A fully human anti-human factor ix monoclonal antibody or antigen-binding fragment thereof and uses thereof

Abstract

The application provides a completely humanized anti-human blood coagulation factor IX monoclonal antibody or an antigen binding fragment thereof and application thereof. The antibody provided by the application has high affinity to human blood coagulation factor IX and significant inhibitory activity, and belongs to a neutralizing antibody, and has a good treatment prospect for thrombus diseases. The functional antibody is directly screened from blood plasma of hemophilia B patients by a mass spectrometry + de novo sequencing technology, and has the advantages of complete human origin, high safety and the like.

Description

Technical Field

[0001] This invention belongs to the field of biopharmaceuticals, specifically relating to a fully human anti-human coagulation factor IX monoclonal antibody or its antigen-binding fragment and its applications. Background Technology

[0002] Thrombotic diseases are a major global health threat, the core of which is the formation of abnormal blood clots in blood vessels that block the blood supply to critical organs. These diseases often lead to sudden death, organ dysfunction, and long-term disability, with higher risks, especially among hospitalized, post-operative, or patients with chronic diseases.

[0003] Existing anticoagulants (such as warfarin and novel oral anticoagulants (NOACs)) are all small-molecule chemical drugs, primarily administered orally, and are effective in preventing and treating thrombosis. However, while these drugs are effective in preventing thrombosis, they inevitably interfere with the body's normal hemostatic function. The resulting bleeding risk is their most significant limitation; mild cases may lead to subcutaneous or gingival bleeding, while severe cases can cause fatal intracranial or gastrointestinal hemorrhage. This high bleeding risk makes many doctors and patients hesitant to prescribe these drugs, especially in the elderly, those with renal insufficiency, or those using combined medications, where severe bleeding events can be fatal, limiting their widespread use. Therefore, there is an urgent clinical need for safer and more effective new drugs. Summary of the Invention

[0004] To address the shortcomings of existing technologies, the present invention aims to provide a fully human anti-human coagulation factor IX monoclonal antibody or its antigen-binding fragment and its applications. This antibody can significantly reduce the risk of bleeding while exhibiting antithrombotic effects. The present invention employs a "mass spectrometry + de novo sequencing" antibody discovery technique. This technique can isolate polyclonal antibodies capable of recognizing antigens from animal or human blood, and then directly sequence these polyclonal antibodies using mass spectrometry and de novo sequencing technology to obtain the complete sequence of a monoclonal antibody with high affinity. The present invention has successfully sequenced a series of antibodies with high affinity for human coagulation factor IX using this antibody discovery method, and this invention provides a fully human anti-human coagulation factor IX monoclonal antibody. This antibody has significant inhibitory activity against human coagulation factor IX, belongs to the neutralizing antibody category, and shows good therapeutic prospects for thrombotic diseases.

[0005] To achieve this objective, the present invention adopts the following technical solution: In a first aspect, the present invention provides a fully human anti-human coagulation factor IX monoclonal antibody or its antigen-binding fragment thereof, wherein the amino acid sequence of CDR1 in the heavy chain variable region of the antibody or its antigen-binding fragment is shown in SEQ ID NO:1, the amino acid sequence of CDR2 is shown in SEQ ID NO:2, and the amino acid sequence of CDR3 is shown in SEQ ID NO:3; and the amino acid sequence of CDR1 in the light chain variable region of the antibody or its antigen-binding fragment is shown in SEQ ID NO:4, the amino acid sequence of CDR2 is shown in SEQ ID NO:5, and the amino acid sequence of CDR3 is shown in SEQ ID NO:6.

[0006] Preferably, the amino acid sequence of the heavy chain variable region of the antibody or its antigen-binding fragment is as shown in SEQ ID NO:7, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:9.

[0007] Preferably, the amino acid sequence of the heavy chain of the antibody or its antigen-binding fragment is as shown in SEQ ID NO:8, and the amino acid sequence of the light chain is as shown in SEQ ID NO:10.

[0008] In this invention, the sequencing steps for the antibody are as follows: This project successfully recruited a hemophilia B patient who lacked a functional coagulation factor IX. After receiving coagulation factor IX infusion therapy, the patient developed antibodies against coagulation factor IX. After drawing blood from the patient's vein and centrifuging the blood, we obtained a plasma sample containing coagulation factor IX antibodies.

[0009] Targeted polyclonal antibody enrichment: The antigen protein (recombinant coagulation factor IX) is immobilized on agarose microspheres and then incubated with plasma. Antibodies in the plasma that can bind to the antigen will attach to the microspheres, thus separating them from other antibodies in the plasma. After eluting the antibodies bound to the microspheres, polyclonal antibodies that can bind to the antigen can be obtained.

[0010] Mass spectrometry acquisition: After appropriate separation of the polyclonal antibody, it is successively reduced, alkylated and digested with enzymes. The resulting peptides are analyzed by mass spectrometry and the data is acquired.

[0011] De novo sequencing: All mass spectrometry data were analyzed using the Novor de novo sequencing platform to obtain the complete amino acid sequences of the antibody light and heavy chains.

[0012] Antibody expression verification: The corresponding gene was synthesized based on the amino acid sequence of the antibody, and the plasmid was transfected into mammalian cells for antibody expression. The purified antibody was verified by ELISA to confirm its affinity for the antigen, and by APTT to confirm its inhibitory activity against coagulation factor IX.

[0013] In this invention, mass spectrometry combined with de novo sequencing is used to directly sequence antibody proteins in plasma. Since these antibody proteins are derived from clinically pre-validated and screened high-titer plasma, direct sequencing of these proteins enables more efficient discovery of fully human antibodies with high affinity.

[0014] This invention directly discovers human antibodies within the human body, referring to the resulting antibodies as "fully human antibodies." These "fully human antibodies" differ from existing "fully human antibodies," such as those discovered in humanized mice. The "fully human antibodies" of this invention exhibit low immunogenicity and superior safety.

[0015] In a second aspect, the present invention provides a nucleic acid molecule that encodes the fully human anti-human coagulation factor IX monoclonal antibody or its antigen-binding fragment as described in the first aspect.

[0016] Thirdly, the present invention provides a recombinant vector containing the nucleic acid molecule described in the second aspect.

[0017] Fourthly, the present invention provides a recombinant cell expressing the fully human anti-human coagulation factor IX monoclonal antibody or its antigen-binding fragment as described in the first aspect, wherein the recombinant cell contains at least one copy of the recombinant vector as described in the third aspect, or wherein the nucleic acid molecule as described in the second aspect is integrated into its genome.

[0018] Fifthly, the present invention provides a kit for detecting human coagulation factor IX, the kit comprising the fully human anti-human coagulation factor IX monoclonal antibody or its antigen-binding fragment as described in the first aspect.

[0019] In a sixth aspect, the present invention provides the use of any one or at least two combinations of the fully human anti-human coagulation factor IX monoclonal antibody or its antigen-binding fragment as described in the first aspect, the nucleic acid molecule as described in the second aspect, the recombinant vector as described in the third aspect, the recombinant cell as described in the fourth aspect, or the kit for detecting human coagulation factor IX as described in the fifth aspect in the preparation of a product for detecting human coagulation factor IX.

[0020] In a seventh aspect, the present invention provides the use of the fully human anti-human coagulation factor IX monoclonal antibody or its antigen-binding fragment described in the first aspect in the preparation of a medicament for treating thrombotic diseases.

[0021] Preferably, the thrombotic disease includes: venous thromboembolism (VTE), arterial thrombotic disease, microvascular thrombotic disease or special types of thrombotic disease, as well as diseases associated with cancer-associated thrombus formation (CAT; or cancer-associated venous thromboembolism (CAT / VTE), or diseases associated with anticoagulation therapy in patients with end-stage renal disease (ESRD).

[0022] Preferably, the venous thromboembolism includes: deep vein thrombosis (DVT), pulmonary embolism (PE), or visceral venous thrombosis.

[0023] Preferably, the arterial thrombotic disease includes: cardiovascular and cerebrovascular diseases (palpitation infarction, ischemic stroke) or peripheral arterial thrombosis; Preferably, the microvascular thrombotic disease includes thrombotic microangiopathy (TMA) or disseminated intravascular coagulation (DIC). Preferably, the specific type of thrombotic disease includes thrombophilia.

[0024] Eighthly, the present invention provides a medicament for treating human thrombotic diseases, the medicament comprising the fully human anti-human coagulation factor IX monoclonal antibody or its antigen-binding fragment as described in the first aspect.

[0025] In a ninth aspect, the present invention provides a bispecific antibody comprising a first binding domain and a second binding domain, wherein the first binding domain comprises the heavy chain variable region and / or light chain variable region of the fully human anti-human coagulation factor IX monoclonal antibody or its antigen-binding fragment as described in the first aspect.

[0026] Compared with the prior art, the present invention has the following beneficial effects: (1) High safety: The antibodies provided by this invention are obtained by direct sequencing from patient plasma. These antibodies are produced by the human immune system and screened by the blood circulation system, thus exhibiting low immunogenicity and superior safety.

[0027] (2) Precise targeting: The neutralizing antibody discovered in this invention can directly inhibit the activity of coagulation factor IX, which will help it to be directly developed into an antibody drug for the treatment of thrombosis. This type of antibody drug specifically targets coagulation factor IX, and can avoid the risk of bleeding while anticoagulating, overcoming the shortcomings of existing therapeutic drugs.

[0028] (3) Mature technology: The present invention uses mass spectrometry + de novo sequencing technology to sequence the antibody protein and successfully obtain the complete amino acid sequence of the antibody. Based on these sequences, the antibody can be produced on a large scale using the recombinant protein expression scheme, which eliminates batch-to-batch differences and reduces production costs. Attached Figure Description

[0029] Figure 1 This is a gel image of purified antibody protein.

[0030] Figure 2 This is the result of verifying the affinity of the antibody for human coagulation factor IX.

[0031] Figure 3 This is the result of antibody coagulation activity verification. Detailed Implementation

[0032] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention in any way.

[0033] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased from legitimate channels.

[0034] Example 1 Antibody sequencing.

[0035] 1. Multi-antibody enrichment.

[0036] The antigen protein was immobilized on agarose microspheres and then incubated with patient plasma overnight at 4°C using a rotating incubator. The agarose microspheres were washed 3-4 times with pre-cooled wash buffer to remove unbound antibodies. After each wash, the agarose microspheres were collected by brief centrifugation. An appropriate amount of glycine elution buffer (e.g., 100-200 µL) was added, gently mixed, and incubated at 4°C for 5-10 minutes. The supernatant was collected after centrifugation, which was the target polyclonal antibody.

[0037] 2. Sample pretreatment.

[0038] Add 4 volumes of acetone (pre-prepared at -20°C) to the target polyclonal antibody, mix well, and incubate at -20°C for at least 1 hour. Then centrifuge at 15000 rpm, 4°C for 15 minutes. Discard the supernatant, open the cap, and air dry for 5 minutes. Dissolve the protein precipitate in 10 μL of 8 M urea (urea solution) (100 mM Tris, pH 8.5). Sonicate for 10 minutes to aid protein dissolution. Add 0.2 μL of 500 mM TCEP (final concentration 5 mM) and react at room temperature for 20 minutes. Add 0.2 μL of 500 mM MIAA (final concentration 10 mM) and react at room temperature for 15 minutes. Add 30 μL of 100 mM Tris, pH 8.5, and dilute the 8 M urea (urea solution) to 2 M. Add trypsin at an enzyme-to-protein mass ratio of 1:(20-100) and digest at 37°C in the dark for 12-16 hours. Add 2.2 μL of 90% formic acid to terminate the enzymatic digestion reaction, with a final formic acid concentration of 5%.

[0039] 3. Mass spectrometry acquisition.

[0040] After the peptides obtained from enzymatic digestion were loaded onto a reversed-phase chromatographic column packed with C18 packing material, they were washed with a solution of 95% water, 5% acetonitrile, and 0.1% formic acid to remove salts from the sample. The column loaded with peptide samples was then connected to a high-performance liquid chromatograph (HPLC), and the peptides on the column were eluted one by one using a 1-hour gradient elution program, which were then sent to a mass spectrometer for detection. The pyrolysis voltage of the mass spectrometer was set to 3.5 kV, the HPLC flow rate was set to 200 nL / min, and the mass spectrometer was set to data-dependent mode. The primary mass spectrometry scan was performed in an orbitrap at a resolution of 60,000 m / z. The primary mass spectrometry scan range was 400–2000 m / z. Each primary scan was followed by eight data-dependent secondary mass spectrometry scans. Peptide fragmentation was performed in a linear ion trap using high-energy collisional dissociation (HCD) mode with a collision energy of 35%. The dynamic exclusion time was set to 30 seconds.

[0041] 4. De novo sequencing.

[0042] Mass spectrometry data analysis was performed using Novor software. This software is capable of de novo sequencing of peptides, accurately identifying the amino acid sequences of peptides without relying on DNA and protein databases. The software was published in 2015; Ma, B. Novor: Real-Time Peptide de Novo Sequencing Software. J. Am. Soc. MassSpectrom. 26, 1885-1894 (2015). https: / / doi.org / 10.1007 / s13361-015-1204-0. After analysis using Novor software, the complete amino acid sequences of the antibody's light and heavy chains were obtained.

[0043] The complete amino acid sequences of the light and heavy chains of the antibodies are shown in Tables 1 and 2.

[0044] Table 1

[0045] Table 2

[0046] Example 2 Antibody expression verification.

[0047] The corresponding gene was synthesized based on the antibody's amino acid sequence and constructed into the pTT5 vector. The plasmid was then transfected into 293F cells for transient expression. The resulting monoclonal antibody protein was purified using conventional protein purification methods. Figure 1 As shown.

[0048] The affinity of the purified monoclonal antibody for the antigen was verified by ELISA. In the ELISA experiment, recombinant coagulation factor IX antigen was first added to a 96-well plate and incubated overnight at 4°C to allow the antigen to coat the plate. The plate was washed 3-5 times with PBS, each time thoroughly washing to remove unbound antigen. 200 µL of blocking buffer (PBS buffer containing 3% BSA) was added to each well. After sealing, the plate was incubated at room temperature for 1 hour to block the surface of wells not coated with antigen, reducing non-specific binding. 100 µL of purified monoclonal antibody was added to each well and incubated at room temperature for 1 hour. The plate was washed 6 times to remove unbound monoclonal antibody. 100 µL of enzyme-labeled secondary antibody solution was added to each well and incubated at room temperature for 1 hour. The plate was washed 6 times to remove unbound enzyme-labeled secondary antibody. 100 µL of enzyme substrate solution (TMB) was added to each well and incubated at room temperature for 10 minutes. 50 µL of stop solution (2 M sulfuric acid solution) was added to each well to stop the reaction. The absorbance was measured at 450 nm using an ELISA reader.

[0049] ELISA experimental results as follows Figure 2 As shown in the figure, antibody AB204313 has a high affinity for human coagulation factor IX.

[0050] Example 3 Functionality verification.

[0051] APTT coagulation activity test.

[0052] The activated partial thromboplastin time (APTT) is a routine test to assess the normal function of various coagulation factors in the intrinsic and common pathways. The APTT test involves mixing normal human plasma with an activator (such as diatomaceous earth), phospholipids, and calcium ions, and recording the number of seconds required for fibrin clot formation.

[0053] The APTT test used a STart 4 fibrinometer (Diagnostica Stago, France), APTT reagents (Siemens, Germany), and normal human plasma (clinical sample). For the APTT test, the instrument and reagents were first preheated to 37°C. The antibody was then diluted with PBS, the APTT program was selected, and the reaction vessel was placed. 40 μL of APTT reagent, 40 μL of normal human plasma, and magnetic beads were added sequentially, and the start button was pressed to initiate bead oscillation. Then, 40 μL of antibody was added, and the timer was started. After 180 seconds, 25 μL of 50 mmol / L CaCl2 was added, and the measurement button on the handle was pressed simultaneously. The oscillation of the magnetic beads continued until the plasma clotted, and the number of seconds recorded on the instrument was observed. The normal reference value is approximately 25-30 seconds.

[0054] The purified antibody AB204313 was added to the reaction vessel at specified concentration gradients (22.5, 45, 90, and 180 μg / mL), and the APTT test was performed according to the above experimental procedure. The time required for plasma coagulation was recorded, and the experimental results were plotted as a line graph. Figure 3 As shown, antibody AB204313 can significantly prolong the coagulation time of normal human plasma, indicating that it has significant inhibitory activity against coagulation factor IX.

[0055] In summary, this invention directly screens for fully human anticoagulation factor IX antibodies from the human immune system. The resulting antibodies are produced by the human immune system and screened by the blood circulation system, thus exhibiting low immunogenicity and superior safety. The resulting antibodies have significant application prospects in the preparation of antithrombotic drugs and are expected to overcome the limitations of traditional anticoagulant drugs.

[0056] The applicant declares that the above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. A fully human anti-human coagulation factor IX monoclonal antibody or its antigen-binding fragment, characterized in that, The amino acid sequences of the heavy chain variable regions of the antibody or its antigen-binding fragment are shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3; the amino acid sequences of the light chain variable regions of the antibody or its antigen-binding fragment are shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:

6.

2. The fully human anti-human coagulation factor IX monoclonal antibody or its antigen-binding fragment according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region of the antibody or its antigen-binding fragment is shown in SEQ ID NO:7, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

9.

3. The fully human anti-human coagulation factor IX monoclonal antibody or its antigen-binding fragment according to claim 1, characterized in that, The amino acid sequence of the heavy chain of the antibody or its antigen-binding fragment is shown in SEQ ID NO:8, and the amino acid sequence of the light chain is shown in SEQ ID NO:

10.

4. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes a fully human anti-human coagulation factor IX monoclonal antibody or its antigen-binding fragment as described in any one of claims 1-3.

5. A recombinant vector, characterized in that, The recombinant vector contains the nucleic acid molecule as described in claim 4.

6. A recombinant cell, characterized in that, The recombinant cells express the fully human anti-human coagulation factor IX monoclonal antibody or its antigen-binding fragment as described in any one of claims 1-3, and the recombinant cells contain at least one copy of the recombinant vector as described in claim 5, or have the nucleic acid molecule as described in claim 4 integrated into their genome.

7. A kit for detecting human coagulation factor IX, characterized in that, The kit comprises a fully human anti-human coagulation factor IX monoclonal antibody or its antigen-binding fragment as described in any one of claims 1-3.

8. Use of any one or a combination of at least two of the fully human anti-human coagulation factor IX monoclonal antibody or its antigen-binding fragment as described in any one of claims 1-3, the nucleic acid molecule as described in claim 4, the recombinant vector as described in claim 5, the recombinant cell as described in claim 6, or the kit for detecting human coagulation factor IX as described in claim 7 in the preparation of a product for detecting human coagulation factor IX.

9. The use of the fully human anti-human coagulation factor IX monoclonal antibody or its antigen-binding fragment as described in any one of claims 1-3 in the preparation of a medicament for treating thrombotic diseases.

10. A drug for treating human thrombotic diseases, characterized in that, The drug comprises a fully human anti-human coagulation factor IX monoclonal antibody or its antigen-binding fragment as described in any one of claims 1-3.

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