Anti-prothrombin / phosphatidylserine antibody, pharmaceutical composition, and use

Abstract

Provided in the present invention is an anti-prothrombin / phosphatidylserine antibody, comprising a heavy chain variable region and a light chain variable region; the heavy chain variable region comprises HCDR1 to HCDR3 having amino acid sequences as shown in SEQ ID NOs: 1-3, respectively, and the light chain variable region comprises LCDR1 to LCDR3 having amino acid sequences as shown in SEQ ID NOs: 4-6, respectively. The anti-prothrombin / phosphatidylserine antibody of the present invention can bind to a prothrombin / phosphatidylserine complex with high affinity, thereby targeting and binding to phosphatidylserine, and has a potential therapeutic effect with respect to tumors, infections, atherosclerosis, ischemia-reperfusion injury, inflammatory bowel disease, Alzheimer's disease, Parkinson's disease, etc.

Description

Antiprothrombin / phosphatidylserine antibody, pharmaceutical composition and use TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to an antiprothrombin / phosphatidylserine antibody, a pharmaceutical composition and use. BACKGROUND

[0002] Phosphatidylserine (PS) is an anionic phospholipid composed of a glycerol backbone and two fatty acid acyl chains of varying length and saturation, as well as a phosphate group and a polar serine group. Different types of phospholipids in the bilayer phospholipid membrane of eukaryotic cells are asymmetrically distributed. Phosphatidylcholine (PC) and sphingomyelin mainly exist in the outer leaflet of the cell membrane, while phosphatidylserine (PS), phosphatidylethanolamine (PE) and phosphatidylinositol (PI) mainly exist in the inner leaflet or cytoplasmic lobule of the cell. PC and PE are zwitterionic phospholipids, with the highest content, accounting for 40-50% and 25% of the total phospholipid of the cell membrane, respectively. PS has a relatively low abundance (3-10% of the total phospholipid). Normal cells maintain the asymmetry of membrane components through ATP-dependent flippase, retaining PS on the inner side of the bilayer phospholipid membrane. In biological processes such as cell apoptosis and platelet activation, PS components are reversed and exposed to the outer side of the cell membrane under the action of scramblases such as transmembrane protein 16F (TMEM16F) and XK-related protein 8 (Xkr8). TMEM16F is a calcium-dependent phospholipid scramblase involved in the inward-to-outward reversal of PS in the membrane of activated platelets. Xkr8 is involved in the migration of PS to the outer surface of the membrane during apoptosis. The study of platelets and red blood cells first revealed the asymmetric distribution of membrane phospholipids in cells. Subsequent studies on more cell types have confirmed that the asymmetry of the inner and outer sides of membrane phospholipids is a universal phenomenon in all normal mammalian cells. The loss of membrane phospholipid asymmetry, especially the outward flipping of PS due to cell damage, activation and apoptosis, can trigger different signal transduction events, thus producing different biological results. Therefore, although PS only accounts for a small part of cell lipids, it plays an important role in various physiological or pathological processes. For example, PS-exposed cells and membrane vesicles will be recognized and phagocytosed by the reticuloendothelial system; at the same time, PS exposed on the membrane surface will bind to coagulation factors, promoting the coagulation cascade, leading to blood clotting and thrombus formation. These two biological functions are important for removing unwanted cells and wound healing.

[0003] Biological systems maintain the internal environment of an organism essentially stable in the face of external challenges through self-regulation. At homeostasis, the number of cells gained through cell division and / or migration inward equals the number of cells lost through cell death and / or migration outward. Apoptosis is a key homeostatic mechanism and the most common regulated cell death program (also including pyroptosis, necroptosis, ferroptosis, entosis, and immunogenic cell death, among others), and the process of phagocytic clearance of apoptotic cells is referred to as “phagocytosis”. Apoptosis controls cell numbers both through cell suicide and through effects on other cells to influence cell, tissue, and organism homeostasis.

[0004] During apoptosis, PS is translocated to the inner leaflet of the cell membrane and flips outside before the loss of membrane integrity of the apoptotic cell, thus providing a “eat me” signal for phagocytes (e.g. macrophages, dendritic cells, or epithelial cells) to be phagocytosed (phagocytosis) and cleared before the apoptotic cell loses membrane integrity and releases enzymes, avoiding the activation of the immune system by stimulating and toxic components such as oxidants into the surrounding tissue and preventing the development of a significant inflammatory response.

[0005] Phagocytes recognize PS in apoptosis through direct interaction with PS receptors and / or indirect interaction through bridging molecules. PS-specific receptors include Stabilin-2, brain-specific angiogenesis inhibitor 1 (BAI1), and the T cell immunoglobulin and mucin (TIM) receptor family. Stabilin-2 is a multifunctional scavenger receptor expressed on macrophages that recognizes PS in a calcium-dependent manner. BAI1 is another important receptor that can act as a phagocytic receptor for apoptotic cells. It is expressed on macrophages and can bind to PS as well as cardiolipin and phosphatidic acid (PA). The TIM receptor family, which includes TIM-1, TIM-3, and TIM-4, has been identified as a pattern recognition receptor that contains a binding region specific for PS. The TIM receptors differ in structure and expression, suggesting that they have different functions in regulating immune responses. TIM-1 has been shown to be expressed on T helper 2 (Th2) cells and acts as a costimulatory molecule for T cell activation. TIM-3 is expressed on T helper 1 (Th1) cells, CD8+ T cells, and a subset of activated CD4+ T cells. There is also expression of TIM-3 on subsets of macrophages and dendritic cells (DCs). Binding of PS to TIM-3 generates an inhibitory signal that leads to apoptosis of these cell types. Direct interaction of PS with TIM-4 can be the most important of this receptor family in apoptosis. TIM-4 is expressed only on antigen-presenting cells (APCs), such as macrophages and DCs, and is a key mediator of apoptotic cell uptake. The binding site for PS is located in the extracellular IgV-like domain of the TIM-4 molecule. Structural analysis indicates that there is a metal ion-dependent binding pocket for PS, and the binding region of TIM-4 specifically recognizes serine groups of PS molecules. The PS-TIM-4 interaction is critical for the uptake and removal of apoptotic cells. Binding of PS to the TIM-4 receptor depends on the surface density and expression level of PS, and the binding of PS to TIM-4 increases with increasing molar percentage of membrane surface PS.

[0006] Because of the important role of PS components on the cell membrane in biological events such as hemostasis and apoptosis, exploring this biological phenomenon will promote the development of treatment strategies for diseases such as bacterial and viral infections, cancer, hemostasis, and autoimmunity.

[0007] Cancer comprises over 100 diseases, caused by genetic mutations that result in uncontrolled proliferation of cells. Normal living cells exist primarily in intracellular lobules at the lipid bilayer membrane of the cell as phosphatidylserine (PS). Due to elevated intracellular calcium ion levels, reduced activity of ATP-dependent phospholipid translocase (flippase) and / or elevated activity of phospholipid scramblase in tumor cells, cancer cells are unable to maintain the asymmetry of PS, resulting in PS exposure on the cell membrane surface. The degree of PS flipping on the cell membrane varies significantly among different types of cancer, and even within the same cancer type. There is a positive correlation between the surface PS of tumors and their malignancy, and studies have shown that radiation exposure induces accelerated PS flipping in tumor cells with low or moderate levels of surface PS in vitro and in vivo, in a time- and dose-dependent manner. In animal experiments, radiation treatment increased surface PS of tumor cells in subcutaneous xenografts in nude mice, a process that can reflect early signs of apoptosis, but can also be independent of the apoptotic program of tumor cells. The appearance of surface PS on tumors provides a target for marker diagnosis and treatment of tumors.

[0008] Tumor cells, while affecting host cells, also depend on a complex support network of "normal" cells, blood vessels, nerves, tissue matrix components, and extracellular signaling factors in the organ tissue for expansion and progression. Due to the excess of tumor proliferation over the necessary supply of oxygen and nutrients, which triggers massive tumor cell death, malignant tumors often exhibit histologically distinct necrotic areas of significant size. In addition to the cell loss and relatively high levels of apoptosis observed in aggressive, fast-growing cancers, it is also noteworthy that slow-growing, highly proliferative tumors can occur, in which the number of tumor cells obtained by mitosis is almost completely balanced by the number of apoptotic cells. Although apoptosis is generally "quiet" from the perspective of acute inflammation, its processes and effects are far from silent. On the contrary, actively dying cells can release powerful signals to the surrounding tissues and cells, with far-reaching effects. Since the proliferation and expansion of tumor cells require the support of the tumor microenvironment (TME), which includes resident and recruited stromal cells and various tissue matrix components. Stromal cells include endothelial cells, fibroblasts, and immune stromal cells such as T and B lymphocytes, NK cells, macrophages and other myeloid cells, mast cells, and platelets. Since the TME is a key supportive feature of all cancers (primary, metastatic, and recurrent), it provides a target for anti-cancer therapy.

[0009] Apoptotic phosphatidylserine (PS) egresses to the cell surface to deliver phagocytic signals, which are bound by serum proteins and opsonins that promote extensive pinocytosis. Under physiological conditions, egressed PS acts as a dominant and evolutionarily conserved immunosuppressive signal that promotes tolerance and prevents local and systemic immune activation. PS exposure on cancer cells also leads to immunosuppression in the tumor microenvironment, enhances the activity of natural killer cells and dendritic cells, and converts tumor-associated macrophages (TAMs) into anti-inflammatory (M2) macrophages. Myeloid-derived cells infiltrating tumor tissues that express PS receptors, including TAMs and TIM receptor family, express immunosuppressive cytokines and suppress anti-tumor immune responses upon binding to PS or PS bridging molecules in the tumor microenvironment. The combined effects of PS and PS receptors can provide a “perfect storm” that exacerbates tumor immune escape. PS exposure on microvesicles (exosomes) from patient tumor samples also inhibits the activation of T cell responses. Furthermore, PS is significantly increased in tumors after chemotherapy and radiotherapy, which further enhances PS-mediated immunosuppression.

[0010] Chemotherapy, radiotherapy, and surgery are the traditional mainstay of cancer treatment, but with a deeper understanding of the role of the immune system in cancer development and progression, immunotherapy has emerged as a new treatment modality for cancer. Immune checkpoint inhibitors are the most thoroughly studied class of immunotherapy to date. The two most common checkpoint inhibition strategies are PD-1 / PD-L1 blockade and CTLA4 inhibition. Physiologically, immune checkpoints maintain appropriate immune responses and protect healthy tissues from immune attack. Macrophage phagocytosis of apoptotic cells in damaged tissues induces immunosuppression, leading to immune tolerance. NK cells and tumor-specific T cells are the two main immune cell types responsible for anti-tumor immunity, but their activity can be inhibited by immune checkpoints such as programmed death-1 (PD-1) and indoleamine 2,3-dioxygenase (IDO). PD-1 is mainly expressed on activated T cells and NK cells, and its ligand is mainly expressed on tumors and myeloid cells, and PD-L2 is mainly expressed on dendritic cells (DCs), which inhibit the killing function of effector immune cells. In addition, IDO converts tryptophan into many immunosuppressive molecules, which subsequently inhibit the function of T cells and NK cells. Recently, immune checkpoint inhibitors (ICIs) have demonstrated their therapeutic effects in cancer.

[0011] Disregulation of PS in the tumor microenvironment as described above suggests that targeting PS can also act as an immune checkpoint inhibitor, roughly in the same way that blocking PD-L1 and CTLA4 prevents inhibitory signals in T cells, inhibition of PS signaling can also prevent PS-mediated tumor immune suppression. Preclinical studies with the naturally occurring PS ligand Annexin A5 (AnxA5) suggest that systemic administration of AnxA5 or other PS ligands can slow tumor progression by blocking the tumor-supporting properties of apoptotic cells and tumor-derived microvesicles. In combination with radiotherapy or chemotherapy, AnxA5 can be used as a natural adjuvant to increase the immunogenicity of dying tumor cells, thereby promoting an anti-tumor immune response. Incubation of apoptotic cells with AnxA5 has been shown to dramatically increase the immunogenicity of these cells. Thus, AnxA5 disruption of PS-derived signals from apoptotic tumor cells can trigger a pro-inflammatory response, leading to a specific immune response against tumor cells. AnxA5 reduces the uptake of apoptotic cells by peritoneal macrophages, increases the uptake of apoptotic cells by DCs, and enhances the immunogenicity of irradiated lymphoma cells in vivo. Monoclonal antibodies targeting PS also show preliminary anti-tumor activity, particularly when used in combination with immune checkpoint blockers (anti-cytotoxic T-lymphocyte-associated protein 4 [anti-CTLA-4] and anti-PD-1).

[0012] Drugs targeting the tumor vasculature have become one of the most commonly used molecular targeted therapies in oncology. Tumor cells cannot obtain sufficient blood supply from vessels more than 100-200 pm away, and effective targeting of the tumor vasculature can cause the death of downstream cancer cells that rely on this vascular supply. Drugs developed against blood vessels have been approved for the treatment of the most common malignancies, such as lung cancer, colorectal cancer, kidney cancer and liver cancer, and primary brain tumors. Since vascular endothelial cells are constantly exposed to the blood circulation, sufficient drug delivery can be ensured. At the same time, compared with cancer cells, vascular endothelial cells are also less likely to mutate, thus producing mutations that are resistant to treatment. There are mainly two strategies for treatment against vascular endothelium, namely anti-angiogenic drugs and vascular damaging drugs (VDA). Anti-angiogenic drugs, such as anti-VEGF monoclonal antibody bevacizumab (Avastin®; Genentech [CA, USA]) and VEGF receptor (VEGFR) tyrosine kinase inhibitors sunitinib (Sutent®; Pfizer [NY, USA]) and sorafenib (Nexavar®; Bayer [Germany] and Onyx [CA, USA]) inhibit peripheral neovascularization of tumors. Currently, no VDA has been approved by the US FDA for cancer treatment.

[0013] Notably, in addition to cancer cells in tumors, vascular endothelial cells in the TME also have a high cell membrane surface PS. Studies suggest that PS is a highly specific marker of functional, living tumor endothelium. Conditions in the tumor microenvironment include many factors that can activate and / or damage tumor vascular endothelial cells (ECs), as follows: (a) tumor-derived interleukin-1 and tumor necrosis factor activate endothelium and induce expression of cell adhesion molecules; (b) reactive oxygen species (ROS) produced by leukocytes adhering to tumor endothelium; (c) products of tumor cell metabolism or ROS produced after reoxygenation following hypoxia. Inflammation cytokines, acidosis, thrombin, hypoxia / reoxygenation, and ROS all induce PS exposure on ECs in vitro. Radiotherapy and chemotherapy are common treatments for malignancies, and radiation and chemotherapeutic drugs also increase surface PS on tumor vessels. This tumor or / and tumor-associated mechanism leading to EC cell membrane surface PS redistribution and eversion also provides a target for treatment of tumor vascular endothelium, triggering tumor-associated endothelial cell death and thus tumor extinction.

[0014] PS has been a unique therapeutic target due to its multiple biological effects. The main experimental PS-targeting agents that have been used for cancer localization and treatment include saposin C-dioleoylphosphatidylserine (SapC-DOPS, a PS-targeting nanovesicle composed of saposin C (SapC, a lysosomal protein) and dioleoylphosphatidylserine (DOPS)), bavituximab, PPSD1 (a PS-binding peptide-peptoid hybrid), and BPRDP056 (a zinc-dipyridyl-SN38 conjugate). Among them, peptoids are potential drug leads targeting various cancer targets. The combination of gemcitabine (GEM) and SapC-DOPS for the treatment of pancreatic ductal adenocarcinoma (PDAC) can be observed with the increase of surface PS as the cell cycle progresses from G1 to G2 / M. At the same time, GEM preferentially targets G1 cells with low surface PS, resulting in an increase in the median surface PS level of PDAC cells; in contrast, SapC-DOPS preferentially targets high surface PS cells mainly in the G2 / M phase. The combination of SapC-DOPS and GEM in the treatment of subcutaneous and orthotopic PDAC tumors in vivo can significantly inhibit tumor growth and improve survival. However, small molecule polypeptides, including the natural PS ligand annexin V (molecular weight 36 KD), are rapidly metabolized into urine from the circulatory system, with a half-life (t 1 / 2) of only 20 minutes or less, thus limiting their clinical application. Artificially prepared human annexin V recombinant homodimer Diannexin has a larger molecular weight (73 KD), which exceeds the kidney filtration threshold, so its half-life in circulation is significantly prolonged (t 1 / 2>2 hours), but cardiac and pulmonary toxicities were found in rats and monkeys receiving treatment in preclinical studies, although such findings have not been observed in human subjects receiving Diannexin.

[0015] Monoclonal antibodies are powerful tools for treating diseases, which induce target cell death through effector functions such as antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) by binding to specific targets through Fab domains and binding to Fc gamma receptors (FcγRs) on the surface of immune cells or complement factors through Fc domains. Another Fc-dependent antibody effector function is antibody-dependent cellular phagocytosis (ADCP), which is a mechanism by which target cells are phagocytosed and degraded by macrophages under the action of FcγR on the surface of macrophages. Therefore, monoclonal antibodies targeting PS are also one of the ideal strategies for tumor treatment.

[0016] Antibodies to PS belong to the broad category of antiphospholipid antibodies (aPLs), which are present in patients with a variety of autoimmune diseases, particularly systemic lupus erythematosus (SLE) and primary antiphospholipid syndrome (APS). However, these autoantibodies are highly heterogeneous, with different target specificities and affinities, recognizing various combinations of phospholipids and / or phospholipid-binding proteins. Although aPLs were initially thought to directly target anionic phospholipids, it is now recognized that aPLs mainly recognize phospholipid-binding proteins that interact with anionic phospholipids, such as β2-glycoprotein I (β2GPI), prothrombin (PT), and annexin V. Some aPLs can recognize both free and phospholipid-bound phospholipid-binding proteins, but some aPLs only recognize phospholipid-binding proteins bound to the surface of phospholipids or the combination of phospholipids and phospholipid-binding proteins. Some aPLs interfere with coagulation and prolong coagulation time in in vitro coagulation tests, and are called lupus anticoagulants (LA). Although LA prolongs coagulation time in in vitro coagulation tests, including activated partial thromboplastin time (APTT) assays, most patients with LA do not show a bleeding tendency, and even some patients suffer from thrombotic diseases. The aPLs that cause LA are divided into two major categories: β2GPI-dependent antibodies and prothrombin (PT)-dependent antibodies. The outer leaflet of the plasma membrane of apoptotic cells contains anionic phospholipids, including phosphatidylserine (PS) and other phospholipid types (such as cardiolipin), which are not normally present on the surface of living cells, thus providing an appropriate microenvironment for phospholipid-binding proteins that interact with phospholipids on the surface of apoptotic cells. Therefore, although the mechanism of natural aPL production in autoimmune diseases is not yet clear, apoptotic cells can be one of the antigens that induce aPLs, and more and more evidence suggests that apoptotic cells are also involved in the initiation and / or maintenance of autoimmunity.

[0017] β2-glycoprotein I and prothrombin are the major phospholipid binding protein types that bind PS and cause the LA phenomenon. β2-glycoprotein I (β2GPI) is a plasma protein, also known as apolipoprotein H, which is a highly glycosylated single chain molecule consisting of 345 amino acid residues (including a 19-amino acid N-terminal signal peptide). Although β2GPI has a high concentration in plasma (0.15-0.30 mg / mL, 3-6 μM), its biological function is not well understood and can be related to triglyceride-rich lipoprotein metabolism, platelet function, and coagulation responses. Prothrombin is also a high concentration protein in circulation (~1.5 uM), which is a single chain vitamin K-dependent glycoprotein containing about 10% glycosyl modifications and 10 carboxyglutamic acid residues, the latter of which are involved in calcium ion binding and are mostly, if not all, required for prothrombin to bind completely to cell membrane phospholipid surfaces. The prothrombin structure can be divided into three domain fragments F1 and F2 and prethrombin 2, with the carboxyglutamic acid residues in the GLA domain in F1 being responsible for binding to phospholipids. Under physiologically more relevant conditions (120 mM NaCl and 3 mM CaCl2), prothrombin binds to PS-containing phospholipid membranes with a strong affinity through its carboxyglutamic acid residues in the GLA domain (the dissociation constant Kd of prothrombin to phospholipids is 10 -8 M ~ 10 -6 M in phospholipid membranes containing different proportions of PS). Fluorescein-labeled PT (FITC-PT) specifically binds to apoptotic Jurkat T cells induced by staurosporine in the presence of calcium chloride (CaCl2), but not to live cells, demonstrating that PT itself is capable of binding to the surface of apoptotic cells, which also constitute the antigenic target of PT-dependent aPLs. Under physiological calcium ion concentrations, the affinity of β2GPI for anionic phospholipids is much lower than that of vitamin K-dependent coagulation factors. In comparison to prothrombin, β2GPI binds to PS-containing phospholipid membranes with a much weaker affinity, with dissociation constants (Kd) of 3.9 and 14 μM for phospholipid membranes containing 20% and 10% PS, respectively, and since the plasma concentration of β2GPI is about 4 μM, β2GPI is only likely to bind very weakly to PS-containing phospholipid membranes under physiological conditions. Studies have shown that aPL antibodies against β2GPI and prothrombin enhance the binding of these protein molecules to negatively charged PS phospholipids.

[0018] Because PS is a lipid molecule, it is not very immunogenic, so most PS-targeting mAbs bind to PS by binding to a phospholipid-binding protein that then binds to PS. 3G4, which was generated by immunizing mice after inducing PS exposure on mouse endothelial cells (ECs) using H2O2, is a mouse mAb that targets PS phospholipids. 3G4 does not directly bind to PS molecules, but it can bind to free β2-glycoprotein 1 (β2GP1) with high affinity, where binding of both Fab binding sites in one 3G4 antibody to a β2GP1 molecule greatly enhances the affinity of β2GP1 for the phospholipid PS. Thus, 3G4 can target anionic phospholipid PS by binding to β2GP1. 3G4 was originally produced in hybridoma supernatant, then converted to mouse IgG2a isotype, and a human IgG1 chimeric version of 3G4 (ch3G4) was constructed by replacing the human Fc domain (trade name Bavituximab). The binding of Bavituximab to PS and β2GP1 blocks PS-mediated immunosuppressive signaling from tumor cells, thereby activating T cell-driven immune pathways. Bavituximab binds to Fcγ receptors on myeloid-derived suppressor cells (MDSCs), M2 macrophages, and immature dendritic cells, leading to increased production of TNFα and IL-12 immune-stimulating cytokines. Thus, it induces differentiation of MDSCs into M1-like macrophages and dendritic cells, thereby inducing tumor-specific cytotoxic T cells. Histological evaluation of tumors treated with 3G4 showed increased infiltration of host immune effector cells (mainly macrophages), with increased vascular collapse, reduced vascularization, and increased central tumor necrosis. Bavituximab-paclitaxel combination therapy has been used in a phase I clinical trial for the treatment of HER2-negative breast cancer. The treatment was well tolerated, with an overall response rate of 85%. However, because β2GP1 is present in high levels in circulation (~200 ug / mL), and Bavituximab binds to free β2GP1 with high affinity, as does the 3G4 mouse mAb, when the concentration of Bavituximab antibody is increased, the antibody will have more opportunities to bind only one β2GP1 molecule rather than form a complex of one antibody with two β2GP1 molecules with high affinity for PS, so the therapeutic window of Bavituximab is limited. At the same time, increasing the concentration of Bavituximab antibody increases the risk of bleeding and thrombosis caused by its anti-β2GP1, which is one of the safety concerns in its therapeutic application.

[0019] Imaging plays an important role in individualized cancer diagnosis and treatment, such as assessing tumor stage, detecting disease recurrence, monitoring treatment progress or post-treatment monitoring. Cancer biomarker targeting strategies show considerable promise in cancer treatment. Cancer biomarkers are also important molecular features of cell phenotypes that help detect cancer, even at an early stage. Biomarkers can be proteins (EGFR and HER2), nucleic acids (miR-2, miR-155, BRCA1, DAPK1 and MGMT), lipids (phosphatidylserine (PS)), glycoproteins (a-fetoprotein and CA125) or carbohydrates (CA19-9). PS has shown promise in developing non-invasive imaging techniques to support the diagnosis and treatment effect evaluation of cancer, inflammation and cardiovascular disease. Due to its biological flexibility, PS is generally superior to other membrane lipids in imaging and treatment, and therefore, labeled monoclonal antibodies or other PS targeting agents are one of the ideal tools for imaging diagnosis and monitoring of tumors.

[0020] In addition to diagnosis and treatment of tumors, PS-associated phospholipid redistribution is also of great significance in infectious diseases. Although "silent clearance" of apoptotic cells is necessary to maintain homeostasis, non-inflammatory apoptosis in some parasitic, viral and bacterial infections can be detrimental to the host. Pathologically, PS-extrusion-associated innate immune suppression has been hijacked by numerous viruses, microorganisms and parasites to protect themselves to facilitate infection and latency. Viruses have evolved many strategies to subvert host cells to ensure their successful infection and replication, among which, viruses mimic apoptotic debris by concentrating PS in their membranes (enveloped viruses) or hiding themselves in cell-derived PS-containing vesicles (non-enveloped viruses) to hijack the immune system's apoptotic recognition and clearance mechanisms by mimicking apoptosis to facilitate viral binding, invasion of cells and evasion of immune surveillance and clearance. Viral apoptosis mimicry has been shown to be a widely used lipid-mediated mechanism of viral cell entry, including enveloped viruses such as vaccinia virus, Pichinde virus, cytomegalovirus, Lassa fever virus, lentivirus, dengue virus, Ebola virus and Marburg virus, and non-enveloped viruses such as SV40, hepatitis A and polio, which all use this mechanism to infect the body. In addition to facilitating uptake and binding, by mimicking the apoptotic mechanism, viruses also inhibit the host innate immune response to enhance their own infection. Apoptosis mimics formed during viral infection, including those from highly pathogenic viruses such as Ebola and dengue, are one of the targets for anti-viral infection. Treatment of animals infected with Pichinde virus with PS-targeting antibodies can protect animals from fatal viral infection in vivo. In vitro studies have shown that the infectivity of HIV-1 to human macrophages is reduced in the presence of the natural ligand of PS, AnxA5; and PS-targeting antibodies effectively inhibit HIV-1 infection of peripheral blood mononuclear cells by promoting the upregulation of chemokines that are known to block receptors used by HIV-1 for cell invasion. PS and the non-phospholipid component of the hepatitis B virus (HBV) envelope are involved in AnxA5 binding and HBV infection. PS-targeting antibodies can bind to a variety of enveloped viruses and enveloped virus-infected cells, including Ebola virus, influenza virus and vaccinia virus. Recent studies have shown that PS is associated with non-enveloped viral infection, suggesting that PS-targeting antibodies can also be used to treat such infections, reducing the mortality rate of influenza and RSV in vivo.

[0021] Similar to viruses, bacteria also exploit phagocytosis in host cells to facilitate intercellular spread. For example, macrophages infected with Listeria monocytogenes package the bacteria in PS-coated vesicles and expel them from the cell membrane, which then facilitate uptake and intercellular spread of Listeria monocytogenes by interacting with TIM-4 on other macrophages. Other bacterial pathogens with similar infection mechanisms include Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium marinum, and Chlamydia, among others. In fact, phagocytosis appears to be an effective mechanism for host control of mycobacterial species, highlighting the delicate balance between host cell defense and pathogen transmission mechanisms.

[0022] In addition to viruses and bacteria, there is growing evidence that various protozoa also use PS for apoptosis mimicry and immune evasion as part of their infection life cycle. Exposure of PS on the surface of parasites plays an important role in host / parasite interactions during the infection process of Trypanosoma cruzi, Trypanosoma brucei, and Leishmania amazonensis, which cause Chagas disease, African trypanosomiasis, and leishmaniasis, respectively. Exposure of PS contributes to the infectivity of the parasites. Therefore, drugs targeting PS will likely inhibit the infection and spread of the parasites.

[0023] During the hemostatic response, platelets are activated on the surface of damaged tissue, and the intracellular Ca2+ level continuously increases in the micromolar range, leading to the activation of scramblase, which rapidly and non-specifically reverses the asymmetric distribution of phospholipid components in the platelet plasma membrane, transferring PS from the inner leaflet to the outer leaflet of the cell phospholipid bilayer membrane, providing a reaction site for components of the coagulation cascade, including coagulation factors IX, VIII, X, V, and prothrombin. The enzyme complex composed of activated coagulation factor X (FXa) and its cofactor, activated coagulation factor V (FVa), bound to the PS on the surface of activated platelets catalyzes the conversion of prothrombin bound to the membrane surface PS into thrombin, which converts fibrinogen into fibrin to promote blood clotting. The natural ligand of PS, Annexin V, can bind to phospholipids, thereby inhibiting the coagulation reaction. Anti-β2GPI and anti-prothrombin antibodies in aPL antibodies can interfere with the coagulation reaction to cause LA, but the mechanism by which anti-β2GPI prolongs the coagulation time in coagulation function tests with low phospholipid levels, such as APTT and dRVVT, is not fully understood, while it is generally believed that anti-prothrombin antibodies prolong coagulation time by competing with coagulation factors for binding sites on phospholipids. Although LA is considered to be associated with thrombosis, some anti-prothrombin antibodies (anti-PS / PT) can not only cause abnormalities in in vitro coagulation tests but also reduce coagulation function in patients, leading to a bleeding tendency.

[0024] Uncontrolled PS externalization on apoptotic, activated and damaged cells can lead to thromboembolic and other pathological consequences. Apoptosis occurs in human atherosclerotic plaques. Endothelial cell apoptosis is thought to be a key step in the transition from stable endothelialized plaques to plaque erosion and thrombosis. Rupture of vulnerable plaques leads to exposure of the lipid core, which is highly enriched in tissue factor (TF) and PS externalized apoptotic microparticles required for thrombosis, leading to acute coronary syndrome and acute cerebrovascular events. Thrombosis is a common complication of cancer. TF released by tumor cells, and PS externalized on tumor cells and tumor vasculature are two key factors that contribute to the risk of cancer-associated thrombosis. Increased apoptosis during cancer therapy elevates TF levels and accessibility of PS, further increasing the risk of thrombosis. Increased TF-positive, PS-externalized cells and microparticles due to cell activation and apoptosis have also been reported in many other pathological states associated with thrombosis, including heparin-induced thrombocytopenia, complicated diabetes mellitus, severe hypertension, end-stage renal disease, antiphospholipid syndrome, systemic lupus erythematosus, sickle cell disease, paroxysmal nocturnal hemoglobinuria, and thrombosis associated with biomaterials. Drugs targeting PS would be beneficial for the prevention and treatment of thrombosis associated with these diseases.

[0025] In the pathogenesis of Alzheimer's disease, the natural ligand of PS, Annexin V, was found to protect choroid plexus cells from amyloid beta (Aβ)-induced toxicity. These cells are part of the special brain structure that produces cerebrospinal fluid (CSF). At the same time, the interaction of PS with Annexin V also reduced the misfolding and fibril formation of alpha-synuclein, which plays a key role in the pathogenesis of Parkinson's disease. Therefore, drugs targeting PS can also play a role in the treatment of Alzheimer's disease and Parkinson's disease.

[0026] Inflammatory bowel diseases (IBD) are chronic relapsing diseases of the gastrointestinal tract, characterized by intestinal inflammation and epithelial damage, known as ulcerative colitis (UC), Crohn's disease (CD) and indeterminate colitis (IC). Inflammatory bowel disease is a chronic disease that can relapse and affect the health and quality of life of patients. The treatment of IBD is mainly anti-inflammatory and immunomodulatory drugs, which can improve disease-related symptoms by inhibiting intestinal inflammation. Inhibition of tumor necrosis factor (TNF) can block the up-regulation of pro-inflammatory signals or molecules by TNF-α, which is a common treatment for IBD in clinical practice. Some patients do not respond well to existing clinical drugs for IBD, and cannot effectively control symptoms and cure. At the same time, long-term use of anti-TNF drugs may cause skin damage, immune response, infection and tumor and other side effects. The infiltration of inflammatory cells plays an important role in the pathogenesis of IBD. The interaction between leukocytes and endothelial cells in the blood is initially induced by adhesion molecules VCAM-1 and ICAM-1, and studies have shown that the expression of VCAM-1 and ICAM-1 in the colon of IBD patients is up-regulated, so blocking the transport of lymphocytes to the intestinal mucosa mediated by adhesion molecules is one of the strategies for developing new IBD treatment drugs. Phosphatidylserine (PS) is asymmetrically distributed in normal cells, and under the stimulation of external conditions such as hypoxia, inflammation and infection, the asymmetry of PS distribution is lost and flipped to the outside of the cell membrane. The above pathophysiological changes are widespread in IBD, and studies have found that there is flipped phosphatidylserine (PS) on the surface of colon capillary endothelial cells. Annexin A5 (ANXA5) with high affinity to PS can bind to the surface of colon capillary endothelial cells. Experimental studies have shown that PS high-affinity wild-type ANXA5 has good targeting to the colon, effectively inhibiting the recruitment, adhesion and infiltration of early inflammatory cells, and relieving TNBS-induced experimental colitis. In contrast, ANXA5 mutants lacking PS binding ability do not accumulate in the colon and have no therapeutic effect on colitis. Mechanism exploration shows that ANXA5 induces TLR4 internalization in PS-dependent endocytosis, down-regulates TLR4 on the surface of endothelial cells, weakens NF-κB activation, inhibits LPS-induced HUVEC (human umbilical vein endothelial cells) activation, and reduces the induced expression of cytokines, chemokines, adhesion molecules VCAM-1 and ICAM-1. Although the application of ANXA5 in IBD animal models confirms that PS is an effective target for the treatment of IBD, the defects in the pharmacokinetics of ANXA5 and its possible accumulation in the liver and kidneys limit the feasibility of its clinical application.

[0027] Ischemia reperfusion injury (IRI) is a common and serious complication of reperfusion after vascular occlusion. Studies suggest that hypoxia caused by ischemia causes phosphatidylserine to evert to the surface of endothelial cells (ECs), allowing inflammatory cells and platelets to attach and aggregate, impeding blood flow. During IRI, mediators are produced that increase vascular permeability, leading to edema, which complicates peripheral vascular surgery, stroke, myocardial infarction, and other clinical conditions, including organ transplantation, resulting in serious consequences such as organ dysfunction.

[0028] Ischemia reperfusion injury is one of the main pathological mechanisms of organ damage caused by hemorrhagic shock, hepatectomy (Pringle maneuver), and temporary clamping of the hepatoduodenal ligament after liver transplantation. Despite improvements in organ preservation, surgical techniques, and immunosuppressive regimens, ischemia reperfusion injury remains an important problem in clinical organ transplantation, and IRI has important implications for early and late dysfunction of liver grafts. Studies using in vivo microscopy on a mouse model of early IRI showed that, unlike the flat endothelial cells (ECs) in the blood sinuses of normal livers, which have a high blood flow rate, within 1-3 hours after ischemia reperfusion, the ECs become rounded and partially obstruct the sinusoidal lumen. At the same time, leukocytes and platelets adhere to the ECs, further impeding blood flow in the sinuses, causing blood flow to almost completely stop. These early pathophysiological changes progress to late IRI, which ultimately leads to hepatocyte damage. Effective inhibition of early IRI events reduces the severity of vascular and end-organ damage. Studies have found that the pathogenesis of liver IRI is related to Kupffer cell activation, pro-inflammatory cytokine release, increased expression of adhesion molecules, leukocyte infiltration, and microthrombosis. Studies have shown that a major early event in the pathogenesis of murine liver IRI is the ejection of phosphatidylserine (PS) from the sinusoidal ECs due to hypoxia and reoxygenation, so targeting PS for treatment is one of the strategies for reducing IRI and organ damage. Animal studies using recombinant human membrane annexin V homodimer (Diannexin), which has a high affinity for PS, showed that Diannexin, by binding to cell surface PS, inhibited the attachment of leukocyte and platelet aggregates to ECs, inhibited early IRI events, maintained sinusoidal blood flow in the mouse liver during ischemia reperfusion, and significantly reduced hepatocyte damage 24 hours later, preventing IRI associated with liver transplantation. Therefore, therapeutic intervention strategies that block the early stages of IRI, which promote leukocyte recruitment and damage microvascular blood flow, to prevent and protect end-organ damage, may be more effective than inhibiting late events (such as leukocyte recruitment, inflammatory mediator formation, or caspase action in apoptosis).

[0029] Primary graft dysfunction (PGD) is a clinical manifestation of acute phase ischemia reperfusion injury (IRI) after lung transplantation, characterized by marked deterioration of gas exchange and chest radiographic infiltrates. PGD is a major cause of early morbidity and mortality, and is associated with decreased long-term survival, impaired physiological function, and the development of chronic lung allograft dysfunction. There is currently no effective treatment for this disease. In the early reperfusion phase, there are a large number of apoptotic cells in human and animal transplanted lungs. The phosphatidylserine (PS) everted in the initial stage of IRI due to hypoxia or early apoptosis in the endothelium can attract the attachment of leukocytes and activated platelets, thereby causing slow blood flow and the development of a local pro-inflammatory and pro-coagulant microenvironment. Therefore, the combination of hypoxia or phosphatidylserine everted on the surface of apoptotic cells becomes one of the ideal strategies for treating IRI after lung transplantation. Studies using recombinant human membrane-associated annexin V homodimer (Diannexin) show that diannexin treatment can reduce the expression of pro-inflammatory cytokines and adhesion molecules in the graft, improve ischemia-reperfusion-induced vascular permeability and alveolar pulmonary fibroprotein deposition, thereby successfully preventing acute lung injury associated with IRI, improving gas exchange in the graft, reducing peak airway pressure, and reducing alveolar fibroprotein deposition. At the same time, studies have also shown that PS-targeted therapy can improve changes related to inflammation, cell death, and fibrinolysis, thereby proving the value of a clinical treatment strategy for shielding phosphatidylserine in the early reperfusion phase of lung transplantation.

[0030] Similar to the above-mentioned liver and lung transplantation, renal ischemia / reperfusion injury (IRI) also often complicates shock, kidney transplantation, and heart and aortic surgery. In addition, phosphatidylserine everted to the cell surface is an important pro-inflammatory signal of cell stress after IRI. In animal models and clinical experimental studies of renal IRI, the recombinant protein Diannexin that can bind to PS was shown to protect the kidney, reduce proximal tubular damage and leukocyte influx, reduce the transcription and expression of the kidney injury markers neutrophil gelatinase-associated lipocalin and kidney injury molecule 1, and improve kidney function.

[0031] Myocardial ischemia-reperfusion injury is also a pathogenic mechanism of myocardial infarction and heart failure. In animal experimental studies targeting PS using Diannexin, Diannexin reduces IRI-induced myocardial cell death and oxidative damage by increasing cell viability and inhibiting apoptosis, ROS, lactate dehydrogenase, malondialdehyde production, and antioxidant SOD activity. Technical solutions

[0032] The technical problem to be solved by the present application is to provide an anti-antithrombin / phosphatidylserine antibody, a pharmaceutical composition and uses.

[0033] The present application provides an anti-prothrombin / phosphatidylserine antibody or antigen binding fragment thereof, the antibody comprising a heavy chain variable region and a light chain variable region; the heavy chain variable region in the antibody comprises HCDR1 to HCDR3, and the light chain variable region comprises LCDR1 to LCDR3, wherein:

[0034] the amino acid sequence of HCDR1 is shown as SEQ ID NO: 1, the amino acid sequence of HCDR2 is shown as SEQ ID NO: 2, and the amino acid sequence of HCDR3 is shown as SEQ ID NO: 3; and

[0035] the amino acid sequence of LCDR1 is shown as SEQ ID NO: 4, the amino acid sequence of LCDR2 is shown as SEQ ID NO: 5, and the amino acid sequence of LCDR3 is shown as SEQ ID NO: 6.

[0036] Further, the prothrombin in the prothrombin / phosphatidylserine in the present application is human prothrombin if not otherwise specified.

[0037] Further, the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO: 7, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO: 8.

[0038] Further, the anti-prothrombin / phosphatidylserine antibody or antigen binding fragment thereof, wherein the anti-prothrombin / phosphatidylserine antibody or antigen binding fragment thereof is selected from the group consisting of Fab, Fab', F(ab')2, Fd, Fv, dAb, complementarity determining region fragment, single-chain antibody, and monoclonal human antibody.

[0039] Further, the anti-prothrombin / phosphatidylserine antibody or antigen binding fragment thereof, wherein the antibody comprises a non-CDR region, and the non-CDR region is from a species other than murine, for example, from a human antibody.

[0040] Further, the anti-prothrombin / phosphatidylserine antibody or antigen binding fragment thereof, wherein the heavy chain constant region of the antibody is a constant region sequence of IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, secreted IgA, IgD, or IgE type; and the light chain constant region is a constant region sequence of Ig kappa or Ig lambda. For example, the amino acid sequence of the heavy chain constant region of the anti-prothrombin / phosphatidylserine antibody is shown as SEQ ID NO: 9, the amino acid sequence of the heavy chain constant region of the anti-prothrombin / phosphatidylserine antibody is shown as SEQ ID NO: 10; and the amino acid sequence of the light chain constant region is shown as SEQ ID NO: 11.

[0041] Another aspect of the application relates to nucleic acid molecules encoding the anti- prothrombin / phosphatidylserine antibody or antigen-binding fragment thereof described in the application, wherein SEQ ID NO: 12 represents a cDNA nucleic acid sequence encoding the light chain variable region of the anti-prothrombin / phosphatidylserine antibody; and wherein SEQ ID NO: 13 represents a cDNA nucleic acid sequence encoding the heavy chain variable region of the anti-prothrombin / phosphatidylserine antibody. The nucleic acid molecules contemplated by the application also include nucleic acid sequences encoding the same protein polypeptides as those encoded by SEQ ID NO: 12 and SEQ ID NO: 13 using a plurality of different types of codons (degenerate codons).

[0042] A further aspect of the application relates to a recombinant vector comprising the nucleic acid molecule of the application.

[0043] A further aspect of the application relates to a host cell comprising the nucleic acid molecule of the application or the recombinant vector described in the application.

[0044] The anti-prothrombin / phosphatidylserine antibody or antigen-binding fragment thereof according to any one of the application for use in the treatment or prevention of a tumor;

[0045] Preferably, the tumor is one or more selected from the group consisting of melanoma, colon cancer, rectal cancer, liver cancer, biliary tract cancer, bronchial cancer, lymphoma, ovarian cancer, esophageal cancer, hematological neoplasms, glioblastoma, lung cancer, prostate cancer, bladder cancer, gastric cancer, breast cancer, brain cancer, pancreatic cancer, thyroid cancer, head and neck cancer, and kidney cancer.

[0046] The anti-prothrombin / phosphatidylserine antibody or antigen-binding fragment thereof according to any one of the application for use in the treatment or prevention of viral, bacterial and parasitic infections;

[0047] Preferably, the virus is one or more selected from the group consisting of enveloped viruses and non-enveloped viruses, including vaccinia virus, Pichinde virus, cytomegalovirus, Lassa fever virus, Lentivirus, Dengue virus, Ebola virus and Marburg virus, and one or more of SV40, Hepatitis A and Polio virus.

[0048] Preferably, the bacteria is one or more of Listeria monocytogenes, Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium marinum, Chlamydia, Candida albicans, Acinetobacter baumannii, Staphylococcus epidermidis, Citrobacter freundii, Klebsiella aerobacter, Klebsiella oxytoca, Escherichia coli, Klebsiella pneumoniae, Enterococcus faecalis, Citrobacter freundii, Candida glabrata, Streptococcus agalactiae, Corynebacterium urealyticum, Staphylococcus aureus, Candida parapsilosis, Candida kefyr, Citrobacter freundii, Citrobacter freundii, Streptococcus oralis, Staphylococcus lugdunensis, Morganella morganii, Candida pseudotropicalis, Acinetobacter baumannii, Candida lusitaniae, Proteus vulgaris, Proteus mirabilis, Acinetobacter johnsonii, Candida tropicalis, Pseudomonas japonica, Staphylococcus haemolyticus, Enterococcus faecium, Stenotrophomonas maltophilia, Pseudomonas aeruginosa, Streptococcus agalactiae, Comamonas odorata, Streptococcus constellatus, Streptococcus anginosus, Burkholderia cenocepacia, Gardnerella vaginalis, Enterobacter cloacae, Serratia marcescens.

[0049] Preferably, the parasite is one or more of Trypanosoma cruzi, Trypanosoma brucei, and Leishmania amazonensis.

[0050] The anti-antithrombin / phosphatidylserine antibody or antigen-binding fragment thereof according to any one of the present application for use in the treatment or prevention of a thrombotic disease.

[0051] Preferably, the thrombotic disease comprises arterial, venous and microcirculatory thrombosis. For example, coronary syndrome, myocardial infarction, stroke, deep vein thrombosis, pulmonary embolism, disseminated intravascular coagulation, heparin-induced thrombocytopenia, complicated diabetes, severe hypertension, end-stage renal disease, antiphospholipid syndrome, systemic lupus erythematosus, sickle cell disease, paroxysmal nocturnal hemoglobinuria, and biomaterial-related thrombosis.

[0052] The anti-antithrombin / phosphatidylserine antibody or antigen-binding fragment thereof according to any one of the present application for use in the treatment or prevention of Alzheimer's disease and Parkinson's disease.

[0053] The anti-antithrombin / phosphatidylserine antibody or antigen-binding fragment thereof according to any one of the present application for use in the treatment or prevention of inflammatory bowel disease, including ulcerative colitis, Crohn's disease and indeterminate colitis.

[0054] The anti-antithrombin / phosphatidylserine antibody or antigen-binding fragment thereof according to any one of the present application for use in the treatment or prevention of atherosclerosis.

[0055] The anti-antithrombin / phosphatidylserine antibody or antigen-binding fragment thereof according to any one of the present application for use in the detection of a tumor. The anti-antithrombin / phosphatidylserine antibody or antigen-binding fragment thereof according to any one of the present application for use in the treatment or prevention of a thrombotic disease.

[0056] The anti-prothrombin / phosphatidylserine antibody or antigen-binding fragment thereof according to any one of the present application for use in improving microcirculation flow and reducing tissue injury.

[0057] Preferably, the use is for tissue injury in ischemia-reperfusion, including coronary artery occlusion, liver transplantation, kidney transplantation, pancreatic islet transplantation, lung transplantation, stroke and vascular occlusion recanalization operation.

[0058] Another aspect of the present application relates to an antibody drug conjugate comprising an antibody or antigen-binding fragment thereof and a small molecule drug, wherein the antibody or antigen-binding fragment thereof is the anti-prothrombin / phosphatidylserine antibody or antigen-binding fragment thereof according to any one of the present application; preferably, the small molecule drug is a small molecule cytotoxic drug; more preferably, the small molecule drug is a tumor chemotherapy drug.

[0059] In some embodiments of the present application, the antibody drug conjugate, wherein the antibody or antigen-binding fragment thereof is linked to the small molecule drug via a linker; for example, the linker is a hydrazone bond, a disulfide bond or a peptide bond.

[0060] Preferably, the molar ratio of the antibody or antigen-binding fragment thereof to the small molecule drug is 1:(2-4), for example 1:2, 1:3 or 1:4.

[0061] Another aspect of the present application relates to a pharmaceutical composition comprising an effective amount of the anti-prothrombin / phosphatidylserine antibody or antigen-binding fragment thereof according to any one of the present application or the antibody drug conjugate according to any one of the present application; optionally, the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients.

[0062] In some embodiments of the present application, the pharmaceutical composition further comprises one or more immune checkpoint inhibitors.

[0063] Preferably, the immune checkpoint inhibitor is an antibody targeting PD-1, PD-L1, CTLA-4, CD47, LAG-3, TIGHT, VISTA, STING, TREM2, PCSK9, TMEM176B, DDR1, ICOS, CD137, GITR and / or OX40.

[0064] Preferably, the antibody is a monoclonal antibody or a bispecific antibody.

[0065] Preferably, the antibody is a blocking monoclonal antibody.

[0066] Preferably, the antibody is an anti-PD-1 blocking monoclonal antibody or an anti-PD-L1 blocking monoclonal antibody.

[0067] In some embodiments of the application, the pharmaceutical composition, wherein the mass ratio of the immune checkpoint inhibitor to the anti-anti-prothrombin / phosphatidylserine antibody or antigen-binding fragment thereof is (1 :5) to (5: 1), preferably (1 :2) to (2: 1), more preferably 1 : 1.

[0068] Still another aspect of the application relates to a pharmaceutical product combination comprising a first pharmaceutical product and a second pharmaceutical product, wherein:

[0069] the first pharmaceutical product comprises the anti-anti-prothrombin / phosphatidylserine antibody or antigen-binding fragment thereof of any one of the application or the antibody drug conjugate of any one of the application;

[0070] the second pharmaceutical product comprises one or more immune checkpoint inhibitors;

[0071] Preferably, the immune checkpoint inhibitor is an antibody targeting PD-1, PD-L1, CTLA-4, CD47, LAG-3, TIGHT, VISTA, STING, TREM2, PCSK9, TMEM176B, DDR1, ICOS, CD137, GITR and / or OX40;

[0072] Preferably, the antibody is a monoclonal antibody or a bispecific antibody;

[0073] Preferably, the antibody is a blocking monoclonal antibody;

[0074] Preferably, the antibody is an anti-PD-1 blocking monoclonal antibody or an anti-PD-L1 blocking monoclonal antibody.

[0075] In some embodiments of the application, the pharmaceutical product combination, wherein,

[0076] the mass ratio of the immune checkpoint inhibitor to the anti-anti-prothrombin / phosphatidylserine antibody or antigen-binding fragment thereof is (1 :5) to (5: 1), preferably (1 :2) to (2: 1), more preferably 1 : 1.

[0077] In some embodiments of the application, the pharmaceutical product combination, wherein,

[0078] the first pharmaceutical product and the second pharmaceutical product independently comprise one or more pharmaceutically acceptable excipients;

[0079] Preferably, further comprising a package insert.

[0080] The present application also provides a nucleic acid antibody, which is carried by a virus or non-virus vector and contains a gene (DNA or mRNA) encoding the anti-antithrombin / phosphatidylserine antibody or antigen-binding fragment thereof. Compared with traditional antibodies, the nucleic acid antibody has the advantages of no need for complex in vitro protein expression and purification process, simple sequence design, and low production cost. The gene (DNA or mRNA) encoding the anti-antithrombin / phosphatidylserine antibody or antigen-binding fragment thereof is introduced into cells, and the corresponding anti-antithrombin / phosphatidylserine antibody or antigen-binding fragment thereof is produced by the cells to exert its function. The DNA antibody is a host cell into which DNA containing the coding sequence of the anti-antithrombin / phosphatidylserine antibody or antigen-binding fragment thereof is introduced by a vector, and the corresponding antibody is expressed after transcription, translation and other processes to exert its function. The main delivery vectors of the DNA antibody include: virus vectors, commonly used adenovirus (AdV) vectors and adeno-associated virus (AAV) vectors, etc.; non-virus vectors such as plasmid DNA (pDNA); mRNA antibody is the mRNA encoding the anti-antithrombin / phosphatidylserine antibody or antigen-binding fragment thereof introduced into the cytoplasm by a vector, and the antibody protein is synthesized by translation to exert its function. Lipid nanoparticle (LNP) is the most commonly used delivery vector in recent years, in addition to gene gun, electroporation, protamine, cationic nanoemulsion and cationic polymer liposome which are also commonly used in the delivery of mRNA.

[0081] In the present application, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art, unless otherwise specified. And the cell culture, molecular genetics, nucleic acid chemistry, immunology laboratory operation steps used herein are the conventional steps widely used in the corresponding field. At the same time, in order to better understand the present application, the definitions and explanations of related terms are provided as follows.

[0082] In the present application, the term "blocking monoclonal antibody" specifically refers to a monoclonal antibody used to block the binding site of the immune checkpoint and its ligand or receptor, such as PD-1 and PD-L1, for tumor immunotherapy.

[0083] As used herein, the term EC50 refers to the concentration for 50% of maximal effect, which refers to the concentration that can cause 50% of the maximum effect.

[0084] As used herein, the term "antibody" refers to an immunoglobulin molecule that is generally composed of two pairs of polypeptide chains (each pair having one "light" (L) chain and one "heavy" (H) chain). Antibody light chains can be assigned to a class, kappa and lambda. Heavy chains can be assigned to a class, mu, delta, gamma, alpha, or epsilon, and define a specificity of an antibody as IgM, IgD, IgG, IgA, and IgE, respectively. Within the light and heavy chains, the variable and constant regions are joined by a "J" region of about 12 or more amino acids, and the heavy chain also contains a "D" region of about 3 or more amino acids. Each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region is comprised of three domains, CH1, CH2 and CH3. Each light chain is composed of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region is comprised of one domain, CL. The constant regions of the antibodies can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. The VH and VL regions can also be subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of each heavy / light chain pair (VH and VL) form the antibody binding site. Assignment of amino acids to each region or domain follows the definition of Bethesda M.d., Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, (1987 and 1991)), or Chothia & Lesk J. Mol. Biol. 1987; 196:901-917; Chothia et al. Nature 1989; 342:878-883, or the IMGT numbering system definition, see Ehrenmann F, Kaas Q, Lefranc M P. IMGT / 3Dstructure-DB and IMGT / DomainGapAlign: a database and a tool for immunoglobulins or antibodies, T cell receptors, MHC, IgSF and MhcSF [J]. Nucleic acids research, 2009; 38(suppl_1):D301-D307.

[0085] The term "antibody" is not limited by any particular method of producing the antibody. For example, it includes recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. The antibody can be of different isotypes, e.g., IgG (e.g., IgGl, IgG2, IgG3, or IgG4 subtypes), IgAl, IgA2, IgD, IgE, or IgM antibodies.

[0086] As used herein, the terms "monoclonal" and "monoclonal antibody" refer to a single antibody or fragment of an antibody from a population of highly homogenous antibody molecules, i.e., a population of antibody molecules that are identical except for possible naturally occurring mutations that can arise during production. A monoclonal antibody has high specificity for a single epitope on an antigen. Polyclonal antibodies are in contrast to monoclonal antibodies and generally comprise at least 2 or more different antibodies, which generally recognize different epitopes on the antigen. Monoclonal antibodies can generally be obtained using the hybridoma technology first reported by Kohler et al. (Kohler, G, Milstein, C. Continuous cultures of fused cells secreting antibody of predefined specificity [J]. Nature, 1975; 256(5517): 495), but can also be obtained using recombinant DNA technology (see, e.g., U.S. Patent 4,816,567).

[0087] As used herein, the term "single chain fragment variable (ScFv)" refers to a molecule comprising an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL) joined by a linker. Wherein the VL and VH domains pair to form a monovalent molecule by a linker that enables it to be produced as a single polypeptide chain (see, e.g., Bird et al, Science 1988; 242:423-426 and Huston et al, Proc. Natl. Acad. Sci. USA 1988; 85:5879-5883). Such scFv molecules can have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of repeating GGGGS amino acid sequences or variants thereof. For example, a linker having the amino acid sequence (GGGGS)4 can be used, but variants thereof can also be used (Holliger et al, Proc. Natl. Acad. Sci. USA 1993; 90:6444-6448). Other linkers useful in the present application are described by Alfthan et al, Protein Eng. 1995; 8:725-731, Choi et al, Eur. J. Immunol. 2001; 31:94-106, Hu et al, Cancer Res. 1996; 56:3055-3061, Kipriyanov et al, J. Mol. Biol. 1999; 293:41-56 and Roovers et al, Cancer Immunology, Immunotherapy, 2001, 50(1):51-59.

[0088] As used herein, the term "isolated" or "isolated" refers to that which is obtained by artificial means from the natural state. If a certain "isolated" substance or component appears in nature, it is possible that the natural environment in which it is located has been changed, or the substance has been separated from the natural environment, or both. For example, a certain polynucleotide or polypeptide naturally present in a certain living animal is not isolated, while a high-purity same polynucleotide or polypeptide isolated from such a natural state is called isolated. The term "isolated" or "isolated" does not exclude the mixing of artificial or synthetic substances, nor does it exclude the presence of other impurities that do not affect the activity of the substance.

[0089] As used herein, the term "vector" refers to a nucleic acid vehicle into which a polynucleotide can be inserted. When the vector is capable of directing the expression of a polynucleotide inserted into it, the vector is referred to as an expression vector. A vector can be introduced into a host cell by transformation, transduction or transfection, and directs the expression of elements carried in the genetic material of the vector in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1 -derived artificial chromosomes (PAC); bacteriophages such as lambda phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papova viruses (such as SV40). A vector can contain a variety of elements that control expression, including but not limited to, promoter sequences, transcriptional initiation sequences, enhancer sequences, selection elements and reporter genes. In addition, a vector can contain a replication origin.

[0090] As used herein, a cell as a host refers to a cell that can be used to introduce a vector, including but not limited to, prokaryotic cells such as E. coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblast cells, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.

[0091] As used herein, the term "specifically binds" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and the antigen against which it is directed. In certain embodiments, an antibody that specifically binds to (or has specificity for) an antigen refers to an antibody that binds to the antigen with an affinity (KD) of less than about 10 -5 M, for example less than about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 M or less.

[0092] As used herein, the term "KD" refers to the dissociation equilibrium constant of a particular antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding, and the higher the affinity between the antibody and the antigen. Typically, an antibody binds to an antigen with an affinity (KD) of less than about 10 -5 M, for example less than about 10 -6 M, 10 -7 M, 10 -8M, 10 -9 M or 10 -10 M or smaller dissociation equilibrium constant (KD) binds to an antigen (e.g., a TMEM176B protein). KD can be determined using methods known to those skilled in the art, for example, using Biacore assay.

[0093] As used herein, the terms "monoclonal antibody" and "monoclonal" have the same meaning and are used interchangeably; the terms "polyclonal antibody" and "polyclonal" have the same meaning and are used interchangeably. Also in the present application, amino acids are generally represented by their commonly known single and three letter codes. For example, alanine can be represented by A or Ala.

[0094] As used herein, the term "pharmaceutically acceptable excipient" or "pharmaceutically acceptable carrier and / or excipient" refers to a carrier and / or excipient that is compatible, in pharmacological and / or physiological terms, with the subject and active ingredient, which is well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to, pH adjusting agents, surfactants, adjuvants, ionic strength enhancers. For example, pH adjusting agents include, but are not limited to, phosphate buffer; surfactants include, but are not limited to, cationic, anionic or non-ionic surfactants, such as Tween-80; ionic strength enhancers include, but are not limited to, sodium chloride.

[0095] As used herein, the term "adjuvant" refers to a non-specific immune enhancer that, when delivered into the body together with or prior to an antigen, can enhance the immune response of the body to the antigen or change the type of immune response. There are many kinds of adjuvants, including but not limited to aluminum adjuvants (e.g., aluminum hydroxide), Freund's adjuvants (e.g., complete Freund's adjuvant and incomplete Freund's adjuvant), Corynebacterium parvum, lipopolysaccharide, cytokines, etc. Freund's adjuvant is the most commonly used adjuvant in animal experiments at present. Aluminum hydroxide adjuvant is more commonly used in clinical experiments.

[0096] As used herein, the term "effective amount" means an amount that is sufficient to achieve or at least partially achieve a desired effect. For example, an effective prophylactic amount is an amount that is sufficient to prevent, arrest, or delay the onset of a disease; an effective therapeutic amount is an amount that is sufficient to cure or at least partially arrest the disease and its complications in an already afflicted patient. Determining such effective amounts is well within the capability of those skilled in the art. For example, an amount effective for a therapeutic use will depend on the severity of the disease to be treated, the general state of the patient's own immune system, the general condition of the patient, e.g., age, weight, and gender, the mode of administration of the drug, and other therapies that the patient may be undergoing, etc.

[0097] In the present application, if not otherwise specified, the "first" (e.g., first drug product) or "second" (e.g., second drug product) is merely for the purpose of distinction in reference, and does not have the meaning of particular order.

[0098] Beneficial effects

[0099] Phospholipid components in eukaryotic cells under physiological conditions are asymmetrically distributed in the phospholipid bilayer membrane, with zwitterionic phospholipids such as phosphatidylcholine (PC) and sphingomyelin mainly existing in the outer leaflet of the cell membrane, while phosphatidylserine (PS), phosphatidylethanolamine (PE) and phosphatidylinositol (PI) mainly existing in the inner leaflet or cytoplasmic leaflet. Various physiological and pathological processes, such as platelet activation, apoptosis, tumors and microbial infections, cause changes in the asymmetric distribution of phospholipid components on both sides of the cell membrane, leading to PS exposure. Phosphatidylserine (PS) externalization is a typical characteristic of apoptosis, which can inhibit phagocytosis by phagocytes and reduce inflammation. Cancer cells cannot maintain the asymmetry of PS on the inner and outer sides of the cell membrane, resulting in the reversal of PS exposure on the cell membrane, which becomes a target for tumor targeted therapy. At the same time, the apoptosis of tumor cells that occurs rapidly and the apoptosis of tumor cells induced by radiotherapy and other immunotherapy also inhibit tumor immunity through the action of PS and PS receptors on the surface of phagocytes. Compared with normal organ tissues, the PS level in the tumor vasculature associated with tumor cells is also elevated, thus becoming a therapeutic target for destroying tumor vascular tissue. Viruses, bacteria and parasites can hijack the body's apoptosis mechanism to inhibit immunity, so preparations targeting PS can also be used for the treatment of infectious diseases. During the body's hemostasis process, platelets are activated, PS is externalized to provide a binding surface for coagulation factors to promote the coagulation cascade, so PS is also a target for anticoagulant and anti-thrombotic therapy. PS is also one of the targets for treating atherosclerosis, Alzheimer's disease and Parkinson's disease.

[0100] PS itself is weakly immunogenic, so anti-PS antibodies usually need to target PS binding proteins to recognize and bind to PS molecules. Both prothrombin and β2GPI are PS binding proteins, but under physiological conditions, prothrombin can bind to the phospholipid membrane surface of exposed PS molecules through the carboxylated glutamic acid residues in the GLA domain of the F1 fragment in the presence of calcium ions, while β2GPI can only weakly bind to PS at physiological ion strength. Prothrombin can bind to the surface of apoptotic cells with exposed PS under physiological conditions, but not to normal living cells. In some anti-phospholipid syndrome patients, there are anti-prothrombin / phosphatidylserine antibodies that bind to prothrombin bound to phospholipids with high affinity, competitively excluding other coagulation factors from binding to the phospholipid PS, thereby prolonging the coagulation time in in vitro coagulation function tests such as activated partial thromboplastin time (APTT) and dilute venom test (dRVVT) with low phospholipid levels, resulting in lupus anticoagulant (LA). An anti-prothrombin / phosphatidylserine antibody involved in the present application also binds to the prothrombin / PS complex with high affinity, thereby targeting PS to exert anti-tumor, infection, anti-thrombus, atherosclerosis, Alzheimer's disease and Parkinson's disease treatment.

[0101] Antithrombin / phosphatidylserine antibodies bind to PS everted exposed tumor cells or tumor vascular endothelium, and then interact with the complement system through its Fc segment to induce complement-dependent cytotoxicity, or interact with Fc receptors on natural killer (NK) cells or macrophages to induce antibody-dependent cellular cytotoxicity (ADCC) or antibody-dependent cellular phagocytosis (ADCP), thereby promoting the killing of tumor cells and tumor endothelial cells by macrophages and the like. Because antibodies have a large molecular weight (~ 150 kDa) and hydrophilicity, the antithrombin / phosphatidylserine antibodies involved in the present application cannot pass through the plasma membrane, so they cannot recognize or bind to intracellular PS, ensuring their tumor cell and tumor vascular targeting. Because phosphatidylserine (PS) everted exposure is a common phenomenon of tumor cells, and PS does not mutate with the occurrence and development of tumor cells, the antithrombin / phosphatidylserine antibodies can effectively kill tumor cells by targeting tumor cell PS. At the same time, radiotherapy and chemotherapy increase the everted exposure of PS on tumor cells, promoting the binding and killing of tumor cells by the PS-targeted antithrombin / phosphatidylserine antibodies involved in the present application, thereby enhancing the anti-cancer effect of monoclonal antibodies. The antithrombin / phosphatidylserine antibodies highly specifically recognize and bind to the molecular target phosphatidylserine (PS) that is continuously exposed on the tumor vascular endothelial cells in the circulating blood, thereby effectively targeting and killing the tumor vasculature, leading to the death of downstream tumor cells supplied by the blood vessels, and achieving the purpose of effectively treating tumors. Because radiotherapy, chemotherapy, and immunotherapy promote the degree of PS everted exposure in tumor endothelial cells, the combination of antithrombin / phosphatidylserine antibodies and classic anti-tumor methods can further destroy the tumor vascular system and improve the effect of tumor treatment.

[0102] Immune checkpoint inhibitors are the most thoroughly studied class of immunotherapies to date. The two most common checkpoint inhibition strategies are PD-1 / PD-L1 blockade and CTLA4 inhibition. Phosphatidylserine (PS) e xpo sure is an important biological feature of tumor cells and also constitutes an important immune checkpoint. Under physiological conditions, normal apoptotic cells are cleared by the body without triggering a severe immune response, but in the case of tumor and some parasitic, viral and bacterial infections, tumor cells or microorganisms hijack the body's mechanism for clearing apoptotic cells, promoting tolerance of tumor cells and pathogenic microorganisms by the immune system, and preventing local and systemic immune activation. The e xpo sure of phosphatidylserine (PS) on the membrane of apoptotic cells and the binding of PS receptors on phagocytes are important mechanisms for the body to clear apoptotic cells without triggering a severe immune response. PS receptors expressed on myeloid-derived cells, including the TAM and TIM receptor families, promote tissue homeostasis after PS or PS bridging molecules are bound, and increase the expression of immunosuppressive cytokines; after dendritic cells (DCs) in the tumor microenvironment bind and take up PS-exposed tumor cells, they remain in an immature state and cannot fully express costimulatory molecules to effectively present antigens, so the e xpo sure of tumor cells inhibits the scope and degree of anti-tumor immune response. The e xpo sure of PS on microvesicles (exosomes) produced by tumor cells also inhibits the activation of T cell responses. In addition, after chemotherapy and radiotherapy, PS in tumors increases significantly, which further enhances PS-mediated immune suppression.

[0103] The anti-antithrombin / prophosphatidylserine antibody involved in the present application is a PS-specific antibody that, by binding to PS exposed on the surface of tumor cells, blocks the binding of PS to PS receptors such as phagocytes in tumor tissue, removes the tumor immune checkpoint inhibition caused by PS, and stimulates a strong anti-tumor immune response. In fact, the efficacy of the anti-antithrombin / prophosphatidylserine antibody involved in the present application in blocking the binding of PS exposed on the surface of tumor cells is similar to that of immune checkpoint inhibitors, and is roughly the same as the way of blocking PD-L1 and CTLA4 to prevent inhibitory signals in T cells. At the same time, the effect of the anti-antithrombin / prophosphatidylserine antibody involved in the present application in shaping a pro-tumor immune microenvironment is better than that of other immune checkpoint blockers such as anti-cytotoxic T lymphocyte-associated protein 4 (anti-CTLA-4) and anti-PD-1 in combination, which will achieve better tumor immunotherapy effect.

[0104] Similarly, the anti-antithrombin / prophosphatidylserine antibody involved in the present application can also reverse the immune suppression caused by viruses, bacteria and microorganisms using the body's apoptotic cell clearance mechanism by binding to PS exposed on the surface of the cell membrane, promote the inhibition and clearance of these pathogenic microorganisms, and thus achieve the purpose of treating infectious diseases.

[0105] The anti-prothrombin / phosphatidylserine antibodies of the present invention can also be used for the specific labeling and imaging of tumors due to their property of specifically binding PS.

[0106] PS ecto-exposure plays an important role in physiological and pathological hemostasis and thrombosis. Upon activation, platelets change the asymmetric composition of phospholipids, and PS exposure binds coagulation factors to promote the coagulation cascade. Uncontrolled PS ecto-exposure on apoptotic cells and tumor cells can also provide phospholipid surfaces for coagulation reactions, leading to thromboembolic and other pathological consequences. Apoptosis of endothelial cells in atherosclerotic plaques is a key step in the transition from stable, endothelialized plaques to plaque erosion and thrombosis. Rupture of vulnerable plaques leads to exposure of the lipid core, which is highly enriched in thrombogenic TF and PS-exposed apoptotic microparticles, leading to acute coronary syndrome and acute cerebrovascular events. Thrombosis is a common complication of cancer, and the ecto-exposure of tissue factor (TF) and PS produced by tumor cells and tumor vascular endothelium are two key factors leading to tumor thrombosis. Increased apoptosis of tumor cells and tumor vascular endothelium during radiotherapy, chemotherapy, and immunotherapy increases the level of TF and the degree of PS ecto-exposure, further increasing the risk of thrombosis. The anti-prothrombin / phosphatidylserine antibodies of the present invention specifically bind PS, competitively hinder coagulation factors, including coagulation factor X, coagulation factor IX, coagulation factor XI, coagulation factor V, coagulation factor VIII, and coagulation factor VII from binding to the ecto-exposed PS molecules, thereby hindering the progression of the coagulation cascade and the generation of thrombin, thus achieving the purpose of preventing and treating thrombosis.

[0107] Based on similar mechanisms of action, the anti-prothrombin / phosphatidylserine antibodies of the present invention can also prevent and treat other diseases and pathological states related to thrombosis, such as cardiovascular and cerebrovascular diseases, heparin-induced thrombocytopenia, complicated diabetes, severe hypertension, end-stage renal disease, antiphospholipid syndrome, systemic lupus erythematosus, sickle cell disease, paroxysmal nocturnal hemoglobinuria, and thrombosis associated with biomaterials.

[0108] The natural ligand of phosphatidylserine (PS), Annexin V, protects choroid plexus cells from amyloid beta (Aβ)-induced toxicity and reduces the misfolding and fibril formation of alpha-synuclein. The anti-prothrombin / phosphatidylserine antibodies of the present invention also target PS, so they will also have beneficial effects on the treatment of Alzheimer's disease and Parkinson's disease.

[0109] In inflammatory bowel diseases (IBD) including ulcerative colitis (UC), Crohn's disease (CD) and indeterminate colitis (IC), inhibition of PS-dependent endocytosis in intestinal endothelial cells can down-regulate TLR4 on the surface of endothelial cells, impair NF-κB activation, and reduce the induced expression of cytokines, chemokines, and adhesion molecules VCAM-1 and ICAM-1. The anti-antithrombin / phosphatidylserine antibodies involved in the present application target PS, and thus can reduce the infiltration of lymphocytes in the intestine through the above mechanisms, achieve the purpose of reducing inflammatory response, and relieve and treat IBD.

[0110] The anti-antithrombin / phosphatidylserine antibodies or antigen-binding fragments thereof according to any one of the present application bind to cell surface PS, inhibit the aggregation of leukocytes and platelets and the attachment of EC, inhibit early IRI events, maintain sinusoidal blood flow in the liver during reperfusion after ischemia, prevent IRI and post-transplant liver function impairment associated with liver transplantation. The anti-antithrombin / phosphatidylserine antibodies or antigen-binding fragments thereof according to any one of the present application can prevent and treat IRI after kidney transplantation, improve kidney function, and improve the success rate of kidney transplantation. The anti-antithrombin / phosphatidylserine antibodies or antigen-binding fragments thereof according to any one of the present application bind to cell surface PS, reduce ischemia-reperfusion injury caused by stroke, ischemic myocardial infarction, and surgical operation occlusion of blood vessel recanalization. BRIEF DESCRIPTION OF DRAWINGS

[0111] FIGS. 1A-B are anti-antithrombin / phosphatidylserine antibody and prothrombin / phosphatidylserine binding tests.

[0112] Figures 2A-B are clotting responses in an anti-antithrombin / phosphatidylserine antibody binding phospholipid inhibition of clotting assay.

[0113] Figure 3 is an anti-antithrombin / phosphatidylserine antibody binding apoptosis cell assay. Embodiments of the application

[0114] The application is further described in connection with specific embodiments. It will be understood, however, that these embodiments are not limiting and are merely intended to illustrate the application and that various modifications or changes in light thereof will occur to those skilled in the art. It is to be understood that within the scope of the appended claims, the application can be practiced otherwise than is specifically enumerated.

[0115] Example 1

[0116] The present example provides an anti-antithrombin / phosphatidylserine antibody, the antibody comprising a heavy chain variable region and a light chain variable region; the heavy chain variable region in the antibody comprising HCDR1 to HCDR3, and the light chain variable region comprising LCDR1 to LCDR3, wherein:

[0117] the amino acid sequence of HCDR1 is as set forth in SEQ ID NO: 1, the amino acid sequence of HCDR2 is as set forth in SEQ ID NO: 2, and the amino acid sequence of HCDR3 is as set forth in SEQ ID NO: 3; and

[0118] the amino acid sequence of LCDR1 is as set forth in SEQ ID NO: 4, the amino acid sequence of LCDR2 is as set forth in SEQ ID NO: 5, and the amino acid sequence of LCDR3 is as set forth in SEQ ID NO: 6.

[0119] the amino acid sequence of the heavy chain variable region is as set forth in SEQ ID NO: 7, and the amino acid sequence of the light chain variable region is as set forth in SEQ ID NO: 8.

[0120] the nucleic acid cDNA sequence encoding the amino acid sequence of the heavy chain variable region is as set forth in SEQ ID NO: 12, and the nucleic acid cDNA sequence encoding the amino acid sequence of the light chain variable region is as set forth in SEQ ID NO: 13.

[0121] Example 2

[0122] Anti-antithrombin / phosphatidylserine antibody binding to antithrombin / phosphatidylserine assay

[0123] The ability of the anti-antithrombin / phosphatidylserine antibody (aPS / PT) to bind PS / PT complex is detected by enzyme-linked immunosorbent assay (ELISA). A plastic microplate with antithrombin / phosphatidylserine complex coated in the microwells is used. The sample to be tested is added to the microwells for incubation to allow the sample to fully react with the immobilized antithrombin / phosphatidylserine antibody complex. After the incubation reaction is completed, the microwells are washed with a washing solution to remove non-specifically bound proteins. Then, horseradish peroxidase (HRP)-labeled anti-human IgG antibody is added to the microwells, and after incubation, the unbound labeled antibody is washed away, and peroxide is added for reaction to develop color. After the color development reaction is completed, the color intensity is measured by an optical density measuring instrument at a single wavelength of 450 nm. A standard curve is established using gradient-diluted human serum anti-antithrombin / phosphatidylserine antibody standard with known content (PS / PT IgG unit range 9.4-150). The binding strength of the recombinant anti-antithrombin / phosphatidylserine antibody with known protein concentration is detected and compared with the standard curve to calculate the binding strength.

[0124] As shown in FIG. 1, the anti-antithrombin / phosphatidylserine antibody involved in the present application can bind to PS / PT complex. When having the same light and heavy chain variable regions, both IgG3 and IgG1 type anti-antithrombin / phosphatidylserine antibodies can bind to PS / PT complex.

[0125] Example 3

[0126] Effect of anti-antithrombin / phosphatidylserine antibody on coagulation cascade reaction by binding to antithrombin and PS

[0127] The anti-prothrombin / phosphatidylserine antibodies involved in the present invention prolong plasma clotting time in in vitro coagulation tests in a phospholipid-dependent manner. In patients with antiphospholipid syndrome (aPL), autoantibodies against phospholipids or phospholipid-binding protein β2GPI or prothrombin can cause prolongation of in vitro coagulation tests, such as activated partial thromboplastin time (APTT), but this coagulation test abnormality can be corrected by the addition of excess phospholipids, and thus the antibodies mediating this coagulation test abnormality are also called lupus anticoagulants (LA). The mechanism of LA occurrence in aPL has not been fully elucidated, and it is generally believed that aPL, particularly autoantibodies associated with prothrombin, cause LA phenomena by competing with coagulation factors for binding sites on anionic phospholipids. The addition of excess phospholipids dilutes aPL, thereby reducing their likelihood of competing with coagulation factors. Anti-β2GPI antibodies are less likely to cause LA phenomena than prothrombin because the affinity of β2GPI for anionic phospholipids is much lower than that of vitamin K-dependent coagulation factors, including prothrombin, coagulation factor X, IX, etc., under physiological calcium ion concentrations. Although the affinity of anti-phospholipid antibodies in aPL is significantly enhanced by binding to β2GPI, it is unlikely that this would compete with the high affinity of vitamin K-dependent coagulation factors. LA is detected by phospholipid-sensitive coagulation assays, including dilute Russell's viper venom time (dRVVT). dRVVT is based on the activation of coagulation factor X (FX) by Russell's viper venom, thereby bypassing the intrinsic and extrinsic coagulation pathways to generate thrombin to clot blood. The coagulation reaction initiated with Russell's viper venom involves three phospholipid-dependent reactions: activation of FX (FXa) to activate coagulation factor V (FV), assembly of the prothrombin complex, and activation of prothrombin by the prothrombin complex. Anti-prothrombin antibodies prolong clotting time by competing with coagulation factors for binding sites on phospholipids. Antibodies against prothrombin enhance the binding of these proteins to negatively charged phospholipids.

[0128] The whole blood of 3.8% sodium citrate anticoagulation is centrifuged at 3000r / min for 15 minutes to separate the plasma, and is incubated with the negative high clay or tannic acid molecules at 37 DEG C in a coagulation instrument to make the coagulation system contact and activate, then 20mM CaCl2 is added to activate the common path, and the coagulation time is recorded. After the antithrombin / phosphatidylserine antibody in the application is added to the reaction system, the APTT coagulation time is significantly prolonged, and is greater than the upper limit of the normal human reference value. The lupus anticoagulant is detected in the coagulation instrument, low-concentration phospholipid (screening test) and high-phospholipid concentration (confirmation test) Russell's viper venom is added to the plasma, the coagulation time is recorded, and the dRVVT screening ratio = dRVVT screening time (s) / dRVVT screening normal value (s); the dRVVT confirmation ratio = dRVVT confirmation time (s) / dRVVT confirmation normal value (s); and the dRVVT standardization ratio = dRVVT screening ratio / dRVVT confirmation ratio. The best critical value of the dRVVT standardization ratio is 1.2.

[0129] As shown in FIG. 2, the antithrombin / phosphatidylserine antibody involved in the application can prolong the coagulation time in the APTT test. After the antithrombin / phosphatidylserine antibody in the application is added to the reaction system, the dRVVT screening time is significantly prolonged, the dRVVT standardization ratio is greater than 1.2 compared with the confirmation time, and it is proved that the antithrombin / phosphatidylserine antibody in the application binds to the thrombin / phosphatidylserine to inhibit the coagulation in the coagulation test.

[0130] Example 4

[0131] Flow cytometry detection of antithrombin / phosphatidylserine antibody binding to apoptotic cells

[0132] Normal cells are hydrophobic in nature because they express phosphatidylserine on the inner membrane (the side facing the cytoplasm), and when the cells undergo apoptosis, the inner membrane is turned into the outer membrane, thereby exposing phosphatidylserine (PS). Annexin V (annexin V) with high affinity for PS binds to cells with exposed PS in a Ca 2+ dependent manner, and the FITC-coupled labeled annexin V is a sensitive probe for flow cytometry analysis of cells undergoing apoptosis, and quantitatively determines the percentage of cells actively undergoing apoptosis in the population.

[0133] The FITC Conjugation Kit - Lightning-Link® (ab102884) provides a simple, fast procedure for covalently labelling antibodies with FITC by targeting primary amine groups (e.g. lysine). With a hands-on time of just 30 seconds, conjugates are ready to use in 3 hours 30 minutes and there is no loss of antibody. This format retains the high affinity of the anti-ATIII / PS antibody for PS and so can be used in flow cytometry with cells undergoing apoptosis.

[0134] Conjugation of FITC to anti-ATIII / PS antibody

[0135] Allow all materials and prepared reagents to equilibrate to room temperature before use.

[0136] 1. Add 1 μL of Modifier reagent per 10 μL of antibody to be labelled and mix gently.

[0137] 2. Remove the cap from the FITC Conjugation Mix vial and pipette the antibody sample (with added modifier) directly onto the lyophilised material. Resuspend gently by pipetting and re-distribute the liquid once or twice.

[0138] 3. Replace the cap on the vial and incubate for 3 hours at room temperature in the dark (20-25°C).

[0139] 4. After 3 hours (or longer) of incubation, add 1 μL of Quencher reagent per 10 μL.

[0140] 5. Use the antibody and mix gently. The conjugate is ready to use after 30 minutes. The conjugate does not need to be purified.

[0141] 6. Preparation of apoptotic cells and flow cytometric detection of FITC-labelled anti-ATIII / PS antibody binding to apoptotic cells

[0142] Cells (0.5 x 106cells) were placed in T25 flasks (quadruplicate for experiments) and 3 T25 flasks for controls (no staining, Annexin V only, FITC labeled anti- prothrombin / phosphatidylserine antibody only). After 6 hours of induction of apoptosis using 5 uM concentration of camptothecin, cells were collected, centrifuged at 500g for 5 minutes and resuspended in PBS to a cell concentration of 2 x 107cells / mL. 1 x 106cells (50 uL) were taken to flow tubes. 2 uL of Annexin V and a final concentration of 3 mM CaCl2were added to the Annexin V samples; 4 uL of FITC labeled anti-prothrombin / phosphatidylserine antibody and a final concentration of 3 mM CaCl2and 1.8 IU of prothrombin complex compound (PCC) were added to the FITC labeled anti-prothrombin / phosphatidylserine antibody samples. HBS was added to all samples to make up to 100 uL. After staining at room temperature in the dark for 25 minutes, 650 uL of 1% paraformaldehyde was added for fixation.

[0143] Cells positive for Annexin V were considered to be apoptotic cells. As can be seen from Figure 3, the FITC labeled anti-prothrombin / phosphatidylserine antibody was shown to have the same property of binding to apoptotic cells as the FITC labeled Annexin V.

Claims

1. An anti-prothrombin / phosphatidylserine antibody or antigen-binding fragment thereof, characterized in that: The antibody comprises a heavy chain variable region and a light chain variable region; the heavy chain variable region in the antibody comprises HCDR1 to HCDR3, and the light chain variable region comprises LCDR1 to LCDR3, wherein: the amino acid sequence of HCDR1 is as shown in SEQ ID NO: 1, the amino acid sequence of HCDR2 is as shown in SEQ ID NO: 2, and the amino acid sequence of HCDR3 is as shown in SEQ ID NO: 3; and the amino acid sequence of LCDR1 is as shown in SEQ ID NO: 4, the amino acid sequence of LCDR2 is as shown in SEQ ID NO: 5, and the amino acid sequence of LCDR3 is as shown in SEQ ID NO:

6.

2. The anti-anti-prothrombin / phosphatidylserine antibody or antigen-binding fragment thereof according to claim 1, characterized in that: the amino acid sequence of the heavy chain variable region 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:

8.

3. The anti-anti-prothrombin / phosphatidylserine antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The anti-antithrombin / phosphatidylserine antibody is an antibody of IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, secreted IgA, IgD or IgE type.

4. The anti-anti-prothrombin / phosphatidylserine antibody or antigen-binding fragment thereof according to claim 1, characterized in that: When the heavy chain of the anti-antithrombin / phosphatidylserine antibody is an IgG3 constant region, the amino acid sequence is as shown in SEQ ID NO: 9; when the heavy chain of the anti-antithrombin / phosphatidylserine antibody is an IgG1 constant region, the amino acid sequence is as shown in SEQ ID NO: 10; and when the light chain of the anti-antithrombin / phosphatidylserine antibody is an Ig Lamda constant region, the amino acid sequence is as shown in SEQ ID NO:

11.

5. The anti-anti-prothrombin / phosphatidylserine antibody or antigen-binding fragment thereof according to claim 1, characterized in that, The anti-antithrombin / phosphatidylserine antibody or antigen-binding fragment thereof is selected from the group consisting of Fab, Fab', F(ab')2, Fd, Fv, dAb, complementarity determining region fragment, single-chain antibody and monoclonal human antibody.

6. The anti-anti-prothrombin / phosphatidylserine antibody or antigen-binding fragment thereof of claim 1, wherein, The anti-antithrombin / phosphatidylserine antibody is PEGylated or non-PEGylated.

7. A nucleic acid molecule, characterized in that: The nucleic acid cDNA sequence encoding the amino acid sequence of the heavy chain variable region of the anti-antithrombin / phosphatidylserine antibody or antigen-binding fragment thereof according to claim 1 or 2 is as shown in SEQ ID NO:

12.

8. A nucleic acid molecule, characterized in that: The nucleic acid cDNA sequence encoding the amino acid sequence of the light chain variable region of the anti-antithrombin / phosphatidylserine antibody or antigen-binding fragment thereof according to claim 1 or 2 is as shown in SEQ ID NO:

13.

9. The nucleic acid molecule of claim 7 or 8, characterized in that: The nucleic acid molecule further comprises a nucleic acid sequence encoding the same protein polypeptide as encoded by SEQ ID NO: 12 and SEQ ID NO: 13 using a plurality of different types of codons.

10. A eukaryotic or prokaryotic recipient cell, characterized in that, The eukaryotic or prokaryotic recipient cell is capable of producing a fragment of the antibody according to claim 1.

11. A pharmaceutical composition for treating or preventing a tumor-related disease, comprising a therapeutically effective amount of the anti-antithrombin / phosphatidylserine antibody or antigen-binding fragment thereof according to any one of claims 1-2, and a pharmaceutically acceptable excipient.

12. A pharmaceutical composition for treating or preventing a disease associated with viral, bacterial and parasitic infection, comprising a therapeutically effective amount of the anti- prothrombin / phosphatidylserine antibody or antigen-binding fragment thereof according to any one of claims 1-2, and a pharmaceutically acceptable excipient.

13. A pharmaceutical composition for treating or preventing a disease associated with thrombosis, comprising a therapeutically effective amount of the anti-prothrombin / phosphatidylserine antibody or antigen-binding fragment thereof according to any one of claims 1-2, and a pharmaceutically acceptable excipient.

14. A pharmaceutical composition for treating or preventing a disease associated with atherosclerosis, cardiovascular disease, stroke, comprising a therapeutically effective amount of the anti-prothrombin / phosphatidylserine antibody or antigen-binding fragment thereof according to any one of claims 1-2, and a pharmaceutically acceptable excipient.

15. A pharmaceutical composition for treating or preventing a disease associated with Alzheimer's disease, comprising a therapeutically effective amount of the anti-prothrombin / phosphatidylserine antibody or antigen-binding fragment thereof according to any one of claims 1-2, and a pharmaceutically acceptable excipient.

16. A pharmaceutical composition for treating or preventing a disease associated with Parkinson's disease, comprising a therapeutically effective amount of the anti-prothrombin / phosphatidylserine antibody or antigen-binding fragment thereof according to any one of claims 1-2, and a pharmaceutically acceptable excipient.

17. A pharmaceutical composition for treating a disease comprising ulcerative colitis, Crohn's disease and indeterminate colitis, comprising a therapeutically effective amount of the anti-prothrombin / phosphatidylserine antibody or antigen-binding fragment thereof according to any one of claims 1-2, and a pharmaceutically acceptable excipient.

18. A pharmaceutical composition for treating a disease associated with ischemia / reperfusion injury, comprising a therapeutically effective amount of the anti-prothrombin / phosphatidylserine antibody or antigen-binding fragment thereof according to any one of claims 1-2, and a pharmaceutically acceptable excipient.

19. A nucleic acid antibody, characterized in that, A viral or non-viral vector carrying a gene encoding the anti-prothrombin / phosphatidylserine antibody or antigen-binding fragment thereof according to any one of claims 1-2.

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