Novel anti-tissue plasminogen activator antibody and application thereof

Abstract

The invention relates to the field of biological detection, in particular to a novel anti-tissue plasminogen activator antibody and application thereof. The antibody has good binding specificity and affinity to TPA, can be used for detecting a sample, and provides more diversified antibody selections for TPA detection and diagnosis or auxiliary diagnosis of thrombosis diseases with TPA as a marker; meanwhile, after TPA therapy, the hemorrhagic risk may be caused by excessive TPA, and the antibody of the invention can be combined with TPA to reduce the hemorrhagic risk.

Description

Technical Field

[0001] The present invention relates to the field of biological detection, and in particular to a novel anti-tissue plasminogen activator antibody and application thereof. Background Art

[0002] Tissue plasminogen activator is a single-chain glycoprotein that is mainly synthesized, secreted, and continuously released into the blood by vascular endothelial cells. It is widely present in various tissues of the body. The liver is the main site of inactivation of tissue plasminogen activator. It has a high affinity for fibrin, and then converts tyrosine plasminogen to plasmin, degrading fibrinogen and some coagulation factors. It is a key substance in the fibrinolytic system.

[0003] When the level of tissue plasminogen activator decreases, it indicates that fibrinolytic activity decreases, which is common in prethrombotic states and thrombotic diseases, such as arterial thrombosis, deep vein thrombosis, ischemic stroke, etc. In clinical practice, tissue plasminogen activator (TPA) therapy is often used to treat thrombotic diseases. TPA is the only effective medical treatment for ischemic stroke and it also reduces the mortality rate of patients with acute myocardial infarction, but TPA treatment significantly increases the risk of severe or fatal bleeding. When the level of tissue plasminogen activator increases, fibrinolytic activity is hyperactive, which often leads to primary and secondary hyperfibrinolysis, such as disseminated intravascular coagulation, acute promyelocytic leukemia, liver disease, coronary heart disease, hyperlipidemia, stress response, etc.

[0004] Therefore, detecting the content of tissue plasminogen activator is of great value for detecting thrombotic diseases. The most commonly used detection method is to utilize the immune reaction of antigen and antibody, achieve high-affinity binding to the protein through highly specific antibodies, and then use biochemical, chemiluminescence and other methods to achieve qualitative and quantitative detection of the protein; in addition, specific antibodies can bind to tissue plasminogen activator to reduce the risk of bleeding caused by its excessive amount, but currently there is little research on specific antibodies for tissue plasminogen activator, which cannot fully meet the needs of the market. Summary of the invention

[0005] The purpose of the present invention is to provide a novel anti-tissue plasminogen activator antibody and its application. The antibody has good binding specificity and affinity for TPA, can be used to detect samples, and provides more diverse antibody selections for the detection of TPA and the diagnosis or auxiliary diagnosis of thrombotic diseases with TPA as a marker; at the same time, after TPA therapy, there may be a risk of bleeding due to excessive TPA, and the antibody of the present invention can bind to TPA to reduce the risk of bleeding.

[0006] To achieve the above object, according to one aspect of the present invention, a novel anti-tissue plasminogen activator antibody is provided, wherein the antibody has the following complementary determining regions:

[0007] Heavy chain complementarity determining region:

[0008] HCDR1, which comprises or consists of the amino acid sequence shown in SEQ ID NO: 1;

[0009] HCDR2, which comprises or consists of the amino acid sequence shown in SEQ ID NO: 2;

[0010] and HCDR3, which comprises or consists of the amino acid sequence shown in SEQ ID NO:3;

[0011] and light chain complementarity determining region:

[0012] LCDR1, which comprises or consists of the amino acid sequence shown in SEQ ID NO:4;

[0013] LCDR2, which comprises or consists of the amino acid sequence shown in SEQ ID NO:5; and

[0014] LCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO:6.

[0015] The complementarity determining regions were determined using the Kabat method.

[0016] "Antibodies" in the present invention include polyclonal antibodies and monoclonal antibodies and antigen compound binding fragments of these antibodies, including Fab, F(ab')2, Fd, Fv, scFv, bispecific antibodies and antibody minimum recognition units, as well as single-chain derivatives of these antibodies and fragments. The types of antibodies can be selected from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD. In addition, the term "antibody" includes naturally occurring antibodies and non-naturally occurring antibodies, including, for example, chimeric, bifunctional and humanized antibodies, and related synthetic isoforms. The term "antibody" can be used interchangeably with "immunoglobulin".

[0017] In the present invention, the heavy chain complementary determining region is represented by HCDR, which includes HCDR1, HCDR2 and HCDR3; the light chain complementary determining region is represented by LCDR, which includes LCDR1, LCDR2 and LCDR3. Commonly used CDR labeling methods in the art include: Kabat numbering scheme, IMGT numbering scheme, Chothia and Lesk numbering scheme, and the new standardized numbering system introduced by Lefranc et al. for all protein sequences of the immunoglobulin superfamily in 1997. Kabat et al. were the first to propose a standardized numbering scheme for immunoglobulin variable regions. Over the past few decades, the accumulation of sequences has led to the creation of the KABATMAN database, and the Kabat numbering scheme is generally considered to be a widely used standard for numbering antibody residues. The present invention uses the Kabat annotation standard to mark the CDR region, but CDR regions marked by other methods also fall within the scope of protection of the present invention.

[0018] In the present invention, the region other than CDR in the antibody heavy chain variable region and light chain variable region is the framework region; wherein the heavy chain framework region can be further subdivided into adjacent regions separated by CDR, comprising HFR1, HFR2, HFR3 and HFR4 framework regions; the light chain framework region can be further subdivided into adjacent regions separated by CDR, comprising LFR1, LFR2, LFR3 and LFR4 framework regions.

[0019] Typically, the variable regions of the heavy and light chains can be obtained by arranging and connecting the following numbered CDRs and FRs in the following combinations: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0020] Preferably, the anti-tissue plasminogen activator antibody is a monoclonal antibody.

[0021] More preferably, the amino acid sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody are the amino acid sequences shown in SEQ ID NO.7 and SEQ ID NO.8, respectively.

[0022] The "variable region" of an antibody in the present invention refers to the amino-terminal domain of the heavy or light chain of an antibody. These domains are usually the most variable parts of an antibody and contain the antigen binding site. The light or heavy chain variable region consists of a framework region interrupted by three hypervariable regions called "complementarity determining regions" or "CDRs". The scope of the framework region and CDR has been precisely defined, for example in Kabat (see Sequences of Proteins of Immunological Interest, E. Kabat et al., U.S. Department of Health and Human Services, (1983)) and Chothia. The framework region of an antibody, i.e., the combined framework region of the constituent light and heavy chains, plays a role in positioning and aligning the CDRs, which are primarily responsible for binding to the antigen. In the present invention, the term "complementarity determining region", "CDR" refers to the highly variable region of the heavy and light chains of immunoglobulins, and refers to the region containing one or more or even all of the major amino acid residues that contribute to the binding affinity of an antibody or antigen-binding fragment to its recognized antigen or epitope. In a specific embodiment of the present invention, CDR refers to the highly variable region of the heavy and light chains of the antibody.

[0023] In an alternative embodiment, the antibody further comprises a constant region.

[0024] In an alternative embodiment, the constant region includes a heavy chain constant region and / or a light chain constant region.

[0025] In an alternative embodiment, the heavy chain constant region is selected from the heavy chain constant region of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD, and the light chain constant region is selected from the κ type or λ type light chain constant region.

[0026] In an alternative embodiment, the species of origin of the constant region is cattle, camel, horse, dairy cow, pig, sheep, goat, rat, mouse, dog, cat, rabbit, donkey, deer, mink, chicken, duck, goose, turkey, fighting cock or human.

[0027] In an alternative embodiment, the species origin of the constant region is mouse.

[0028] In another aspect, the present invention provides an isolated nucleic acid molecule, which is DNA or RNA, encoding the anti-tissue plasminogen activator antibody.

[0029] In another aspect, the present invention provides a vector comprising the isolated nucleic acid molecule.

[0030] In another aspect, the present invention provides a host cell transformed with the vector.

[0031] In another aspect, the present invention also provides a method for preparing the tissue plasminogen activator antibody.

[0032] In another aspect, the present invention also provides a composition comprising the anti-tissue plasminogen activator antibody.

[0033] On the other hand, the present invention also provides the use of the anti-tissue plasminogen activator antibody for preparing a drug for treating or preventing TPA-induced bleeding.

[0034] In another aspect, the present invention also provides a method for treating or preventing TPA-induced bleeding, comprising administering an effective amount of the anti-tissue plasminogen activator antibody to a subject in need thereof.

[0035] As used herein, "treat" refers to ameliorating the disease or condition (i.e., slowing or arresting or reducing the development of the disease or at least one clinical symptom thereof); or alleviating or alleviating at least one physical parameter, including those that may not be perceptible to the patient; or regulating the disease or condition physically (e.g., stabilizing perceptible symptoms), physiologically (e.g., stabilizing physical parameters), or both or physiologically; or preventing or delaying the onset or development or progression of the disease or condition.

[0036] "Preventing" a condition or disorder means delaying or preventing the onset or reducing the severity of the condition or disorder as assessed by the presence or extent of one or more symptoms of the condition or disorder.

[0037] The term "subject" refers to an animal. Generally, an animal is a mammal. A subject also refers to, for example, primates (e.g., humans), cows, sheep, goats, horses, dogs, cats, rabbits, rats, mice, fish, birds, etc. In certain embodiments, the subject is a primate. In yet other embodiments, the subject is a human.

[0038] The "effective amount" of the present invention refers to an amount that is effective to achieve the desired therapeutic result at the required dosage and for the required period of time. The therapeutically effective amount of an antibody or antibody portion can vary according to a variety of factors such as the disease state, the age, sex and weight of the subject, and the ability of the antibody or antibody portion to stimulate the desired response in the subject. The dosage unit form as used herein refers to a physically discrete unit suitable as a single dose for a mammalian subject to be treated; each unit contains a predetermined amount of active compound, which is calculated to produce the desired therapeutic effect when combined with the required pharmaceutical carrier.

[0039] Beneficial effects:

[0040] The present invention provides a highly specific anti-tissue plasminogen activator antibody and its use:

[0041] (1) The antibody can specifically bind to TPA, providing antibody selection for the detection of TPA;

[0042] (2) This antibody provides more diverse antibody options for the diagnosis or auxiliary diagnosis of thrombotic diseases using TPA as a marker;

[0043] (3) The antibody specifically binds to TPA and can bind to TPA to reduce the hemorrhagic risk caused by TPA therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0045] Figure 1 : Binding of anti-TPA monoclonal antibody to TPA;

[0046] Figure 2 : The therapeutic effect of anti-TPA antibodies, among which, Figure 2 A is the amount of bleeding, Figure 2 B is the bleeding time. DETAILED DESCRIPTION

[0047] The embodiments of the present invention will be described in detail below in conjunction with the examples, but it will be appreciated by those skilled in the art that the following examples are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. If no specific conditions are specified in the examples, the conditions are carried out according to normal conditions or the conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be obtained commercially.

[0048] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry, and immunology, which are within the capabilities of a skilled artisan. This technique is fully explained in the literature, such as Molecular Cloning: A Laboratory Manual, 2nd Edition (Sambrook et al., 1989); Oligonucleotide Synthesis (MJ Gait, ed., 1984); Animal Cell Culture (RI Freshney, ed., 1987); Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (DM Weir and CC Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, eds., 1987); Current Protocols in Molecular Biology (FM Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction (PCR: The Polymerase Chain Reaction) (Academic Press, Inc.); Reaction" (Mullis et al., eds., 1994); and Current Protocols in Immunology (JE Coligan et al., eds., 1991), each of which is expressly incorporated herein by reference.

[0049] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0050] Example 1: Expression and purification of recombinant protein TPA

[0051] The human TPA gene sequence was cloned into a prokaryotic expression vector to construct a prokaryotic expression plasmid. The expression plasmid was transformed into E. coli BL21 and cultured on an LB plate containing antibiotics. A single clone was picked and inoculated into LB medium. After the bacterial solution OD value reached 0.6, IPTG was added to induce protein expression. After 16 hours of induction, the bacterial solution was centrifuged and collected. After the bacterial cells were broken, the supernatant was collected by centrifugation, and the antigen protein was purified by ion exchange and molecular sieve.

[0052] After SDS-PAGE electrophoresis confirmed that the protein band size was correct, no dragging or mixed bands were generated, and the purity reached more than 90%, it could be used for the preparation of monoclonal antibodies.

[0053] Example 2: Mouse immunization and antibody detection

[0054] Select 5 6-8 week old SPF female BALB / c mice, mix and emulsify Freund's complete adjuvant and 2 mg / ml antigen protein in equal volumes. Use the emulsified antigen to immunize 6-8 week old SPF female BALB / c mice, and inject 40 μg of antigen protein into each mouse by subcutaneous injection in the foot or back. Two weeks after the first immunization, mix and emulsify the antigen protein with Freund's incomplete adjuvant, and inject 40 μg of antigen protein into each mouse by subcutaneous injection in the foot or back. Two weeks later, collect blood through the tail vein, collect the supernatant by centrifugation, and test the serum titer by ELISA. Immunize and test the serum titer every two weeks. After two immunizations, the serum titer is as high as 2.0 or above after a million-fold dilution. Screening serum titer 10 6 Lymphocytes were isolated from the above mice and used for cell fusion.

[0055] Example 3: Cell fusion and positive hybridoma cell screening and subcloning

[0056] The lymphocytes of isolated immune mice were fused with cultured SP2 / 0 cells by PEG1500 or electrofusion. The fused cells were cultured in HAT-1640 medium containing 20% ​​FBS serum. After 6 days of culturing the fused cells, the original culture medium was aspirated and 200 μl of fresh HAT medium was added. The culture supernatant was taken on the second day and the positive wells were screened by microplate ELISA. The ELISA method uses an ELISA detection plate. The preparation conditions of the ELISA detection plate are as follows: the TPA antigen amount per well is coated at 50 ng, the coating liquid is phosphate buffer, and the blocking liquid is 5% skim milk powder; the positive wells are replaced with fresh culture medium again, and the culture supernatant is tested again by ELISA after 1 day of culture; the wells that are positive in both tests are selected for the first cloning culture, that is, by limiting dilution, each well of the 96-well plate contains only 1 cell on average; after 7 days, the cell culture supernatant of the cloning culture plate is tested by ELISA to select the positive wells. The culture supernatant of the positive wells is then subjected to affinity screening. The specific method is as follows: the TPA antigen is coated on the ELISA test plate, the cell culture supernatant is first incubated with the antigen for 1 hour, the antibody in the culture supernatant will bind to the free antigen, and then the incubated culture supernatant is added to the antigen-coated ELISA test plate. The unbound free antibody in the culture supernatant will bind to the coated antigen on the ELISA test plate and remain on the plate. Finally, an enzyme-labeled secondary antibody is added for color development. A positive result indicates that the antibody binds to the coated antigen. The higher the positive value, the more free antibodies remain after the supernatant is incubated with the antigen, that is, the higher the relative affinity. Select the clone wells with high relative affinity to continue cloning until the clone well test results are 100% positive. Generally, 2-4 clonings are required. The cloned cell lines are expanded and finally frozen in liquid nitrogen.

[0057] Example 4 Production and purification of monoclonal antibodies

[0058] Two groups of 6-8 week old BALB / c mice were selected and intraperitoneally injected with 500 μL paraffin oil to suppress the immune response of the mice. One week after the injection, the cell line described in Example 3 was intraperitoneally injected into one group of mice at a cell density of about 1×10 6 After two weeks, the ascites was collected. The collected ascites was precipitated with ammonium sulfate and purified with protein A affinity to obtain the target antibody, and the purification effect was identified by PAGE electrophoresis.

[0059] Example 5: Identification of subtypes of monoclonal antibodies and gene sequence cloning

[0060] The SBA Clonotyping System-HRP kit from Southern Biothech was used to identify the subtypes of the heavy and light chains of the monoclonal antibody according to the instructions. The specific procedures were as follows:

[0061] 1. Dilute the capture antibody to 1 μg / mL with coating solution (0.05M carbonate and bicarbonate buffer at pH 9.5), add 100 μL / well to the ELISA plate, and coat overnight at 4°C. Wash the plate three times with PBS buffer (washing solution) containing 0.05% Tween-20.

[0062] 2. Dilute the culture supernatant of the hybridoma cells to be tested with diluent (1% BSA, 0.1% PBST) at a ratio of 1:1, add 100 μL / well to the ELISA plate, and incubate at 37°C for 30 minutes. Dilute the corresponding enzyme-labeled antibodies (Ig-HRP, IgG1-HRP, IgG2a-HRP, IgG2b-HRP, IgG3-HRP, IgM-HRP, kappa-HRP, lamda-HRP) at a ratio of 1:3000 with diluent.

[0063] 3. After washing the plate three times with the plate washing solution, add 100 μL of diluted enzyme-labeled antibody to each well and incubate at 37°C for 30 minutes. After washing the plate three times again, add the color developing solution, and after about 5 minutes (depending on the strength of the reaction), add 2M sulfuric acid to terminate the reaction and read the OD450 absorbance value.

[0064] After identification, the antibody was found to be an IgG1 mouse monoclonal antibody. Based on the antibody subtype results, the antibody gene sequence was cloned using a mature technical route. Hybridoma cells with good growth were collected, and total RNA of hybridoma cells was extracted using Trizol from Thermo Company. The mRNA was reverse transcribed into cDNA according to the instructions of PrimeScript II ReverseTranscriptase from Takara Company, and then frozen at -20°C for future use.

[0065] The specific operation process of reverse transcription is as follows:

[0066] First, prepare a 10 μL total volume of template RNA / primer DNA premix, including 1 μL 10 μM specific primer, 4 μL 2.5 mM dNTP mixture, and 5 μL RNA (total amount less than 5 μg). After mixing, treat at 65°C for 5 minutes and immediately put on ice.

[0067] Add 4 μL 5× PrimeScript II buffer, 20 units of RNase inhibitor, and 1 μL PrimeScript II RTase (200 units) to the premix, mix well, react at 42°C for 60 minutes, then at 70°C for 15 minutes, and then cool on ice for later use.

[0068] Using cDNA as a template, the antibody gene amplification primers reported in the literature were used to perform amplification attempts independently, and the primers that can efficiently amplify the antibody gene were screened out. PCR was performed according to the operating scheme of the Phanta Max Super-Fidelity DNA Polymerase instruction manual of Nanjing Novozymes. The PCR reaction system is:

[0069] 25μL 2×Phanta

[0070] 1 μL dNTP

[0071] 4μL 10uM primer pair

[0072] 4 μL hybridoma cell cDNA

[0073] 1μL DNA polymerase

[0074] 15 μL dd HO

[0075] The total reaction volume was 50 μL. The amplification conditions were: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 30 s; annealing at 56°C for 30 s; extension at 72°C for 2 min. The PCR product was recovered by gel according to the instructions of OMEGA Gel Extraction Kit from OMEGA. After the amplified product was linked to the T vector, the antibody gene sequence was obtained by plasmid sequencing.

[0076] Example 6: Identification of anti-TPA monoclonal antibody activity

[0077] TPA amidolytic activity was detected using 500 μM of the chromogenic substrate S2288. Plasminogen activation by TPA was determined by monitoring the amidolytic activity of plasmin with 500 μM S2251. The experiment was carried out in Tris-Nacl buffer (50 mM Tris-HCl, 100 mM NaCl, pH 7.4) at 37°C. Plasminogen was pretreated with aprotinin-agarose beads at 4°C for 4 hours to remove contaminating plasmin. In both assays, the absorbance at 405 nm (A405 nm) was continuously recorded. The amidolytic activity of TPA was determined by the initial slope of the absorbance at A405 nm over time. The activation rate of plasminogen by TPA was determined by the change in absorbance per square meter second when the net change in absorbance at A405 nm was less than 0.1 in the initial stage of the reaction. In some cases, anti-TPA antibodies were cultured with TPA to detect their effects on TPA activity or plasminogen activation.

[0078] Example 7: Determination of the binding of anti-TPA monoclonal antibodies to TPA

[0079] The binding constant of TPA and anti-TPA mAb was evaluated by saturation binding experiment using ELISA method. In brief, 7.5 μg / ml of purified anti-TPA monoclonal antibody in PBS was coated on microplate at room temperature for 1 hour, and then blocked with 1% BSA in PBS. Then, different concentrations of human TPA (0-4 μg / ml) were loaded on human serum pre-quenched with 20 μM PPACK and 200 kallikrein inhibitor units aprotinin. Integrated TPA was detected using HRP-coupled anti-human TPA polyclonal antibody, followed by TMB substrate. The reaction was monitored at A370nm within the dynamic range of the microplate reader. Binding constant was calculated using GraphpadPrism software (LaJolla, CA).

[0080] Combined with the situation Figure 1 As shown, it can be seen that the binding effect is good compared with the control.

[0081] Example 8 Therapeutic Effects of Anti-TPA Antibodies

[0082] TPA-induced cerebral hemorrhage is a serious complication of TPA therapy for ischemic stroke. Mice were treated with a standard TPA dose for 2.5 hours, followed by an equimolar dose of anti-TPA antibody or blank. Bleeding time was measured after TPA infusion. Six hours after embolization, anesthetized mice were euthanized, infused, and the brains were harvested for examination of hemorrhage and infarction. Figure 2 A shows the total percentage of cerebral hemorrhage, Figure 2 B shows the bleeding time. It can be seen that after TPA treatment, the administration of anti-TPA antibodies can specifically inactivate TPA and significantly reduce the amount of cerebral hemorrhage and bleeding time.

[0083] The specific method is as follows: Adult mice were anesthetized with a mixture of 1.5-2% isoflurane and oxygen. Rectal temperature was maintained at 37°C using a thermostat-controlled heating pad. The left common carotid artery was isolated after a neck incision, and the external carotid, thyroid, and occipital arteries were ligated. Temporary microvessels were placed on the carotid and occipital arteries. A small arteriotomy was performed in the external carotid artery to retrogradely insert a PES catheter containing 125I-fibrinogen (~5000 cpm / 2μl). The PES catheter containing the clot was inserted into the left carotid artery and passed into the external cerebral carotid artery up to the origin of the middle external carotid artery (MAC). Thrombi were formed in 100μl saline solution at a rate of 0.45ml / min. Regional cerebral perfusion was evaluated using continuous laser-Doppler monitoring to ensure adequate embolization (perfusion decreased to <20% of the pre-ischemic baseline). The right jugular vein was cannulated to administer drugs. Mice were injected with a 20% bolus, 50% TPA (10 mg / kg for 2.5 hours to achieve local ischemia) over 30 minutes. After the bolus TPA, mice were treated with intravenous injection of stoichiometric units of anti-TPA antibody. Bleeding time was measured for 20 minutes. After TPA injection and monitoring for 30 minutes, the tail bleeding volume and bleeding time were measured in 3 mL of bath saline solution pre-warmed at 37°C for 5 minutes.

[0084] Finally, it should be noted that although the above embodiments have been described in the specification and drawings of this application, this does not limit the scope of patent protection of this application. All technical solutions generated by replacing or modifying equivalent structures or equivalent processes based on the essential concept of this application using the contents recorded in the specification and drawings of this application, as well as directly or indirectly implementing the technical solutions of the above embodiments in other related technical fields, are included in the scope of patent protection of this application.

Claims

1. A novel anti-tissue plasminogen activator antibody, characterized in that: The antibody has the following complementarity determining regions: Heavy chain complementarity determining region: HCDR1, which comprises or consists of the amino acid sequence shown in SEQ ID NO: 1; HCDR2, which comprises or consists of the amino acid sequence shown in SEQ ID NO: 2; and HCDR3, which comprises or consists of the amino acid sequence shown in SEQ ID NO:3; and light chain complementarity determining region: LCDR1, which comprises or consists of the amino acid sequence shown in SEQ ID NO:4; LCDR2, which comprises or consists of the amino acid sequence shown in SEQ ID NO:5; and LCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO:

6.

2. The anti-tissue plasminogen activator antibody according to claim 1, characterized in that The anti-tissue plasminogen activator antibody is a monoclonal antibody.

3. The anti-tissue plasminogen activator antibody according to claim 1 or 2, characterized in that: The amino acid sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody are the amino acid sequences shown in SEQ ID NO.7 and SEQ ID NO.8, respectively.

4. An isolated nucleic acid molecule, characterized in that The nucleic acid molecule is DNA or RNA, which encodes the anti-tissue plasminogen activator antibody according to any one of claims 1 to 3.

5. A carrier, characterized in that It comprises the isolated nucleic acid molecule described in claim 4.

6. A host cell, characterized in that It is transformed with the vector described in claim 5.

7. A method for preparing the tissue plasminogen activator antibody according to any one of claims 1 to 3.

8. A composition, characterized in that The composition comprises the anti-tissue plasminogen activator antibody according to any one of claims 1 to 3.

9. Use of the anti-tissue plasminogen activator antibody according to any one of claims 1 to 3 for preparing a medicament for treating or preventing TPA-induced bleeding.

10. A method for treating or preventing TPA-induced bleeding, characterized in that: The method comprises administering an effective amount of the anti-tissue plasminogen activator antibody according to any one of claims 1 to 3 to a subject in need thereof.

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