Monoclonal antibody having anticoagulant activity and use thereof

Abstract

The present invention relates to a monoclonal antibody having an anticoagulant activity and the use thereof. The antibody or a fragment comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region of the antibody or fragment has an amino acid sequence as shown in SEQ ID NO: 1, and the heavy chain variable region has an amino acid sequence as shown in SEQ ID NO: 3. The monoclonal antibody of the present invention has the effect of inhibiting the activity of a common coagulation pathway in the coagulation cascade reaction, and can be used in the prevention and treatment of thrombotic diseases.

Description

A monoclonal antibody with anticoagulant activity and its application Technical Field

[0001] This invention belongs to the field of thrombotic disease treatment, and specifically relates to a monoclonal antibody with anticoagulant activity and its application. Background Technology

[0002] Coagulation is a vital mechanism for maintaining life. Following tissue damage, cells release tissue factor (TF), which binds to circulating activated factor VII (FVIIa), initiating a coagulation cascade via the tissue factor pathway. FVIIa / TF activates factor X (FX) to become activated factor X (FXa), which has enzymatic activity. Simultaneously, it catalyzes a small amount of factor IX to become activated factor IX (FIXa). FXa, with the assistance of activated factor V (FVa), converts prothrombin to thrombin. However, this entire coagulation process is rapidly terminated by the binding of tissue factor pathway inhibitor (TFPI) to FVIIa / TF, resulting in only a trace amount of thrombin. When the vascular endothelium is damaged, blood comes into contact with the negatively charged surface of the subendothelial tissue, triggering the activation of coagulation factor XII (FXIIa). Activated FXIIa, with the participation of prokallikrein, initiates the intrinsic coagulation pathway. FXIIa specifically activates coagulation factor XI (FXI) into its enzymatic active form, activating factor XI (FXIa). FXIa further catalyzes coagulation factor IX to become activated factor IX (FIXa). The latter, along with its coenzyme-activated coagulation factor VIII (FVIIIa), forms an enzyme complex that activates FX, amplifying the coagulation reaction and ultimately leading to the generation of a large amount of thrombin. This thrombin converts circulating fibrinogen into insoluble fibrin, thus causing blood coagulation. The trace amounts of thrombin produced in the tissue factor pathway activate coagulation factor V (FV) and coagulation factor VIII (FVIII), which are called FVa and FVIIIa, thereby promoting the amplification of the coagulation reaction. Thrombin can also catalyze the conversion of coagulation factor XI from proenzyme to enzyme, thereby further enhancing the coagulation reaction.In the aforementioned coagulation cascade, both the intrinsic coagulation pathway and the tissue factor coagulation pathway ultimately activate FX (focal factor), which promotes blood coagulation through the activation of prothrombin via FXa / FVa. This stage of the coagulation reaction is also known as the common pathway. Coagulation requires the participation of phospholipids. During hemostasis in vivo, coagulation factors bind to the phospholipid surface provided by activated platelets, thus enabling efficient coagulation. Calcium ions also play an important role in coagulation; many coagulation factor molecules contain calcium ion binding sites. Calcium ions promote the binding of coagulation factors to phospholipids and participate in maintaining the enzymatic conformation of coagulation factors.

[0003] In coagulation function tests, prothrombin time (PT) is activated through the extrinsic and common pathways of the coagulation system by adding tissue factor, calcium ions, and phospholipids to sodium citrate-anticoagulated plasma. Activated partial thromboplastin time (APTT), on the other hand, is measured by incubating citrate-anticoagulated plasma with phospholipids and negatively charged activators, first activating the intrinsic coagulation pathway, and then adding calcium ions to promote the common pathway coagulation reaction. Deficiency of coagulation factor VII leads to prolonged PT, while deficiencies in intrinsic coagulation pathway components, such as coagulation factors XII, XI, IX, and VIII, lead to prolonged APTT. Defects in coagulation factors X and V and prothrombin in the common pathway can prolong both APTT and PT.

[0004] Thrombotic diseases are one of the major threats to human health. Inhibiting the coagulation activity of components in the coagulation pathway is one of the ideal strategies for highly effective anticoagulation therapy.

[0005] Monoclonal antibodies, small molecule inhibitors, or siRNAs that inhibit coagulation factor expression have shown good efficacy in thrombosis prevention. The discovery and preparation of monoclonal antibodies with anticoagulant activity, particularly against coagulation factors in the coagulation pathway, will greatly contribute to the clinical treatment and prevention of thrombotic diseases. Technical issues

[0006] The technical problem to be solved by the present invention is to provide a monoclonal antibody with anticoagulant activity and its application. The monoclonal antibody has the function of inhibiting the activity of the common coagulation pathway in the coagulation cascade reaction and can be applied to the prevention and treatment of thrombotic diseases. Technical solutions

[0007] This invention provides a monoclonal antibody or antigen-binding fragment thereof with anticoagulant activity, wherein the antibody or fragment includes a light chain variable region and a heavy chain variable region, the amino acid sequence of the light chain variable region of the antibody or fragment is shown in SEQ ID NO:1; and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:3.

[0008] Furthermore, the variable region of the light chain encodes the cDNA nucleotide sequence as shown in SEQ ID NO:2; the variable region of the heavy chain encodes the cDNA nucleotide sequence as shown in SEQ ID NO:4.

[0009] Furthermore, the monoclonal antibody is an antibody of IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, secretory IgA, IgD, or IgE.

[0010] Furthermore, the antigen-binding fragment is one or more of scFv, Fab, F(ab)2, and F(ab')2.

[0011] The present invention also provides eukaryotic or prokaryotic cells capable of producing the aforementioned antibody or antigen-binding fragments.

[0012] The present invention also provides the use of antibodies or antigen-binding fragments in the preparation of medicaments for the treatment or prevention of thrombotic diseases.

[0013] The present invention also provides a pharmaceutical composition for treating or preventing thrombotic diseases, comprising an effective therapeutic amount of the antibody or antigen-binding fragment having anticoagulant activity, and pharmaceutically acceptable excipients.

[0014] The present invention also provides a gene therapy drug for treating or preventing thrombotic diseases, comprising encoding a cDNA nucleotide sequence and a viral vector or non-viral vector packaging the encoding cDNA nucleotide sequence.

[0015] The monoclonal antibody provided by this invention can specifically inhibit the activity of the common coagulation pathway, thereby preventing and treating arterial and venous thrombosis and microvascular thrombosis in different locations.

[0016] After vascular integrity is compromised, the human body initiates the coagulation response through both the contact activation pathway and the tissue factor pathway. In the contact activation pathway, coagulation factors XII, XI, and prokinin are activated on negatively charged surfaces and generate activated coagulation factor IX (FIXa) through a cascade reaction. The enzyme complex formed by FIXa, its cofactor, and activated coagulation factor VIII (FVIIIa), along with the complex formed by activated factor VII (FVIIa) and tissue factor (TF) in the tissue factor pathway, cleaves coagulation factor X (FX) to become activated coagulation factor X (FXa), activating the common coagulation pathway. In the common coagulation pathway, FXa and activated coagulation factor V (FVa) catalyze prothrombin to form thrombin, which in turn cleaves fibrinogen into water-insoluble fibrin, leading to blood coagulation. The activity of the intrinsic coagulation pathway can be detected by activated partial thromboplastin time (APTT), while the activity of the tissue factor pathway can be measured by prothrombin time (PT). Both PT and APTT can be used to detect coagulation activity in the common pathway.

[0017] Traditional anticoagulants, such as heparin and warfarin, act on multiple components of the contact activation pathway, histogenetic pathway, and common coagulation pathway, lacking specificity in their anticoagulant effect. Furthermore, they can deplete coagulation potential by reducing the expression of coagulation factors, leading to a bleeding risk. Novel oral anticoagulants (NOACs), such as rivaroxaban, apixaban, edoxaban, and dabegatran etexilate, inhibit the activity of coagulation factors in the common pathway, but their half-lives are generally short, mostly only a few hours, requiring frequent dosing and causing significant fluctuations in drug concentration. While treating and preventing thrombosis, excessive coagulation inhibition often leads to side effects such as bleeding. The monoclonal antibody involved in this invention specifically acts on the common coagulation pathway to inhibit coagulation activity. When added to normal human plasma samples, it can simultaneously prolong the coagulation time in both APTT and PT tests in coagulation function tests. Although the monoclonal antibody involved in this invention inhibits coagulation activity by delaying the coagulation cascade, it does not completely deplete the body's coagulation potential. At the same time, due to the long half-life of the antibody, the drug concentration can remain relatively stable for a long period of time. Therefore, while effectively inhibiting coagulation activity to prevent and treat thrombosis, the antibody reduces the risk of bleeding caused by anticoagulation therapy, thus enabling safer and more effective prevention and treatment of thrombotic diseases.

[0018] The monoclonal antibody involved in this invention has a long in vivo half-life. The monoclonal antibody involved in this invention can inhibit coagulation activity for a longer period of time, thereby improving the cost of treatment and quality of life for patients. In some cases, the anticoagulant monoclonal antibody molecule can be molecularly modified to prolong its half-life in the human body. The method of prolonging its half-life can be to modify it by applying a chemical reaction to bind PEG molecules to the anticoagulant monoclonal antibody molecule, or by applying other molecules for binding modification. PEG modification can be achieved by activating an amino acid residue in the truncated hinge region of scFv, Fab, or Fab'2 molecules, as described in the (PEG)-lysylmaleimide scheme in the paper by Chapman AP et al. (Chapman AP, et al., 1999, Nature Biotechnology 17, 780-783). Details of other alternative methods can be found in many publications, such as Knight DM, et al., 2004, Platelets 15(7):409-18 and US Patent US5824784.

[0019] The monoclonal antibody involved in this invention is a fully human framework. The amino acid sequences, as well as their encoding DNA and mRNA sequences and corresponding cDNA sequences, are inferred directly or indirectly according to the principles of nucleic acid amino acid coding and translation. The direct methods include, but are not limited to, genomic DNA cloning, cDNA, cDNA libraries, single-cell sequencing, and transcriptome sequencing. The indirect methods include, but are not limited to, partially synthesized or de novo synthesized nucleic acid sequences, including DNA, mRNA, and cDNA, based on biological information provided by GeneBank or other publications and databases. Nucleic acid synthesis techniques, including DNA, mRNA, and cDNA, include, but are not limited to PCR-based nucleic acid synthesis methods.

[0020] The thrombotic diseases mentioned may include, but are not limited to: (1) prevention and treatment of hereditary and acquired thrombophilia; (2) prevention and treatment of ischemic stroke; (3) prevention and treatment of myocardial infarction; (4) prevention and treatment of coronary vascular diseases; (5) prevention and treatment of deep vein thrombosis after surgery; (6) prevention and treatment of portal vein and mesenteric arteriovenous thrombosis; (7) prevention and treatment of pulmonary embolism; (8) prevention and treatment of deep vein and superficial vein thrombosis; and (9) prevention and treatment of arterial thrombosis. (10) Prevention and treatment of intracranial venous sinus thrombosis; (11) Treatment of estrogen- and progesterone-related thrombosis and hypercoagulability; (12) Prevention and treatment of hypercoagulability and thrombosis during pregnancy, preconception and perinatal and lactation periods; (13) Prevention and treatment of tumor-related hypercoagulability and thrombosis; (14) Anticoagulation and thrombosis prevention in kidney disease and dialysis treatment; (15) Anticoagulation therapy during extracorporeal circulation, ECMO and other treatment processes; (16) Prevention and treatment of hypercoagulability caused by other reasons.

[0021] The anticoagulant monoclonal antibody or antigen-binding fragment of this invention can be produced by recombinant expression in host cells using genes encoding light and heavy chains. The host cells can be mammalian cells, or yeast, bacteria, insects, or other cell types. The light and heavy chains of the recombinantly produced anticoagulant monoclonal antibody can be expressed transiently or stably. Both expression strategies involving the cDNA fragments encoding the light and heavy chains of the anticoagulant monoclonal antibody include transfection (in mammalian and yeast systems) or transformation (in mammalian, yeast, insect, and bacterial systems, etc.) of host cells with one or more expression vectors containing cDNA fragments encoding the antibody light and heavy chains, thereby expressing the components of the anticoagulant monoclonal antibody, including the light and heavy chains, in the host cells. A preferred expression method is secretion into a culture medium, from which the antibody can be recovered using methods well-known to those skilled in the art, such as chromatography. The cDNA encoding the genes for producing the anticoagulant monoclonal antibody and its components is prepared using standard gene synthesis methods. The encoding cDNA is cloned into an expression vector, and the anticoagulant monoclonal antibody and its components are expressed after introduction into host cells with the assistance of appropriate gene expression regulatory elements.

[0022] The anticoagulant monoclonal antibody fragments in this invention can be prepared into single-chain Fv using molecular cloning techniques. Using PCR and nucleic acid synthesis methods, the Fab fragment encoding the anticoagulant monoclonal antibody is used to prepare VH- and VL-DNA coding region nucleic acid fragments. Then, the (Gly4)3 coding region is used as a linker to connect the two fragments into a complete molecule, allowing the VH and VL sequences to be expressed in E. coli as a single-chain Fv protein using the method described by McCafferty et al., 1990, Nature 348:552-554.

[0023] To recombinantly express the anticoagulant monoclonal antibody or antibody fragments described in this invention, the coding nucleic acid sequences of the light chain and heavy chain, or the fragments to be prepared, can be inserted into an expression vector. The expression vector must also contain gene expression regulatory sequences, such as promoters, enhancers, and 3' untranslated regions. The nucleic acid fragments encoding the light chain and heavy chain can be inserted into two separate vectors or into the same vector. The vector used to express the anticoagulant monoclonal antibody of this invention should also provide a signal peptide sequence for each expression frame, thereby promoting the secretion of the recombinantly expressed protein polypeptide from the host cell into the culture medium. The expression vector should have at least one origin of replication and one or more selection marker genes for the spontaneous amplification of the expression vector and for screening host cell clones expressing antibody molecules or fragments thereof.

[0024] The host cells used to express and prepare the anticoagulant monoclonal antibody or its antigen-binding fragment as described in this invention, such as Fab or scFv, include CHO cell lines with or without DHFR, mammalian-derived cell lines such as HEK293, insect cells, fungi, or bacteria.

[0025] The main steps and methods for the recovery and purification of anticoagulant monoclonal antibodies or their antigen-binding fragments, such as Fab or scFv, after recombinant expression production as described in this invention mainly include: (1) obtaining anticoagulant monoclonal antibodies or their antigen-binding fragments expressed in cells or secreted into the culture medium after cell lysis or collecting them in the cell supernatant, and removing cell nuclei, cytoplasmic debris, and impurities such as lipids after centrifugation and filtration. (2) using chromatographic techniques, including but not limited to affinity chromatography, ion exchange, molecular sieving, and ultrafiltration or dialysis, to enrich and purify recombinant protein molecules. Methods such as Protein A / G gel affinity chromatography; molecular sieving and ion exchange chromatography techniques can be used in combination before and / or after affinity chromatography to purify recombinantly expressed antibody molecules or their fragments, thereby obtaining high-purity anticoagulant monoclonal antibodies or their antigen-binding fragments.

[0026] The anticoagulant monoclonal antibody and its components of the present invention can be formulated into dosage forms suitable for clinical treatment and prevention of thrombosis and hypercoagulability, comprising an anticoagulant monoclonal antibody molecule, such as an antibody or antibody fragment, and one or more carrier substances acceptable for human use in clinical treatment and prevention, such as solvents, dispersion media, encapsulating agents, antibiotics, antifungal substances, isotonic substances, absorption-delaying substances, and other substances that can be safely applied to the human body. Acceptable carrier substances include one or more of the following: water, salts, phosphate buffers, dextran, glycerol, ethanol, etc., which can be used alone or in combination. Preferred isotonic agents are sugars, polyethanols such as mannitol, sorbito, or sodium chloride. Trehalose is the most preferred isotonic agent. The carrier may further contain trace amounts of auxiliary substances, such as wetting agents, emulsifiers, preservatives, or buffers, which help improve the shelf life or efficacy of the molecule.

[0027] The anticoagulant monoclonal antibody of the present invention can be prepared into various dosage forms, such as injections or infusions, dispersions, or suspensions. The preferred embodiment of the present invention is an injection or infusion, and the routes of administration include, but are not limited to, subcutaneous, intradermal, intramuscular, intravenous injection, and continuous intravenous infusion.

[0028] The encoding nucleic acid sequences of the anticoagulant monoclonal antibody molecules and their fragments of the present invention can be applied to gene therapy for the prevention and treatment of hypercoagulability and thrombosis. The vectors used for gene therapy can be non-viral or viral vectors. Non-viral vectors include, but are not limited to, liposomes. Viral vectors include adeno-associated virus vectors, adenovirus vectors, lentiviral vectors, retroviral vectors, etc. The anticoagulant monoclonal antibody molecules or their fragments are packaged into viral vectors using molecular cloning technology. These vectors are characterized by their ability to express the anticoagulant monoclonal antibody molecules or their fragments, and the ability to transfer the expressed gene into human tissue cells for in vivo expression. In vivo tissue cells include, but are not limited to, liver cells, blood cells, endothelial cells, etc. Beneficial effects

[0029] The anticoagulant monoclonal antibody of this invention inhibits the body's coagulation cascade reaction, effectively reducing coagulation activity and preventing and treating thrombotic diseases. Because the half-life of the monoclonal antibody can reach several weeks, the frequency of treatment is reduced, improving the patient's quality of life. While delaying the coagulation cascade reaction and inhibiting coagulation activity to prevent and treat thrombosis, the anticoagulant monoclonal antibody of this invention does not deplete the body's coagulation potential, reducing the risk of bleeding associated with anticoagulation therapy, thus enabling safer and more effective prevention and treatment of thrombotic diseases. Attached Figure Description

[0030] Figure 1 shows the expression vectors for transient or stable expression of the monoclonal antibodies of the present invention in mammalian cells (such as CHO, HEK293, etc.).

[0031] Figure 2 shows the dual transcriptomic expression vectors for transient or stable expression of the monoclonal antibodies of the present invention in mammalian cells (such as CHO, HEK293, etc.).

[0032] Figure 3 shows the inhibitory effect of the monoclonal antibody of the present invention on blood coagulation function by detecting IgG1 and IgG2 subtypes using partially activated thrombin time (APTT).

[0033] Figure 4 shows the inhibitory effect of the monoclonal antibody of the present invention on blood clotting function by detecting IgG1 and IgG2 subtypes through prothrombin time. Embodiments of the present invention

[0034] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0035] Example 1

[0036] Preparation of monoclonal antibodies with anticoagulant activity:

[0037] The heavy and light chains of the anticoagulant monoclonal antibody were expressed separately using pCDNA3.1 as an expression vector. These were then co-transfected into CHO cells to obtain a stable cell line containing the anticoagulant monoclonal antibody. The coding sequences for the variable regions of the heavy and light chains of the anticoagulant monoclonal antibody were cloned into the pCDNA3.1 expression vector. The construction details are shown in Figure 2. The amino acid sequence of the variable region of the light chain is shown in SEQ ID NO:1; the amino acid sequence of the variable region of the heavy chain is shown in SEQ ID NO:3; the nucleotide sequence of the cDNA encoded by the variable region of the light chain is shown in SEQ ID NO:2; and the nucleotide sequence of the cDNA encoded by the variable region of the heavy chain is shown in SEQ ID NO:4. The heavy and light chain expression vectors of the anticoagulant monoclonal antibody were transiently transfected into CHO cells. Transfection could be performed by electroporation, LipFamine 2000, or other transfection reagents, with three transfections performed simultaneously. Transfected cells were cultured under 50 nM G418 pressure for 2 weeks, allowing untransfected cells to die and transfected cell colonies to form. Transfected cell colonies were sequentially transferred to 24-well, 12-well, 6-well, and 6cm / 10cm culture plates for amplification. The obtained cells were cryopreserved or amplified for antibody expression identification. Selected cell lines were cultured in suspension in EX302 serum-free medium (JRH product), and their expression levels were further determined by ELISA, and cell growth rates were observed. Based on the antibody concentration in the harvested supernatant and acceptable growth characteristics, two optimal cell lines were selected for batch shake-flask culture in EX302 serum-free medium. Protein A HPLC analysis showed that each cell line produced full-length antibody molecules in yields ranging from 10 to 33 pg / cell / day. After isolating and purifying mRNA using an Invitrogen mRNA purification kit, reverse transcription was performed using standard methods. The obtained cDNA was cloned into pUC57, and positive clones were identified by colony PCR. DNA sequencing confirmed that both clones contained full-length light and heavy chain coding regions. The amino acid sequences deduced from the DNA sequencing results were consistent with expectations. The recombinant antibody was purified using the GE Healthcare Select / Capto S / Capto Q protocol.

[0038] Example 2

[0039] The anticoagulant activity of the monoclonal antibody of Example 1 was determined by applying the partially activated thrombin time (APTT) assay:

[0040] Plasma samples were obtained by centrifuging sodium citrate-anticoagulated blood samples at 2000g for 20 minutes. Twenty normal human samples were mixed to form the normal sample to be tested. The plasma sample was mixed with negatively charged substances, such as kaolin or tannic acid, and phospholipids, and incubated at 37°C for 3–5 minutes. Calcium ions were then added, and the time required for plasma coagulation was recorded. As shown in Figure 3, monoclonal antibodies with anticoagulant activity of IgG1 and IgG2 subtypes expressed from CHO cells were purified by affinity chromatography and added to the plasma sample. The APTT test showed a concentration-dependent prolongation of clotting time, demonstrating their anticoagulant biological activity and exhibiting a concentration-dependent inhibitory effect on coagulation function.

[0041] Example 3

[0042] The anticoagulant activity of the monoclonal antibody of Example 1 was determined by applying a prothrombin time (PT) test:

[0043] Plasma samples were obtained by centrifuging sodium citrate-anticoagulated blood specimens at 2000g for 20 minutes. Twenty normal human samples were then mixed to form the normal sample for testing. Tissue factor, phospholipids, and calcium ions were added to the plasma samples, and the coagulation time was recorded. As shown in Figure 4, monoclonal antibodies with anticoagulant activity from CHO cells, after affinity chromatography purification, were added to the plasma samples. The PT test showed a concentration-dependent prolongation of clotting time, demonstrating their anticoagulant biological activity and exhibiting a concentration-dependent inhibitory effect on coagulation function.

Claims

1. A monoclonal antibody or antigen-binding fragment thereof with anticoagulant activity, said antibody or fragment comprising a light chain variable region and a heavy chain variable region, characterized in that: The amino acid sequence of the variable region of the light chain of the antibody or fragment is shown in SEQ ID NO:1; the amino acid sequence of the variable region of the heavy chain is shown in SEQ ID NO:

3.

2. The antibody or fragment according to claim 1, characterized in that: The variable region of the light chain encodes the cDNA nucleotide sequence as shown in SEQ ID NO:2; the variable region of the heavy chain encodes the cDNA nucleotide sequence as shown in SEQ ID NO:

4.

3. The antibody or fragment according to claim 1, characterized in that: The monoclonal antibody is an antibody of IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, secretory IgA, IgD, or IgE.

4. The antibody or fragment according to claim 1, characterized in that: The antigen-binding fragment is one or more of scFv, Fab, F(ab)2 and F(ab')2.

5. Eukaryotic or prokaryotic cells, characterized in that, It can produce the antibody or antigen-binding fragment as described in claim 1.

6. The use of the antibody or antigen-binding fragment as described in any one of claims 1-4 in the preparation of a medicament for treating or preventing thrombotic diseases.

7. A pharmaceutical composition for treating or preventing thrombotic diseases, comprising an effective therapeutic amount of an antibody or antigen-binding fragment having anticoagulant activity as described in any one of claims 1-4, and pharmaceutically acceptable excipients.

8. A gene therapy drug for treating or preventing thrombotic diseases, comprising the cDNA nucleotide sequence encoded by claim 2 and a viral or non-viral vector packaging the cDNA nucleotide sequence.

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