Antibodies and antigen-binding peptides as factor XIA inhibitors and uses thereof

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 to U.S. Provisional Application No. 63 / 135,016, filed January 8, 2021, U.S. Provisional Application No. 63 / 148,767, filed February 12, 2021, U.S. Provisional Application No. 63 / 152,595, filed February 23, 2021, and U.S. Provisional Application No. 63 / 153,045, filed February 24, 2021, each of which is incorporated by reference in its entirety herein.

[0002] (Reference to sequence listing) This application contains a sequence listing with file name "055920-553P01US_Sequence_Lising.txt" (approximately 316,130 bytes, created on December 23, 2020, submitted electronically in ASCII format), which is incorporated by reference in its entirety. [Background technology]

[0003] Thromboembolic disorders are a major cause of death in developed countries, despite the availability of anticoagulants (e.g., dabigatran, apixaban, rivaroxaban, warfarin (COUMADIN®), heparin, low molecular weight heparins (LMWHs), and synthetic pentasaccharides) and platelet drugs (e.g., aspirin and clopidogrel (PLAVIX®)). The discovery and development of safe and effective oral anticoagulants for the prevention and treatment of a broad range of thromboembolic disorders remains important. One approach is to reduce thrombin generation, aiming to inhibit the coagulation of factor XIa (FXIa). FXIa is a plasma serine protease involved in blood coagulation control, and in vivo, the binding of tissue factor (TF) to factor VII (FVII) promotes the production of factor VIIa (FVIIa). The resulting TF:FVIIa complex activates factors IX (FIX) and X (FX), which leads to the production of factor Xa (FXa). The produced FXa catalyzes the conversion of prothrombin to small amounts of thrombin, which is then inhibited by tissue factor pathway inhibitor (TFPI). Catalytic amounts of thrombin further promote clotting by activating factors V, VIII, and XI. The sudden increase in thrombin converts fibrinogen to fibrin, a polymer that forms the structural framework of blood clots, and activates platelets, the key cellular component of clotting. Thus, FXIa plays a key role in this chain of events, making it an attractive target for antithrombotic therapy.

[0004] Plasma prekallikrein is a zymogen of trypsin-like serine protease, and is present in plasma at 35-50μg / mL. It is close to the structure of factor XI (FXI), and the overall amino acid sequence of plasma kallikrein is 58% identical to FXI. Plasma kallikrein is thought to play an important role in many inflammatory diseases. The main inhibitor of plasma kallikrein is serpin C1 esterase inhibitor. Patients with hereditary angioedema (HAE) who are deficient in the gene for C1 esterase inhibitor suffer from intermittent swelling of the face, hands, throat, gastrointestinal tract, and genitals. Blisters formed during sudden onset contain a lot of plasma kallikrein. Plasma kallikrein hydrolyzes high molecular weight kininogen and releases 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 abundant in patients with advanced diabetic macular edema. Recently, it was published that plasma kallikrein is involved in retinal vascular dysfunction in diabetic rats. In addition, administration of the plasma kallikrein inhibitor ASP-440 improved both retinal vascular permeability abnormalities and retinal blood flow abnormalities in diabetic rats. Thus, 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 considered targets of plasma kallikrein inhibitors. To date, no small molecule synthetic plasma kallikrein inhibitors have been approved for medical use. As reported with ecallantide, large protein plasma kallikrein inhibitors carry the 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, which is incorporated herein by reference. These selective FXIa inhibitors or dual inhibitors of FXIa / plasma kallikrein (e.g., compounds provided in the present invention) are developed based on their excellent antithrombotic effects with little or no risk of bleeding. However, even in clinical trials in which such FXIa inhibitors are administered to patients, bleeding may occur in rare cases. In humans, bleeding due to factor XI deficiency may occur, especially in cases of trauma to tissues with strong fibrinolytic activity (e.g., the oral pharynx and urinary tract).

[0007] There are methods to promote hemostasis, such as coagulation factor preparations and recombinant activated factor VII, which are mainly approved for use in hemophilia patients, but can also be envisaged for use in bleeding patients treated with thrombin or FXa inhibitors when specific antagonists cannot be used. However, these methods carry the risk of thrombus formation. Therefore, there is an urgent need to develop compounds that can quickly restore the antithrombotic effect of the selective FXIa inhibitors or dual FXIa / plasma kallikrein inhibitors (e.g., compounds of the present disclosure) without the risk of thrombus formation for patients with severe bleeding or those who require emergency surgery.

[0008] SUMMARY OF THE PRESENTINVENTION The present invention discloses novel antibodies or antigen-binding peptides that specifically bind to selective FXIa inhibitors and / or dual inhibitors of FXIa / plasma kallikrein. The present invention further discloses a method for reducing the antithrombotic effect of FXIa inhibitors by administering to a subject a pharmacologic effective amount of the antibodies or antigen-binding peptides disclosed herein. The present invention further discloses detection reagents and methods for detecting the level of FXIa inhibition in a biological sample.

[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 selected from the group consisting of: (a) 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; (b) a VH-CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 22; or (c) VH-CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 23 to 28 and at least one VL is: (d) a VL-CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 29 to 37; (e) a VL-CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 38 to 43; or (f) VL-CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 44 to 51 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 to 99; 1. An isolated antigen-binding peptide comprising:

[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, and wherein the amino acid sequences of VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3 each have a sequence selected from the group consisting of: (a) respective SEQ ID NOs: 1, 13, 23, 29, 38, and 44; (b) respective SEQ ID NOs: 1, 14, 23, 29, 38, and 45; (c) respective SEQ ID NOs: 1, 13, 24, 30, 38, and 45; (d) respective SEQ ID NOs: 1, 13, 24, 29, 39, and 45; (e) respective SEQ ID NOs: 1, 14, 25, 29, 38, and 46; (f) respective SEQ ID NOs: 2, 13, 26, 31, 40, and 47; (g) respective SEQ ID NOs: 3, 15, 24, 32, 40, and 47; (h) respective SEQ ID NOs: 4, 16, 24, 29, 38, and 46; (i) respective SEQ ID NOs: 5, 15, 24, 29, 38, and 46; (j) respective SEQ ID NOs: 1, 14, 24, 29, 38, and 46; (k) respective SEQ ID NOs: 6, 13, 24, 31, 40, and 47; (l) respective SEQ ID NOs: 3, 15, 24, 32, 41, and 48; (m) respective SEQ ID NOs: 1, 14, 24, 33, 38, and 49; (n) respective SEQ ID NOs: 1, 14, 26, 29, 38, and 46; (o) respective SEQ ID NOs: 7, 17, 26, 29, 38, and 46; (p) respective SEQ ID NOs: 8, 17, 24, 34, 38, and 46; (q) respective SEQ ID NOs: 1, 17, 26, 29, 38, and 46; (r) respective SEQ ID NOs: 1, 17, 26, 35, 38, and 46; (s) respective SEQ ID NOs: 1, 17, 24, 33, 38, and 49; (t) respective SEQ ID NOs: 9, 14, 26, 29, 38, and 46; (u) respective SEQ ID NOs: 9, 14, 26, 35, 38, and 46; (v) respective SEQ ID NOs: 9, 17, 24, 29, 38, and 46; (w) respective SEQ ID NOs: 9, 17, 24, 35, 38, and 46; (x) respective SEQ ID NOs: 9, 17, 24, 34, 38, and 46; (y) respective SEQ ID NOs: 9, 14, 24, 29, 38, and 46; (z) respective SEQ ID NOs: 9, 18, 26, 35, 38, and 46; (aa) respective SEQ ID NOs: 8, 14, 24, 29, 38, and 46; (bb) SEQ ID NOs: 8, 17, 26, 29, 38, and 46, respectively; (cc) respective SEQ ID NOs: 9, 19, 26, 29, 38, and 46; (dd) respective SEQ ID NOs: 9, 17, 26, 34, 38, and 46; (ee) respective SEQ ID NOs: 10, 20, 27, 36, 42, and 50; (ff) respective SEQ ID NOs: 11, 21, 28, 37, 43, and 51; (gg) respective SEQ ID NOs: 12, 22, 26, 33, 38, and 46; (hh) respective SEQ ID NOs: 12, 17, 26, 33, 38, and 46; (ii) SEQ ID NOs: 9, 17, 26, 33, 38, and 46, respectively; and (jj) A variant of (a) to (ii), wherein any amino acid sequence contains one, two, or three conservative substitutions.

[0012] Specific embodiment 4: The 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: (a) SEQ ID NO:52 and SEQ ID NO:84, respectively; (b) SEQ ID NO:53 and SEQ ID NO:85, respectively; (c) SEQ ID NO:54 and SEQ ID NO:86, respectively; (d) SEQ ID NO:54 and SEQ ID NO:87, respectively; (e) SEQ ID NO:55 and SEQ ID NO:88, respectively; (f) SEQ ID NO:56 and SEQ ID NO:89, respectively; (g) SEQ ID NO:57 and SEQ ID NO:90, respectively; (h) SEQ ID NO:58 and SEQ ID NO:88, respectively; (i) SEQ ID NO:59 and SEQ ID NO:88, respectively; (j) SEQ ID NO:60 and SEQ ID NO:91, respectively; (k) SEQ ID NO:61 and SEQ ID NO:89, respectively; (l) SEQ ID NO:57 and SEQ ID NO:92, respectively; (m) SEQ ID NO:60 and SEQ ID NO:93, respectively; (n) SEQ ID NO:60 and SEQ ID NO:88, respectively; (o) SEQ ID NO:62 and SEQ ID NO:88, respectively; (p) SEQ ID NO:63 and SEQ ID NO:88, respectively; (q) SEQ ID NO:64 and SEQ ID NO:88, respectively; (r) SEQ ID NO:65 and SEQ ID NO:94, respectively; (s) SEQ ID NO:66 and SEQ ID NO:88, respectively; (t) SEQ ID NO:66 and SEQ ID NO:95, respectively; (u) SEQ ID NO:67 and SEQ ID NO:88, respectively; (v) SEQ ID NO:68 and SEQ ID NO:93, respectively; (w) SEQ ID NO:69 and SEQ ID NO:88, respectively; (x) SEQ ID NO:69 and SEQ ID NO:95, respectively; (y) SEQ ID NO:70 and SEQ ID NO:88, respectively; (z) SEQ ID NO:70 and SEQ ID NO:95, respectively; (aa) SEQ ID NO:71 and SEQ ID NO:88, respectively; (bb) SEQ ID NO:71 and SEQ ID NO:94, respectively; (cc) SEQ ID NO:72 and SEQ ID NO:88, respectively; (dd) SEQ ID NO:73 and SEQ ID NO:95, respectively; (ee) SEQ ID NO:74 and SEQ ID NO:88, respectively; (ff) SEQ ID NO:75 and SEQ ID NO:88, respectively; (gg) SEQ ID NO:76 and SEQ ID NO:88, respectively; (hh) SEQ ID NO:77 and SEQ ID NO:94, respectively; (ii) SEQ ID NO:78 and SEQ ID NO:96, respectively; (jj) SEQ ID NO:79 and SEQ ID NO:97, respectively; (kk) SEQ ID NO: 80 and SEQ ID NO: 98, respectively; (ll) SEQ ID NO:81 and SEQ ID NO:99, respectively; (mm) SEQ ID NO:81 and SEQ ID NO:98, respectively; (nn) SEQ ID NO:82 and SEQ ID NO:99, respectively; (oo) SEQ ID NO: 83 and SEQ ID NO: 98, respectively; and (pp) A variant of (a) to (oo) containing 1, 2, 3, or 4 conservative substitutions.

[0013] Specific embodiment 5: an isolated antigen-binding peptide of any one of the preceding 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 preceding embodiments, comprising the formula (I): [ka] [In the formula, R 1 is C 1-4 is alkyl; R 2 is independently selected from F, Cl, CF3, CHF2, CH2F, CH3; R 3 is independently selected from CF3, CHF2, CH2F, and CH3; R 4 is H; and R 5 are independently selected from F and Cl. or a stereoisomer or tautomer thereof.

[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 of any one of the preceding embodiments is an antibody.

[0018] Specific embodiment 10: An isolated antigen-binding peptide of any one of the preceding embodiments, wherein the antigen-binding peptide is a Fab, Fab', F(ab')2, Fd, single chain Fv or scFv, disulfide-linked 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: (a) SEQ ID NO:100 and SEQ ID NO:160, respectively; (b) SEQ ID NO:101 and SEQ ID NO:160, respectively; (c) SEQ ID NO:102 and SEQ ID NO:161, respectively; (d) SEQ ID NO:103 and SEQ ID NO:161, respectively; (e) SEQ ID NO:104 and SEQ ID NO:162, respectively; (f) SEQ ID NO:105 and SEQ ID NO:162, respectively; (g) SEQ ID NO:104 and SEQ ID NO:163, respectively; (h) SEQ ID NO:105 and SEQ ID NO:163, respectively; (i) SEQ ID NO:106 and SEQ ID NO:164, respectively; (j) SEQ ID NO:107 and SEQ ID NO:164, respectively; (k) SEQ ID NO:108 and SEQ ID NO:165, respectively; (l) SEQ ID NO:109 and SEQ ID NO:165, respectively; (m) SEQ ID NO:110 and SEQ ID NO:166, respectively; (n) SEQ ID NO:111 and SEQ ID NO:166, respectively; (o) SEQ ID NO:112 and SEQ ID NO:164, respectively; (p) SEQ ID NO:113 and SEQ ID NO:164, respectively; (q) SEQ ID NO:114 and SEQ ID NO:164, respectively; (r) SEQ ID NO:115 and SEQ ID NO:164, respectively; (s) SEQ ID NO:116 and SEQ ID NO:167, respectively; (t) SEQ ID NO:117 and SEQ ID NO:167, respectively; (u) SEQ ID NO:118 and SEQ ID NO:165, respectively; (v) SEQ ID NO:119 and SEQ ID NO:165, respectively; (w) SEQ ID NO:110 and SEQ ID NO:168, respectively; (x) SEQ ID NO:111 and SEQ ID NO:168, respectively; (y) SEQ ID NO:116 and SEQ ID NO:169, respectively; (z) SEQ ID NO:117 and SEQ ID NO:169, respectively; (aa) SEQ ID NO:116 and SEQ ID NO:164, respectively; (bb) SEQ ID NO:117 and SEQ ID NO:164, respectively; (cc) SEQ ID NO:120 and SEQ ID NO:164, respectively; (dd) SEQ ID NO:121 and SEQ ID NO:164, respectively; (ee) SEQ ID NO:122 and SEQ ID NO:164, respectively; (ff) SEQ ID NO:123 and SEQ ID NO:164, respectively; (gg) SEQ ID NO:124 and SEQ ID NO:164, respectively; (hh) SEQ ID NO:125 and SEQ ID NO:164, respectively; (ii) SEQ ID NO:126 and SEQ ID NO:170, respectively; (jj) SEQ ID NO:127 and SEQ ID NO:170, respectively; (kk) SEQ ID NO:128 and SEQ ID NO:164, respectively; (ll) SEQ ID NO:129 and SEQ ID NO:164, respectively; (mm) SEQ ID NO:128 and SEQ ID NO:171, respectively; (nn) SEQ ID NO:129 and SEQ ID NO:171, respectively; (oo) SEQ ID NO:130 and SEQ ID NO:164, respectively; (pp) SEQ ID NO: 131 and SEQ ID NO: 164, respectively; (qq) SEQ ID NO:132 and SEQ ID NO:169, respectively; (rr) SEQ ID NO:133 and SEQ ID NO:169, respectively; (ss) SEQ ID NO:134 and SEQ ID NO:164, respectively; (tt) SEQ ID NO:135 and SEQ ID NO:164, respectively; (uu) SEQ ID NO:134 and SEQ ID NO:171, respectively; (vv) SEQ ID NO:135 and SEQ ID NO:171, respectively; (ww) SEQ ID NO:136 and SEQ ID NO:164, respectively; (xx) SEQ ID NO:137 and SEQ ID NO:164, respectively; (yy) SEQ ID NO:136 and SEQ ID NO:171, respectively; (zz) SEQ ID NO:137 and SEQ ID NO:171, respectively; (aaa) SEQ ID NO:138 and SEQ ID NO:164, respectively; (bbb) SEQ ID NO:139 and SEQ ID NO:164, respectively; (ccc) SEQ ID NO:138 and SEQ ID NO:170, respectively; (ddd) SEQ ID NO:139 and SEQ ID NO:170, respectively; (eee) SEQ ID NO:140 and SEQ ID NO:164, respectively; (fff) SEQ ID NO:141 and SEQ ID NO:164, respectively; (ggg) SEQ ID NO:142 and SEQ ID NO:171, respectively; (hhh) SEQ ID NO:143 and SEQ ID NO:171, respectively; (iii) SEQ ID NO:144 and SEQ ID NO:164, respectively; (jjj) SEQ ID NO:145 and SEQ ID NO:164, respectively; (kkk) SEQ ID NO:146 and SEQ ID NO:164, respectively; (lll) SEQ ID NO:147 and SEQ ID NO:164, respectively; (mmm) SEQ ID NO:148 and SEQ ID NO:164, respectively; (nnn) SEQ ID NO:149 and SEQ ID NO:164, respectively; (ooo) SEQ ID NO:150 and SEQ ID NO:170, respectively; (ppp) SEQ ID NO: 151 and SEQ ID NO: 170, respectively; (qqq) SEQ ID NO:152 and SEQ ID NO:172, respectively; (rrr) SEQ ID NO:153 and SEQ ID NO:172, respectively; (sss) SEQ ID NO:154 and SEQ ID NO:173, respectively; (ttt) SEQ ID NO:155 and SEQ ID NO:173, respectively; (uuu) SEQ ID NO:156 and SEQ ID NO:174, respectively; (vvv) SEQ ID NO:157 and SEQ ID NO:174, respectively; (www) SEQ ID NO:158 and SEQ ID NO:175, respectively; (xxx) SEQ ID NO:159 and SEQ ID NO:175, respectively; (yyy) SEQ ID NO:158 and SEQ ID NO:174, respectively; and (zzz) SEQ ID NO: 159 and SEQ ID NO: 174, respectively 5. The isolated antigen-binding peptide of any one of the preceding embodiments, having a sequence selected from the group consisting of: [ka] It specifically binds to the compound.

[0020] Specific embodiment 12: (a) SEQ ID NO:176 and SEQ ID NO:160, respectively; (b) SEQ ID NO:177 and SEQ ID NO:160, respectively; (c) SEQ ID NO:178 and SEQ ID NO:160, respectively; (d) SEQ ID NO:179 and SEQ ID NO:160, respectively; (e) SEQ ID NO:180 and SEQ ID NO:164, respectively; (f) SEQ ID NO:181 and SEQ ID NO:164, respectively; (g) SEQ ID NO:182 and SEQ ID NO:164, respectively; (h) SEQ ID NO:183 and SEQ ID NO:164, respectively; (i) SEQ ID NO:184 and SEQ ID NO:163, respectively; (j) SEQ ID NO:185 and SEQ ID NO:163, respectively; (k) SEQ ID NO:186 and SEQ ID NO:163, respectively; (l) SEQ ID NO:187 and SEQ ID NO:163, respectively; (m) SEQ ID NO:184 and SEQ ID NO:162, respectively; (n) SEQ ID NO: 185 and SEQ ID NO: 162, respectively; (o) SEQ ID NO:186 and SEQ ID NO:162, respectively; (p) SEQ ID NO:187 and SEQ ID NO:162, respectively; (q) SEQ ID NO:188 and SEQ ID NO:165, respectively; (r) SEQ ID NO:189 and SEQ ID NO:165, respectively; (s) SEQ ID NO:190 and SEQ ID NO:165, respectively; (t) SEQ ID NO:191 and SEQ ID NO:165, respectively; (u) SEQ ID NO:192 and SEQ ID NO:161, respectively; (v) SEQ ID NO:193 and SEQ ID NO:161, respectively; (w) SEQ ID NO: 194 and SEQ ID NO: 161, respectively; and (x) SEQ ID NO: 195 and SEQ ID NO: 161, respectively wherein the isolated antigen-binding peptide specifically binds to a 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: The 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: The 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: The 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: The 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: The 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: The 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: The 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 a compound of formula (II). [ka]

[0037] Specific embodiment 29: The 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: The 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 a compound of formula (II). [ka]

[0041] Specific embodiment 33: The 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 a 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 according to any one of embodiments 1 to 34.

[0044] Specific embodiment 36: An isolated vector comprising the polynucleotide of embodiment 35.

[0045] Specific embodiment 37: An isolated host cell comprising the vector of embodiment 36.

[0046] Specific embodiment 38: A method for producing an antigen-binding peptide or Fab fragment antibody or a tandem Fab fragment antibody, comprising: (a) culturing the host cell of embodiment 37 under conditions that promote protein production, such that the host cell produces the antigen-binding peptide or Fab fragment antibody or tandem Fab fragment antibody; and (b) isolating said antigen-binding peptide or Fab fragment antibody or tandem Fab fragment antibody from the culture medium of (a).

[0047] Specific embodiment 39: A detection reagent comprising 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, and a detectable label.

[0048] Specific embodiment 40: The detection reagent of embodiment 39, wherein the isolated antigen-binding peptide or the isolated Fab fragment antibody or the isolated tandem Fab fragment antibody is linked 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, comprising administering to a subject in need thereof a pharma- ceutical effective amount of 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. [ka] Where: R 1 is C 1-4 is alkyl; R 2 is independently selected from F, Cl, CF3, CHF2, CH2F, CH3; R 3 is independently selected from CF3, CHF2, CH2F, and CH3; R 4 is H; and R 5 is independently selected from F and Cl.

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

[0051] Specific embodiment 43: The method of embodiment 41 or 42, wherein the pharma- ceutical effective amount of the isolated antigen-binding peptide or isolated Fab fragment antibody or isolated tandem Fab fragment antibody comprises an antigen-binding peptide or Fab fragment antibody or tandem Fab fragment antibody in a molar ratio of at least about 1:1 relative to the administered amount of the compound of formula (I) or (II), or in a molar ratio of at least about 1:1 relative to the amount of the compound of formula (I) or (II) present in the subject.

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

[0053] Specific embodiment 45: A method according to any one of embodiments 41 to 44, wherein the isolated antigen-binding peptide or the isolated Fab fragment antibody or the isolated tandem Fab fragment antibody 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 a stereoisomer, tautomer, or a pharma- ceutically acceptable salt thereof in a biological sample. [ka] Where: R 1 is C 1-4 is alkyl; R 2 is independently selected from F, Cl, CF3, CHF2, CH2F, CH3; R 3 is independently selected from CF3, CHF2, CH2F, and CH3; R 4 is H; and R 5 is independently selected from F and Cl. The above method is as follows: (a) contacting a biological sample with the isolated antigen-binding peptide or isolated Fab fragment antibody or isolated tandem Fab fragment antibody according to any one of embodiments 1 to 34; and (b) detecting the amount of a complex bound by the compound and the isolated antigen-binding peptide or the isolated Fab fragment antibody or the isolated tandem Fab fragment antibody. It is characterized by:

[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 the isolated antigen-binding peptide or the isolated Fab fragment antibody or the 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 immunoassay.

[0059] Specific embodiment 51: The method according to any one of embodiments 47 to 50, wherein the biological sample comprises urine, stool, saliva, whole blood, plasma, organ tissue, hair, skin, cells, or cell cultures.

[0060] Specific embodiment 52: A method for binding a compound of formula (I) or a stereoisomer or tautomer thereof in a subject receiving a therapeutically effective amount of a compound of formula (I) or a stereoisomer or tautomer thereof, comprising administering to the subject a pharma- ceutical effective amount of an isolated antigen-binding peptide or an isolated Fab fragment antibody or an isolated tandem Fab fragment antibody according to any one of claims 1 to 34. [ka] Where: R 1 is C 1-4 is alkyl; R 2 is independently selected from F, Cl, CF3, CHF2, CH2F, CH3; R 3 is independently selected from CF3, CHF2, CH2F, and CH3; R 4 is H; and R 5 is 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 any one of embodiments 52 or 53, wherein the pharma- ceutical effective amount of the isolated antigen-binding peptide or isolated Fab fragment antibody or isolated tandem Fab fragment antibody comprises an antigen-binding peptide or Fab fragment antibody or tandem Fab fragment antibody in a molar ratio of at least about 1:1 relative to the administered amount of the compound of formula (I) or (II), or in a molar ratio of at least about 1:1 relative to the amount of the compound of formula (I) or (II) present in the subject.

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

[0064] Specific embodiment 56: A method according to any one of embodiments 52 to 55, wherein the isolated antigen-binding peptide or the isolated Fab fragment antibody or the isolated tandem Fab fragment antibody 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 description of the drawings]

[0066] [Figure 1] Figures 1A and 1B show a comparison of the 26D5 mAb sequence with the closest 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. The Kabat numbering and Kabat definitions of HCDR2 and HCDR3 are shown. HCDR1 is shown with the Kabat and AbM definitions. Figure 1A shows SEQ ID NOs: 205, 83 and 220, respectively, in order of expression. Figure 1B shows a comparison of the 26D5 VK sequence with the germline sequences of IGKV1-12 and IGKJ4. The Kabat numbering and Kabat definitions of the CDRs are shown. Figure 1B shows SEQ ID NOs: 206, 98 and 207, respectively, in order of expression.

[0067] [Diagram 2] Figure 2 shows the positions of 26D5-GV-Q mAb randomized in the mutation scan. Kabat numbering indicates each position randomized in the mutation scan. The Kabat definitions of LCDR1-3, HCDR2-3 (where the last 6 amino acids are missing from HCDR2); and the AbM definition of HCDR1 are shown. Figure 2 shows SEQ ID NOs: 33, 46, 208-209, and 26 from the top, respectively.

[0068] [Diagram 3] Figures 3A-E show heat map scans showing the positions where mutations occur in the binding site of the 26D5-GV-Q mAb antibody (favorable, neutral, unfavorable). 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 amino acid sequences of the subsequent generations of the affinity-matured 26D5-GV-Q antibody. Kabat was used for the definition and numbering of CDRs. Figure 4A shows the alignment of the heavy chain variable region (SEQ ID NOs: 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, respectively). Figure 4B shows the alignment of the light chain variable region (SEQ ID NOs: 98, 99, 88, 88, 88, 88, 88, 88, 88, 88, 93, 93, 94, 95, 88, 95, 88, 94, 95, 88, 94, 95, and 88, respectively).

[0070] [Diagram 5] Figure 5 shows the positions of 26D5-295-B08 mAb randomized in the mutation scan. The numbering according to Kabat indicates each position randomized in the mutation scan. Figure 5 shows SEQ ID NOs: 210 to 215 from the top.

[0071] [Figure 6] Figures 6A-F show heat map scans depicting the locations of mutations in the CDRs and adjacent framework regions of the antibody binding site of 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 amino acid positions of 26D5-295-B08 mAb that were randomized to create a library of complexes (Figure 7A: partial complex library (sequence numbers 216 to 219 from the top, respectively); Figure 7B: doped complex library (sequence number 215)).

[0073] [Figure 8] Figures 8A and 8B show the alignment of the amino acid sequences of the progeny of affinity-matured 26D5-295-B08 mAb. Kabat was used for the definition and numbering of CDRs. Figure 8A shows the alignment of the heavy chain variable region (SEQ ID NOs: 83, 82, 60, 54, 52, 54, 60, 53, 61, 56, 59, 57, 57, 58, and 55, respectively). Figure 8B shows the alignment of the light chain variable region (SEQ ID NOs: 98, 99, 88, 86, 84, 87, 91, 85, 89, 89, 88, 92, 90, 88, and 88, respectively).

[0074] [Figure 9] Figure 9 shows the results of a chromogenic enzyme assay to determine the amount of antibody required to restore 50% or more of Compound A activity. Factor XIa enzyme activity (y-axis) was plotted as a function of representative affinity matured mAb concentration (x-axis) at constant concentrations of Factor XIa substrate and Compound A. The top graph shows data obtained with parental 26D5 mAb IgG1f (SEQ ID NOs: 83 and 98; also referred to herein as P1-072224) (shown in the figure as "26D5 mAb"). The bottom graph shows data obtained with 26D5-296-G07 mAb IgG1f (SEQ ID NOs: 65 and 94; also referred to herein as P1-073056) (shown in the figure as "26D5-296-G07 mAb").

[0075] [Figure 10] FIG. 10 shows a surface plasmon resonance (SPR) graph depicting the binding affinity of the parent 26D5 mAb (P1-072224) to various concentrations of Compound A (denoted as "26D5" in the figure).

[0076] [Figure 11] FIG. 11 shows surface plasmon resonance (SPR) graphs depicting 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] FIG. 12 shows surface plasmon resonance (SPR) graphs depicting 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] FIG. 13 shows time resolved fluorescence-fluorescence resonance energy transfer (TR-FRET) data from competition of (A) the mAb form of 26D5-GVR-Q-FT-Fab-LONG, (B) the mAb form of 26D5-295-B08-Fab-LONG, and (C) the mAb form of 26D5-75747-348-D07-Fab-LONG against compound 5, respectively.

[0079] [Figure 14] FIG. 14 shows time resolved fluorescence-fluorescence resonance energy transfer (TR-FRET) data from competition data for the indicated antibodies and Fab fragment antibodies against compound 5, respectively.

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

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

[0082] [Figure 17] FIG. 17 depicts the structure of 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 reversal 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 plasma concentration of unbound Compound A in the presence or absence of Fab fragment antibody 26D5-75616-348-F10-Fab-SHORT. Human plasma clotting time (aPTT) is plotted as a function of plasma concentration of Compound A in the absence (closed circles) and presence (open circles) of neutralizing Fab fragment antibody 26D5-75616-348-F10-Fab-SHORT.

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

[0086] [Figure 21] FIG. 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) followed 20 minutes later by intravenous administration of Fab fragment antibody 26D5-75616-348-F10-Fab-SHORT (160 mg / kg).

[0087] [Figure 22] Figure 22 shows the pharmacokinetics (plasma concentration in rats) of Fab fragment antibody 26D5-75616-348-F10-Fab-SHORT and tandem Fab fragment antibody 26D5-75616-348-F10-TanFab after intravenous administration of 10 mg / kg over 10 minutes. TANDEM FAB (○) shows data for tandem Fab fragment antibody 26D5-75616-348-F10-TanFab. FAB (△) shows data for 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 Compound A (0.4 mg / kg) was administered intravenously over 10 minutes by infusion, followed 20 minutes later by tandem Fab fragment antibody 26D5-75616-348-F10-TanFab (40 mg / kg) administered intravenously over 10 minutes by infusion. TANDEM Fab (○) shows data for tandem Fab fragment antibody 26D5-75616-348-F10-TanFab.

[0089] [Figure 24] Figure 24 shows the reversal of the anticoagulant effect of Compound A by neutralizing tandem Fab fragment antibody 26D5-75616-348-F10-TanFab. Human plasma clotting time (aPTT) was plotted as a function of the plasma concentration of Compound A and the plasma concentration of neutralizing tandem Fab fragment antibody 26D5-75616-348-F10-TanFab. "Tandem Fab" refers to tandem Fab fragment antibody 26D5-75616-348-F10-TanFab. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0090] The present invention provides novel antibodies or antigen-binding peptides that bind selective FXIa inhibitors and / or dual inhibitors of FXIa / plasma kallikrein. As used herein, a FXIa inhibitor is a compound of formula (I) that can inhibit the activity or function of FXIa. Thus, in some embodiments, the antigen-binding peptides disclosed herein (such as, but not limited to, antibodies or fragment antibodies) specifically bind to a compound of formula (I) or a stereoisomer or tautomer thereof. In some embodiments, the R 1 is C 1-4 R in formula (I) is alkyl; 2 is independently selected from F, Cl, CF3, CHF2, CH2F, CH3; R in formula (I) 3 is independently selected from CF, CHF, CHF, and CH; R in formula (I) 4 is H; and R of formula (I) 5 is independently selected from F and Cl. In certain embodiments, the antigen-binding peptides disclosed herein (such as, but not limited to, antibodies or antibody fragments) specifically bind to a compound of formula (II). As used herein, the term compound of formula (I) or (II) includes all compounds having formula (I) or (II), or stereoisomers or tautomers thereof. [ka]

[0091] In certain embodiments, the antigen conjugated to the antigen-binding peptide of the present invention is a compound of formula (II) (also referred to herein as Compound A and known as milvexian). Milvexian is a high affinity and highly selective, direct acting, reversible small molecule therapeutic agent that binds to and inhibits activated human coagulation factor XI (FXIa). The chemical name of milvexian is (5R,9S)-9-(4-(5-chloro-2-(4-chloro-1H-1,2,3-triazol-1-yl)phenyl)-6-oxopyrimidin-1(6H)-yl)-21-(difluoromethyl)-5-methyl-21H-3-aza-1(4,2)-pyridina-2(5,4)-pyrazolacyclononaphan-4-one. Milvexian and methods for making milvexian are described in U.S. Patent No. 9,453,018, the entire contents 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 proenzyme FXI. FXI is then physiologically activated by FXIIa and thrombin. See Mohammed B. et al. Thromb Res., 161:94-105 (2018), which is 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 molecule compounds, proteins, polypeptides, peptides, carbohydrates, polynucleotides, lipids, or any portion or combination thereof.

[0094] In some embodiments, the antigen-binding peptides of the invention are antibodies, or any of the following, including but not limited to, (Fab), Fab', F(ab')2, Fd, single chain Fv or scFv, disulfide-linked 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.

[0095] In some embodiments, the antigen-binding peptides of the invention (e.g., antibodies or Fab fragments) may be isolated. As used herein, the term "isolated" means that the nucleic acid, peptide or protein is separated from the original environment, e.g., the cell or organism in which it was produced, or the fluid in which the nucleic acid, peptide or protein naturally occurs. In the context of peptides or proteins with novel, non-naturally occurring amino acid sequences, an "isolated" peptide or protein means that the protein or peptide is separated from the engineered cells that produce them. A peptide or protein is considered isolated for the purposes of the present invention if it is not in the cell or original environment in which it is produced, even if the peptide or protein is a component of a mixture or composition, such as a pharmaceutical formulation.

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

[0097] In one specific embodiment of the present invention, a 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 an embodiment, 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 tandem Fabs are disclosed in Table 4 of the present application. As used herein, the terms "tandem Fab", "tandem Fab fragment antibody" and "TanFab fragment antibody" are used interchangeably.

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

[0099] The variable regions of the heavy and light chains generally each consist of four framework regions (FRs) linked by three complementarity determining regions (CDRs), also known as hypervariable regions. The CDRs of each chain are adjacent to each other through the FRs and contribute to the formation of the antigen-binding domain of the antibody together with the CDRs of the other chain. 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 interspecies sequence diversity; and (2) a method based on crystal structure analysis of antigen-antibody complexes. Furthermore, those skilled in the art may use a combination of these two methods to determine CDRs. The CDRs of each chain are numbered from the amino terminus to the carboxy terminus as CDR1, CDR2, and CDR3.

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

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

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

[0103] The Kabat numbering system is commonly used to refer to residues in the heavy chain variable region or light chain variable region domains (approximately residues 1-107 for the light chain and 1-113 for 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)). Antigen-interacting residues in the CDRs can also be determined by crystal structure analysis of the antigen-antibody complex.

[0104] As used herein, the terms "polypeptide," "peptide," and "protein" are used interchangeably to refer to a polymer of amino acids of any length. An amino acid polymer may be linear or branched and may contain modified amino acids. It may also be interrupted by non-amino acids. The term also includes amino acid polymers that are naturally or artificially modified. In some embodiments, the amino acid polymer is modified by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other treatment or modification. In some embodiments, the amino acid polymer is modified by conjugation with a labeling component. Also included within this definition are peptides having one or more analogs of amino acids known to those of skill in the art as well as non-naturally occurring amino acids.

[0105] "Specifically binds" (or "specific binding") is a term well known to those of skill in the art and generally means that the antigen-binding site of an antigen-binding peptide (such as, but not limited to, an antibody or antibody fragment) specifically recognizes an antigen through the antigen-binding domain, and the binding means that there is at least some complementarity between the antigen-binding domain and the antigen. As stated in this definition, an antigen-binding peptide (such as, but not limited to, an antibody or antibody fragment) is said to "specifically bind" to an epitope of an antigen through its antigen-binding domain and binds more readily 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 set forth in Table 1. In some embodiments, the antibody or fragment antibody of the present invention disclosed herein specifically binds to a FXIa inhibitor of the present disclosure (e.g., but not limited to, a FXIa inhibitor of formula (I)) and comprises a VH and a VL, each of which has CDRs that independently make up to four (i.e., 0, 1, 2, 3, or 4) conservative amino acid substitutions from the corresponding CDRs set forth in Table 1. Table 1: Amino acid sequences of the CDRs of the variable heavy (VH) and light (VL) chains [Table 1] [Table 2] [Table 3] [Table 4] [Table 5]

[0107] In some embodiments, antibodies or antibody fragments of the invention comprise a combination of VH and VL amino acid sequences set forth in Table 2. In some embodiments, antibodies or antibody fragments of the invention comprise a combination of partial heavy chain amino acid sequences and full-length light chain amino acid sequences set forth in Table 3. In some embodiments, antibodies or antibody fragments of the invention comprise a combination of tandem partial heavy chain amino acid sequences and full-length light chain amino acid sequences set forth in Table 4.

[0108] In some embodiments, the antibody or antibody fragment 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 antibody fragment of the present invention comprises both a variable light chain and a variable heavy chain as described herein. In some embodiments, the antibody or antibody fragment of the present invention comprises one variable heavy chain paired with one variable light chain as described herein. In some embodiments, the antibody or antibody fragment 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 antibody fragment 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 antibody fragments having VH and VL sequences that are at least about 80%, 85%, 89%, 90%, 95%, or 99% identical to the VH and VL sequences disclosed in Table 2 herein. Table 2: Variable heavy and light chain amino acid sequences (underlined regions are CDRs, non-underlined regions are framework regions) [Table 6] [Table 7] [Table 8] [Table 9] [Table 10] [Table 11]

[0110] The present invention also includes antibodies or antibody fragments having partial heavy chain 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 and full-length light chain amino acid sequences disclosed in Table 3. The present invention also includes antibodies or antibody fragments (e.g., Fab fragment antibodies) that include or are composed of any combination of any one of the N-terminal portions of the heavy chains in Table 3 and any one of the full-length light chains in Table 3. The present invention also includes antibodies or antibody fragments (e.g., Fab fragment antibodies) that include or are composed of any pair of the N-terminal portion of the heavy chain and full-length light chain shown in Table 3. The present invention also includes antibodies or antibody fragments (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 antibody fragments (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 antibody fragments (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 antibody fragments (eg, Fab fragment antibodies) consisting of the sequences of SEQ ID NO:106 and SEQ ID NO:164. Table 3: Fab sequences (non-bolded parts are variable regions of CDRs (underlined) and framework regions (not underlined); bolded 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 antibody fragments having tandem partial heavy chain 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 and full-length light chain amino acid sequences disclosed in Table 4. Table 4: Tandem Fab sequences (non-bolded parts are variable regions of CDRs (underlined) and framework regions (not underlined); bolded amino acids are constant regions of the Fab region, heavy and light chains, respectively; double underlined parts are linker sequences) [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 an art-recognized meaning and can be calculated using published techniques (see, e.g., 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)). There are many methods for measuring 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)). Commonly used methods for determining 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 Carillo, H. and Lipton, D., SIAM J. Applied Math. 48:1073 (1988).Methods for aligning 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 (which uses the Smith-Waterman local homology algorithm (Advances in Applied Mathematics 2:482 489 (1981))).

[0113] For example, a polynucleotide is at least 95% "identical" to a reference sequence of nucleotides, respectively, meaning that the nucleotide sequence of the polynucleotide is identical to the reference sequence. However, the polynucleotide sequence may contain up to 5 mutations per 100 nucleotides compared to the reference sequence of nucleotides. For example, to obtain a polynucleotide having a nucleotide sequence that is at least 95% identical to the reference sequence of nucleotides, up to 5% of the nucleotides in the reference sequence may be deleted or replaced by another nucleotide. Alternatively, multiple nucleotides up to a total of 5% of the reference sequence may be inserted into the reference sequence.

[0114] As a practical matter, 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 known computer programs. The method of determining the best overall match between a query sequence and a reference sequence, also called global alignment, can be determined using the FASTDB computer program based on the algorithm of Brutlag et al. (Comp. App. Biosci. 6:237-245 (1990)). In conventional nucleotide sequence alignment, both the query sequence and the reference sequence are DNA sequences. However, it can also be compared with RNA sequences by converting U to T. The result of global alignment is expressed as percent identity. In one embodiment of the invention, the parameters used in FASTDB alignment of DNA sequences to calculate percent 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, Window Size=500 (or whichever is shorter for the length of the nucleotide sequences of interest).

[0115] When the reference sequence is shorter than the query sequence, not due to internal sequence deletion, but due to deletion of the 5' or 3' end, manual correction is essential to obtain the result. This is because the FASTDB program does not take into account the deletion of the 5' and 3' ends of the reference sequence when calculating the identity (%). For reference sequences that are truncated at the 5' or 3' end with respect 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 are not matched / aligned compared to the query sequence as a percentage of the total bases of the query sequence. This percentage (%) is then subtracted from the identity (%) calculated by the FASTDB program or the like using specific parameters to obtain the final identity (%) score. The corrected score is the one used for the purpose of the present invention. Only bases other than the 5' and 3' ends of the reference sequence displayed by the FASTDB alignment that are not matched / aligned with the query sequence 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 the percent 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 at the 5' end. The 10 unpaired bases are 10% of the sequence (number of bases at the 5' and 3' ends that are not matched / number of bases in the entire query sequence), so 10% is subtracted from the percent identity score calculated by the FASTDB program. If the remaining 90 bases are perfectly matched, the final percent identity will be 90%. As another example of comparing a 100-base query sequence with a 90-base reference sequence, if the deletion is an internal sequence deletion, there are no bases at the 5' or 3' ends in the reference sequence that are not matched / aligned with the query sequence. In this case, the percent identity calculated by FASTDB is not manually corrected. Again, only bases at the 5' and 3' ends of the reference sequence that are not matched / aligned with the query sequence are manually corrected.

[0117] For example, a polypeptide having an amino acid sequence that is at least 95% "identical" to an 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 subject polypeptide sequence may contain up to 5 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 replaced by another amino acid. These changes in the reference sequence may occur at the amino terminus, carboxy terminus, or anywhere between these termini of the reference amino acid sequence, either individually or interspersed in one or more contiguous portions.

[0118] As a practical matter, 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 be conventionally determined using known computer programs. A preferred method of determining the best overall match between a query sequence (a sequence of the present invention) and a reference sequence, also referred to as global alignment, can be determined using the FASTDB computer program mentioned above. In sequence alignment, both the query sequence and the reference sequence are amino acid sequences. The result of the global alignment is percent identity. In one embodiment of the invention, the parameters used in FASTDB alignment of amino acid sequences to calculate percent 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 whichever is shorter for the amino acid sequences of interest).

[0119] When the reference sequence is shorter than the query sequence due to N- or C-terminal deletions, rather than internal sequence deletions, manual correction is essential to obtain results. This is because the FASTDB program does not take into account the N- and C-terminal deletions of the reference sequence when calculating the identity percentage. For reference sequences truncated at the N- or C-terminus, the identity percentage is corrected by calculating the number of residues at the N- and C-terminus of the reference sequence that are not matched / aligned compared to the corresponding residues of the query sequence as a percentage of the total bases of the query sequence. Whether or not a residue is matched / aligned is determined by the result of sequence alignment in FASTDB. This percentage is then subtracted from the identity percentage calculated by the above FASTDB program with specific parameters to obtain the final identity percentage score. This final identity percentage score is the one used for the purposes of the present invention. Only the bases at the N- and C-terminus of the reference sequence that are not matched / aligned with the query sequence are considered to manually adjust the identity percentage score. That is, residues of the query sequence are located other than the farthest N- and C-terminal residues in the reference sequence.

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

[0121] Within the disclosed percent identity ranges, the present invention also relates to substitution variants of the disclosed polypeptides of the present invention. Substitution variants include polypeptides in which one or more amino acid residues are deleted and replaced by another residue. In certain embodiments, the percent identity disclosed above is for the entire sequence of the identified specific sequence, with the amino acid residues that are not substituted and are not subject to substitution being the CDRs of the sequence, and the amino acid residues that are subject to substitution being the framework regions of the sequence. For example, in certain embodiments, when an antigen-binding peptide of the present invention (such as, but not limited to, an antibody or a fragment antibody) comprises 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, the CDR regions of the VH are constant and the framework regions may vary. However, the overall percent identity with SEQ ID NO: 64 is within the scope of the embodiments. The mutations are substitutions of a conservative nature, but in certain embodiments of the present invention, non-conservative substitutions are also included. Conservative substitutions in the present invention may be defined as shown in Tables 5-7 below. Amino acids are classified according to their physical properties and contribution to the secondary and tertiary structure of proteins. Conservative substitutions are recognized by those skilled in the art as those in which one amino acid is replaced with another amino acid that has similar properties. Examples of conservative substitutions are shown below. [Table 44] Alternatively, the amino acids may be grouped as set forth in Lehninger (1975) Biochemistry, Second Edition; Worth Publishers, pp. 71-77, as set forth below. [Table 45]

[0122] Further examples of conservative substitutions are given below. [Table 46]

[0123] In some embodiments of the antibody or fragment antibody of the present invention, the CH1 domain comprises a partial heavy chain constant region having the amino acid sequence of 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 comprises a partial heavy chain constant region having the amino acid sequence of ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTH (SEQ ID NO: 203). The present invention also includes antibodies or antibody fragments having a CH1 domain that 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 antibody fragments of the present invention, the CL domain comprises a light chain constant region having the amino acid sequence of RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 204). The present invention also includes antibodies or antibody fragments having a CL domain that 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 antibody fragment of the present invention comprises one or more heavy chain N-terminal portions and a full-length light chain, respectively, as described herein. In some embodiments, the antigen-binding peptide (such as, but not limited to, an antibody or antibody fragment) comprises both the heavy chain N-terminal portion and the full-length light chain sequence as described herein. In some embodiments, the antibody or antibody fragment of the present invention comprises the N-terminal portion of one heavy chain paired with one full-length light chain as described herein. In some embodiments, the antibody or antibody fragment 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 antibody fragment 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 N-terminal portions of the two heavy chains are linked via a linker.

[0125] Table 4 shows the heavy and light chain sequences of examples of tandem Fabs of the invention. In some embodiments, the heavy chain of the tandem Fab comprises the N-terminal portion of one or two antibody heavy chains linked via a linker, and the light chain of the tandem Fab comprises the full-length light chain (VL-CL) of an antibody. In some embodiments, the heavy chain of the tandem Fab can be represented as "VH-CH1-linker-CH1-VH or VH-CH1-linker-VH-CH1".

[0126] The linker included in the present invention can be any suitable molecule of various structures. In certain embodiments, the linker is a polypeptide linker. The polypeptide linker can be of various lengths. In some embodiments, the linker is a polypeptide having about 20 or less amino acids. Examples of polypeptide linker sequences are disclosed in Table 8 and Table 4 in the double underlined portion. [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 (such as, but not limited to, an antibody or fragment antibody) 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: 1-12; a VH-CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 13-22; or a VH-CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 23-28. In some embodiments, the at least one VL comprises at least one of a VL-CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 29-37; a VL-CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 38-43; or a VL-CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 44-51. In some embodiments, an antigen-binding peptide of the invention (e.g., but not limited to, an antibody or fragment antibody) comprises a VH-CDR1, a VH-CDR2, a VH-CDR3, a VL-CDR1, a VL-CDR2, and a VL-CDR3 with 1, 2, 3, or 4 conservative substitutions.

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

[0130] In some embodiments, at least one VH region and at least one VL region disclosed in Table 2 herein also includes a variant sequence having 1, 2, 3, or 4 conservative substitutions.

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

[0132] The present invention further includes polynucleotides having nucleic acid sequences encoding all or part of the antigen-binding peptides disclosed herein (eg, but not limited to, antibodies or antibody fragments).

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

[0134] The present invention also includes polynucleotides having a sequence 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% identical to any of the polynucleotides of the present disclosure. The present invention further provides polynucleotide variants that encode fragments, analogs, and derivatives of the antigen-binding peptides of the present disclosure (e.g., but not limited to, antibodies or antibody fragments). Polynucleotide variants can include mutations in coding regions, non-coding regions, or both. In some embodiments, polynucleotide variants include mutations that result in silent mutations, insertions, or deletions, but do not alter the properties or activities of the encoded polypeptide. In some embodiments, polynucleotide variants are obtained by silent mutations due to the degeneracy of the genetic code. Polynucleotide variants can be obtained for a variety of reasons, such as for optimizing expression of codons for a particular host.

[0135] In certain embodiments, the polynucleotides of the present invention have a coding sequence for a mature polypeptide linked in-frame to a polynucleotide that encodes a polypeptide that aids in the expression and secretion of the polypeptide from, for example, a host cell. In some embodiments, the mature polypeptide is an antigen-binding peptide of the present disclosure (e.g., but not limited to, an antibody or a fragment antibody). In certain embodiments, the polynucleotide has a sequence that encodes a signal polypeptide sequence that functions as a secretory sequence to control the transport of the polypeptide from the cell. A polypeptide with a signal sequence is a precursor protein, which can be degraded by the host cell to form the mature polypeptide. The polynucleotide can also encode a precursor protein that includes 5' amino acid residues added to the mature protein. A mature polypeptide with a prosequence is a precursor protein, which is an inactive protein. Once the prosequence is degraded, an active mature protein remains. In certain embodiments, the polynucleotide has a coding sequence for a mature polypeptide linked in-frame to a marker sequence that can, for example, allow purification of the encoded polypeptide.

[0136] In some embodiments, the present invention provides a vector comprising any one of the polynucleotides disclosed herein. As used herein, the term "vector" refers to a construct capable of transporting and expressing one or more polynucleotides, proteins, or sequences of interest in a host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or naked RNA expression vectors, plasmids, cosmids, or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells (e.g., production cells).

[0137] The present invention further provides a host cell comprising the vector disclosed herein. In some embodiments, the host cell is an isolated cell. In some embodiments, the isolated host cell produces the antigen-binding peptide (e.g., but not limited to, an antibody or antibody fragment) disclosed herein. Suitable host cells include prokaryotes, yeast, insect, or higher eukaryotic cells. Prokaryotes include gram-negative or gram-positive bacteria (e.g., E. coli or Bacillus). Higher eukaryotic cells include established cell lines of mammalian origin as described below. Cell-free translation systems may also be used. Suitable cloning and expression vectors for bacterial, fungal, yeast, and mammalian cell hosts are generally known to those skilled in the art. Various mammalian or insect cell culture systems may also be utilized to express recombinant proteins. Expression of recombinant proteins in mammalian cells is generally because such recombinant proteins are correctly folded, appropriately modified, and fully functional. Examples of suitable mammalian host cell lines include monkey kidney cell lines COS-7, L cells, C127, 3T3, Chinese hamster ovary (CHO), HeLa, and BHK cell lines. Additionally, baculovirus systems for producing heterologous proteins in insect cells are commonly known to those skilled in the art.

[0138] The antigen-binding peptides of the invention (such as, but not limited to, antibodies or antibody fragments) obtained from the transformed host can be purified by any suitable method, including chromatography (e.g., ion exchange, affinity and size exclusion column chromatography), centrifugation, differential lysis, or any other standard technique used to purify proteins. Affinity tags (e.g., hexahistidine (SEQ ID NO:221), maltose binding domain, influenza coat sequence and glutathione-S-transferase) can be attached to the protein to facilitate purification through an appropriate affinity column. Isolated proteins can be physically characterized using techniques such as proteolysis, nuclear magnetic resonance, mass spectrometry and x-ray crystallography. Purification methods for antibodies and other proteins are generally known to those of skill in the art.

[0139] In certain embodiments, the present invention provides a method for producing an antigen-binding peptide (e.g., but not limited to, an antibody or antibody fragment) of the present disclosure. In examples, the method comprises: (a) culturing a host cell as disclosed above under culture conditions that promote production of a protein such that the host cell produces the antigen-binding peptide (e.g., but not limited to, an antibody or antibody fragment); and (b) isolating the antigen-binding peptide (e.g., but not limited to, an antibody or antibody fragment) from the cultured cell. The antigen-binding peptide (e.g., but not limited to, an antibody or antibody fragment) of the present invention can be obtained using methods for producing antigen-binding peptides that are generally known to those of skill in the art.

[0140] In some embodiments, the antigen-binding peptides disclosed herein (e.g., but not limited to, antibodies or antibody fragments) are used as detection reagents. In some embodiments, the antigen-binding peptides (e.g., but not limited to, antibodies or antibody fragments) are detectably labeled. As used herein, the term "label" refers to a detectable compound that is directly or indirectly attached to the antigen-binding peptide (e.g., but not limited to, antibodies or antibody fragments). The label may be detectable by itself (e.g., radioisotope label or fluorescent label) or, in the case of an enzymatic label, may catalyze a chemical change of a detectable substrate. In certain embodiments, the label is selected from the group consisting of immunofluorescent label, chemiluminescent label, phosphorescent label, enzymatic label, isotopic label, avidin / biotin, gold colloid, colored particle, and magnetic particle.

[0141] In some embodiments, the antigen-binding peptide (such as, but not limited to, an antibody or a fragment antibody) forms a complex bound to 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 using a variety of methods. In some embodiments, it is detected by immunological assay.

[0142] Immunological assay as used herein refers to any assay that utilizes the specificity of antibody-antigen binding in vitro or in vivo. In some embodiments, the assay can be used to confirm the presence or absence of a target molecule in a biological sample. In some embodiments, the assay can be used to measure the amount or level of a target molecule. In some embodiments, the target molecule is an immune complex of an antigen-binding peptide (such as, but not limited to, an antibody or a fragment antibody) bound to a compound of formula (I) or (II) in vitro or in vivo. In some embodiments, the target molecule is a compound of formula (I) or (II) itself. In some embodiments, the immunological assay includes, but is not limited to, radioimmunoassay, immunohistochemical staining, chemiluminescent immunoassay (CLIA), enzyme immunoassay (EIA) or enzyme-linked immunosorbent assay (ELISA), western blotting, enumeration immunoassay, flow cytometry, fluorescent immunoassay, and fluorescence activated cell sorting (FACS).

[0143] Biological sample as used herein may be any sample obtained from a patient. In some embodiments, biological sample is urine, stool, saliva, whole blood, plasma, organ tissue, hair, skin, cell, or cell culture. In some embodiments, biological sample is liquid. In some embodiments, biological sample may be fixed by fixative. For example, aldehyde-based fixative (e.g. formalin (formaldehyde) and glutaraldehyde) is commonly used.

[0144] The present invention further provides a method for suppressing the antithrombotic effect of a FXIa inhibitor or a dual inhibitor of FXIa and plasma kallikrein in a subject in need thereof. In some embodiments, the present invention relates to a method for suppressing the antithrombotic effect of a FXIa inhibitor. In certain embodiments, the FXIa inhibitor is a compound of formula (I) or (II). In some embodiments, the method comprises administering to the subject a pharma- ceutical effective amount of an antigen-binding peptide (e.g., but not limited to, an antibody or an antibody fragment) disclosed herein. In some embodiments, the antigen-binding peptide (e.g., but not limited to, an antibody or an antibody fragment) can bind with high affinity to a compound of formula (I) or (II) and suppress its antithrombotic effect in vitro or in vivo. In some embodiments, binding of the antigen-binding peptide (e.g., but not limited to, an antibody or an antibody fragment) to a 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 a FXIa inhibitor (eg, 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, non-human primates, etc. 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 a treatment disclosed herein. The terms "subject" and "patient" are used interchangeably herein.

[0146] For example, the terms "treatment" or "treating" are used interchangeably and refer to a method of treatment that cures, slows, inhibits, reduces, reverses or resolves the effects of, and / or halts the progression of, a condition. As used herein, the term "treatment" is used to refer to receiving at least one of the antigen-binding peptides of the invention (e.g., but not limited to, an antibody or antibody fragment). The term "prevent" or "avoid risk" is used to refer to a prophylactic method that prevents the onset of a condition of interest and / or slows its progression, or reduces the risk of developing an abnormal condition compared to a person not receiving treatment. Thus, subjects in need of treatment include those who already have the condition (e.g., thrombosis), those who are susceptible to developing the condition, and those in whom the condition is to be prevented.

[0147] The present invention also provides pharmaceutical compositions comprising the antigen-binding peptides of the present disclosure (e.g., but not limited to, antibodies or antibody fragments). In some embodiments, the pharmaceutical compositions of the present invention include therapeutic and / or prophylactic compositions. In some embodiments, the pharmaceutical compositions comprise a therapeutically effective dose of the antigen-binding peptides (e.g., but not limited to, antibodies or antibody fragments) and a pharma- ceutically acceptable carrier or excipient. Such pharma-ceutically 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, flow agents, lubricants, adsorbents, vehicles, sweeteners, flavors, colorants, odorants, salts (the substances of the present invention themselves may be used in the form of pharma-ceutically acceptable salts), buffers, coating agents, and antioxidants. Examples of excipients include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, sucrose, sorbitol, and any combination thereof. In some embodiments, the pharma-ceutically acceptable excipient is an inactive ingredient. However, it is understood that some pharma- ceutically acceptable excipients may affect the manufacture, quality, safety, or efficacy of a pharmaceutical composition. In some embodiments, a pharmaceutical composition may also contain a therapeutically active agent in addition to an antigen-binding peptide of the invention (e.g., but not limited to, an antibody or antibody fragment).

[0148] The pharmaceutical compositions of the invention may be presented in dosage or multi-dose containers, such as sealed ampoules and vials, and stored in a freeze-dried condition requiring only the addition of a sterile injectable liquid carrier, such as water or saline, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets.

[0149] Pharmaceutical compositions can be administered in a convenient and suitable manner according to the purpose.In some embodiments, pharmaceutical compositions can be administered by parenteral route.In some embodiments, parenteral administration can be intravenous administration, intraperitoneal administration, intramuscular administration, intratumoral administration, subcutaneous administration, intranasal administration, or intradermal administration.

[0150] Pharmaceutical compositions for transdermal administration may be formulated as discrete patches intended to remain in intimate adhesion to the epidermis of the recipient for a prolonged period of time, for example, the active ingredient may be released from the patch by iontophoresis, as generally described in Pharmaceutical Research, 3(6):318 (1986).

[0151] Pharmaceutical compositions for nasal administration wherein the carrier is a solid include coarse powder having a particle size of, for example, 20 to 500μ, and these are administered by inhalation, i.e., rapid inhalation through the nose from a container of the powder held close to the nose. Compositions wherein the carrier is a liquid, suitable for administration as a nasal spray or nasal drops, include aqueous or oily solutions of the active ingredient.

[0152] Pharmaceutical compositions for parenteral administration may include aqueous and non-aqueous sterile injection solutions, including aqueous and non-aqueous suspensions that may contain antioxidants, buffers, bacteriostatic agents, and solutes to make the preparation substantially isotonic with the blood of the intended recipient; and suspending agents and thickening agents.Excipients that can be used for injection solutions include, for example, water, alcohol, polyol, glycerin, and vegetable oils.

[0153] As used herein, the terms "effective amount", "therapeutically effective amount" and "pharmaceutical effective amount" are used interchangeably and refer to a dose sufficient to achieve a physiological effect. In some embodiments, a pharmaceutical effective amount of an antigen-binding peptide (e.g., but not limited to, an antibody or an antibody fragment) disclosed herein refers to an amount of an antigen-binding peptide (e.g., but not limited to, an antibody or an antibody fragment) that is effective to reduce or neutralize the antithrombotic effect of the compound of the present disclosure in a subject in need thereof. In some embodiments, the antithrombotic effect of a compound of Formula (I) or (II) can be immediately restored by administering a pharmaceutical effective amount of an antigen-binding peptide (e.g., but not limited to, an antibody or an antibody fragment) to a subject experiencing severe bleeding. In some embodiments, administration of a pharmaceutical effective amount of an antigen-binding peptide (e.g., but not limited to, an antibody or an antibody fragment) has a limited duration of action. In certain embodiments, a single administration of a pharmaceutical effective amount of an antigen-binding peptide (e.g., but not limited to, an antibody or an antibody fragment) has a sufficient duration of action to restore the antithrombotic effect of a compound of Formula (I) or (II). On the other hand, a single administration of a pharmacologic effective amount of an antigen-binding peptide (e.g., but not limited to, an antibody or antibody fragment) has a sufficiently short duration of action that antithrombotic therapy can be resumed immediately after administration of the single pharmacologic effective amount, minimizing the time during which the risk of thromboembolism is increased.

[0154] In some embodiments, the pharma- ceutical effective amount may be determined empirically and routinely for the above-mentioned purposes. For example, in some embodiments, the dose of the compound of formula (II) is about 25 mg (once a day) to about 375 mg (twice a day). In some embodiments, the pharma- ceutical effective amount of the antigen-binding peptide (e.g., but not limited to, an antibody or a fragment antibody) is about 25 mg once a day. In some embodiments, the pharma- ceutical effective amount of the antigen-binding peptide (e.g., but not limited to, an antibody or a fragment antibody) is about 50 mg once a day. In some embodiments, the pharma- ceutical effective amount of the antigen-binding peptide (e.g., but not limited to, an antibody or a fragment antibody) is about 75 mg once a day. In some embodiments, the pharma- ceutical effective amount of the antigen-binding peptide (e.g., but not limited to, an antibody or a fragment antibody) is about 100 mg once a day. In some embodiments, the pharma- ceutical effective amount of the antigen-binding peptide (e.g., but not limited to, an antibody or a fragment antibody) is about 125 mg once a day. In some embodiments, the pharma- ceutical effective amount of the antigen-binding peptide (e.g., but not limited to, an antibody or antibody fragment) is about 150 mg once a day. In some embodiments, the pharma- ceutical effective amount of the antigen-binding peptide (e.g., but not limited to, an antibody or antibody fragment) is about 175 mg once a day. In some embodiments, the pharma- ceutical effective amount of the antigen-binding peptide (e.g., but not limited to, an antibody or antibody fragment) is about 200 mg once a day. In some embodiments, the pharma- ceutical effective amount of the antigen-binding peptide (e.g., but not limited to, an antibody or antibody fragment) is about 375 mg once a day.

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

[0156] In some embodiments of the present invention, the pharma- ceutical effective amount of an antigen-binding peptide (e.g., but not limited to, an antibody or antibody fragment) is determined primarily in relation to the amount of a compound of Formula (I) or (II) previously administered. In some embodiments, the pharma- ceutical effective amount includes at least about a 1:1 molar ratio of the antigen-binding peptide (e.g., but not limited to, an antibody or antibody fragment) to the amount of a compound of Formula (I) or (II) previously administered to the subject. In some embodiments, the pharma- ceutical effective amount includes at least about a 2:1 to about 10:1 molar ratio of the antigen-binding peptide (e.g., but not limited to, an antibody or antibody fragment) to the amount of a compound of Formula (I) or (II) previously administered to the subject. In some embodiments, a pharma- ceutical effective amount includes 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 of an antigen-binding peptide (e.g., but not limited to, an antibody or antibody fragment) relative to the amount of a compound of Formula (I) or (II) previously administered to the subject.

[0157] In some embodiments, a medicamentically effective amount includes an antigen-binding peptide (e.g., but not limited to, an antibody or antibody fragment) in a molar ratio of at least about 1:1 to the amount of a compound of Formula (I) or (II) in a subject. In some embodiments, a medicamentically effective amount includes an antigen-binding peptide (e.g., but not limited to, an antibody or antibody fragment) in a molar ratio of at least about 2:1 to about 10:1 to the amount of a compound of Formula (I) or (II) in a subject. In some embodiments, a medicamentically effective amount includes an antigen-binding peptide (e.g., but not limited to, an antibody or antibody fragment) 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 a compound of Formula (I) or (II) in a subject.

[0158] In some embodiments, the pharma- ceutical effective amount of antigen-binding peptide (e.g., but not limited to, antibody or antibody fragment) is calculated by mass ratio. For example, the molecular weight (MW) of the antigen-binding peptide (e.g., but not limited to, antibody or antibody fragment) may be about 75 times the MW of the compound of formula (II). In this example, the equivalent molar of antigen-binding peptide (e.g., but not limited to, antibody or antibody fragment) to about 100 mg of the compound of formula (II) is about 7.5 g. The molar masses of substances are described herein, so that one skilled in the art can easily calculate the mass ratio of antigen-binding peptide (e.g., but not limited to, antibody or antibody fragment) to the compound of formula (I) or (II).

[0159] In some embodiments, the dose of the antigen-binding peptide (such as, but not limited to, an antibody or antibody fragment) is determined in a clinical trial. Prior to the two studies, computational models and simulations were performed that incorporate (1) human pharmacokinetic and human pharmacological information (obtained from Phase 1 studies), (2) binding kinetics, and (3) predicted human PK parameters.

[0160] The antigen-binding peptides of the present invention (e.g., but not limited to, antibodies or antibody fragments) may be administered simultaneously or after administration of a compound of Formula (I) or (II). In some embodiments, the antigen-binding peptides (e.g., but not limited to, antibodies or antibody fragments) are administered simultaneously with administration of a compound of Formula (I) or (II). In some embodiments, the antigen-binding peptides (e.g., but not limited to, antibodies or antibody fragments) are administered immediately after administration of a compound of Formula (I) or (II). In some embodiments, the antigen-binding peptides (e.g., but not limited to, antibodies or antibody fragments) are administered about 30 minutes after the start of administration of a compound of Formula (I) or (II). In some embodiments, the antigen-binding peptides (e.g., but not limited to, antibodies or antibody fragments) are administered about 20 minutes after the completion of administration of a compound of Formula (I) or (II). However, the antigen-binding peptides (e.g., but not limited to, antibodies or antibody fragments) and the compound of Formula (I) or (II) may be administered simultaneously or sequentially in any order as deemed appropriate by one of skill in the art.

[0161] The antigen-binding peptides of the present invention (e.g., but not limited to, antibodies or antibody fragments) may be administered by any route deemed appropriate by one of skill in the art. In certain embodiments, the antigen-binding peptides (e.g., but not limited to, antibodies or antibody fragments) are administered intravenously, intramuscularly, or subcutaneously. In some embodiments, the antigen-binding peptides (e.g., but not limited to, antibodies or antibody fragments) are administered once a day. In some embodiments, the antigen-binding peptides (e.g., but not limited to, antibodies or antibody fragments) are administered one or more times a day. In some embodiments, the antigen-binding peptides (e.g., but not limited to, antibodies or antibody fragments) are administered over a period of about 10 minutes. In some embodiments, the antigen-binding peptides (e.g., but not limited to, antibodies or antibody fragments) are administered over a period of less than about 10 minutes. In some embodiments, the antigen-binding peptides (e.g., but not limited to, antibodies or antibody fragments) are administered over a period of about 10 minutes or more.

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

[0163] The scope of the present disclosure should not be limited by any of the above-described exemplary embodiments, but should be defined only in accordance with the following claims and their equivalents.

[0164] The contents of all references (including literature references, patents, patent applications, and websites) that may be cited in this application are expressly incorporated herein in their entirety for any purpose, as if referenced.

[0165] Working Example Example 1: Production of human monoclonal antibodies against compound A using transgenic mice expressing human antibody genes - Production of antigens and related compounds

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

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

[0168] To a flask (100 mL) containing a white suspension of tert-butyl 1-(4-chloro-2-(6-hydroxypyrimidin-4-yl)phenyl)-1H-1,2,3-triazole-4-carboxylate (105 mg, 0.28 mmol) in acetonitrile (3.7 mL), HATU (1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate, 117 mg, 0.31 mmol) and DBU (1,8-diazabicyclo[5.4.0]undec-7-ene, 55.0 μL, 0.37 mmol) were added. The resulting clear yellow solution was stirred at room temperature for 5 min. (5R,9S)-9-Amino-21-(difluoromethyl)-5-methyl-21H-3-aza-1(4,2)-pyridin-2(5,4)-pyrazolacyclononaphan-4-one (94 mg, 0.281 mmol) was added and the resulting suspension was stirred at room temperature for 3 h, at which point it was concentrated to dryness. The resulting residue was dissolved in EtOAc (1 mL) and loaded onto an ISCO column (40 g). The product was eluted with a linear gradient of EtOAc / hexanes (0% to 100%) over 35 min. The product eluted exactly at 100% EtOAc. tert-Butyl 1-(4-chloro-2-(1-((5R,9S)-21-(difluoromethyl)-5-methyl-4-oxo-21H-3-aza-1(4,2)-pyridina-2(5,4)-pyrazolacyclononaphan-9-yl)-6-oxo-1,6-dihydropyrimidin-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)-pyrazolacyclononaphan-9-yl)-6-oxo-1,6-dihydropyrimidin-4-yl)phenyl)-1H-1,2,3-triazole-4-carboxylic acid hydrochloride (compound 1A) [ka] tert-Butyl 1-(4-chloro-2-(1-((5R,9S)-21-(difluoromethyl)-5-methyl-4-oxo-21H-3-aza-1(4,2)-pyridina-2(5,4)-pyrazolacyclononaphan-9-yl)-6-oxo-1,6-dihydropyrimidin-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 h, at which point deprotection was complete as indicated by LCMS. The reaction mixture was concentrated to dryness and further dried under vacuum overnight. 1-(4-chloro-2-(1-((5R,9S)-21-(difluoromethyl)-5-methyl-4-oxo-21H-3-aza-1(4,2)-pyridin-2(5,4)-pyrazolacyclononaphan-9-yl)-6-oxo-1,6-dihydropyrimidin-4-yl)phenyl)-1H-1,2,3-triazole-4-carboxylic acid 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)-pyrazolacyclononaphan-9-yl)-6-oxo-1,6-dihydropyrimidin-4-yl)phenyl)-1H-1,2,3-triazole-4-carboxamido)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)-pyrazolacylcyclononaphan-9-yl)-6-oxo-1,6-dihydropyrimidin-4-yl)phenyl)-1H-1,2,3-triazole-4-carboxylic acid 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 (benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate, 27.8 mg, 0.063 mmol) was added and the mixture was stirred for 4 h at room temperature. The resulting residue was concentrated to dryness and then diluted with CHCl (4 mL) and TFA (trifluoroacetic acid, 2 mL). The reaction mixture was stirred for 2 h at room temperature and then concentrated to dryness to give a residue. 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-triazol-1-yl)phenyl)-6-oxopyrimidin-1(6H)-yl)-5-methyl-4-oxo-21H-3-aza-1(4,2)-pyridin-2(3,4)-pyrazolacyclononaphan-21-yl)ethoxy)ethoxy)ethoxy)propanoic acid (compound 2); Synthesis of 3-(2-(2-(2-((5R,9S)-9-(4-(5-chloro-2-(4-chloro-1H-1,2,3-triazol-1-yl)phenyl)-6-oxopyrimidin-1(6H)-yl)-5-methyl-4-oxo-21H-3-aza-1(4,2)-pyridin-2(5,4)-pyrazolacyclononaphan-21-yl)ethoxy)ethoxy)ethoxy)propanoic acid (compound 3) [ka] (5R,9S)-9-(4-(5-chloro-2-(4-chloro-1H-1,2,3-triazol-1-yl)phenyl)-6-oxopyrimidin-1(6H)-yl)-5-methyl-21H-3-aza-1(4,2)-pyridina-2(5,4)-pyrazolacylnonaphan-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 h. The obtained 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] Compound 2, compound 3 and compound 1 were conjugated to BSA and KLH for immunological 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 mixing 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 in the dark at room temperature for 3 hours and then dialyzed against 5 L of DPBS (1x, Lonza, cat#17-512Q).

[0174] Condensation to BSA : A sample of 2 mg 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 mixing by vortexing. Then 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 (added ratio compound:carrier:activator=10:1:2). The sample was incubated at room temperature for 3 hours in the dark, then dialyzed against 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-triazol-1-yl)phenyl)-6-oxopyrimidin-1(6H)-yl)-5-methyl-4-oxo-21H-3-aza-1(4,2)-pyridin-2(3,4)-pyrazolacyclononaphan-21-yl)-12-oxo-3,6,9-trioxa-13-azapentadecan-15-yl)-5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-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]imidazol-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 h. 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 transgenic mice expressing human antibody genes - Immunization of mice

[0178] Human anti-compound A antibodies were produced by immunizing a mouse strain (HCo42:01 KCo5:01 [J / K]) transgenic for human Ig genes (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) in Ribi adjuvant (RA). Immunization was performed by injecting the immunogen mixture in Ribi adjuvant into the footpad. Mice were immunized every 3–5 days for a total of seven times over a three-week period, with a prefusion booster inoculation given the day before tissue collection, followed by lymph node removal. In vivo experiments were performed in accordance with the regulations of the Bristol-Myers Squibb Company Animal Care and Use Committee. Lymph nodes were removed from three immunized mice, homogenized, and stored in liquid. Mouse myeloma cells (SP2 / 0-Ag14 (ATCC CRL-1581) TM Hybridomas were produced by electrofusion with 6938.6938.26D5.D12 (referred to herein as "26D5"), a hybridoma subclone with the 6938 fusion cell line, which showed the highest ability to produce fully human antibodies.

[0179] Example 3: Affinity Maturation of Antibodies Affinity maturation was performed on human mAb 26D5 to improve its affinity for compound A. First, the sequence of mAb 26D5 was compared to the closest human germline V and J gene sequences (Figure 1A-B). To reduce the risk of immunogenicity in humans, the germline-different framework regions were mutated or reverted back to the germline sequences to generate human mAb 26D5-GV-Q (this fully human antibody form is also referred to as P1-072226), which retained similar binding to compound A. mAb 26D5-GV-Q (P1-072226) was used as the starting point for a mutational scan to measure the relative fitness of all possible amino acid substitutions at the antibody CDR positions shown in Figure 2. The "deep mutation scan" approach has been 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, we created libraries of scFvs (single chain) with amino acid substitutions using NNK oligonucleotides in the CDRs shown in Figure 2. To create this library of single chain mutants, we designed multiple oligonucleotides, each containing an NNK codon (where N=A, C, G, T, and K=G, T) at a single position. By using these degenerate codons, we can code for all 20 naturally occurring amino acids (and a stop codon) at the position where the NNK codon was incorporated. Kabat definitions were used for all CDRs except for HCDR1, which was defined by AbM (Abhinandan and Martin, Mol. Immunol. 45:3832, 2008; Swindells et al., J. Mol. Biol. 429:356, 2017). Amino acids 60-65 of HCDR2 were not analyzed.Next, the first round of DNA library was transcribed and translated in vitro 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). Among any scFvs that bound to compound 5 (biotin-labeled compound 2), those that interacted with streptavidin magnetic particles and were eluted were selected and amplified by PCR. Finally, both the DNA library created at the beginning and the eluted DNA encoding the scFv that bound to compound 5 were sequenced by next-generation sequencing (NGS).

[0180] To analyze the NGS data, the sequences read from both forward and reverse directions by NGS were organized using FLASH (Magoc and Salzberg, Bioinformatics 27:2957, 2011) and classified into distinct ranges according to the mutation position and the identity of the mutated amino acid. All sequences with low quality and multiple mutations were excluded during the analysis. The enrichment efficiency (ER) was then calculated by dividing the frequency of each sequence before selection by the frequency after selection. In other words, the enrichment efficiency is the number of specific sequence variants in the compound 5 binding sample divided by the number of specific sequence variants in the initial library. This was then normalized to the enrichment efficiency of the parental 26D5-GV-Q mAb. In this way, the effect of each single amino acid substitution in the CDR region described herein on the binding to compound 5 (and thus the binding to compound A) was evaluated. Figure 3A shows a heat map of LCDR1 generated using the analysis of the mutation scan data, illustrating the relationship between the sequence and activity of single amino acid substitutions. In general, the error in this method is about 2-fold. In other words, if the enrichment efficiency (ER) value is 0.5 to 2, it is considered to be a general substitution, that is, a substitution that maintains the binding property to compound 5. An ER value of 2 or more is considered to be favorable for binding, and an ER value of less than 0.5 is considered to be poor for binding. Figures 3B to 3E show heat maps of CDRs other than LCDR1.

[0181] Based on the enrichment efficiency calculated from the NGS data, variable region genes containing single and multiple favorable amino acid substitutions were synthesized and cloned into an IgG expression vector together with the Fc region of human IgG1f and the human LCκ region (CK). The vector was transiently introduced into Expi293 HEK cells at a small scale (2 mL of medium) and purified using a protein A filter plate. The functional activity of the IgG protein was evaluated by SPR (surface plasmon resonance) (see data below). The sequences of the antibodies of interest identified according to this evaluation are shown in Figure 4A-B.

[0182] The progeny 26D5-295-B08 was selected for further affinity maturation using three different library designs. In the first design, the residues shown in Figure 5 were randomized and 26D5-295-B08 was subjected to a second mutation scan. In this second mutation scan, the non-CDR portions defined by Kabat and / or AbM were randomized around the portion adjacent to compound A in the co-crystal structure. Heat maps obtained from the mutation scan of 26D5-295-B08 are shown in Figures 6A-F. Variable region genes containing single and multiple favorable amino acid substitutions were synthesized as described above and cloned into IgG1f expression vectors for use in the assays. mAbs of interest identified from a 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, in which the target portion was further randomized (Figures 7A-B). The partial library was designed to include one or two substitutions for all amino acids (except cysteine ​​and methionine) in the CDR and adjacent framework regions, including other modifications as described in Figure 7A. Sequences containing chemical disorders were also removed from the design and synthesized from an oligonucleotide chip (Twist Bioscience). The doped library was randomized intensively for HCDR3 and the surrounding framework region residues so that two or more mutations were generated in one CDR. The DNA oligonucleotides encoding this library were created by "doping" a randomized region based on the 26D5-295-B08 DNA sequence. Each nucleotide in the randomized region contained 70% of the DNA base of 26D5-295-B08 and 10% of other bases. For example, if the DNA of 26D5-295-B08 has a G at a particular position, the doped nucleotide has 70% G, 10% A, 10% C, and 10% T at that position. While the region of interest is more randomized, it is still biased toward the original parent sequence library. The oligonucleotides are used to generate a library of partial DNA scFvs and a library of doped DNA scFvs. These libraries are selected using the mRNA display method as in the protocol above, but later with off-rates and multiple rounds in succession. The resulting sequences are sequenced using NGS, and the data is analyzed to select variable regions for synthesis and cloning into IgG expression vectors for testing. The mAbs of interest 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 from the doped library as the antibody of interest.8A-B show sequences of interest identified from affinity matured 26D5-295-B08 mAb.

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

[0185] Example 4: Identification of mAbs by chromogenic enzyme assay The ability of mAbs to maintain FXIa enzymatic activity in the presence of Compound A was used as an assay to screen for improved antibody affinity. In this assay, S-2366 chromogenic peptide (Chromogenix) was used as a 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 Compound A or a control compound for 10 minutes at 37°C. The chromogenic substrate (S-2366) was then added to a final concentration of 0.5 mM, and human FXIa enzyme (Haematologic Techologies, Inc.; HCZIA-0160) was added to 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 herein is performed over a significant measurement range. The plate was immediately read (OD405 nm, 37 °C) using a SPECTRAmax (Molecular Devices) to measure the rate of substrate hydrolysis. Signals were normalized to 0% activity (FXIa enzyme and compound A added) and 100% activity (FXIa enzyme added without inhibitor). The EC 50 The results of the example are shown in FIG.

[0186] Example 5: Production of recombinant α-compound AFab fragment antibodies The selected antibodies were cloned as untagged Fab fragment antibodies in the pTT5 vector of the Expi293 expression system (Fab sequences are shown in SEQ ID NO:). Optimized DNA sequences for mammalian expression were obtained from GenScript. For 1 L scale expression, cells (900 mL, 3x10 6 0.25 mg each of heavy and light chain DNA constructs were seeded in Opti-MEM TM (50 mL). TM (4.1mL) / Opti-MEM TM Incubate the 150 mL at room temperature for 5 minutes, then add 50 mL of the DNA / Opti-MEM mixture. TM The mixture was added and incubated for 20 min at room temperature. A total of 100 mL of this transfection mixture was added to the cell culture medium (900 mL) described above and placed on a shaker (125 rpm, 8% CO2 in air) at 37°C. On day 1, the product was fed with VPA (2 mM) and CHO CD EfficientFeed B (50 mL). Cell viability was analyzed and the product was obtained on day 5. The average cell viability / cell density was 80% / 6x10 6The concentration of cells / mL was 1.5 mg / mL. The product was centrifuged (2,000 rpm) for 20 min at 4°C and the conditioned medium supernatant was filtered through a 0.2 μm filter. A column of rProteinA Sepharose FF (30 mL) was washed with 2 CV (column volumes) of 6 M guanidine, 2 CV of 0.033 M hydrochloric acid, and then equilibrated with 1x PBS (Dulbecco). The pH of the supernatant was ensured to be >7.0 and then loaded onto the rProteinA Sepharose column at 10 mL / min. All progenies of 1873.6938.26D5.D12, including affinity matured variants, bound rProteinA through framework regions by interaction with Protein A. The column was washed with 1xDPBS until baseline was reached, then with 80 mM sodium acetate (pH 2.8) and collected in a vessel filled with Tris HCl (pH 8.0, ~20 mL) so that the proteins were neutralized during elution. The column was then neutralized with 1xDPBS. The eluted sample was concentrated to <10mL and loaded onto an S200 26 / 600 column equilibrated with 1xDPBS at 2.5mL / min. Fractions (5mL) were collected at 2.5mL / min and analyzed by SDS-PAGE and chromatograms. Purified Fab fragment antibodies were obtained at an average of 150-250mg / L.

[0187] Example 6: Surface Plasmon Resonance (SPR) (Biacore TM ) analysis to identify mAbs and Fabs The hybridoma expressing the parent 26D5 mAb (VH SEQ ID NO: 83 and VL SEQ ID NO: 98 in the form of IgG1f, P1-072224) and the affinity matured mAb for Compound A were analyzed by SPR (Biacore) using binding to Protein A. TM) was investigated. 1xPBS (phosphate buffered saline, pH 7.4) containing 0.05% Tween 20 and 2% DMSO was used as the running buffer. Binding assays were performed at 37°C. Protein A was coated onto a sensor chip (CM5, S series, Cytiva, Cat. No. 29149603) at high density (Protein A RU: 2000+). Protein A was immobilized using standard amine coupling method as recommended by the manufacturer. Affinity matured antibodies at a concentration of 2ug / mL were then bound to the Protein A surface at a flow rate of 3μL / min for 2 minutes. Compound A at concentrations ranging from 100 to 3 nM (100, 50, 25, 12.5, 6.25, 3.125 nm) (Figure 10) and screening concentrations ranging from 100 to 3 nM (100, 33, 11 nm) were then injected onto the bound antibody at a flow rate of 100 μL / min for 2 min and allowed to dissociate for at least 450 s. The chip surface was regenerated by a pulse of 10 mM glycine (pH 1.5) for 40 s each cycle. Background binding to the Protein A surface alone was used to subtract nonspecific binding. All experiments were performed on a Biacore T200 surface plasmon resonance using Biacore T200 control software v.2 and data analysis was performed using Biacore T200 evaluation software v3.1. For ranking, only apparent affinities were determined because dissociation rates were too slow to be measured by Biacore. Example binding assay data are shown in Figure 10 and Table 10. The mAb names in Table 10 refer to mAbs in the form of IgG1f with the heavy and light chain sequences of the corresponding Fabs in Table 3. [Table 49] [Table 50]

[0188] The binding of the first affinity matured Fab fragment antibody to the compound A binding version was measured by SPR (Biacore) using a sensor chip (CM5, S series, Cytiva, Cat. No. 29149603) coated with BSA pre-bound with compound 2, compound 3 or compound 1. TM ) were investigated. The immobilization levels were 150-250 RU. The running buffer was 1xPBS (phosphate buffered saline, pH 7.4) with 0.05% Tween 20, and the experiments were performed at 25°C. Fab fragment antibodies at concentrations between 200 and 0.8 nM were injected onto the BSA-compound coated surface at a flow rate of 30 μL / min for 2 min. The Fab fragment antibodies were then allowed to dissociate for 15 min. The chip surface was regenerated with a 1 min pulse of 10 mM glycine (pH 2) and a 1 min pulse of NaOH (50 mM) for each cycle. All experiments were performed on the same equipment and analyzed with the same software as above. For ranking, only apparent affinities were determined, since the dissociation rates were too slow to be measured by Biacore. Comparative experiments were also performed with selected mAbs (see Figure 11). The binding assay data for the example are shown in Figure 11, and the binding assay data for compound 2-BSA are shown in Table 11. The sequences of the Fabs in Table 11 are shown in Table 3. [ka]

[0189] Binding of the first and second rounds of affinity matured Fab fragment antibodies to the compound A binding version was measured using SPR (Biacore) with a biotin CAP sensor chip (S series) and kit reagents (Cytiva, Cat No. 28920234). TM) were investigated. Biotin-CAP chips were hydrated overnight with buffer. A 50% aqueous solution of biotin-CAP reagent was flowed over the chip surface for 150 seconds at a flow rate of 2 μL / min. 0.25 ug / mL of compound 5 (biotin-labeled compound 2) was bound to the biotin-CAP surface with a 20-second pulse at a flow rate of 10 μL / min. The running buffer was 1xPBS (phosphate-buffered saline, pH 7.4) with 0.05% Tween 20, and the experiments were performed at 25°C or 37°C. Fab fragment antibodies at concentrations of 100-3 nM were injected over the biotin-CAP-compound 5 surface for 3 minutes at a flow rate of 30 μL / min. The Fab fragment antibodies were then allowed to dissociate for 11.7 minutes. The chip surface was regenerated with a pulse of 6M guanidine-HCl / NaOH (250 mM) for 2 minutes each cycle. All experiments were performed on the same equipment and analyzed with the same software as described above. In ranking, only apparent affinity was determined because the dissociation rates were too slow to be measured by Biacore. Binding assay data for the example is shown in FIG. 12, and binding assay data for TanFab fragment antibodies is shown in Table 12. The sequences of TanFab 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-fluorescence resonance energy transfer) assay was developed to rank the rate of dissociation of Compound A from α-Compound A mAb equilibrated at 37°C. The assay buffer was HBS-N (10 mM HEPES, 150 mM NaCl, pH 7.4; GE Healthcare), 0.1% (w / v) BSA (bovine serum albumin; Sigma), 2% DMSO (dimethyl sulfoxide; Sigma). All reagents were prepared in assay buffer and evenly dispensed into a white 384-well microplate to a final volume of 20 μL. The final concentrations were 4 nM of compound 5 (biotin-labeled compound 2), 100–0.1 nM of 7 drops of human α-compound A antibody, 0.1 nM of europium-labeled α-mouse IgG (LANCE Eu-W1024; PerkinElmer), 5 nM of streptavidin-D2 (Cisbio), and 4 nM of mAb 26D5 VH_A10G_Y33A_S53P_M89V_G95A; VK_W32N_H38Q (P1-075621, second optimization of parent strain 26D5-295-B08 in the form of Fc of mouse IgG). Compound 5 and α-compound A antibody were first dropped onto the microplate, and the first incubation (37 °C, 1 h, 1000 rpm) was performed to encourage the antibody to bind to the compound. After the first incubation, europium α-mouse IgG, streptavidin-D2, and 26D5-mouse IgG (P1-075621) were added in sequence, and the plate was then 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 min (T0) and after 24 h (T24). For each antibody titration, the FRET signal was converted to % inhibition (100-((FRET at antibody concentration / FRET at 0 nM)*100) relative to wells without α-Compound A antibody. The % inhibition for the antibody titrations was plotted using TIBCO Spotfire (v.7) and IC was calculated using a 4-parameter model curve fitting. 50 The IC at each time point was determined. 50, and IC compared to T0 50 Curve shift was indicated. 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 26D5-mouse IgG1 (P1-075621) competitor to bind, producing a FRET signal that is detected in the assay.

[0191] Competitive FRET performance data from affinity optimization screening of α-compound A 26D5 is shown in FIG. 13. Three human antibodies were represented: 1) 26D5-GVR-Q-FT-Fab-LONG in the form of an IgG1f mAb (also referred to herein as P1-072226, except that R31 in 26D5-GVR-Q-FT HC is S31 in P1-072226 HC; top graph), 2) 26D5-295-B08-Fab-LONG, a progeny of the first optimization round in the form of an IgG1f mAb (also referred to herein as P1-072963; middle graph), and 3) 26D5-75747-348-D07-Fab-LONG, a progeny of the second optimization round in the form of an IgG1f mAb (also referred to herein as P1-075747; bottom graph). On the titration curves of each antibody, IC 50 are indicated by asterisks. IC between T0 and T24 50 The difference between the IC10 and IC20 values ​​was taken as Δ and is shown in each graph. The parent 26D5-GVR-Q-FT-Fab-LONG mAb had the largest IC10 from T0 to T24. 50 The IC shift of 63 nM was observed. 50 The shift in the IC of T24 was smaller (P1-072963 = 20.5 nM, P1-075747 = 4.3 nm). 50 and I.C. 50 Antibodies were identified by their improved off-rate of Compound A versus parental mAb 26D5-GVR-Q-FT-Fab-LONG and mAb 26D5-295-B08 (P1-072963) at 37° C., ranked by shift (Δ).

[0192] The competitive TR-FRET assay defined above was further modified to evaluate the progeny of the 26D5 antibody, reconstituted and optimized as a Fab. The titration range of the α-Compound A antibody and Fab was extended to 250-0.244 nM (11 titrations), with each concentration replicated four times. To further ensure equilibrium was reached in the assay, the incubation time was increased to 48 h, with an IC of T48. 50 and I.C. 50 Shifts were measured. Percent inhibition titrations were plotted in Graphpad Prism (v.8) and fitted with a four-parameter model. All other experimental conditions were identical to the antibody TF-FRET assay.

[0193] Competition FRET data from affinity optimized α-Compound A progeny 26D5 is shown in Figure 14 and Table 13 in comparison to the human antibody and Fab. Each graph in Figure 14 shows FRET data for α-Compound A progeny 26D5 overlaid with an antibody (mAb, solid line, O) and Fab (dashed line, △) with identical sequences. The 26D5 parental Fab (Fab 26D5-GV-Q; also referred to herein as P1-073708) and the first round optimized progeny Fab 26D5-295-C08 (also referred to herein as P1-074468-1) both exhibited T48 IC 50 Shifts of at least 26 nM with significant IC 50 The second round of affinity maturation resulted in optimized progeny that dissociated slowly from Compound A at 37°C, with minimal IC of T48, in both antibody and Fab forms. 50 The FRET data is shown in Table 13, where the mAb / Fab names 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 A portion of the purified Fab (20 μg) was injected and eluted over 20 min using an Agilent 1260 HPLC column (Shodex K403-4F, mobile phase: 100 mM sodium phosphate, 150 mM sodium chloride, pH 7.3, flow rate 0.3 mL / min). Gel filtration standards confirmed that most Fabs were at least 98% monomeric, with recoveries of >75%.

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

[0196] Example 10: Kinetic Exclusion Assay (KinExA) The solution affinity of the disclosed Fab fragment antibodies for Compound A was measured by kinetic exclusion (KinExA). Duplicate titrations of Compound A were performed with 100, 200, and 300 pM of 26D5 affinity matured Fab fragment antibody (equilibrated for 24-72 hours). The relative concentration of unbound 26D5 affinity matured Fab fragment antibody was determined 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 identical to the kinetic analysis to determine the association rate of the complex, except that a single tube of mixture (200 pM Fab and 400 pM Compound A) was prepared and no equilibration was performed. The results are shown in Table 15 below. DSC thermostability 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. The Fab fragment antibody 26D5-75616-348-F10-Fab-SHORT (SEQ ID NO: 106 and SEQ ID NO: 164; see Table 3) was selected for X-ray crystallography and in vivo experiments described below in Example 12. [Table 54]

[0197] Example 11: Crystallization of Fab Fab26D5-GVR-Q-FT-Fab-SHORT with affinity tag GGHHHHHHH (SEQ ID NO:222) was concentrated to 10 mg / mL in DPBS (Dulbecco's Phosphate Buffered Saline). The protein was combined with a 5-fold molar excess of Compound A and incubated overnight at 4°C. The complex was crystallized by vapor diffusion. The drop consisted of 1 μL of complex and 1 μL of reservoir solution. The reservoir solution consisted of 20 g of PEG 3350 dissolved in water for a total volume of 100 mL and 20 mM sodium citrate, which is not a buffer. Crystals were made by adding 2.5 μL of a 40% PEG400:40% glycerol (v / v) mixture and 7.5 μL of reservoir solution to the drop solution in a serial dilution for rapid cooling with liquid nitrogen.

[0198] Fab fragment antibody 26D5-75616-348-F10-Fab-SHORT was concentrated to 20 mg / mL in DPBS buffer. The protein was combined with a 5-fold molar excess of compound A and incubated overnight at 4°C. The complex was crystallized by vapor diffusion. The drop consisted of 1 μL of complex and 1 μL of reservoir solution. The reservoir solution for crystallization 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. Crystals were generated by adding serial dilutions of 2.5 μL of a 40% PEG400:40% glycerol (v / v) mixture and 7.5 μL of reservoir solution to the drop for rapid cooling in liquid nitrogen.

[0199] The data were obtained using PILATUS 6M (Advanced Photon Source, beamline: 17-ID). Data were processed with 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] and with 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 26D5-GVR-Q-FT-Fab-SHORT / compound A crystal was one asymmetric unit per complex, with space group P212121 and unit cell edges a=57.7 Å; b=75.1 Å; c=84.7 Å. The data were treated isotropically and extended by 1.47 Å, but to preserve the data, cuts were made with ellipsoids extended by 1.38 Å for a*, 1.44 Å for b*, and 1.32 Å for c*. Molecular replacement with PHASER (McCoy, AJ, Grosse-Kunstleve, RW, Adams, PD, Winn, MD, Storoni, LC & Read, RJ (2007). Phaser Crystallographic Software. J. Appl. Crystallogr. 40, 658-674.) was used to identify the CL:CH1 model from the PDB 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), the VL model from the PDB 4PY7 (Wyrzucki, A., Dreyfus, C., Kohler, I., Steck, M., Wilson, IA, The structure was determined using the VH model (Wensley, B. Structure of a Lysozyme Fab Complex, unpublished) obtained from the PDB 4TSA (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 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 shows clear electron density for compound A. The ligand was 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) and geometrically constrained 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 model building with Coot (Emsley, P., Lokhamp, B., Scott, WG & Cowtan, K. (2010). Features and Development of Coot. Acta Crystallogr Sect. D 66, 486-501) and refinement with 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 refined structure of the 26D5-GVR-Q-FT-Fab-SHORT / Compound A complex.

[0201] The crystals of 26D5-75616-348-F10-Fab-SHORT / compound A were one asymmetric unit per four complexes, in space group P1, with unit cell edges a=64.8Å; b=84.9Å; c=100.9Å; α=83.4°; β=88.4°; γ=67.9°. The data were treated isotropically and extended by 2.7Å, but to preserve the data, they were cut with ellipsoids extending 0.880a*+0.436b*-0.189c* by 1.91Å, 0.063a*+0.898b*-0.436c* by 2.20Å, and 0.093a*+0.382b*+0.920c* by 2.98Å. The structure was determined by molecular replacement 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.) using the CL:CH1, VL and VH models (excluding CDR-H3) from the Fab fragment antibody 26D5-GVR-Q-FT-Fab-SHORT. The first electron density map shows the electron density for compound A.Model building was performed using Coot (Emsley, P., Lokhamp, B., Scott, WG & Cowtan, K. (2010). Features and Development of Coot. Acta Crystallogr Sect. D 66, 486-501) and automated NCS restraints were used in 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 with automated NCS and target-structure restraints in BUSTER. Acta Crystallogr Sect. D 68, 368-380). Figure 17 shows the refined structure of the 26D5-75616-348-F10-Fab-SHORT / compound A complex.

[0202] DSC thermostability analysis and KinExA showed 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 the 1:1 stoichiometric binding between Compound A and the Fab fragment antibody, and the binding mechanism between the heavy and light chains of the Fab fragment antibody.

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

[0204] In vitro experiments The effect of 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 the Fab fragment with plasma containing a known amount of Compound A. Plasma concentrations of Compound A were selected that significantly increased the aPTT, where a significant increase can be defined as an increase in clotting time of at least 20% compared to the clotting time (e.g., aPTT) in the absence of the factor XIa inhibitor. Antibodies or Fab fragment antibodies capable of binding to factor XIa inhibitors in plasma reduce the ability of the factor XIa inhibitor to bind to factor XIa, resulting in a shortening of the clotting time compared to the clotting 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 stored healthy human plasma at the same concentrations of 8000, 4000, 2000, 1000, 500, 250, 125, 62.5, 31.3 and 15.6 nM. Compound A-containing plasma, Fab fragment antibody 26D5-75616-348-F10-Fab-SHORT-containing plasma and normal plasma were combined and adjusted to various concentrations (including compound A in the absence of Fab fragment antibody 26D5-75616-348-F10-Fab-SHORT:26D5-75616-348-F10-Fab-SHORT molar ratios of 5:5, 5:4, 5:3 and 5:2, etc.).

[0206] Activated partial thromboplastin time (aPTT) (i.e., human plasma clotting time) was determined for each sample using ACTIN® FS (Siemens / Dade-Behring) according to the accompanying 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 warmed to 37°C for 1 minute. ACTIN® FS (0.05 mL) was added to the plasma and incubated for an additional 3 minutes. Calcium chloride (25 mM, 0.05 mL) was added to the reaction to initiate clotting. Clotting time refers to the time (in seconds) from the moment calcium chloride was added until a clot was detected.

[0207] Figure 18 shows the 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 the plasma clotting time (aPTT) in the absence (closed circles) and presence (open circles) of the neutralizing Fab fragment antibody 26D5-75616-348-F10-Fab-SHORT as a function of the concentration of the free factor XIa inhibitor (Compound A). The clotting time (aPTT) was 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 various concentrations and molar ratios of Compound A and Fab fragment antibody 26D5-75616-348-F10-Fab-SHORT were processed and the concentrations of bound and unbound 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 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 Fab fragment antibody 26D5-75616-348-F10-Fab-SHORT.

[0210] Free Compound A in plasma was obtained by ultrafiltration (Centrifree®, MilliporeSigma, molecular weight cutoff: 30 kDa). Plasma (0.5 mL) was added to the top of an ultrafiltration column (Centrifree®, 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] A portion of the plasma and the plasma ultrafiltrate was frozen at -80°C in a polypropylene tube. The total concentrations of Compound A in the plasma and free Compound A in the plasma ultrafiltrate were measured by liquid chromatography tandem mass spectrometry (LC / MS) analysis. Samples for LC / MS analysis were prepared by 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 solution of 50% water and 0.5% formic acid in methanol (20 μL) was added. The plate was covered and mixed on a shaker at 95°C for 20 minutes. An internal standard [10 nM, stable isotope (13C,15N) labeled compound A] and acetonitrile (80 μL) containing 1% formic acid were added to the above solution to precipitate proteins. The plate was mixed by vortexing at room temperature for an additional 15 minutes, then centrifuged at 3,600 rpm for 5 minutes. A portion of the supernatant (100 μL) was transferred to an injection plate (96 well, 0.3 mL). The supernatant (5 μL) was injected into an Ultra Performance LC System (Waters® Acquity UPLC) tandem mass spectrometer coupled to a QTRAP MS / MS (AB Sciex 6500). The substances were separated using a C18 column (Waters HSS T3, 2X50mm, 1.8μm) at 60℃ with a gradient (flow rate 0.7mL / 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 multiple reaction monitoring (MRM) in positive electrospray ionization mode. + The MRM transitions observed were 626→319 for compound A and 630→323 for isotope-labeled compound A. The lower limit of quantification was 0.5 nM.

[0213] The concentration of Fab fragment antibody 26D5-75616-348-F10-Fab-SHORT in plasma was determined as follows: The total plasma concentration of both Fab fragment antibody 26D5-75616-348-F10-Fab-SHORT and Fab fragment antibody 26D5-75616-348-F10-Fab-SHORT not bound to Compound A in plasma was measured by ligand binding assay using a Gyrolab® automated microfluidic system (Gyros Protein Technologies AB). Biotinylated mouse anti-human kappa (SouthernBiotech, AL) was used as the molecule that binds all Fab fragment antibodies 26D5-75616-348-F10-Fab-SHORT. Samples, standards, and QCs were brought up to a final base 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 Gyrolab® Bioaffy 200 CD (Gyros Protein Technologies AB) was used. After the final washing step, all bound Fab fragment antibody 26D5-75616-348-F10-Fab-SHORT was detected using Alexa Fluor® 647-labeled mouse anti-human Ig kappa light chain mAb clone G20-361 (BD Catalog No. 555861, Lot No. 8333691). Fluorescence intensity was measured in Gyrolab® and the total concentration of Fab fragment antibody 26D5-75616-348-F10-Fab-SHORT in the plasma samples was calculated using a 4-parameter logistic curve generated from the standard substance of Fab fragment antibody 26D5-75616-348-F10-Fab-SHORT. The calibration curve range for the total 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. Quality control samples were prepared at plasma concentrations of 20000, 7500, and 750 ng / mL.Standards and QCs were analyzed in each experiment to ensure an acceptable assay, and the assay performed within acceptable limits (%CVs for standards and QCs <20% and QC recoveries within ±20% of nominal values).

[0214] Compound 5 was used as a molecule to bind the Fab fragment antibody 26D5-75616-348-F10-Fab-SHORT that was not bound to Compound A. Samples, standards, and QCs were brought up to a final base 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 Ig kappa light chain mAb clone G20-361 (BD Catalog No. 555861, Lot No. 8333691). The fluorescence intensity was measured in Gyrolab® and the concentration of "active / free" Fab (26D5-75616-348-F10-Fab-SHORT) in plasma samples was calculated using a 4-parameter logistic curve generated from a standard of Fab fragment antibody 26D5-75616-348-F10-Fab-SHORT. The calibration range of "active / free" 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. Quality control samples were prepared at plasma concentrations of 20000, 7500, and 750 ng / mL. Standards and QCs were analyzed in each experiment to ensure an acceptable assay. The assays were performed within acceptable limits (%CVs for standards and QCs less than 20% and QC recoveries within ±20% of apparent values).

[0215] In vivo experiments In vivo experiments were performed in accordance with the regulations of the Animal Care and Use Committee of the Bristol-Myers Squibb Company. Indwelling catheters were placed in the central ear artery (for blood sampling) and marginal ear vein (for substance administration) of rabbits (male, New Zealand White, 2-4 kg). A fixed amount of compound A (1.0 mg / kg) was administered intravenously by infusion over 10 minutes. Twenty minutes after the infusion of compound A was completed, a fixed amount of Fab fragment antibody 26D5-75616-348-F10-Fab-SHORT (160 mg / kg) was administered intravenously by infusion over 10 minutes. The administered Fab fragment antibody 26D5-75616-348-F10-Fab-SHORT was administered at a molar concentration that was apparently 2-fold excess of compound A. Blood samples (1.5 mL) were collected before administration of Compound A, at the end of Compound A infusion, immediately before administration of Fab fragment antibody, at the end of administration of Fab fragment antibody, and at various intervals after administration of Fab fragment antibody and up to 24 hours after the start of administration of Compound A. Blood samples were added to 3.8% sodium citrate (0.167 mL) in polypropylene tubes, mixed thoroughly by inversion at least twice, and placed on ice. Plasma was isolated within 1 hour of blood collection by centrifuging whole blood 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 by activated partial thromboplastin time (aPTT). aPTT was determined using ACTIN® FS (Siemens / Dade-Behring) according to the attached instructions. Plasma (0.05 mL) was warmed at 37°C for 1 min. ACTIN® FS (0.05 mL) was added to the plasma and incubated for an additional 3 min. Calcium chloride (25 mM, 0.05 mL) was added to the reaction system to initiate coagulation. The coagulation time refers to the time (seconds) from the moment calcium chloride was added until a clot was detected.

[0217] After administration of Compound A in vivo, rabbit plasma aPTT increased approximately 2-fold compared to baseline. Subsequent administration of Fab fragment antibody 26D5-75616-348-F10-Fab-SHORT 20 minutes later resulted in the rabbit plasma aPTT returning to baseline, which persisted for over 12 hours. Figure 20 shows the plasma clotting time (aPTT) of rabbits intravenously administered Compound A (1 mg / kg) 20 minutes followed by Fab fragment antibody 26D5-75616-348-F10-Fab-SHORT (160 mg / kg). Results are the average of three rabbits.

[0218] Plasma concentrations of Compound A (total and free) and Fab fragment antibody 26D5-75616-348-F10-Fab-SHORT were determined as described above. PK parameters were obtained from noncompartmental analysis of plasma concentration versus 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. Area under the plasma concentration-time curve (AUC[0-T]) was calculated using a combination of linear and log-trapezoidal methods. Total body clearance (CL), volume of distribution at steady state (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 at which concentrations were quantifiable.

[0219] After administration of 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 administration of 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 vascular by the law of mass action. The plasma concentration of free Compound A in rabbits remained less than 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 administered 20 minutes intravenously with Compound A (1 mg / kg) followed by Fab fragment antibody 26D5-75616-348-F10-Fab-SHORT (160 mg / kg). Results are the average of three rabbits.

[0220] Example 13: Pharmacokinetics of tandem Fab fragment antibodies 26D5-75616-348-F10-TanFab (tandem Fab heavy chain SEQ ID NO: 180, tandem Fab light chain SEQ ID NO: 164) was produced and purified according to common methods known to those skilled in the art, as described in Example 5 above.

[0221] Pharmacokinetics in rats In vivo experiments were performed in accordance with the regulations of the Animal Care and Use Committee of the Bristol-Myers Squibb Company. An indwelling catheter was placed in the jugular vein (for blood sampling and substance administration) of rats (male, 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 over 10 min. Blood samples (0.2 mL) were taken at the end of the infusion and at various intervals up to 48 h after the start of administration. Blood samples were added to EDTA in polypropylene tubes, mixed thoroughly by inversion at least twice, and placed on ice. Plasma was isolated within 1 h of blood collection by centrifugation of whole blood at 1,500 x g for at least 10 min. The concentrations of Fab fragment antibody 26D5-75616-348-F10-Fab-SHORT and 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 not bound to Compound A in plasma, and the plasma concentrations of 26D5-75616-348-F10-Fab-SHORT and 26D5-75616-348-F10-TanFab were measured by ligand binding assay using a Gyrolab® automated microfluidic system (Gyros Protein Technologies AB). Biotinylated mouse anti-human kappa (SouthernBiotech Cat No 9230-08, Lot No K5613-X088) was used as the binding molecule for all 26D5-75616-348-F10-Fab-SHORT and 26D5-75616-348-F10-TanFab. Samples, standards and QCs were brought up to a final base concentration of 10% plasma / 1xPTB (1%BSA / 0.05%Tween20 / PBS) and loaded onto 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, total bound 26D5-75616-348-F10-Fab-SHORT and 26D5-75616-348-F10-TanFab were detected using Alexa Fluor™ 647-labeled mouse anti-human Ig kappa light chain mAb clone G20-361 (Becton Dickinson Cat No 555861, Lot No 833694). Fluorescence intensity was measured in a Gyrolab™ using a 4-parameter logistic curve generated 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 calibration range for 26D5-75616-348-F10-Fab-SHORT and 26D5-75616-348-F10-TanFab combined was 10-25000ng / mL in plasma. The upper and lower limits of quantification were 25000 and 10ng / mL. Quality control samples were prepared at plasma concentrations of 20000, 7500, 750, 75 and 30ng / mL. Standards and QCs were analyzed in each experiment to ensure an acceptable assay. The assays were performed within the acceptable range (%CVs for standards and QCs <20% and QC recoveries within ±20% of apparent values).

[0223] Compound 5 was used as a molecule to conjugate 26D5-75616-348-F10-Fab-SHORT and 26D5-75616-348-F10-TanFab that were not conjugated with Compound A. Samples, standards, and QCs were brought up to a final base concentration of 10% plasma / 1xPTB (1%BSA / 0.05%Tween20 / PBS) and loaded onto a Gyrolab™ automated microfluidic system. The 3-Step-2-Wash Wizard method of the Gyrolab™ Bioaffy 200 CD was used. After a 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 Ig kappa light chain mAb clone G20-361 (Becton Dickinson Cat No 555861, Lot No 8333694). Fluorescence intensity was measured with Gyrolab® and the concentrations of "active / free" 26D5-75616-348-F10-Fab-SHORT and 26D5-75616-348-F10-TanFab in plasma samples were calculated using a 4-parameter logistic curve constructed from standard substances of 26D5-75616-348-F10-Fab-SHORT and 26D5-75616-348-F10-TanFab. The calibration curve range of "active / free" 26D5-75616-348-F10-Fab-SHORT and 26D5-75616-348-F10-TanFab was 10-25000ng / mL (in plasma). The upper and lower limits of quantification were 25000 and 10ng / mL. Quality control samples were prepared at plasma concentrations of 20000, 7500, 750, 75 and 30 ng / mL. Standards and QCs were analyzed in each experiment to ensure an acceptable assay. The assay performed within acceptable limits (%CVs for standards and QCs <20% and QC recoveries within ±20% of nominal values).

[0224] FIG. 22 shows the pharmacokinetic data obtained.

[0225] Pharmacokinetics in rabbits In vivo experiments were performed in accordance with the regulations of the Bristol-Myers Squibb Company Institutional Animal Care and Use Committee. Indwelling catheters were placed in the femoral artery and femoral vein (for blood sampling) and the marginal ear vein (for substance administration) of rabbits (male, New Zealand White, 2-4 kg). A fixed amount of compound A (0.4 mg / kg, 0.64 μM / kg) was administered intravenously over 10 minutes. Twenty minutes after the completion of the infusion of compound A, a fixed amount of the tandem Fab fragment antibody 26D5-75616-348-F10-TanFab (40 mg / kg, 0.43 μM / kg) was administered intravenously over 10 minutes. Given the 2:1 binding capacity, the administered tandem Fab fragment antibody 26D5-75616-348-F10-TanFab was administered at an apparent molar excess of 1.34 times over the administered Compound A (2*0.43 / 0.64). Blood samples (1.5 mL) were taken 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 at various intervals 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 3.8% sodium citrate (0.167 mL) in polypropylene tubes, mixed thoroughly by inversion at least twice, and placed on ice. Plasma was isolated within 1 hour of blood collection by centrifuging whole blood 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 plasma ultrafiltrate was frozen at -80°C in polypropylene tubes. The total concentrations of Compound A in plasma and free Compound A in the plasma ultrafiltrate were 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 solution of 50% water and 0.5% formic acid in methanol (20 μL) was added. The plate was covered and mixed for 20 min on a shaker at 95°C. Acetonitrile (80 μL) containing an internal standard [1 μM] and 1% formic acid was added to the above solution to precipitate proteins. The plate was mixed by vortexing for an additional 15 min at room temperature and then centrifuged at 3,700 rpm for 8 min. A portion of the supernatant (100 μL) was transferred to an injection plate (96 well, 0.3 mL). A portion of the supernatant (3 μL) was injected into an Ultra Performance LC System (Waters® Acquity iClass UPLC) tandem mass spectrometer coupled with a Quadrapole MS / MS (Thermo Quantiva). The substances were separated using a C18 column (Waters HSS T3, 2X50mm, 1.8μm) at 40℃ with a gradient (flow rate 0.6mL / min) consisting of two buffer solutions (A: water + 5mM ammonium formate, 0.1% formic acid; B: acetonitrile + 0.1% formic acid). The precursor (M+H) was analyzed using multiple reaction monitoring (MRM) in positive electrospray ionization mode. + The MRM transitions observed for compound A were 626.3→319.1, and for the internal standard, 474.3→269. The lower limit of quantification was 0.5 nM.

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

[0229] FIG. 23 shows the pharmacokinetic data obtained.

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

Claims

1. 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 the at least one VH has the following structure: (a) a VH complementarity determining region 1 (VH-CDR1) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-12; (b) a VH-CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 13-22; or (c) a VH-CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 23 to 28; and at least one VL is selected from the following: (d) a VL-CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 29-37; (e) a VL-CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 38-43; or (f) a VL-CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 44 to 51; An isolated antigen-binding peptide comprising at least one of the following:

2. below: (a) at least one heavy chain variable region having an amino acid sequence selected from the group consisting of SEQ ID NOs: 52-83; 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.

1. An isolated antigen-binding peptide comprising:

3. 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 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, wherein the amino acid sequences of VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3 are as follows, respectively: (a) respective SEQ ID NOs: 1, 13, 23, 29, 38, and 44; (b) respective SEQ ID NOs: 1, 14, 23, 29, 38, and 45; (c) respective SEQ ID NOs: 1, 13, 24, 30, 38, and 45; (d) respective SEQ ID NOs: 1, 13, 24, 29, 39, and 45; (e) respective SEQ ID NOs: 1, 14, 25, 29, 38, and 46; (f) respective SEQ ID NOs: 2, 13, 26, 31, 40, and 47; (g) respective SEQ ID NOs: 3, 15, 24, 32, 40, and 47; (h) respective SEQ ID NOs: 4, 16, 24, 29, 38, and 46; (i) respective SEQ ID NOs: 5, 15, 24, 29, 38, and 46; (j) respective SEQ ID NOs: 1, 14, 24, 29, 38, and 46; (k) respective SEQ ID NOs: 6, 13, 24, 31, 40, and 47; (l) respective SEQ ID NOs: 3, 15, 24, 32, 41, and 48; (m) respective SEQ ID NOs: 1, 14, 24, 33, 38, and 49; (n) respective SEQ ID NOs: 1, 14, 26, 29, 38, and 46; (o) respective SEQ ID NOs: 7, 17, 26, 29, 38, and 46; (p) respective SEQ ID NOs: 8, 17, 24, 34, 38, and 46; (q) respective SEQ ID NOs: 1, 17, 26, 29, 38, and 46; (r) respective SEQ ID NOs: 1, 17, 26, 35, 38, and 46; (s) respective SEQ ID NOs: 1, 17, 24, 33, 38, and 49; (t) respective SEQ ID NOs: 9, 14, 26, 29, 38, and 46; (u) respective SEQ ID NOs: 9, 14, 26, 35, 38, and 46; (v) respective SEQ ID NOs: 9, 17, 24, 29, 38, and 46; (w) respective SEQ ID NOs: 9, 17, 24, 35, 38, and 46; (x) respective SEQ ID NOs: 9, 17, 24, 34, 38, and 46; (y) respective SEQ ID NOs: 9, 14, 24, 29, 38, and 46; (z) respective SEQ ID NOs: 9, 18, 26, 35, 38, and 46; (aa) respective SEQ ID NOs: 8, 14, 24, 29, 38, and 46; (bb) SEQ ID NOs: 8, 17, 26, 29, 38, and 46, respectively; (cc) respective SEQ ID NOs: 9, 19, 26, 29, 38, and 46; (dd) respective SEQ ID NOs: 9, 17, 26, 34, 38, and 46; (ee) respective SEQ ID NOs: 10, 20, 27, 36, 42, and 50; (ff) respective SEQ ID NOs: 11, 21, 28, 37, 43, and 51; (gg) respective SEQ ID NOs: 12, 22, 26, 33, 38, and 46; (hh) respective SEQ ID NOs: 12, 17, 26, 33, 38, and 46; (ii) SEQ ID NOs: 9, 17, 26, 33, 38, and 46, respectively; and (jj) A variant of (a) to (ii), wherein any amino acid sequence contains one, two, or three conservative substitutions.

2. An isolated antigen-binding peptide having a sequence selected from the group consisting of:

4. At least one VH region and at least one VL region, respectively, (a) SEQ ID NO:52 and SEQ ID NO:84, respectively; (b) SEQ ID NO:53 and SEQ ID NO:85, respectively; (c) SEQ ID NO:54 and SEQ ID NO:86, respectively; (d) SEQ ID NO:54 and SEQ ID NO:87, respectively; (e) SEQ ID NO:55 and SEQ ID NO:88, respectively; (f) SEQ ID NO:56 and SEQ ID NO:89, respectively; (g) SEQ ID NO:57 and SEQ ID NO:90, respectively; (h) SEQ ID NO:58 and SEQ ID NO:88, respectively; (i) SEQ ID NO:59 and SEQ ID NO:88, respectively; (j) SEQ ID NO:60 and SEQ ID NO:91, respectively; (k) SEQ ID NO:61 and SEQ ID NO:89, respectively; (l) SEQ ID NO:57 and SEQ ID NO:92, respectively; (m) SEQ ID NO:60 and SEQ ID NO:93, respectively; (n) SEQ ID NO:60 and SEQ ID NO:88, respectively; (o) SEQ ID NO:62 and SEQ ID NO:88, respectively; (p) SEQ ID NO:63 and SEQ ID NO:88, respectively; (q) SEQ ID NO:64 and SEQ ID NO:88, respectively; (r) SEQ ID NO:65 and SEQ ID NO:94, respectively; (s) SEQ ID NO:66 and SEQ ID NO:88, respectively; (t) SEQ ID NO:66 and SEQ ID NO:95, respectively; (u) SEQ ID NO:67 and SEQ ID NO:88, respectively; (v) SEQ ID NO:68 and SEQ ID NO:93, respectively; (w) SEQ ID NO:69 and SEQ ID NO:88, respectively; (x) SEQ ID NO:69 and SEQ ID NO:95, respectively; (y) SEQ ID NO:70 and SEQ ID NO:88, respectively; (z) SEQ ID NO:70 and SEQ ID NO:95, respectively; (aa) SEQ ID NO:71 and SEQ ID NO:88, respectively; (bb) SEQ ID NO:71 and SEQ ID NO:94, respectively; (cc) SEQ ID NO:72 and SEQ ID NO:88, respectively; (dd) SEQ ID NO:73 and SEQ ID NO:95, respectively; (ee) SEQ ID NO:74 and SEQ ID NO:88, respectively; (ff) SEQ ID NO:75 and SEQ ID NO:88, respectively; (gg) SEQ ID NO:76 and SEQ ID NO:88, respectively; (hh) SEQ ID NO:77 and SEQ ID NO:94, respectively; (ii) SEQ ID NO:78 and SEQ ID NO:96, respectively; (jj) SEQ ID NO:79 and SEQ ID NO:97, respectively; (kk) SEQ ID NO: 80 and SEQ ID NO: 98, respectively; (ll) SEQ ID NO:81 and SEQ ID NO:99, respectively; (mm) SEQ ID NO:81 and SEQ ID NO:98, respectively; (nn) SEQ ID NO:82 and SEQ ID NO:99, respectively; (oo) SEQ ID NO: 83 and SEQ ID NO: 98, respectively; and (pp) A variant of (a) to (oo) containing conservative substitutions of 1, 2, 3, or 4 amino acids.

4. The isolated antigen-binding peptide of claim 3, having an amino acid sequence selected from the group consisting of:

5. The isolated antigen-binding peptide specifically has the formula (I): 【Chemistry 1】 [In the formula, R 1 is C 1-4 is alkyl; R 2 are independently F, Cl, and CF 3 , CHF 2 , C.H. 2 F, C.H. 3 Selected from; R 3 is independently CF 3 , CHF 2 , C.H. 2 F, and C.H. 3 Selected from; R 4 is H; and R 5 are independently selected from F and Cl.

2. The isolated antigen-binding peptide of claim 1, which binds to a compound represented by the formula:

6. The compound has the formula (II): 【Chemistry 2】 6. The isolated antigen-binding peptide of claim 5, having the following structure:

7. The isolated antigen-binding peptide may be an antibody, Fab, Fab', F(ab') 2 , Fd, single chain Fv or scFv, disulfide-linked Fv, V-NAR domain, IgNar, intrabody, IgGACH2, minibody, F(ab') 3 , tetrabodies, triabodies, bispecific antibodies, single domain antibodies, DVD-Ig, Fcab, mAb 2 , (scFv) 2 2. The isolated antigen-binding peptide of claim 1, which is a Fc, scFv-Fc, or a tandem Fab.

8. below: (a) SEQ ID NO:100 and SEQ ID NO:160, respectively; (b) SEQ ID NO:101 and SEQ ID NO:160, respectively; (c) SEQ ID NO:102 and SEQ ID NO:161, respectively; (d) SEQ ID NO:103 and SEQ ID NO:161, respectively; (e) SEQ ID NO:104 and SEQ ID NO:162, respectively; (f) SEQ ID NO:105 and SEQ ID NO:162, respectively; (g) SEQ ID NO:104 and SEQ ID NO:163, respectively; (h) SEQ ID NO:105 and SEQ ID NO:163, respectively; (i) SEQ ID NO:106 and SEQ ID NO:164, respectively; (j) SEQ ID NO:107 and SEQ ID NO:164, respectively; (k) SEQ ID NO:108 and SEQ ID NO:165, respectively; (l) SEQ ID NO:109 and SEQ ID NO:165, respectively; (m) SEQ ID NO:110 and SEQ ID NO:166, respectively; (n) SEQ ID NO:111 and SEQ ID NO:166, respectively; (o) SEQ ID NO:112 and SEQ ID NO:164, respectively; (p) SEQ ID NO:113 and SEQ ID NO:164, respectively; (q) SEQ ID NO:114 and SEQ ID NO:164, respectively; (r) SEQ ID NO:115 and SEQ ID NO:164, respectively; (s) SEQ ID NO:116 and SEQ ID NO:167, respectively; (t) SEQ ID NO:117 and SEQ ID NO:167, respectively; (u) SEQ ID NO:118 and SEQ ID NO:165, respectively; (v) SEQ ID NO:119 and SEQ ID NO:165, respectively; (w) SEQ ID NO:110 and SEQ ID NO:168, respectively; (x) SEQ ID NO:111 and SEQ ID NO:168, respectively; (y) SEQ ID NO:116 and SEQ ID NO:169, respectively; (z) SEQ ID NO:117 and SEQ ID NO:169, respectively; (aa) SEQ ID NO:116 and SEQ ID NO:164, respectively; (bb) SEQ ID NO:117 and SEQ ID NO:164, respectively; (cc) SEQ ID NO:120 and SEQ ID NO:164, respectively; (dd) SEQ ID NO:121 and SEQ ID NO:164, respectively; (ee) SEQ ID NO:122 and SEQ ID NO:164, respectively; (ff) SEQ ID NO:123 and SEQ ID NO:164, respectively; (gg) SEQ ID NO:124 and SEQ ID NO:164, respectively; (hh) SEQ ID NO:125 and SEQ ID NO:164, respectively; (ii) SEQ ID NO:126 and SEQ ID NO:170, respectively; (jj) SEQ ID NO:127 and SEQ ID NO:170, respectively; (kk) SEQ ID NO:128 and SEQ ID NO:164, respectively; (ll) SEQ ID NO:129 and SEQ ID NO:164, respectively; (mm) SEQ ID NO:128 and SEQ ID NO:171, respectively; (nn) SEQ ID NO:129 and SEQ ID NO:171, respectively; (oo) SEQ ID NO:130 and SEQ ID NO:164, respectively; (pp) SEQ ID NO: 131 and SEQ ID NO: 164, respectively; (qq) SEQ ID NO:132 and SEQ ID NO:169, respectively; (rr) SEQ ID NO:133 and SEQ ID NO:169, respectively; (ss) SEQ ID NO:134 and SEQ ID NO:164, respectively; (tt) SEQ ID NO:135 and SEQ ID NO:164, respectively; (uu) SEQ ID NO:134 and SEQ ID NO:171, respectively; (vv) SEQ ID NO:135 and SEQ ID NO:171, respectively; (ww) SEQ ID NO:136 and SEQ ID NO:164, respectively; (xx) SEQ ID NO:137 and SEQ ID NO:164, respectively; (yy) SEQ ID NO:136 and SEQ ID NO:171, respectively; (zz) SEQ ID NO:137 and SEQ ID NO:171, respectively; (aaa) SEQ ID NO:138 and SEQ ID NO:164, respectively; (bbb) SEQ ID NO:139 and SEQ ID NO:164, respectively; (ccc) SEQ ID NO:138 and SEQ ID NO:170, respectively; (ddd) SEQ ID NO:139 and SEQ ID NO:170, respectively; (eee) SEQ ID NO:140 and SEQ ID NO:164, respectively; (fff) SEQ ID NO:141 and SEQ ID NO:164, respectively; (ggg) SEQ ID NO:142 and SEQ ID NO:171, respectively; (hhh) SEQ ID NO:143 and SEQ ID NO:171, respectively; (iii) SEQ ID NO:144 and SEQ ID NO:164, respectively; (jjj) SEQ ID NO:145 and SEQ ID NO:164, respectively; (kkk) SEQ ID NO:146 and SEQ ID NO:164, respectively; (lll) SEQ ID NO:147 and SEQ ID NO:164, respectively; (mmm) SEQ ID NO:148 and SEQ ID NO:164, respectively; (nnn) SEQ ID NO:149 and SEQ ID NO:164, respectively; (ooo) SEQ ID NO:150 and SEQ ID NO:170, respectively; (ppp) SEQ ID NO: 151 and SEQ ID NO: 170, respectively; (qqq) SEQ ID NO:152 and SEQ ID NO:172, respectively; (rrr) SEQ ID NO:153 and SEQ ID NO:172, respectively; (sss) SEQ ID NO:154 and SEQ ID NO:173, respectively; (ttt) SEQ ID NO:155 and SEQ ID NO:173, respectively; (uuu) SEQ ID NO:156 and SEQ ID NO:174, respectively; (vvv) SEQ ID NO:157 and SEQ ID NO:174, respectively; (www) SEQ ID NO:158 and SEQ ID NO:175, respectively; (xxx) SEQ ID NO:159 and SEQ ID NO:175, respectively; (yyy) SEQ ID NO:158 and SEQ ID NO:174, respectively; (zzz) SEQ ID NO:159 and SEQ ID NO:174, respectively; (aaaa) SEQ ID NO:176 and SEQ ID NO:160, respectively; (bbbb) SEQ ID NO:177 and SEQ ID NO:160, respectively; (cccc) SEQ ID NO:178 and SEQ ID NO:160, respectively; (dddd) SEQ ID NO:179 and SEQ ID NO:160, respectively; (eeee) SEQ ID NO:180 and SEQ ID NO:164, respectively; (ffff) SEQ ID NO:181 and SEQ ID NO:164, respectively; (gggg) SEQ ID NO:182 and SEQ ID NO:164, respectively; (hhhh) SEQ ID NO:183 and SEQ ID NO:164, respectively; (iiii) SEQ ID NO:184 and SEQ ID NO:163, respectively; (jjjj) SEQ ID NO:185 and SEQ ID NO:163, respectively; (kkkk) SEQ ID NO:186 and SEQ ID NO:163, respectively; (llll) SEQ ID NO: 187 and SEQ ID NO: 163, respectively; (mmmm) SEQ ID NO:184 and SEQ ID NO:162, respectively; (nnnn) SEQ ID NO:185 and SEQ ID NO:162, respectively; (oooo) SEQ ID NO:186 and SEQ ID NO:162, respectively; (pppp) SEQ ID NO: 187 and SEQ ID NO: 162, respectively; (qqqq) SEQ ID NO:188 and SEQ ID NO:165, respectively; (rrrr) SEQ ID NO:189 and SEQ ID NO:165, respectively; (ssss) SEQ ID NO:190 and SEQ ID NO:165, respectively; (tttt) SEQ ID NO:191 and SEQ ID NO:165, respectively; (uuuu) SEQ ID NO:192 and SEQ ID NO:161, respectively; (vvvv) SEQ ID NO:193 and SEQ ID NO:161, respectively; (www) SEQ ID NO: 194 and SEQ ID NO: 161, respectively; and (xxxx) SEQ ID NO: 195 and SEQ ID NO: 161, respectively 2. The isolated antigen-binding peptide of claim 1, having a sequence selected from the group consisting of: 【Chemistry 3】 An isolated antigen-binding peptide that binds to a compound of formula (I).

9. 9. The isolated antigen-binding peptide of claim 8, having the sequences of SEQ ID NO: 106 and SEQ ID NO: 164, respectively.

10. The isolated antigen-binding peptide of claim 9, which is a Fab fragment antibody.

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

12. The isolated antigen-binding peptide of claim 11, which is a tandem Fab fragment antibody.

13. 2. An isolated polynucleotide having a nucleic acid sequence encoding the antigen-binding peptide of claim 1.

14. A vector comprising the isolated polynucleotide of claim 13.

15. A host cell comprising the vector of claim 14.

16. 16. A method for producing an antigen-binding peptide, comprising: (a) culturing a host cell under culture conditions promoting protein production, such that the host cell of claim 15 produces the antigen-binding peptide; and (b) isolating the antigen-binding peptide from the culture medium of (a).

17. A detection reagent comprising the isolated antigen-binding peptide of claim 1 and a detectable label.

18. Administering to a subject in need thereof a pharmacologic effective amount of an isolated antigen-binding peptide of claim 1, 【Chemistry 4】 [In the formula, R 1 is C 1-4 is alkyl; R 2 are independently F, Cl, and CF 3 , CHF 2 , C.H. 2 F, C.H. 3 Selected from; R 3 is independently CF 3 , CHF 2 , C.H. 2 F, and C.H. 3 Selected from; R 4 is H; and R 5 are independently selected from F and Cl.

4. A method for reducing the antithrombotic effect of a compound of formula (I) or a stereoisomer or tautomer thereof.

19. Formula (I) 【Chemistry 5】 [In the formula, R 1 is C 1-4 is alkyl; R 2 are independently F, Cl, and CF 3 , CHF 2 , C.H. 2 F, C.H. 3 Selected from; R 3 is independently CF 3 , CHF 2 , C.H. 2 F, and C.H. 3 Selected from; R 4 is H; and R 5 are independently selected from F and Cl. or a stereoisomer, tautomer, or pharma- ceutically acceptable salt thereof in a biological sample, comprising the steps of: (a) contacting a biological sample with the isolated antigen-binding peptide of claim 1; and (b) detecting the level of a complex bound by the compound and the isolated antigen-binding peptide. A method comprising:

20. A therapeutically effective amount of formula (I) 【Chemistry 6】 [In the formula, R 1 is C 1-4 is alkyl; R 2 are independently F, Cl, and CF 3 , CHF 2 , C.H. 2 F, C.H. 3 Selected from; R 3 is independently CF 3 , CHF 2 , C.H. 2 F, and C.H. 3 Selected from; R 4 is H; and R 5 are independently selected from F and Cl.

2. A method for binding a compound of formula (I) or a stereoisomer or tautomer thereof, comprising administering to a subject taking a compound of formula (I) or a stereoisomer or tautomer thereof a pharma- ceutical effective amount of an isolated antigen-binding peptide of claim 1.

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