[0162] Furthermore, the present invention provides a method for detecting the level of a compound of formula (I) or (II) in a biological sample. In some embodiments, the method is characterized by contacting the biological sample with an antigen-binding peptide (e.g., an antibody or fragment antibody, but not limited to the following). In some embodiments, the method is characterized by detecting the level of a complex of the compound of formula (I) or (II) and an antigen-binding peptide (e.g., an antibody or fragment antibody, but not limited to the following). In some embodiments, the method is characterized by contacting the biological sample with an antigen-binding peptide (e.g., an antibody or fragment antibody, but not limited to the following), and then detecting the level of a complex of the compound and an antigen-binding peptide (e.g., an antibody or fragment antibody, but not limited to the following).

[0163] The scope of this disclosure should not be limited to any exemplary embodiments described above, but should be defined solely by the following claims and their equivalents.

[0164] All citations that may be referenced herein (including references, patents, patent applications, and websites) are expressly incorporated herein in their entirety for any purpose, as they are cited. This disclosure relates, for example, to the following: [Section 1] An isolated antigen-binding peptide comprising at least one heavy chain variable region (VH) and at least one light chain variable region (VL), wherein at least one VH is: (a) VH complementarity determination region 1 (VH-CDR1) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 12; (b) VH-CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 13-22; or (c) VH-CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs. 23-28; and It includes at least one of the following, and at least one VL is: (d) VL-CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs. 29-37; (e) VL-CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs. 38-43; or (f) VL-CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 44-51 It includes at least one of the following. [Section 2] below: (a) At least one heavy chain variable region having an amino acid sequence selected from the group consisting of SEQ ID NOs. 52 to 83; and (b) At least one light chain variable region having an amino acid sequence selected from the group consisting of SEQ ID NOs: 84-99 Isolated antigen-binding peptides containing [the specified substance]. [Section 3] An isolated antigen-binding peptide comprising at least one heavy chain variable region (VH) and at least one light chain variable region (VL), wherein the VH comprises three complementarity-determining regions (CDRs): VH-CDR1, VH-CDR2, and VH-CDR3, and the VL comprises three CDRs: VL-CDR1, VL-CDR2, and VL-CDR3, where the amino acid sequences of VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3 are as follows, respectively: (a) Sequence numbers for each: 1, 13, 23, 29, 38, and 44; (b) Sequence numbers for each: 1, 14, 23, 29, 38, and 45; (c) Sequence numbers for each: 1, 13, 24, 30, 38, and 45; (d) Sequence numbers for each: 1, 13, 24, 29, 39, and 45; (e) Sequence numbers for each: 1, 14, 25, 29, 38, and 46; (f) Sequence numbers for each: 2, 13, 26, 31, 40, and 47; (g) Sequence numbers for each: 3, 15, 24, 32, 40, and 47; (h) Sequence numbers for each: 4, 16, 24, 29, 38, and 46; (i) Sequence numbers for each: 5, 15, 24, 29, 38, and 46; (j) Sequence numbers for each: 1, 14, 24, 29, 38, and 46; (k) Sequence numbers for each: 6, 13, 24, 31, 40, and 47; (l) Sequence numbers for each: 3, 15, 24, 32, 41, and 48; (m) Sequence numbers for each: 1, 14, 24, 33, 38, and 49; (n) Sequence numbers for each: 1, 14, 26, 29, 38, and 46; (o) Sequence numbers for each: 7, 17, 26, 29, 38, and 46; (p) Sequence numbers: 8, 17, 24, 34, 38, and 46; (q) Sequence numbers for each: 1, 17, 26, 29, 38, and 46; (r) Sequence numbers for each: 1, 17, 26, 35, 38, and 46; (s) Sequence numbers for each: 1, 17, 24, 33, 38, and 49; (t) Sequence numbers: 9, 14, 26, 29, 38, and 46; (u) Sequence numbers: 9, 14, 26, 35, 38, and 46; (v) Sequence numbers for each: 9, 17, 24, 29, 38, and 46; (w) Sequence numbers: 9, 17, 24, 35, 38, and 46; (x) Sequence numbers for each: 9, 17, 24, 34, 38, and 46; (y) Sequence numbers for each: 9, 14, 24, 29, 38, and 46; (z) Sequence numbers: 9, 18, 26, 35, 38, and 46; (aa) Sequence numbers for each: 8, 14, 24, 29, 38, and 46; (bb) Sequence numbers: 8, 17, 26, 29, 38, and 46; (cc) Sequence numbers for each: 9, 19, 26, 29, 38, and 46; (dd) Sequence numbers for each: 9, 17, 26, 34, 38, and 46; (ee) Sequence numbers: 10, 20, 27, 36, 42, and 50; (ff) Sequence numbers: 11, 21, 28, 37, 43, and 51; (gg) Sequence numbers for each: 12, 22, 26, 33, 38, and 46; (hh) Sequence numbers for each: 12, 17, 26, 33, 38, and 46; (ii) Each sequence number: 9, 17, 26, 33, 38, and 46; and (jj) Variants of (a)-(ii) in which any amino acid sequence contains one, two, or three conserved substitutions. It has a sequence selected from the group consisting of the following. [Section 4] At least one VH region and at least one VL region are, respectively, as follows: (a) Sequence ID 52 and Sequence ID 84, respectively; (b) Sequence ID 53 and Sequence ID 85, respectively; (c) Sequence IDs 54 and 86, respectively; (d) Sequence IDs 54 and 87, respectively; (e) Sequence IDs 55 and 88, respectively; (f) Sequence ID 56 and Sequence ID 89, respectively; (g) Sequence IDs 57 and 90, respectively; (h) Sequence IDs 58 and 88, respectively; (i) Sequence ID 59 and Sequence ID 88, respectively; (j) Sequence IDs 60 and 91, respectively; (k) Sequence IDs 61 and 89, respectively; (l) Sequence ID 57 and Sequence ID 92, respectively; (m) Sequence IDs 60 and 93, respectively; (n) Sequence ID 60 and Sequence ID 88, respectively; (o) Sequence IDs 62 and 88, respectively; (p) Sequence IDs 63 and 88, respectively; (q) Sequence ID 64 and Sequence ID 88, respectively; (r) Sequence IDs 65 and 94, respectively; (s) Sequence ID 66 and Sequence ID 88, respectively; (t) Sequence ID 66 and Sequence ID 95, respectively; (u) Sequence IDs 67 and 88, respectively; (v) Sequence IDs 68 and 93, respectively; (w) Sequence IDs 69 and 88, respectively; (x) Sequence ID 69 and Sequence ID 95, respectively; (y) Sequence IDs 70 and 88, respectively; (z) Sequence IDs 70 and 95, respectively; (aa) Sequence IDs 71 and 88, respectively; (bb) Sequence IDs 71 and 94, respectively; (cc) Sequence IDs 72 and 88, respectively; (dd) Sequence IDs 73 and 95, respectively; (ee) Sequence IDs 74 and 88, respectively; (ff) Sequence IDs 75 and 88, respectively; (gg) Sequence IDs 76 and 88, respectively; (hh) Sequence IDs 77 and 94, respectively; (ii) Sequence IDs 78 and 96, respectively; (jj) Sequence IDs 79 and 97, respectively; (kk) Sequence IDs 80 and 98 respectively; (ll) Sequence IDs 81 and 99, respectively; (mm) Sequence IDs 81 and 98, respectively; (nn) Sequence IDs 82 and 99, respectively; (oo) Sequence ID 83 and Sequence ID 98 respectively; and (pp) Variants of (a)~(oo) including a conservative substitution of 1, 2, 3, or 4 amino acids. An isolated antigen-binding peptide as described in item 3, having an amino acid sequence selected from the group consisting of the following. [Section 5] An isolated antigen-binding peptide as described in any one of the above items, each containing two heavy chain variable regions paired with one light chain variable region. [Section 6] Furthermore, the isolated antigen-binding peptide described in item 5 comprises a polypeptide linker having a sequence selected from sequence numbers 196-199. [Section 7] The isolated antigen-binding peptide specifically conforms to formula (I): [C1] TIFF0007863566000082.tif58160 [In the formula, R 1 C 1-4 It is alkyl; R 2 These are independently F, Cl, and CF. 3 CHF 2 CH 2 F, CH 3 Selected from; R 3 CF is independent 3 CHF 2 CH 2 F and CH 3 Selected from; R 4 is H; and R 5 [It is independently selected from F and Cl.] An isolated antigen-binding peptide according to any one of the above items, which binds to the compound shown by, or its stereoisomer or tautomer. [Section 8] The compound is given by formula (II): [C2] TIFF0007863566000083.tif58160 The isolated antigen-binding peptide described in item 7. [Section 9] The isolated antigen-binding peptide described in any one of the above items is an antibody. [Section 10] The aforementioned isolated antigen-binding peptides are Fab, Fab', and F(ab'). 2 Fd, single-chain Fv or scFv, disulfide-bonded Fv, V-NAR domain, IgNar, intra-body, IgGACH2, mini-body, F(ab') 3 Tetrabody, triabody, bispecific antibody, single-domain antibody, DVD-Ig, FCab, mAb 2 (scFv) 2 An isolated antigen-binding peptide as described in any one of the above items, which is scFv-Fc or tandem Fab. [Section 11] below: (a) Sequence ID 100 and Sequence ID 160, respectively; (b) Sequence ID 101 and Sequence ID 160, respectively; (c) Sequence IDs 102 and 161, respectively; (d) Sequence IDs 103 and 161, respectively; (e) Sequence ID 104 and Sequence ID 162, respectively; (f) Sequence IDs 105 and 162, respectively; (g) Sequence IDs 104 and 163, respectively; (h) Sequence IDs 105 and 163, respectively; (i) Sequence ID 106 and Sequence ID 164, respectively; (j) Sequence ID 107 and Sequence ID 164, respectively; (k) Sequence ID 108 and Sequence ID 165, respectively; (l) Sequence ID 109 and Sequence ID 165, respectively; (m) Sequence IDs 110 and 166, respectively; (n) Sequence ID 111 and Sequence ID 166, respectively; (o) Sequence IDs 112 and 164, respectively; (p) Sequence IDs 113 and 164, respectively; (q) Sequence IDs 114 and 164, respectively; (r) Sequence IDs 115 and 164, respectively; (s) Sequence IDs 116 and 167, respectively; (t) Sequence IDs 117 and 167, respectively; (u) Sequence ID 118 and Sequence ID 165, respectively; (v) Sequence IDs 119 and 165, respectively; (w) Sequence IDs 110 and 168, respectively; (x) Sequence ID 111 and Sequence ID 168, respectively; (y) Sequence IDs 116 and 169, respectively; (z) Sequence IDs 117 and 169, respectively; (aa) Sequence ID 116 and Sequence ID 164, respectively; (bb) Sequence IDs 117 and 164, respectively; (cc) Sequence IDs 120 and 164, respectively; (dd) Sequence IDs 121 and 164, respectively; (ee) Sequence IDs 122 and 164, respectively; (ff) Sequence IDs 123 and 164, respectively; (gg) Sequence IDs 124 and 164, respectively; (hh) Sequence IDs 125 and 164, respectively; (ii) Sequence IDs 126 and 170, respectively; (jj) Sequence IDs 127 and 170, respectively; (kk) Sequence IDs 128 and 164, respectively; (ll) Sequence IDs 129 and 164, respectively; (mm) Sequence IDs 128 and 171, respectively; (nn) Sequence IDs 129 and 171, respectively; (oo) Sequence IDs 130 and 164, respectively; (pp) Sequence IDs 131 and 164, respectively; (qq) Sequence IDs 132 and 169, respectively; (rr) Sequence IDs 133 and 169, respectively; (ss) Sequence IDs 134 and 164, respectively; (tt) Sequence IDs 135 and 164, respectively; (uu) Sequence IDs 134 and 171, respectively; (vv) Sequence IDs 135 and 171, respectively; (ww) Sequence IDs 136 and 164, respectively; (xx) Sequence IDs 137 and 164, respectively; (yy) Sequence IDs 136 and 171, respectively; (zz) Sequence IDs 137 and 171, respectively; (aaa) Sequence ID 138 and Sequence ID 164, respectively; (bbb) Sequence IDs 139 and 164, respectively; (ccc) Sequence ID 138 and Sequence ID 170, respectively; (ddd) Sequence ID 139 and Sequence ID 170, respectively; (eee) Sequence IDs 140 and 164, respectively; (fff) Sequence IDs 141 and 164, respectively; (ggg) Sequence IDs 142 and 171, respectively; (hhh) Sequence IDs 143 and 171, respectively; (iii) Sequence ID 144 and Sequence ID 164, respectively; (jjj) Sequence IDs 145 and 164, respectively; (kkk) Sequence IDs 146 and 164, respectively; (lll) Sequence IDs 147 and 164, respectively; (mmm) Sequence IDs 148 and 164, respectively; (nnn) Sequence IDs 149 and 164, respectively; (ooo) Sequence ID 150 and Sequence ID 170, respectively; (ppp) Sequence IDs 151 and 170, respectively; (qqq) Sequence IDs 152 and 172, respectively; (rrr) Sequence IDs 153 and 172, respectively; (sss) Sequence IDs 154 and 173 respectively; (ttt) Sequence IDs 155 and 173, respectively; (uuu) Sequence IDs 156 and 174, respectively; (vvv) Sequence IDs 157 and 174, respectively; (www) Sequence ID 158 and Sequence ID 175, respectively; (xxx) Sequence IDs 159 and 175, respectively; (yyy) Sequence ID 158 and Sequence ID 174 respectively; and (zzz) Sequence IDs 159 and 174 respectively An isolated antigen-binding peptide according to any one of the above items, having a sequence selected from the group consisting of, wherein the isolated antigen-binding peptide specifically corresponds to formula (II) [C3] TIFF0007863566000084.tif58160 It binds to the compound. [Section 12] below: (a) Sequence ID 176 and Sequence ID 160, respectively; (b) Sequence ID 177 and Sequence ID 160, respectively; (c) Sequence ID 178 and Sequence ID 160, respectively; (d) Sequence IDs 179 and 160, respectively; (e) Sequence ID 180 and Sequence ID 164, respectively; (f) Sequence IDs 181 and 164, respectively; (g) Sequence IDs 182 and 164, respectively; (h) Sequence IDs 183 and 164, respectively; (i) Sequence ID 184 and Sequence ID 163, respectively; (j) Sequence ID 185 and Sequence ID 163, respectively; (k) Sequence ID 186 and Sequence ID 163, respectively; (l) Sequence IDs 187 and 163, respectively; (m) Sequence IDs 184 and 162, respectively; (n) Sequence ID 185 and Sequence ID 162, respectively; (o) Sequence IDs 186 and 162, respectively; (p) Sequence IDs 187 and 162, respectively; (q) Sequence ID 188 and Sequence ID 165, respectively; (r) Sequence IDs 189 and 165, respectively; (s) Sequence ID 190 and Sequence ID 165, respectively; (t) Sequence IDs 191 and 165, respectively; (u) Sequence IDs 192 and 161, respectively; (v) Sequence IDs 193 and 161, respectively; (w) Sequence ID 194 and Sequence ID 161 respectively; and (x) Sequence IDs 195 and 161 respectively An isolated antigen-binding peptide according to any one of the above items, having a sequence selected from the group consisting of the following, and formula (II) [C4] TIFF0007863566000085.tif58160 An isolated antigen-binding peptide that specifically binds to the compound. [Section 13] An isolated antigen-binding peptide according to item 11, having the sequences of SEQ ID NO: 106 and SEQ ID NO: 164, respectively, of formula (II) [5] TIFF0007863566000086.tif58160 An isolated antigen-binding peptide that specifically binds to the compound. [Section 14] An isolated Fab fragment antibody having the sequences of SEQ ID NO: 106 and SEQ ID NO: 164, respectively, and formula (II) [6] TIFF0007863566000087.tif58160 An isolated Fab fragment antibody that specifically binds to the compound. [Section 15] An isolated antigen-binding peptide according to item 12, having the sequences of SEQ ID NO: 180 and SEQ ID NO: 164, respectively, and formula (II) [7] TIFF0007863566000088.tif58160 An isolated antigen-binding peptide that specifically binds to the compound. [Section 16] An isolated tandem Fab fragment antibody having the sequences of SEQ ID NO: 180 and SEQ ID NO: 164, respectively, wherein formula (II) [8] TIFF0007863566000089.tif58160 An isolated tandem Fab fragment antibody that specifically binds to the compound. [Section 17] An isolated antigen-binding peptide according to item 12, having the sequences of SEQ ID NO: 181 and SEQ ID NO: 164, respectively, and formula (II) [9] TIFF0007863566000090.tif58160 An isolated antigen-binding peptide that specifically binds to the compound. [Section 18] An isolated tandem Fab fragment antibody having the sequences of SEQ ID NO: 181 and SEQ ID NO: 164, respectively, wherein formula (II) [C10] TIFF0007863566000091.tif58160 An isolated tandem Fab fragment antibody that specifically binds to the compound. [Section 19] An isolated antigen-binding peptide according to item 12, having the sequences of SEQ ID NO: 182 and SEQ ID NO: 164, respectively, and formula (II) [C11] TIFF0007863566000092.tif58160 An isolated antigen-binding peptide that specifically binds to the compound. [Section 20] An isolated tandem Fab fragment antibody having sequences of SEQ ID NO: 182 and SEQ ID NO: 164, respectively, wherein formula (II) [C12] TIFF0007863566000093.tif58160 An isolated tandem Fab fragment antibody that specifically binds to the compound. [Section 21] An isolated antigen-binding peptide according to item 12, having the sequences of SEQ ID NO: 183 and SEQ ID NO: 164, respectively, and formula (II) [C13] TIFF0007863566000094.tif58160 An isolated antigen-binding peptide that specifically binds to the compound. [Section 22] An isolated tandem Fab fragment antibody having sequences of SEQ ID NO: 183 and SEQ ID NO: 164, respectively, wherein formula (II) [C14] TIFF0007863566000095.tif58160 An isolated tandem Fab fragment antibody that specifically binds to the compound. [Section 23] An isolated antigen-binding peptide according to item 12, having the sequences of SEQ ID NO: 176 and SEQ ID NO: 160, respectively, and formula (II) [C15] TIFF0007863566000096.tif58160 An isolated antigen-binding peptide that specifically binds to the compound. [Section 24] An isolated tandem Fab fragment antibody having sequences of SEQ ID NO: 176 and SEQ ID NO: 160, respectively, wherein formula (II) [C16] TIFF0007863566000097.tif58160 An isolated tandem Fab fragment antibody that specifically binds to the compound. [Section 25] An isolated antigen-binding peptide according to item 12, having the sequences of SEQ ID NO: 177 and SEQ ID NO: 160, respectively, and formula (II) [C17] TIFF0007863566000098.tif58160 An isolated antigen-binding peptide that specifically binds to the compound. [Section 26] An isolated tandem Fab fragment antibody having sequences of SEQ ID NO: 177 and SEQ ID NO: 160, respectively, and formula (II) [C18] TIFF0007863566000099.tif58160 An isolated tandem Fab fragment antibody that specifically binds to the compound. [Section 27] An isolated antigen-binding peptide according to item 12, having the sequences of SEQ ID NO: 184 and SEQ ID NO: 162, respectively, and formula (II) [C19] TIFF0007863566000100.tif58160 An isolated antigen-binding peptide that specifically binds to the compound. [Section 28] An isolated tandem Fab fragment antibody having sequences of SEQ ID NO: 184 and SEQ ID NO: 162, respectively, and formula (II)

[20] TIFF0007863566000101.tif58160 An isolated tandem Fab fragment antibody that specifically binds to the compound. [Section 29] An isolated antigen-binding peptide according to item 12, having the sequences of SEQ ID NO: 184 and SEQ ID NO: 163, respectively, and formula (II)

[21] TIFF0007863566000102.tif58160 An isolated antigen-binding peptide that specifically binds to the compound. [Section 30] An isolated tandem Fab fragment antibody having sequences of SEQ ID NO: 184 and SEQ ID NO: 163, respectively, wherein formula (II) [C22] TIFF0007863566000103.tif58160 An isolated tandem Fab fragment antibody that specifically binds to the compound. [Section 31] An isolated antigen-binding peptide according to item 12, having the sequences of SEQ ID NO: 188 and SEQ ID NO: 165, respectively, and formula (II)

[23] TIFF0007863566000104.tif58160 An isolated antigen-binding peptide that specifically binds to the compound. [Section 32] An isolated tandem Fab fragment antibody having sequences of SEQ ID NO: 188 and SEQ ID NO: 165, respectively, and formula (II)

[24] TIFF0007863566000105.tif58160 An isolated tandem Fab fragment antibody that specifically binds to the compound. [Section 33] An isolated antigen-binding peptide of item 12 having the sequences of SEQ ID NO: 192 and SEQ ID NO: 161, respectively, and formula (II)

[25] TIFF0007863566000106.tif58160 An isolated antigen-binding peptide that specifically binds to the compound. [Section 34] An isolated tandem Fab fragment antibody having the sequences of SEQ ID NO: 192 and SEQ ID NO: 161, respectively, and formula (II)

[26] TIFF0007863566000107.tif58160 An isolated tandem Fab fragment antibody that specifically binds to the compound. [Section 35] An isolated polynucleotide having a nucleic acid sequence encoding an antigen-binding peptide or a Fab fragment antibody or a tandem Fab fragment antibody, as described in any one of items 1 to 34. [Section 36] A vector comprising the isolated polynucleotide described in item 35. [Section 37] A host cell containing the vector described in item 36. [Section 38] A method for producing an antigen-binding peptide, a Fab fragment antibody, or a tandem Fab fragment antibody, characterized by (a) culturing host cells under conditions that promote protein production such that the host cells described in item 37 produce an antigen-binding peptide, a Fab fragment antibody, or a tandem Fab fragment antibody, and (b) isolating the antigen-binding peptide, Fab fragment antibody, or tandem Fab fragment antibody from the culture medium in (a). [Section 39] A detection reagent comprising an isolated antigen-binding peptide or isolated Fab fragment antibody or isolated tandem Fab fragment antibody as described in any one of items 1 to 34, and a detectable label. [Section 40] The detection reagent according to item 39, wherein an isolated antigen-binding peptide or an isolated Fab fragment antibody or an isolated tandem Fab fragment antibody is conjugated to a detectable label. [Section 41] Formula (I) is characterized by administering a pharmaceutically effective amount of an isolated antigen-binding peptide or isolated Fab fragment antibody or isolated tandem Fab fragment antibody described in any one of items 1 to 34 to the target subject.

[27] TIFF0007863566000108.tif64160 [In the formula, R 1 C 1-4 It is alkyl; R 2 These are independently F, Cl, and CF. 3 CHF 2 CH 2 F, CH 3 Selected from; R 3 CF is independent 3 CHF 2 CH 2 F and CH 3 Selected from; R 4 is H; and R 5 [It is independently selected from F and Cl.] A method for reducing the antithrombotic effect of a compound or its stereoisomers or tautomers. [Section 42] The compound of formula (I) is formula (II):

[28] TIFF0007863566000109.tif58160 The method according to item 41, wherein the method is having the characteristics of item 41. [Section 43] The method according to item 41 or 42, wherein a pharmaceutically effective amount of isolated antigen-binding peptide or isolated Fab fragment antibody or isolated tandem Fab fragment antibody comprises the antigen-binding peptide or Fab fragment antibody or tandem Fab fragment antibody in a molar ratio of at least about 1:1 with respect to the dose of the compound of formula (I) or (II), or in a molar ratio of at least about 1:1 with respect to the amount of the compound of formula (I) or (II) in the subject. [Section 44] The method according to any one of claims 41 to 43, wherein an isolated antigen-binding peptide or an isolated Fab fragment antibody or an isolated tandem Fab fragment antibody is administered concurrently with or after the administration of a compound of formula (I) or (II). [Section 45] The method according to any one of claims 41 to 44, wherein an isolated antigen-binding peptide or an isolated Fab fragment antibody or an isolated tandem Fab fragment antibody is administered intravenously, intramuscularly, or subcutaneously. [Section 46] The method described in any one of items 41 to 45, wherein the subject is a human. [Section 47] Equation (I) [C29] TIFF0007863566000110.tif64160 [In the formula, R 1 C 1-4 It is alkyl; R 2 These are independently F, Cl, and CF. 3 CHF 2 CH 2 F, CH 3 Selected from; R 3 CF is independent 3 CHF 2 CH 2 F and CH 3 Selected from; R 4 is H; and R 5 [It is independently selected from F and Cl.] A method for detecting the level of a compound or its stereoisomers, tautomers, or pharmaceutically acceptable salts in a biological sample, (a) Contacting a biological sample with an isolated antigen-binding peptide or isolated Fab fragment antibody or isolated tandem Fab fragment antibody as described in any one of items 1 to 34, and (b) Detect the level of the complex to which the compound and the isolated antigen-binding peptide or isolated Fab fragment antibody or isolated tandem Fab fragment antibody are bound. A method characterized by the following. [Section 48] The compound of formula (I) is formula (II):

[30] TIFF0007863566000111.tif58160 The method according to paragraph 47, having the following characteristics. [Section 49] The method according to item 47 or 48, wherein an isolated antigen-binding peptide or an isolated Fab fragment antibody or an isolated tandem Fab fragment antibody is labeled. [Section 50] The method according to any one of items 47 to 49, wherein detection is performed by an immunoassay. [Section 51] The method according to any one of items 47 to 50, wherein the biological sample includes urine, feces, saliva, whole blood, plasma, organ tissue, hair, skin, cells, or cell cultures. [Section 52] Formula (I) for the therapeutically effective dose

[31] TIFF0007863566000112.tif64160 [In the formula, R 1 C 1-4 It is alkyl; R 2 These are independently F, Cl, and CF. 3 CHF 2 CH 2 F, CH 3 Selected from; R 3 CF is independent 3 CHF 2 CH 2 F and CH 3 Selected from; R 4 is H; and R 5 [It is independently selected from F and Cl.] A method for conjugating a compound of formula (I) or its stereoisomer or tautomer, characterized in that a subject taking the compound or its stereoisomer or tautomer is administered an isolated antigen-binding peptide or isolated Fab fragment antibody or isolated tandem Fab fragment antibody described in any one of items 1 to 34 of the pharmaceutically effective amount section. [Section 53] The compound of formula (I) is formula (II):

[32] TIFF0007863566000113.tif58160 The method described in paragraph 52, which has [Section 54] The method according to item 52 or 53, wherein a pharmaceutically effective amount of isolated antigen-binding peptide or isolated Fab fragment antibody or isolated tandem Fab fragment antibody comprises the antigen-binding peptide or Fab fragment antibody or tandem Fab fragment antibody in a molar ratio of at least about 1:1 with respect to the dose of the compound of formula (I) or (II), or in a molar ratio of at least about 1:1 with respect to the amount of the compound of formula (I) or (II) in the subject. [Section 55] The method according to any one of claims 52 to 54, wherein an isolated antigen-binding peptide or an isolated Fab fragment antibody or an isolated tandem Fab fragment antibody is administered concurrently with or after the administration of a compound of formula (I) or (II). [Section 56] The method according to any one of claims 52 to 55, wherein an isolated antigen-binding peptide or an isolated Fab fragment antibody or an isolated tandem Fab fragment antibody is administered intravenously, intramuscularly, or subcutaneously. [Section 57] The method described in any one of items 52 to 56, wherein the subject is a human.

[0165] Examples Example 1: Production of human monoclonal antibodies against compound A using genetically modified mice expressing human antibody genes - Production of antigen and related compounds

[0166] A fully human monoclonal antibody against compound A, a specific inhibitor of factor XIa, was obtained by immunizing genetically modified mice with keyhole limpet hemocyanin (KLH) conjugated with compound A, as shown below. [ka]

[0167] Example 1A. Synthesis of Compound 3 Step 1: Synthesis of tert-butyl1-(4-chloro-2-(1-((5R,9S)-21-(difluoromethyl)-5-methyl-4-oxo-21H-3-aza-1(4,2)-pyridina-2(5,4)-pyrazolacyclonononaphan-9-yl)-6-oxo-1,6-dihydropyrimidine-4-yl)phenyl)-1H-1,2,3-triazole-4-carboxylate (Compound 1) [ka]

[0168] To a 100 mL flask containing a white suspension of tert-butyl 1-(4-chloro-2-(6-hydroxypyrimidine-4-yl)phenyl)-1H-1,2,3-triazole-4-carboxylate (105 mg, 0.28 mmol) / acetonitrile (3.7 mL), HATU (1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxidehexafluorophosphate, 117 mg, 0.31 mmol) and DBU (1,8-diazabicyclo[5.4.0]undeca-7-ene, 55.0 μL, 0.37 mmol) were added. The resulting clear yellow solution was stirred at room temperature for 5 minutes. (5R,9S)-9-amino-21-(difluoromethyl)-5-methyl-21H-3-aza-1(4,2)-pyridina-2(5,4)-pyrazolacyclonononaphan-4-one (94 mg, 0.281 mmol) was added, and the resulting suspension was stirred at room temperature for 3 hours, at which point it was concentrated and dried. The resulting residue was dissolved in ELISA (1 mL) and loaded onto an ISCO column (40 g). The product was eluted over 35 minutes with a linear gradient of ELISA / hexane (0% to 100%). The product eluted exactly at 100% ELISA. tert-butyl1-(4-chloro-2-(1-((5R,9S)-21-(difluoromethyl)-5-methyl-4-oxo-21H-3-aza-1(4,2)-pyridina-2(5,4)-pyrazolacyclonononaphan-9-yl)-6-oxo-1,6-dihydropyrimidine-4-yl)phenyl)-1H-1,2,3-triazole-4-carboxylate (161 mg, 0.233 mmol, 83% yield) was isolated as a white solid.

[0169] Step 2: Synthesis of 1-(4-chloro-2-(1-((5R,9S)-21-(difluoromethyl)-5-methyl-4-oxo-21H-3-aza-1(4,2)-pyridina-2(5,4)-pyrazolacyclonononaphan-9-yl)-6-oxo-1,6-dihydropyrimidine-4-yl)phenyl)-1H-1,2,3-triazole-4-carboxylate hydrochloride (compound 1A) [ka] tert-butyl1-(4-chloro-2-(1-((5R,9S)-21-(difluoromethyl)-5-methyl-4-oxo-21H-3-aza-1(4,2)-pyridina-2(5,4)-pyrazolacyclonononaphan-9-yl)-6-oxo-1,6-dihydropyrimidine-4-yl)phenyl)-1H-1,2,3-triazole-4-carboxylate (161 mg, 0.233 mmol) was dissolved in HCl / dioxane (3 mL, 12.00 mmol) and stirred for 2 hours. At that point, completion of deprotection was indicated by LC-MS. The reaction mixture was concentrated and dried, and then dried under reduced pressure overnight. 1-(4-chloro-2-(1-((5R,9S)-21-(difluoromethyl)-5-methyl-4-oxo-21H-3-aza-1(4,2)-pyridina-2(5,4)-pyrazolacyclonononaphan-9-yl)-6-oxo-1,6-dihydropyrimidine-4-yl)phenyl)-1H-1,2,3-triazole-4-carboxylate hydrochloride (150 mg, 0.223 mmol, 96% yield) was isolated as a pale yellow solid.

[0170] Step 3: Synthesis of 3-(2-(2-(1-(4-chloro-2-(1-((5R,9S)-21-(difluoromethyl)-5-methyl-4-oxo-21H-3-aza-1(4,2)-pyridina-2(5,4)-pyrazolacyclonononaphan-9-yl)-6-oxo-1,6-dihydropyrimidine-4-yl)phenyl)-1H-1,2,3-triazole-4-carboxamide)ethoxy)ethoxy)propanoic acid (compound 1B) [ka] 1-(4-chloro-2-(1-((5R,9S)-21-(difluoromethyl)-5-methyl-4-oxo-21H-3-aza-1(4,2)-pyridina-2(5,4)-pyrazolacyclonononaphan-9-yl)-6-oxo-1,6-dihydropyrimidine-4-yl)phenyl)-1H-1,2,3-triazole-4-carboxylate hydrochloride (40 mg, 0.063 mmol), tert-butyl 3-(2-(2-aminoethoxy)ethoxy)propanoate (14.67 mg, 0.063 mmol), and triethylamine (8.77 μL, 0.063 mmol) were dissolved in DMF (N,N-dimethylformamide, 2 mL). BOP (benzotriazole-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate, 27.8 mg, 0.063 mmol) was added, and the mixture was stirred at room temperature for 4 hours. The resulting residue was concentrated and dried, and then diluted with CH2Cl2 (4 mL) and TFA (trifluoroacetic acid, 2 mL). This reaction mixture was stirred at room temperature for 2 hours, and then concentrated to dryness until a residue was obtained. Compound 1 was purified by preparative HPLC. Preparative HPLC - Column = Sunfire Prep C18 OBD 5μ (30x100mm) Solvent A = 10% MeOH, 90% water, 10 mM ammonium acetate Solvent B = 90% MeOH, 10% water, 10 mM ammonium acetate Linear gradient from 25%B to 100%B

[0171] Example 1B. Synthesis of Compounds 2 and 3 3-(2-(2-(2-((5R,9S)-9-(4-(5-chloro-2-(4-chloro-1H-1,2,3-triazole-1-yl)phenyl)-6-oxopyrimidine-1(6H)-yl)-5-methyl-4-oxo-21H-3-aza-1(4,2)-pyridina-2(3,4)-pyrazolacyclonononaphan-21-yl)ethoxy)ethoxy)ethoxy)propanoic acid (compound 2) and Synthesis of 3-(2-(2-(2-((5R,9S)-9-(4-(5-chloro-2-(4-chloro-1H-1,2,3-triazole-1-yl)phenyl)-6-oxopyrimidine-1(6H)-yl)-5-methyl-4-oxo-21H-3-aza-1(4,2)-pyridina-2(5,4)-pyrazolacyclonononaphan-21-yl)ethoxy)ethoxy)ethoxy)propanoic acid (compound 3) [ka] (5R,9S)-9-(4-(5-chloro-2-(4-chloro-1H-1,2,3-triazole-1-yl)phenyl)-6-oxopyrimidine-1(6H)-yl)-5-methyl-21H-3-aza-1(4,2)-pyridina-2(5,4)-pyrazolacyclonononaphane-4-one (compound 4) (70 mg, 0.121 mmol), tert-butyl 3-(2-(2-(2-bromoethoxy)ethoxy)ethoxy)propanoate (41.4 mg, 0.121 mmol), and cesium carbonate (39.6 mg, 0.121 mmol) were heated in DMF (N,N-dimethylformamide, 3 mL) at 60°C for 1 hour, and then cooled to room temperature. The reaction mixture was filtered and then concentrated to dryness. The resulting residue was diluted with CH2Cl2 (4 mL) and TFA (2 mL), and then stirred at room temperature for 1 hour. Compounds 2 and 3 were purified by preparative HPLC. Preparative HPLC - Column = Sunfire Prep C18 OBD 5μ (30x100mm) Solvent A = 10% MeOH, 90% water, 10 mM ammonium acetate Solvent B = 90% MeOH, 10% water, 10 mM ammonium acetate Linear gradient from 25%B to 100%B Compound 2 (40 mg, 0.051 mmol, 41.8% yield) and Compound 3 (15 mg, 0.019 mmol, 15.51% yield) were isolated as white solids.

[0172] Compounds 2, 3, and 1 were conjugated to BSA and KLH for immunoassay and ELISA screening.

[0173] Condensation to KLH A sample (2 mg) of compound 1, 2, or 3 was dissolved in DMSO (90 μL), followed by the addition of MES buffer (390 μL), and mixed by vortexing. Then, 200 μL of KLH (stock solution, 10 mg / mL) was added to the mixture. Finally, 50 μL of EDC (stock solution, 20 mg / mL) was added. The sample was incubated at room temperature in the dark for 3 hours, and then dialyzed with 5 L of DPBS (1x, Lonza, cat#17-512Q).

[0174] Condensation to BSA A 2 mg sample of compound (compound 2, compound 3, or compound 1) was dissolved in DMSO (200 μL), followed by the addition of MES buffer (200 μL, MES pH 4.7), and mixed by vortexing. Next, 400 μL of BSA (stock solution 5 mg / mL) was added to the mixture. Finally, 50 μL of EDC (stock solution 20 mg / mL) was added (the ratio of added compounds:carrier:activator = 10:1:2). The sample was incubated at room temperature in the dark for 3 hours, and then dialyzed with 5 L of DPBS (1x, Lonza, cat#17-512Q).

[0175] Compound 5 (biotin-labeled compound 2) has the following structure. [ka]

[0176] Synthesis of N-(1-((5R,9S)-9-(4-(5-chloro-2-(4-chloro-1H-1,2,3-triazole-1-yl)phenyl)-6-oxopyrimidine-1(6H)-yl)-5-methyl-4-oxo-21H-3-aza-1(4,2)-pyridina-2(3,4)-pyrazolacyclonononaphan-21-yl)-12-oxo-3,6,9-trioxa-13-azapentadecane-15-yl)-5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazole-4-yl)pentanamide (compound 5) [ka] Compound 2 (16 mg, 0.02 mmol), N-(2-aminoethyl)-5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazole-4-yl)pentanamide (5.9 mg, 0.02 mmol), biotin, and triethylamine (2.9 μL, 0.02 mmol) were dissolved in DMF (2 mL). BOP (9.1 mg, 0.02 mmol) was added, and the reaction mixture was stirred at room temperature for 4 hours. The reaction mixture was then purified by preparative HPLC. Preparative HPLC - Column = Sunfire Prep C18 OBD 5μ (30x100mm) Solvent A = 10% MeOH, 90% water, 10 mM ammonium acetate Solvent B = 90% MeOH, 10% water, 10 mM ammonium acetate Linear gradient from 25%B to 100%B Compound 5 (18 mg, 80%) was isolated as a white solid.

[0177] Example 2: Production of human monoclonal antibodies against compound A using genetically modified mice expressing human antibody genes - Immunization of mice

[0178] Human anti-compound A antibody was produced by immunizing a mouse strain (HCo42:01 KCo5:01 [J / K]) into which the human Ig gene had been introduced (Lonberg, Handbook of Experimental Pharmacology 113:49, 1994; Lonberg et al. Nature 368:856, 1994). The immunogen was a mixture of three KLH-compound complexes (compound 1-KLH, compound 2-KLH, and compound 3-KLH) with Ribi adjuvant (RA). Immunization was performed by injecting the immunogen mixture in Ribi adjuvant into the sole of the foot. Immunization of mice was performed a total of seven times every 3-5 days over three weeks, and a pre-fusion booster was administered the day before tissue collection, after which lymph nodes were excised. In vivo experiments were conducted in accordance with the rules of the Bristol-Myers Squibb Company's Animal Welfare and Use Committee. Lymph nodes were extracted from three immunized mice, homogenized, and stored in liquid. Mouse myeloma cells (SP2 / 0-Ag14(ATCC CRL-1581) TM Hybridomas were produced by electrical fusion with )). The fused cells were allowed to stand for 7 days in a multi-well plate containing selective HAT medium, and then screened by ELISA to determine which cells bound to the antigen. Based on these results, hybridoma clones 1H2, 9C8, 24H1, and 26D5 were selected from fusion cell 6938 for further analysis, subcloned, and sequenced. The subcloned hybridomas were expanded into 400 mL of culture medium for purification. The secreted fully human antibodies were purified by protein A affinity chromatography. From the hybridoma subclones, 1873.6938.26D5.D12 (referred to herein as "26D5"), which had the highest capacity to produce fully human antibodies, was ultimately used for affinity maturation.

[0179] Example 3: Antibody affinity maturation To improve affinity for compound A, affinity maturation was performed using human mAb 26D5. First, the sequence of mAb 26D5 was compared with the closest human germline V and J gene sequences (Figure 1A-B). To minimize the risk of immunogenicity in humans, different framework regions were mutated depending on the germline, or the germline sequence was restored to produce human mAb 26D5-GV-Q (this fully human antibody form is also referred to as P1-072226) that retained similar binding to compound A. Using mAb 26D5-GV-Q (P1-072226) as the starting point for mutation scanning, all possible amino acid substitutions and their relative fitness were measured at the CDR position of the antibody shown in Figure 2. The "deep mutation scanning" technique is described in Araya et al., Trends in Biotechnology 29:435, 2001; Forsynth et al., mAbs 5:523, 2013; and Wrenbeck et al., Curr. Opin. Struct. Biol. 45:36, 2017. First, libraries of scFv (single-chain) mutants with amino acid substitutions were created using NNK oligonucleotides in the CDR shown in Figure 2. In creating these single-chain mutant libraries, multiple oligonucleotides were designed, each containing an NNK codon (where N=A, C, G, T, and K=G, T) at one location. By using these degenerate codons, it is possible to encode all 20 naturally occurring amino acids (and stop codons) at the location where the NNK codon is incorporated. Except for HCDR1, which was analyzed using the AbM definition (Abhinandan and Martin, Mol. Immunol. 45:3832, 2008; Swindells et al., J. Mol. Biol. 429:356, 2017), all CDRs were analyzed using the Kabat definition. Furthermore, amino acids 60-65 of HCDR2 were not analyzed.Next, the first in vitro transcription and translation of the DNA library was performed by binding the encoding mRNA to its own scFv protein molecule via puromycin using the mRNA display method (Xu et al., Chemistry & Biology 9: 933, 2002; Roberts and Szostak, Proc. Natl. Acad. Sci. USA 94:12297, 1997; Kurz et al., Nucleic Acids Res. 28: E83, 2000). Of the scFv bound to compound 5 (biotin-labeled compound 2), the scFv that interacted with streptavidin magnetic particles and eluted were selected and amplified by PCR. Finally, both the initially prepared DNA library and the eluted DNA encoding the scFv bound to compound 5 were sequenced by next-generation sequencing (NGS).

[0180] To analyze the NGS data, sequences read from both sides in forward and reverse directions by NGS were organized using FLASH (Magoc and Salzberg, Bioinformatics 27:2957, 2011) and classified into segments according to the mutation site and the identity of the mutated amino acid. During the analysis, all low-quality sequences and sequences with multiple mutations were excluded. Next, the enrichment efficiency (ER) was calculated by dividing the frequency of each sequence before selection by the frequency after selection. In other words, enrichment efficiency is the number of specific sequence variants in the compound 5-bound sample divided by the number of specific sequence variants in the original library. This was then normalized to the enrichment efficiency of the parent strain 26D5-GV-Q mAb. This method was used to evaluate the effect of each amino acid substitution on binding to compound 5 in the CDR region described herein (and thus inferred binding to compound A). Figure 3A shows a heatmap of LCDR1 prepared using the analysis of mutation scan data, illustrating the relationship between the sequence and activity of a single amino acid substitution. Generally, the error in this method is approximately 2x. In other words, if the concentration efficiency (ER) value is between 0.5 and 2, it is considered a typical substitution, i.e., a substitution that maintains the binding properties to compound 5. An ER value of 2 or higher indicates easy binding, while an ER value of less than 0.5 indicates difficult binding. Figures 3B to 3E show heatmaps of CDRs other than LCDR1.

[0181] Based on enrichment efficiencies calculated from NGS data, variable region genes containing one or more preferred amino acid substitutions were synthesized and replicated in IgG expression vectors along with the Fc region and human LCκ region (CK) of human IgG1f. The vectors were transiently introduced into Expi293 HEK cells in small-scale (2 mL of medium) and purified using a protein A filter plate. The functional activity of the IgG protein was evaluated by surface plasmon resonance (SPR) (see data below). The sequences of the target antibodies identified according to this evaluation are shown in Figures 4A-B.

[0182] To perform further affinity maturation using three different library designs, the progeny 26D5-295-B08 was selected. In the first design, the residues shown in Figure 5 were randomized, and a second mutation scan was performed on 26D5-295-B08. In this second mutation scan, the non-CDR portion as defined by Kabat and / or AbM was randomized, focusing on the region adjacent to compound A in the cocrystal structure. Heatmaps obtained from the mutation scan of 26D5-295-B08 are shown in Figures 6A-F. As described above, variable region genes containing one and more preferred amino acid substitutions were synthesized and replicated into IgG1f expression vectors for use in assays. The target mAbs identified from the mutation scan of 26D5-295-B08 include 26D5-75202-343-A09, 26D5-75229-343-A10, 26D5-75203-343-B09, 26D5-75017-343-F04, and 26D5-75214-343-F06 (see Tables 1-3 for sequences). All human antibodies disclosed in the examples are in the form of IgG1f (e.g., SEQ ID NO: 200) and human LCκ (e.g., SEQ ID NO: 204) unless otherwise noted.

[0183] Furthermore, two complex libraries (partial and doped) were constructed based on 26D5-295-B08, with the target region further sufficiently randomized (Figures 7A-B). The partial library was designed to contain one or two substitutions for all amino acids (except cysteine ​​and methionine) in the CDR and adjacent framework region, including other modifications shown in Figure 7A. Sequences containing chemical interference were removed from the design and synthesized from oligonucleotide chips (Twist Bioscience). The doped library was intensively randomized to HCDR3 and surrounding framework region residues so that two or more mutations occurred in a single CDR. DNA oligonucleotides encoding this library were prepared by "doping" the randomized region based on the 26D5-295-B08 DNA sequence. Each nucleotide within the randomized region contained 70% of the 26D5-295-B08 DNA bases and 10% of other bases. For example, if the DNA 26D5-295-B08 has G at a specific position, the doped nucleotide will have 70% G, 10% A, 10% C, and 10% T at that position. The target region is more randomized, but still biased towards the original parent sequence library. Oligonucleotides are used to produce a library of partial DNA scFv and a library of doped DNA scFv. These libraries are selected using mRNA display as per the protocol above, but later includes off-rates and was performed multiple times consecutively. The resulting sequences were sequenced using NGS, and the data were analyzed to select variable regions for replication to synthetic and test IgG expression vectors. The target mAbs identified from a portion of the library include 26D5-75592-348-A04, 26D5-75768-348-A10, 26D5-75576-348-B03, 26D5-75746-348-C07, 26D5-75747-348-D07, and 26D5-75602-348-F04 (see Tables 1-3 for sequences). The mAb 26D5-75616-348-F10 was identified as the target antibody from a doped library.Figures 8A and 8B show the target sequence identified from the affinity-matured mAb of 26D5-295-B08.

[0184] To confirm results on a small scale, the expression vector of the target antibody was transiently introduced into Expi293 HEK cells in a 340 mL scale. The cells were purified using a pre-packed POROS A column (20 mL), the buffer was replaced with PBS using an Amicon 30K MWCO filter, and the samples were aseptically filtered through a PES filter (0.2 μm). The samples were then aliquoted and stored at -80°C. The molecular weight of each sample was confirmed by LC / MS and identified using analytical SEC.

[0185] Example 4: Identification of mAbs by chromogenic enzyme assay The activity of mAbs that maintain FXIa enzyme activity in the presence of compound A was used as an assay to screen for improved antibody affinity. In this assay, the S-2366 chromogenic peptide (Chromogenix) was used as the substrate for the factor XIa enzyme. Each test mAb was serially diluted from 100 nM to 1.5 nM and incubated with 2.5 nM of compound A or a control compound at 37°C for 10 minutes. Then, the chromogenic substrate (S-2366) was added at a final concentration of 0.5 mM, and human FXIa enzyme (Haematologic Technologies, Inc.; HCZIA-0160) was added at a final concentration of 0.2 nM. Compound A at a concentration of 2.5 nM inhibited ~90% of FXIa activity, i.e., almost all FXIa activity. The assay described herein was performed within a significant measurement range. The plates were immediately read using SPECTRAmax (Molecular Devices) (OD405nm, 37°C) to measure the hydrolysis rate of the substrate. The signals were normalized to 0% activity (with FXIa enzyme and compound A added) and 100% activity (with FXIa enzyme added without the inhibitor). The EC of the antibody that restored more than 50% of the inhibitory activity of compound A was measured. 50 This was determined. The results of the example are shown in Figure 9.

[0186] Example 5: Production of recombinant α-compound AFab fragment antibody The selected antibody was replicated as an untagged Fab fragment antibody using the pTT5 vector of the Expi293 expression system (see SEQ ID NO: Fab sequence). The DNA sequence optimized for mammalian expression was obtained from GenScript. For expression on a 1L scale, cells (900mL, 3x10) were used. 6 Cells ( / mL) were seeded into a 2L flask (Corning). 0.25 mg each of heavy-chain and light-chain DNA constructs were added to Opti-MEM. TM (50 mL) was added. ExpiFectamine TM (4.1 mL) / Opti-MEM TM Incubate (150 mL) at room temperature for 5 minutes, then add 50 mL of that 150 mL to DNA / Opti-MEM. TM The mixture was added and incubated at room temperature for 20 minutes. This total of 100 mL of transfection mixture was added to the aforementioned cell culture medium (900 mL) and placed in a shaker (125 rpm, 28% CO2 in air) at 37°C. On day 1, the product was given VPA (2 mM) and CHO CD EfficientFeed B (50 mL). Cell viability was analyzed, and the product was obtained on day 5. The mean cell viability / cell density was 80% / 6x10⁻⁶. 6The cell count was 1 / mL. The product was centrifuged at 4°C for 20 minutes (2,000 rpm), and the supernatant of the acclimatization medium was filtered through a 0.2 μm filter. A 30 mL rProteinA Sepharose FF column was washed with 2 CV (column volume) of 6 M guanidine and 2 CV of 0.033 M hydrochloric acid, and then equilibrated with 1x PBS (Dulbecco). Ensured that the pH of the supernatant was >7.0, it was then loaded onto the rProteinA Sepharose column at 10 mL / min. All progeny of 1873.6938.26D5.D12, including affinity matured variants, bound to rProteinA via the framework region through interaction with protein A. The column was washed with 1x DPBS until baseline was reached, then with 80 mM sodium acetate (pH 2.8), and collected in a container filled with Tris HCl (pH 8.0, ~20 mL) so that the protein would be neutralized during elution. The column was then neutralized with 1x DPBS. The eluted sample was concentrated to <10 mL and loaded onto an S200 26 / 600 column equilibrated with 1x DPBS at 2.5 mL / min. A fraction (5 mL) was collected at 2.5 mL / min and analyzed by SDS-PAGE and chromatography. The purified Fab fragment antibody yielded an average of 150–250 mg / L.

[0187] Example 6: Surface Plasmon Resonance (SPR) (Biacore) TM Identification of mAbs and Fabs through analysis Hybridomas expressing the parental 26D5 mAb (in the form of IgG1f, VH SEQ ID NO: 83 and VL SEQ ID NO: 98, P1-072224) matured in binding and affinity to compound A, and SPR (Biacore) was used to utilize the binding to protein A. TMThe assay was performed using the following method. 1x PBS (phosphate-buffered saline, pH 7.4) containing 0.05% Tween 20 and 2% DMSO was used as the running buffer. The binding assay was performed at 37°C. Protein A was densely coated onto a sensor tip (CM5, S series, Cytiva, Cat. No. 29149603) (RU of protein A: 2000+). Protein A was immobilized using the manufacturer's recommended standard amine coupling method. Subsequently, a 2 ug / mL affinity mature antibody was conjugated to the surface of protein A at a flow rate of 3 μL / min for 2 minutes. Next, compound A at concentrations ranging from 100 to 3 nM (100, 50, 25, 12.5, 6.25, 3.125 nm) (Figure 10) and compound A at screening concentrations ranging from 100 to 3 nM (100, 33, 11 nm) were injected onto the bound antibody at a flow rate of 100 μL / min for 2 minutes, and dissociation was allowed for at least 450 seconds. The chip surface was regenerated by a 40-second pulse of 10 mM glycine (pH 1.5) in each cycle. Background of binding only to the protein A surface was used to subtract nonspecific binding. All experiments were performed using Biacore T200 surface plasmon resonance with Biacore T200 control software v.2, and data analysis was performed using Biacore T200 evaluation software v3.1. For ranking, since the dissociation rate was very slow and could not be measured with Biacore, only clear affinity was determined. The binding assay data for the examples are shown in Figure 10 and Table 10. The mAb names in Table 10 represent mAbs in the form of IgG1f having the H and L chain sequences of the corresponding Fab in Table 3. [Table 49] [Table 50]

[0188] Using a sensor chip (CM5, S series, Cytiva, Cat. No. 29149603) pre-coated with BSA bound to compound 2, compound 3, or compound 1, the first affinity-matured Fab fragment antibody was bound to the compound A-bound version via SPR (Biacore). TM The study was conducted using the following method. The fixation level was 150-250 RU. 1x PBS (phosphate-buffered saline, pH 7.4) with 0.05% Tween 20 added was used as the running buffer, and the experiment was performed at 25°C. Fab fragment antibodies at concentrations between 200 and 0.8 nM were injected into a BSA-compound coated surface at a flow rate of 30 μL / min for 2 minutes. The Fab fragment antibodies were then dissociated for 15 minutes. The chip surface was regenerated with a 1-minute pulse of 10 mM glycine (pH 2) and a 1-minute pulse of NaOH (50 mM) in each cycle. All experiments were performed using the same instrument and software as described above. For ranking, the dissociation rate was very slow and could not be measured with Biacore, so only clear affinity was determined. Comparative experiments were also performed similarly with the selected mAbs (see Figure 11). Figure 11 shows the binding assay data for the examples, and Table 11 shows the binding assay data for compound 2-BSA. The sequence of Fab in Table 11 is shown in Table 3. [ka]

[0189] The first and second affinity-matured Fab fragment antibody binding to the compound A-bound version was performed using a biotin CAP sensor chip (S series) and kit reagent (Cytiva, Cat No. 28920234) with SPR (Biacore). TMThe biotin CAP chip was investigated using the following method. The biotin CAP chip was hydrated overnight with buffer. A 50% aqueous solution of biotin CAP reagent was flowed over the chip surface at a flow rate of 2 μL / min for 150 seconds. Compound 5 (biotin-labeled compound 2) at a concentration of 0.25 ug / mL was bound to the biotin CAP surface with a pulse at a flow rate of 10 μL / min for 20 seconds. Experiments were conducted at 25°C or 37°C using 1x PBS (phosphate-buffered saline, pH 7.4) with 0.05% Tween 20 added to the running buffer. Fab fragment antibody at concentrations of 100-3 nM was injected onto the biotin CAP-compound 5 surface at a flow rate of 30 μL / min for 3 minutes. The Fab fragment antibody was then dissociated for 11.7 minutes. The chip surface was regenerated with a 2-minute pulse of 6 M guanidine-HCl / NaOH (250 mM) in each cycle. All experiments were performed using the same instrument and analyzed with the same software as described above. In ranking the antibodies, the dissociation rate was extremely slow and could not be measured with Biacore, so only clear affinity was determined. Figure 12 shows the binding assay data for the examples, and Table 12 shows the binding assay data for the TanFab fragment antibody. The TanFab sequences in Table 12 are shown in Table 4. [Table 51]

[0190] Example 7: Identification of mAbs and Fabs by TR-FRET assay A competitive TR-FRET (time-resolved fluorescence-resonance energy transfer) assay was developed to rank the rates of dissociation of compound A from α-compound A mAb, which reaches equilibrium at 37°C. The assay buffers used were HBS-N (10 mM HEPES, 150 mM NaCl, pH 7.4; GE Healthcare), 0.1% (w / v) BSA (bovine serum albumin; Sigma), and 2% DMSO (dimethyl sulfoxide; Sigma). All reagents were prepared in the assay buffers and uniformly dispensed into white 384 microplates to a final volume of 20 μL / well. The final concentrations were 4 nM for compound 5 (biotin-labeled compound 2), 100-0.1 nM for 7 drops of human α-compound A antibody, 0.1 nM for europium-labeled α-mouse IgG (LANCE Eu-W1024; PerkinElmer), 5 nM for streptavidin-D2 (Cisbio), and 4 nM for mAb 26D5 VH_A10G_Y33A_S53P_M89V_G95A; VK_W32N_H38Q (P1-075621, the second optimization of the parent strain 26D5-295-B08, which was converted to the Fc form of mouse IgG). First, compound 5 and α-compound A antibody were dropped onto a microplate, and the first incubation (37°C, 1 hour, 1000 rpm) was performed to promote antibody binding to the compounds. After the first incubation, europium α-mouse IgG, streptavidin-D2, and 26D5-mouse IgG (P1-075621) were added sequentially, and the plate was incubated again (37°C, 1000 rpm). The microplate was read using an EnVision plate reader (PerkinElmer), and the measured FRET signal was defined as [665 nm] / [620 nm]*10,000. The microplate was read at 30 minutes (T0) and 24 hours (T24). In the titration of each antibody, the FRET signal was converted to the inhibition rate (%) (100 - ((FRET at antibody concentration / FRET at 0 nM)*100) compared to the well without α-compound A antibody. The inhibition rate % in antibody titration was plotted using TIBCO Spotfire (v.7), and IC was performed using a four-variable model curve fitting. 50 The IC at each point in time was decided. 50, and IC compared with T0 50 A curve shift was shown. IC 50 The curve shift indicates that as the assay reaches equilibrium, the α-compound A antibody dissociates from the d-biotin-labeled compound, which then allows the competing molecule 26D5-mouse IgG1 (P1-075621) to bind, emitting the FRET signal detected in the assay.

[0191] Figure 13 shows the competitive FRET data obtained from affinity optimization screening of α-compound A 26D5. Three representative human antibodies were used: 1) 26D5-GVR-Q-FT-Fab-LONG (also referred to herein as P1-072226, except that R31 in 26D5-GVR-Q-FT HC is S31 in P1-072226 HC; see upper graph), 2) 26D5-295-B08-Fab-LONG (also referred to herein as P1-072963; see middle graph), a descendant of the first optimization of the IgG1f mAb form, and 3) 26D5-75747-348-D07-Fab-LONG (also referred to herein as P1-075747; see lower graph), a descendant of the second optimization of the IgG1f mAb form. The IC25 titration curves for each antibody were shown. 50 These are indicated by an asterisk. IC between T0 and T24 50 The difference is denoted as Δ, and its value is shown in each graph. The parent strain 26D5-GVR-Q-FT-Fab-LONG mAb represents the largest IC from T0 to T24. 50 The shift was 63 nM. With each repeated affinity maturation, the dissociation rate of compound A improved in the next progeny, and the IC of T0~T24 50 The shift was reduced (P1-072963=20.5nM, P1-075747=4.3nm). α-compound A antibody was used in the IC of T24. 50 and IC 50 The antibodies were identified by ranking them by shift (Δ) and measuring the dissociation rate of compound A against the parental strains mAb 26D5-GVR-Q-FT-Fab-LONG and mAb 26D5-295-B08 (P1-072963), which improved at 37°C.

[0192] The competitive TR-FRET assay defined above was reconstructed as Fab and further modified to evaluate the progeny of the optimized 26D5 antibody. The titration range for α-compound A antibody and Fab was expanded to 250–0.244 nM (11 drops of titration), and each concentration was reproduced four times. Furthermore, to ensure equilibrium was reached in the assay, the incubation time was set to 48 hours, and T48 IC was performed. 50 and IC 50 The shift was measured. The inhibition % titration was plotted using Graphpad Prism (v.8) and fitted with a four-variable model. All other experimental conditions were the same as those for the antibody TF-FRET assay.

[0193] Competitive FRET data obtained from affinity-optimized α-compound A progeny 26D5 are shown in Figure 14 and Table 13, compared with human antibodies and Fab. Each graph in Figure 14 shows the FRET data of α-compound A progeny 26D5, with antibodies (mAb, solid line, ○) and Fab (dashed line, △) having the same sequence superimposed. The Fab of the 26D5 parent strain (Fab 26D5-GV-Q; also referred to herein as P1-073708) and the first optimized progeny Fab26D5-295-C08 (also referred to herein as P1-074468-1) showed T48 IC 50 The shift is at least 26 nM, and the IC is remarkable. 50 A shift was observed. After a second affinity maturation, the optimized progeny slowly dissociated from compound A at 37°C in both antibody and Fab forms, with minimal T48 IC. 50 The shifts are shown. The FRET data is shown in Table 13, and the mAb / Fab names in Table 13 refer to the corresponding sequences in Tables 1-3. All mAbs listed in Table 13 are in the form of human IgG1f / LCκ. [Table 52]

[0194] Example 8: Gel Filtration Using an Agilent 1260 HPLC (Shodex K403-4F, mobile phase: 100 mM sodium phosphate, 150 mM sodium chloride, pH 7.3, flow rate 0.3 mL / min), a portion of the purified Fab (20 μg) was injected and eluted over 20 minutes. Gel filtration using standard substances confirmed that at least 98% of the Fab was monomer, and the recovery rate exceeded 75%.

[0195] Example 9: Thermal stability analysis of Fab Thermal stability analysis was performed on 20 μM Fab using UNcle™ / Tagg (UNchained Labs) in the presence or absence of 50 μM compound A (or control compound). The capillary was scanned at 0.5 °C / min in the range of 25–90 °C, and selected Fab was further analyzed by differential scanning calorimetry using MicroCal VP-Capillary DSC (Malvern). Samples were dissolved in a suitable buffer and loaded together with 10 μM Fab in the presence or absence of 15 μM compound A (or control compound). The sample was scanned in the range of 15–110 °C at a rate of 60 °C / Hr (Filter period: 16 sec, Gain: None). Software provided by the manufacturers of UNcle and Capillary DSC (Malvern) was used for all sample analyses. Figure 15 and Table 14 below show representative DSC results. The sequences of the Fab in Table 14 are shown in Table 3. [Table 53]

[0196] Example 10: Kinetic Exclusion Method (KinExA) The solution affinity of the Fab fragment antibody of this disclosure to compound A was measured by kinetic exclusion (KinExA). Double titration of compound A was performed using 100, 200, and 300 pM 26D5 affinity mature Fab fragment antibodies (equilibriumization over 24–72 hours). The relative concentration of unbound 26D5 affinity mature Fab fragment antibody was detected by capturing the antibody with compound 5 bound to streptavidin-coated beads, followed by detection with a fluorescently labeled antibody that recognizes human IgG Fab. The assay was the same as the kinetic analysis used to determine the association rate of the complex, except that one test tube of the mixture (200 pM Fab and 400 pM compound A) was prepared and equilibration was not performed. The results are shown in Table 15 below. DSC thermal stability analysis and KinExA revealed that two Fabs, 26D5-75229-343-A10-Fab-SHORT and 26D5-75616-348-F10-Fab-SHORT, were the two most preferred Fabs. For the X-ray crystallography and in vivo experiments described in Example 12 below, the Fab fragment antibody 26D5-75616-348-F10-Fab-SHORT (SEQ ID NO: 106 and SEQ ID NO: 164; see Table 3) was selected. [Table 54]

[0197] Example 11: Crystallization of Fab Fab26D5-GVR-Q-FT-Fab-SHORT, possessing the affinity tag GGHHHHHHH (SEQ ID NO: 222), was concentrated to 10 mg / mL in DPBS (Dulbeccio phosphate-buffered saline). This protein was conjugated with a 5-fold excess of compound A and incubated overnight at 4°C. The complex was crystallized by vapor diffusion. Each dropper consisted of 1 μL of the complex and 1 μL of the reservoir solution. The reservoir solution consisted of 100 mL of a 20 g PEG 3350 solution in water and 20 mM sodium citrate (not a buffer). Crystals were prepared by serially diluting 2.5 μL of a 40% PEG400:40% glycerol (v / v) mixture and 7.5 μL of the reservoir solution and adding them to the dropper solution for rapid cooling with liquid nitrogen.

[0198] The Fab fragment antibody 26D5-75616-348-F10-Fab-SHORT was concentrated to 20 mg / mL in DPBS buffer. This protein was conjugated with compound A in excess at a 5-fold molar concentration and incubated overnight at 4°C. This complex was crystallized by vapor diffusion. Each drop consisted of 1 μL of the complex and 1 μL of the reservoir solution. The crystallization reservoir solution consisted of 100 mM CAPS (N-cyclohexyl-3-aminopropanesulfonic acid, pH 10.5), 200 mM lithium sulfate, 1.2 M sodium phosphate, and 0.8 M potassium phosphate. To rapidly cool the crystals with liquid nitrogen, 2.5 μL of a 40% PEG400:40% glycerol (v / v) mixture and 7.5 μL of the reservoir solution were added to the dropwise solution in serial dilutions.

[0199] Data was obtained using PILATUS 6M (Advanced Photon Source, beamline: 17-ID). The data was processed using autoPROC [Vonrhein, C., Flensburg, C., Keller, P., Sharff, A., Smart, O., Paciorek, W., Womack, T. & Bricogne, G. (2011). Data processing and analysis with the autoPROC toolbox. Acta Crystallogr. Sect. D 67, 293-302], as well as the basic processing software XDS, the scaling software XSCALE, and the anisotropic data software STARANISO [W. Kabsch (2010). XDS. Acta Crystallogr. Sect. D 66, 125-132 and W. Kabsch (2010). Integration, scaling, space-group assignment and post-refinement. Acta Crystallogr. Sect. D 66, 133-144; STARANISO (Tickle, IJ, Flensburg, C., Keller, P., Paciorek, W., Sharff, A., Vonrhein, C., Bricogne, G. (2018). STARANISO (available on the internet at staraniso.globalphasing.org / cgi-bin / staraniso.cgi) Cambridge, United Kingdom: Global Phasing Ltd.

[0200] The crystal of compound A (26D5-GVR-Q-FT-Fab-SHORT) consisted of one asymmetric unit per complex, with a space group of P212121 and unit cell edges of a=57.7Å; b=75.1Å; c=84.7Å. The data was expanded by 1.47Å by isotropic processing, but to preserve the data, it was cut with ellipsoids that expanded a* by 1.38Å, b* by 1.44Å, and c* by 1.32Å. Molecular substitution using PHASER (McCoy, AJ, Grosse-Kunstleve, RW, Adams, PD, Winn, MD, Storoni, LC & Read, RJ (2007). Phaser Crystallographic Software. J. Appl. Crystallogr. 40, 658-674.) yielded the CL:CH1 model from PDB's 2O5X (Verdino, P., Aldag, C., Hilvert, D., Wilson, IA (2008) Closely Related Antibody Receptors Exploit Fundamentally Different Strategies for Steroid Recognition. Proc. Natl. Acad. Sci., USA 105, 11725-11730), and the VL model from PDB's 4PY7 (Wyrzucki, A., Dreyfus, C., Kohler, I., Steck, M., Wilson, IA, The structure was determined using Hangartner, L. (2014). Alternative Recognition of the Conserved Stem Epitope In Influenza A Virus Hemagglutinin By A VH3-30-Encoded Heterosubtypic Antibody. J. Virol. 88, 7083-7092), and the VH model obtained from the PDB's 4TSA (Wensley, B. Structure of a Lysozyme Fab Complex, unpublished). All CDRs (complementarity-determining regions) were removed from the VH and VL models.The initial electron density map clearly shows the electron density of compound A. The ligands, initially positioned using RHOFIT (Womack, TO, Smart, OS, Sharff, A., Flensburg, C., Keller, P., Paciorek, W, Vonrhein, C. and Bricogne, G., Global Phasing, Ltd., Cambridge, United Kingdom), were geometrically restricted using GRADE (Smart, OS, Womack, TO, Sharff, A., Flensburg, C., Keller, P., Paciorek, W., Vonrhein, C. and Bricogne, G., Global Phasing, Ltd., Cambridge, United Kingdom). The structure was improved by alternating between model construction using Coot (Emsley, P., Lokhamp, B., Scott, WG & Cowtan, K. (2010). Features and Development of Coot. Acta Crystallogr Sect. D 66, 486-501) and refinement using autoBUSTER (Bricogne, G., Blanc, E., Brandl, M., Flensburg, C , Keller, P., Paciorek, W., Roversi, P, Sharff, A., Smart, O., Vonrhein, C , Womack, T. BUSTER version 2.11.7. Global Phasing, Ltd., Cambridge, United Kingdom). Figure 16 shows the precise structure of the 26D5-GVR-Q-FT-Fab-SHORT / compound A complex.

[0201] The crystal of compound A (26D5-75616-348-F10-Fab-SHORT) consisted of one asymmetric unit for every four complexes, with space group P1 and unit cell edges a=64.8Å; b=84.9Å; c=100.9Å; α=83.4°; β=88.4°; γ=67.9°. The data was expanded by 2.7Å by isotropic processing, but to preserve the data, it was cut by ellipsoids expanding by 1.91Å at 0.880a*+0.436b*-0.189c*, 2.20Å at 0.063a*+0.898b*-0.436c*, and 2.98Å at 0.093a*+0.382b*+0.920c*. The structures were determined using CL:CH1, VL, and VH models (excluding CDR-H3) obtained from the Fab fragment antibody 26D5-GVR-Q-FT-Fab-SHORT by molecular substitution using a phaser (McCoy, AJ, Grosse-Kunstleve, RW, Adams, PD, Winn, MD, Storoni, LC & Read, RJ (2007). Phaser Crystallographic Software. J. Appl. Crystallogr. 40, 658-674). The first electron density map shows the electron density of compound A.Model construction using Coot (Emsley, P., Lokhamp, B., Scott, WG & Cowtan, K. (2010). Features and Development of Coot. Acta Crystallogr Sect. D 66, 486-501), and autoBUSTER (Bricogne, G., Blanc, E., Brandl, M., Flensburg, C., Keller, P., Paciorek, W., Roversi, P, Sharff, A., Smart, O., Vonrhein, C. & Womack, T. BUSTER version 2.11.7. Global Phasing, Ltd., Cambridge, United Kingdom and Smart, OS Womack, TO, Flensburg, C., Keller, P., Paciorek, W., Sharff, A., Vonrhein, C. & Bricogne, G. (2012). Exploiting structure similarity in The structure was improved by alternating refinement using automated NCS and target-structure restraints in BUSTER (Acta Crystallogr Sect. D 68, 368-380). Figure 17 shows the precise structure of the 26D5-75616-348-F10-Fab-SHORT / compound A complex.

[0202] DSC thermal stability analysis and KinExA revealed that 26D5-75229-343-A10-Fab-SHORT and 26D5-75616-348-F10-Fab-SHORT were the two most preferred Fabs. X-ray crystallography of the Fab fragment antibody 26D5-75616-348-F10-Fab-SHORT demonstrated a 1:1 stoichiometric binding ratio between compound A and the Fab fragment antibody, and revealed the binding mechanism between the heavy and light chains of the Fab fragment antibody.

[0203] Example 12: Plasma binding test of Fab fragment antibody A study was conducted to evaluate the efficacy of 26D5-75616-348-F10-Fab-SHORT against the anticoagulant effect of milbexian in plasma.

[0204] In vitro experiment The effect of the Fab fragment antibody 26D5-75616-348-F10-Fab-SHORT in neutralizing the in vitro anticoagulant activity of compound A, a factor XIa inhibitor, was determined by combining Fab fragment with plasma containing a known amount of compound A. Plasma concentrations of compound A that significantly increased aPTT were selected. A significant increase can be defined as a coagulation time that is at least 20% longer compared to the coagulation time (e.g., aPTT) in the absence of the factor XIa inhibitor. Antibodies or Fab fragment antibodies that can bind to factor XIa inhibitors in plasma reduce the binding ability of the factor XIa inhibitor to factor XIa, resulting in a shortened coagulation time compared to the coagulation time (e.g., aPTT) in the absence of the antibody or Fab fragment antibody.

[0205] Compound A was added to stored healthy human plasma at concentrations of 8000, 4000, 2000, 1000, 500, 250, 125, 62.5, 31.3, and 15.6 nM. Fab fragment antibody 26D5-75616-348-F10-Fab-SHORT was added to the same stored healthy human plasma at concentrations of 8000, 4000, 2000, 1000, 500, 250, 125, 62.5, 31.3, and 15.6 nM. Plasma containing compound A, plasma containing Fab fragment antibody 26D5-75616-348-F10-Fab-SHORT, and normal plasma were combined and adjusted to various concentrations (molar ratios of compound A (including compound A in the absence of Fab fragment antibody 26D5-75616-348-F10-Fab-SHORT): 26D5-75616-348-F10-Fab-SHORT: 5:5, 5:4, 5:3, and 5:2, etc.).

[0206] Using ACTIN® FS (Siemens / Dade-Behring), the activated partial thromboplastin time (aPTT), i.e., human plasma coagulation time, was determined for each sample according to the provided instructions. For a description of the aPTT assay, see Goodnight, SH et al., "Screening Tests of Hemostasis", Disorders of Thrombosis and Hemostasis: A Clinical Guide, 2nd Edition, pp. 41-51, McGraw-Hill, New York (2001). Plasma (0.05 mL) was incubated at 37°C for 1 minute. ACTIN® FS (0.05 mL) was added to the plasma and incubated for a further 3 minutes. Calcium chloride (25 mM, 0.05 mL) was added to the reaction system to initiate coagulation. Coagulation time refers to the time (in seconds) from the moment calcium chloride is added until a thrombus is detected.

[0207] Figure 18 shows plasma clotting time (aPTT) as a function of the concentration of the factor XIa inhibitor (i.e., compound A) and the concentration of the neutralizing Fab fragment antibody 26D5-75616-348-F10-Fab-SHORT. Figure 19 shows plasma clotting time (aPTT) as a function of the concentration of free factor XIa inhibitor (compound A) in the absence (black circles) and presence (white circles) of the neutralizing Fab fragment antibody 26D5-75616-348-F10-Fab-SHORT. Clotting time (aPTT) is plotted as a function of the plasma concentration of free compound A in the absence and presence of the Fab fragment antibody 26D5-75616-348-F10-Fab-SHORT.

[0208] Human plasma samples containing compound A and Fab fragment antibody 26D5-75616-348-F10-Fab-SHORT at various concentrations and molar ratios were processed, and the concentrations of conjugated and unconjugated compound A and Fab fragment antibody 26D5-75616-348-F10-Fab-SHORT in each sample were determined, as detailed below.

[0209] The total concentration of compound A in plasma refers to the sum of free compound A, compound A bound to plasma proteins, and compound A bound to the Fab fragment antibody 26D5-75616-348-F10-Fab-SHORT. Free compound A / unbound compound A refers to compound A that is not bound to plasma proteins or the Fab fragment antibody 26D5-75616-348-F10-Fab-SHORT.

[0210] Free compound A was obtained from plasma by ultrafiltration (Centrifree (trademark), MilliporeSigma, molecular weight cutoff: 30 kDa). 0.5 mL of plasma was added to the top of an ultrafiltration column (Centrifree (trademark), Ultracel PL membrane, REF# 4104; MilliporeSigma). The column was placed in a centrifuge (SORVALL SLA-3000; Thermo Scientific) and centrifuged at 2,000 x g for 20 minutes (SORVALL RC 6 Plus; Thermo Scientific), after which the ultrafiltrate was collected.

[0211] Plasma and a portion of the plasma ultrafiltrate were frozen at -80°C in polypropylene tubes. The total concentration of compound A in the plasma and free compound A in the plasma ultrafiltrate was measured by liquid chromatography-tandem mass spectrometry (LC / MS). Samples for LC / MS analysis were prepared using the protein precipitation method described below.

[0212] A portion of the biological sample (20 μL) was transferred to a 96-well deep plate (1.2 mL, round-bottom, polypropylene). A methanol solution containing 50% water and 0.5% formic acid (20 μL) was added. The plate was covered and mixed in a shaker at 95°C for 20 minutes. An acetonitrile solution containing an internal standard [10 nM, stable isotope (13C,15N) labeled compound A] and 1% formic acid (80 μL) was added to the above solution to precipitate the protein. The plate was further vortexed at room temperature for 15 minutes, and then centrifuged at 3,600 rpm for 5 minutes. A portion of the supernatant (100 μL) was transferred to an injection plate (96 wells, 0.3 mL). The supernatant (5 μL) was injected into an Ultra Performance LC System (Waters® Acquity UPLC) tandem mass spectrometer connected to a QTRAP MS / MS (AB Sciex 6500). The substances to be measured were separated using a C18 column (Waters HSS T3, 2 x 50 mm, 1.8 μm) at 60°C using a gradient (flow rate 0.7 mL / min) consisting of two buffer solutions (A: water + 0.1% formic acid; B: acetonitrile + 0.1% formic acid). The precursor (M+H) was analyzed using positive electrospray ionization mode multiple reaction monitoring (MRM). + The substance was detected. MRM transitions were observed in compound A (626→319) and in isotope-labeled compound A (630→323). The limit of quantification was 0.5 nM.

[0213] The plasma concentration of the Fab fragment antibody 26D5-75616-348-F10-Fab-SHORT was determined as follows: The total plasma concentration of both the Fab fragment antibody 26D5-75616-348-F10-Fab-SHORT and the Fab fragment antibody 26D5-75616-348-F10-Fab-SHORT not bound to compound A was measured by ligand binding assay using a Gyrolab® automated microfluidic system (Gyros Protein Technologies AB). Biotinylated mouse anti-human κ (SouthernBiotech, AL) was used as the molecule to bind to all Fab fragment antibodies 26D5-75616-348-F10-Fab-SHORT. Samples, standards, and QCs were increased to the final baseline concentration of 10% plasma / 1xPTB (1% BSA / 0.05% Tween20 / PBS) and loaded into the Gyrolab® automated microfluidic system. The 3-step-2-wash wizard method of the Gyrolab (trademark) Bioaffy 200 CD (Gyros Protein Technologies AB) was used. After the final wash step, all conjugated Fab fragment antibodies 26D5-75616-348-F10-Fab-SHORT were detected using clone G20-361 (BD Catalog No. 555861, Lot No. 8333691) of Alexa Fluor (trademark) 647-labeled mouse anti-human Igκ light chain mAb. The fluorescence intensity was measured using Gyrolab (trademark) with a 4-coefficient logistic curve prepared from a standard of Fab fragment antibody 26D5-75616-348-F10-Fab-SHORT, and the total concentration of Fab fragment antibody 26D5-75616-348-F10-Fab-SHORT in the plasma sample was calculated. The total calibration curve range for the Fab fragment antibody 26D5-75616-348-F10-Fab-SHORT was 250–25000 ng / mL (in plasma). The upper and lower limits of quantification were 25000 and 250 ng / mL, respectively. Quality control samples were prepared at plasma concentrations of 20000, 7500, and 750 ng / mL.To ensure that the assays were reliably acceptable, the reference material and quality control (QC) were analyzed in each experiment. The assays were performed within acceptable limits (%CV of the reference material and QC was less than 20%, and QC recovery was within ±20% of the apparent value).

[0214] Compound 5 was used as a molecule to bind to the Fab fragment antibody 26D5-75616-348-F10-Fab-SHORT, which was not bound to compound A. Samples, standards, and QC were increased to the final baseline concentration of 10% plasma / 1xPTB (1% BSA / 0.05% Tween20 / PBS) and loaded into a Gyrolab® automated microfluidic system. The 3-step-2-wash wizard method of the Gyrolab® Bioaffy 200 CD was used. After the final wash step, the bound “active / free” Fab fragment antibody 26D5-75616-348-F10-Fab-SHORT was detected using Alexa Fluor® 647-labeled mouse anti-human Igκ light chain mAb clone G20-361 (BD Catalog No. 555861, Lot No. 8333691). The fluorescence intensity was measured using Gyrolab (trademark registered) with a four-coefficient logistic curve prepared from a standard substance of the Fab fragment antibody 26D5-75616-348-F10-Fab-SHORT, and the concentration of "active / free" Fab (26D5-75616-348-F10-Fab-SHORT) in plasma samples was calculated. The calibration curve range for the "active / free" Fab fragment antibody 26D5-75616-348-F10-Fab-SHORT was 250 to 25000 ng / mL (in plasma). The upper and lower limits of quantification were 25000 and 250 ng / mL, respectively. Quality control samples were prepared at plasma concentrations of 20000, 7500, and 750 ng / mL. Standard substances and QC were analyzed in each experiment to ensure that the assay was reliably acceptable. The assay was performed within acceptable limits (%CV of the standard substance and QC was less than 20%, and QC recovery was within ±20% of the apparent value).

[0215] In vivo experiment In vivo experiments were conducted in accordance with the rules of the Animal Welfare and Management Committee within Bristol-Myers Squibb Company. Indwelling catheters were placed in the central auricular artery (for blood collection) and marginal auricular vein (for substance administration) of male rabbits (New Zealand White breed, 2-4 kg). A fixed amount of compound A (1.0 mg / kg) was administered intravenously by drip infusion over 10 minutes. Twenty minutes after the completion of the drip infusion of compound A, a fixed amount of Fab fragment antibody 26D5-75616-348-F10-Fab-SHORT (160 mg / kg) was administered intravenously by drip infusion over 10 minutes. The administered Fab fragment antibody 26D5-75616-348-F10-Fab-SHORT appeared to be administered at a molar concentration twice that of compound A. Blood samples (1.5 mL) were collected at various intervals: before administration of compound A, at the end of compound A infusion, immediately before administration of Fab fragment antibody, at the end of Fab fragment antibody administration, and after administration of Fab fragment antibody and up to 24 hours after the start of compound A administration. The blood samples were added to 3.8% sodium citrate (0.167 mL) in a polypropylene tube, thoroughly mixed by inverting at least twice, and left to stand on ice. Within one hour of blood collection, plasma was isolated by centrifugation of whole blood at at least 1,500 x g for at least 10 minutes. Free compound A was obtained by ultrafiltration as described above.

[0216] The anticoagulant effect of compound A was evaluated using activated partial thromboplastin time (aPTT). aPTT was determined using ACTIN® FS (Siemens / Dade-Behring) according to the provided instructions. Plasma (0.05 mL) was incubated at 37°C for 1 minute. ACTIN® FS (0.05 mL) was added to the plasma and incubated for a further 3 minutes. Calcium chloride (25 mM, 0.05 mL) was added to the reaction system to initiate coagulation. Coagulation time refers to the time (in seconds) from the moment calcium chloride was added until a thrombus was detected.

[0217] In vivo administration of compound A resulted in approximately a twofold increase in the plasma aPTT of rabbits compared to baseline. Subsequently, administration of the Fab fragment antibody 26D5-75616-348-F10-Fab-SHORT 20 minutes later restored the rabbits' plasma aPTT to baseline, a state that lasted for more than 12 hours. Figure 20 shows the plasma coagulation time (aPTT) of rabbits 20 minutes after intravenous administration of compound A (1 mg / kg) followed by intravenous administration of the Fab fragment antibody 26D5-75616-348-F10-Fab-SHORT (160 mg / kg). The results are the average of three rabbits.

[0218] Plasma concentrations of compound A (total and free) and the Fab fragment antibody 26D5-75616-348-F10-Fab-SHORT were determined as described above. PK parameters were obtained from non-compartmental analysis data of plasma concentration and time (Phoenix WinNonlin software, Version 6.4, Pharsight Corporation, Mountain View, CA). Values ​​below the lower limit of quantification were not used in the calculations. The area under the blood concentration-time curve (AUC [0-T]) was calculated using a combination of linear and log-trapezoidal methods. Systemic clearance (CL), steady-state volume of distribution (Vss), half-life (T-HALF), and mean residence time (MRT) after IV administration were estimated. T-HALF was estimated using at least three time points where the concentration could be quantified.

[0219] After administering compound A (1 mg / kg) to rabbits, the plasma concentration of compound A was 4.3 μM, and the plasma concentration of free compound A was 290 nM. After administering Fab fragment antibody 26D5-75616-348-F10-Fab-SHORT (160 mg / kg), the plasma concentration of compound A was 14 μM, and the plasma concentration of unbound compound A was less than 0.2 nM. The decrease in the plasma concentration of unbound compound A is due to the high binding affinity of Fab fragment antibody 26D5-75616-348-F10-Fab-SHORT. The plasma concentration of compound A increases mainly due to the distribution of Fab fragment antibody 26D5-75616-348-F10-Fab-SHORT in the blood vessels and the redistribution of compound A from the extravascular to the blood vessels according to the law of mass action. The plasma concentration of free compound A in rabbits remained below 10 nM for more than 12 hours. Figure 21 shows the plasma concentrations of Fab fragment antibody 26D5-75616-348-F10-Fab-SHORT, compound A, and free compound A in rabbits that were intravenously administered Fab fragment antibody 26D5-75616-348-F10-Fab-SHORT (160 mg / kg) 20 minutes after intravenous administration of compound A (1 mg / kg). The results are the average of three rabbits.

[0220] Example 13: Pharmacokinetics of Tandem Fab Fragment Antibody 26D5-75616-348-F10-TanFab (Tandem Fab heavy chain SEQ ID NO: 180, Tandem Fab light chain SEQ ID NO: 164) was prepared and purified according to a general method known to those skilled in the art, similar to Example 5 above.

[0221] Pharmacokinetics in rats In vivo experiments were conducted in accordance with the rules of the Animal Welfare and Management Committee within Bristol-Myers Squibb Company. Indwelling catheters were placed in the jugular veins (for blood collection and substance administration) of male rats (Sprague-Dawley, 0.2-0.4 kg). A fixed amount of Fab fragment antibody 26D5-75616-348-F10-Fab-SHORT and tandem Fab fragment antibody 26D5-75616-348-F10-TanFab (10 mg / kg each) were administered intravenously by drip infusion over 10 minutes. Blood samples (0.2 mL) were collected at the end of the infusion and at various intervals up to 48 hours after the start of administration. The blood samples were added to EDTA in a polypropylene tube, thoroughly mixed by inverting at least twice, and left to stand on ice. Within 1 hour of blood collection, plasma was isolated by centrifugation of whole blood at at least 1,500 x g for at least 10 minutes. The concentrations of the Fab fragment antibody 26D5-75616-348-F10-Fab-SHORT and the tandem Fab fragment antibody 26D5-75616-348-F10-TanFab in plasma were determined as follows.

[0222] The concentrations of 26D5-75616-348-F10-Fab-SHORT and 26D5-75616-348-F10-TanFab in plasma were determined as follows: The total plasma concentration of both 26D5-75616-348-F10-Fab-SHORT and 26D5-75616-348-F10-TanFab that were not bound to compound A in plasma, as well as the plasma concentrations of 26D5-75616-348-F10-Fab-SHORT and 26D5-75616-348-F10-TanFab, were measured by ligand binding assays using a Gyrolab (trademark) automated microfluidic system (Gyros Protein Technologies AB). Biotinylated mouse anti-human κ (SouthernBiotech Cat No 9230-08, Lot No K5613-X088) was used as the molecule to bind to all 26D5-75616-348-F10-Fab-SHORT and 26D5-75616-348-F10-TanFab molecules. Samples, standards, and QC were increased to the final baseline concentration of 10% plasma / 1xPTB (1% BSA / 0.05% Tween20 / PBS) and loaded into a Gyrolab® automated microfluidic system. The 3-step-2-wash wizard method of the Gyrolab® Bioaffy 200 CD (Gyros Protein Technologies AB) was used. After the final washing step, all bound 26D5-75616-348-F10-Fab-SHORT and 26D5-75616-348-F10-TanFab were detected using Alexa Fluor® 647-labeled mouse anti-human Igκ light chain mAb clone G20-361 (Becton Dickinson Cat No. 555861, Lot No. 833694). Fluorescence intensity was measured using a Gyrolab® system with four-coefficient logistic curves prepared from standards of 26D5-75616-348-F10-Fab-SHORT and 26D5-75616-348-F10-TanFab, and the total concentration of 26D5-75616-348-F10-Fab-SHORT and 26D5-75616-348-F10-TanFab in the plasma samples was calculated.The combined calibration curve range for 26D5-75616-348-F10-Fab-SHORT and 26D5-75616-348-F10-TanFab was 10–25000 ng / mL (in plasma). The upper and lower limits of quantification were 25000 and 10 ng / mL, respectively. Quality control samples were prepared at plasma concentrations of 20000, 7500, 750, 75, and 30 ng / mL. Standard materials and QC were analyzed in each experiment to ensure acceptable assays. Assays were performed within acceptable limits (%CV of standard materials and QC less than 20%, and QC recovery within ±20% of the apparent value).

[0223] Compound 5 was used as a molecule to bind to 26D5-75616-348-F10-Fab-SHORT and 26D5-75616-348-F10-TanFab, which were not bound to Compound A. Samples, standards, and QCs were increased to the final baseline concentration of 10% plasma / 1xPTB (1% BSA / 0.05% Tween20 / PBS) and loaded into a Gyrolab® automated microfluidic system. The 3-step-2-wash wizard method of the Gyrolab® Bioaffy 200 CD was used. After the final washing step, bound "active / free" 26D5-75616-348-F10-Fab-SHORT and 26D5-75616-348-F10-TanFab were detected using Alexa Fluor® 647-labeled mouse anti-human Igκ light chain mAb clone G20-361 (Becton Dickinson Cat No. 555861, Lot No. 8333694). Using four-coefficient logistic curves prepared from the 26D5-75616-348-F10-Fab-SHORT and 26D5-75616-348-F10-TanFab standards, fluorescence intensity was measured using Gyrolab (trademark registered), and the concentrations of "active / free" 26D5-75616-348-F10-Fab-SHORT and 26D5-75616-348-F10-TanFab in plasma samples were calculated. The calibration curve range for "active / free" 26D5-75616-348-F10-Fab-SHORT and 26D5-75616-348-F10-TanFab was 10 to 25000 ng / mL (in plasma). The upper and lower limits of quantification were 25000 and 10 ng / mL, respectively. Quality control samples were prepared at plasma concentrations of 20000, 7500, 750, 75, and 30 ng / mL. Reference materials and QC were analyzed in each experiment to ensure acceptable assays. Assays were performed within acceptable limits (%CV of reference materials and QC less than 20%, and QC recovery within ±20% of the apparent value).

[0224] Figure 22 shows the obtained pharmacokinetic data.

[0225] Pharmacokinetics in Rabbits In vivo experiments were conducted in accordance with the rules of the Animal Welfare and Management Committee within Bristol-Myers Squibb Company. Indwelling catheters were placed in the femoral artery and femoral vein (for blood collection) and the marginal auricular vein (for substance administration) of male rabbits (New Zealand White breed, 2-4 kg). A fixed amount of compound A (0.4 mg / kg, 0.64 μM / kg) was administered intravenously by drip infusion over 10 minutes. Twenty minutes after the completion of the drip infusion of compound A, a fixed amount of tandem Fab fragment antibody 26D5-75616-348-F10-TanFab (40 mg / kg, 0.43 μM / kg) was administered intravenously by drip infusion over 10 minutes. Considering that the administered tandem Fab fragment antibody 26D5-75616-348-F10-TanFab has a 2:1 binding affinity, it was administered at an apparent 1.34-fold excess molar concentration relative to the administered compound A (2*0.43 / 0.64). Blood samples (1.5 mL) were collected at various intervals: before administration of compound A, at the end of the infusion of compound A, immediately before administration of tandem Fab fragment antibody 26D5-75616-348-F10-TanFab, at the end of administration of tandem Fab fragment antibody 26D5-75616-348-F10-TanFab, and after administration of tandem Fab fragment antibody 26D5-75616-348-F10-TanFab and up to 24 hours after the start of administration of compound A.

[0226] Blood samples were added to 0.167 mL of 3.8% sodium citrate in a polypropylene tube, thoroughly mixed by inverting the tube at least twice, and then allowed to stand on ice. Within one hour of blood collection, plasma was isolated by centrifugation of whole blood at at least 1,500 x g for at least 10 minutes. Free compound A was obtained by ultrafiltration as described above. A portion of the plasma and the plasma ultrafiltrate was frozen in a polypropylene tube at -80°C. The total concentration of compound A in the plasma and the free compound A in the plasma ultrafiltrate was measured by liquid chromatography-tandem mass spectrometry (LC / MS) analysis. Samples for LC / MS analysis were prepared by the protein precipitation method described below.

[0227] A portion of the biological sample (20 μL) was transferred to a 96-well deep plate (1.2 mL, round-bottom, polypropylene). A methanol solution containing 50% water and 0.5% formic acid (20 μL) was added. The plate was covered and mixed in a 95°C shaker for 20 minutes. An acetonitrile solution containing an internal standard [1 μM] and 1% formic acid (80 μL) was added to the above solution to precipitate the protein. The plate was further vortexed at room temperature for 15 minutes, and then centrifuged at 3,700 rpm for 8 minutes. A portion of the supernatant (100 μL) was transferred to an injection plate (96 wells, 0.3 mL). The supernatant (3 μL) was injected into an Ultra Performance LC System (Waters® Acquity iClass UPLC) tandem mass spectrometer connected to a Quadrapole MS / MS (Thermo Quantiva). The substances to be measured were separated using a C18 column (Waters HSS T3, 2 x 50 mm, 1.8 μm) at 40°C using a gradient (flow rate 0.6 mL / min) consisting of two buffer solutions (A: water + 5 mM ammonium formate, 0.1% formic acid; B: acetonitrile + 0.1% formic acid). The precursor (M+H) was analyzed using positive electrospray ionization mode multiple reaction monitoring (MRM). + The substance was detected. MRM transitions were observed in compound A (626.3 → 319.1) and in the internal standard (474.3 → 269). The limit of quantification was 0.5 nM.

[0228] In this example, the concentration of the tandem Fab fragment antibody 26D5-75616-348-F10-TanFab in plasma was determined as described above.

[0229] Figure 23 shows the obtained pharmacokinetic data.

[0230] In vitro experiment Compound A was added to stored healthy human plasma at a concentration of 2000 nM. The tandem Fab fragment antibody 26D5-75616-348-F10-TanFab was added to stored healthy human plasma at a concentration of 1000 nM. Plasma containing compound A, plasma containing 26D5-75616-348-F10-TanFab, and normal plasma were combined to adjust various concentrations (molar ratios of compound A (including compound A in the absence of 26D5-75616-348-F10-TanFab):26D5-75616-348-F10-TanFab of 2:1, 2:0.8, 2:0.6, and 2:0.4, etc.). The activated partial rhomboplastin time (aPTT) (i.e., human plasma coagulation time) in each sample was determined as in Example 12 described above. Figure 24 shows the obtained coagulation plasma data.

Claims

1. An isolated antigen-binding peptide comprising at least one heavy chain variable region (VH) and at least one light chain variable region (VL), The VH comprises three complementarity-determining regions (CDRs): VH-CDR1, VH-CDR2, and VH-CDR3, and the VL comprises three CDRs: VL-CDR1, VL-CDR2, and VL-CDR3, where the amino acid sequences of VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3 are as follows: (a) Sequence numbers for each: 1, 13, 23, 29, 38, and 44; (b) Sequence numbers for each: 1, 14, 23, 29, 38, and 45; (c) Sequence numbers for each: 1, 13, 24, 30, 38, and 45; (d) Sequence numbers for each: 1, 13, 24, 29, 39, and 45; (e) Sequence numbers for each: 1, 14, 25, 29, 38, and 46; (f) Sequence numbers for each: 2, 13, 26, 31, 40, and 47; (g) Sequence numbers for each: 3, 15, 24, 32, 40, and 47; (h) Sequence numbers for each: 4, 16, 24, 29, 38, and 46; (i) Sequence numbers for each: 5, 15, 24, 29, 38, and 46; (j) Sequence numbers for each: 1, 14, 24, 29, 38, and 46; (l) Sequence numbers for each: 3, 15, 24, 32, 41, and 48; (n) Sequence numbers for each: 1, 14, 26, 29, 38, and 46; (o) Sequence numbers for each: 7, 17, 26, 29, 38, and 46; (p) Sequence numbers: 8, 17, 24, 34, 38, and 46; (q) Sequence numbers for each: 1, 17, 26, 29, 38, and 46; (r) Sequence numbers for each: 1, 17, 26, 35, 38, and 46; (s) Sequence numbers for each: 1, 17, 24, 33, 38, and 49; (t) Sequence numbers: 9, 14, 26, 29, 38, and 46; (u) Sequence numbers: 9, 14, 26, 35, 38, and 46; (v) Sequence numbers for each: 9, 17, 24, 29, 38, and 46; (w) Sequence numbers: 9, 17, 24, 35, 38, and 46; (x) Sequence numbers for each: 9, 17, 24, 34, 38, and 46; (y) Sequence numbers for each: 9, 14, 24, 29, 38, and 46; (z) Sequence numbers: 9, 18, 26, 35, 38, and 46; (aa) Sequence numbers for each: 8, 14, 24, 29, 38, and 46; (bb) Sequence numbers: 8, 17, 26, 29, 38, and 46; (cc) Sequence numbers for each: 9, 19, 26, 29, 38, and 46; (dd) Sequence numbers for each: 9, 17, 26, 34, 38, and 46; (ee) Sequence numbers: 10, 20, 27, 36, 42, and 50; (ff) Sequence numbers: 11, 21, 28, 37, 43, and 51; (gg) Sequence numbers for each: 12, 22, 26, 33, 38, and 46; (hh) Sequence numbers for each: 12, 17, 26, 33, 38, and 46; and (ii) Sequence numbers for each: 9, 17, 26, 33, 38, and 46; It has a sequence selected from the group consisting of, The antigen-binding peptide is of formula (I): 【Chemistry 1】 [In the formula, R1 is a C1-4 alkyl group; R2 is independently selected from F, Cl, CF3, CHF2, CH2F, and CH3; R 3 is independently selected from CF 3, CHF 2, CH 2 F, and CH 3; R 4 is H; and R 5 is independently selected from F and Cl. The compound shown, or its stereoisomer or tautomer, specifically binds to the compound shown, Isolated antigen-binding peptide.

2. An isolated antigen-binding peptide according to Claim 1, wherein at least one VH region and at least one VL region are, respectively: (a) Sequence ID 52 and Sequence ID 84, respectively; (b) Sequence ID 53 and Sequence ID 85, respectively; (c) Sequence IDs 54 and 86, respectively; (d) Sequence IDs 54 and 87, respectively; (e) Sequence IDs 55 and 88, respectively; (f) Sequence ID 56 and Sequence ID 89, respectively; (g) Sequence IDs 57 and 90, respectively; (h) Sequence IDs 58 and 88, respectively; (i) Sequence ID 59 and Sequence ID 88, respectively; (j) Sequence IDs 60 and 91, respectively; (l) Sequence ID 57 and Sequence ID 92, respectively; (n) Sequence ID 60 and Sequence ID 88, respectively; (o) Sequence IDs 62 and 88, respectively; (p) Sequence IDs 63 and 88, respectively; (q) Sequence ID 64 and Sequence ID 88, respectively; (r) Sequence IDs 65 and 94, respectively; (s) Sequence ID 66 and Sequence ID 88, respectively; (t) Sequence ID 66 and Sequence ID 95, respectively; (u) Sequence IDs 67 and 88, respectively; (v) Sequence IDs 68 and 93, respectively; (w) Sequence IDs 69 and 88, respectively; (x) Sequence ID 69 and Sequence ID 95, respectively; (y) Sequence IDs 70 and 88, respectively; (z) Sequence IDs 70 and 95, respectively; (bb) Sequence IDs 71 and 94, respectively; (cc) Sequence IDs 72 and 88, respectively; (dd) Sequence IDs 73 and 95, respectively; (ee) Sequence IDs 74 and 88, respectively; (ff) Sequence IDs 75 and 88, respectively; (gg) Sequence IDs 76 and 88, respectively; (hh) Sequence IDs 77 and 94, respectively; (ii) Sequence IDs 78 and 96, respectively; (jj) Sequence IDs 79 and 97, respectively; (kk) Sequence IDs 80 and 98 respectively; (ll) Sequence IDs 81 and 99, respectively; (mm) Sequence IDs 81 and 98 respectively; and (oo) Sequence IDs 83 and 98 respectively; Having an amino acid sequence selected from the group consisting of, Isolated antigen-binding peptide.

3. The compound is given by formula (II): 【Chemistry 2】 An isolated antigen-binding peptide according to claim 1 or 2, which is a compound represented by .

4. The aforementioned isolated antigen-binding peptides are antibodies, Fab, Fab', and F(ab'). 2 single-chain Fv or scFv, disulfide bond Fv, intra-body, mini-body, F(ab') 3 Tetrabody, triabody, bispecific antibody, DVD-Ig, mAb 2 (scFv) 2 An isolated antigen-binding peptide according to any one of claims 1 to 3, which is scFv-Fc or tandem Fab.

5. below: (a) Sequence ID 100 and Sequence ID 160, respectively; (b) Sequence ID 101 and Sequence ID 160, respectively; (c) Sequence IDs 102 and 161, respectively; (d) Sequence IDs 103 and 161, respectively; (e) Sequence ID 104 and Sequence ID 162, respectively; (f) Sequence IDs 105 and 162, respectively; (g) Sequence IDs 104 and 163, respectively; (h) Sequence IDs 105 and 163, respectively; (i) Sequence ID 106 and Sequence ID 164, respectively; (j) Sequence ID 107 and Sequence ID 164, respectively; (k) Sequence ID 108 and Sequence ID 165, respectively; (l) Sequence ID 109 and Sequence ID 165, respectively; (m) Sequence IDs 110 and 166, respectively; (n) Sequence ID 111 and Sequence ID 166, respectively; (o) Sequence IDs 112 and 164, respectively; (p) Sequence IDs 113 and 164, respectively; (q) Sequence IDs 114 and 164, respectively; (r) Sequence IDs 115 and 164, respectively; (s) Sequence IDs 116 and 167, respectively; (t) Sequence IDs 117 and 167, respectively; (w) Sequence IDs 110 and 168, respectively; (x) Sequence ID 111 and Sequence ID 168, respectively; (aa) Sequence ID 116 and Sequence ID 164, respectively; (bb) Sequence IDs 117 and 164, respectively; (cc) Sequence IDs 120 and 164, respectively; (dd) Sequence IDs 121 and 164, respectively; (ee) Sequence IDs 122 and 164, respectively; (ff) Sequence IDs 123 and 164, respectively; (gg) Sequence IDs 124 and 164, respectively; (hh) Sequence IDs 125 and 164, respectively; (ii) Sequence IDs 126 and 170, respectively; (jj) Sequence IDs 127 and 170, respectively; (kk) Sequence IDs 128 and 164, respectively; (ll) Sequence IDs 129 and 164, respectively; (mm) Sequence IDs 128 and 171, respectively; (nn) Sequence IDs 129 and 171, respectively; (oo) Sequence IDs 130 and 164, respectively; (pp) Sequence IDs 131 and 164, respectively; (qq) Sequence IDs 132 and 169, respectively; (rr) Sequence IDs 133 and 169, respectively; (ss) Sequence IDs 134 and 164, respectively; (tt) Sequence IDs 135 and 164, respectively; (uu) Sequence IDs 134 and 171, respectively; (vv) Sequence IDs 135 and 171, respectively; (ww) Sequence IDs 136 and 164, respectively; (xx) Sequence IDs 137 and 164, respectively; (yy) Sequence IDs 136 and 171, respectively; (zz) Sequence IDs 137 and 171, respectively; (ccc) Sequence ID 138 and Sequence ID 170, respectively; (ddd) Sequence ID 139 and Sequence ID 170, respectively; (eee) Sequence IDs 140 and 164, respectively; (fff) Sequence IDs 141 and 164, respectively; (ggg) Sequence IDs 142 and 171, respectively; (hhh) Sequence IDs 143 and 171, respectively; (iii) Sequence ID 144 and Sequence ID 164, respectively; (jjj) Sequence IDs 145 and 164, respectively; (kkk) Sequence IDs 146 and 164, respectively; (lll) Sequence IDs 147 and 164, respectively; (mmm) Sequence IDs 148 and 164, respectively; (nnn) Sequence IDs 149 and 164, respectively; (ooo) Sequence ID 150 and Sequence ID 170, respectively; (ppp) Sequence IDs 151 and 170, respectively; (qqq) Sequence IDs 152 and 172, respectively; (rrr) Sequence IDs 153 and 172, respectively; (sss) Sequence IDs 154 and 173 respectively; (ttt) Sequence IDs 155 and 173, respectively; (uuu) Sequence IDs 156 and 174, respectively; (vvv) Sequence IDs 157 and 174, respectively; (www) Sequence ID 158 and Sequence ID 175, respectively; (xxx) Sequence IDs 159 and 175, respectively; (yyy) Sequence ID 158 and Sequence ID 174, respectively; (zzz) Sequence IDs 159 and 174, respectively; (aaaa) Sequence ID 176 and Sequence ID 160, respectively; (bbbb) Sequence IDs 177 and 160, respectively; (cccc) Sequence ID 178 and Sequence ID 160, respectively; (dddd) Sequence ID 179 and Sequence ID 160, respectively; (eeee) Sequence IDs 180 and 164, respectively; (ffff) Sequence IDs 181 and 164, respectively; (gggg) Sequence IDs 182 and 164, respectively; (hhhh) Sequence IDs 183 and 164, respectively; (iiii) Sequence ID 184 and Sequence ID 163, respectively; (jjjj) Sequence IDs 185 and 163, respectively; (kkkk) Sequence ID 186 and Sequence ID 163, respectively; (llll) Sequence IDs 187 and 163 respectively; (mmmm) Sequence ID 184 and Sequence ID 162, respectively; (nnnn) Sequence IDs 185 and 162, respectively; (oooo) Sequence ID 186 and Sequence ID 162, respectively; (pppp) Sequence IDs 187 and 162, respectively; (qqqq) Sequence IDs 188 and 165, respectively; (rrrr) Sequence ID 189 and Sequence ID 165, respectively; (ssss) Sequence IDs 190 and 165, respectively; (tttt) Sequence IDs 191 and 165, respectively; (uuuu) Sequence IDs 192 and 161 respectively; (vvvv) Sequence ID 193 and Sequence ID 161, respectively; (www) Sequence ID 194 and Sequence ID 161 respectively; and (xxxx) Sequence ID 195 and Sequence ID 161 respectively An isolated antigen-binding peptide according to any one of claims 1 to 4, having a sequence selected from the group consisting of, wherein the isolated antigen-binding peptide specifically comprises formula (II) 【Transformation 3】 An isolated antigen-binding peptide that binds to the compound.

6. The isolated antigen-binding peptide according to claim 5, having the sequences of SEQ ID NO: 106 and SEQ ID NO: 164, respectively.

7. The isolated antigen-binding peptide according to claim 6, which is a Fab fragment antibody.

8. The isolated antigen-binding peptide according to claim 5, having the sequences of SEQ ID NO: 180 and SEQ ID NO: 164, respectively.

9. The isolated antigen-binding peptide according to claim 8, which is a tandem Fab fragment antibody.

10. An isolated polynucleotide having a nucleic acid sequence encoding an antigen-binding peptide according to any one of claims 1 to 9.

11. A vector comprising the isolated polynucleotide described in claim 10.

12. A host cell comprising the vector according to claim 11.

13. A method for producing an antigen-binding peptide, comprising: (a) culturing host cells under conditions that promote protein production such that the host cells described in claim 12 produce an antigen-binding peptide; and (b) isolating the antigen-binding peptide from the culture medium in (a).

14. A detection reagent comprising an isolated antigen-binding peptide and a detectable label according to any one of claims 1 to 9.

15. Formula (I) 【Chemistry 4】 [In the formula, R 1 C 1-4 It is alkyl; R 2 is independently selected from F, Cl, CF 3 , CHF 2 , CH 2 F, CH 3 ; and is selected from R 3 CF is independent 3 CHF 2 CH 2 F and CH 3 Selected from; R 4 is H; and R 5 [F and Cl are selected independently.] A pharmaceutical composition comprising an isolated antigen-binding peptide according to any one of claims 1 to 9, for reducing the antithrombotic effect of the compound in a subject taking the compound or its stereoisomer or tautomer.

16. Equation (I) 【Transformation 5】 [In the formula, R 1 C 1-4 It is alkyl; R 2 These are independently F, Cl, and CF. 3 CHF 2 CH 2 F, CH 3 Selected from; R 3 CF is independent 3 CHF 2 CH 2 F and CH 3 Selected from; R 4 is H; and R 5 [F and Cl are selected independently.] A method for detecting the level of a compound or its stereoisomers, tautomers, or pharmaceutically acceptable salts in a biological sample, (a) Contacting a biological sample in vitro with an isolated antigen-binding peptide according to any one of claims 1 to 9, and (b) To detect the level of the complex to which the compound and the isolated antigen-binding peptide are bound. Methods that include...

17. Formula (I) for the therapeutically effective dose 【Transformation 6】 [In the formula, R 1 C 1-4 It is alkyl; R 2 These are independently F, Cl, and CF. 3 CHF 2 CH 2 F, CH 3 Selected from; R 3 CF is independent 3 CHF 2 CH 2 F and CH 3 Selected from; R 4 is H; and R 5 [F and Cl are selected independently.] A pharmaceutical composition comprising an isolated antigen-binding peptide according to any one of claims 1 to 9 for binding the compound or its stereoisomer or tautomer to a subject who is taking the compound or its stereoisomer or tautomer.

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