Method for detecting biological activity of anticoagulants

Abstract

The present disclosure relates to a method for detecting biological activity of an anticoagulant drug. Specifically, the present disclosure relates to a method for detecting biological activity of an anti-FXI / FXIa antibody, which uses an enzyme label meter to replace a coagulation meter in a traditional method for analyzing biological activity of the anti-FXI / FXIa antibody, and simplifies experimental operation, shortens experimental period, and improves detection efficiency.

Description

TECHNICAL FIELD

[0001] The present disclosure relates to the field of biological activity detection of antibody drugs, more particularly to an in vitro biological activity detection method of antibodies against Factor XI (FXI) or activated form XIa (FXIa) and antigen-binding fragments thereof. BACKGROUND

[0002] Thrombotic diseases not only have high morbidity, but also have high mortality and disability. Anticoagulant drugs are effective in preventing and treating thrombotic diseases. The coagulation cascade is a process in which a series of coagulation factors are activated in succession, and ultimately fibrin is formed. The coagulation cascade is initiated by the intrinsic pathway (also known as the contact activation pathway) and the extrinsic pathway (also known as the tissue factor pathway), and then proceeds through the common pathway to form fibrin. FXI is essential for maintaining the intrinsic pathway and plays a key role in the amplification of the coagulation cascade. Therefore, drugs targeting FXI can block the intrinsic pathway and inhibit the amplification of the coagulation cascade, thereby playing an anti-thrombosis role.

[0003] High levels of coagulation factor XI in vivo are associated with thrombotic events, and anti-FXI / FXIa antibodies are an effective drug for preventing or treating thrombosis, which prevents the intrinsic coagulation pathway by binding to coagulation factor XI or its activated form XIa in the blood, thereby achieving the purpose of anticoagulation. As one of the methods for evaluating or controlling the quality of antibody drugs, it is of great significance to determine the in vitro biological activity of anti-FXI / FXIa monoclonal antibodies.

[0004] Anti-FXI / FXIa antibody biological activity assays usually use activated partial thromboplastin time (aPTT) method. The aPTT method usually uses a coagulation instrument to determine the coagulation time of a series of concentrations of antibodies, plots the curve of antibody concentration versus coagulation time, and then calculates the biological activity of the test product. Determining the biological activity of antibodies with a coagulation instrument requires batch determination of a series of concentrations of samples and control products, which often takes a long time to determine one sample. For an 8-channel coagulation instrument, it takes about 2 hours, and most biological drug analysis laboratories do not have a coagulation instrument, while an enzyme-labeled instrument is a commonly used instrument in laboratories. In the present disclosure, we use an enzyme-labeled instrument instead of a coagulation instrument, and according to the different degrees of coagulation of a series of concentrations of antibodies at the same action time, we determine the transmittance OD value. When the plasma does not coagulate, the transmittance OD value is the smallest; when the plasma completely coagulates, the transmittance OD value reaches the maximum; and when the plasma is in the coagulation process, the transmittance OD value is linearly correlated with the coagulation time within a certain range (R 2 >0.99, see the accompanying drawings Figure 1, 2). Thus, the OD value of the coagulation process can reflect the degree of plasma coagulation. By determining the coagulation OD value of the antibody series concentration at a certain reaction time, the dose-effect response curve of the antibody series concentration and the OD value is drawn, which reflects the anticoagulant effect of the anti-FXI / FXIa antibody. By comparison with the standard (or control), the biological activity of the anti-FXI / FXIa antibody test sample can be calculated. The determination of a sample (containing a control) by the microplate reader method only takes 20 minutes, which is much shorter than the coagulometer method. When the number of samples is large, the former has more obvious advantages.

[0005] The purpose of the present disclosure is to provide an anti-FXI / FXIa antibody biological activity detection method, which uses a microplate reader to replace the coagulometer of the traditional method to analyze the biological activity of the anti-FXI / FXIa antibody, and simplifies the experimental operation, shortens the experimental period, and improves the detection efficiency. SUMMARY

[0006] The present disclosure relates to an anti-FXI / FXIa antibody activity detection method with short operation time, high accuracy, and good repeatability.

[0007] The present disclosure provides an anti-coagulant drug activity detection method, which comprises the following steps:

[0008] (a) diluting the test sample and the control sample into a series of concentrations with buffer solution in proportion;

[0009] (b) adding plasma and incubating;

[0010] (c) adding aPTT reagent and incubating;

[0011] (d) adding CaCl2; and

[0012] (e) reading the OD value on the microplate reader, and calculating the relative biological activity of the test sample according to the detection data.

[0013] In some embodiments, the above-mentioned anti-coagulant drug is an antibody, such as an anti-FXI / FXIa antibody, an anti-FXII / FXIIa antibody, an anti-FIX antibody, an anti-FX antibody, an anti-FVII antibody, an anti-FVIII antibody, an anti-platelet glycoprotein IIb / IIIa receptor antibody.

[0014] In some embodiments, the above-mentioned test sample and control sample are anti-coagulant drugs, such as an anti-FXI / FXIa antibody, an anti-FXII / FXIIa antibody, an anti-FIX antibody, an anti-FX antibody, an anti-FVII antibody, an anti-FVIII antibody, an anti-platelet glycoprotein IIb / IIIa receptor antibody, heparin, aspirin, hirudin, colchicine, tirofiban, cilostazol, and tioclomine.

[0015] In some embodiments, the test and control articles are the same anticoagulant drug, e.g., the same anti-FXI / FXIa antibody.

[0016] In some embodiments, the activity detection method further comprises step (f) adding an EDTA solution to terminate the reaction, wherein step (f) is between steps (d) and (e).

[0017] In some embodiments, further, the concentration of the EDTA solution in step (f) is about 15 mM to about 50 mM, e.g., about 15 mM to about 45 mM, about 15 mM to about 40 mM, about 15 mM to about 35 mM, about 15 mM to about 30 mM, about 15 mM to about 25 mM, about 20 mM to about 50 mM, about 20 mM to about 45 mM, about 20 mM to about 35 mM, about 20 mM to about 30 mM, about 17 mM to about 27 mM, about 25 mM to about 30 mM, about 25 mM to about 35 mM. In some non-limiting embodiments, the concentration of the EDTA solution in step (f) is about 15 mM, about 20 mM, about 21 mM, about 22 mM, about 23 mM, about 24 mM, about 25 mM, about 26 mM, about 27 mM, about 28 mM, about 29 mM, about 30 mM, about 40 mM, or about 50 mM. In some embodiments, further, the concentration of the EDTA solution in step (f) is about 25 mM.

[0018] In some embodiments, the activity detection method of any of the above, wherein the buffer in step (a) is a Tris-HCl buffer.

[0019] In some embodiments, the activity detection method of any of the above, wherein the concentration of the buffer in step (a) is about 20 mM to about 100 mM, e.g., about 20 mM to about 90 mM, about 30 mM to about 80 mM, about 40 mM to about 70 mM, about 45 mM to about 60 mM, about 50 mM to about 55 mM, about 25 mM to about 95 mM, about 35 mM to about 85 mM, about 45 mM to about 75 mM, about 55 mM to about 65 mM, about 20 mM to about 70 mM, about 20 mM to about 50 mM, about 30 mM to about 60 mM, about 40 mM to about 60 mM, about 45 mM to about 55 mM. In some non-limiting embodiments, wherein the concentration of the buffer in step (a) is 20 mM, about 30 mM, about 40 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, or about 100 mM. In some embodiments, the activity detection method of any of the above, wherein the concentration of the buffer in step (a) is about 50 mM.

[0020] In some embodiments, the method of detecting activity as in any of the above, wherein the pH of the buffer in step (a) is about 6.0 to about 8.5, for example, about 6.0 to about 8.0, about 6.0 to about 7.5, about 6.5 to about 8.5, about 6.5 to about 8.0, about 6.5 to about 7.5, about 7.0 to about 8.0, about 7.0 to about 7.5, about 7.0 to about 7.4, about 7.2 to about 7.4 (7.3 ± 0.1). In some non-limiting embodiments, the pH of the buffer in step (a) is about 6.0, about 6.5, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8.0, about 8.5. In some embodiments, the method of detecting activity as in any of the above, wherein the pH of the buffer in step (a) is 7.3 ± 0.1.

[0021] In some embodiments, the method of detecting activity as in any of the above, wherein the buffer in step (a) can contain a colored substance including, but not limited to, trypan blue, methylene blue, patent blue, isothianol blue, toluidine blue, alkali blue, eosin, crystal violet, neutral red, Janus green B, safranin, and the colored substance does not affect the reaction of the antibody with the antigen and the coagulation process. In some embodiments, further, the colored substance includes, but is not limited to, trypan blue.

[0022] Further, the colored substance in the buffer in step (a) is about 0.001% to about 0.005%, for example, 0.001%, about 0.002%, about 0.003%, about 0.004%, about 0.005%.

[0023] In some embodiments, the method of detecting activity as in any of the above, wherein the test sample and the control sample in step (a) are diluted with the buffer in a dilution ratio selected from any one or more of 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:16, 1:32, 1:64, 1:128.

[0024] In some embodiments, the method of detecting activity as described in any of the above, wherein the dilution in step (a) is to a series of concentrations, which can include, but are not limited to, about 200 pg / mL, about 100 pg / mL, about 50 pg / mL, about 25 pg / mL, about 12.5 pg / mL, about 6.25 pg / mL, about 3.13 pg / mL, about 1.56 pg / mL, about 0.781 pg / mL, about 0.391 pg / mL, about 0.195 pg / mL, about 0.0977 pg / mL. In some embodiments, some of the concentrations can be added or removed, such as the highest and / or lowest concentration points: 200 pg / mL, 0.0977 pg / mL, noting that the addition or removal of concentrations does not affect the integrity of the final curve fit.

[0025] In some embodiments, the method of detecting activity as described in any of the above, wherein the plasma in step (b) can be, but is not limited to, human plasma.

[0026] In some embodiments, the method of detecting activity as described in any of the above, wherein an equal volume of aPTT reagent is added in step (c).

[0027] In some embodiments, the method of detecting activity as described in any of the above, wherein the incubation conditions in steps (b) and (c) are about 20 °C to about 37 °C, such as about 20 °C, about 23 °C, about 25 °C, about 27 °C, about 30 °C, about 32 °C, about 35 °C, about 37 °C; about 500 rpm to about 2000 rpm, such as about 500 rpm, 800 rpm, 900 rpm, 1000 rpm, 1100 rpm, 1200 rpm, 1500 rpm, 2000 rpm; for 1 min to 30 min, such as 1 min to 30 min, 1 min to 20 min, 1 min to 10 min, 3 min to 10 min.

[0028] In some specific embodiments, the method of detecting activity as described in any of the above, wherein the incubation conditions in step (b) are about 25 °C ± 2 °C, about 1000 rpm for 10 min.

[0029] In some specific embodiments, the method of detecting activity as described in any of the above, wherein the incubation conditions in step (c) are about 25 °C ± 2 °C, about 1000 rpm for 3 min.

[0030] In some embodiments, the method of detecting activity as described in any of the above, wherein the concentration of the CaCl2solution in step (d) is about 1 mM to about 30 mM, for example about 1.0 mM, about 5.0 mM, about 10.0 mM, about 11.0 mM, about 12.0 mM, about 12.1 mM, about 12.2 mM, about 12.3 mM, about 12.4 mM, about 12.5 mM, about 12.6 mM, about 12.7 mM, about 12.8 mM, about 12.9 mM, about 13.0 mM, about 15.0 mM, about 20.0 mM, about 30.0 mM. In some embodiments, the method of detecting activity as described in any of the above, wherein the concentration of the CaCl2solution in step (d) is about 12.5 mM.

[0031] In some embodiments, the method of detecting activity as described in any of the above, wherein the OD value in step (e) is obtained at 60 seconds to 180 seconds reading at 340 ± 20 nm, for example 60 seconds at 340 nm, 90 seconds at 340 nm, 120 seconds at 340 nm, 150 seconds at 340 nm, 180 seconds at 340 nm, 60 seconds at 320 nm, 90 seconds at 320 nm, 120 seconds at 320 nm, 150 seconds at 320 nm, 180 seconds at 320 nm, 60 seconds at 360 nm, 90 seconds at 360 nm, 120 seconds at 360 nm, 150 seconds at 360 nm, 180 seconds at 360 nm, but not limited thereto. In some embodiments, the method of detecting activity as described in any of the above, wherein the OD value in step (e) is obtained at 90 seconds reading at 340 nm.

[0032] In some embodiments, the method of detecting activity as described in any of the above, wherein the relative biological activity of the test sample in step (e) is calculated by fitting the detection data to a four-parameter equation, calculating the EC 50 values of the test sample and the control, respectively, and then calculating the relative biological activity of the test sample according to the ratio of the EC 50 value of the control to the EC 50 value of the test sample.

[0033] In some embodiments, the activity detection method of any of the above, wherein the volumes of the test and control samples, plasma, aPTT reagent, CaCl2solution, and EDTA solution in steps (a) through (d) and (f) are the same, and the volumes are about 20 to about 50 μΐ^ / well, e.g., about 25 to about 50 μΐ^ / well, about 30 to about 50 μΐ^ / well, about 35 to about 50 μΐ^ / well, about 40 to about 50 μΐ^ / well, about 20 to about 45 μΐ^ / well, about 25 to about 40 μΐ^ / well, about 30 to about 35 μΐ^ / well, about 20 to about 30 μΐ^ / well. In some non-limiting embodiments, the volumes are about 20 μΐ^ / well, about 25 μΐ^ / well, about 30 μΐ^ / well, about 35 μΐ^ / well, about 40 μΐ^ / well, about 45 μΐ^ / well, about 50 μΐ^ / well. In some embodiments, the activity detection method of any of the above, wherein the volumes of the test and control samples, plasma, aPTT reagent, CaCl2solution, and EDTA solution in steps (a) through (d) and (f) are about 25 μΐ^ / well.

[0034] In some embodiments, the activity detection method of any of the above, wherein the anti-FXI / FXIa antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein:

[0035] the heavy chain variable region comprises HCDR1, HCDR2, HCDR3 as set forth in SEQ ID NOs: 7, 8, 9, respectively, and the light chain variable region comprises LCDR1, LCDR2, LCDR3 as set forth in SEQ ID NOs: 10, 11, 12, respectively.

[0036] In some embodiments, wherein the anti-FXI / FXIa antibody or antigen-binding fragment thereof can be selected from a murine antibody, a chimeric antibody, a humanized antibody, e.g., a humanized antibody.

[0037] In alternative embodiments, wherein the light and heavy chain FR sequences on the humanized anti-FXI / FXIa antibody light and heavy chain variable regions are derived from the FRs of human germline light and heavy chains, respectively, or mutated sequences thereof.

[0038] In some embodiments, the anti-FXI / FXIa antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein:

[0039] the heavy chain variable region sequence is SEQ ID NO: 5 or a sequence at least 80%, at least 85%, at least 90% identical thereto, and the light chain variable region sequence is SEQ ID NO: 6 or a sequence at least 80%, at least 85%, at least 90% identical thereto;

[0040] Heavy chain variable region sequences such as SEQ ID NO: 13 or sequences having at least 80%, at least 85%, or at least 90% identity with it; light chain variable region sequences such as SEQ ID NO: 14 or sequences having at least 80%, at least 85%, or at least 90% identity with it.

[0041] The heavy chain variable region sequence is such as SEQ ID NO: 15 or a sequence having at least 80%, at least 85%, or at least 90% identity with it; the light chain variable region sequence is such as SEQ ID NO: 16 or a sequence having at least 80%, at least 85%, or at least 90% identity with it.

[0042] Heavy chain variable region sequences such as SEQ ID NO: 17 or sequences having at least 80%, at least 85%, or at least 90% identity with it; light chain variable region sequences such as SEQ ID NO: 16 or sequences having at least 80%, at least 85%, or at least 90% identity with it.

[0043] In some specific embodiments, the anti-FXI / FXIa antibody or its antigen-binding fragment comprises an antibody heavy chain variable region and a light chain variable region, wherein: the heavy chain variable region sequence is shown in SEQ ID NO: 17, and the light chain variable region sequence is shown in SEQ ID NO: 16.

[0044] In some embodiments, the anti-FXI / FXIa antibody or its antigen-binding fragment further comprises a heavy chain constant region and a light chain constant region. In optional embodiments, the heavy chain constant region is selected from the constant regions of human IgG1, IgG2, IgG3, IgG4, IgG4P (i.e., the S241P mutant of IgG4), and the light chain constant region is selected from the constant regions of human κ and λ chains. The sequence of the IgG4PFc (i.e., IgG4 Fc containing S241P) is, for example, shown in SEQ ID NO: 19. In some embodiments, the sequence of the heavy chain CH1 is as shown in SEQ ID NO: 18 or a sequence having at least 80%, at least 85%, or at least 90% identity with it, and / or the sequence of the light chain constant region is as shown in SEQ ID NO: 20 or a sequence having at least 80%, at least 85%, or at least 90% identity with it.

[0045] In some embodiments, the anti-FXI / FXIa antibody or its antigen-binding fragment comprises a heavy chain and a light chain, wherein: the heavy chain has the sequence shown in SEQ ID NO: 21 or a sequence having at least 80%, at least 85%, or at least 90% identity with it, and the light chain has the sequence shown in SEQ ID NO: 22 or a sequence having at least 80%, at least 85%, or at least 90% identity with it.

[0046] In the present disclosure, the "at least 90% identity" encompasses at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity.

[0047] In some embodiments, the anti-FXI / FXIa antibody comprises an antibody heavy chain and a light chain, wherein: the heavy chain sequence is set forth in SEQ ID NO: 21, and the light chain sequence is set forth in SEQ ID NO: 22.

[0048] In some embodiments, the method for detecting activity of an anti-FXI / FXIa antibody, wherein the antigen-binding fragment of the anti-FXI / FXIa antibody is a Fab, Fv, sFv, Fab', F(ab')2, linear antibody, single-chain antibody, scFv, sdAb, sdFv, nanobody, peptibody, domain antibody, and multi-specific antibody (bispecific antibody, diabody, triabody and tetrabody, tandem di-scFv, tandem tri-scFv), for example, specifically a scFv, Fv, Fab or Fab' fragment.

[0049] The present disclosure provides a method for detecting activity of an anti-FXI / FXIa antibody, comprising the steps of:

[0050] (a) diluting the test sample and the control sample into a series of concentrations with a buffer;

[0051] (b) adding plasma and incubating;

[0052] (c) adding aPTT reagent and incubating;

[0053] (d) adding CaCl2; and

[0054] (e) reading OD value on a microplate reader, and calculating the relative biological activity of the test sample according to the detection data;

[0055] wherein the test sample and the control sample are anti-FXI / FXIa antibodies;

[0056] Optionally, step (f) adding EDTA solution to terminate the reaction can be further included, wherein step (f) is located between steps (d) and (e).

[0057] In a first embodiment, the method comprises the steps of:

[0058] (a) diluting the test sample and the control sample into a series of concentrations with a buffer;

[0059] (b) adding plasma, incubating;

[0060] (c) adding aPTT reagent, incubating;

[0061] (d) adding about 12.5 mM CaCl2; and

[0062] (e) reading OD value at 340 nm for 90 seconds on a microplate reader, and calculating the relative biological activity of the test sample according to the detection data.

[0063] wherein the test sample and the control sample are anti-FXI / FXIa antibodies;

[0064] Optionally, step (f) adding EDTA solution to terminate the reaction can be further included, which is between steps (d) and (e), wherein the concentration of the EDTA solution is about 15 mM to about 50 mM, preferably about 25 mM;

[0065] Optionally, the buffer in step (a) can contain about 0.001% to about 0.005% trypan blue as a colored substance, preferably about 0.002%;

[0066] Optionally, the dilution ratio in step (a) is selected from any one or more of 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:16, 1:32, 1:64, 1:128;

[0067] Optionally, the incubation conditions in steps (b) and (c) are about 20°C to about 37°C, and about 500 rpm to about 2000 rpm for 1 min to 30 min;

[0068] Optionally, the test sample and the control sample, the plasma, the aPTT reagent, the CaCl2 solution and the EDTA solution in steps (a) to (d) and (f) are added in the same volume, which is about 20 to about 50 μL / well, preferably about 25 μL / well.

[0069] Optionally, the relative biological activity of the test sample in step (e) is calculated by fitting the detection data with a four-parameter equation, calculating the EC50 value of the test sample and the control sample respectively, and then calculating the relative biological activity of the test sample according to the ratio of the EC50 value of the control sample to the EC50 value of the test sample.

[0070] Optionally, the anti-FXI / FXIa antibody comprises a heavy chain variable region and a light chain variable region, wherein: the heavy chain variable region comprises HCDR1, HCDR2, HCDR3 as set forth in SEQ ID NO: 7, 8, 9, respectively, and the light chain variable region comprises LCDR1, LCDR2, LCDR3 as set forth in SEQ ID NO: 10, 11, 12, respectively.

[0071] In a specific embodiment, the method comprises the following steps:

[0072] (a) diluting the test sample and the control sample into a series of concentrations with about 50 mM Tris-HCl buffer solution with a pH value of about 7.3±0.1 in proportion;

[0073] (b) adding plasma, incubating at about 25℃±2℃, about 1000 rpm for 10 min;

[0074] (c) adding aPTT reagent, incubating at about 25℃±2℃, about 1000 rpm for 3 min;

[0075] (d) adding about 1 mM to about 30 mM CaCl2 solution, preferably about 12.5 mM; and

[0076] (e) reading OD value at 340 nm for 90 seconds on a microplate reader, and calculating the relative biological activity of the test sample according to the detection data.

[0077] wherein the test sample and the control sample are anti-FXI / FXIa antibodies;

[0078] Optionally, step (f) adding EDTA solution to terminate the reaction can be further included, which is located between steps (d) and (e) in step (f), wherein the concentration of the EDTA solution is about 15 mM to about 50 mM, preferably about 25 mM;

[0079] Optionally, the buffer solution in step (a) can contain about 0.001% to about 0.005% trypan blue as a colored substance, preferably about 0.002%;

[0080] Optionally, the dilution ratio in step (a) is selected from any one or more of 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:16, 1:32, 1:64, 1:128;

[0081] Optionally, the test sample and the control sample, plasma, aPTT reagent, CaCl2 solution and EDTA solution in steps (a) to (d) and (f) are added in the same volume, and the added volume is about 20 to about 50 μL / well, preferably about 25 μL / well.

[0082] Optionally, the relative biological activity of the test sample in step (e) is calculated by fitting the detection data to a four-parameter equation, calculating the EC50 value of the test sample and the control sample respectively, and then calculating the relative biological activity of the test sample according to the ratio of the EC50 value of the control sample to the EC50 value of the test sample.

[0083] Optionally, the anti-FXI / FXIa antibody comprises a heavy chain variable region and a light chain variable region, wherein: the heavy chain variable region comprises HCDR1, HCDR2, HCDR3 as shown in SEQ ID NO: 7, 8, 9 respectively, and the light chain variable region comprises LCDR1, LCDR2, LCDR3 as shown in SEQ ID NO: 10, 11, 12 respectively.

[0084] In a specific embodiment, the method comprises the following steps:

[0085] (a) diluting the test sample and the control sample into a series of concentrations with a 50 mM Tris-HCl buffer having a pH value of about 7.3±0.1 in a proportion;

[0086] (b) adding plasma, incubating at about 25℃±2℃ and a rotation speed of about 1000 rpm for 10 min;

[0087] (c) adding aPTT reagent, incubating at about 25℃±2℃ and a rotation speed of about 1000 rpm for 3 min;

[0088] (d) adding a 12.5 mM CaCl2 solution; and

[0089] (e) reading the OD value at 340±20 nm for 60-180 seconds on an enzyme marker, and calculating the relative biological activity of the test sample according to the detection data;

[0090] Optionally, the anti-FXI / FXIa antibody comprises a heavy chain variable region and a light chain variable region, wherein: the heavy chain variable region comprises HCDR1, HCDR2, HCDR3 as shown in SEQ ID NO: 7, 8, 9 respectively, and the light chain variable region comprises LCDR1, LCDR2, LCDR3 as shown in SEQ ID NO: 10, 11, 12 respectively.

[0091] Optionally, step (f) of adding an EDTA solution to terminate the reaction can be further included, and step (f) is located between steps (d) and (e), wherein the concentration of the EDTA solution is about 15 mM to about 50 mM, preferably about 25 mM;

[0092] Optionally, the buffer in step (a) can contain about 0.001% to about 0.005% trypan blue as a colored substance, preferably about 0.002%;

[0093] Optionally, the dilution ratio in step (a) is selected from any one or more of 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:16, 1:32, 1:64, 1:128;

[0094] Optionally, the volumes of the test sample and the control sample, the plasma, the aPTT reagent, the CaCl2 solution and the EDTA solution in steps (a) to (d) and (f) are added in the same volume, which is about 20 to about 50 μL / well, preferably about 25 μL / well.

[0095] Optionally, the relative biological activity of the test sample in step (e) is calculated by fitting the detection data to a four-parameter equation, calculating the EC 50 values of the test sample and the control sample, respectively, and calculating the relative biological activity of the test sample according to the ratio of the EC 50 value of the control sample to the EC 50 value of the test sample.

[0096] Optionally, the anti-FXI / FXIa antibody comprises a heavy chain variable region and a light chain variable region, wherein: the heavy chain variable region comprises HCDR1, HCDR2, HCDR3 as shown in SEQ ID NO: 7, 8, 9, respectively, and the light chain variable region comprises LCDR1, LCDR2, LCDR3 as shown in SEQ ID NO: 10, 11, 12, respectively.

[0097] In a specific embodiment, the method comprises the following steps:

[0098] (a) diluting the test sample and the control sample with about 50 mM Tris-HCl buffer having a pH value of about 7.3 ± 0.1 into a series of concentrations at a dilution ratio selected from any one or more of 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:16, 1:32, 1:64, 1:128;

[0099] (b) adding the plasma, incubating at about 25°C ± 2°C and a rotation speed of about 1000 rpm for 10 min;

[0100] (c) adding the aPTT reagent, incubating at about 25°C ± 2°C and a rotation speed of about 1000 rpm for 3 min;

[0101] (d) adding about 12.5 mM CaCl2 solution; and

[0102] (e) reading the OD value at 340 nm for 90 seconds on the microplate reader, fitting the detection data to a four-parameter equation, calculating the EC 50 values of the test sample and the control sample, respectively, and calculating the relative biological activity of the test sample according to the ratio of the EC 50 value of the control sample to the EC 50 value of the test sample.

[0103] wherein the test sample and the control sample are anti-FXI / FXIa antibodies.

[0104] Optionally, step (f) adding EDTA solution to terminate the reaction can be further included, said step (f) is located between steps (d) and (e), wherein the concentration of the EDTA solution is about 15 mM to about 50 mM, preferably about 25 mM;

[0105] Optionally, the buffer in step (a) can contain about 0.001% to about 0.005% trypan blue as a colored substance, preferably about 0.002%;

[0106] Optionally, the serial concentrations after dilution in step (a) are selected from any one or more of about 200 μg / mL, about 100 μg / mL, about 50 μg / mL, about 25 μg / mL, about 12.5 μg / mL, about 6.25 μg / mL, about 3.13 μg / mL, about 1.56 μg / mL, about 0.781 μg / mL, about 0.391 μg / mL, about 0.195 μg / mL, about 0.0977 μg / mL;

[0107] Optionally, the volumes of the test sample and control, plasma, aPTT reagent, CaCl2 solution and EDTA solution added in steps (a) to (d) and (f) are the same, said added volume is about 20 to about 50 μL / well, preferably about 25 μL / well.

[0108] Optionally, the anti-FXI / FXIa antibody comprises a heavy chain variable region and a light chain variable region, wherein: the heavy chain variable region comprises HCDR1, HCDR2, HCDR3 as set forth in SEQ ID NO: 7, 8, 9, respectively, and the light chain variable region comprises LCDR1, LCDR2, LCDR3 as set forth in SEQ ID NO: 10, 11, 12, respectively.

[0109] In a particular embodiment, the method comprises the following steps:

[0110] (a) diluting the test sample and control with about 20 mM to about 100 mM Tris-HCl buffer having a pH value of about 6.0 to about 8.5 into serial concentrations;

[0111] (b) adding plasma, incubating at about 20°C to about 37°C for 1 min to 30 min at a rotation speed of about 1000 rpm;

[0112] (c) adding aPTT reagent, incubating at about 20°C to about 37°C for 1 min to 30 min at a rotation speed of about 1000 rpm;

[0113] (d) adding about 1 mM to about 30 mM CaCl2 solution; and

[0114] (e) reading OD value at 340±20nm for 60-180 seconds on microplate reader, and calculating the relative biological activity of the test sample according to the detection data;

[0115] wherein the test sample and the control sample are anti-FXI / FXIa antibodies;

[0116] Optionally, step (f) adding EDTA solution to terminate the reaction can be further included, which is located between steps (d) and (e) in step (f), wherein the concentration of the EDTA solution is about 15mM to about 50mM, preferably about 25mM.

[0117] Optionally, the buffer in step (a) can contain about 0.001% to about 0.005% trypan blue as a colored substance, preferably about 0.002%;

[0118] Optionally, the dilution ratio in step (a) is selected from any one or more of 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:16, 1:32, 1:64, 1:128.

[0119] Optionally, the test sample and the control sample, the plasma, the aPTT reagent, the CaCl2 solution and the EDTA solution in steps (a) to (d) and (f) are added in the same volume, which is about 20 to about 50μL / well, preferably about 25μL / well.

[0120] Optionally, the relative biological activity of the test sample in step (e) is calculated by fitting the detection data with a four-parameter equation, and then calculating the EC 50 values of the test sample and the control sample, respectively, and then calculating the relative biological activity of the test sample according to the ratio of the EC 50 value of the control sample to the EC 50 value of the test sample.

[0121] Optionally, the anti-FXI / FXIa antibody comprises a heavy chain variable region and a light chain variable region, wherein: the heavy chain variable region comprises HCDR1, HCDR2, HCDR3 as shown in SEQ ID NO: 7, 8, 9, respectively, and the light chain variable region comprises LCDR1, LCDR2, LCDR3 as shown in SEQ ID NO: 10, 11, 12, respectively.

[0122] The anti-FXI / FXIa antibody used in the present disclosure includes but is not limited to the anti-FXI / FXIa antibody in the above specific embodiments.

[0123] The data processing in the present disclosure adopts a four-parameter equation for fitting, and the regression equation is: Y=(A-D) / [1+(X / C) B+ D. Wherein A is the asymptote estimate of the curve; B is the slope of the curve; C is the EC 50 value (pg / mL); D: asymptote estimate of the curve. The bioactivity of the sample to be tested is calculated according to the following formula: Bioactivity of the sample to be tested (%) = (EC 50 of the control) / (EC 50 of the sample to be tested) x 100.

[0124] The present disclosure also provides a method for determining the biological activity of anti-FXI / FXIa antibody and its application in quality control of anti-FXI / FXIa monoclonal antibody.

[0125] The present disclosure determines the biological activity of anti-FXI / FXIa antibody by in vitro enzyme-linked immunosorbent assay with competitive inhibition, which has the advantages of simple operation, high accuracy, and more controllable quality.

[0126] The detection method provided by the present disclosure meets the requirements of specificity, precision, linearity and range, accuracy, and durability in verification, and can be effectively applied to the detection of biological activity of anti-FXI / FXIa antibody.

[0127] The present disclosure also provides a kit or product, which comprises plasma, aPTT reagent, CaCl2 solution, and an enzyme label meter in any of the foregoing detection methods.

[0128] The present disclosure also provides the application of the foregoing any of the activity detection methods, kits or products of anticoagulant drugs in quality release. BRIEF DESCRIPTION OF DRAWINGS

[0129] Figure 1 : In vitro FXIIa-mediated FXI activation enzyme activity inhibition test of anti-FXI / FXIa antibody.

[0130] Figure 2: aPTT / PT anticoagulant activity detection of human blood, wherein, Figure 2A , 2B are respectively the aPTT and PT test results of anti-FXI / FXIa antibody on human blood.

[0131] Figure 3: aPTT / PT anticoagulant activity detection of monkey blood, wherein, Figure 3A , 3B are respectively the aPTT and PT test results of anti-FXI / FXIa antibody on monkey blood.

[0132] Figure 4: animal experiment detection of anti-FXI / FXIa antibody on cynomolgus monkeys, wherein, Figure 4A is the aPTT, plasma drug concentration, FXI:C%, and free FXI change curve of antibody 3882 in cynomolgus monkeys, Figure 4BThrombin generation inhibition of antibody 3882 in cynomolgus monkeys, Figure 4C and Figure 4D Results of bleeding time assay and PT assay for safety testing of antibody 3882 in cynomolgus monkeys.

[0133] Figure 5: Results of in vivo pharmacodynamic (PD) experiments with anti-FXI / FXIa antibodies in cynomolgus monkeys, wherein, Figure 5A Results of APTT (s) assay, Figure 5B Results of FXI:C (%) assay, 3882 (1 mg / kg) both intravenously and subcutaneously, control BAY1213790 (1 mg / kg) intravenously.

[0134] Figure 6 aPTT activation coagulation kinetic curves.

[0135] Figure 7 Linear regression analysis of OD values of transmitted light versus reaction time during coagulation.

[0136] Figure 8 Coagulation curves of different concentrations of anti-FXI / FXIa antibodies.

[0137] Figure 9 Bioactivity assay of anti-FXI / FXIa antibodies.

[0138] Figure 10 Bioactivity assay of anti-FXI / FXIa antibodies (specificity evaluation).

[0139] Figure 11 Linear regression analysis of measured bioactivity versus theoretical bioactivity of anti-FXI / FXIa antibodies (accuracy evaluation). DETAILED DESCRIPTION

[0140] Terms

[0141] For the purposes of the present disclosure, certain technical and scientific terms are specifically defined below. Unless specifically defined herein, all other technical and scientific terms used have the meanings that are commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0142] The three letter code and one letter code for amino acids used in the present disclosure are as described in J. Biol. Chem, 243, p 3558 (1968).

[0143] "Factor XI" is also referred to herein as "Coagulation Factor XI", "FXI", or "fXI", which is a two-chain glycoprotein with a molecular weight of about 160 kilodaltons (kD). The two chains can be identical disulfide-linked polypeptides with a molecular weight of about 80,000 daltons. FXI contains four "apple domains" (A1-A4 from the N-terminus, heavy chain) and a C-terminal catalytic domain (light chain). Without wishing to be bound by a particular theory, it is believed that the four apple domains contain FXI binding sites for other proteins, such as Al for thrombin; A2 for HK, A3 for Factor IX (FIX), GPIb, and heparin, A4 for FXIIa. FXI can be converted to its active form, coagulation factor XIa (FXIa), by Factor XIIa (FXIIa). The 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.

[0144] "Antibody" as described herein is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies; monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), full-length antibodies and antibody fragments (or antigen binding fragments, or antigen binding portions), as long as they exhibit the desired antigen-binding activity. An antibody can refer to an immunoglobulin, a naturally occurring intact antibody is a four polypeptide chain structure connected by interchain disulfide bonds, consisting of two identical heavy chains and two identical light chains. The immunoglobulin heavy chain constant region has different amino acid compositions and arrangement orders, so its antigenicity is different. Accordingly, immunoglobulins can be divided into five categories, or called isotypes of immunoglobulins, namely IgM, IgD, IgG, IgA and IgE, and the corresponding heavy chains are μ chain, δ chain, γ chain, α chain and ε chain, respectively. The same class of Ig can be divided into different subclasses according to the differences in amino acid composition of the hinge region, the number and position of heavy chain disulfide bonds, such as IgG can be divided into IgG1, IgG2, IgG3, IgG4. The light chain is divided into κ chain or λ chain through the constant region. Each of the five types of Ig can have κ chain or λ chain.

[0145] The sequences of the heavy and light chains of an antibody near the N-terminus are highly variable, forming the variable region (Fv region); the remaining amino acid sequences near the C-terminus are relatively stable, forming the constant region. The variable region includes three hypervariable regions (HVR) and four relatively conserved framework regions (FR). The three hypervariable regions determine the specificity of the antibody, also known as the complementarity determining region (CDR). Each light chain variable region (LCVR) and heavy chain variable region (HCVR) is composed of three CDR regions and four FR regions, arranged in the order of FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 from the amino terminal to the carboxyl terminal. The three CDR regions of the light chain are referred to as LCDR1, LCDR2, and LCDR3; the three CDR regions of the heavy chain are referred to as HCDR1, HCDR2, and HCDR3. The CDR amino acid residues of the LCVR region and HCVR region of the antibody or antigen binding fragment described in the present disclosure are in accordance with the known Kabat numbering rules (LCDR1-3, HCDR2-3), or in accordance with the numbering rules of kabat and chothia (HCDR1).

[0146] The "antibody" of the present disclosure includes not only full-length antibodies, but also antigen binding fragments capable of binding to antigens.

[0147] The term "antigen binding fragment" or "functional fragment" refers to one or more fragments of an antibody that retain its ability to specifically bind to an antigen. It has been shown that the antigen binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term "antigen binding fragment" include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region, (iii) a Fd fragment consisting of the VH and CHI domains; (iv) a Fv fragment consisting of the VH and VL domains of a single arm of an antibody; (v) a dsFv, a stabilized antigen binding fragment formed by the VH and VL domains of an antibody via an interchain disulfide bond; (vi) diabodies, bispecific antibodies and multispecific antibodies comprising a scFv, a dsFv, a Fab, and the like fragments. Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be produced as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see, e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci USA 85:5879-5883). Such single chain antibodies are also included in the term "antigen binding fragment" of an antibody. Such antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same fashion as are intact antibodies. Antigen binding moieties can be produced by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact immunoglobulin. The antibody can be an antibody of different isotype, e.g., an IgG (e.g., IgGl, IgG2, IgG3 or IgG4 subtypes), IgAl, IgA2, IgD, IgE or IgM antibody.

[0148] The term "binds to FXI / FXIa" of the present disclosure refers to the ability of an antibody or antigen binding fragment of the present disclosure to interact with FXI / FXIa or an epitope thereof, which can be of human origin. The term "antigen binding site" of the present disclosure refers to a three-dimensional space on an antigen recognized by an antibody or antigen binding fragment of the present disclosure.

[0149] The term "specifically binds" refers to as determined by techniques available in the art, e.g., a competitive ELISA, a radioimmunoassay, or a determination, or determination. The term also applies to the situation when, for example, the antigen binding domain of an antibody of the present disclosure is specific for a particular epitope carried by a number of antigens, in which case the antibody carrying the antigen binding domain is capable of specifically binding to a plurality of antigens carrying the epitope.

[0150] The term "test sample" refers to a sample to be tested, to be identified. In the present disclosure, the test sample includes an antibody sample to be tested for biological activity produced in industrial production, specifically including an anti-FXI / FXIa antibody stock solution or finished product.

[0151] The term "sample buffer" refers to a buffer used to dilute a sample or other reagent.

[0152] The term "release" refers to the ability of an intermediate product and / or a finished product to achieve an acceptable quality according to process data.

[0153] The experimental methods in the following examples were generally carried out according to conventional conditions or as suggested by the manufacturer, unless otherwise specified. The materials and reagents used in the following examples were generally obtained from commercial suppliers, unless otherwise noted. The experimental methods and materials described herein are illustrative only.

[0154] The details of one or more embodiments of the present disclosure are set forth in the accompanying description below. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, the methods and materials described below are presented for the purpose of illustration. Other features, objects, and advantages of the disclosure will be apparent from the description and from the claims. In the specification and the claims, unless otherwise indicated, the singular forms "a", "an", and "the" include plural referents. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. All patents and publications cited in this specification are incorporated by reference. The following examples are presented to more fully describe optional embodiments of the disclosure. These examples should not be construed as limiting the scope of the disclosure, as defined by the claims, in any manner.

[0155] Examples

[0156] The present disclosure is further described by the following examples, which do not limit the scope of the disclosure. Unless otherwise indicated, the experimental methods in the examples were carried out according to conventional conditions, such as Antibody Techniques Laboratory Manual, Cold Spring Harbor, Molecular Cloning Manual, or as suggested by the manufacturer of the reagent or product. Unless otherwise indicated, the reagents used in the examples were commercially available reagents.

[0157] Materials and General Methods

[0158] Reagents and Materials

[0159] Preparation, purification, etc. of anti-FXI / FXIa antibody (control or test sample) refer to the following Examples 1 to 3; activated partial thromboplastin time assay kit, standard human plasma, 25 mM CaCl2solution were purchased from Dade Behring; Tris base was purchased from Sigma-Aldrich; EDTA was purchased from National Pharmaceutical Group Chemical Reagent Co., Ltd.; 96-well plate was purchased from Greiner Bio-One.

[0160] Apparatus

[0161] Multifunctional enzyme label instrument (model SpectraMax M5e) was purchased from Molecular Devices; micro-pore plate constant temperature mixing device (model PHMP-4) was purchased from Grant-bio

[0162] Samples

[0163] Sample diluent [50 mM Tris-HCl buffer (pH 7.3, containing 0.002% trypan blue)]: Tris 0.60 g, 0.4% trypan blue 0.5 mL, purified water to 100 mL, adjust pH to 7.3±0.1 with 5M HCl.

[0164] 12.5 mM CaCl2solution: 25 mM CaCl2solution mixed with equal volume of ultrapure water.

[0165] Standard human plasma: according to the product instruction, reconstitute on the day of experiment. Add 1 mL ultrapure water to each bottle of standard human plasma lyophilized product, shake gently to dissolve completely.

[0166] 25 mM EDTA solution: disodium EDTA 0.465 g, purified water to 500 mL, adjust pH to 7.3±0.1 with 5M HCl.

[0167] Example 1, screening and preparation of hybridoma monoclonal antibody against FXI / FXIa

[0168] 1.1, preparation of human FXI / FXIa antigen and detection protein

[0169] Human FXI / FXIa protein (Uniprot Acc No. P03951) was purchased from Enzyme research laboratories (FXI: Cat. HFXI1111; FXIa: HFXIa 1111a) as the antigen and detection protein involved in the present disclosure. The following FXI / FXIa antigens not specifically stated refer to human FXI / FXIa.

[0170] > Amino acid sequence of human FXI / FXIa:

[0171] ECVTQLLKDTCFEGGDITTVFTPSAKYCQVVCTYHPRCLLFTFTAESPSEDPTRWFTCVLKDSVTETLPRVNRTAAISGYSFKQCSHQISACNKDIYVDLDMKGINYNSSVAKSAQECQERCTDDVHCHFFTYATRQFPSLEHRNICLLKHTQTGTPTRITKLDKVVSGFSLKSCALSNLACIRDIFPNTVFADSNIDSVMAPDAFVCGRICTHHPGCLFFTFFSQEWPKESQRNLCLLKTSESGLPSTRIKKSKALSGFSLQSCRHSIPVFCHSSFYHDTDFLGEELDIVAAKSHEACQKLCTNAVRCQFFTYTPAQASCNEGKGKCYLKLSSNGSPTKILHGRGGISGYTLRLCKMDNECTTKIKPRIVGGTASVRGEWPWQVTLHTTSPTQRHLCGGSIIGNQWILTAAHCFYGVESPKILRVYSGILNQSEIKEDTSFFGVQEIIIHDQYKMAESGYDIALLKLETTVNYTDSQRPICLPSKGDRNVIYTDCWVTGWGYRKLRDKIQNTLQKAKIPLVTNEECQKRYRGHKITHKMICAGYREGGKDACKGDSGGPLSCKHNEVWHLVGITSWGEGCAQRERPGVYTNVVEYVDWILEKTQAV

[0172] (SEQ ID NO: 1)

[0173] FXIa specific polypeptide was also coupled to KLH for mouse immunization. The FXIa specific polypeptide sequence is as follows:

[0174] CFYGVESPKILRVYSGIL (SEQ ID NO: 2)

[0175] CGYRKLRDKIQNTLQKAKIPL (SEQ ID NO: 3)

[0176] CGVQEIIIHDQYKMAESGYDI (SEQ ID NO: 4)

[0177] 1.2. Purification of FXI / FXIa related recombinant proteins, and hybridoma antibodies, recombinant antibodies

[0178] First, the supernatant of the mouse hybridoma is separated and purified by Protein G affinity chromatography, and then the fusion protein or antibody with human Fc tag is extracted by Protein A affinity chromatography, and further purified by anion chromatography.

[0179] 1.3, antibody screening

[0180] Take 5 SJL white mice and 5 Balb / c white mice, respectively, and immunize with 25 μg of FXIa antigen and 3 KLH-coupled polypeptides mixed with adjuvant. The time is 0, 14, 35 days. On day 0, 25 μg of emulsified antigen was injected intraperitoneally (IP) per mouse. On days 14 and 35, 12.5 ug / mouse was injected. On days 21 and 42, blood was taken, and the antibody titer in the mouse serum was determined by ELISA method. After the 4th-5th immunization, the mice with high antibody titer and titer tending to plateau in the serum were selected for spleen cell fusion.

[0181] The spleen lymphocytes were fused with myeloma cells Sp2 / 0-Ag14 cells by optimized PEG-mediated fusion steps to obtain hybridoma cells. According to the growth density of the hybridoma cells, the hybridoma culture supernatant was purified, cell binding experiment and cell blocking experiment by combined ELISA method, and the positive well cells were timely amplified, frozen and preserved, and sequenced. The hybridoma clones were obtained by further screening of the hybridoma cells by ELISA method, and then the antibodies were prepared and purified.

[0182] A positive clone 3807 was obtained, and the corresponding antibody variable region amino acid sequence is as follows:

[0183] >3807-VH:

[0184] EVQLQQSGAELVRPGTSVKLSCIASGFNIK DDYMH WVKQRPEQGLEWIG WIDPENGDTEYASKFQG KATITADTSANTAYLQFSSLTSEDTAVYYCLY GNFYYFDY WGQGTTLTVSS

[0185] SEQ ID NO: 5

[0186] >3807-VL:

[0187] QIVLTQSPAIMSASPGEKVTMTC SASSSINYMH WYQQKPGTSPKRWIY DTSKLAS GVPARFSGSGSGTSYSLTISSMEAEDAATYYC HQRSFSPLT FGAGTKLELK

[0188] SEQ ID NO: 6

[0189] Table 1. Heavy and light chain CDR region sequences of antibody 3807

[0190]

[0191]

[0192] Example 2, Humanization and immunogenicity modification of anti-FXI / FXIa hybridoma antibodies

[0193] 2.1, Humanization

[0194] By performing three-dimensional homology modeling on antibody molecule 3807, combined with the results of alignment of the V-base human germline sequence database, the IMGT human antibody heavy chain variable region germline gene database, the high homology heavy chain variable region germline gene of the selected and screened antibody was selected as a template, and the CDR of the mouse-derived monoclonal antibody was transplanted into the corresponding human module. The transplanted antibody was again subjected to three-dimensional structure simulation and analysis, and specific sites in the FR region that affect the structure and morphology of the CDR region were subjected to restoration mutation. Among them, the amino acid residues are determined and annotated by the Kabat numbering system.

[0195] The light chain template of the humanized framework of hybridoma clone 3807 is IGKV3-11*01, and the heavy chain template is IGHV1-69-2*01, and the humanized variable region sequence is as follows (underlined is CDR):

[0196] >3807 VH-CDR graft

[0197] QVQLVQSGAEVKKPGSSVKVSCKASGYTFT DDYMH WVRQAPGQGLEWMG WIDPENGDTEYASKFQG RVTITADESTSTAYMELSSLRSEDTAVYYCAR GNFYYFDY WGQGTTVTVSS

[0198] SEQ ID NO: 13

[0199] >3807 VL-CDR graft

[0200] EIVLTQSPATLSLSPGERATLSC SASSSINYMH WYQQKPGQAPRLLIY DTSKLAS GIPARFSGSGSGTDFTLTISSLEPEDFAVYYC HQRSFSPLT FGQGTKLEIK

[0201] SEQ ID NO: 14

[0202] The back-mutation sites and mutation modes of hybridoma clone 3807 are designed as shown in Table 2.

[0203] Table 2. Sequences of each heavy chain variable region and light chain variable region

[0204]

[0205] The specific sequences of 3807-VH1 and 3807-VL3 are as follows.

[0206] >3807-VH1:

[0207] QVQLVQSGAEVKKPGSSVKVSCKASGFNIK DDYMH WVRQAPGQGLEWMG WIDPENGDTEYASKFQG RVTITADESTSTAYMELSSLRSEDTAVYYCLY GNFYYFDY WGQGTTVTVSS

[0208] SEQ ID NO: 15

[0209] >3807-VL3:

[0210] EIVLTQSPATLSLSPGERATLSC SASSSINYMH WYQQKPGQAPKRWIY DTSKLAS GVPARFSGSGSGTDFTLTISSLEPEDFAVYYC HQRSFSPLT FGQGTKLEIK

[0211] SEQ ID NO: 16

[0212] The VH of antibody 3871 is 3807-VH1, and the VL is 3807-VL3.

[0213] 2.2, Immunogenicity modification

[0214] The antibody 3871 is genetically modified to have higher stability and lower immunogenicity. Only the FR region of the heavy chain variable region is modified, and the light chain variable region is not changed.

[0215] The sequence of the heavy chain variable region of the humanized antibody 3871 genetic modification is as follows (the light chain is not changed):

[0216] >3807-VH1 (GTFS)

[0217] QVQLVQSGAEVKKPGSSVKVSCKASGGTFS DDYMH WVRQAPGQGLEWMG WIDPENGDTEYASKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCLY GNFYYFDY WGQGTTVTVSS

[0218] SEQ ID NO: 17

[0219] The VH of antibody 3882 is 3807-VH1 (GTFS) and the VL is 3807-VL3.

[0220] The above heavy chain variable regions were fused to a humanized antibody heavy chain CH1 (SEQ ID NO: 18) and human antibody IgG4 Fc (with S241P mutation) (SEQ ID NO: 19) for the heavy chain and humanized kappa (SEQ ID NO: 20) for the light chain to form recombinant chimeric antibodies for subsequent testing.

[0221] > Human antibody heavy chain CH1:

[0222] ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRV

[0223] SEQ ID NO: 18

[0224] > Human antibody IgG4 PFc (i.e. with S241P mutation):

[0225] ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0226] SEQ ID NO: 19

[0227] > Human antibody light chain CK:

[0228] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0229] SEQ ID NO: 20

[0230] The full-length sequence of the 3882 example antibody is shown.

[0231] >3882-HC:

[0232] QVQLVQSGAEVKKPGSSVKVSCKASGGTFSDDYMHWVRQAPGQGLEWMGWIDPENGDTEYASKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCLYGNFYYFDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0233] SEQ ID NO: 21

[0234] >3882-LC:

[0235] EIVLTQSPATLSLSPGERATLSCSASSSINYMHWYQQKPGQAPKRWIYDTSKLASGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCHQRSFSPLTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0236] SEQ ID NO: 22

[0237] The expression vector for preparing the antibody was prepared by a conventional method, CHO cells were transfected, isolated, purified, tested, and the target antibody was obtained.

[0238] Example 3, verification of the function of anti-FXI / FXIa antibody

[0239] 3.1, affinity determination

[0240] The Biacore method was used. A certain amount of antibody to be tested was captured by affinity with a Protein A biosensor chip (Cat. # 29127556, GE), and then a series of concentration gradients of human FXI / FXIa were flowed through the chip surface, and the binding and dissociation curves were obtained by real-time detection of the reaction signal using a Biacore instrument (Biacore T200, GE). After each cycle of dissociation was completed, the biosensor chip was washed and regenerated with a regeneration solution provided in the human anti-capture kit or a pH 1.5 glycine-hydrochloric acid regeneration solution (Cat. # BR-1003-54, GE). The buffer used in the experiment was HBS-EP+10x buffer solution (Cat. # BR-1006-69, GE) diluted to 1x with D.I. Water (pH 7.4)

[0241] The data obtained from the experiment were fitted with the (1:1) Langmuir model using BIAevaluation version 4.1, GE software, and the affinity values were obtained. The test results are shown in Table 3.

[0242] Table 3. Affinity determination of anti-FXI / FXIa antibody

[0243] Antibody Antigen ka (1 / Ms) kd (1 / s) KD (M) 3871 FXI 1.32E+06 2.66E-05 2.00E-11 3882 FXI 1.973E+6 5.846E-5 2.963E-11 3807 FXIa 1.59E+06 2.35E-04 1.48E-10 3871 FXIa 1.24E+06 7.08E-04 5.70E-10 3882 FXIa 1.06E+06 3.87E-04 3.64E-10

[0244] 3.2, in vitro FXIIa-mediated FXI activation enzyme activity inhibition test

[0245] The ability of the anti-FXI / FXIa antibodies to block FXIIa cleavage of FXI was detected by measuring the FXI cleavage in the presence of the antibodies.

[0246] The SpectraMax M5 microplate reader was pre-set to 37°C, and the 384-well plate was pre-chilled on ice before adding 10ul of FXI (12ug / mL), 10ul of FXIIa (7.8ug / mL), 10ul of the candidate antibody (300ug / mL), 10ul of Dextran (100ug / mL) diluted with buffer (20mM HEPES, pH 7.4, 150mM NaCl and 0.1% BSA) respectively, incubated at 37°C for 60min, then chilled on ice for 5min, and then 10ul of S-2366 (8mM) (Chromogenix, S821090) was added before detection by the microplate reader. The results are shown in Figure 1 , wherein human IgG isotype was used as negative control (NC).

[0247] 3.3, aPTT / PT anticoagulant activity detection of human blood / monkey blood

[0248] Human blood / monkey blood was freshly collected in sodium citrate tubes, centrifuged at 3000rpm for 15min, and the upper plasma was taken.

[0249] The activated partial thromboplastin time (aPTT) in the presence of different concentrations of candidate antibodies (diluted in PBS) was determined using the Sysmex kit. The candidate antibody to be tested was incubated with plasma at 37°C for 3min, and then the coagulation was initiated by adding 25mM calcium chloride reagent, and the time when coagulation occurred was determined. The concentration of the candidate antibody that prolonged the aPTT by 50% (aPTT1.5) and the half maximal effective concentration (EC50) were determined. The test results of some antibodies are shown in Figure 2A , Figure 3A and Table 4. 1209 is another anti-FXI / FXIa antibody obtained by screening in the present application.

[0250] The prothrombin time (PT) in the presence of different concentrations of candidate antibodies (diluted in PBS) was determined using the Sysmex kit. The candidate antibody to be tested was incubated with plasma at 37°C for 3min, and then the coagulation was initiated by adding thromboplastin, and the time when coagulation occurred was determined. The test results of some antibodies are shown in Figure 2B and Figure 3B .

[0251] Table 4. aPTT / PT determination of anti-FXI / FXIa antibodies

[0252] Antibody Plasma aPTT 1.5 (ug / mL) EC 50 (ug / mL) 3882 Human blood 0.91 1.24 3882 Monkey blood 0.98 1.25

[0253] 3.4. Pharmacokinetics (PK) / pharmacodynamics (PD) test in cynomolgus monkeys

[0254] Normal adult male cynomolgus monkeys (body weight in the range of 4.0-4.7 kg) were assigned into groups according to body weight (body weight of the 1st, 4th, 5th into one group, and body weight of the 2nd, 3rd, 6th into another group), 3 monkeys in each group, observed for 14 days.

[0255] Blood samples were collected with sodium citrate blood collection tubes one day before administration as pre-dose samples, and aPTT, PT, plasma drug concentration, plasma active Factor XI (FXI) ratio (FXI:C%, i.e. the ratio of FXI with coagulation activity) and plasma free FXI concentration were determined; at the same time, the bleeding time of each animal was determined. Among the two groups of animals, one group was blank without administration; the other group was administered 3882 at 5 mg / kg body weight (mg / kg, mpk). The drug was dissolved in phosphate buffer (PBS) at a concentration of 5 mg / mL, and administered by intravenous bolus injection.

[0256] The blank control group was observed for bleeding time at 15 minutes (min) and 3 hours (h) after administration; and the plasma was collected and aPTT and PT were observed according to the above method at 15 min, 3 h, 6 h and 1 day (d) after administration. The administration group was observed for bleeding time at 15 min, 3 h, 2 d, 4 d, 1 week (w), 2 w and 3 w after administration; and the plasma was collected and aPTT, PT, plasma drug concentration, plasma FXI:C% and plasma free FXI concentration were observed according to the above method at 15 min, 3 h, 6 h, 1 d, 2 d, 4 d, 1 w, 2 w, 3 w, 4 w, 5 w and 6 w after administration. Both groups of animals were subjected to AV-shunt thrombosis 1 d after administration, and the thrombosis time was 10 min, and the net weight of the thrombus was measured after thrombosis.

[0257] Among them, aPTT, PT and plasma FXI:C% were determined by coagulometer and corresponding kit, plasma free FXI concentration and plasma drug concentration were determined by ELISA method. The thrombus weight was compared between the administration group and the control group, and the statistical analysis was performed by t test.

[0258] The results of antibody test are shown in Table 2. Figure 4A - Figure 4D Among them, the negative control (NC) was not administered. The results showed that 3882 well inhibited thrombus formation, and at the same time, prolonged the endogenous coagulation time, but had no significant effect on the bleeding time and exogenous coagulation time of the animals, and the PK results showed that the half-life was about 20 days.

[0259] 3.5. Pharmacodynamics (PD) test in cynomolgus monkeys

[0260] Normal adult male cynomolgus monkeys (body weight in the range of 7-9 kg) 6, were randomly divided into 3 groups, respectively: BAY1213790 intravenous administration of 1 mg / kg group; 3882 intravenous administration of 1 mg / kg group; 3882 subcutaneous administration of 1 mg / kg group. Intravenous administration group animals before administration and after administration of 5 min, 1 h, 1 d, 2 d, 3 d, 5 d, 1 w, 2 w, 3 w, 4 w, subcutaneous administration group before administration and after administration of 1 h, 1 d, 2 d, 3 d, 5 d, 1 w, 2 w, 3 w, 4 w, with sodium citrate blood collection tube to collect blood samples, with coagulation instrument and corresponding kit to determine aPTT and plasma FXI:C%.

[0261] Test results see Figure 5A , 5B. The results show that 3882 subcutaneous administration and intravenous administration can significantly prolong the intrinsic coagulation time and inhibit the activity of FXI. Under the condition of equal dose administration, compared with BAY1213790, 3882 can maintain longer prolongation of intrinsic coagulation time and stronger inhibition of FXI.

[0262] Heavy chain of BAY1213790:

[0263] EVQLLESGGGLVQPGGSLRLSCAASGFTFSQYGMDWVRQAPGKGLEWVSGIGPSGGSTVYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRGGPYYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 23)

[0264] Light chain of BAY1213790:

[0265] DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPS RFSGSGSGTDFTFTISSLQPEDIATYYCQQADSFPVTFGGGTKVEIKRTVAAPSVFIFPPS DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 24)

[0266] The control or test sample in the following examples is the same anti-FXI / FXIa antibody, i.e., the 3882 antibody.

[0267] Example 4, Coagulation curve of different anti-FXI / FXIa antibody concentrations

[0268] The anti-FXI / FXIa antibody (3882 antibody) was diluted with 50 mM TB (pH 7.3, 0.002% trypan blue) buffer to a series of concentrations: 6.25 μg / mL, 1.56 μg / mL, 0.391 μg / mL, 0.0977 μg / mL, 0.0488 μg / mL, and 0 μg / mL (TB), 25 μL of each was added to a 96-well plate, and an equal volume of standard human plasma was added, and incubated at 25°C, 1000 rpm for 10 min. Then 25 μL / well of aPTT reagent was added, and incubated at 25°C, 1000 rpm for 3 min; 25 μL / well of 12.5 mM CaCl2 solution was added to start the coagulation process; immediately scan the kinetic curve at 340 nm on the microplate reader.

[0269] Figure 6 、 Figure 7 and Figure 8 The results show that the coagulation curves of the TB buffer and each concentration of the antibody are "S" shaped, and the OD values of the baseline of the curve and the end point of complete coagulation reach the same level; as the concentration of the antibody increases, the coagulation initiation time and the time to reach the midpoint of coagulation are prolonged, which is consistent with the anticoagulant effect of the antibody.

[0270] Example 5, Determination of the biological activity of the anti-FXI / FXIa antibody

[0271] The test sample and control sample were pre-diluted to 200 μg / mL with 50 mM TB (pH 7.3, 0.002% trypan blue) buffer, then serially diluted by 2-fold gradient for 8-10 times in a dilution plate, 9-11 concentration sample solutions were obtained, each concentration was set in duplicate, 25 μL of each was added to a 96-well half-hole plate, an equal volume of standard human plasma was added, and the mixture was incubated at 25°C and 1000 rpm for 10 min, and a control blank hole with only buffer was set. Then 25 μL of aPTT reagent was added per well, and the mixture was incubated at 25°C and 1000 rpm for 3 min; 25 μL of 12.5 mM CaCl2 solution was added to each well to start the coagulation process; the mixture was incubated at 25°C for 60 seconds, and then 25 μL of 25 mM EDTA solution was added to each well; the control blank hole was zeroed on a microplate reader, and the OD value at 340 nm was read. The logarithm of the concentration was taken as the abscissa, and the OD value was taken as the ordinate, a four-parameter equation was selected for fitting, a curve was drawn, and the EC 50 value of the sample was calculated.

[0272] Figure 9 The results showed that the four-parameter curve R 2 values of the control sample and the test sample were 0.991 and 0.992, respectively, indicating that the curve fitting was good; the EC 50 value of the control sample was 1.969 μg / mL, and the EC 50 value of the test sample was 1.827 μg / mL, and the relative biological activity of the test sample was 108%.

[0273] Example 6, Biological activity determination of anti-FXI / FXIa antibody (specificity evaluation)

[0274] The test sample (anti-FXI / FXIa antibody), test sample buffer and irrelevant antibody (an antibody targeting a non-FXI / FXIa antigen of IgG4 subtype, same as the test sample) were determined. The test sample buffer was diluted according to the same method as the test sample, and the irrelevant antibody was diluted according to its protein content, and the determination was performed on the same 96-well plate as the test sample.

[0275] Figure 10 The results showed that the test sample showed a clear dose-effect curve, and the test sample buffer and the irrelevant antibody showed no obvious dose-effect curve, indicating that the method had good specificity for the biological activity determination of anti-FXI / FXIa antibody.

[0276] Example 7, Biological activity determination of anti-FXI / FXIa antibody (precision evaluation)

[0277] Two experimenters independently determined the same sample (anti-FXI / FXIa antibody) on different days for 3 times, and the RSD (%) of the biological activity values of 6 times was calculated.

[0278] Table 5 Precision test results

[0279]

[0280] The results of Table 5 show that the RSD of the bioactivity of the test sample anti-FXI / FXIa antibody determined by two experimenters for 6 times is 14.7%, indicating that the precision of the method is good.

[0281] Example 8, determination of bioactivity of anti-FXI / FXIa antibody (evaluation of accuracy)

[0282] The anti-FXI / FXIa antibody sample was diluted to 400 μg / mL, 300 μg / mL, 200 μg / mL, 140 μg / mL and 100 μg / mL, respectively, to prepare samples with theoretical bioactivity of 200%, 150%, 100%, 70% and 50%, respectively, and then detected after serial gradient dilution at 1:2, and the recovery rate of the 5 groups of samples was calculated (recovery rate = measured bioactivity / theoretical bioactivity x 100%).

[0283] Table 6 results of accuracy test

[0284] Sample Name Theoretical Biological Activity (%) Measured Biological Activity (%) Relative Error (%) Recovery 200% Anti-FXI / FXIa Antibody 200 177 -11.5 88.5％ 150% Anti-FXI / FXIa Antibody 150 155 3.3 103.3％ 100% Anti-FXI / FXIa Antibody 100 110 10.0 110.0％ 70% Anti-FXI / FXIa Antibody 70 70 0.0 100.0％ 50% Anti-FXI / FXIa Antibody 50 mL 51 2.0 102.0％

[0285] Note: The control sample is the same as the 100% anti-FXI / FXIa antibody sample.

[0286] The results show that the recovery rates of the 5 concentration points with theoretical bioactivity of 200%, 150%, 100%, 70% and 50% are all between 80% and 120%, indicating good accuracy. Linear regression analysis of the measured bioactivity value and the theoretical bioactivity value shows that R 2 is 0.967, indicating good linear fitting, see Figure 11 .

[0287] Example 9, comparison of determination of bioactivity of anti-FXI / FXIa antibody by ELISA and coagulation instrument

[0288] The control sample used in this example has a concentration of 43.8 mg / mL, and the 4 test samples have concentrations of 43.8 mg / mL, 62.6 mg / mL, 6.1 mg / mL and 12.1 mg / mL, respectively, and are all stored at 2-8°C.

[0289] Microplate Reader Method: The test sample and control sample were diluted with 50 mM TB (pH 7.3, 0.002% trypan blue) in a 2-fold gradient, and 11 sample solutions were obtained at concentrations of 200 μg / mL, 100 μg / mL, 50 μg / mL, 25 μg / mL, 12.5 μg / mL, 6.25 μg / mL, 3.13 μg / mL, 1.56 μg / mL, 0.781 μg / mL, 0.391 μg / mL, and 0.195 μg / mL. The diluted sample was taken 25 μL / well and added to the 96-well plate, and 25 μL / well of the diluted solution was added to the NC well and the blank control well. Each concentration was set in duplicate. The standard human plasma was added to the sample and NC well (no addition to the blank control well), 25 μL / well, and the microplate was placed in a microplate incubator at 1000 rpm, 25°C±2°C for 10 min. The aPTT reagent was added to the sample and NC well (no addition to the blank control well), 25 μL / well, and the microplate was placed in a microplate incubator at 1000 rpm, 25°C±2°C for 3 min. The 12.5 mM CaCl2 solution was added to the sample and NC well (no addition to the blank control well), 25 μL / well. After the addition of the 12.5 mM CaCl2 solution, the final concentrations of the sample in each well were 50 μg / mL, 25 μg / mL, 12.5 μg / mL, 6.25 μg / mL, 3.13 μg / mL, 1.56 μg / mL, 0.781 μg / mL, 0.391 μg / mL, 0.195 μg / mL, 0.0977 μg / mL, and 0.0488 μg / mL, respectively. The microplate was placed on the microplate reader tray, and the OD value at 340 nm was read after 90 seconds with the blank control well as the zero reference. The four-parameter curve of the OD value versus the antibody concentration was fitted, and the antibody EC 50 value and relative biological activity were calculated.

[0290] Coagulation Instrument Method: The test sample and control sample were diluted with 50 mM TB (pH 7.3, 0.002% trypan blue) in a gradient, and 10 sample solutions were obtained at concentrations ranging from 200 μg / mL to 390.625 ng / mL. The diluted sample was mixed with an equal volume of standard human plasma, and incubated at 37°C for 3 min. The coagulation tube was placed in the coagulation instrument, and aPTT coagulation analysis was performed according to the conventional method to determine the coagulation time of each concentration of antibody. The coagulation time versus antibody concentration curve was plotted, and the antibody EC 50 value and relative biological activity were calculated.

[0291] Table 7 Comparison of the results of the determination of the biological activity of the anti-FXI / FXIa antibody by the microplate reader method and the coagulation instrument method

[0292]

[0293] The results showed that the bioactivity results of enzyme-labeled instrument method were consistent with those of coagulation instrument method, and the recoveries were 90.0%-100.0% and 82.0%-114.3% respectively, both of which could reach a good level (all between 80%-120%). The enzyme-labeled instrument method for determining the bioactivity of anti-FXI / FXIa antibody could reach the same accuracy as the coagulation instrument method, and could be used as a method for quality control of anti-FXI / FXIa antibody samples.

Claims

1. A method for detecting biological activity of an anticoagulant drug, comprising the following steps: (a) diluting a test sample and a control sample with a buffer to a series of concentrations; (b) adding plasma and incubating; (c) adding aPTT reagent and incubating; (d) adding a CaCl 2 solution; and (e) reading OD values on an enzyme label meter, and calculating the relative biological activity of the test sample according to the detection data; wherein the anticoagulant drug is an anti-FXI / FXIa antibody; the anti-FXI / FXIa antibody comprises an antibody heavy chain variable region and a light chain variable region, the sequence of the heavy chain variable region is shown as SEQ ID NO: 17, and the sequence of the light chain variable region is shown as SEQ ID NO:

16. The method further comprises a step (f) of adding an EDTA solution to terminate the reaction, which is located between steps (d) and (e). 3.The method of claim 2, wherein the concentration of the EDTA solution is 15 mM to 50 mM. The pH value of the buffer in step (a) is 6.0 to 8.

5. 5.The method of claim 1, wherein the buffer in step (a) is a Tris-HCl buffer. 6.The method of claim 5, wherein the concentration of the Tris-HCl buffer is 20 mM to 100 mM. 7.The method of claim 1, wherein the buffer in step (a) contains a colored substance, and the colored substance does not affect the reaction of the antibody with the antigen and the blood coagulation process. wherein 8.The method of claim 7, wherein the colored substance is trypan blue.

2. The method of claim 1, wherein, 9.The method of claim 7, wherein the content of the colored substance is 0.001% to 0.005%. 10.The method of claim 1, wherein the dilution ratio in step (a) is selected from any one or more of 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:16, 1:32, 1:64, and 1:

128.

4. The method of claim 1, wherein, The incubation conditions in steps (b) and (c) are 20℃ to 37℃, a rotation speed of 500 rpm to 1000 rpm, and an incubation time of 1 min to 30 min. 12.The method of claim 1, wherein the concentration of the CaCl 2 solution in step (d) is 1 mM to 30 mM. 13.The method of claim 1, wherein the OD values in step (e) are obtained by reading at 340±20 nm for 60 seconds to 180 seconds. The anti-FXI / FXIa antibody comprises a heavy chain and a light chain, wherein the sequence of the heavy chain is shown as SEQ ID NO: 21, and the sequence of the light chain is shown as SEQ ID NO:

22. 16.A method for detecting the activity of an anti-FXI / FXIa antibody, comprising the following steps: (a) diluting a test sample and a control sample with a 20 mM to 100 mM Tris-HCl buffer having a pH value of 6.0 to 8.5 to a series of concentrations; ​ ​ 11. The method of claim 1, wherein, ​ ​ ​ 14. The method of claim 1, wherein, The relative biological activity of the test sample in step (e) is calculated by fitting the test data to a four parameter equation, calculating the EC 50 values for the test sample and control sample, respectively, and calculating the relative biological activity of the test sample based on the ratio of the control sample EC 50 value to the test sample EC 50 value.

15. The method according to any one of claims 1 to 14, characterized in that, ​ ​ ​ (b) adding plasma, incubating at 25°C to 37°C for 1 min to 30 min at 500 rpm to 2000 rpm; (c) adding aPTT reagent, incubating at 25°C to 37°C for 1 min to 30 min at 500 rpm to 2000 rpm; (d) adding 1 mM to 30 mM CaCl2solution; and (e) reading OD value at 340 ± 20 nm for 60 s to 180 s on a microplate reader, and calculating the relative biological activity of the test sample according to the detection data; wherein, the test sample and the control sample are anti-FXI / FXIa antibodies; the anti-FXI / FXIa antibodies comprise an antibody heavy chain variable region and a light chain variable region, the heavy chain variable region sequence is shown as SEQ ID NO: 17, and the light chain variable region sequence is shown as SEQ ID NO:

16.

17. The method of claim 16, further comprising step (f) adding EDTA solution to terminate the reaction, the step (f) is between steps (d) and (e), wherein the concentration of the EDTA solution is 15 mM to 50 mM.

18. The method of claim 16, wherein the buffer in step (a) contains 0.001% to 0.005% trypan blue as a colored substance.

19. The method of claim 16, wherein the dilution ratio in step (a) is selected from any one or more of 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:16, 1:32, 1:64, 1:

128.

20. The method of claim 16, wherein the volumes of the test sample and the control sample, the plasma, the aPTT reagent, the CaCl2solution, and the EDTA solution added in steps (a) to (d) and (f) are the same, and the added volume is 20 to 50 μL / well.

21. The method of claim 16, wherein the relative biological activity of the test sample in step (e) is calculated by fitting the test data to a four parameter equation, calculating the EC50 values for the test sample and the control sample, respectively, and calculating the relative biological activity of the test sample based on the ratio of the EC50 value of the control sample to the EC50 value of the test sample. 50 50 50 value of the test sample.​​ 22. The method of any one of claims 16 to 21, wherein, the anti-FXI / FXIa antibodies comprise a heavy chain and a light chain, wherein: the heavy chain sequence is shown as SEQ ID NO: 21, and the light chain sequence is shown as SEQ ID NO: 22.

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