Methods for detecting anti-drug antibodies against factor XI and / or factor XIa antibodies

Abstract

The present disclosure relates to methods for detecting and measuring anti-drug antibodies (ADAs) against factor XI and / or factor XIa therapeutic antibodies, for example, in subjects undergoing treatment with said factor XI and / or factor XIa therapeutic antibodies. Antibodies that bind to factor XI and / or factor XIa are being investigated. The present disclosure enhances the development of such antibodies and provides additional clinical methods, including dosing regimens, for treating patients with specific thromboembolic disorders with desirable safety and efficacy.

Description

Technical Field

[0001] Cross - reference to related applications This application claims the benefit and priority of U.S. Provisional Patent Application No. 63 / 127,536, filed on December 18, 2020, the entire disclosure of which is incorporated herein by reference for all purposes.

[0002] Sequence listing This application was electronically filed in ASCII format and contains a sequence listing that is incorporated herein by reference in its entirety. The ASCII copy was created on December 14, 2021, has the name ATD - 010WO_ST25.txt, and is 39,185 bytes in size.

[0003] Field of the present disclosure The present disclosure generally relates to methods for detecting and measuring anti - drug antibodies (ADAs) against factor XI and / or factor XIa therapeutic antibodies, for example, in subjects undergoing treatment with the factor XI and / or factor XIa therapeutic antibodies.

Background Art

[0004] Background There is a large unmet medical need for a safer therapy that reduces thromboembolic complications, such as stroke, systemic embolism, cognitive decline, and mortality, with efficacy equivalent to or improved over existing therapies and a lower risk of bleeding. Factor XI (FXI) is a serine protease that functions in both the intrinsic and extrinsic coagulation pathways. Factor XI exists as a homodimer in zymogenous form; cleavage of the R369-I370 peptide bond activates factor XI (factor XIa, FXIa). While FXI plays a minor role in normal hemostasis in high-tissue-factor environments, it plays a crucial role in thrombosis. Genetic factor XI deficiency is associated with a reduced incidence of ischemic stroke and venous thromboembolic events (Salomon et al. 2008; Salomon, et al. (2011) Thromb Haemost.; 105:269-73). Hemorrhagic events in individuals with factor XI deficiency are infrequent, often mild, and caused by injury or trauma, and very rarely affect critical organs (Salomon et al 2011). Antibodies that bind to factor XI and / or factor XIa have been studied. For example, WO2016 / 207858 describes one such anti-factor XI and / or factor XIa antibody, disclosed as antibody 1 in Table 1 of this application. This disclosure enhances these developments and provides further clinical methods, including drug regimens, to treat patients with specific thromboembolic disorders with desired safety and efficacy. Furthermore, this disclosure enhances previous developments in the art by providing formulations containing such FXI and / or FXIa antibodies that are sufficiently stable and appropriate for administration to patients. [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] International Publication No. 2016 / 207858 [Non-patent literature]

[0006] [Non-Patent Document 1] Salomon, et al. (2011) Thromb Haemost.; 105:269-73 [Overview of the Initiative] [Means for solving the problem]

[0007] Summary of this disclosure This disclosure relates to a method for detecting and measuring an anti-factor XI and / or activated factor XI (factor XIa) antibody or its antigen-binding fragment.

[0008] Accordingly, in one embodiment, a method for detecting an anti-drug antibody (ADA) against anti-factor XI and / or anti-factor XIa antibody or its antigen-binding fragment is provided herein, comprising the steps of: (a) incubating a sample with an acid to dissociate anti-factor XI and / or anti-factor XIa antibody-antigen complexes and / or anti-factor XI and / or anti-factor XIa antibody-ADA complexes present in the sample to create an acid digest; (b) incubating the acid digest on a plate coated with anti-factor XI and / or anti-factor XIa antibody or its antigen-binding fragment; (c) neutralizing the acid digest; and (d) detecting the presence of ADA using a ruthenium-labeled detection cocktail.

[0009] In some embodiments, the sample is a sample derived from the subject. In some embodiments, the sample is selected from the group consisting of blood, plasma, or serum derived from the subject. In some embodiments, the method includes a first step of preparing the sample.

[0010] In some embodiments, the acid is selected from the group consisting of acetic acid, citric acid, phosphoric acid, and mixtures thereof. In some embodiments, the acid is acetic acid. In some embodiments, the concentration of acetic acid is approximately 300 mM.

[0011] In some embodiments, the antigen is factor XI and / or factor XIa. In some embodiments, the plate is coated with streptavidin. In some embodiments, the concentration of anti-factor XI and / or anti-factor XIa antibody or its antigen-binding fragment on the plate is selected from the group consisting of about 0.1 μg / ml, about 0.25 μg / ml, about 0.5 μg / ml, about 0.75 μg / ml, and about 1 μg / ml. In some embodiments, the concentration of anti-factor XI and / or anti-factor XIa antibody or its antigen-binding fragment on the plate is about 0.25 μg / ml.

[0012] In some embodiments, a neutralization step allows ADA to bind to anti-factor XI and / or anti-factor XIa antibodies or their antigen-binding fragments on the coated plate. In some embodiments, the neutralization step uses a base with a pH of approximately 8.0. In some embodiments, the neutralization step uses a base selected from the group consisting of Tris, phosphates, HEPES, triethanolamine, and mixtures thereof. In some embodiments, the base is Tris.

[0013] In some embodiments, the ruthenium-labeled detection cocktail includes an antibody. In some embodiments, the antibody is selected from the group consisting of anti-human IgG, anti-human IgM, anti-human IgE, anti-rabbit Ig, and any combination thereof. In some embodiments, the detection step specifically detects ADA and does not detect factor XI and / or factor XIa.

[0014] In some embodiments, the method further includes a washing step after the incubation step.

[0015] In some embodiments, the concentration of the anti-factor XI and / or anti-factor XIa antibody or its antigen-binding fragment is approximately 0.25 μg / mL.

[0016] In some embodiments, the anti-factor XI and / or anti-factor XIa antibody or its antigen-binding fragment includes a heavy chain variable region (VH) comprising the complementarity-determining regions HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 9 or 29, and a light chain variable region (VL) comprising the complementarity-determining regions LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 19 or 39.

[0017] In some embodiments, the anti-factor XI and / or anti-factor XIa antibody or its antigen-binding fragment is i. heavy chain variable region CDR1 of SEQ ID NO: 23; heavy chain variable region CDR2 of SEQ ID NO: 24; heavy chain variable region CDR3 of SEQ ID NO: 25; light chain variable region CDR1 of SEQ ID NO: 33; light chain variable region CDR2 of SEQ ID NO: 34; and light chain variable region CDR3 of SEQ ID NO: 35; ii. heavy chain variable region CDR1 of SEQ ID NO: 26; heavy chain variable region CDR2 of SEQ ID NO: 27; heavy chain variable region CDR3 of SEQ ID NO: 28; light chain variable region CDR1 of SEQ ID NO: 36; light chain variable region CDR2 of SEQ ID NO: 37; iii. Light chain variable region CDR3 of SEQ ID NO: 38; iii. Heavy chain variable region CDR1 of SEQ ID NO: 43; Heavy chain variable region CDR2 of SEQ ID NO: 44; Heavy chain variable region CDR3 of SEQ ID NO: 45; Light chain variable region CDR1 of SEQ ID NO: 47; Light chain variable region CDR2 of SEQ ID NO: 37; and Light chain variable region CDR3 of SEQ ID NO: 15; or iv. Heavy chain variable region CDR1 of SEQ ID NO: 46; Heavy chain variable region CDR2 of SEQ ID NO: 4; Heavy chain variable region CDR3 of SEQ ID NO: 5; Light chain variable region CDR1 of SEQ ID NO: 33; Light chain variable region CDR2 of SEQ ID NO: 14; and Light chain variable region CDR3 of SEQ ID NO: 15.

[0018] In some embodiments, the anti-factor XI and / or anti-factor XIa antibody or its antigen-binding fragment comprises a heavy chain variable region (VH) selected from the group consisting of SEQ ID NOs: 9 and 29, and a VH having 90% identity thereto; and a light chain variable region (VL) selected from the group consisting of SEQ ID NOs: 19 and 39, and a VL having 90% identity thereto. In certain embodiments, the anti-factor XI and / or anti-factor XIa antibody or its antigen-binding fragment comprises a heavy chain variable region (VH) selected from the group consisting of SEQ ID NOs: 9 and 29; and a light chain variable region (VL) selected from the group consisting of SEQ ID NOs: 19 and 39.

[0019] In some embodiments, the anti-factor XI and / or anti-factor XIa antibody comprises a heavy chain containing the amino acid sequence of SEQ ID NOs. 31, 11 and a heavy chain having 90% identity thereto; and a light chain containing the amino acid sequence of SEQ ID NOs. 41, 21 and a light chain having 90% identity thereto. In a particular embodiment, the anti-factor XI and / or anti-factor XIa antibody comprises a heavy chain containing the amino acid sequence of SEQ ID NOs. 31 and a light chain containing the amino acid sequence of SEQ ID NOs. 41.

[0020] In some embodiments, the anti-factor XI and / or anti-factor XIa antibody is a human monoclonal antibody. In some embodiments, the anti-factor XI and / or anti-factor XIa antibody is a human IgG1 isotype. In some embodiments, the anti-factor XI and / or anti-factor XIa antibody contains D265A and P329A substitutions within the Fc domain.

[0021] In another aspect, a method for detecting an anti-drug antibody (ADA) against an anti-factor XI and / or anti-factor XIa antibody or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises: i. heavy chain variable region CDR1 of SEQ ID NO: 23; heavy chain variable region CDR2 of SEQ ID NO: 24; heavy chain variable region CDR3 of SEQ ID NO: 25; light chain variable region CDR1 of SEQ ID NO: 33; light chain variable region CDR2 of SEQ ID NO: 34; and light chain variable region CDR3 of SEQ ID NO: 35; ii. heavy chain variable region CDR1 of SEQ ID NO: 26; heavy chain variable region CDR2 of SEQ ID NO: 27; heavy chain variable region CDR3 of SEQ ID NO: 28; light chain variable region CDR1 of SEQ ID NO: 36; light chain variable region CDR2 of SEQ ID NO: 37; and light chain variable region CDR3 of SEQ ID NO: 38; iii. heavy chain variable region CDR1 of SEQ ID NO: 43; heavy chain variable region CDR2 of SEQ ID NO: 44; heavy chain variable region CDR3 of SEQ ID NO: 45; light chain variable region CDR1 of SEQ ID NO: 47; light chain variable region CDR2 of SEQ ID NO: 37; and light chain variable region CDR3 of SEQ ID NO: 15; or iv. heavy chain variable region CDR1 of SEQ ID NO: 46; heavy chain variable region CDR2 of SEQ ID NO: 4; heavy chain variable region CDR3 of SEQ ID NO: 5; light chain variable region CDR1 of SEQ ID NO: 33; light chain variable region CDR2 of SEQ ID NO: 14; and light chain variable region CDR3 of SEQ ID NO: 15, and the method comprises: (a) incubating the sample with an acid to dissociate any anti-factor XI and / or anti-factor XIa antibody-antigen complexes present in the sample and / or to dissociate any anti-factor XI and / or anti-factor XIa antibody-ADA complexes, thereby creating an acid digest; (b) incubating the acid digest on a plate coated with an anti-factor XI and / or anti-factor XIa antibody or an antigen-binding fragment thereof; (c) neutralizing the acid digest; and (d) detecting the presence of ADA using a ruthenium-labeled detection cocktail.

[0022] In some embodiments, the sample is a sample derived from a subject. In some embodiments, the sample is selected from the group consisting of blood, plasma, or serum derived from a subject. In some embodiments, the method includes an initial step of preparing the sample.

[0023] In some embodiments, the acid is selected from the group consisting of acetic acid, citric acid, phosphoric acid, and mixtures thereof. In some embodiments, the acid is acetic acid. In some embodiments, the concentration of acetic acid is about 300 mM.

[0024] In some embodiments, the antigen is factor XI and / or factor XIa. In some embodiments, the plate is coated with streptavidin. In some embodiments, the concentration of the anti-factor XI and / or anti-factor XIa antibody or antigen-binding fragment thereof on the plate is selected from the group consisting of about 0.1 μg / ml, about 0.25 μg / ml, about 0.5 μg / ml, about 0.75 μg / ml, and about 1 μg / ml. In some embodiments, the concentration of the anti-factor XI and / or anti-factor XIa antibody or antigen-binding fragment thereof on the plate is about 0.25 μg / ml.

[0025] In some embodiments, the neutralizing step enables ADA to bind to the anti-factor XI and / or anti-factor XIa antibody or antigen-binding fragment thereof on the coated plate. In some embodiments, the neutralizing step uses a base at a pH of about 8.0. In some embodiments, the neutralizing step uses a base selected from the group consisting of Tris, phosphate, HEPES, triethanolamine, and mixtures thereof. In some embodiments, the base is Tris.

[0026] In some embodiments, the ruthenium-labeled detection cocktail contains an antibody. In some embodiments, the antibody is selected from the group consisting of anti-human IgG, anti-human IgM, anti-human IgE, anti-rabbit Ig, and any combination thereof. In some embodiments, the detecting step specifically detects ADA and does not detect factor XI and / or factor XIa.

[0027] In some embodiments, the method further includes a washing step after the incubating step.

[0028] In some embodiments, the concentration of the anti-factor XI and / or anti-factor XIa antibody or its antigen-binding fragment is approximately 0.25 μg / mL.

[0029] In some embodiments, the anti-factor XI and / or anti-factor XIa antibody or its antigen-binding fragment comprises a heavy chain variable region (VH) selected from the group consisting of SEQ ID NOs: 9 and 29, and a VH having 90% identity thereto; and a light chain variable region (VL) selected from the group consisting of SEQ ID NOs: 19 and 39, and a VL having 90% identity thereto. In certain embodiments, the anti-factor XI and / or anti-factor XIa antibody or its antigen-binding fragment comprises a heavy chain variable region (VH) selected from the group consisting of SEQ ID NOs: 9 and 29; and a light chain variable region (VL) selected from the group consisting of SEQ ID NOs: 19 and 39.

[0030] In some embodiments, the anti-factor XI and / or anti-factor XIa antibody comprises a heavy chain containing the amino acid sequence of SEQ ID NOs. 31, 11 and a heavy chain having 90% identity thereto; and a light chain containing the amino acid sequence of SEQ ID NOs. 41, 21 and a light chain having 90% identity thereto. In a particular embodiment, the anti-factor XI and / or anti-factor XIa antibody comprises a heavy chain containing the amino acid sequence of SEQ ID NOs. 31 and a light chain containing the amino acid sequence of SEQ ID NOs. 41.

[0031] In some embodiments, the anti-factor XI and / or anti-factor XIa antibody is a human monoclonal antibody. In some embodiments, the anti-factor XI and / or anti-factor XIa antibody is a human IgG1 isotype. In some embodiments, the anti-factor XI and / or anti-factor XIa antibody contains D265A and P329A substitutions within the Fc domain.

[0032] Other embodiments and details of this disclosure are presented below in this specification. [Brief explanation of the drawing]

[0033] [Figure 1]Figure 1 is a schematic diagram of the first bridging assay for detecting anti-drug antibodies (ADAs) in cynomolgus monkey samples.

[0034] [Figure 2] Figure 2 is a schematic diagram of a modified bridging assay involving a factor XI / factor XIa depletion step for detecting ADA in cynomolgus monkey samples.

[0035] [Figure 3] Figure 3 is a schematic diagram of a modified bridging assay with ruthenium-labeled anti-monkey Ig for detecting ADA in cynomolgus monkey samples.

[0036] [Figure 4] Figure 4 is a schematic diagram of the ADA assay for cynomolgus monkey samples.

[0037] [Figure 5] Figure 5 is a schematic diagram of the ADA assay for human samples. [Modes for carrying out the invention]

[0038] Detailed explanation definition To facilitate understanding of the present invention, several terms and phrases are defined below.

[0039] The terms “a” and “an” as used herein mean “one or more” and include multiple terms unless otherwise appropriate in the context.

[0040] As used herein, the terms “FXI protein,” “FXI antigen,” and “FXI” are used interchangeably and refer to factor XI protein in different species. Factor XI is the mammalian plasma coagulation factor XI, a glycoprotein present in human plasma as zymogen at concentrations of 25–30 nM, which, when converted to an active serine protease by limited protein hydrolysis, is involved in the endogenous pathway of blood coagulation.

[0041] The terms "FXIa protein," "FXIa antigen," and "FXIa" are used interchangeably and refer to the activated FXI protein in different species. Zymogen factor XI is converted to its active form, coagulation factor XIa (FXIa), either via the contact phase of blood coagulation or through thrombin-mediated activation on the platelet surface. During this activation of factor XI, the peptide bonds within each of the two chains are cleaved, resulting in activated factor XIa, a serine protease composed of two heavy chains and two light chains held together by disulfide bonds. This serine protease FXIa converts coagulation factor IX to IXa, which then activates coagulation factor X (Xa). Xa can then mediate coagulation factor II / thrombin activation. For example, human FXI has the sequence listed in Table 1 (Sequence ID 1), which has been reported and documented in previous publications and literature (Mandle RJ Jr, et al. (1979) Blood; 54 (4): 850; NCBI Reference Sequence: AAA51985).

[0042] For the purposes of this disclosure, the terms “FXI” and “FXIa” (etc.) encompass mutants and variants of the native FXI and FXIa proteins that have substantially the same amino acid sequence as the native primary structure (amino acid sequence) described in the above report, respectively.

[0043] The terms “catalytic domain,” “serine protease catalytic domain,” and similar terms, as used herein, refer to amino acids Ile370–Val607, counting from Glu1 at the N-terminus of the mature protein in circulation. This can also be described as C-terminal residues 388–625 of FXI. As used herein, the term “active site” refers to the catalytic triplicate structure composed of amino acids His413, Asp462, and Ser557 (Bane and Gailani (2014) Drug Disc. 19 (9)).

[0044] As used herein, the term “antibody” refers to the entire antibody and any antigen-binding fragment (i.e., “antigen-binding portion”) or single chain. The entire antibody is a glycoprotein composed of at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region consists of three domains, CH1, CH2, and CH3. Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region consists of one domain, CL. The VH and VL regions can be further subdivided into a highly variable region called the complementarity-determining region (CDR) and more conserved regions interspersed between them, called the framework region (FR). Each VH and VL consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with the antigen. The constant region of the antibody can mediate the binding of immunoglobulins to host tissues or factors, including cells of various immune systems (e.g., effector cells) and the first component (Clq) of the classical complement system. In some specific embodiments, the antibody may be a monoclonal antibody, a human antibody, a humanized antibody, a camelized antibody, or a chimeric antibody. The antibody may be of any isotype (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass.

[0045] The CDR of the antigen-binding site can be determined by the methods described in Kabat et al., J. Biol. Chem. 252, 6609-6616 (1977) and Kabat et al., Sequences of protein of immunological interest. (1991), Chothia et al., J. Mol. Biol. 196: 901-917 (1987), and MacCallum et al., J. Mol. Biol. 262: 732-745 (1996). The CDR determined under these definitions generally includes duplicates or subsets of amino acid residues when compared to one another. In certain embodiments, the term "CDR" refers to the CDR as defined by MacCallum et al., J. Mol. Biol. 262: 732-745 (1996) and Martin A., Protein Sequence and Structure Analysis of Antibody Variable Domains, in Antibody Engineering, Kontermann and Dubel, eds., Chapter 31, pp. 422-439, Springer-Verlag, Berlin (2001). In certain embodiments, the term "CDR" refers to the CDR as defined by Kabat et al., J. Biol. Chem. 252, 6609-6616 (1977) and Kabat et al., Sequences of protein of immunological interest. (1991). In certain embodiments, the heavy chain CDR and light chain CDR of an antibody are defined using different conventions. For example, in certain embodiments, the heavy chain CDR is defined according to MacCallum (above), and the light chain CDR is defined according to Kabat (above). CDRH1, CDRH2, and CDRH3 represent heavy chain CDRs, while CDRL1, CDRL2, and CDRL3 represent light chain CDRs.

[0046] As used herein, the terms “drug delivery formulation” or “intravenous drug delivery formulation” refer to a pharmaceutical formulation comprising a combination of an active agent and an inactive or active carrier, which is particularly suitable for use in vivo or ex vivo for diagnosis or treatment.

[0047] As used herein, the terms “subject” and “patient” refer to organisms treated by the methods and compositions described herein. Such organisms may preferably include, but are not limited to, mammals (e.g., murites, apes, equids, bovines, pigs, primates, canines, felines, etc.), and more preferably include humans. In certain embodiments, the subject is a cynomolgus macaque. In certain embodiments, the subject is a human.

[0048] "Thromboembolic disorders" or similar terms, as used herein, refer to any number of conditions or diseases in which the endogenous and / or common coagulation pathway is ectopically activated or not spontaneously deactivated (e.g., without therapeutic means). These conditions include, but are not limited to, thromboembolic stroke and other types of ischemic stroke, atrial fibrillation, prevention of stroke in atrial fibrillation (SPAF), deep vein thrombosis, venous thromboembolism, and pulmonary embolism. These may also include catheter-related thrombosis (e.g., Hickman catheter in tumor patients) in which a thrombus is formed by a catheter, as well as prevention and management of extracorporeal membrane oxygenation (ECMO) in which a blood clot is formed by a tube and membrane oxygenator.

[0049] When used herein, “thromboembolic disorder” or similar terms may also refer to any of the following which can be prevented or treated using any number of the anti-FXI and / or FXIa antibodies or antigen-binding fragments of this disclosure: - Thromboembolism in subjects suspected of or confirmed to have cardiac arrhythmias such as paroxysmal, persistent, or permanent atrial fibrillation or atrial flutter; - Prevention of stroke due to atrial fibrillation (SPAF), this subgroup consists of AF patients undergoing percutaneous coronary intervention (PCI); - Management of acute venous thromboembolic events (VTE) and prevention of long-term secondary VTE in patients at high risk of bleeding; - The target is children; venous thromboembolism (pediatric VTE); - Prevention of cerebral and cardiovascular events or non-disabling strokes during secondary prevention after transient ischemic attack (TIA), and prevention of thromboembolic events in heart failure with sinus rhythm; - Hemorrhagic stroke; - Thromboembolism in patients undergoing electrical cardioversion for blood clot formation in the left atrium and cardiac arrhythmias; - Thrombosis before, during, and after ablation procedures for cardiac arrhythmias; - Venous thrombosis, which includes, but is not exclusive, treatment and secondary prevention of deep or superficial vein thrombosis in the lower or upper members, thrombosis in the abdominal and thoracic veins, sinus thrombosis, and thrombosis of the jugular vein; - Catheters, pacemaker wires, synthetic arterial grafts; thrombosis on any artificial surface in a vein or artery, such as mechanical or biological heart valves or left ventricular assist devices; - Pulmonary embolism in patients with or without venous thrombosis; - Chronic thromboembolic pulmonary hypertension (CTEPH); - Arterial thrombosis in ruptured atherosclerotic lesions, thrombosis in intra-arterial prostheses or catheters, and thrombosis in seemingly normal arteries, including, but not limited to, acute coronary syndrome, ST-elevation myocardial infarction, non-ST-elevation myocardial infarction, unstable angina, stent thrombosis, thrombosis of any artificial surface in the arterial system, and pulmonary artery thrombosis in subjects with or without pulmonary hypertension; - Thrombosis and thromboembolism in patients undergoing percutaneous coronary intervention (PCI); - Cardiogenic stroke and cryptogenic stroke; - Non-central nervous system embolism (non-CNS systemic embolism); - Thrombosis in patients with invasive and non-invasive cancerous malignancies; - Thrombosis in an indwelling catheter; - Thrombosis and thromboembolism in critically ill patients; - Cardiac thrombosis and thromboembolism, including but not limited to cardiac thrombosis after myocardial infarction, ventricular aneurysm, myocardial fibrosis, cardiomegaly and heart failure, myocarditis, and cardiac thrombosis associated with conditions such as artificial heart surface; - Thromboembolism in patients with or without atrial fibrillation and valvular heart disease; - Thromboembolism of mechanical or biological prosthetic valves; - Thromboembolism in patients who have acquired native or artificial heart patches, arterial or venous conduits after cardiac repair of simple or complex cardiac malformations; - Venous thrombosis and thromboembolism following knee replacement, total hip replacement, and orthopedic surgery, thoracic surgery, or abdominal surgery; - Arterial or venous thrombosis following neurosurgical procedures, including intracranial and spinal cord interventions; -Congenital or acquired thrombotic tendencies, including, but not exclusively, factor V Leiden, prothrombin mutations, antithrombin III, protein C and protein S deficiencies, factor XIII mutations, familial abnormal fibrinogenemia, congenital plasminogen deficiency, elevated factor XI levels, sickle cell anemia, antiphospholipid syndrome, autoimmune diseases, chronic bowel disease, nephrotic syndrome, hemolytic uremic disease, myeloproliferative disorders, disseminated intravascular coagulation, paroxysmal nocturnal hemoglobinuria and heparin-induced thrombocytopenia; - Thrombosis and thromboembolism in chronic kidney disease; as well as - Thrombosis and thromboembolism in patients undergoing hemodialysis and patients receiving extracorporeal membrane oxygenation (ECMO).

[0050] As used herein, the terms “trough” or “trough level” refer to the lowest concentration of the drug reached before the next dose of the drug is administered.

[0051] The terms “treat,” “treating,” or “treatment,” and other grammatical equivalents, as used in this disclosure, are intended to include, and may include, alleviate, reduce, improve or prevent a disease, condition or symptom; prevent additional symptoms; improve or prevent the underlying metabolic cause of a symptom; inhibit a disease or condition, e.g., suppress the onset of a disease or condition; alleviate a disease or condition; cause regression of a disease or condition; alleviate a condition caused by a disease or condition; or halt the symptoms of a disease or condition; and include preventive measures. The terms further include achieving therapeutic and / or preventive benefits. Therapeutic benefit means the eradication or improvement of the underlying disorder being treated. The therapeutic benefit is also achieved by the eradication or improvement of one or more physiological symptoms associated with the underlying disorder, and therefore improvement is observed in the patient, even though the patient may still be suffering from the underlying disorder.

[0052] As used herein, the term "vial" refers to a container for holding a drug. In some embodiments, a vial may be a vial, bag, pen, or syringe.

[0053] As used herein, the term “agent” refers to the anti-factor XI / XIa antibody described herein, for example, antibody 1 disclosed in Table 1, as well as excipients, for example, histidine buffers, sugars, and polysorbates.

[0054] The term "approximately" refers to any minimum change in the concentration or amount of the active ingredient that does not alter the efficacy of the active ingredient in the preparation of a formulation and in the treatment of a disease or disorder. In certain embodiments, the term "approximately" may encompass ±5%, ±10%, or ±15% of a specified numerical value or data point.

[0055] A range may be expressed in this disclosure as "about" one specific value and / or "about" another specific value. Where such a range is expressed, another aspect includes "about" one specific value and / or other specific values. Similarly, where a value is expressed as an approximation by the preceding use of "about," it is understood that a specific value forms another aspect. Furthermore, it is understood that the endpoint for each range can have meaning both in relation to other endpoints and independently of other endpoints. It is also understood that a large number of values ​​are disclosed in this disclosure, and that each value is disclosed not only as the value itself but also "about" its specific value. Throughout this application, it is understood that the data is presented in a large number of different formats, and that this data represents ranges at endpoints and starting points as well as any combination of data points. For example, if a specific data point "10" and a specific data point "15" are disclosed, it is understood that not only the range between 10 and 15 is disclosed, but also the ranges greater than 10 and 15, greater than or equal to 10 and 15, less than 10 and 15, less than or equal to 10 and 15, and equal to 10 and 15. It is also understood that each unit between two specific units is disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0056] Throughout this description, where a composition is described as having, containing, or comprising certain components, or where a process and method is described as having, containing, or comprising certain steps, it is intended that there exist compositions of the present invention that are essentially composed of or comprise the described components, and that there exist processes and methods of the present invention that are essentially composed of or comprise the described process steps.

[0057] As a general rule, compositions for which percentages are specified are percentages by weight unless otherwise specified. Furthermore, if a variable is not defined, it is governed by the previous definition of that variable. Anti-factor XI and / or activated factor XI (factor XIa) antibody

[0058] In some embodiments, the Disclosure provides a method for detecting ADA against anti-factor XI and / or anti-factor XIa antibodies or their antigen-binding fragments that bind to human, rabbit, baboon, and cynomolgus monkey FXI and / or FXIa. In certain embodiments, the anti-factor XI and / or anti-factor XIa antibody may comprise a heavy chain variable domain (VH) having the amino acid sequence of SEQ ID NO: 9 or 29. In certain embodiments, the antibody comprises a VH having the amino acid sequence of SEQ ID NO: 29. The Disclosure also provides a method for detecting ADA against anti-factor XI and / or anti-factor XIa antibodies or their antigen-binding fragments, wherein the antibody specifically binds to the FXI and / or FXIa proteins and comprises a VH CDR having the amino acid sequence of any one of the VH CDRs listed in Table 1 below. In particular, the present disclosure provides a method for detecting ADA against anti-factor XI and / or anti-factor XIa antibodies or antigen-binding fragments thereof, wherein the antibody specifically binds to FXI and / or FXIa proteins (e.g., human, rabbit, baboon, and cynomolgus monkey FXI and / or FXIa) and comprises (or consists of) one, two, three, or more VH CDRs having any of the amino acid sequences of the VH CDRs listed in Table 1 below (see PCT International Patent Application No. PCT / IB2016 / 053790, filed on 24 June 2016 and published as WO2016 / 207858, which is incorporated herein by reference in its entirety).

[0059] In some embodiments, the Disclosure provides a method for detecting ADA against an anti-factor XI and / or anti-factor XIa antibody or its antigen-binding fragment, wherein the antibody specifically binds to FXI and / or FXIa proteins (e.g., human, rabbit, baboon, and cynomolgus monkey FXI and / or FXIa) and comprises a light chain variable domain (VL) having the amino acid sequence of SEQ ID NO: 19 or 39. In certain embodiments, the antibody comprises a VL having the amino acid sequence of SEQ ID NO: 39. The Disclosure also provides a method for detecting ADA against an anti-factor XI and / or anti-factor XIa antibody or its antigen-binding fragment, wherein the antibody specifically binds to FXI and / or FXIa proteins (e.g., human, rabbit, baboon, and cynomolgus monkey FXI and / or FXIa) and comprises a VL CDR having the amino acid sequence of any one of the VL CDRs listed in Table 1 below. In particular, the present disclosure provides a method for detecting ADA against an anti-factor XI and / or anti-factor XIa antibody or its antigen-binding fragment, wherein the antibody specifically binds to an FXIa protein (e.g., human, rabbit, baboon, and cynomolgus monkey FXI and / or FXIa) and comprises (or consists of) one, two, three or more VL CDRs having any of the amino acid sequences of the VL CDRs listed in Table 1 below.

[0060] In some embodiments, other anti-factor XI and / or anti-factor XIa antibodies are used in the methods for detecting ADA described herein (e.g., methods for detecting ADA against anti-factor XI and / or anti-factor XIa antibodies or their antigen-binding fragments), and they may contain amino acids that are mutated but still have at least 60, 70, 80, 85, 90, or 95 percent CDR region identity with respect to the CDR region shown in the sequences listed in Table 1. In some embodiments, the antibody contains a mutant amino acid sequence in which one or fewer, two or fewer, three or fewer, four or five or fewer amino acids in the CDR region are mutated compared to the CDR region shown in the sequences listed in Table 1. Table 1-1 Table 1-2 Table 1-3 Table 1-4 Table 1-5 Table 1-6 Table 1-7 Table 1-8 Table 1-9 Table 1-10 Table 1-11 Table 1-12 Table 1-13

[0061] In some embodiments, the anti-factor XI and / or anti-factor XIa antibodies used in the methods for detecting ADA described herein (e.g., methods for detecting ADA against anti-factor XI and / or anti-factor XIa antibodies or their antigen-binding fragments) include an amino acid sequence in which the amino acids are mutated but still have at least 60, 65, 70, 75, 80, 85, 90, or 95 percent identity with the sequences listed in Table 1. In some embodiments, the anti-factor XI and / or anti-factor XIa antibodies include a mutant amino acid sequence in which one or fewer, two or fewer, three or fewer, four or five or fewer amino acids in the variable region are mutated compared to the variable region shown in the sequences listed in Table 1, but while retaining substantially the same antigen-binding activity.

[0062] Since each of these antibodies can bind to FXI and / or FXIa, the VH, VL, full-length light chain, and full-length heavy chain sequences (amino acid sequences and nucleotide sequences encoding amino acid sequences) can be “mixed and harmonized” to create other FXI and / or FXIa-binding antibodies of the present disclosure. Such “mixed and harmonized” FXI and / or FXIa-binding antibodies can be tested using binding assays known in the art (e.g., ELISA and other assays described in the Examples section). When mixing and harmonizing these chains, the VH sequence from a particular VH / VL pair should be replaced with a structurally similar VH sequence. Similarly, the full-length heavy chain sequence from a particular full-length heavy chain / full-length light chain pair should be replaced with a structurally similar full-length heavy chain sequence. Similarly, the VL sequence from a particular VH / VL pair should be replaced with a structurally similar VL sequence. Similarly, the full-length light chain sequence from a particular full-length heavy chain / full-length light chain pair should be replaced with a structurally similar full-length light chain sequence.

[0063] Accordingly, for use in the methods described herein (for example, a method for detecting ADA against anti-factor XI and / or anti-factor XIa antibodies or their antigen-binding fragments), the present disclosure provides an isolated antibody or its antigen-binding region having a heavy chain variable domain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 9 and 29, and a light chain variable domain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 19 and 39, wherein the antibody specifically binds to FXI and / or FXIa (e.g., human, rabbit, baboon, and cynomolgus monkey FXIa).

[0064] In certain embodiments, the disclosure provides isolated antibodies or antigen-binding fragments thereof having a heavy chain variable domain and a light chain variable domain, each comprising amino acid sequences selected from SEQ ID NOs. 9 and 29; or 19 and 39, respectively, for use in a method for detecting ADA against anti-factor XI and / or anti-factor XIa antibodies or antigen-binding fragments thereof.

[0065] In certain embodiments for use in the methods described herein (for example, a method for detecting ADA against an anti-factor XI and / or anti-factor XIa antibody or its antigen-binding fragment), the antibody or its antigen-binding fragment provided herein, which specifically binds to human FXI and / or FXIa, comprises a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 9 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 19.

[0066] In certain embodiments for use in the methods described herein (for example, a method for detecting ADA against an anti-factor XI and / or anti-factor XIa antibody or its antigen-binding fragment), the antibody or its antigen-binding fragment provided herein, which specifically binds to human FXI and / or FXIa, comprises a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 29 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 39.

[0067] In certain embodiments for use in the methods described herein (for example, a method for detecting ADA against anti-factor XI and / or anti-factor XIa antibodies or their antigen-binding fragments), the Disclosure provides (i) an isolated antibody having a full-length heavy chain containing an amino acid sequence optimized for expression in mammalian cells, selected from the group consisting of SEQ ID NOs: 11 or 31, and a full-length light chain containing an amino acid sequence optimized for expression in mammalian cells, selected from the group consisting of SEQ ID NOs: 21 or 41; or (ii) a functional protein having the antigen-binding moiety thereof. In certain embodiments, the Disclosure provides an isolated antibody having a heavy chain and a light chain containing amino acid sequences selected from SEQ ID NOs: 11 and 31; or 21 and 41, respectively, for use in a method for detecting ADA against anti-factor XI and / or anti-factor XIa antibodies or their antigen-binding fragments.

[0068] In certain embodiments for use in the methods described herein (for example, a method for detecting ADA against an anti-factor XI and / or anti-factor XIa antibody or its antigen-binding fragment), the antibody or its antigen-binding fragment provided herein, which specifically binds to human FXI and / or FXIa, comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 11 and a light chain containing the amino acid sequence of SEQ ID NO: 21.

[0069] In certain embodiments for use in the methods described herein (for example, a method for detecting ADA against an anti-factor XI and / or anti-factor XIa antibody or its antigen-binding fragment), the antibody or its antigen-binding fragment provided herein, which specifically binds to human FXI and / or FXIa, comprises a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 31 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 41.

[0070] The terms “complementarity-determining region” and “CDR” as used herein refer to sequences of amino acids within the antibody variable region that confer antigen specificity and binding affinity. Generally, there are three CDRs (HCDR1, HCDR2, HCDR3) in each heavy chain variable region and three CDRs (LCDR1, LCDR2, LCDR3) in each light chain variable region.

[0071] The precise amino acid sequence boundaries of a given CDR can be easily determined using one of the following schemes, including the "combination" schemes: Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD ("Kabat" numbering scheme), Al-Lazikani et al., (1997) JMB 273, 927-948 ("Chothia" numbering scheme), Lefranc et al., (2003) Dev. Comp. Immunol., 27, 55-77 ("IMGT" numbering scheme).

[0072] For example, under Kabat, the CDR amino acid residues of antibody 2 are numbered as follows: 31-35 (HCDR1), 50-66 (HCDR2), and 99-111 (HCDR3) within the heavy chain variable domain (VH), and 22-35 (LCDR1), 51-57 (LCDR2), and 90-100 (LCDR3) within the light chain variable domain (VL). Under Chothia, the CDR amino acids in VH are numbered as 26-32 (HCDR1), 52-57 (HCDR2), and 99-111 (HCDR3), and the amino acid residues in VL are numbered as 25-33 (LCDR1), 51-53 (LCDR2), and 92-99 (LCDR3). By combining the CDR definitions of both Kabat and Chothia, the CDR in human VH consists of amino acid residues 26-35 (HCDR1), 50-66 (HCDR2), and 99-111 (HCDR3), and in human VL consists of amino acid residues 22-35 (LCDR1), 51-57 (LCDR2), and 90-100 (LCDR3). By combining the CDR definitions of both Kabat and Chothia, the "combined" CDR consists of amino acid residues 26-35 (HCDR1), 50-66 (HCDR2), and 99-108 (HCDR3) in human VH, and in human VL consists of amino acid residues 24-38 (LCDR1), 54-60 (LCDR2), and 93-101 (LCDR3). As another example, under IMGT, the CDR amino acid residues in the heavy chain variable domain (VH) are numbered 26-33 (HCDR1), 51-58 (HCDR2), and 97-108 (HCDR3), and the CDR amino acid residues in the light chain variable domain (VL) are numbered 27-36 (LCDR1), 54-56 (LCDR2), and 93-101 (LCDR3). Table 1 presents exemplary Kabat, Chothia, combinations, and HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 for anti-FXI / FXIa antibodies, e.g., antibody 2 and antibody 1, according to IMGT. In another embodiment, the present disclosure provides FXIa-binding antibodies comprising the heavy and light chain CDR1, CDR2, and CDR3, or combinations thereof, as described in Table 1.The amino acid sequences of the antibody's VH CDR1 are shown in SEQ ID NOs: 3 and 23. The amino acid sequences of the antibody's VH CDR2 are shown in SEQ ID NOs: 4 and 24. The amino acid sequences of the antibody's VH CDR3 are shown in SEQ ID NOs: 5 and 25. The amino acid sequences of the antibody's VL CDR1 are shown in SEQ ID NOs: 13 and 33. The amino acid sequences of the antibody's VL CDR2 are shown in SEQ ID NOs: 14 and 34. The amino acid sequences of the antibody's VL CDR3 are shown in SEQ ID NOs: 15 and 35. These CDR regions were delineated using the Kabat system.

[0073] Alternatively, when defined using the Chothia system (Al-Lazikani et al., (1997) JMB 273, 927-948), the amino acid sequences of the antibody's VH CDR1 are shown in SEQ ID NOs. 6 and 26. The amino acid sequences of the antibody's VH CDR2 are shown in SEQ ID NOs. 7 and 27. The amino acid sequences of the antibody's VH CDR3 are shown in SEQ ID NOs. 8 and 28. The amino acid sequences of the antibody's VL CDR1 are shown in SEQ ID NOs. 16 and 36. The amino acid sequences of the antibody's VL CDR2 are shown in SEQ ID NOs. 17 and 37. The amino acid sequences of the antibody's VL CDR3 are shown in SEQ ID NOs. 18 and 38.

[0074] Alternatively, when defined using a combination system, the amino acid sequence of the antibody's VH CDR1 is shown in SEQ ID NO: 46. The amino acid sequence of the antibody's VH CDR2 is shown in SEQ ID NO: 4. The amino acid sequence of the antibody's VH CDR3 is shown in SEQ ID NO: 5. The amino acid sequence of the antibody's VL CDR1 is shown in SEQ ID NO: 33. The amino acid sequence of the antibody's VL CDR2 is shown in SEQ ID NO: 14. The amino acid sequence of the antibody's VL CDR3 is shown in SEQ ID NO: 15.

[0075] Alternatively, if defined using the IMGT numbering scheme, the amino acid sequence of the antibody's VH CDR1 is shown in SEQ ID NO: 43. The amino acid sequence of the antibody's VH CDR2 is shown in SEQ ID NO: 44. The amino acid sequence of the antibody's VH CDR3 is shown in SEQ ID NO: 45. The amino acid sequence of the antibody's VL CDR1 is shown in SEQ ID NO: 47. The amino acid sequence of the antibody's VL CDR2 is shown in SEQ ID NO: 37. The amino acid sequence of the antibody's VL CDR3 is shown in SEQ ID NO: 15.

[0076] Given that each of these antibodies can bind to FXI and / or FXIa, and that the antigen-binding specificity is primarily provided by the CDR1, 2, and 3 regions, the VH CDR1, 2, and 3 sequences and the VL CDR1, 2, and 3 sequences can be “mixed and harmonized” (i.e., CDRs from different antibodies can be mixed and harmonized to create other FXI and / or FXIa-binding molecules of this disclosure, but preferably each antibody contains both VH CDR1, 2, and 3 and VL CDR1, 2, and 3). Such “mixed and harmonized” FXI and / or FXIa-binding antibodies can be tested using binding assays and examples known in the art (e.g., ELISA, SET, BIACORE® assays). When mixing and harmonizing VH CDR sequences, the CDR1, CDR2, and / or CDR3 sequences from a particular VH sequence should be replaced with one or more structurally similar CDR sequences. Similarly, when mixing and harmonizing VL CDR sequences, the CDR1, CDR2, and / or CDR3 sequences from a particular VL sequence should be replaced with one or more structurally similar CDR sequences. It will be readily apparent to those skilled in the art that novel VH and VL sequences can be created by replacing one or more VH and / or VL CDR region sequences with structurally similar sequences from the CDR sequences shown herein with respect to the monoclonal antibodies of this disclosure. In addition to the above, in one embodiment, the antigen-binding fragment of the antibody described herein may include VH CDR1, 2, and 3, or VL CDR1, 2, and 3, where the fragment binds to FXI and / or FXIa as a single variable domain. Note that the CDR sequences of antibody 1 and antibody 2 are identical.

[0077] In certain embodiments of this disclosure, for use in a method for detecting ADA against anti-factor XI and / or anti-factor XIa antibodies or their antigen-binding fragments, the antibody or its antigen-binding fragment may have the heavy and light chain sequences of Fab as described in Table 1. More specifically, the antibody or its antigen-binding fragment may have the heavy and light chain sequences of antibody 2 and antibody 1.

[0078] In certain embodiments of this disclosure, the antibody or its antigen-binding fragment that specifically binds to FXI and / or FXIa, for use in a method for detecting ADA against anti-factor XI and / or anti-factor XIa antibodies or their antigen-binding fragments, includes heavy chain variable region CDR1, heavy chain variable region CDR2, heavy chain variable region CDR3, light chain variable region CDR1, light chain variable region CDR2, and light chain variable region CDR3 as defined by Kabat and listed in Table 1. For example, in certain embodiments of this disclosure, the antibody or its antigen-binding fragment that specifically binds to FXI and / or FXIa, for use in a method for detecting ADA against anti-factor XI and / or anti-factor XIa antibodies or their antigen-binding fragments, includes heavy chain variable region CDR1, heavy chain variable region CDR2, heavy chain variable region CDR3, light chain variable region CDR1, light chain variable region CDR2, and light chain variable region CDR3 as defined by Chothia and listed in Table 1. In other embodiments, the antibody or its antigen-binding fragment that specifically binds to FXI and / or FXIa, for use in a method for detecting ADA against anti-factor XI and / or anti-factor XIa antibodies or their antigen-binding fragments, is defined by a combination system and includes heavy chain variable region CDR1, heavy chain variable region CDR2, heavy chain variable region CDR3, light chain variable region CDR1, light chain variable region CDR2, and light chain variable region CDR3, as listed in Table 1. In certain embodiments of the present disclosure, the antibody or its antigen-binding fragment that specifically binds to FXI and / or FXIa, for use in a method for detecting ADA against anti-factor XI and / or anti-factor XIa antibodies or their antigen-binding fragments, is defined by IMGT and includes heavy chain variable region CDR1, heavy chain variable region CDR2, heavy chain variable region CDR3, light chain variable region CDR1, light chain variable region CDR2, and light chain variable region CDR3, as listed in Table 1.

[0079] In certain embodiments for use in the methods described herein (for example, for use in methods for detecting ADA against anti-factor XI and / or anti-factor XIa antibodies or their antigen-binding fragments), the Disclosure includes antibodies that specifically bind to FXI and / or FXIa, comprising the heavy chain variable region CDR1 of SEQ ID NO: 3; heavy chain variable region CDR2 of SEQ ID NO: 4; heavy chain variable region CDR3 of SEQ ID NO: 5; light chain variable region CDR1 of SEQ ID NO: 13; light chain variable region CDR2 of SEQ ID NO: 14; and light chain variable region CDR3 of SEQ ID NO: 15.

[0080] In certain embodiments, the Disclosure includes antibodies that specifically bind to FXI and / or FXIa, comprising the heavy chain variable region CDR1 of SEQ ID NO: 23; heavy chain variable region CDR2 of SEQ ID NO: 24; heavy chain variable region CDR3 of SEQ ID NO: 25; light chain variable region CDR1 of SEQ ID NO: 33; light chain variable region CDR2 of SEQ ID NO: 34; and light chain variable region CDR3 of SEQ ID NO: 35, for use in a method for detecting ADA against anti-factor XI and / or anti-factor XIa antibodies or their antigen-binding fragments.

[0081] In certain embodiments, the Disclosure includes antibodies that specifically bind to FXI and / or FXIa, comprising the heavy chain variable region CDR1 of SEQ ID NO: 6; heavy chain variable region CDR2 of SEQ ID NO: 7; heavy chain variable region CDR3 of SEQ ID NO: 8; light chain variable region CDR1 of SEQ ID NO: 16; light chain variable region CDR2 of SEQ ID NO: 17; and light chain variable region CDR3 of SEQ ID NO: 18, for use in a method for detecting ADA against anti-factor XI and / or anti-factor XIa antibodies or their antigen-binding fragments.

[0082] In certain embodiments, the Disclosure includes antibodies that specifically bind to FXI and / or FXIa, comprising the heavy chain variable region CDR1 of SEQ ID NO: 26; the heavy chain variable region CDR2 of SEQ ID NO: 27; the heavy chain variable region CDR3 of SEQ ID NO: 28; the light chain variable region CDR1 of SEQ ID NO: 36; the light chain variable region CDR2 of SEQ ID NO: 37; and the light chain variable region CDR3 of SEQ ID NO: 38, for use in a method for detecting ADA against anti-factor XI and / or anti-factor XIa antibodies or their antigen-binding fragments.

[0083] In certain embodiments, the present invention provides an antibody that specifically binds to FXI and / or FXIa, comprising the heavy chain variable region CDR1 of SEQ ID NO: 43; heavy chain variable region CDR2 of SEQ ID NO: 44; heavy chain variable region CDR3 of SEQ ID NO: 45; light chain variable region CDR1 of SEQ ID NO: 47; light chain variable region CDR2 of SEQ ID NO: 37; and light chain variable region CDR3 of SEQ ID NO: 15, for use in a method for detecting ADA against anti-factor XI and / or anti-factor XIa antibodies or their antigen-binding fragments.

[0084] In certain embodiments, the present invention provides an antibody that specifically binds to FXI and / or FXIa, comprising the heavy chain variable region CDR1 of SEQ ID NO: 46; heavy chain variable region CDR2 of SEQ ID NO: 4; heavy chain variable region CDR3 of SEQ ID NO: 5; light chain variable region CDR1 of SEQ ID NO: 33; light chain variable region CDR2 of SEQ ID NO: 14; and light chain variable region CDR3 of SEQ ID NO: 15, for use in a method for detecting ADA against anti-factor XI and / or anti-factor XIa antibodies or their antigen-binding fragments.

[0085] In certain embodiments, the Disclosure includes antibodies or antigen-binding fragments that specifically bind to FXI and / or FXIa, as listed in Table 1, for use in a method for detecting ADA against anti-factor XI and / or anti-factor XIa antibodies or their antigen-binding fragments. In certain embodiments for use in the method described herein, the antibodies or antigen-binding fragments that bind to FXI and / or FXIa for use in a method for detecting ADA against anti-factor XI and / or anti-factor XIa antibodies or their antigen-binding fragments are antibody 2 and antibody 1.

[0086] As used herein, a human antibody includes a heavy or light chain variable region or a full-length heavy or light chain that is "product of" or "derived from" a specific germline sequence, if the variable region or full-length chain of the antibody is obtained from a system using a human germline immunoglobulin gene. Such a system includes immunizing transgenic mice having a human immunoglobulin gene with an antigen of interest, or screening a human immunoglobulin gene library displayed on phages with an antigen of interest. A human antibody that is "product of" or "derived from" a human germline immunoglobulin sequence can be identified as such by comparing the amino acid sequence of the human antibody with the amino acid sequence of a human germline immunoglobulin and selecting the human germline immunoglobulin sequence that is closest in sequence to the human antibody sequence (i.e., has the highest % identity).

[0087] Human antibodies that are "products of" or "derived from" a particular human germline immunoglobulin sequence may contain amino acid differences compared to the germline sequence, for example, due to the intentional introduction of naturally occurring somatic mutations or site-directed mutations. However, within the VH or VL framework region, selected human antibodies generally contain amino acid residues that are at least 90% identical to the amino acid sequence encoded by the human germline immunoglobulin gene, thereby identifying the human antibody as human when compared to germline immunoglobulin amino acid sequences of other species (e.g., mouse germline sequences). In certain particular cases, the amino acid sequence of a human antibody may be at least 60%, 70%, 80%, 90%, or at least 95%, or even at least 96%, 97%, 98%, or 99% identical to the amino acid sequence encoded by the germline immunoglobulin gene.

[0088] Generally, recombinant human antibodies exhibit a difference of 10 or fewer amino acids from the amino acid sequence encoded by a human germline immunoglobulin gene within the VH or VL framework region. In certain cases, human antibodies may exhibit a difference of 5 or fewer amino acids, or even 4 or fewer, 3 or fewer, 2 or fewer, or 1 or fewer amino acids from the amino acid sequence encoded by a germline immunoglobulin gene. Examples of human germline immunoglobulin genes, but not limited to these, include the variable domain germline fragments listed below, as well as DP47 and DPK9. homologous antibodies

[0089] In further embodiments for use in the methods described herein (for example, a method for detecting ADA against anti-factor XI and / or anti-factor XIa antibodies or their antigen-binding fragments), the Disclosure provides an antibody or its antigen-binding fragment comprising an amino acid sequence homologous to the sequences listed in Table 1 (e.g., SEQ ID NOs. 29, 31, 39, or 41), which binds to FXI and / or FXIa proteins (e.g., human, rabbit, baboon, and cynomolgus monkey FXIa) and retains desired functional properties of the antibodies listed in Table 1, such as antibody 2 and antibody 1. In certain embodiments, such homologous antibodies retain the CDR amino acid sequences listed in Table 1 (e.g., Kabat CDR, Chothia CDR, IMGT CDR, or combined CDR).

[0090] For example, in some embodiments, the Disclosure provides an isolated antibody or a functional antigen-binding fragment thereof, comprising a heavy chain variable domain and a light chain variable domain, for use in a method for detecting ADA against an anti-factor XI and / or anti-factor XIa antibody or its antigen-binding fragment, wherein the heavy chain variable domain comprises an amino acid sequence that is at least 80%, at least 90%, or at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 9 and 29, and the light chain variable domain comprises an amino acid sequence that is at least 80%, at least 90%, or at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 19 and 39, and the antibody specifically binds to FXI and / or FXIa (e.g., human, rabbit, baboon, and cynomolgus monkey FXIa). In a particular embodiment, for use in a method for detecting ADA against an anti-factor XI and / or anti-factor XIa antibody or its antigen-binding fragment, the isolated antibody or its functional antigen-binding fragment comprises a heavy-chain variable domain and a light-chain variable domain, wherein the heavy-chain variable domain comprises an amino acid sequence that is at least 80%, at least 90%, or at least 95% identical to the amino acid sequence of SEQ ID NO: 9, and the light-chain variable domain comprises an amino acid sequence that is at least 80%, at least 90%, or at least 95% identical to the amino acid sequence of SEQ ID NO: 19, and the antibody specifically binds to FXI and / or FXIa (e.g., human, rabbit, baboon, and cynomolgus monkey FXIa).In a particular embodiment, for use in a method for detecting ADA against an anti-factor XI and / or anti-factor XIa antibody or its antigen-binding fragment, the isolated antibody or its functional antigen-binding fragment comprises a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises an amino acid sequence that is at least 80%, at least 90%, or at least 95% identical to the amino acid sequence of SEQ ID NO: 29, and the light chain variable domain comprises an amino acid sequence that is at least 80%, at least 90%, or at least 95% identical to the amino acid sequence of SEQ ID NO: 39, and the antibody specifically binds to FXI and / or FXIa (e.g., human, rabbit, baboon, and cynomolgus monkey FXIa). In certain embodiments of this disclosure, the heavy and light chain sequences of an antibody for use in a method for detecting ADA against anti-factor XI and / or anti-factor XIa antibody or its antigen-binding fragment include the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 sequences as defined by Kabat, for example, SEQ ID NOs. 3, 4, 5, 13, 14, and 15, respectively. In certain embodiments of this disclosure, the heavy and light chain sequences of an antibody for use in a method for detecting ADA against anti-factor XI and / or anti-factor XIa antibody or its antigen-binding fragment include the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 sequences as defined by Chothia, for example, SEQ ID NOs. 6, 7, 8, 16, 17, and 18, respectively. In a particular embodiment, the heavy and light chain sequences of an antibody for use in a method for detecting ADA against anti-factor XI and / or anti-factor XIa antibodies or their antigen-binding fragments include HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 sequences defined by a combination system, for example, SEQ ID NOs. 46, 4, 5, 33, 14, and 15, respectively.In a particular embodiment, the heavy and light chain sequences of an antibody for use in a method for detecting ADA against an anti-factor XI and / or anti-factor XIa antibody or its antigen-binding fragment include the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 sequences as defined by IMGT, for example, SEQ ID NOs. 43, 44, 45, 47, 37, and 15, respectively.

[0091] In other embodiments for use in the methods described herein (e.g., a method for detecting ADA against anti-factor XI and / or anti-factor XIa antibodies or their antigen-binding fragments), the VH and / or VL amino acid sequences of anti-factor XI and / or anti-factor XIa antibodies may be 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequences listed in Table 1. In other embodiments, the VH and / or VL amino acid sequences may be identical except for amino acid substitutions at one or fewer, two or fewer, three or fewer, four or five or fewer amino acid positions. Antibodies having VH and VL regions with high (e.g., 80% or more) identity to the VH and VL regions of those listed in Table 1 can be obtained by mutagenesis (e.g., site-directed or PCR-mediated mutagenesis) of nucleic acid molecules encoding SEQ ID NOs. 10 or 30 and SEQ ID NOs. 20 and 40, respectively, and then testing the encoded modified antibodies for retention of function using the functional assays described herein.

[0092] In other embodiments for use in the methods described herein (e.g., a method for detecting ADA against anti-factor XI and / or anti-factor XIa antibodies or their antigen-binding fragments), the full-length heavy chain and / or full-length light chain amino acid sequences of anti-factor XI and / or anti-factor XIa antibodies may be 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequences listed in Table 1 (e.g., SEQ ID NOs. 11 and / or 21, or 31 and / or 41). Antibodies having full-length heavy chains and full-length light chains with high (e.g., 80% or more) identity to the full-length heavy chain of either SEQ ID NOs. 11 or 31, and the full-length light chain of either SEQ ID NOs. 21 or 41, can be obtained by mutagenesis (e.g., site-directed or PCR-mediated mutagenesis) of the nucleic acid molecules encoding such polypeptides, and then testing the encoded modified antibodies for retention of function using the functional assays described herein.

[0093] In one embodiment, the present invention provides an isolated antibody or a functional antigen-binding fragment thereof, comprising a heavy chain and a light chain, for use in a method for detecting ADA against an anti-factor XI and / or anti-factor XIa antibody or its antigen-binding fragment, wherein the heavy chain comprises an amino acid sequence that is at least 80%, at least 90%, or at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 11 and 31, and the light chain comprises an amino acid sequence that is at least 80%, at least 90%, or at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 21 and 41, and the antibody specifically binds to FXI and / or FXIa (e.g., human, rabbit, baboon, and cynomolgus monkey FXIa). In one embodiment, for use in a method for detecting ADA against an anti-factor XI and / or anti-factor XIa antibody or its antigen-binding fragment, the isolated antibody or its functional antigen-binding fragment comprises a heavy chain and a light chain, where the heavy chain comprises an amino acid sequence that is at least 80%, at least 90%, or at least 95% identical to the amino acid sequence of SEQ ID NO: 11, and the light chain comprises an amino acid sequence that is at least 80%, at least 90%, or at least 95% identical to the amino acid sequence of SEQ ID NO: 21, and the antibody specifically binds to FXI and / or FXIa (e.g., human, rabbit, baboon, and cynomolgus monkey FXIa). In a particular embodiment, for use in a method for detecting ADA against anti-factor XI and / or anti-factor XIa antibodies or their antigen-binding fragments, an isolated antibody or its functional antigen-binding fragment comprises a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence that is at least 80%, at least 90%, or at least 95% identical to the amino acid sequence of SEQ ID NO: 31, and the light chain comprises an amino acid sequence that is at least 80%, at least 90%, or at least 95% identical to the amino acid sequence of SEQ ID NO: 41, and the antibody specifically binds to FXI and / or FXIa (e.g., human, rabbit, baboon, and cynomolgus monkey FXIa).In certain embodiments of this disclosure, the heavy and light chain sequences further include the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 sequences as defined by Kabat, for example, SEQ ID NOs. 3, 4, 5, 13, 14, and 15, respectively. In certain embodiments of this disclosure, the heavy and light chain sequences of an antibody or its functional antigen-binding fragment for use in a method for detecting ADA against an anti-factor XI and / or anti-factor XIa antibody or its antigen-binding fragment include the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 sequences as defined by Chothia, for example, SEQ ID NOs. 6, 7, 8, 16, 17, and 18, respectively. In certain embodiments, the heavy and light chain sequences of an antibody or its functional antigen-binding fragment for use in a method for detecting ADA against anti-factor XI and / or anti-factor XIa antibody or its antigen-binding fragment include the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 sequences defined by the combination system, for example, SEQ ID NOs. 46, 4, 5, 33, 14, and 15, respectively. In certain embodiments, the heavy and light chain sequences of an antibody or its functional antigen-binding fragment for use in a method for detecting ADA against anti-factor XI and / or anti-factor XIa antibody or its antigen-binding fragment include the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 sequences defined by IMGT, for example, SEQ ID NOs. 43, 44, 45, 47, 37, and 15, respectively.

[0094] In other embodiments for use in the methods described herein, the full-length heavy chain and / or full-length light chain nucleotide sequences may be 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequences listed in Table 1 (e.g., SEQ ID NOs. 12 and / or 22, or 32 and / or 42).

[0095] In other embodiments for use in the methods described herein, the nucleotide sequences of the variable region of the heavy chain and / or the variable region of the light chain may be 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequences listed in Table 1 (e.g., SEQ ID NOs. 10 and / or 20, or 30 and / or 40).

[0096] As used herein, percent identity between two sequences is a function of the number of identical positions shared by those sequences, taking into account the number of gaps that need to be introduced to optimally align the two sequences and the length of each gap (i.e., % identity is equal to the number of identical positions / the total number of positions × 100). Sequence comparison and determination of percent identity between two sequences can be achieved using mathematical algorithms, as described in the non-restrictive examples below.

[0097] The isolated anti-FXI and / or anti-FXIa antibodies or their antigen-binding fragments described herein may be monoclonal antibodies, human or humanized antibodies, chimeric antibodies, single-chain antibodies, Fab fragments, Fv fragments, F(ab')2 fragments, or scFv fragments, and / or IgG isotypes (e.g., IgG1 such as human IgG1). In certain embodiments, the anti-FXI and / or anti-FXIa antibodies described herein are recombinant human antibodies. In certain embodiments, the anti-FXI and / or anti-FXIa antibodies described herein are human IgG1 / lambda(λ) antibodies. In certain embodiments, the anti-FXI and / or anti-FXIa antibodies described herein are human IgG1 / lambda(λ) antibodies containing human Fc domains, e.g., D265A and / or P329A substitutions, that have been engineered to reduce the potential for effector function (e.g., ADCC and / or CDC).

[0098] In addition, or instead, to identify relevant sequences, for example, the protein sequences of this disclosure can be used as further "query sequences" to perform searches of public databases. For example, such searches can be performed using the BLAST program (version 2.0) described in Altschul, et al., 1990 J. Mol. Biol. 215: 403-10. Antibodies with Conservative Modifications

[0099] In certain other embodiments, the antibody of this disclosure for use in the methods described herein (e.g., a method for detecting ADA against an anti-factor XI and / or anti-factor XIa antibody or its antigen-binding fragment) has a heavy chain variable region comprising CDR1, CDR2, and CDR3 sequences and a light chain variable region comprising CDR1, CDR2, and CDR3 sequences, wherein one or more of these CDR sequences have a specific amino acid sequence based on the antibody described herein or a conserved modification thereof, and the antibody retains the desired functional properties of the FXIa-binding antibody of this disclosure.

[0100] Accordingly, for use in the methods described herein, in some embodiments, the Disclosure relates to an isolated antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising CDR1, CDR2, and CDR3 sequences and a light chain variable region comprising CDR1, CDR2, and CDR3 sequences, wherein the heavy chain variable region CDR1 amino acid sequence is selected from the group consisting of SEQ ID NOs: 3 and 23 and their conserved modifications, the heavy chain variable region CDR2 amino acid sequence is selected from the group consisting of SEQ ID NOs: 4 and 24 and their conserved modifications, and the heavy chain variable region CDR3 amino acid sequence The present invention provides an isolated antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment is specifically bound to FXIa, and is selected from the group consisting of SEQ ID NOs. 5 and 25 and their conservative modifications, the CDR1 amino acid sequence of the light chain variable region is selected from the group consisting of SEQ ID NOs. 13 and 33 and their conservative modifications, the CDR2 amino acid sequence of the light chain variable region is selected from the group consisting of SEQ ID NOs. 14 and 34 and their conservative modifications, and the CDR3 amino acid sequence of the light chain variable region is selected from the group consisting of SEQ ID NOs. 15 and 35 and their conservative modifications.

[0101] In one embodiment, the present invention provides an isolated antibody or antigen-binding fragment comprising a heavy chain variable region containing CDR1, CDR2, and CDR3 sequences and a light chain variable region containing CDR1, CDR2, and CDR3 sequences, wherein the CDR1 amino acid sequence of the heavy chain variable region is selected from the group consisting of those listed in Table 1 and their conservative modifications, the CDR2 amino acid sequence of the heavy chain variable region is selected from the group consisting of those listed in Table 1 and their conservative modifications, the CDR3 amino acid sequence of the heavy chain variable region is selected from the group consisting of those listed in Table 1 and their conservative modifications, the CDR1 amino acid sequence of the light chain variable region is selected from the group consisting of those listed in Table 1 and their conservative modifications, the CDR2 amino acid sequence of the light chain variable region is selected from the group consisting of those listed in Table 1 and their conservative modifications, and the antibody or antigen-binding fragment specifically binds to FXIa.

[0102] In other embodiments for use in the methods described herein, the antibodies of the Disclosure are optimized for expression in mammalian cells and have a full-length heavy chain sequence and a full-length light chain sequence, wherein one or more of these sequences have a specific amino acid sequence based on the antibodies described herein or their conserved modifications, and the antibodies retain the desired functional properties of the FXIa-binding antibodies of the Disclosure. Accordingly, the Disclosure provides isolated antibodies comprising a full-length heavy chain and a full-length light chain, optimized for expression in mammalian cells, wherein the full-length heavy chain has an amino acid sequence selected from the group of SEQ ID NOs: 11 or 31 and its conserved modifications, and the full-length light chain has an amino acid sequence selected from the group of SEQ ID NOs: 21 or 41 and its conserved modifications, and the antibodies specifically bind to FXI and / or FXIa (e.g., human, rabbit, baboon, and cynomolgus monkey FXIa). The same epitope-binding antibody

[0103] In some embodiments, the Disclosure provides antibodies that compete for the same epitopes with the FXI and / or FXIa-binding antibodies listed in Table 1 for use in the methods described herein (e.g., methods for detecting ADA against anti-factor XI and / or anti-factor XIa antibodies or their antigen-binding fragments). Therefore, additional antibodies can be identified based on their ability to compete with other antibodies of the Disclosure in an FXI and / or FXIa binding assay (e.g., by binding to the same or overlapping epitopes, thereby statistically and competitively inhibiting binding). The ability of a test antibody to inhibit the binding of the antibodies of the Disclosure to the FXI and / or FXIa proteins demonstrates that the test antibody may compete with that antibody for binding to FXI and / or FXIa; such antibodies, according to a non-limiting theory, bind to the same or related (e.g., structurally similar or spatially proximal) epitopes on the FXI and / or FXIa proteins as the antibody it competes with. In certain embodiments, the antibody that binds to the same epitopes on FXI and / or FXIa as the antibody of this disclosure is a human monoclonal antibody. Such a human monoclonal antibody can be prepared and isolated as described herein.

[0104] As used herein, an antibody is “competing” for binding if, in the presence of the competing antibody at equimolar concentrations, the competing antibody binds to the same FXI and / or FXIa epitopes as the antibody or antigen-binding fragment of the Disclosure (e.g., antibody 1 or antibody 2) and inhibits the binding of the antibody or antigen-binding fragment of the Disclosure to FXI and / or FXIa by more than 50% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%). This can be determined, for example, by any method well known to those skilled in the art in a competitive binding assay.

[0105] When used herein, an antibody or its antigen-binding fragment does not “compete” with the FXI and / or FXIa antibody or antigen-binding fragment of the Disclosure (e.g., antibody 1 or antibody 2) unless the competing antibody or its antigen-binding fragment binds to the same FXI and / or FXIa epitope as, or to overlapping FXI and / or FXIa epitopes. As used herein, a competing antibody or its antigen-binding fragment does not include (i) sterically blocking the binding of the antibody or antigen-binding fragment of the Disclosure to its target (for example, the competing antibody binding to a nearby, non-overlapping FXI and / or FXIa epitope, thereby physically preventing the binding of the antibody or antigen-binding fragment of the Disclosure to its target); and / or (ii) binding to a different, non-overlapping FXI and / or FXIa epitope, thereby inducing a conformational change in the FXI and / or FXIa protein, resulting in the inability of the FXI and / or FXIa antibody or antigen-binding fragment of the Disclosure to the protein in a manner that would occur if the conformational change were no longer present. Engineered and modified antibodies

[0106] In some embodiments, for use in the methods described herein (e.g., methods for detecting ADA against anti-factor XI and / or anti-factor XIa antibodies or their antigen-binding fragments), the antibodies of the Disclosure may further be prepared by using an antibody having one or more of the VH and / or VL sequences shown herein as a starting material for engineering a modified antibody, which may have altered properties from the starting antibody. An antibody can be engineering by modifying one or more residues within one or both of the variable regions (i.e., VH and / or VL), for example, within one or more CDR regions and / or one or more framework regions. In addition, or instead, an antibody can be engineering by modifying residues within the constant region(s) to change the effector function(s) of the antibody(s).

[0107] One type of engineering manipulation of variable regions that can be performed is CDR grafting. Antibodies predominantly interact with target antigens through amino acid residues located in six heavy-chain and light-chain complementarity-determining regions (CDRs). For this reason, the amino acid sequences within CDRs exhibit greater variability among individual antibodies than the sequences outside the CDRs. Since CDR sequences are involved in the vast majority of antibody-antigen interactions, it is possible to express recombinant antibodies that mimic the characteristics of specific naturally occurring antibodies by constructing expression vectors that include the CDR sequence of that specific naturally occurring antibody grafted onto framework sequences derived from different antibodies with different characteristics (see, for example, Riechmann, L. et al., 1998 Nature 332: 323-327; Jones, P. et al., 1986 Nature 321: 522-525; Queen, C. et al., 1989 Proc. Natl. Acad., USA 86: 10029-10033; U.S. Patent No. 5,225,539 for Winter, and U.S. Patent Nos. 5,530,101, 5,585,089, 5,693,762 and 6,180,370 for Queen et al.).

[0108] Accordingly, another embodiment of the present disclosure relates to an isolated antibody or antigen-binding fragment for use in a method for detecting ADA against an anti-factor XI and / or anti-factor XIa antibody or its antigen-binding fragment, comprising a heavy chain variable region including a CDR1 sequence having an amino acid sequence selected from the group consisting of SEQ ID NOs: 3 and 23; a CDR2 sequence having an amino acid sequence selected from the group consisting of SEQ ID NOs: 4 and 24; a CDR3 sequence having an amino acid sequence selected from the group consisting of SEQ ID NOs: 5 and 25; and a light chain variable region including a CDR1 sequence having an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 and 33; a CDR2 sequence having an amino acid sequence selected from the group consisting of SEQ ID NOs: 14 and 34; and a CDR3 sequence having an amino acid sequence selected from the group consisting of SEQ ID NOs: 15 and 35. Accordingly, such an antibody contains VH and VL CDR sequences of a monoclonal antibody, but nevertheless may contain framework sequences different from those of these antibodies.

[0109] Such framework sequences can be obtained from public DNA databases containing germline antibody gene sequences or from publicly available references. For example, germline DNA sequences of human heavy and light chain variable region genes can be found in the "VBase" human germline sequence database, as well as in Kabat, EA, et al., 1991 Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242; Tomlinson, IM, et al., 1992 J. Mol. Biol. 227: 776-798; and Cox, JPL et al., 1994 Eur. J Immunol. 24: 827-836, the contents of which are explicitly incorporated herein by reference.

[0110] Examples of framework sequences for use with the antibodies of this disclosure are those structurally similar to the framework sequences used by selected antibodies of this disclosure, e.g., consensus sequences and / or framework sequences used by monoclonal antibodies of this disclosure. The VH CDR1, 2, and 3 sequences, and the VL CDR1, 2, and 3 sequences, can be grafted onto framework regions having sequences identical to those found in the germline immunoglobulin genes from which the framework sequences originate, or the CDR sequences can be grafted onto framework regions containing one or more mutations compared to the germline sequences. For example, in certain cases, it has been found beneficial to mutate residues within the framework region so as to maintain or enhance the antigen-binding ability of the antibody (see, e.g., U.S. Patents 5,530,101; 5,585,089; 5,693,762 and 6,180,370 to Queen et al.). Frameworks that can be used as scaffolds for building the antibodies and antigen-binding fragments described herein include, but are not limited to, VH1A, VH1B, VH3, Vk1, Vk2, and Vk2.

[0111] Accordingly, another embodiment of the present disclosure for use in the method described herein (for example, a method for detecting ADA against anti-factor XI and / or anti-factor XIa antibodies or their antigen-binding fragments) relates to an isolated FXIa-binding antibody or its antigen-binding fragment, comprising a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 9 and 29 or an amino acid sequence having one, two, three, four or five amino acid substitutions, deletions or additions within the framework region of such a sequence, and further comprising a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 19 or 39 or an amino acid sequence having one, two, three, four or five amino acid substitutions, deletions or additions within the framework region of such a sequence.

[0112] Another type of variable region modification involves mutating amino acid residues within the VH and / or VL CDR1, CDR2 and / or CDR3 regions to thereby improve one or more binding properties (e.g., affinity) of the antibody of interest, which is known as "affinity maturation." Mutations (one or more) can be introduced by site-directed mutagenesis or PCR-mediated mutagenesis, and their effects on antibody binding or other functional properties of interest can be evaluated in in vitro or in vivo assays as described herein and presented in the Examples section. Conservative modifications (as described above) can be introduced. Mutations may be amino acid substitutions, additions, or deletions. Furthermore, generally, one, two, three, four, or five residues within the CDR region are changed.

[0113] Therefore, in another embodiment for use in the method described herein (for example, a method for detecting ADA against anti-factor XI and / or anti-factor XIa antibodies or their antigen-binding fragments), the disclosure has a VH CDR1 region having an amino acid sequence selected from the group having SEQ ID NOs: 3 and 23 or an amino acid sequence having one, two, three, four, or five amino acid substitutions, deletions, or additions compared to SEQ ID NOs: 3 and 23; a VH CDR2 region having an amino acid sequence selected from the group having SEQ ID NOs: 4 and 24 or an amino acid sequence having one, two, three, four, or five amino acid substitutions, deletions, or additions compared to SEQ ID NOs: 4 and 24; a heavy chain variable region having a VH CDR3 region having an amino acid sequence selected from the group having SEQ ID NOs: 5 and 25 or an amino acid sequence having one, two, three, four, or five amino acid substitutions, deletions, or additions compared to SEQ ID NOs: 5 and 25; and a VL region having an amino acid sequence selected from the group having SEQ ID NOs: 13 and 33 or an amino acid sequence having one, two, three, four, or five amino acid substitutions, deletions, or additions compared to SEQ ID NOs: 13 and 33. The present invention provides an isolated FXIa-binding antibody or its antigen-binding fragment, comprising: a CDR1 region; a VL CDR2 region having an amino acid sequence selected from the group consisting of SEQ ID NOs: 14 and 34, or an amino acid sequence having one, two, three, four, or five amino acid substitutions, deletions, or additions compared to SEQ ID NOs: 14 and 34; and a VL CDR3 region having an amino acid sequence selected from the group consisting of SEQ ID NOs: 15 and 35, or an amino acid sequence having one, two, three, four, or five amino acid substitutions, deletions, or additions compared to SEQ ID NOs: 15 and 35.

[0114] Therefore, in another embodiment for use in the method described herein (for example, a method for detecting ADA against anti-factor XI and / or anti-factor XIa antibodies or their antigen-binding fragments), the disclosure has a VH CDR1 region having an amino acid sequence selected from the group having SEQ ID NOs: 6 and 26 or an amino acid sequence having one, two, three, four, or five amino acid substitutions, deletions, or additions compared to SEQ ID NOs: 6 and 26; a VH CDR2 region having an amino acid sequence selected from the group having SEQ ID NOs: 7 and 27 or an amino acid sequence having one, two, three, four, or five amino acid substitutions, deletions, or additions compared to SEQ ID NOs: 7 and 27; a VH CDR3 region having an amino acid sequence selected from the group having SEQ ID NOs: 8 and 28 or an amino acid sequence having one, two, three, four, or five amino acid substitutions, deletions, or additions compared to SEQ ID NOs: 8 and 28; and a VL region having an amino acid sequence selected from the group having SEQ ID NOs: 16 and 36 or an amino acid sequence having one, two, three, four, or five amino acid substitutions, deletions, or additions compared to SEQ ID NOs: 16 and 36. The present invention provides an isolated FXIa-binding antibody or its antigen-binding fragment, comprising: a CDR1 region; a VL CDR2 region having an amino acid sequence selected from the group consisting of SEQ ID NOs: 17 and 37, or an amino acid sequence having one, two, three, four, or five amino acid substitutions, deletions, or additions compared to SEQ ID NOs: 17 and 37; and a VL CDR3 region having an amino acid sequence selected from the group consisting of SEQ ID NOs: 18 and 38, or an amino acid sequence having one, two, three, four, or five amino acid substitutions, deletions, or additions compared to SEQ ID NOs: 18 and 38. Antibodies with extended half-lives

[0115] In some embodiments, this disclosure provides antibodies with extended in vivo half-lives that specifically bind to the FXIa protein for use in methods described herein (e.g., methods for detecting ADA against anti-factor XI and / or anti-factor XIa antibodies or their antigen-binding fragments). Extended half-live anti-factor XI and / or anti-factor XIa antibodies may be meaningful in methods for detecting ADA if anti-factor XI and / or anti-factor XIa antibodies are used in methods for treating patients, and therefore the physiological and clinical correlation of ADA methods and therapeutic anti-factor XI and / or anti-factor XIa antibodies is beneficial for treating or maintaining a patient's disease or disorder.

[0116] Many factors can influence the in vivo half-life of a protein. For example, renal filtration, hepatic metabolism, degradation by proteases, and immunogenic responses (e.g., antibody-mediated protein neutralization and uptake by macrophages and dendritic cells). The half-life of the antibodies of this disclosure can be extended using various strategies. For example, by chemical coupling with polyethylene glycol (PEG), reCODE PEG, antibody scaffolds, polysialic acid (PSA), hydroxyethyl starch (HES), albumin-binding ligands, and carbohydrate shields; by gene fusion and transfer with proteins that bind to serum proteins such as albumin, IgG, FcRn; by coupling (genetically or chemically) with other binding moieties that bind to serum proteins, such as nanobodies, Fab, DARPin, avimer, aphibodies, and antikalin; by gene fusion with rPEG, albumin, albumin domains, albumin-binding proteins, and Fc; or by incorporation into nanocarriers, slow-release formulations, or medical devices.

[0117] To prolong the in vivo serum circulation of antibodies, inert polymer molecules such as high molecular weight PEG can be attached to antibodies or fragments of the antibody, with or without a multifunctional linker, either through site-specific conjugation of PEG to the N-terminus or C-terminus of the antibody, or via epsilon-amino groups present on lysine residues. To pegyrate an antibody, the antibody or fragment is typically reacted with polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions that one or more PEG groups attach to the antibody or antibody fragment. Pegylation can be carried out by acylation or alkylation reactions with a reactive PEG molecule (or a similar reactive water-soluble polymer). As used herein, the term "polyethylene glycol" is intended to encompass any form of PEG used to derivatize other proteins, such as mono(C1-C10) alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol-maleimide. In certain embodiments, the antibody to be pegyrated is an unglycosylated antibody. Linear or branched polymer derivatization is used to minimize the loss of biological activity. Conjugation can be closely monitored by SDS-PAGE and mass spectrometry to ensure proper conjugation of PEG molecules to the antibody. Unreacted PEG can be separated from the antibody-PEG conjugate by size exclusion or ion-exchange chromatography. Antibodies derivatized with PEG can be tested for binding activity and in vivo efficacy using methods well known to those skilled in the art, for example, by immunoassays described herein. Methods for pegyrating proteins are known in the art and can be applied to the antibodies of this disclosure. See, for example, EP 0 154 316 by Nishimura et al. and EP 0 401 384 by Ishikawa et al.

[0118] Another modified PEG technique is Reconstituted Chemical Orthogonal Directional Manipulation (ReCODE PEG), which incorporates chemically specified side chains into biosynthetic proteins via a reconstituted system containing tRNA synthetase and tRNA. This technique makes it possible to incorporate more than 30 new amino acids into biosynthetic proteins in E. coli, yeast, and mammalian cells. The tRNA allows the non-native amino acid to be incorporated at any location where an amber codon is located, and the amber is converted from a stop codon to a codon that signals the incorporation of a chemically specified amino acid.

[0119] Recombinant PEG (rPEG) technology can also be used to extend the serum half-life. This technology involves fusing an unstructured protein tail of 300-600 amino acids with an existing pharmaceutical protein. Since the apparent molecular weight of such an unstructured protein chain is approximately 15 times its actual molecular weight, the serum half-life of the protein is significantly increased. In contrast to conventional PEGylation, which requires chemical conjugation and repurification, the manufacturing process is significantly simplified and the product is homogeneous.

[0120] Another technique is polysialylation, which uses the natural polymer polysialic acid (PSA) to extend the activity lifetime and improve the stability of therapeutic peptides and proteins. PSA is a polymer of sialic acid (sugar). When used for protein and therapeutic peptide drug delivery, polysialic acid provides a protective microenvironment for the conjugation. This increases the activity lifetime of therapeutic proteins in circulation and prevents their recognition by the immune system. PSA polymers are found naturally in the human body. PSA polymers have been utilized by certain bacteria, which have evolved over millions of years, to coat their walls. As a result, these naturally polysialylated bacteria were able to reinforce the body's defense system through molecular mimicry. PSA, the ultimate natural stealth technology, can be easily produced in large quantities with predetermined physical characteristics by such bacteria. Bacterial PSA is chemically identical to PSA in the human body and is therefore completely non-immunogenic even when coupled with proteins.

[0121] Another technique involves the use of hydroxyethyl starch ("HES") derivatives conjugated with antibodies. HES is a modified natural polymer derived from waxy corn starch and can be metabolized by enzymes in the body. HES solutions are typically administered to replenish deficient blood volume and improve the hydrodynamic properties of the blood. HESification of antibodies can extend their circulating half-life by increasing molecular stability and reducing renal clearance, resulting in increased biological activity. A wide range of HES antibody conjugates can be customized by varying various parameters, such as the molecular weight of HES.

[0122] Antibodies with increased in vivo half-lives can also be produced by introducing one or more amino acid modifications (i.e., substitutions, insertions, or deletions) into the IgG constant domain or its FcRn-binding fragment (preferably an Fc or hinged Fc domain fragment). See, for example, International Publication No. WO98 / 23289; International Publication No. WO97 / 34631; and U.S. Patent No. 6,277,375.

[0123] Furthermore, antibodies can be conjugated with albumin (e.g., human serum albumin; HSA) to produce antibodies or antibody fragments that are more stable in vivo or have a longer half-life in vivo. This technique is well known in the art. See, for example, International Patent Publications WO93 / 15199, WO93 / 15200, and WO01 / 77137; and European Patent Publication EP 413,622. In addition, with respect to the bispecific antibodies described above, the specificity of the antibody can be designed such that one binding domain of the antibody binds to FXIa, while the second binding domain of the antibody binds to serum albumin, preferably HSA.

[0124] Strategies for increasing half-life are particularly useful for nanobodies, fibronectin-based conjugates, and other antibodies or proteins for which an increase in half-life in vivo is desired. Antibody conjugate

[0125] In some embodiments, the Disclosure provides an antibody or fragment thereof that specifically binds to the FXIa protein for use in the methods described herein (e.g., a method for detecting ADA against anti-factor XI and / or anti-factor XIa antibodies or their antigen-binding fragments), and that is fused or chemically conjugated (including both covalent and non-covalent conjugations) by recombination with a heterologous protein or polypeptide (or fragment thereof, preferably a polypeptide of at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90 or at least 100 amino acids) to produce a fusion protein. In particular, the Disclosure provides a fusion protein comprising an antigen-binding fragment of the antibody described herein (e.g., Fab fragment, Fd fragment, Fv fragment, F(ab)2 fragment, VH domain, VH CDR, VL domain or VL CDR) and a heterologous protein, polypeptide, or peptide for use in a method for detecting ADA against anti-factor XI and / or anti-factor XIa antibodies or their antigen-binding fragments. Methods for fusing or conjugating proteins, polypeptides, or peptides with antibodies or antibody fragments are known in the art. For example, U.S. Patents Nos. 5,336,603, 5,622,929, 5,359,046, 5,349,053, 5,447,851, and 5,112,946; European Patents Nos. EP307,434 and EP367,166; International Publications WO96 / 04388 and WO91 / 06570; Ashkenazi et al., 1991, Proc. Natl. Acad. Sci. USA 88: 10535-10539; Zheng et al., 1995, J. Immunol. 154: 5590-5600; and Vil et al., 1992, Proc. Natl. Acad. Sci. USA 89: See 11337-11341.

[0126] Additional fusion proteins can be generated by techniques such as gene shuffling, motif shuffling, exon shuffling, and / or codon shuffling (collectively referred to as "DNA shuffling"). DNA shuffling can be used to alter the activity of the antibody or fragment of the disclosure (e.g., an antibody or fragment with higher affinity and a lower dissociation rate). See, in general, U.S. Patents No. 5,605,793, 5,811,238, 5,830,721, 5,834,252, and 5,837,458; Patent et al., 1997, Curr. Opinion Biotechnol. 8: 724-33; Harayama, 1998, Trends Biotechnol. 16 (2): 76-82; Hansson, et al., 1999, J. Mol. Biol. 287: 265-76; and Lorenzo and Blasco, 1998, Biotechniques 24 (2): 308-313 (these patents and publications are incorporated by reference in their entirety). An antibody or a fragment thereof, or an antibody or fragment encoding such an antibody, can be modified before recombination by subjecting it to error-prone PCR, random nucleotide insertion, or other methods of random mutagenesis. A polynucleotide encoding an antibody or fragment thereof that specifically binds to the FXIa protein can be recombined using one or more components, motifs, sections, parts, domains, fragments, etc., of one or more heterologous molecules.

[0127] Furthermore, to facilitate purification, antibodies or fragments thereof can be fused with marker sequences such as peptides. In certain embodiments, the marker amino acid sequence is, among other things, a hexahistidine peptide (SEQ ID NO: 48), such as the tag provided in the pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, CA, 91311), many of which are commercially available. As described in Gentz ​​et al., 1989, Proc. Natl. Acad. Sci. USA 86: 821-824, for example, hexahistidine (SEQ ID NO: 48) leads to convenient purification of the fusion protein. Other peptide tags useful for purification include, but are not limited to, hemagglutinin ("HA") tags corresponding to epitopes derived from influenza hemagglutinin protein (Wilson et al., 1984, Cell 37: 767), and "flag" tags.

[0128] In other embodiments, antibodies for detecting ADA can be conjugated with diagnostic or detectable active substances. Such detections may involve: antibodies conjugating various enzymes, e.g., horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; prosthetic groups, e.g., streptavidin / biotin and avidin / biotin; and fluorescent materials, e.g., umbelliferone, fluorescein, fluorescein isothiocyanate (fluorescein). Examples include isothiocynate, rhodamine, dichlorotriazinylamine fluorescein, dansilchloride or phycoerythrin; luminescent materials, e.g., luminol, etc.; bioluminescent materials, e.g., luciferase, luciferin, and aequorin, etc.; radioactive materials, e.g., iodine (131I, 125I, 123I, and 121I), carbon (14C), sulfur (35S), tritium (3H), indium (115In, 113In, 112In, and 111In), technetium (99Tc), thallium (201Ti), gallium (68 This can be achieved by coupling with detectable materials, including Ga (67Ga), palladium (103Pd), molybdenum (99Mo), xenon (133Xe), fluorine (18F), 153Sm, 177Lu, 159Gd, 149Pm, 140La, 175Yb, 166Ho, 90Y, 47Sc, 186Re, 188Re, 142Pr, 105Rh, 97Ru, 68Ge, 57Co, 65Zn, 85Sr, 32P, 153Gd, 169Yb, 51Cr, 54Mn, 75Se, 113Sn, and 117Tin, as well as positron-emitting metals used in various positron emission tomography techniques, and non-radioactive paramagnetic metal ions. In certain embodiments, an antibody for detecting ADA is included in a ruthenium-labeled detection cocktail.

[0129] In some embodiments, the disclosure further encompasses the use of anti-factor XI and / or anti-factor XIa antibodies or fragments thereof conjugated with a therapeutic moiety for use in methods for detecting ADA against anti-factor XI and / or anti-factor XIa antibodies or their antigen-binding fragments. The antibody or fragment thereof can be conjugated with a therapeutic moiety such as a cytotoxin, e.g., a cell growth inhibitor or cell disruptor, a therapeutic agent, or a radioactive metal ion, e.g., an alpha-emitter. The cytotoxin or cytotoxic agent includes any active substance that is harmful to cells.

[0130] Furthermore, antibodies or fragments thereof can be conjugated with therapeutic or drug portions that modify a given biological response, and such conjugated antibodies can be used in methods for detecting ADA against anti-factor XI and / or anti-factor XIa antibodies or their antigen-binding fragments. The therapeutic or drug portion is not construed to be limited to classical chemotherapeutic agents. For example, the drug portion may be a protein, peptide, or polypeptide having the desired biological activity. Such proteins may include, for example, toxins such as abrin, lysine A, Pseudomonas exotoxin, cholera toxin, or diphtheria toxin; proteins such as tumor necrosis factor, α-interferon, β-interferon, nerve growth factor, platelet-derived growth factor, tissue plasminogen activator, apoptotic activators, anti-angiogenic agents, etc.; or biological reaction modifiers such as lymphokines, etc.

[0131] Furthermore, antibodies can be conjugated with radioactive metal ions useful for conjugation to polypeptides, including, but not limited to, 131In, 131LU, 131Y, 131Ho, and 131Sm, such as alpha-emitters, e.g., 213Bi, or macrocyclic chelating agent therapeutic moieties. In certain embodiments, the macrocyclic chelating agent is 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA), which can be attached to the antibody via a linker molecule. Such linker molecules are generally known in the art and are described by reference in Denardo et al., 1998, Clin Cancer Res. 4 (10): 2483-90; Peterson et al., 1999, Bioconjug. Chem. 10 (4): 553-7; and Zimmerman et al., 1999, Nucl. Med. Biol. 26 (8): 943-50.

[0132] Techniques for conjugating therapeutic components with antibodies are well-known. For example, Arnon et al., “Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy”, in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review”, in Monoclonal Antibodies 84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); “Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer See "Therapy", in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., 1982, Immunol. Rev. 62: 119-58.

[0133] Antibodies can also be attached to a solid support, which is particularly useful for immunoassays or purification of target antigens. Examples of such solid supports, though not limited to these, include glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene. Detection and measurement of anti-drug antibodies (ADAs)

[0134] The inventors have developed a novel method for the qualitative and / or quantitative detection of anti-XI / XIa factor ADA from a sample. This method is effective in reducing and eliminating interference problems caused by the presence of drugs or targets in ADA detection. Assays on ADA

[0135] Accordingly, a method for detecting an anti-drug antibody (ADA) against an anti-factor XI and / or anti-factor XIa antibody or its antigen-binding fragment is described herein, comprising the steps of: incubating a sample with an acid to dissociate anti-factor XI and / or anti-factor XIa antibody-antigen complexes and / or anti-factor XI and / or anti-factor XIa antibody-ADA complexes present in the sample to create an acid digest; incubating the acid digest on a plate coated with an anti-factor XI and / or anti-factor XIa antibody or its antigen-binding fragment; neutralizing the acid digest; and detecting the presence of ADA using a ruthenium-labeled detection cocktail. In certain embodiments, the anti-factor XI and / or anti-factor XIa antibody is antibody 1.

[0136] In some embodiments, the sample is a sample derived from a subject, e.g., a human subject. In some embodiments, the sample is selected from the group consisting of blood, plasma, or serum obtained from a subject, e.g., a human subject. In some embodiments, the sample is not obtained from a subject. In some embodiments, the sample is an in vitro sample prepared for testing, e.g., a sample containing a drug, a target, and ADA. In certain embodiments of the method, the method includes a first step of preparing the sample.

[0137] Suitable acids for dissociating anti-factor XI and / or anti-factor XIa antibody-antigen complexes, and / or anti-factor XI and / or anti-factor XIa antibody-ADA complexes include, but are not limited to, acetic acid, propionic acid, lactic acid, malic acid, tartaric acid, citric acid, or phosphoric acid, or mixtures thereof. In some embodiments, the acid is selected from the group consisting of acetic acid, citric acid, phosphoric acid, and mixtures thereof. In certain embodiments, the acid is acetic acid. It is intended that the pH of a particular acid or combination of acids can be adjusted to a desired pH, for example, using methods known in the art. The concentration of the acid, e.g., acetic acid, may be about 50 mM, about 100 mM, about 150 mM, about 200 mM, about 250 mM, about 300 mM, about 350 mM, about 400 mM, or about 500 mM. In certain embodiments, the concentration of the acid, e.g., acetic acid, may be about 300 mM.

[0138] The sample can be incubated with the acid for a desired length of time for dissociation. For example, the sample can be incubated with the acid for about 5 minutes, about 10 minutes, about 15 minutes, about 30 minutes, about 45 minutes, or about 60 minutes. In a particular embodiment, the sample is incubated with the acid for about 10 minutes. In a particular embodiment, the sample is incubated with the acid at room temperature. It is intended herein that the incubation temperature may affect the required incubation time; that is, a longer incubation time will be required if the sample is incubated at a temperature lower than room temperature, and a shorter incubation time will be required if the sample is incubated at a temperature higher than room temperature.

[0139] Assay plates can first be coated with a molecule (e.g., a protein) to enhance the affinity of the drug (e.g., antibody 1) to the plate. In certain embodiments, the plate is coated with streptavidin and the drug is biotin-labeled. In certain embodiments, the plate is coated with nickel and the drug is given a histidine tag (e.g., 6x-His). In certain embodiments, the plate is coated with an antibody against a small molecule peptide tag and the drug is modified (e.g., genetically or chemically) to express the tag (e.g., V5, Flag, Myc, HA, GST, GFP, etc.). Alternative techniques for enhancing affinity for drug / protein are known in the art.

[0140] The concentration of the anti-factor XI and / or anti-factor XIa antibody or its antigen-binding fragment may vary depending on the assay conditions. In some embodiments, the concentration of the anti-factor XI and / or anti-factor XIa antibody or its antigen-binding fragment (e.g., antibody 1) is between approximately 0.05 μg / ml and approximately 0.45 μg / ml, between approximately 0.10 μg / ml and approximately 0.40 μg / ml, between approximately 0.15 μg / ml and approximately 0.35 μg / ml, or between approximately 0.20 μg / ml and approximately 0.30 μg / ml. In certain embodiments, the concentration of the anti-factor XI and / or anti-factor XIa antibody or its antigen-binding fragment (e.g., antibody 1) is selected from the group consisting of 0.1 μg / ml, 0.25 μg / ml, 0.5 μg / ml, 0.75 μg / ml, and 1 μg / ml. In a particular embodiment, the concentration of the anti-factor XI and / or anti-factor XIa antibody or its antigen-binding fragment (e.g., antibody 1) is approximately 0.25 μg / ml.

[0141] In some embodiments, the method includes a neutralization step, which enables ADA to bind to anti-factor XI and / or anti-factor XIa antibodies or their antigen-binding fragments (e.g., antibody 1) on a coated plate. In certain embodiments, the neutralization step uses a base with a pH of approximately 8.0. It is intended herein that the pH of a particular base or combination of bases can be adjusted to a desired pH for neutralization, for example, using methods known in the art.

[0142] Suitable bases for the neutralization step include, but are not limited to, Tris, phosphates, CAPS, CHAPS, EDTA, EGTA, HEPES, PIPES, MOPS, tricine, glycine, histidine, triethanolamine, and mixtures thereof. In certain embodiments, the base is selected from the group consisting of Tris, phosphates, HEPES, triethanolamine, and mixtures thereof. In certain embodiments, the base is Tris. In certain embodiments, the base is Tris at a pH of approximately 8.0.

[0143] In some embodiments, ADA is detected using a ruthenium-labeled detection cocktail. In certain embodiments, the ruthenium-labeled detection cocktail comprises an antibody. In certain embodiments, the antibody is anti-human IgG, anti-human IgM, anti-human IgE, anti-rabbit Ig, or any combination thereof. In certain embodiments, the ruthenium-labeled detection cocktail comprises ruthenium-labeled anti-human IgG. In certain embodiments, the ruthenium-labeled detection cocktail comprises ruthenium-labeled anti-human IgM. In certain embodiments, the ruthenium-labeled detection cocktail comprises ruthenium-labeled anti-human IgE. In certain embodiments, the ruthenium-labeled detection cocktail comprises ruthenium-labeled anti-rabbit Ig. In certain embodiments, the ruthenium-labeled detection cocktail comprises a combination of the ruthenium-labeled antibodies.

[0144] In some embodiments, the method includes a washing step after any of the above steps. In certain embodiments, the method includes a washing step after the incubation step. The washing step can be carried out using a suitable washing buffer, such as Tris-HCl buffered saline (TBS), TBS Tween® 20, phosphate-buffered saline (PBS), PBS Tween 20, etc. In some embodiments, the washing step is carried out using PBS Tween 20. In certain embodiments, the washing step is carried out using PBS 0.05% Tween 20.

[0145] In some embodiments, the plate is blocked before the sample is added. Exemplary blocking buffers may include, but are not limited to, bovine serum albumin (BSA), milk, goat serum, fetal bovine serum (FBS), horse serum (HS), or casein. In certain embodiments, the blocking buffer is BSA in phosphate-buffered saline (PBS-T) with tween-20. In certain embodiments, the concentrations of BSA in PBS-T are about 1%, about 2.5%, about 5%, about 7.5%, and about 10%. In certain embodiments, the concentration of BSA in PBS-T is about 5%.

[0146] As shown in Figure 1, in some embodiments of the assay described herein, a cocktail comprising biotin-labeled antibody 1, ruthenium-labeled antibody 1, and anti-antibody 1 antibody 3 is incubated with a sample (e.g., blood, plasma, or serum) and added to a streptavidin-coated plate 4. In certain embodiments, a detectable signal (e.g., a chemiluminescent signal, e.g., light 5) is generated in proportion to the concentration of anti-antibody 1 antibody. In certain embodiments, the sample (e.g., blood, plasma, or serum) is derived from a cynomolgus monkey.

[0147] As shown in Figure 2, in some embodiments of the assay described herein, a sample (e.g., blood, plasma, or serum) containing antibody 1 10, anti-antibody 1 11, homodimer FXI and / or FXIa 12, anti-antibody 1-FXI / FXIa complex 13, and anti-antibody 1-antibody 1 complex 14 is incubated with beads (e.g., streptavidin beads) 15 coupled with anti-FXI antibody 16 to form a depleted sample. The depleted sample is then incubated with acid 17 to dissociate the antibody 1-anti-antibody 1 complex present in the depleted sample to form a dissociated sample. The dissociated sample is incubated with a cocktail containing biotin-labeled antibody 1 18, ruthenium-labeled antibody 1 19, and anti-antibody 1 antibody, and added to a streptavidin-coated plate 20. In certain embodiments, a detectable signal (e.g., chemiluminescent signal, e.g., light) is generated in proportion to the concentration of anti-antibody 1 antibody. In certain embodiments, the sample (e.g., blood, plasma, or serum) is derived from a cynomolgus monkey.

[0148] As shown in Figure 3, in some embodiments of the assay described herein, a cocktail comprising biotin-labeled antibody 1 30, ruthenium-labeled anti-monkey immunoglobulin 31, and anti-antibody 1 antibody 32 is incubated with a sample (e.g., blood, plasma, or serum) containing homodimer FXI and / or FXIa 33 and added to a streptavidin-coated plate 34. In certain embodiments, a detectable signal (e.g., a chemiluminescent signal, e.g., light 35) is generated in proportion to the concentration of anti-antibody 1 antibody. In certain embodiments, the endogenous FXI / FXIa dimer is not detected by the ruthenium-labeled anti-monkey immunoglobulin. In certain embodiments, the sample (e.g., blood, plasma, or serum) is derived from cynomolgus monkeys.

[0149] As shown in Figure 4, in some embodiments of the assay described herein, a sample (e.g., blood, plasma, or serum) containing antibody 1 40, anti-antibody 1 41, homodimer FXI and / or FXIa 42, antibody 1-FXI / FXIa complex 43, and anti-antibody 1-antibody 1 complex 44 is incubated with acid 45 to dissociate the antibody 1-antibody 1 complex present in the sample and form an acid digest. The acid digest is incubated with a cocktail containing biotin-labeled antibody 1, ruthenium-labeled anti-monkey or anti-rabbit immunoglobulin 46, and anti-antibody 1 antibody, and added to a high-binding plate (e.g., high-binding ELISA plate 47). In certain embodiments, a detectable signal (e.g., chemiluminescent signal, e.g., light 48) is generated in proportion to the concentration of anti-antibody 1 antibody. In certain embodiments, the endogenous FXI / FXIa dimer is not detected by the ruthenium-labeled anti-monkey or anti-rabbit immunoglobulin. In certain embodiments, the sample (e.g., blood, plasma, or serum) is derived from a cynomolgus monkey.

[0150] As shown in Figure 5, in some embodiments of the assay described herein, a sample (e.g., blood, plasma, or serum) containing antibody 1 50, anti-antibody 1 51, homodimer FXI and / or FXIa 52, antibody 1-FXI / FXIa complex 53, and anti-antibody 1-antibody 1 complex 54 is incubated with acid 55 to dissociate the antibody 1-antibody 1 complex and / or antibody 1-FXI / FXIa complex present in the sample and form an acid digest. The acid digest is added to a plate 56 coated with antibody 1 and neutralized by adding a suitable base 57 (e.g., Tris). A ruthenium-labeled detection cocktail 58 containing ruthenium-labeled anti-human IgG, anti-human IgM, ruthenium-labeled anti-human IgE, and ruthenium-labeled anti-rabbit Ig is provided. In certain embodiments, a detectable signal (e.g., a chemiluminescent signal, e.g., light) is generated in proportion to the concentration of anti-antibody 1. In certain embodiments, the endogenous FXI / FXIa dimer is not detected by the ruthenium-labeled detection cocktail. In certain embodiments, antibody 1 is not detected by the ruthenium-labeled detection cocktail. In certain embodiments, the sample (e.g., blood, plasma, or serum) is of human origin. [Examples]

[0151] The disclosure described herein in general terms is merely illustrative of certain aspects and embodiments of the disclosure and does not limit the scope of the disclosure in any way, as will be more readily understood by referring to the following examples. (Example 1) Development of factor XI and / or factor XIa anti-drug antibody assays (ADA) for cynomolgus monkey samples.

[0152] First, the presence of anti-factor XI and anti-factor XIa (FXI / FXIa) anti-drug antibodies (ADAs) in cynomolgus monkey serum was assayed using a standard bridging assay (Figure 1). A cocktail containing biotin-labeled antibody 1, ruthenium-labeled antibody 1, and antibody 1ADA was added to a streptavidin-coated plate. Light generation from the chemiluminescence reaction was proportional to the amount of ADA. However, this method resulted in a high percentage of false positives because the endogenous protein-targeted homodimer FXI / FXIa bridged to the labeled antibody 1.

[0153] Therefore, we hypothesized that introducing a depletion step for the endogenous homodimeric FXI / FXIa target would reduce the high false-positive rate. Accordingly, we added two further steps: depletion of endogenous FXI / FXIa using beads coated with anti-FXI / FXIa, followed by acid dissociation of the drug from the antibody, before the bridging assay described above (Figure 2). However, the depletion step using anti-FXI antibody was inefficient, and no improvement in target interference in serum was detected. Therefore, we tested a novel assay format using ruthenium-labeled anti-monkey Ig as the detector (Figure 3). As shown in Table 2 below, a significant improvement in FXI interference was revealed by the determination of the signal-to-noise ratio. [Table 2]

[0154] A schematic diagram of the final assay format for ADA detection in cynomolgus monkeys is shown in Figure 4. A summary of the assay validation parameters is shown in Table 3. The results for drug resistance and target interference are summarized in Table 4. [Table 3] [Table 4] (Example 2) Development of Factor XI and / or Factor XIa anti-drug antibody assays (ADAs) for human samples.

[0155] The assay for cynomolgus monkeys was adapted for validation in human serum. It was assumed that the antibody 1 assay should be performed in Fab format to avoid nonspecific binding by anti-human Ig. Antibody 1 was digested using pepsin or papain to generate the F(ab')2 fragment. However, the resulting Fc fragment produced an unacceptably elevated background. Negative purification to retain the Fc fragment produced the F(ab')2 fragment in very low yield and was unreactive. Therefore, it was decided to use the full-length antibody format.

[0156] A schematic diagram of the assay configuration is shown in Figure 5. Briefly, a 96-well plate was coated with antibody 1 at a concentration of 0.25 μg / ml and subsequently blocked with 5% BSA in PBS-T. To dissociate the anti-factor XI and / or anti-factor XIa antibody-antigen complexes and the anti-factor XI and / or anti-factor XIa antibody-ADA complexes, the samples were incubated with 300 mM acetic acid for 10 minutes, thereby forming acid digests. The blocked plates were washed, and 1 M Tris, pH 8.0 was added to neutralize the acid. The acid digests were then incubated on the antibody 1-coated plates. A detection cocktail of anti-human IgG / IgM, ruthenium-labeled anti-human IgE, and ruthenium-labeled anti-rabbit Ig was added. The plates were washed after incubation with the acid digests. Antibody 1ADA was then detected by chemiluminescence quantification.

[0157] We tested the three-tiered ADA assay conditions recommended by the FDA: the 5% false-positive cutpoint in the screening assay, the specificity of the response to the drug in the confirmatory assay, and the semi-quantitative estimation of ADA concentration in the titer assay.

[0158] The results of the screening assay are summarized in Table 5. Plates were coated with a ruthenium-labeled detection cocktail at a concentration of 0.25 μg / mL: 0.1 μg / mL anti-rabbit, 0.01 μg / mL anti-human IgG / M, and 0.01 μg / mL anti-human IgE. Plates were blocked using a buffer consisting of 5% bovine serum albumin (BSA) in PBS-T. The assay results demonstrate high sensitivity and minimal background, FXI, and drug interference. [Table 5]

[0159] The results of the confirmatory assay are summarized in Table 6. As shown, sensitivity was established down to at least 1.23 ng / mL. [Table 6-1] [Table 6-2]

[0160] The results of the drug tolerance titer measurement are summarized in Table 7. The values ​​shown are the signal-to-noise ratio. [Table 7]

Claims

1. 1. A method for detecting anti-drug antibodies (ADA) against an anti-factor XI and / or anti-factor XIa antibody or an antigen-binding fragment thereof, comprising: (a) incubating the sample with acid to dissociate anti-factor XI and / or anti-factor XIa antibody-antigen complexes and / or anti-factor XI and / or anti-factor XIa antibody-ADA complexes present in the sample to create an acid digest; (b) incubating the acid digest on a plate coated with the anti-factor XI and / or anti-factor XIa antibody or antigen-binding fragment thereof; (c) neutralizing the acid digest; (d) detecting the presence of said ADA using a ruthenium-labeled detection cocktail; A method comprising:

2. 2. The method of claim 1, wherein the sample is selected from the group consisting of human blood, plasma, or serum. (a) an initial step of preparing the sample; and / or (b) after said incubating step, a washing step The method of claim 1 or 2, further comprising: (a) the acid is selected from the group consisting of acetic acid, citric acid, phosphoric acid, and mixtures thereof; (b) the neutralizing step is optionally with a base selected from the group consisting of Tris, phosphate, HEPES, triethanolamine, and mixtures thereof, at a pH of about 8.0; and / or (c) the ruthenium-labeled detection cocktail comprises a detection antibody; 4. The method according to any one of claims 1 to 3. (a) the acid is acetic acid, optionally at a concentration of about 300 mM; and / or (b) the base is Tris; The method according to claim 4.

6. The method of any one of claims 1 to 5, wherein the plate is coated with streptavidin.

7. The method of claim 1, wherein the anti-factor XI and / or anti-factor XIa antibody or antigen-binding fragment thereof is coated onto the plate in a buffer at a concentration selected from the group consisting of about 0.1 μg / mL, about 0.25 μg / mL, about 0.5 μg / mL, about 0.75 μg / mL, and about 1 μg / mL, preferably about 0.25 μg / mL.

8. 8. The method of claim 4, wherein the detection antibody is selected from the group consisting of anti-human IgG, anti-human IgM, anti-human IgE, anti-rabbit Ig, and any combination thereof.

9. The anti-factor XI and / or anti-factor XIa antibody or antigen-binding fragment thereof is (i) a heavy chain variable region CDR1 of SEQ ID NO: 23; a heavy chain variable region CDR2 of SEQ ID NO: 24; a heavy chain variable region CDR3 of SEQ ID NO: 25; a light chain variable region CDR1 of SEQ ID NO: 33; a light chain variable region CDR2 of SEQ ID NO: 34; and a light chain variable region CDR3 of SEQ ID NO: 35; (ii) a heavy chain variable region CDR1 of SEQ ID NO: 26; a heavy chain variable region CDR2 of SEQ ID NO: 27; a heavy chain variable region CDR3 of SEQ ID NO: 28; a light chain variable region CDR1 of SEQ ID NO: 36; a light chain variable region CDR2 of SEQ ID NO: 37; and a light chain variable region CDR3 of SEQ ID NO: 38; (iii) a heavy chain variable region CDR1 of SEQ ID NO: 43; a heavy chain variable region CDR2 of SEQ ID NO: 44; a heavy chain variable region CDR3 of SEQ ID NO: 45; a light chain variable region CDR1 of SEQ ID NO: 47; a light chain variable region CDR2 of SEQ ID NO: 37; and a light chain variable region CDR3 of SEQ ID NO: 15; or (iv) a heavy chain variable region CDR1 of SEQ ID NO: 46; a heavy chain variable region CDR2 of SEQ ID NO: 4; a heavy chain variable region CDR3 of SEQ ID NO: 5; a light chain variable region CDR1 of SEQ ID NO: 33; a light chain variable region CDR2 of SEQ ID NO: 14; and a light chain variable region CDR3 of SEQ ID NO:

15. The method according to any one of claims 1 to 8, comprising:

10. The anti-factor XI and / or anti-factor XIa antibody or antigen-binding fragment thereof is (a) a heavy chain variable region (VH) selected from the group consisting of SEQ ID NOs: 9, 29, and a VH having 90% identity thereto; and a light chain variable region (VL) selected from the group consisting of SEQ ID NOs: 19, 39, and a VL having 90% identity thereto; or (b) a heavy chain variable region (VH) selected from the group consisting of SEQ ID NOs: 9 and 29; and a light chain variable region (VL) selected from the group consisting of SEQ ID NOs: 19 and 39. The method according to any one of claims 1 to 9, comprising:

11. The anti-factor XI and / or anti-factor XIa antibody is (a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 31, 11, or a heavy chain having 90% identity thereto; and a light chain comprising the amino acid sequence of SEQ ID NO: 41, 21, or a light chain having 90% identity thereto; or (b) a heavy chain comprising the amino acid sequence of SEQ ID NO: 31 and a light chain comprising the amino acid sequence of SEQ ID NO:

41. The method according to any one of claims 1 to 10, comprising:

12. The method of any one of claims 1 to 11, wherein the anti-factor XI and / or anti-factor XIa antibody is a human monoclonal antibody.

13. 13. The method of claim 12, wherein the anti-factor XI and / or anti-factor XIa antibody is of human IgG1 isotype and comprises an antibody Fc domain optionally containing a D265A substitution and a P329A substitution.

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