WO2020029179A1 - Anti-factor xi antibodies - Google Patents

Anti-factor xi antibodies Download PDF

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Publication number
WO2020029179A1
WO2020029179A1 PCT/CN2018/099638 CN2018099638W WO2020029179A1 WO 2020029179 A1 WO2020029179 A1 WO 2020029179A1 CN 2018099638 W CN2018099638 W CN 2018099638W WO 2020029179 A1 WO2020029179 A1 WO 2020029179A1
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Prior art keywords
seq
antibody
fxi
antibodies
thrombosis
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PCT/CN2018/099638
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English (en)
French (fr)
Inventor
Wenyi Wang
Quan Yu
Xiaowu Liu
John Liuzhong Xu
Zhiqiang Du
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Shanghai Benemae Pharmaceutical Corporation
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Priority to BR112021002472-7A priority Critical patent/BR112021002472A2/pt
Priority to KR1020217006922A priority patent/KR20210042352A/ko
Priority to CN202111467365.1A priority patent/CN114478782B/zh
Priority to CN202310301532.8A priority patent/CN116554334A/zh
Priority to CN201880098606.XA priority patent/CN113227150B/zh
Priority to CN202111466510.4A priority patent/CN114478781B/zh
Priority to EP18929685.8A priority patent/EP3833692A1/en
Priority to PCT/CN2018/099638 priority patent/WO2020029179A1/en
Priority to AU2018436195A priority patent/AU2018436195A1/en
Priority to CA3108708A priority patent/CA3108708A1/en
Priority to JP2021506623A priority patent/JP2021534098A/ja
Priority to MX2021001613A priority patent/MX2021001613A/es
Priority to US16/537,427 priority patent/US11958911B2/en
Publication of WO2020029179A1 publication Critical patent/WO2020029179A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/36—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against blood coagulation factors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • This disclosure relates to antibodies capable of binding to the coagulation factor XI (FXI) and/or its activated form factor XIa (FXIa) , and to fragments of FXI and/or FXIa, and uses thereof, including uses as anticoagulation agents for treating thrombosis that do not compromise hemostasis.
  • FXI coagulation factor XI
  • FXIa activated form factor XIa
  • Thrombosis is a condition that involves blood clotting in a blood vessel, thereby blocking or obstructing blood flow in the affected area. This condition can lead to serious complications if the blood clots travel along the circulatory system to a crucial body part such as heart, brain, and lungs, causing heart attack, stroke, pulmonary embolism, etc.
  • Thrombosis is the major cause of most strokes and myocardial infarctions, deep vein thrombosis (DVT) , pulmonary embolism, and other cardiovascular events.
  • DVT deep vein thrombosis
  • 1, 2 Thrombosis can be treated or prevented by anticoagulants such as low-molecular-weight heparin, warfarin, and Factor Xa direct inhibitors.
  • anticoagulants such as low-molecular-weight heparin, warfarin, and Factor Xa direct inhibitors.
  • the most common adverse effect of these currently available therapies is impairing haemostasis.
  • antibodies that bind to coagulation factor XI (FXI) and/or its activated form factor XIa (FXIa) , and to fragments of FXI and/or FXIa.
  • the antibodies are monoclonal antibodies.
  • the antibodies are recombinant antibodies.
  • the antibodies are humanized antibodies.
  • the antibodies are immunologically active portions of immunoglobulin molecules, e.g., Fabs, Fvs, or scFvs.
  • the antibodies bind to the A3 domain of FXI and/or FXIa.
  • the antibodies include one or more CDRs consisting of or comprising the amino acid sequences of SEQ ID NOs: 11-16, 27-32, 43-48, 59-64, 75-80, 91-96, 107-112, 123-128, 139-144, 155-160, 171-176, and 187-192.
  • the pharmaceutical composition comprises one or more anti-FXI and/or anti-FXIa antibodies as disclosed herein.
  • the pharmaceutical composition further comprises one or more pharmaceutically acceptable adjuvants, carriers, excipients, preservatives, or a combination thereof.
  • nucleic acid encoding an anti-FXI and/or anti-FXIa antibody as disclosed herein, or a functional fragment of either antibody, as well as a vector comprising the nucleic acid, and a host cell comprising the vector.
  • the vector is an expression vector that is capable of producing the antibody or a functional fragment thereof encoded by the nucleic acid in a host cell.
  • kits comprising one or more anti-FXI and/or anti-FXIa antibodies as disclosed herein for use in treating and/or preventing thrombosis and/or complications or conditions associated with thrombosis.
  • the kit comprises a pharmaceutical composition comprising one or more anti-FXI and/or anti-FXIa antibodies as disclosed herein for use in treating and/or preventing thrombosis and/or complications or conditions associated with thrombosis.
  • the kit further comprises instructions for use.
  • the method includes administering to a subject in need thereof a therapeutically effective amount of one or more anti-FXI and/or anti-FXIa antibodies as disclosed herein.
  • the method includes administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition containing an anti-FXI antibody, an anti-FXIa antibody, or a functional fragment of either antibody.
  • an anti-FXI and/or anti-FXIa antibody as disclosed herein formulating a medicament for treating and/or preventing thrombosis and/or complications or conditions associated with thrombosis.
  • an anti-FXI and/or anti-FXIa antibody as disclosed herein.
  • the method entails the steps of transforming a host cell with a vector comprising a nucleic acid encoding the antibody, and expressing the antibody in the host cell.
  • the method can further include purifying the expressed antibody from the host cell.
  • the purified antibody can be subjected to modifications such that the modified recombinant antibody retains the activity of the corresponding human antibody.
  • an antibody disclosed herein can be produced from culturing a hybridoma.
  • Figures 1A-1E illustrate the effects of five anti-FXI antibodies via APTT assay in human plasma.
  • Human plasma supplemented with five different antibodies at a concentration ranging from 0 to 400 nM were tested in an APTT assay as described in Example 3.
  • the five antibodies tested included 19F6 (A) , 34F8 (B) , 42A5 (C) , 1A6 (D) , and 14E11 (E) .
  • Antibodies 1A6 and 14E11 were used as positive controls in this experiment.
  • Figures 2A-2C illustrate the effects of antibodies 19F6 (A) , 34F8 (B) , and 42A5 (C) on the APTT assay in monkey plasma.
  • Figures 3A-3F illustrates SPR sensorgrams for FXI binding to immobilized h-19F6 (A) , h-34F8 (B) , and h-42A5 (C) , as well as SPR sensorgrams for FXIa binding to immobilized h-19F6 (D) , h-34F8 (E) , and h-42A5 (F) .
  • Data were fit with 1: 1 binding model, and curve fits at test concentrations of FXI (0.005 -1 ng/mL) are shown overlaid on the sensorgrams. Each curve indicates a different test concentration of FXI or FXIa.
  • Figures 4A-4C illustrate the concentration-response curves of antibodies h-19F6 (A) , h-34F8 (B) , and h-42A5 (C) inhibiting human FXIa from hydrolyzing S-2366.
  • Figures 5A-5B illustrate the inhibitory effects of antibodies h-19F6 (A) and h-42A5 (B) on FXIa-mediated activation of FIX to FIXa.
  • Human FIX 200 nM was incubated with FXIa (5 nM) in PBS with 5 mM CaCl 2 at room temperature with 1 ⁇ M h-19F6 or h-42A5. At the indicated intervals, samples were collected and the FIX as well as FIXa was determined by Western blots using goat anti-human FIX IgG (Affinity Biologicals) .
  • Figures 5C-5D illustrate the inhibitory effects of antibodies h-19F6 (C) and h-42A5 (D) on FXIIa-mediated activation of FXI to FXIa.
  • Human FXI 500 nM was incubated with FXIIa (50 nM) in the presence of 1 ⁇ M of h-19F6 or h-42A5.
  • FXI, as well as FXIa light chain, which represents FXIa production, at indicated time points was determined by Western blots.
  • a human IgG4 (1 ⁇ M) was used as the control.
  • Figures 6A-6C illustrate the effects of antibodies h-34F8, h-19F6, and h-42A5 on APTT in cynomolgus monkeys.
  • the monkeys were intravenously administered with indicated doses of h-34F8 (A) , h-19F6 (B) , and h-42A5 (C) .
  • Ex vivo clotting time APTT was determined at pre-dose (time 0) , and 0.5, 1, 3, 6, 12, and 24 hours post-dose.
  • Figures 7A-7C illustrate the effects of antibodies h-34F8, h-19F6, and h-42A5 on PT in cynomolgus monkeys.
  • Monkeys were intravenously administered with the indicated doses of h-34F8 (A) , h-19F6 (B) , and h-42A5 (C) .
  • Ex vivo clotting time PT was determined at pre-dose (time 0) , and 0.5, 1, 3, 6, 12, and 24 hours post-dose.
  • Figures 8A-8C illustrate the effects of antibodies h-34F8, h-19F6, and h-42A5 on AV shunt thrombosis in cynomolgus monkeys.
  • Figures 9A-9C illustrate the effects of antibodies h-34F8, h-19F6, and h-42A5 on bleeding time in cynomolgus monkeys.
  • Figures 10A-10B illustrate the antithrombotic effects of antibodies h-34F8, h-19F6, and h-42A5.
  • the time to 80%thrombotic occlusion (A) and to 100%thrombotic occlusion (B) were determined by monitoring the blood flow velocity.
  • Figures 11A-11D illustrate that the treatment with antibodies h-34F8, h-19F6, or h-42A5 did not prolong the bleeding time in monkeys.
  • the individual bleeding time in h-34F8, h-19F6, and h-42A5 treated group is shown in (A) , (B) and (C) , respectively.
  • the bleed time change upon vehicle, h-34F8, h-19F6, or h-42A5 treatment is shown in (D) .
  • Figures 12A-12B illustrate the effects of antibodies h-34F8, h-19F6, and h-42A5 on clotting times of monkey plasma.
  • the APTT changes and PT changes are shown in (A) and (B) , respectively. **P ⁇ 0.01 and ***P ⁇ 0.001 vs. vehicle.
  • Figure 13 illustrates the amino acid sequence of human FXI (SEQ ID NO: 203) .
  • Figures 14A-14B illustrate the effects of modified antibodies h-19F6 (A) , and h-42A5 (B) on APTT in cynomolgus monkeys.
  • the monkeys were intravenously administered with indicated doses of modified h-19F6 and h-42A5.
  • Ex vivo clotting time APTT was determined at pre-dose (time 0) , and 0.5, 2, 6, 12, 24, 48, 96, 168, 240, and 336 hours post-dose.
  • Figures 15A-15B illustrate the effects of modified antibodies h-19F6 (A) , and h-42A5 (B) on PT in cynomolgus monkeys.
  • Monkeys were intravenously administered with the indicated doses of modified h-19F6 and h-42A5.
  • Ex vivo clotting time PT was determined at pre-dose (time 0) , and 0.5, 2, 6, 12, 24, 48, 96, 168, 240, and 336 hours post-dose.
  • Figures 16A-16B illustrate the effects of h-19F6 and h-42A5 on APTT and PT in human plasma.
  • Figure 16A shows the effects of h-19F6 and h-42A5 on APTT in human plasma.
  • Figure 16B shows the effects of h-19F6 and h-42A5 on PT in human plasma.
  • Figure 17 shows the binding specificity of test antibodies to human FXI.
  • 10 ⁇ L of human standard plasma or FXI-deficient plasma were served as FXI-positive and FXI-negative controls.
  • Figure 18 shows the effects of h-19F6 and h-42A5 in AV shunt thrombosis models on bleeding times recorded at pre-dose and 1-hour post-dose.
  • Figures 19A-19D show the binding properties of h-19F6 and h-42A5 to human FXI.
  • Figure 19A shows sensorgrams for h-19F6 captured on a sensor chip subjected to flows of indicated concentrations of FXI.
  • Figure 19B shows sensorgrams for h-42A5 captured on a sensor chip subjected to flows of indicated concentrations of FXI.
  • Figure 19C shows antibodies captured when test antibodies (5 ⁇ g/mL) flew through a sensor chip immobilized with equal amounts of 4 mutant FXIs in which the A1, A2, A3, or A4 domain was replaced with the corresponding domain from prekallikrein. A reported anti-FXI antibody, O1A6, was also tested a positive control.
  • Figure 19D shows that FXI was immobilized on a sensor chip.
  • H-19F6 and h-42A5 (5 ⁇ g/ml) were successively injected into flow cells on the sensor surface at a flow rate of 30 ⁇ l/minute, and the response change was monitored. The experiment was performed twice, and a representative result is depicted.
  • Figures 20A-20B show the binding properties of h-19F6 and h-42A5 to human FXIa.
  • Figure 20A shows sensorgrams for h-19F6 captured on a sensor chip subjected to flows of indicated concentrations of FXIa.
  • Figure 20B shows sensorgrams for h-42A5 captured on a sensor chip subjected to flows of indicated concentrations of FXIa.
  • the intrinsic pathway also called the contact activation pathway, is initiated by contact with a surface interface and results in activation of FXII.
  • the intrinsic pathway also involves FXI, FIX and FVIII.
  • the extrinsic pathway also called the tissue factor (TF) pathway, is initiated by vascular injury and results in the formation of an activated complex of TF-FVIIa. These two pathways meet and activate the common pathway, leading to conversion of prothrombin to thrombin and eventually the formation of cross-linked fibrin clot.
  • An ideal anticoagulant should be efficacious in preventing thrombosis without compromising haemostasis.
  • FXI can be activated by factor XIIa via the intrinsic pathway to FXIa, which in turn activates factor IX.
  • Epidemiological studies have suggested that FXI deficiency in humans is associated with decreased risk of venous thromboembolism and stroke, whereas increased FXI levels are associated with increased risk. 9-11
  • FXI-deficient humans show a very low bleeding tendency. 12, 13
  • mice deficient in FXI are protected against many types of thrombosis without increased bleeding.
  • small-molecule inhibitors, antibodies and antisense oligonucleotides that inhibit FXI have demonstrated antithrombotic properties with no bleeding risk in many animal models of thrombosis.
  • the antibodies disclosed herein binds to FXI and/or FXIa and target the intrinsic pathway of blood coagulation.
  • the structure of FXI and FXI’s involvement in blood coagulation have been reported in various publications. 33
  • FXI-deficient mice have been studied by many research teams and have displayed remarkable antithrombotic phenotypes in several models, including FeCl 3 -induced arterial and deep vein thrombosis models, a pulmonary embolism model, and a cerebral artery occlusion model. 14, 17, 22, 23
  • VTE venous thromboembolism
  • FXI thromboembolism
  • FXI-deficient mice do not show excessive bleeding, as their tail-bleeding times are comparable to those of wild-type animals. 23, 24 In addition, severely FXI-deficient patients do not exhibit spontaneous bleeding, although they may display a variable bleeding tendency during surgical operations. 12, 13 Combination of two or more anti-thrombotics are widely used clinically. A previous study showed that aspirin caused a similar bleeding tendency in wild-type and FXI-deficient mice, suggesting that targeting FXI might still be safe even in the presence of other anti-thrombotic therapies. 14
  • FXI/FXIa is a safe drug target for treating thrombosis-related diseases without compromising haemostasis.
  • many approaches have been applied to target FXI/FXIa for developing therapeutics for treating thrombosis, such as antibodies, oligonucleotides, and small-molecule inhibitors.
  • antibody-type blockers of FXI/FXIa were generated.
  • the advantages of antibodies include fast-acting properties and a low frequency of dosing, and a major weakness of antibodies is their potential immunogenicity. 25 At least two test antibodies were humanized before conducting in vivo studies.
  • FXI/FXIa inhibitors have prolonged APTT and exhibited antithrombotic effects in different models.
  • Anti-FXI antibody 14E11 increased APTT by approximately 1.3-fold and reduced thrombosis in exteriorized femoral arteriovenous shunts in baboons. 17
  • An antisense oligonucleotide inhibiting FXI expression reduced plasma FXI levels by approximately 50%and decreased thrombus formation in baboons. 26, 27
  • an orally bioavailable small-molecule FXIa inhibitor, ONO-5450598 significantly inhibited thrombosis formation in monkey models of thrombosis.
  • Bleeding risk is the most concerning issue in developing antithrombotic agents. As previously mentioned, FXI-deficient patients may show a bleeding tendency under surgical settings. It is unclear to what extent plasma FXI activity inhibition is still safe in terms of bleeding risk. As demonstrated in the working examples, the bleeding risk of intensive inhibition of FXI/FXIa by h-19F6 and h-42A5 was tested in the same monkeys used in thrombosis experiments. In AV shunt thrombosis animals, no bleeding tendency was observed as the treating dose of h-19F6 or h-42A5 escalated, suggesting that bleeding risk may be independent of the extent of FXI inhibition.
  • antibodies that bind to FXI, FXIa, and/or a fragment of FXI or FXIa and inhibit the formation of blood clot are capable of binding to FXI, FXIa, and/or a fragment of FXI or FXIa (e.g., a fragment comprising the A3 domain) and exhibiting an inhibitory effect at a concentration that is much lower than the maximum safety dose.
  • a dose of the antibody between 0.1 mg/kg i.v. and 3 mg/kg i.v. exhibits an inhibitory effect on conversion of FXI to FXIa in cynomolgus monkeys.
  • the antibodies disclosed herein can be used as anticoagulation agents with superior safety due to their minimal risk of causing bleeding versus conventional anticoagulation agents such as heparin.
  • anti-human FXI antibodies were generated by immunizing rats with human FXI to identify antibodies with anticoagulation properties. A dozen such antibodies were identified, and some of which were humanized for further development.
  • the humanized rat anti-human FXI antibodies such as h-19F6 and h-42A5 antibodies, were characterized in vitro and in vivo. In the in vitro studies, the humanized antibodies inhibited activated FXI (FXIa) -mediated hydrolysis of factor IX but not factor XIIa-induced FXI activation.
  • the binding properties of the antibodies to FXI were determined, and the dissociation constants (KD) for h-19F6 and h-42A5 were 22 pM and 35 pM, respectively. These two antibodies bind different sites in the A3 domain of FXI.
  • two distinct primate thrombosis models were used to evaluate the anti-thrombotic effects and bleeding risks of the humanized antibodies.
  • arteriovenous (AV) shunt thrombosis models both antibodies dose-dependently decreased thrombus formation without causing bleeding.
  • AV arteriovenous
  • FeCl3-induced thrombosis models both antibodies extended the time to thrombosis-mediated vessel occlusion, and neither antibody increased bleeding. The two antibodies showed anti-thrombotic efficacy without compromising haemostasis in primates, further confirming that targeting FXI can be used for treating thrombosis.
  • composition or method includes at least the recited elements.
  • consisting essentially of means that the composition or method includes the recited elements, and may further include one or more additional elements that do not materially affect the novel and basic characteristics of the composition or method.
  • a composition consisting essentially of recited elements may include those recited elements plus one or more trace contaminants from the isolation and purification method, pharmaceutically acceptable carriers such as phosphate buffered saline, preservatives, and the like.
  • Consisting of means the composition or method includes only the recited elements. Embodiments defined by each of the transitional terms are within the scope of this invention.
  • an antibody refers to an immunoglobulin molecule or an immunologically active portion thereof that specifically binds to, or is immunologically reactive with a particular antigen, for example, FXI, FXIa, or a particular domain or fragment of FXI or FXIa, e.g., the A3 domain.
  • an antibody for use in the present methods, compositions, and kits is a full-length immunoglobulin molecule, which comprises two heavy chains and two light chains, with each heavy and light chain containing three complementary determining regions (CDRs) .
  • antibody in addition to natural antibodies, also includes genetically engineered or otherwise modified forms of immunoglobulins, such as synthetic antibodies, intrabodies, chimeric antibodies, fully human antibodies, humanized antibodies, peptibodies and heteroconjugate antibodies (e.g., bispecific antibodies, multispecific antibodies, dual-specific antibodies, anti-idiotypic antibodies, diabodies, triabodies, and tetrabodies) .
  • the antibodies disclosed herein can be monoclonal antibodies or polyclonal antibodies.
  • an antibody is an immunologically active portion of an immunoglobulin molecule
  • the antibody may be, for example, a Fab, Fab’, Fv, Fab’F (ab’) 2 , disulfide-linked Fv, single chain Fv antibody (scFv) , single domain antibody (dAb) , or diabody.
  • the antibodies disclosed herein, including those that are immunologically active portion of an immunoglobulin molecule, retain the ability to bind a specific antigen, for example FXI or FXIa, or to bind a specific fragment of FXI or FXIa such as the A3 domain.
  • the anti-FXI and/or anti-FXIa antibodies disclosed herein have undergone post-translational modifications such as phosphorylation, methylation, acetylation, ubiquitination, nitrosylation, glycosylation, or lipidation associated with expression in a mammalian cell line, including a human or a non-human host cell.
  • post-translational modifications such as phosphorylation, methylation, acetylation, ubiquitination, nitrosylation, glycosylation, or lipidation associated with expression in a mammalian cell line, including a human or a non-human host cell.
  • polynucleotides or nucleic acids encoding the anti-FXI and/or anti-FXIa antibodies disclosed herein include DNA, mRNA, cDNA, plasmid DNA.
  • the nucleic acid encoding the antibody or a functional fragment thereof disclosed herein can be cloned into a vector, such as a pTT5 mammalian expression vector, which may further include a promoter and/or other transcriptional or translational control elements such that the nucleic acid can be expressed to produce the antibody or the functional fragment thereof.
  • nucleic acid (DNA) and/or amino acid (PRT) sequences including the sequences of the VH and VL and CDRs, of some examples of the antibodies disclosed herein are listed in Table 1 below.
  • humanized anti-FXI and/or anti-FXIa antibodies are provided in certain embodiments herein.
  • Various techniques are known in the art for humanizing antibodies from non-human species such that the antibodies are modified to increase their similarity to antibodies naturally occurring in humans.
  • Six CDRs are present in each antigen binding domain of a natural antibody. These CDRs are short, non-contiguous sequences of amino acids that are specifically positioned to form the antigen binding domain as the antibody assumes its three dimensional configuration.
  • the remainder of the amino acids in the antigen binding domains referred to as “framework” regions, show less inter-molecular variability and form a scaffold to allow correct positioning of the CDRs.
  • the antibodies or fragments disclosed herein have conserved sequences for CDR3 regions.
  • humanization of the antibodies disclosed herein can be accomplished by CDR grafting of monoclonal antibodies produced by immunizing mice or rats.
  • the CDRs of a mouse monoclonal antibody can be grafted into a human framework, which is subsequently joined to a human constant region to obtain a humanized antibody.
  • the human germline antibody sequence database, the protein data bank (PDB) , the INN (International Nonproprietary Names) database, and other suitable databases can be searched and the most similar frameworks to the antibodies can be identified by the search.
  • some back mutations to the donor residues are made in the human acceptor frameworks.
  • the variable regions are linked to a human IgG constant region.
  • human IgG1, IgG2, IgG3 and IgG4 Fc domains can be used. It is within the purview of one of ordinary skill in the art to humanize a monoclonal antibody produced by a non-human species based on the existing technology.
  • the antibodies provided herein include variants of the sequences disclosed herein that contain one or more mutations in their amino acid sequences while retaining binding affinity for FXI, FXIa, and/or a fragment thereof (e.g., a fragment comprising the A3 domain) .
  • the antibodies include a variable region having an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%identical to a sequence selected from the group consisting of SEQ ID NOs: 9, 10, 25, 26, 41, 42, 57, 58, 73, 74, 89, 90, 105, 106, 121, 122, 137, 138, 153, 154, 169, 170, 185, 186, and 197-209, or a fragment thereof that retains binding affinity for FXI, FXIa, and/or a fragment thereof.
  • nucleic acids encoding antibodies that bind to FXI, FXIa, and/or a fragment thereof (e.g., a fragment comprising the A3 domain) .
  • the nucleic acids encoding the antibodies include a variable region having a nucleic acid sequence that is at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%identical to a sequence selected from the group consisting of SEQ ID NOs: 1, 2, 17, 18, 33, 34, 49, 50, 65, 66, 81, 82, 97, 98, 113, 114, 129, 130, 145, 146, 161, 162, 177, and 178, or a fragment thereof that encodes a polypeptide with binding affinity for FXI, FXIa, and/or a fragment thereof.
  • the antibodies are further subjected to a strategic Chemistry, Manufacturing, and Control (CMC) development such that the novel antibodies such as monoclonal antibodies or humanized monoclonal antibodies disclosed herein are advanced from discovery to human clinical trials, and then to the pharmaceutical market.
  • CMC strategic Chemistry, Manufacturing, and Control
  • the modifications further improve the properties of the antibodies without compromising the immunological functions of the antibodies.
  • a CMC modified antibody is more stable under various temperatures (e.g., 4°C, 25°C, and 37°C) for an extended period of time (e.g., 3 days, 7 days, 14 days and 28 days) and under repeated freeze/thaw cycles (e.g., -40°C/25°C for up to 5 cycles) comparing to the unmodified antibody.
  • the CMC modified antibodies have an acceptable solubility.
  • certain amino acids can be potential oxidation and glycosylation sites. These amino acid residues at the potential oxidation, deamidation, or glycosylation sites may be mutated and additional residues in the proximity can also be mutated and/or optimized to maintain the 3D structure and function of a particular antibody.
  • one or more amino acid residues in a CDR region having the potential of oxidation, deamidation, or glycosylation are mutated to improve the stability of the antibody or a fragment thereof without compromising the immunological functions.
  • one or more Met residues in a CDR region having the potential of oxidation are mutated.
  • one or more Asn residues in a CDR region having the potential of deamidation are mutated.
  • the antibodies disclosed herein can be formulated into pharmaceutical compositions.
  • the pharmaceutical compositions may further comprise one or more pharmaceutically acceptable carriers, excipients, preservatives, or a combination thereof.
  • the pharmaceutical compositions can have various formulations, e.g., injectable formulations, lyophilized formulations, liquid formulations, etc. Depending on the formulation and administration route, one would select suitable additives, such as adjuvants, carriers, excipients, preservatives. 34
  • the pharmaceutical composition can be included in a kit with an instruction for using the composition.
  • a method of treating and/or preventing thrombosis in a subject suffering from thrombosis and/or at an elevated risk of developing thrombosis is also provided.
  • a method of inhibiting the formation of blood clots in a subject entail administering a therapeutically effective amount of an anti-FXI and/or FXIa antibody provided herein to intervene in the intrinsic pathway.
  • these methods comprise administering a pharmaceutical composition comprising an anti-FXI and/or anti-FXIa antibody as provided herein to the subject.
  • thrombosis causes or is associated with a number of complications or conditions, such as embolic stroke, venous thrombosis such as venous thromboembolism (VTE) , deep vein thrombosis (DVT) , and pulmonary embolism (PE) , arterial thrombosis such as acute coronary syndrome (ACS) , coronary artery disease (CAD) , and peripheral artery disease (PAD) .
  • embolic stroke venous thrombosis such as venous thromboembolism (VTE) , deep vein thrombosis (DVT) , and pulmonary embolism (PE)
  • VTE venous thromboembolism
  • VVT deep vein thrombosis
  • PE pulmonary embolism
  • PE pulmonary embolism
  • ACS acute coronary syndrome
  • CAD coronary artery disease
  • PAD peripheral artery disease
  • thrombosis Other conditions associated with thrombosis include, for example, high risk of VTE in surgical patients, immobilized patients, patients with cancer, patients with heart failure, pregnant patients, or patients having other medical conditions that may cause thrombosis.
  • the methods disclosed herein relate to a preventive anticoagulant therapy, that is, thromboprophylaxis. These methods entail administering to a subject suffering from a thrombosis-related complication disclosed above a therapeutically effective amount of an anti-FXI and/or FXIa antibody as disclosed herein or a therapeutically effective amount of a pharmaceutical composition comprising the anti-FXI and/or FXIa antibody.
  • the antibody or pharmaceutical composition can be administered either alone or in combination with any other therapy for treating or preventing the thrombosis-related complications or conditions.
  • the antibodies disclosed herein can be used as a secondary therapy in combination with other therapeutic agents for treating sepsis, such as antibiotics.
  • the term “subject” refers to mammalian subject, preferably a human.
  • a "subject in need thereof” refers to a subject who has been diagnosed with thrombosis or complications or conditions associated with thrombosis, or is at an elevated risk of developing thrombosis or complications or conditions associated with thrombosis.
  • the phrases “subject” and “patient” are used interchangeably herein.
  • treat, ” “treating, ” and “treatment” as used herein with regard to a condition refers to alleviating the condition partially or entirely, preventing the condition, decreasing the likelihood of occurrence or recurrence of the condition, slowing the progression or development of the condition, or eliminating, reducing, or slowing the development of one or more symptoms associated with the condition.
  • treating may refer to preventing or slowing the existing blood clot from growing larger, and/or preventing or slowing the formation of blood clot.
  • the term “treat, ” “treating, ” or “treatment” means that the subject has a reduced number or size of blood clots comparing to a subject without being administered with the antibodies or functional fragments thereof. In some embodiments, the term “treat, ” “treating, ” or “treatment” means that one or more symptoms of thrombosis and/or thrombosis-related conditions or complications are alleviated in a subject receiving an antibody or pharmaceutical composition as disclosed herein comparing to a subject who does not receive such treatment.
  • a “therapeutically effective amount” of an antibody or pharmaceutical composition as used herein is an amount of the antibody or pharmaceutical composition that produces a desired therapeutic effect in a subject, such as treating and/or preventing thrombosis.
  • the therapeutically effective amount is an amount of the antibody or pharmaceutical composition that yields maximum therapeutic effect.
  • the therapeutically effective amount yields a therapeutic effect that is less than the maximum therapeutic effect.
  • a therapeutically effective amount may be an amount that produces a therapeutic effect while avoiding one or more side effects associated with a dosage that yields maximum therapeutic effect.
  • a therapeutically effective amount for a particular composition will vary based on a variety of factors, including but not limited to the characteristics of the therapeutic composition (e.g., activity, pharmacokinetics, pharmacodynamics, and bioavailability) , the physiological condition of the subject (e.g., age, body weight, sex, disease type and stage, medical history, general physical condition, responsiveness to a given dosage, and other present medications) , the nature of any pharmaceutically acceptable carriers, excipients, and preservatives in the composition, and the route of administration.
  • the characteristics of the therapeutic composition e.g., activity, pharmacokinetics, pharmacodynamics, and bioavailability
  • the physiological condition of the subject e.g., age, body weight, sex, disease type and stage, medical history, general physical condition, responsiveness to a given dosage, and other present medications
  • the nature of any pharmaceutically acceptable carriers, excipients, and preservatives in the composition e.g., the nature of any pharmaceutically acceptable
  • a therapeutically effective amount of an antibody disclosed herein is in the range from about 0.01 mg/kg to about 30 mg/kg, from about 0.1 mg/kg to about 10 mg/kg, from about 1 mg/kg to about 5 mg/kg.
  • the antibody or pharmaceutical composition can be administered continuously or intermittently, for an immediate release, controlled release or sustained release. Additionally, the antibody or pharmaceutical composition can be administered three times a day, twice a day, or once a day for a period of 3 days, 5 days, 7 days, 10 days, 2 weeks, 3 weeks, or 4 weeks. The antibody or pharmaceutical composition may be administered over a pre-determined time period. Alternatively, the antibody or pharmaceutical composition may be administered until a particular therapeutic benchmark is reached. In certain embodiments, the methods provided herein include a step of evaluating one or more therapeutic benchmarks to determine whether to continue administration of the antibody or pharmaceutical composition.
  • these methods entail the steps of cloning a nucleic acid encoding an anti-FXI and/or anti-FXIa antibody into a vector, transforming a host cell with the vector, and culturing the host cell to express the antibody.
  • the expressed antibody can be purified from the host cell using any known technique.
  • Various expression vectors such as pTT5 vector, and pcDNA3 vector, as well as various host cell lines such as CHO cells (e.g. CHO-K1 and ExpiCHO) , and HEK193T cells, can be used.
  • antibodies produced by the method disclosed above may have been subjected to one or more post-translational modifications.
  • Antibody preparation Animal immunization and hybridoma screening were performed at Genscript Inc. (Nanjing, China) , and the procedures that were applied to animals in this protocol were approved by the Genscript Institutional Animal Care and Use Committee. The experiment was performed in accordance with the approved guidelines. Wistar rats were immunized with human FXI, and splenocytes from animals with a good immune response were collected for the preparation of hybridomas, which were subjected to subcloning by limiting dilution. Finally, several monoclonal hybridoma clones expressing the desired anti-FXI antibodies, including 19F6, h-34F8 and 42A5, were obtained by using ELISA and functional screening.
  • 19F6, h-34F8 and 42A5 were subjected to humanization, resulting in three humanized antibodies, h-19F6 , h-34F8 and h-42A5, in an IgG4 form. These three humanized antibodies were produced in a transient mammalian expression system and purified by Protein G chromatography.
  • APTT Activated partial thromboplastin time
  • PT prothrombin time
  • monkey plasma diluted with an equal volume of phosphate-buffered saline (PBS) was used instead of the above mentioned human plasma-antibody mixture.
  • FXI activation by FXIIa Human FXI (500 nM) was pre-incubated at room temperature with 1 ⁇ M control IgG4 or h-19F6 or h-34F8 or h-42A5 in PBS for 5 minutes. At time zero, FXIIa, HK, and kaolin were added so that the final concentrations were FXI (250 nM) , FXIIa (50 nM) , HK (100 nM) , and kaolin (0.5 mg/mL) . At 0, 30, 60, 120 min intervals, 50- ⁇ L samples were collected into dodecyl sulfate sample buffer.
  • FXIa-mediated FIX activation Human FIX (200 nM) was incubated with FXIa (5 nM) in PBS containing 5 mM CaCl 2 at room temperature in the presence of 1 ⁇ M control lgG4, h-19F6, h-34F8, or h-42A5. At intervals of 0, 15, 30, 45, and 60 min, 50- ⁇ L samples were collected into dodecyl sulfate sample buffer. Samples were size-fractionated on 10%non-reducing gels and transferred to polyvinylidene fluoride membranes. Western blotting was performed to determine the FIX as well as FIXa levels using goat anti-human FIX IgG (Affinity Biologicals) . The image results were acquired using a ChemiDocMP Imaging System with Image Lab Software (Bio-Rad) .
  • SPR Surface plasmon resonance
  • test antibodies on FXI were first generated by replacing each apple domain (A1, A2, A3, and A4) with the corresponding domains from human prekallikrein. Equal amounts of each mutant were immobilized on a CM5 sensor chip, and test antibodies (33.3nM) were allowed to flow through the chip for 180 s for association and then for 1200 s for dissociation. The amounts of each antibody captured were recorded in response units (RU) using the Biacore Evaluation Software.
  • Epitope binding results of the test antibodies were also analyzed using the Biacore T200 system. Briefly, wild-type FXI with 6 ⁇ His tag was pre-immobilized on a CM5 sensor chip (GE Healthcare) , and h-19F6, h-34F8, or h-42A5 (5 ⁇ g/ml) was successively injected into flow cells on the sensor surface at a flow rate of 30 ⁇ l/minute to monitor the change in response.
  • Plasma samples were diluted with an equal volume of phosphate buffered saline (pH 7.4) and then subjected to APTT and PT analysis on an automatic analyser (CA660, Sysmex Inc. ) .
  • AV shunt thrombosis and bleeding time test A 30-min post-test antibody treatment was administered via intravenous bolus in cynomolgus monkeys. A tail vein bleeding time test was then performed, followed by thrombosis induction. Thrombosis was induced by connecting a shunt device between the femoral arterial and femoral venous cannulas containing a pre-weighed 10-cm-long thread. Blood was allowed to flow through the shunt for 10 min. The thrombus formed on the thread was weighed. Immediately after the removal of the shunt, blood samples were collected, and the next higher level of test antibody was administered. This bleeding/thrombosis process was carried out four times to dose the vehicle and three escalating levels (0.1, 0.3, 1 mg/kg) of test antibody in the same animal.
  • a 2-mL syringe was inserted into the tail vein of the animals.
  • the elapsed time was recorded manually as the bleeding time.
  • Ferric chloride (FeCl 3 ) -induced thrombosis and bleeding time test This animal experiment was performed at PharmaLegacy Laboratories Inc. (Shanghai, China) , and the procedures that were applied to animals in this protocol were approved by the PharmaLegacy Institutional Animal Care and Use Committee. The experiment was performed in accordance with the approved guidelines. Cynomolgus monkeys were pre-anaesthetized with 1.5 mg/kg of Zoletil, intubated, and ventilated with a respirator. Anaesthesia was maintained with isoflurane. Blood pressure, heart rate, and body temperature were monitored throughout the entire procedure.
  • the vehicle or 0.3 mg/kg of h-19F6, h-34F8, or h-42A5 was administered through a limb vein 2 hours before FeCl 3 application.
  • the left femoral artery was exposed and isolated via blunt dissection.
  • a Doppler flow probe was set up on the artery, and blood flow was continuously recorded.
  • blood flow was measured for at least 5 minutes.
  • two pieces of filter paper pre-soaked with 40%FeCl 3 were applied to the adventitial surface of the vessel upstream of the probe for 10 minutes. After the filter paper was removed, the site of application was washed with saline. Blood flow was continuously measured until it decreased to 0.
  • test antibodies (h-19F6, h-42A5, and 14E11) were first biotinylated using EZ-Link TM Sulfo-NHS-LC-Biotinylation Kit (Cat No. 21435, Thermo Fisher Inc. ) . These antibodies (25 ⁇ g each) were incubated with 200 ⁇ L of human standard plasma (Siemens Inc. ) or FXI-deficient plasma (Hyphen Biomed Inc. ) for 1h. Then 50 ⁇ L of Streptavidin-coated beads (Dynabeads TM M-280 Streptavidin, Thermo Fisher Inc.
  • mice and Wistar rats were immunized with human FXI, and splenocytes from the animals with good immune response were collected for the preparation of hybridomas, which were subjected to subcloning by limiting dilution. Twelve monoclonal hybridoma clones expressing desired anti-FXI antibodies 3G12, 5B2, 7C9, 7F1, 13F4, 19F6, 21F12, 34F8, 38E4, 42A5, 42F4, and 45H1 were obtained by using capture ELISA and functional screening.
  • V L and V H variable region of the light (V L ) and heavy chain (V H ) of these antibodies.
  • cDNAs encoding V L and V H were cloned from the corresponding hybridoma cells by standard RT-PCR procedures.
  • the V L and V H sequences of exemplary antibodies, including the sequences of CDRs, are shown in Table 1.
  • Example 3 Determination of anti-coagulation activity in human plasma using activated partial thromboplastin time (APTT) assay and prothrombin time (PT) assay
  • APTT assay measures the activity of the intrinsic and common pathways of coagulation; whereas PT assay measures the activity of the extrinsic and common pathways of coagulation.
  • the antibodies tested in these experiments were 19F6, 34F8, 42A5, 1A6 and 14E11. Antibodies 1A6 and 14E11 were used as positive controls in this experiment.
  • the sequences of the variable regions of the control antibodies were obtained from U.S. Patent No. 8,388,959 and U.S. Patent Application Publication No. 2013/0171144 and reformatted to IgG4. These antibodies were then expressed using ExpiCHO cell system.
  • the APTT assay and PT assay were performed as described above.
  • Example 4 Determination of the anti-coagulation activity in the plasma of non-human species using activated partial thromboplastin time (APTT) assay
  • the affinity of anti-FXI/FXIa antibodies to FXI/FXIa were determined using surface plasmon resonance (SPR) technology performed on the BIAcore T200 instrument.
  • the humanized antibodies were constructed by linking the variable regions of the antibodies disclosed herein to human IgG4 Fc domain and the recombinants were expressed in CHO cells. These antibodies were captured onto a Biacore CM5 sensor chip that was pre-immobilized with an anti-human IgG antibody.
  • the dissociation constants (K D ) of h-19F6, h-34F8, and h-42A5 to FXI and FXIa were calculated and detailed in Table 4.
  • the affinities of each antibody to FXI and FXIa are considered to be the same since the difference between them is less than 10 times.
  • the binding sites of 19F6 and 42A5 on FXI were determined using the SPR technology. Briefly, human IgG capture antibody was pre-immobilized on a Biacore CM5 sensor chip, and recombinant h-19F6 or h-42A5 was captured by flowing through the chip. An equal amount (15 relative units) of h-19F6 and h-42A5 was captured through adjustment of the antibody flowing time. Then wild type FXI or chimeric FXI in which individual apple domain was replaced with the corresponding domain from the human prekallikrein (FXI/PK chimeras) was allowed to flow through the chip for 180 seconds for association with h-19F6 or h-42A5, followed by a time period of 1800 seconds for dissociation.
  • FXI/PK chimeras human prekallikrein
  • the binding data was analyzed in a high performance kinetic mode as only one concentration of FXI, wild-type or chimeric, was tested in the SPR assay. Results showed that both h-19F6 and h-42A5 bound FXI as well as FXI/PK chimeras except when the A3 domain of FXI was replaced with the corresponding PK domain, indicating that part or the complete epitope of h-19F6 and h-42A5 on FXI is located in the A3 domain.
  • Human FXIa activity was determined by measuring the cleavage of a specific, chromogenic substrate, S-2366 (Diapharma Inc. ) .
  • S-2366 Diapharma Inc.
  • antibodies h-19F6, h-34F8 and h-42A5 were pre-incubated for 5 minutes at room temperature with a final concentration of 5 nM of FXIa in PBS (phosphate buffer saline) .
  • PBS phosphate buffer saline
  • S-2366 phosphate buffer saline
  • S-2366 phosphate buffer saline
  • Data were analyzed using the GraphPad Prism software and are shown in Figure 4.
  • the calculated apparent Ki for h-19F6, h-34F8, and h-42A5 are 0.67, 2.08, and 1.43 nM, respectively. Therefore, all three antibodies tested exhibited satisfying inhibitory effects on FXIa at a relatively low concentration.
  • the FXIa-mediated FIX activation was performed as described above.
  • Anti-FXI antibodies may modulate the intrinsic pathway by inhibiting FXI activation and/or by inhibiting FXIa activity.
  • both h-19F6 and h-42A5 reduced FIX activation in a concentration-dependent manner.
  • the inhibitory effect of these two antibodies on FXIa activity was further confirmed by using a chromogenic substrate of FXIa, S-2366. Both antibodies concentration-dependently inhibited the hydrolysis of S-2366 ( Figure 4) .
  • Example 10 Evaluation of the effects of anti-FXI antibodies on clotting time in cynomolgus monkeys
  • APTT test 50 ⁇ L of diluted plasma sample and 25 ⁇ L of APTT reagent (SMN 10445709, Symens Inc. ) were mixed and incubated at 37 °C for 4 min. Then 25 ⁇ L of CaCl 2 Solution (25 mM, SMN 10446232, Symens Inc. ) was added and time to clot formation was determined.
  • APTT reagent 25 mM, SMN 10446232, Symens Inc.
  • PT test 50 ⁇ L of diluted plasma sample was mixed with equal volume of PT reagent (SMN 10446442, Symens Inc. ) and incubated at 37°C and time to clot formation was determined. All three antibodies tested demonstrated dose-dependently increased APTT as shown in Figure 6 and none of them affected PT as shown in Figure 7.
  • Example 11 Evaluation of the effects of anti-FXI antibodies in arteriovenous (AV) shunt thrombosis and tail vein bleeding models in cynomolgus monkeys
  • Both thrombosis and bleeding time were assessed in the same animal for multiple doses of each antibody tested.
  • the antibodies included in this experiment were h-34F8, h-19F6, and h42A5. Briefly, bleeding time and thrombosis were sequentially evaluated at pre-dose and 30 minutes following each administration of the antibody. The bleeding/thrombosis assessments were conducted four times: pre-dose and post-dose at three escalating dose levels (0.1, 0.3 and 1 mg/kg) .
  • a shunt device containing a pre-weighed 10-cm long silk thread was applied to connecting the femoral arterial and femoral venous cannulae, and blood was allowed to flow through the shunt for 10 minutes. Then the thread was removed from the shunt and weighed again. Clot weight on the thread was calculated as the difference of the thread weight before and after blood flow.
  • a 2-mL syringe was inserted into the tail vein of the animals.
  • the elapsed time was recorded manually as the bleeding time.
  • Example 12 Evaluation of the effects of anti-FXI antibodies on ferric chloride–induced artery thrombosis and template bleeding time in cynomolgus monkeys
  • Cynomolgus monkeys were pre-anesthetized with 1.5 mg/kg of Zoletil, intubated, and ventilated with a respirator. Anesthesia was maintained with isoflurane. The blood pressure, heart rate, and body temperature were monitored throughout the entire procedure. The antibodies tested, including h-34F8, h-19F6, and h-42A5, or the vehicle control were administered through limb vein by injection 2 hours before FeCl 3 application. The left femoral artery was exposed and isolated via blunt dissection. A Doppler flow probe was set up on the artery and the blood flow was continuously recorded. Before applying FeCl 3 , the blood flow was measured for at least 5 minutes.
  • Example 13 Evaluation of the effects of modified anti-FXI antibodies on clotting time in cynomolgus monkeys for an extended period of time
  • modified antibodies disclosed herein did not show any adverse effects of prolonged bleeding while effectively inhibiting the intrinsic pathway of coagulation for an extended period of time, up to 14 days.
  • Example 14 Effects on clotting times of standard human plasma
  • Example 15 Binding properties of h-19F6 and h-42A5 to FXI
  • the affinities of h-19F6 and h-42A5 to FXI were determined using surface plasmon resonance (SPR) technology.
  • the test antibodies were captured on a sensor chip, and then indicated concentrations of FXI were allowed to flow through the chip.
  • Sensorgrams for h-19F6 ( Figure 19A) and h-42A5 ( Figure 19B) were obtained.
  • the dissociation constants for h-19F6 and h-42A5 were 22 and 36 pM, respectively ( Figures 19A and 19B) .
  • FXI is a homodimer consisting of 4 tandem apple domains (A1-4) and a catalytic domain.
  • A1-4 tandem apple domains
  • Four mutants of FXI were generated by replacing each apple domain with corresponding domains from human prekallikrein and tested the binding properties of h-19F6 or h-42A5 to the 4 mutants of FXI using SPR.
  • Equal amounts of the 4 mutant FXIs in which the A1, A2, A3, or A4 domain was replaced with the corresponding domain from prekallikrein were immobilized on a sensor chip, and test antibodies (5 ⁇ g/mL) were allowed to flow through the chip for association. The amounts of each antibody captured were recorded.
  • Example 16 Binding properties of h-19F6 and h-42A5 to FXIa
  • the antibodies bound to FXIa with the good affinities with which they bound to FXI ( Figures 20A and 20B) .
  • the affinities of h-19F6 and h-42A5 to FXI were determined using surface plasmon resonance (SPR) technology.
  • the dissociation constants for h-19F6 and h-42A5 were 26 and 81 pM, respectively ( Figures 20A and 20B) .
  • the test antibodies were captured on a sensor chip, and then indicated concentrations of FXIa were allowed to flow through the chip. Sensorgrams for h-19F6 ( Figure 20A) and h-42A5 ( Figure 20B) were obtained.

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WO2018145533A1 (zh) * 2017-02-10 2018-08-16 上海仁会生物制药股份有限公司 抗凝血因子xi抗体

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BR112021002472A2 (pt) 2021-07-27
MX2021001613A (es) 2021-04-28
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CN114478782B (zh) 2024-04-02
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AU2018436195A1 (en) 2021-02-18
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