Use of Anti-adrenomedullin antibody in prevention or treatment of cerebral stroke

Abstract

The present application discloses a use of an anti-adrenomedullin (ADM) antibody in the preparation of a drug for preventing or treating cerebral stroke. The antibody or a fragment thereof specifically binds to the first to twenty-first amino acid sequences of the N-terminus of human ADM, the first to twenty-first amino acid sequences of the N-terminus of the human ADM are as shown in SEQ ID NO: 2, and the monoclonal antibody or the fragment exhibits an affinity for ADM with a KD value of less than 10-10M. In the present disclosure, an anti-ADM monoclonal antibody is screened for by the hybridoma technology, and by means of humanization and expression of the antibody and panning of a fully human single-stranded phage antibody library, a fully human anti-ADM monoclonal antibody is obtained. On this basis, in the present application, the activity of higher binding to the N-terminus of human ADM is ultimately obtained by screening an affinity maturation library. The anti-ADM non-neutralizing antibody of the present application can be used in the development of a drug for preventing or treating cerebral stroke.

Description

Application of anti-adrenomedullin antibodies in the prevention or treatment of stroke

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This disclosure claims priority to Chinese Patent Application No. 2024100581319, filed on January 16, 2024, and the entire text of the above-mentioned Chinese Patent Application is incorporated herein by reference. Technical Field

[0003] The present disclosure relates to the field of biomedical technology, and in particular to the use of anti-adrenomedullin antibodies in preventing or treating stroke. Background Art

[0004] Adrenomedullin (ADM) is a 6 kDa polypeptide composed of 52 amino acids. It was first discovered in pheochromocytomas in 1993 and has since been found to be widely secreted and expressed in vascular epithelial cells and vascular smooth muscle cells. The adrenomedullin gene is located on chromosome 11. After translation, the preprohormone is progressively cleaved enzymatically to form the calcitonin gene-related peptide receptor-like receptor (CRLR). Heterodimers consisting of CRLR and RAMP2 or RAMP3 are the receptors for adrenomedullin. Due to receptor-mediated endocytosis and the action of proteases, its half-life is only 22 minutes.

[0005] Adrenomedullin maintains endothelial cell integrity and strengthens the barrier. This is achieved through the following two aspects: (1) enhancing the activity of the GTPase Rac1, increasing the production of cortical actin and stress fibers; (2) reducing myosin light chain kinase-induced actomyosin contraction by inhibiting the RhoA / ROCK pathway. Adrenomedullin also has vasodilation and blood pressure lowering effects. This is achieved through the following two aspects: (1) releasing NO through the PI3K / Akt pathway, activating cyclic guanosine monophosphate (cGMP) / activated protein kinase K (PKG); (2) by binding to vascular smooth muscle, resulting in an increase in the concentration of cyclic adenosine monophosphate (cAMP) / activated protein kinase A (PKA), which causes relaxation of smooth muscle cells through phosphorylation.

[0006] In healthy individuals, adrenomedullin concentrations are extremely low, around 10 pg / mL. It can flexibly shuttle between blood vessels, regulating vasodilation and maintaining endothelial cell barrier function. In patients with septic shock, adrenomedullin concentrations are 5-6 times higher than normal and are directly correlated with disease severity and prognosis. In patients with septic shock, inflammation weakens the vascular barrier function, and vasodilation leads to further lowering of blood pressure.

[0007] Cerebral stroke, also known as "stroke" or "cerebral vascular accident" (CVA), is an acute cerebrovascular disease caused by sudden rupture of a cerebral blood vessel or blockage of a vascular blockage, resulting in brain tissue damage. Non-neutralizing antibodies against adrenomedullin confine adrenomedullin to the blood vessels, correcting the intravascular barrier function, weakening the extracellular vasodilation effect, and increasing the half-life of adrenomedullin in plasma. These antibodies are expected to be used in the development of drugs for stroke indications. Therefore, there is an urgent need to develop non-neutralizing antibodies with a high affinity for adrenomedullin, and to explore the development of effective drugs for the prevention or treatment of stroke. Summary of the Invention

[0008] In order to address the deficiencies of the prior art, the present disclosure aims to provide an anti-adrenomedullin (anti-ADM) antibody or a fragment thereof, which can specifically and highly bind to the 1st to 21st amino acid sequence of the N-terminus of human adrenomedullin (ADM), effectively preventing or treating stroke, and is used for drug development.

[0009] To achieve the above objectives, in a first aspect, the present disclosure provides the use of an anti-adrenomedullin (anti-ADM) antibody or a fragment thereof in the preparation of a drug for preventing or treating stroke, wherein the antibody or fragment thereof specifically binds to the N-terminal 1-21 amino acid sequence of human adrenomedullin (ADM), the N-terminal 1-21 amino acid sequence of human ADM is as shown in SEQ ID NO: 2, and the monoclonal antibody or fragment exhibits a KD value of less than 10- 10 M's affinity.

[0010] Wherein, the fragment includes Fab, Fab', F(ab)2, Fv fragment, F(ab')2, scFv, di-scFv, VHH and / or dAb.

[0011] Wherein, the types of antibodies include IgG, IgA, IgM, IgD or IgE.

[0012] In an alternative embodiment, the antibody comprises,

[0013] (a) The heavy chain variable region of the antibody comprises the following CDR sequences:

[0014] (i) GYTFTX1Y, wherein X1 is selected from S, Q or H,

[0015] (ii) SX2YX3GX4, wherein X2 is selected from A or P, X3 is selected from N, Q, S or T, and X4 is selected from N or K,

[0016] (iii) EGRX5GGSFX6I, wherein X5 is selected from S or W, and X6 is selected from D or N; and

[0017] (b) The light chain variable region of the antibody comprises the following CDR sequence:

[0018] (i)RAX7X8GIX9X 10 YLA, wherein X7 is selected from S or A, X8 is selected from Q or E, X9 is selected from S or G, X 10 Select from S or E,

[0019] (ii)DX 11 SX 12 X 13 X 14 X 15 , where X 11 Select A, V or T, X 12 Selected from N, I or D, X 13 Select from L or V, X 14 Select from E or D, X 15 Select from T or A,

[0020] (iii)QQYDX 16 LX 17 LX 18 , where X 16 Select from N or D, X 17 Select from P or D, X 18 Select from T or S.

[0021] In another alternative embodiment, the antibody comprises,

[0022] (a) The heavy chain variable region of the antibody comprises the following CDR sequences:

[0023] (i) GYAFTTF (as shown in SEQ ID NO:11),

[0024] (ii) NTYSRV (shown in SEQ ID NO: 12)

[0025] (iii) GYGGEGGLGF (as shown in SEQ ID NO: 13); and

[0026] (b) The light chain variable region of the antibody comprises the following CDR sequence:

[0027] (i) RSSQSIIDSDGNTYLE (as shown in SEQ ID NO: 14),

[0028] (ii) KVSNRFS (as shown in SEQ ID NO: 15),

[0029] (iii) FQGSHFPYT (as shown in SEQ ID NO: 16).

[0030] In a second aspect, the present disclosure provides an anti-adrenomedullin (anti-ADM) antibody or a fragment thereof, wherein the antibody comprises a heavy chain amino acid sequence as shown in any one of SEQ ID NOs: 62-68, and the antibody comprises a light chain amino acid sequence as shown in any one of SEQ ID NOs: 69-76;

[0031] Preferably, the antibody is selected from any one of the following combinations:

[0032] (1) comprising the heavy chain amino acid sequence of SEQ ID NO: 67 and the light chain amino acid sequence of SEQ ID NO: 72; or

[0033] (2) comprising the heavy chain amino acid sequence of SEQ ID NO: 62 and the light chain amino acid sequence of SEQ ID NO: 69; or

[0034] (3) comprising the heavy chain amino acid sequence of SEQ ID NO: 63 and the light chain amino acid sequence of SEQ ID NO: 69; or

[0035] (4) comprising the heavy chain amino acid sequence of SEQ ID NO: 64 and the light chain amino acid sequence of SEQ ID NO: 69; or

[0036] (5) comprising the heavy chain amino acid sequence of SEQ ID NO: 65 and the light chain amino acid sequence of SEQ ID NO: 70; or

[0037] (6) comprising the heavy chain amino acid sequence of SEQ ID NO: 65 and the light chain amino acid sequence of SEQ ID NO: 71; or

[0038] (7) comprising the heavy chain amino acid sequence of SEQ ID NO: 66 and the light chain amino acid sequence of SEQ ID NO: 70; or

[0039] (8) comprising the heavy chain amino acid sequence of SEQ ID NO: 65 and the light chain amino acid sequence of SEQ ID NO: 72; or

[0040] (9) comprising the heavy chain amino acid sequence of SEQ ID NO: 65 and the light chain amino acid sequence of SEQ ID NO: 73; or

[0041] (10) comprising the heavy chain amino acid sequence of SEQ ID NO: 68 and the light chain amino acid sequence of SEQ ID NO: 72; or

[0042] (11) comprising the heavy chain amino acid sequence of SEQ ID NO: 68 and the light chain amino acid sequence of SEQ ID NO: 74; or

[0043] (12) comprising the heavy chain amino acid sequence of SEQ ID NO: 67 and the light chain amino acid sequence of SEQ ID NO: 75; or

[0044] (13) comprising a heavy chain amino acid sequence as shown in SEQ ID NO:65 and a light chain amino acid sequence as shown in SEQ ID NO:76.

[0045] In a third aspect, the present disclosure provides a nucleic acid molecule encoding the anti-adrenomedullin (anti-ADM) antibody or a fragment thereof as described in the second aspect.

[0046] In a fourth aspect, the present disclosure provides an expression vector comprising the nucleic acid molecule described in the third aspect.

[0047] Preferably, the vector is selected from a plasmid expression vector, a lentiviral expression vector, an adenoviral expression vector, an adeno-associated viral expression vector or a transposable vector.

[0048] More preferably, the plasmid vector is pcDNA3.4.

[0049] In a fifth aspect, the present disclosure provides a host cell comprising the nucleic acid molecule described in the third aspect or the expression vector described in the fourth aspect, wherein the host cell is selected from a hamster cell, a human cell or a mouse cell.

[0050] Preferably, the hamster cells are selected from CHO cells or BHK cells.

[0051] Preferably, the human cells are selected from Expi293f cells, HEK293 cells, HT-1080 cells, PER.C6 cells, CAP cells, HKB-11 cells or HuH-7 cells.

[0052] Preferably, the mouse cells are selected from NS0 cells or Sp2 / 0 cells.

[0053] In a sixth aspect, the present disclosure provides a method for preparing the anti-adrenomedullin (anti-ADM) antibody or a fragment thereof as described in the second aspect, comprising culturing the host cell as described in the fifth aspect, and then isolating and obtaining the anti-adrenomedullin (anti-ADM) antibody or a fragment thereof.

[0054] In a seventh aspect, the present disclosure provides a pharmaceutical composition comprising the anti-adrenomedullin (anti-ADM) antibody or a fragment thereof described in the second aspect, or the anti-adrenomedullin (anti-ADM) antibody or a fragment thereof prepared by the preparation method described in the fifth aspect.

[0055] In an alternative embodiment, the anti-adrenomedullin (anti-ADM) antibody or fragment thereof is used in combination with at least one other pharmaceutical ingredient.

[0056] In an optional embodiment, the other drug compound is selected from a separate pharmaceutical dosage form of a neurotransmitter release regulator, a neuroreceptor ligand or agonist or antagonist, a GLP-1R agonist, a calcium channel agent, an acid ion channel agent, an immunomodulator, an antiplatelet drug, an anticoagulant drug, an anti-atherosclerotic drug, a thrombolytic drug, a neuroprotective drug, a vasopressor, a TNF-α-antibody, an antibiotic or other central nervous system reactive antibody.

[0057] In an optional embodiment, the anti-adrenomedullin (anti-ADM) antibody or a fragment thereof is used in combination with at least one other pharmaceutical component such as edaravone, butylphthalide, troxerutin, citicoline, piracetam, aspirin, alteplase, atorvastatin and GLP-1R agonist.

[0058] In an eighth aspect, the present disclosure provides a reagent or a kit comprising the anti-adrenomedullin (anti-ADM) antibody or a fragment thereof described in the second aspect, the anti-adrenomedullin (anti-ADM) antibody or a fragment thereof prepared by the preparation method described in the fifth aspect, or the pharmaceutical composition described in the seventh aspect.

[0059] In a ninth aspect, the present disclosure provides a method for preventing or treating stroke, comprising administering to a subject the anti-adrenomedullin (anti-ADM) antibody or a fragment thereof described in the second aspect, the anti-adrenomedullin (anti-ADM) antibody or a fragment thereof prepared by the preparation method described in the fifth aspect, the pharmaceutical composition described in the seventh aspect, or the reagent described in the eighth aspect.

[0060] In the tenth aspect, the present disclosure provides an anti-adrenomedullin (anti-ADM) antibody or a fragment thereof as described in the second aspect for preventing or treating stroke, an anti-adrenomedullin (anti-ADM) antibody or a fragment thereof prepared by the preparation method described in the fifth aspect, the pharmaceutical composition as described in the seventh aspect, or the reagent as described in the eighth aspect.

[0061] Beneficial technical effects of the present disclosure:

[0062] The anti-ADM non-neutralizing antibodies disclosed herein exhibit high affinity for ADM and can be used in the development of drugs for the prevention or treatment of stroke.

[0063] Furthermore, animal experiments have shown that the anti-ADM monoclonal antibody provided by the present disclosure has a preventive and therapeutic effect on stroke mice that is comparable to that of the existing drug Cerebrolysin, and can significantly improve the survival status of stroke mice. BRIEF DESCRIPTION OF THE DRAWINGS

[0064] Figure 1 is a schematic diagram of anti-ADM antibodies blocking hADM-induced cAMP production in CHOK1 / CRLR / RAMP3 cells;

[0065] FIG2 is a graph showing the effect of anti-ADM antibodies on the survival rate of mice with LPS-induced sepsis;

[0066] FIG3 shows the effects of different drug administration groups on cerebral infarction area in MCAO mice;

[0067] FIG4 shows the effects of different drug administration groups on the cerebral infarction area in MCAO mice. DETAILED DESCRIPTION

[0068] I. Definition

[0069] In this disclosure, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and chemistry used herein are those commonly used in the respective fields. To facilitate a better understanding of this disclosure, definitions and explanations of relevant terms are provided below.

[0070] For purposes of clarity and conciseness, features are described herein as part of the same or separate embodiments; however, it will be understood that the scope of the present disclosure may include embodiments having a combination of all or some of the described features.

[0071] The term "antibody" or "antibody fragment" refers to a non-neutralizing antibody against adrenomedullin or a fragment thereof that is capable of binding to ADM and is therefore directed against ADM and may therefore be referred to as an "anti-ADM antibody" or "anti-ADM antibody fragment."

[0072] In this context, for the purpose of simplification, antibodies or antibody fragments having "non-neutralizing anti-ADM activity" collectively referred to as "non-neutralizing" anti-ADM antibodies or antibody fragments (which, for example, block less than 80% of the biological activity of ADM) are defined as:

[0073] One or more molecules that bind to ADM, upon addition to a culture of a eukaryotic cell line expressing a functional human recombinant ADM receptor consisting of CRLR (calcitonin receptor-like receptor) and RAMP3 (receptor activity modifying protein 3), reduce the amount of cAMP produced by the cell line through the action of a synthetic human ADM peptide added in parallel, wherein the added synthetic human ADM is added in an amount that, in the absence of the non-neutralizing antibodies to be analyzed, results in a half-maximal stimulation of cAMP synthesis, and wherein the binding of the molecule to ADM does not result in a reduction in cAMP of more than 80%, even when the non-neutralizing molecule capable of binding to ADM to be analyzed is added in an amount that is 10 times greater than the amount required to obtain the maximal reduction in cAMP that can be obtained using the non-neutralizing antibodies to be analyzed.

[0074] The same definition applies to other ranges: 95%, 90%, 50%, etc.

[0075] Biological activity is defined as the effect that a substance exhibits upon its interaction with a living organism, tissue, organ, or functional unit in vivo or in vitro (e.g., in an assay). In the case of ADM biological activity, this could be the effect of ADM in a human recombinant adrenomedullin receptor cAMP functional assay. Thus, according to the present disclosure, biological activity is defined by the adrenomedullin receptor cAMP functional assay.

[0076] To determine ADM bioactivity in such an assay, the following steps may be performed:

[0077] Dose response curves were performed using ADM in the human recombinant adrenomedullin receptor cAMP functional assay.

[0078] The ADM concentration that gives half-maximal cAMP stimulation can be calculated.

[0079] At constant half-maximal cAMP stimulation, ADM concentration dose response curves (up to a final concentration of 100 μg / ml) were performed with either the ADM stabilizing antibody or the adrenomedullin stabilizing antibody fragment, respectively.

[0080] A maximum inhibition of 50% in the ADM bioassay indicates that the anti-ADM antibody or anti-adrenomedullin antibody fragment, respectively, blocked 50% of the baseline bioactivity. A maximum inhibition of 80% in the ADM bioassay indicates that the anti-ADM antibody or anti-adrenomedullin antibody fragment, respectively, blocked 80% of the ADM bioactivity. This means that no more than 80% of the ADM bioactivity was blocked. This means that approximately 20% of residual ADM bioactivity still existed.

[0081] However, throughout this specification and in the above context, in conjunction with the anti-ADM antibodies and anti-ADM antibody fragments disclosed herein, the expression "blocking the biological activity of ADM" should be understood as only reducing the biological activity of ADM, preferably reducing the ADM biological activity from 100% to 20% of the remaining ADM biological activity at a maximum, preferably reducing the ADM biological activity from 100% to 50% of the remaining ADM biological activity, but in any case there is still ADM biological activity that can be measured as described above.

[0082] Herein, an anti-adrenomedullin (ADM) antibody is an antibody that specifically binds to ADM, and an anti-adrenomedullin antibody fragment is a fragment of an ADM antibody, wherein the fragment specifically binds to ADM. Specific binding to ADM also allows for binding to other antigens. This means that this specificity does not preclude the antibody from cross-reacting with polypeptides other than the polypeptide that elicited the antibody. This also applies to the specificity of the anti-ADM antibodies or fragments thereof disclosed herein.

[0083] The non-neutralizing anti-ADM antibodies or non-neutralizing anti-ADM antibody fragments of the present disclosure offer significant therapeutic advantages over neutralizing anti-ADM antibodies or neutralizing anti-ADM antibody fragments.

[0084] The antibodies disclosed herein are proteins comprising one or more polypeptides that are substantially encoded by immunoglobulin genes and that specifically bind to an antigen. Recognized immunoglobulin genes include kappa, lambda, alpha (IgA), gamma (IgG1, IgG2, IgG3, IgG4), delta (IgD), epsilon (IgE), and mu (IgM) constant region genes, as well as numerous immunoglobulin variable region genes. Full-length immunoglobulin light chains are typically about 25 KDa or 214 amino acids in length. Full-length immunoglobulin heavy chains are typically about 50 KDa or 446 amino acids in length. The light chain is encoded by a variable region gene (about 110 amino acids in length) at the NH2-terminus and a kappa or lambda constant region gene at the COOH-terminus. The heavy chain is also encoded by a variable region gene (about 116 amino acids in length) and one of the other constant region genes.

[0085] The basic structural unit of an antibody is typically a tetramer consisting of two pairs of identical immunoglobulin chains, each pair having a light chain and a heavy chain. In each pair, the light chain and heavy chain variable regions bind to the antigen, while the constant region mediates the effector or function. Immunoglobulins also exist in a variety of other forms, including, for example, Fv, Fab and F(ab')2, as well as bifunctional hybrid antibodies and single-chain antibodies. The immunoglobulin light or heavy chain variable region includes a framework region interrupted by three hypervariable regions, also known as complementary determining regions (CDR's). As noted above, CDRs are primarily responsible for binding to the epitope of the antigen. Immune complexes are antibodies such as monoclonal antibodies, chimeric antibodies, humanized antibodies or human antibodies or functional antibody fragments that specifically bind to the antigen.

[0086] In the art, antibody CDRs can be defined using a variety of methods, such as the Kabat definition rules based on sequence variability, the Chothia definition rules based on the position of structural loop regions, and concepts based on the IMGT Ontology. In the present disclosure, the amino acid sequences of the VL and VH of the anti-ADM antibodies are encoded according to the Chothia encoding rules, and the light chain CDRs 1 to 3 (LCDRs 1 to 3) and heavy chain CDRs 1 to 3 (HCDRs 1 to 3) of the anti-ADM antibodies are defined according to the Chothia definitions.

[0087] Chimeric antibodies are antibodies in which the light and heavy chain genes are constructed from immunoglobulin variable and constant region genes belonging to different species by genetic engineering. For example, the variable segments from mouse monoclonal antibody genes can be linked to human constant segments such as κ and γ1 or γ3. In one example, a therapeutic chimeric antibody is a hybrid protein consisting of a variable domain or antigen-binding domain from a mouse antibody and a constant or effector domain from a human antibody, although variable regions can be generated using other mammalian species or by molecular techniques.

[0088] A "humanized" immunoglobulin is an immunoglobulin that includes a human framework region and one or more CDRs from a non-human (such as mouse, rat, or synthetic) immunoglobulin. The non-human immunoglobulin that provides the CDRs is called the "donor," and the human immunoglobulin that provides the framework is called the "acceptor."

[0089] In one embodiment, all CDRs in the humanized immunoglobulin are derived from a donor immunoglobulin. Constant regions need not be present, but if present, they must be substantially identical to human immunoglobulin constant regions, i.e., at least about 85% to 90%, such as about 95% identical or more identical. Thus, all parts of the humanized immunoglobulin, except possibly the CDRs, are substantially identical to corresponding parts of the natural human immunoglobulin sequence. A "humanized antibody" is an antibody comprising a humanized light chain and a humanized heavy chain immunoglobulin. A humanized antibody can bind to the same antigen as the donor antibody that provides the CDRs. The acceptor framework of a humanized immunoglobulin or antibody may have a limited number of amino acid substitutions from the donor framework. Humanized or other monoclonal antibodies may have other conservative amino acid substitutions that have substantially no effect on antigen binding or other immunoglobulin functions. Humanized immunoglobulins can be constructed by genetic engineering.

[0090] Human antibodies are antibodies in which the light and heavy chain genes are derived from humans. Human antibodies can be produced by immortalizing human B cells that secrete the antibody of interest. Immortality can be achieved, for example, by EBV infection or by fusing human B cells with myeloma or hybridoma cells to produce tri-hybridoma cells. Human antibodies can also be produced by phage display methods or selected from human combinatorial monoclonal antibody libraries. Human antibodies can also be produced using transgenic animals carrying human immunoglobulin genes.

[0091] Therefore, the anti-ADM antibody may have any form known in the art, such as human antibodies, monoclonal antibodies, humanized antibodies, chimeric antibodies, and CDR-grafted antibodies.

[0092] In preferred embodiments, the antibodies of the present disclosure are recombinantly produced antibodies, such as IgG, or antibody fragments, such as chemically coupled antibodies (fragment antigen binding), containing at least the F-variable domains of the heavy and / or light chains.

[0093] Therefore, in a preferred embodiment of the present disclosure, the anti-Adrenomedullin monoclonal antibody fragments described in the present disclosure include Fab, Fab', F(ab)2, Fv fragment, F(ab')2, scFv, di-scFv, VHH and / or dAb.

[0094] In a preferred embodiment of the present disclosure, the antibodies of the present disclosure can be produced as follows:

[0095] Mice were immunized with a synthetic N-terminal 16 amino acid polypeptide of human ADM, a N-terminal 21 amino acid polypeptide of human ADM, or a N-terminal 19 amino acid polypeptide of mouse ADM (abbreviated as YY-19, SEQ ID NO: 3) and a conjugate as immunogens. Blood was collected one week after the last immunization, and the titer of serum anti-YY-21 was determined by ELISA. After the mice with high serum titers were immunized with immunogen pulses, spleen cells were collected for fusion. After spleen cell fusion and hybridoma clone screening, hybridoma clones were obtained. After affinity determination, the hybridoma clones showed a KD value of less than 10 for ADM. -10 M's affinity.

[0096] In this disclosure, the terms "KD," "KD," or "KD" are used interchangeably and generally refer to the equilibrium dissociation constant of an antibody-antigen interaction. "KD," as used in this disclosure, is the ratio of the dissociation rate constant (kdis, also known as "off-rate (koff)" or "kd") to the association rate constant (kon, also known as "association rate (kon)" or "ka").

[0097] In this disclosure, the term "specific binding" refers to a non-random binding reaction between two molecules, such as an antibody and its antigen. The strength or affinity of a specific binding interaction can be measured in terms of the equilibrium dissociation constant (KD) or half-maximal effect concentration (EC 50 )express.

[0098] The specific binding properties between two molecules can be determined using methods known in the art. One method involves measuring the rates of formation and dissociation of the antigen binding site / antigen complex. Both the "association rate constant" (ka or kon) and the "dissociation rate constant" (kdis or koff) can be calculated from the concentration and the actual rates of association and dissociation (see Malmqvist M, Nature, 1993, 361:186-187). The ratio of kdis / kon is equal to the dissociation constant, KD (see Davies et al., Annual Rev Biochem, 1990;59:439-473). KD, kon, and kdis values ​​can be measured using any valid method. In certain embodiments, the dissociation constant can be measured using bioluminescence interferometry (e.g., the ForteBio Octet method). Surface plasmon resonance techniques (e.g., Biacore) or Kinexa can also be used to measure the dissociation constant.

[0099] In this disclosure, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector is capable of expressing a protein encoded by the inserted polynucleotide, the vector is referred to as an expression vector. A vector can be introduced into a host cell through transformation, transduction, or transfection, so that the genetic material elements it carries are expressed 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 (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs); 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, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomas (such as SV40). A vector can contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, a vector may also contain a replication initiation site.

[0100] Expression and cloning vectors contain nucleic acid sequences that enable the vector to replicate in one or more selected host cells. Typically, in cloning vectors, this sequence is one that enables the vector to replicate independently of the host chromosomal DNA, and it includes an origin of replication or an autonomous replication sequence. The term "expression vector" as used herein refers to a vector comprising a recombinant polynucleotide, which comprises an expression regulatory sequence operably linked to the nucleotide sequence to be expressed. The expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be provided by host cells or in vitro expression systems. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses).

[0101] In the present disclosure, the term "host cell" refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia 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 fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.

[0102] In this disclosure, the term "pharmaceutically acceptable" means that when the molecule itself, molecule fragment or composition is appropriately administered to an animal or human, it does not produce adverse, allergic or other untoward reactions. Specific examples of some substances that can serve as pharmaceutically acceptable carriers or components thereof include sugars (such as lactose), starch, cellulose and its derivatives, vegetable oils, gelatin, polyols (such as propylene glycol), alginic acid, etc.

[0103] In the present disclosure, the term "prevention" refers to a method implemented in order to prevent or delay the occurrence of a disease or disorder or symptom (e.g., a disease or disorder related to coagulation or thromboembolism) in a subject. As used herein, the term "treatment" refers to a method implemented in order to obtain a beneficial or desired clinical result. For the purposes of the present invention, beneficial or desired clinical results include, but are not limited to, alleviating symptoms, reducing the scope of the disease, stabilizing (i.e., no longer worsening) the state of the disease, delaying or slowing the development of the disease, improving or alleviating the state of the disease, alleviating symptoms (whether partially or completely), alleviating or improving prognosis, reducing or inhibiting the recurrence of the disease, etc., whether detectable or undetectable. In addition, "treatment" can also refer to prolonging survival compared to the expected survival (if not receiving treatment).

[0104] In this disclosure, the term "subject" refers to a mammal, such as a primate mammal, such as a human. In certain embodiments, the subject (eg, human) suffers from a disease or condition related to coagulation or thromboembolism, or is at risk of suffering from the above disease.

[0105] In the present disclosure, the term "effective amount" refers to an amount sufficient to obtain or at least partially obtain the desired effect. For example, an effective amount for preventing a disease (e.g., a disease or condition related to coagulation or thromboembolism) refers to an amount sufficient to prevent, stop, or delay the occurrence of a disease (e.g., a disease or condition related to coagulation or thromboembolism); an effective amount for treating a disease refers to an amount sufficient to cure or at least partially stop the disease and its complications in a patient already suffering from the disease. Determining such an effective amount is well within the capabilities of those skilled in the art. For example, an amount effective for therapeutic use will depend on the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general condition such as age, weight and sex, the mode of administration of the drug, and other treatments administered simultaneously, etc.

[0106] In this disclosure, the term "stroke" is well known in the art. Stroke can be occlusive (due to closure of a blood vessel) or hemorrhagic (due to bleeding from a blood vessel). The term "ischemic" as used herein refers to the lack of blood supply and oxygen that occurs when the autoregulatory dilation of resistance vessels cannot compensate for the reduced perfusion pressure distal to an abnormally narrowed (constricted) blood vessel. Although most occlusive strokes are caused by atherosclerosis and thrombosis, and most hemorrhagic strokes are associated with hypertension or aneurysms, either type of stroke may occur at any age due to a variety of causes, including heart disease, trauma, infection, tumors, blood dyscrasias, vascular malformations, immune disorders, and exogenous toxins.

[0107] Unless otherwise indicated, in this application, the term "stroke" generally refers to ischemic stroke, which is usually caused by a decrease in blood flow to the brain or part thereof, which causes insufficient oxygen supply to brain cells. In particular, due to the death of brain cells, stroke can cause irreversible tissue damage. The symptoms of stroke are well known in the art. For example, stroke symptoms include sudden numbness or weakness in the face, arms or legs (particularly on one side of the body), sudden confusion, difficulty speaking or understanding, sudden loss of sight in one or both eyes, and sudden difficulty walking, dizziness, loss of balance or coordination. Ischemic stroke may be caused by atherothrombosis or cerebral aorta embolism, by coagulation disorders or non-tumor vascular diseases, or by cardiac ischemia that causes a decrease in total blood flow. For example, atherothrombotic stroke, cardiogenic stroke and lacunar stroke, atrial fibrillation can also cause cardiogenic stroke (also commonly referred to as embolic or thromboembolic stroke).

[0108] In the present disclosure, the term "Enibarcimab" refers to Anti-Adrenomedullin, which is a humanized murine monoclonal immunoglobulin G1 (IgG1) antibody targeting the vascular protective peptide adrenomedullin, with a molecular weight of 145.5 KD.

[0109] II. Examples

[0110] In one aspect, the present disclosure provides the use of an anti-adrenomedullin (anti-ADM) antibody or a fragment thereof in the preparation of a drug for preventing or treating stroke, wherein the antibody or fragment thereof specifically binds to the N-terminal 1-21 amino acid sequence of human adrenomedullin (ADM), the N-terminal 1-21 amino acid sequence of human ADM is as shown in SEQ ID NO: 2, and the monoclonal antibody or fragment exhibits a KD value for ADM of less than 10- 10 M's affinity.

[0111] In another aspect, the present disclosure provides a method for preventing or treating stroke, comprising administering to a subject a therapeutically effective amount of an anti-adrenomedullin (anti-ADM) antibody or a fragment thereof, wherein the antibody or fragment thereof specifically binds to the N-terminal 1-21 amino acid sequence of human adrenomedullin (ADM), the N-terminal 1-21 amino acid sequence of human ADM being as shown in SEQ ID NO: 2, and the monoclonal antibody or fragment exhibits a KD value for ADM of less than 10- 10 M's affinity.

[0112] In another aspect, the present disclosure provides an anti-adrenomedullin (anti-ADM) antibody or a fragment thereof for preventing or treating stroke, wherein the antibody or fragment thereof specifically binds to the N-terminal 1-21 amino acid sequence of human adrenomedullin (ADM), the N-terminal 1-21 amino acid sequence of human ADM being as shown in SEQ ID NO: 2, and the monoclonal antibody or fragment exhibits a KD value for ADM of less than 10- 10 M's affinity.

[0113] In certain embodiments, the fragment comprises a Fab, a Fab', a F(ab)2, an Fv fragment, a F(ab')2, a scFv, a di-scFv, a VHH, and / or a dAb.

[0114] In certain embodiments, the antibody type comprises IgG, IgA, IgM, IgD, or IgE.

[0115] In certain embodiments, hybridoma clones are prepared and screened by immunizing mice, wherein the heavy chain variable region of the hybridoma clones comprises:

[0116] (i) a CDR1 sequence having the formula: GYAFTTF (as shown in SEQ ID NO: 11),

[0117] (ii) a CDR2 sequence having the formula: NTYSRV (as shown in SEQ ID NO: 12),

[0118] (iii) a CDR3 sequence having the formula: GYGGEGGLGF (as shown in SEQ ID NO: 13), and

[0119] The light chain variable region of the hybridoma clone comprises:

[0120] (i) a CDR1 sequence having the formula: RSSQSIIDSDGNTYLE (as shown in SEQ ID NO: 14),

[0121] (ii) a CDR2 sequence having the formula: KVSNRFS (as shown in SEQ ID NO: 15),

[0122] (iii) a CDR3 sequence having the following formula: FQGSHFPYT (as shown in SEQ ID NO: 16).

[0123] In certain embodiments of the present disclosure, the hybridoma clone comprises a heavy chain variable region sequence as shown in SEQ ID NO:9, and the hybridoma clone comprises a light chain variable region sequence as shown in SEQ ID NO:10.

[0124] In some embodiments, humanization of anti-ADM antibodies can be performed according to the following scheme:

[0125] Through sequence alignment, the human antibody germline gene with the highest homology was selected as the humanization design framework. The heavy chain variable region of the hybridoma clone was subjected to CDR grafting and back mutation to obtain the humanized heavy chain variable region sequence. The light chain variable region of the hybridoma clone was subjected to CDR grafting and back mutation to obtain the humanized light chain variable region sequence.

[0126] Thus, in certain embodiments of the present disclosure, the heavy chain variable region of the humanized antibody comprises:

[0127] (i) a CDR1 sequence having the formula: GYAFTTF (as shown in SEQ ID NO: 11),

[0128] (ii) a CDR2 sequence having the formula: NTYSRV (as shown in SEQ ID NO: 12),

[0129] (iii) a CDR3 sequence having the formula: GYGGEGGLGF (as shown in SEQ ID NO: 13), and

[0130] The light chain variable region of the humanized antibody comprises:

[0131] (i) a CDR1 sequence having the formula: RSSQSIIDSDGNTYLE (as shown in SEQ ID NO: 14),

[0132] (ii) a CDR2 sequence having the formula: KVSNRFS (as shown in SEQ ID NO: 15),

[0133] (iii) a CDR3 sequence having the following formula: FQGSHFPYT (as shown in SEQ ID NO: 16).

[0134] In certain embodiments of the present disclosure, the humanized antibody comprises a heavy chain variable region sequence as shown in SEQ ID NO: 17, and the humanized antibody comprises a light chain variable region sequence as shown in any one of SEQ ID NO: 18 or 19.

[0135] In certain embodiments of the present disclosure, the humanized antibody comprises a heavy chain variable region sequence as shown in SEQ ID NO: 17, and the humanized antibody comprises a light chain variable region sequence as shown in SEQ ID NO: 18.

[0136] In certain embodiments of the present disclosure, the humanized antibody comprises a heavy chain variable region sequence as shown in SEQ ID NO: 17, and the humanized antibody comprises a light chain variable region sequence as shown in SEQ ID NO: 19.

[0137] In certain embodiments of the present disclosure, the monoclonal antibody further comprises an antibody light chain constant region, wherein the light chain constant region comprises the amino acid sequence shown in SEQ ID NO: 20.

[0138] In certain embodiments of the present disclosure, the monoclonal antibody further comprises an antibody heavy chain constant region, wherein the heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO: 21.

[0139] The light chain variable region sequence and the light chain constant region of the monoclonal antibody described in the present disclosure are combined to form the antibody light chain, and the heavy chain variable region sequence and the heavy chain constant region of the monoclonal antibody described in the present disclosure are combined to form the antibody heavy chain.

[0140] Thus, in certain embodiments of the present disclosure, the monoclonal antibody comprises a heavy chain sequence as shown in any one of SEQ ID NOs: 23, 25, and 27, and the monoclonal antibody comprises a light chain sequence as shown in any one of SEQ ID NOs: 22, 24, and 26.

[0141] In certain embodiments of the present disclosure, the monoclonal antibody comprises a heavy chain sequence as shown in SEQ ID NO: 23, and the monoclonal antibody comprises a light chain sequence as shown in SEQ ID NO: 22.

[0142] In certain embodiments of the present disclosure, the monoclonal antibody comprises a heavy chain sequence as shown in SEQ ID NO: 25, and the monoclonal antibody comprises a light chain sequence as shown in SEQ ID NO: 24.

[0143] In certain embodiments of the present disclosure, the monoclonal antibody comprises a heavy chain sequence as shown in SEQ ID NO: 27, and the monoclonal antibody comprises a light chain sequence as shown in SEQ ID NO: 26.

[0144] After codon optimization, gene synthesis was performed and the synthesized gene fragment was cloned into an expression vector. After expression plasmid amplification and plasmid extraction, the two plasmids were co-transfected into Expi293F or CHO-K1 cells for transient antibody expression. After expression, the antibody was purified by Protein A affinity chromatography. Affinity determination showed that the humanized anti-ADM antibody exhibited a KD value of less than 10 for ADM. -10 M's affinity.

[0145] In the present disclosure, "monoclonal antibody" generally refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations and / or post-translational modifications (e.g., isomerization, amidation) that may be present in minor amounts. For example, in certain embodiments of the present disclosure, the anti-adrenomedullin monoclonal antibody contains a post-translational modification (PTM) site of NG.

[0146] The present disclosure can also screen anti-ADM antibodies by screening a fully human single-chain phage antibody library.

[0147] Thus, in a preferred embodiment of the present disclosure, in the anti-adrenomedullin antibody or fragment thereof,

[0148] (a) The heavy chain variable region of the antibody comprises:

[0149] (i) a CDR1 sequence having the formula: GYTFTSY (as shown in SEQ ID NO: 43),

[0150] (ii) a CDR2 sequence having the formula: SAYNGN (as shown in SEQ ID NO: 44),

[0151] (iii) a CDR3 sequence having the formula: EGRSGGSFDI (as shown in SEQ ID NO: 45), and

[0152] (b) the light chain variable region of the antibody comprises:

[0153] (i) a CDR1 sequence having the formula: RASQGISSYLA (as shown in SEQ ID NO: 46),

[0154] (ii) a CDR2 sequence having the formula: DASNLET (as shown in SEQ ID NO: 47),

[0155] (iii) a CDR3 sequence having the following formula: QQYDNLPLT (as shown in SEQ ID NO: 48).

[0156] In certain embodiments of the present disclosure, the post-translational modification (PTM) site of NG contained in the anti-adrenomedullin antibody is site-directedly mutated to QG to eliminate deamidation isomerization.

[0157] Thus, in certain embodiments of the present disclosure, in the anti-adrenomedullin antibody or fragment thereof,

[0158] (a) The heavy chain variable region of the antibody comprises:

[0159] (i) a CDR1 sequence having the formula: GYTFTSY (as shown in SEQ ID NO: 43),

[0160] (ii) a CDR2 sequence having the formula: SAYQGN (as shown in SEQ ID NO: 49),

[0161] (iii) a CDR3 sequence having the formula: EGRSGGSFDI (as shown in SEQ ID NO: 45), and

[0162] (b) the light chain variable region of the antibody comprises:

[0163] (i) a CDR1 sequence having the formula: RASQGISSYLA (as shown in SEQ ID NO: 46),

[0164] (ii) a CDR2 sequence having the formula: DASNLET (as shown in SEQ ID NO: 47),

[0165] (iii) a CDR3 sequence having the following formula: QQYDNLPLT (as shown in SEQ ID NO: 48).

[0166] In certain embodiments of the present disclosure, the antibody comprises a heavy chain variable region sequence as shown in any one of SEQ ID NOs: 28 and 29, and the antibody comprises a light chain variable region sequence as shown in SEQ ID NO: 35.

[0167] In certain embodiments of the present disclosure, the humanized antibody comprises a heavy chain variable region sequence as shown in SEQ ID NO: 28, and the humanized antibody comprises a light chain variable region sequence as shown in SEQ ID NO: 35.

[0168] In certain embodiments of the present disclosure, the humanized antibody comprises a heavy chain variable region sequence as shown in SEQ ID NO: 29, and the humanized antibody comprises a light chain variable region sequence as shown in SEQ ID NO: 35.

[0169] In order to improve the affinity of fully human antibodies to human ADM, the present disclosure also designed and constructed a fully human antibody affinity maturation library. Through screening of the fully human antibody affinity maturation library and identification of monoclonal clones, the present disclosure obtained different fully human anti-ADM antibodies, all of which showed KD values ​​for ADM of less than 10 -10 M's affinity.

[0170] Therefore, in a preferred embodiment of the present disclosure, in the anti-adrenomedullin antibody or fragment thereof:

[0171] (a) The heavy chain variable region of the antibody comprises:

[0172] (i) a CDR1 sequence having the formula: GYTFTX1Y, wherein X1 is selected from S, Q or H,

[0173] (ii) a CDR2 sequence having the formula: SX2YX3GX4, wherein X2 is selected from A or P, X3 is selected from N, Q, S or T, and X4 is selected from N or K,

[0174] (iii) a CDR3 sequence having the formula: EGRX5GGSFX6I, wherein X5 is selected from S or W, and X6 is selected from D or N; and

[0175] (b) The light chain variable region of the antibody comprises the following CDR sequence:

[0176] (i) a CDR1 sequence having the formula: RAX7X8GIX9X 10 YLA, wherein X7 is selected from S or A, X8 is selected from Q or E, X9 is selected from S or G, X 10 Select from S or E,

[0177] (ii) a CDR2 sequence having the formula: DX 11 SX 12 X 13 X 14 X 15 , where X 11 Select A, V or T, X 12 Selected from N, I or D, X13 Select from L or V, X 14 Select from E or D, X 15 Select from T or A,

[0178] (iii) a CDR3 sequence having the formula: QQYDX 16 LX 17 LX 18 , where X 16 Select from N or D, X 17 Select from P or D, X 18 Select from T or S.

[0179] In a further preferred embodiment of the present disclosure, in the anti-adrenomedullin antibody or fragment thereof,

[0180] (1) The heavy chain variable region of the antibody comprises:

[0181] (i) a CDR1 sequence having the formula: GYTFTSY (as shown in SEQ ID NO: 43),

[0182] (ii) a CDR2 sequence having the formula: SAYNGN (as shown in SEQ ID NO: 44),

[0183] (iii) a CDR3 sequence having the formula: EGRSGGSFDI (as shown in SEQ ID NO: 45); and

[0184] (2) The light chain variable region of the antibody comprises:

[0185] (i) a CDR1 sequence having the formula: RASQGISSYLA (as shown in SEQ ID NO: 46),

[0186] (ii) a CDR2 sequence having the formula: DASNLET (as shown in SEQ ID NO: 47),

[0187] (iii) a CDR3 sequence having the following formula: QQYDNLPLT (as shown in SEQ ID NO: 48).

[0188] In a further preferred embodiment of the present disclosure, in the anti-adrenomedullin antibody or fragment thereof,

[0189] (1) The heavy chain variable region of the antibody comprises:

[0190] (i) a CDR1 sequence having the formula: GYTFTSY (as shown in SEQ ID NO: 43),

[0191] (ii) a CDR2 sequence having the formula: SAYQGN (as shown in SEQ ID NO: 49),

[0192] (iii) a CDR3 sequence having the formula: EGRSGGSFDI (as shown in SEQ ID NO: 45); and

[0193] (2) The light chain variable region of the antibody comprises:

[0194] (i) a CDR1 sequence having the formula: RASQGISSYLA (as shown in SEQ ID NO: 46),

[0195] (ii) a CDR2 sequence having the formula: DASNLET (as shown in SEQ ID NO: 47),

[0196] (iii) a CDR3 sequence having the following formula: QQYDNLPLT (as shown in SEQ ID NO: 48).

[0197] In a further preferred embodiment of the present disclosure, in the anti-adrenomedullin antibody or fragment thereof,

[0198] (a) The heavy chain variable region of the antibody comprises:

[0199] (i) a CDR1 sequence having the formula: GYTFTSY (as shown in SEQ ID NO: 43),

[0200] (ii) a CDR2 sequence having the formula: SAYQGN (as shown in SEQ ID NO: 49),

[0201] (iii) a CDR3 sequence having the formula: EGRWGGSFNI (as shown in SEQ ID NO: 50), and

[0202] (b) the light chain variable region of the antibody comprises:

[0203] (i) a CDR1 sequence having the formula: RASQGISSYLA (as shown in SEQ ID NO: 46),

[0204] (ii) a CDR2 sequence having the formula: DASNLET (as shown in SEQ ID NO: 47),

[0205] (iii) a CDR3 sequence having the following formula: QQYDNLPLT (as shown in SEQ ID NO: 48).

[0206] In a further preferred embodiment of the present disclosure, in the anti-adrenomedullin antibody or fragment thereof,

[0207] (a) The heavy chain variable region of the antibody comprises:

[0208] (i) a CDR1 sequence having the formula: GYTFTQY (as shown in SEQ ID NO: 51),

[0209] (ii) a CDR2 sequence having the formula: SAYQGN (as shown in SEQ ID NO: 49),

[0210] (iii) a CDR3 sequence having the formula: EGRWGGSFNI (as shown in SEQ ID NO: 50), and

[0211] (b) the light chain variable region of the antibody comprises:

[0212] (i) a CDR1 sequence having the formula: RASEGISEYLA (as shown in SEQ ID NO: 52),

[0213] (ii) a CDR2 sequence having the formula: DASNLET (as shown in SEQ ID NO: 47),

[0214] (iii) a CDR3 sequence having the following formula: QQYDNLPLT (as shown in SEQ ID NO: 48).

[0215] In a further preferred embodiment of the present disclosure, in the anti-adrenomedullin antibody or fragment thereof,

[0216] (a) The heavy chain variable region of the antibody comprises:

[0217] (i) a CDR1 sequence having the formula: GYTFTQY (as shown in SEQ ID NO: 51),

[0218] (ii) a CDR2 sequence having the formula: SAYQGN (as shown in SEQ ID NO: 49),

[0219] (iii) a CDR3 sequence having the formula: EGRWGGSFNI (as shown in SEQ ID NO: 50), and

[0220] (b) the light chain variable region of the antibody comprises:

[0221] (i) a CDR1 sequence having the formula: RAAEGIGSYLA (as shown in SEQ ID NO: 53),

[0222] (ii) a CDR2 sequence having the formula: DASNLET (as shown in SEQ ID NO: 47),

[0223] (iii) a CDR3 sequence having the following formula: QQYDNLPLT (as shown in SEQ ID NO: 48).

[0224] In a further preferred embodiment of the present disclosure, in the anti-adrenomedullin antibody or fragment thereof,

[0225] (a) The heavy chain variable region of the antibody comprises:

[0226] (i) a CDR1 sequence having the formula: GYTFTSY (as shown in SEQ ID NO: 43),

[0227] (ii) a CDR2 sequence having the formula: SPYSGN (as shown in SEQ ID NO: 54),

[0228] (iii) a CDR3 sequence having the formula: EGRWGGSFNI (as shown in SEQ ID NO: 50), and

[0229] (b) the light chain variable region of the antibody comprises:

[0230] (i) a CDR1 sequence having the formula: RASEGISEYLA (as shown in SEQ ID NO: 52),

[0231] (ii) a CDR2 sequence having the formula: DASNLET (as shown in SEQ ID NO: 47),

[0232] (iii) a CDR3 sequence having the following formula: QQYDNLPLT (as shown in SEQ ID NO: 48).

[0233] In a further preferred embodiment of the present disclosure, in the anti-adrenomedullin antibody or fragment thereof,

[0234] (a) The heavy chain variable region of the antibody comprises:

[0235] (i) a CDR1 sequence having the formula: GYTFTHY (as shown in SEQ ID NO: 55),

[0236] (ii) a CDR2 sequence having the formula: SAYQGN (as shown in SEQ ID NO: 49),

[0237] (iii) a CDR3 sequence having the formula: EGRWGGSFNI (as shown in SEQ ID NO: 50), and

[0238] (b) the light chain variable region of the antibody comprises:

[0239] (i) a CDR1 sequence having the formula: RASQGISSYLA (as shown in SEQ ID NO: 46),

[0240] (ii) a CDR2 sequence having the formula: DVSILDA (as shown in SEQ ID NO: 56),

[0241] (iii) a CDR3 sequence having the following formula: QQYDNLPLT (as shown in SEQ ID NO: 48).

[0242] In a further preferred embodiment of the present disclosure, in the anti-adrenomedullin antibody or fragment thereof,

[0243] (a) The heavy chain variable region of the monoclonal antibody comprises:

[0244] (i) a CDR1 sequence having the formula: GYTFTQY (as shown in SEQ ID NO: 51),

[0245] (ii) a CDR2 sequence having the formula: SAYQGN (as shown in SEQ ID NO: 49),

[0246] (iii) a CDR3 sequence having the formula: EGRWGGSFNI (as shown in SEQ ID NO: 50), and

[0247] (b) the light chain variable region of the antibody comprises:

[0248] (i) a CDR1 sequence having the formula: RASQGISSYLA (as shown in SEQ ID NO: 46),

[0249] (ii) a CDR2 sequence having the formula: DVSILDA (as shown in SEQ ID NO: 56),

[0250] (iii) a CDR3 sequence having the following formula: QQYDNLPLT (as shown in SEQ ID NO: 48).

[0251] In a further preferred embodiment of the present disclosure, in the anti-adrenomedullin antibody or fragment thereof,

[0252] (a) The heavy chain variable region of the antibody comprises:

[0253] (i) a CDR1 sequence having the formula: GYTFTQY (as shown in SEQ ID NO: 51),

[0254] (ii) a CDR2 sequence having the formula: SAYQGN (as shown in SEQ ID NO: 49),

[0255] (iii) a CDR3 sequence having the formula: EGRWGGSFNI (as shown in SEQ ID NO: 50), and

[0256] (b) the light chain variable region of the antibody comprises:

[0257] (i) a CDR1 sequence having the formula: RASQGISSYLA (as shown in SEQ ID NO: 46),

[0258] (ii) a CDR2 sequence having the formula: DASNVDT (as shown in SEQ ID NO: 57),

[0259] (iii) a CDR3 sequence having the following formula: QQYDNLPLT (as shown in SEQ ID NO: 48).

[0260] In a further preferred embodiment of the present disclosure, in the anti-adrenomedullin antibody or fragment thereof,

[0261] (a) The heavy chain variable region of the antibody comprises:

[0262] (i) a CDR1 sequence having the formula: GYTFTSY (as shown in SEQ ID NO: 43),

[0263] (ii) a CDR2 sequence having the formula: SPYTGK (as shown in SEQ ID NO: 58),

[0264] (iii) a CDR3 sequence having the formula: EGRWGGSFNI (as shown in SEQ ID NO: 50), and

[0265] (b) the light chain variable region of the antibody comprises:

[0266] (i) a CDR1 sequence having the formula: RASQGISSYLA (as shown in SEQ ID NO: 46),

[0267] (ii) a CDR2 sequence having the formula: DVSILDA (as shown in SEQ ID NO: 56),

[0268] (iii) a CDR3 sequence having the following formula: QQYDNLPLT (as shown in SEQ ID NO: 48).

[0269] In a further preferred embodiment of the present disclosure, in the anti-adrenomedullin antibody or fragment thereof,

[0270] (a) The heavy chain variable region of the antibody comprises:

[0271] (i) a CDR1 sequence having the formula: GYTFTSY (as shown in SEQ ID NO: 43),

[0272] (ii) a CDR2 sequence having the formula: SPYTGK (as shown in SEQ ID NO: 58),

[0273] (iii) a CDR3 sequence having the formula: EGRWGGSFNI (as shown in SEQ ID NO: 50), and

[0274] (b) the light chain variable region of the antibody comprises:

[0275] (i) a CDR1 sequence having the formula: RASQGISSYLA (as shown in SEQ ID NO: 46),

[0276] (ii) a CDR2 sequence having the formula: DTSDLDT (as shown in SEQ ID NO: 59),

[0277] (iii) a CDR3 sequence having the following formula: QQYDNLPLT (as shown in SEQ ID NO: 48).

[0278] In a further preferred embodiment of the present disclosure, in the anti-adrenomedullin antibody or fragment thereof,

[0279] (a) The heavy chain variable region of the antibody comprises:

[0280] (i) a CDR1 sequence having the formula: GYTFTHY (as shown in SEQ ID NO: 55),

[0281] (ii) a CDR2 sequence having the formula: SAYQGN (as shown in SEQ ID NO: 49),

[0282] (iii) a CDR3 sequence having the formula: EGRWGGSFNI (as shown in SEQ ID NO: 50), and

[0283] (b) the light chain variable region of the antibody comprises:

[0284] (i) a CDR1 sequence having the formula: RASQGISSYLA (as shown in SEQ ID NO: 46),

[0285] (ii) a CDR2 sequence having the formula: DASNLET (as shown in SEQ ID NO: 47),

[0286] (iii) a CDR3 sequence having the formula: QQYDDLDLT (as shown in SEQ ID NO: 60).

[0287] In a further preferred embodiment of the present disclosure, in the anti-adrenomedullin antibody or fragment thereof,

[0288] (a) The heavy chain variable region of the antibody comprises:

[0289] (i) a CDR1 sequence having the formula: GYTFTQY (as shown in SEQ ID NO: 51),

[0290] (ii) a CDR2 sequence having the formula: SAYQGN (as shown in SEQ ID NO: 49),

[0291] (iii) a CDR3 sequence having the formula: EGRWGGSFNI (as shown in SEQ ID NO: 50), and

[0292] (b) the light chain variable region of the antibody comprises:

[0293] (i) a CDR1 sequence having the formula: RASQGISSYLA (as shown in SEQ ID NO: 46),

[0294] (ii) a CDR2 sequence having the formula: DASNLET (as shown in SEQ ID NO: 47),

[0295] (iii) a CDR3 sequence having the following formula: QQYDDLPLS (as shown in SEQ ID NO: 61).

[0296] In addition, the present disclosure also obtains the heavy chain variable region sequence and light chain variable region sequence of the antibody with improved affinity.

[0297] Thus, in a preferred embodiment of the present disclosure, in the anti-adrenomedullin antibody or fragment thereof, the antibody comprises a heavy chain variable region sequence as shown in any one of SEQ ID NOs: 28 to 34, and the antibody comprises a light chain variable region sequence as shown in any one of SEQ ID NOs: 35 to 42.

[0298] In certain embodiments of the present disclosure, the humanized antibody comprises a heavy chain variable region sequence as shown in SEQ ID NO: 28, and the humanized antibody comprises a light chain variable region sequence as shown in SEQ ID NO: 35.

[0299] In certain embodiments of the present disclosure, the humanized antibody comprises a heavy chain variable region sequence as shown in SEQ ID NO: 29, and the humanized antibody comprises a light chain variable region sequence as shown in SEQ ID NO: 35.

[0300] In certain embodiments of the present disclosure, the humanized antibody comprises a heavy chain variable region sequence as shown in SEQ ID NO: 30, and the humanized antibody comprises a light chain variable region sequence as shown in SEQ ID NO: 35.

[0301] In certain embodiments of the present disclosure, the humanized antibody comprises a heavy chain variable region sequence as shown in SEQ ID NO: 31, and the humanized antibody comprises a light chain variable region sequence as shown in SEQ ID NO: 36.

[0302] In certain embodiments of the present disclosure, the humanized antibody comprises a heavy chain variable region sequence as shown in SEQ ID NO: 31, and the humanized antibody comprises a light chain variable region sequence as shown in SEQ ID NO: 37.

[0303] In certain embodiments of the present disclosure, the humanized antibody comprises a heavy chain variable region sequence as shown in SEQ ID NO: 32, and the humanized antibody comprises a light chain variable region sequence as shown in SEQ ID NO: 36.

[0304] In certain embodiments of the present disclosure, the humanized antibody comprises a heavy chain variable region sequence as shown in SEQ ID NO: 33, and the humanized antibody comprises a light chain variable region sequence as shown in SEQ ID NO: 38.

[0305] In certain embodiments of the present disclosure, the humanized antibody comprises a heavy chain variable region sequence as shown in SEQ ID NO:31, and the humanized antibody comprises a light chain variable region sequence as shown in SEQ ID NO:38.

[0306] In certain embodiments of the present disclosure, the humanized antibody comprises a heavy chain variable region sequence as shown in SEQ ID NO:31, and the humanized antibody comprises a light chain variable region sequence as shown in SEQ ID NO:39.

[0307] In certain embodiments of the present disclosure, the humanized antibody comprises a heavy chain variable region sequence as shown in SEQ ID NO: 34, and the humanized antibody comprises a light chain variable region sequence as shown in SEQ ID NO: 38.

[0308] In certain embodiments of the present disclosure, the humanized antibody comprises a heavy chain variable region sequence as shown in SEQ ID NO: 34, and the humanized antibody comprises a light chain variable region sequence as shown in SEQ ID NO: 40.

[0309] In certain embodiments of the present disclosure, the humanized antibody comprises a heavy chain variable region sequence as shown in SEQ ID NO: 33, and the humanized antibody comprises a light chain variable region sequence as shown in SEQ ID NO: 41.

[0310] In certain embodiments of the present disclosure, the humanized antibody comprises a heavy chain variable region sequence as shown in SEQ ID NO:31, and the humanized antibody comprises a light chain variable region sequence as shown in SEQ ID NO:42.

[0311] In certain embodiments of the present disclosure, the antibody further comprises an antibody light chain constant region comprising the amino acid sequence shown in SEQ ID NO: 20.

[0312] In certain embodiments of the present disclosure, the antibody further comprises an antibody heavy chain constant region comprising the amino acid sequence shown in SEQ ID NO: 21.

[0313] The antibody light chain variable region sequence and the light chain constant region are combined to form the antibody light chain, and the antibody heavy chain variable region sequence and the heavy chain constant region are combined to form the antibody heavy chain.

[0314] In certain embodiments of the present disclosure, the antibody comprises a heavy chain amino acid sequence as shown in any one of SEQ ID NOs: 64-68, and the antibody comprises a light chain amino acid sequence as shown in any one of SEQ ID NOs: 69-76;

[0315] The antibody preferably comprises a heavy chain amino acid sequence as shown in SEQ ID NO: 62 and a light chain amino acid sequence as shown in SEQ ID NO: 69;

[0316] The antibody preferably comprises a heavy chain amino acid sequence as shown in SEQ ID NO: 63 and a light chain amino acid sequence as shown in SEQ ID NO: 69;

[0317] The antibody preferably comprises a heavy chain amino acid sequence as shown in SEQ ID NO: 64 and a light chain amino acid sequence as shown in SEQ ID NO: 69;

[0318] The antibody preferably comprises a heavy chain amino acid sequence as shown in SEQ ID NO: 65 and a light chain amino acid sequence as shown in SEQ ID NO: 70;

[0319] The antibody preferably comprises a heavy chain amino acid sequence as shown in SEQ ID NO: 65 and a light chain amino acid sequence as shown in SEQ ID NO: 71;

[0320] The antibody preferably comprises a heavy chain amino acid sequence as shown in SEQ ID NO: 66 and a light chain amino acid sequence as shown in SEQ ID NO: 70;

[0321] The antibody preferably comprises a heavy chain amino acid sequence as shown in SEQ ID NO: 67 and a light chain amino acid sequence as shown in SEQ ID NO: 72;

[0322] The antibody preferably comprises a heavy chain amino acid sequence as shown in SEQ ID NO: 65 and a light chain amino acid sequence as shown in SEQ ID NO: 72;

[0323] The antibody preferably comprises a heavy chain amino acid sequence as shown in SEQ ID NO: 65 and a light chain amino acid sequence as shown in SEQ ID NO: 73;

[0324] The antibody preferably comprises a heavy chain amino acid sequence as shown in SEQ ID NO: 68 and a light chain amino acid sequence as shown in SEQ ID NO: 72;

[0325] The antibody preferably comprises a heavy chain amino acid sequence as shown in SEQ ID NO: 68 and a light chain amino acid sequence as shown in SEQ ID NO: 74;

[0326] The antibody preferably comprises a heavy chain amino acid sequence as shown in SEQ ID NO: 67 and a light chain amino acid sequence as shown in SEQ ID NO: 75;

[0327] The antibody preferably comprises a heavy chain amino acid sequence as shown in SEQ ID NO:65 and a light chain amino acid sequence as shown in SEQ ID NO:76.

[0328] After codon optimization, gene synthesis is performed, and the synthesized gene fragment is cloned into the expression vector pcDNA3.4. After expression and purification in CHO cells, affinity-matured monoclonal antibodies can be obtained. Affinity testing shows that the anti-ADM antibody exhibits a KD value for ADM of less than 10-10M.

[0329] In another aspect, the present disclosure provides the use of the anti-adrenomedullin antibody or fragment thereof described in the present disclosure in the preparation of a drug for preventing or treating stroke, wherein the stroke includes ischemic stroke or hemorrhagic stroke.

[0330] In certain embodiments, the antibody comprises a heavy chain amino acid sequence as shown in any one of SEQ ID NOs: 62-68, and the antibody comprises a light chain amino acid sequence as shown in any one of SEQ ID NOs: 69-76;

[0331] The antibody preferably comprises a heavy chain amino acid sequence as shown in SEQ ID NO: 62 and a light chain amino acid sequence as shown in SEQ ID NO: 69;

[0332] The antibody preferably comprises a heavy chain amino acid sequence as shown in SEQ ID NO: 63 and a light chain amino acid sequence as shown in SEQ ID NO: 69;

[0333] The antibody preferably comprises a heavy chain amino acid sequence as shown in SEQ ID NO: 64 and a light chain amino acid sequence as shown in SEQ ID NO: 69;

[0334] The antibody preferably comprises a heavy chain amino acid sequence as shown in SEQ ID NO: 65 and a light chain amino acid sequence as shown in SEQ ID NO: 70;

[0335] The antibody preferably comprises a heavy chain amino acid sequence as shown in SEQ ID NO: 65 and a light chain amino acid sequence as shown in SEQ ID NO: 71;

[0336] The antibody preferably comprises a heavy chain amino acid sequence as shown in SEQ ID NO: 66 and a light chain amino acid sequence as shown in SEQ ID NO: 70;

[0337] The antibody preferably comprises a heavy chain amino acid sequence as shown in SEQ ID NO: 67 and a light chain amino acid sequence as shown in SEQ ID NO: 72;

[0338] The antibody preferably comprises a heavy chain amino acid sequence as shown in SEQ ID NO: 65 and a light chain amino acid sequence as shown in SEQ ID NO: 72;

[0339] The antibody preferably comprises a heavy chain amino acid sequence as shown in SEQ ID NO: 65 and a light chain amino acid sequence as shown in SEQ ID NO: 73;

[0340] The antibody preferably comprises a heavy chain amino acid sequence as shown in SEQ ID NO: 68 and a light chain amino acid sequence as shown in SEQ ID NO: 72;

[0341] The antibody preferably comprises a heavy chain amino acid sequence as shown in SEQ ID NO: 68 and a light chain amino acid sequence as shown in SEQ ID NO: 74;

[0342] The antibody preferably comprises a heavy chain amino acid sequence as shown in SEQ ID NO: 67 and a light chain amino acid sequence as shown in SEQ ID NO: 75;

[0343] The antibody preferably comprises a heavy chain amino acid sequence as shown in SEQ ID NO:65 and a light chain amino acid sequence as shown in SEQ ID NO:76.

[0344] In certain embodiments, the antibody type comprises IgG, IgA, IgM, IgD, or IgE.

[0345] In another aspect, the present disclosure provides a nucleic acid molecule encoding the anti-adrenomedullin antibody or a fragment thereof described in the present disclosure.

[0346] In another aspect, the present disclosure provides an expression vector comprising the nucleic acid molecule described in the present disclosure, wherein the vector pcDNA3.4 is preferred.

[0347] In another aspect, the present disclosure provides a host cell comprising the nucleic acid molecule or expression vector described in the present disclosure, wherein a CHO cell is preferred.

[0348] In another aspect, the present disclosure provides a method for preparing the anti-adrenomedullin (anti-ADM) antibody or fragment thereof described in the present disclosure, comprising culturing the aforementioned host cells and then isolating and obtaining the adrenomedullin (anti-ADM) antibody or fragment thereof.

[0349] In another aspect, the present disclosure provides a pharmaceutical composition comprising the anti-adrenomedullin antibody or fragment thereof according to the present disclosure.

[0350] In a preferred embodiment of the present disclosure, the anti-adrenomedullin antibody or fragment thereof is used in combination with at least one additional pharmaceutical component.

[0351] In further preferred embodiments of the present disclosure, the pharmaceutical compound is selected from a single pharmaceutical dosage form of a neurotransmitter release modulator, a neuroreceptor ligand or agonist or antagonist, a GLP-1R agonist, a calcium channel agent, an acid ion channel agent, an immunomodulator, an antiplatelet drug, an anticoagulant drug, an anti-atherosclerotic drug, a thrombolytic drug, a neuroprotective drug, a vasopressor, a TNF-α-antibody, an antibiotic, or other central nervous system reactive antibody. In some preferred embodiments, the thrombolytic drug is alteplase. In some preferred embodiments, the neuroprotective drug is edaravone. In some preferred embodiments, the drug component is Enibarcimab, Cefixime, Edaravone, Butylphthalide, Troxerutin, Citicoline, Piracetam, Aspirin, Alteplase, Atorvastatin or a GLP-1R agonist, that is, the anti-adrenomedullin antibody or its fragment is used in combination with Enibarcimab, Cefixime, Edaravone, Butylphthalide, Troxerutin, Citicoline, Piracetam, Aspirin, Alteplase, Atorvastatin or a GLP-1R agonist.

[0352] In another aspect, the present disclosure provides a reagent or kit comprising the anti-adrenomedullin antibody or fragment thereof, or the pharmaceutical composition described in the present disclosure.

[0353] In a preferred embodiment of the present disclosure, the dosage form of the agent includes injection or powder.

[0354] In a further preferred embodiment of the present disclosure, the injection comprises a sterile or sterilized solution, water injection, oil injection or powder injection; and the powder comprises a lyophilized powder.

[0355] In another aspect, the present disclosure provides a method for preventing or treating stroke, comprising administering to a subject a therapeutically effective amount of the aforementioned anti-adrenomedullin (anti-ADM) antibody or fragment thereof, the anti-adrenomedullin (anti-ADM) antibody or fragment thereof prepared by the aforementioned preparation method, the aforementioned pharmaceutical composition, or the aforementioned reagent.

[0356] In another aspect, the present disclosure provides the aforementioned anti-adrenomedullin (anti-ADM) antibody or fragment thereof for preventing or treating stroke, the anti-adrenomedullin (anti-ADM) antibody or fragment thereof prepared by the aforementioned preparation method, the aforementioned pharmaceutical composition, or the aforementioned reagent or kit.

[0357] In another aspect, the present disclosure provides the aforementioned anti-adrenomedullin (anti-ADM) antibody or fragment thereof, the anti-adrenomedullin (anti-ADM) antibody or fragment thereof prepared by the aforementioned preparation method, the aforementioned pharmaceutical composition, or the aforementioned reagent or kit for use in preparing a drug for preventing or treating stroke.

[0358] In the preferred embodiment of the present disclosure, the mode of administration for preventing or treating stroke includes any suitable method known in the art, including but not limited to, oral, oral, sublingual, ophthalmic, topical, parenteral, rectal, intrathecal, intracytoplasmic reticulum, inguinal, intravesical, topical (such as, powder, ointment or drops), or nasal route. However, for many therapeutic uses, preferred route of administration / mode is parenteral administration (such as intravenous injection, subcutaneous injection, intraperitoneal injection, intramuscular injection). Technicians will understand that route of administration and / or mode will change according to the intended purpose. In a preferred embodiment, the antibody of the present invention or its antigen-binding fragment, pharmaceutical composition are given by intravenous infusion or injection.

[0359] In a preferred embodiment of the present disclosure, the dosage is 0.25 mg / kg-10 mg / kg; specifically 0.25 mg / kg, 0.5 mg / kg, 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg; more preferably 0.5 mg / kg-8 mg / kg; and even more preferably 2 mg / kg.

[0360] The present disclosure is described in more detail below with reference to specific examples, which are, however, for illustrative purposes only and do not limit the present disclosure. The reagents and biological materials described in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0361] Example 1: Screening of anti-ADM monoclonal clones using hybridoma technology

[0362] 1.1 Immunization of mice

[0363] Mice were immunized with a synthetic peptide conjugated with the N-terminal 16 amino acids of human ADM (YY-16, SEQ ID NO: 1), the N-terminal 21 amino acids of human ADM (YY-21, SEQ ID NO: 2), or the N-terminal 19 amino acids of mouse ADM (YY-19, SEQ ID NO: 3) and KLH (Sigma, H8283). For the initial immunization, the immunogen was emulsified with Freund's complete adjuvant at a 1:1 ratio and injected intraperitoneally into 6- to 8-week-old female Balb / c mice, SJL mice, or SD rats at a dose of 100 μg. Booster immunizations were then administered every 2-3 weeks with 50 μg of immunogen in Freund's incomplete adjuvant. Blood was collected after 3-4 immunizations for titer determination. Serum anti-YY-21 titers were determined by ELISA one week after the final immunization. Mice with high serum titers were immunized by intraperitoneal injection of 50 μg of immunogen, and spleen cells of the animals were taken for fusion on the third day.

[0364] 1.2 Spleen cell fusion

[0365] After euthanasia, the mice were dissected, the spleens removed, and the cells were ground and collected. The cells were suspended in 5 mL of red blood cell lysis buffer and incubated at 4°C for 5 minutes. The reaction was terminated with DMEM + 10% FBS. After centrifugation, the spleen cells were resuspended in 40 mL of DMEM and allowed to stand for 2-3 minutes. The supernatant was then transferred to another 50 mL centrifuge tube. A mixture of SP2 / 0 and spleen cells was prepared at a ratio of 1:2 and centrifuged. The supernatant was thoroughly aspirated, the cell pellet was mixed, and the mixed cells were washed twice with DMEM. The cells were resuspended in electrofusion buffer and added to the electrofusion chamber. After the electrofusion process was completed, the fused cells were allowed to stand for 5 minutes before being added to DMEM + 10% FBS + 1× HAT selection medium. The cell suspension was plated into a 96-well cell culture plate and incubated in a 37°C, 75% humidity, 5% CO2 incubator for 7-9 days.

[0366] 1.3 Screening of hybridoma clones

[0367] YY-21 or YY-19 was diluted to 1.0 μg / mL in PBS and added to a 96-well plate (Corning, 9018) at 100 μL / well. Coating was allowed to proceed overnight at 4°C. The next day, the ELISA plate was washed three times with wash buffer (PBS + 0.05% Tween 20) on an automated plate washer. 300 μL of blocking buffer (PBS + 0.05% Tween 20 + 1% BSA) was added to each well and blocked at room temperature for 1 hour. The plate was then washed three times with wash buffer on an automated plate washer. Hybridoma supernatant was added to each well of the ELISA plate and incubated at room temperature for 1 hour. The plate was then washed three times as described above. Goat Anti-mouse IgG Fc-HRP (Sigma, A0168) or Goat Anti-rat IgG-HRP (Sigma, A5795) was diluted 1:5000 in blocking buffer and 100 μL was added to each well. The plates were incubated at room temperature for 1 hour. Then wash the plate 3 times according to the above method. Add 100 μL / well TMB substrate solution and incubate at room temperature for 10 minutes, then add 50 μL 1.0M hydrochloric acid to each well to terminate the reaction. 450 The plate was read using a nm reader. Positive cells were selected for subcloning and subclone screening until a stable hybridoma cell line secreting monoclonal antibodies that bind to YY-21 and YY-19 was obtained. Through binding experiments, hybridoma clone 40E12 was identified. The cell line was frozen and then produced on a small scale in 50 mL using serum-free medium. The clones were purified on a protein A column for subsequent characterization.

[0368] 1.4 Affinity determination of hybridoma clone 40E12

[0369] The affinity of candidate antibodies for biotinylated human ADM (referred to as human ADM-C-biotin, the human ADM sequence is shown in SEQ ID NO: 4) and mouse ADM (referred to as mouse ADM-C-biotin, the mouse ADM sequence is shown in SEQ ID NO: 5) was determined using the Octet RED96e (Fortebio). Antigens and antibodies were diluted in 1× PBST at a working concentration of 2 μg / ml for antigen and 100 nM for antibody.

[0370] First, the sample was added to a 96-well plate (Greiner bio-one, 655209) at a volume of 200 μL / well. The software parameters were then set, with the plate temperature set to 30°C and the frequency of collecting standard kinetic signals at 5.0 Hz. Next, the streptavidin sensor (Fortébio, Cat. No. 18-5020) was pre-wetted with 1× PBST for 10 minutes before detection. Each cycle consisted of the following steps: 1) immersion in buffer for 180 seconds to stabilize the baseline; 2) antigen solidification for 10 seconds to allow the antigen to bind to the sensor, with the antigen binding amount controlled between 0.5 and 1.0 nm; 3) sensor immersion in buffer for 180 seconds; 4) antigen and antibody binding for 180 seconds; and 5) antigen-antibody dissociation for 10 minutes.

[0371] Fortebio's Data Analysis 12.0 software was used to measure the association rate (Kon) and dissociation rate (Koff) of the antigen-antibody in a 1:1 binding mode. The equilibrium dissociation constant (KD) of the antibody was calculated based on this. The results are shown in Table 1.

[0372] Table 1 Equilibrium dissociation constant (KD) of candidate antibody 40E12

[0373] 1.5 Sequencing of hybridoma clone 40E12

[0374] Hybridoma monoclonal cell lines were cultured and 5×10 6Total RNA was extracted from hybridoma cells using the Trizol method. The cDNA obtained after reverse transcription was subjected to a G-addition reaction using terminal transferase. DNA containing the variable region sequence was then amplified using a VH primer (amino acid sequence shown in SEQ ID NO: 6), a VK primer (amino acid sequence shown in SEQ ID NO: 7), and a polyC primer (amino acid sequence shown in SEQ ID NO: 8). TA cloning was performed, and sequencing yielded the murine hybridoma clone 40E12 heavy chain variable region (40E12VH) and 40E12 light chain variable region (40E12VK) sequences shown in SEQ ID NO: 9 and SEQ ID NO: 10, respectively. The light and heavy chain variable regions were numbered according to Chothia. The amino acid sequences of HCDR1, HCDR2, and HCDR3 of clone 40E12 using the Chothia definition are shown in SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:13, respectively. The amino acid sequences of LCDR1, LCDR2, and LCDR3 are shown in SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:16, respectively.

[0375] Example 2: Humanization and expression of anti-ADM antibodies

[0376] 2.1 Humanization of anti-ADM antibodies

[0377] Through sequence alignment, the human antibody germline gene with the highest homology was selected as the humanization design framework.

[0378] The heavy chain variable region was constructed using the human antibody germline gene sequences IGHV7-4-1*02 and IGHJ6*01 as the framework, and 40E12 VH (also referred to as 2004hzVH0) was subjected to CDR grafting and backmutation to obtain the humanized heavy chain variable region sequence 2004hzVH9 (amino acid sequence set forth in SEQ ID NO: 17). The light chain variable region was constructed using the human antibody germline gene sequences IGKV2-30*02 and IGKJ2*01 as the framework, and 40E12 VK (also referred to as 2004hzVK0) was subjected to CDR grafting and backmutation to obtain the humanized light chain variable region sequences 2004hzVK7 (amino acid sequence set forth in SEQ ID NO: 18) and 2004hzVK9 (amino acid sequence set forth in SEQ ID NO: 19).

[0379] 2.2 Expression of anti-ADM antibodies

[0380] The 2004hzVK7 and 2004hzVK9 sequences were combined with the human light chain constant region (CL, amino acid sequence as shown in SEQ ID NO: 20) to form the antibody light chain (amino acid sequence as shown in SEQ ID NO: 78), and the 2004hzVH9 sequence was combined with the human heavy chain constant region (CH, amino acid sequence as shown in SEQ ID NO: 21) to form the antibody heavy chain (amino acid sequence as shown in SEQ ID NO: 77). 2004hzVH0 and 2004hzVK0 were respectively combined with the human heavy chain constant region and the human light chain constant region and paired to form the chimeric antibody 2004hz00 (the amino acid sequences of their light and heavy chains are shown in SEQ ID NOs: 22 and 23); 2004hzVH9 and 2004hzVK7 were respectively combined with the human heavy chain constant region and the human light chain constant region and paired to form the humanized antibody 2004hz97 (the amino acid sequences of their light and heavy chains are shown in SEQ ID NOs: 24 and 25); 2004hzVH9 and 2004hzVK9 were respectively combined with the human heavy chain constant region and the human light chain constant region and paired to form the humanized antibody 2004hz99 (the amino acid sequences of their light and heavy chains are shown in SEQ ID NOs: 26 and 27).

[0381] After codon optimization, gene synthesis was performed and the synthesized gene fragment was cloned into the expression vector pcDNA3.4 (Life Technologies). Expression plasmid amplification and plasmid extraction (Qiagen, The two plasmids were co-transfected into Expi293F (ThermoFisher Scientific, A14527) or CHO-K1 cells (ECACC catalog no. 85051005) using the PCR-polymerase chain reaction (PCR) Plasmid Maxi Kit, Cat. No. 12362. Transient expression of the antibody was performed according to the supplier's Expi293F or CHO-K1 expression system protocol. After expression, the antibody was purified by Protein A affinity chromatography, eluted with citrate buffer (pH 3.4), and the absorbance at 280 nm was read using a NanoDrop instrument. The antibody was then dialyzed and collected for later use.

[0382] The antibody expression results are shown in Table 2, indicating that anti-ADM antibodies can be expressed using the above method.

[0383] Table 2 Expression levels of candidate antibodies

[0384] Example 3: Affinity determination of anti-ADM antibodies

[0385] Octet RED96e (Fortebio) was used to determine the affinity of antibodies 2004hz00, 2004hz97, and 2004hz99 to biotinylated human and mouse ADM (human ADM catalog number: 894757, mouse ADM catalog number: 894758, synthesized by GLBiochem). Antigens and antibodies were diluted with 1×PBST (1×PBS: Sangon, B548117-0500; 0.02% Tween 20: Sigma, P1379). The antibody concentration was 100 nM, and the antigen concentration was 2 μg / mL. The candidate antibody sample was added to a 96-well plate (Greiner bio-one, 655209) at 200 μL / well. The software parameters were set to 30°C and 5.0 Hz for collecting standard kinetic signals. The SA sensor (Fortebio, catalog number: 18-5020) was pre-wetted with 1×PBST for 10 minutes before testing on the instrument.

[0386] Each cycle includes the following steps: 1) immersion in buffer for 60 seconds; 2) detection of non-specific binding of the antigen to the sensor; 3) regeneration with 10 mM glycine solution at pH 1.7; 4) immersion in buffer for 60 seconds; 5) antigen immobilization on the sensor for 10 seconds; 6) sensor immersion in buffer for 180 seconds; 7) antigen binding to antibody for 180 seconds; 8) antigen-antibody dissociation for 600 seconds; 9) sensor regeneration.

[0387] Fortebio's Data Analysis 12.0 software was used to measure the association rate (Kon) and dissociation rate (Koff) of a 1:1 antigen-antibody binding reaction, and the equilibrium dissociation constant (KD) of the antibody was calculated. The results are shown in Tables 3 and 4, respectively. As shown in Tables 3 and 4, the affinities of the humanized antibodies 2004hz97 and 2004hz99 for human and mouse ADM are comparable to that of the chimeric antibody 2004hz00.

[0388] Table 3 Affinity of candidate antibodies to human ADM

[0389] Table 4 Affinity of candidate antibodies to mouse ADM

[0390] Example 4: Evaluation of physicochemical properties of humanized antibodies

[0391] The expression levels and affinities of antibodies 2004hz97 and 2004hz99 were ideal, and they were selected as candidate molecules for further physicochemical drugability evaluation, as detailed below.

[0392] 4.1 SEC-HPLC purity analysis

[0393] Adjust the sample concentration to 1 mg / mL, centrifuge, transfer the supernatant to a sample vial, and place it on the HPLC sample tray. Chromatographic conditions were as follows: TSK G3000SWxl column; detection wavelength, 280 nm; column temperature, 25°C; sample chamber temperature, 5°C; flow rate, 0.5 mL / min. After equilibration of the column with the mobile phase (200 mM phosphate buffer, pH 6.8), the sample was injected and analyzed. Data were analyzed using chromatography software. Peak area normalization was used to calculate the peak area percentage of each peak. A higher percentage indicates higher antibody purity.

[0394] 4.2 HIC-HPLC analysis

[0395] The sample concentration was adjusted to 1 mg / mL, and the supernatant was collected for analysis. The chromatographic conditions were set as follows: chromatographic column, MAbPac TM HIC-10; detection wavelength, 214 nm; column temperature, 30°C; sample chamber temperature, 5°C; flow rate, 0.8 mL / min. Gradient elution was performed using mobile phase A (50 mM phosphate buffer / 1 M ammonium sulfate, pH 7.0) and mobile phase B (50 mM phosphate buffer, pH 7.0). The retention time of the main peak was recorded; shorter peak elution times indicate stronger hydrophilicity of the antibody.

[0396] 4.3 Melting temperature (Tm) value analysis

[0397] According to Protein Thermal Shift TM According to the Starter Kit instructions, add 13 μL of the test solution to a PCR tube, add 5 μL of Protein Thermal Shift™ Buffer, and add 2 μL of 10× staining solution to a reaction volume of 20 μL. Mix thoroughly and centrifuge at 12,000 rpm for 5 minutes to remove bubbles. Place the test sample in a PCR instrument for analysis and record the sample's Tm value. A higher Tm value indicates better thermal stability of the antibody.

[0398] 4.4 Isoelectric focusing (iCIEF) analysis

[0399] The sample solution was added to the following thoroughly mixed solution: 70 μL of 1% methylcellulose (MC), 80 μL of 5M urea, 8 μL of ampholyte Pharmalyte pH 3-10, and 2 μL each of pI markers 5.5 and 9.5. An appropriate volume of ultrapure water was added to bring the total to 200 μL and mixed thoroughly. Centrifuge the solution and remove the supernatant for analysis. After analysis, the result file was imported into ChromPerfect software for chromatographic integration, calculation of the isoelectric point and percentage of each peak, and analysis of the charge variant distribution of the candidate antibody.

[0400] 4.5 nrCE-SDS

[0401] Dilute the sample to 4 mg / mL with ultrapure water, take 25 μL, add 75 μL of SDS sample buffer and 5 μL of 0.25 mol L-1 iodoacetamide (IAM), mix thoroughly, heat at 70°C for 10 min, and transfer 90 μL of the supernatant to a sample vial for analysis. Capillary electrophoresis was performed using a Beckman PA800 Plus capillary electrophoresis system with uncoated capillaries (total length 31 cm, effective length 21 cm). Detection conditions were: separation voltage 15 kV, capillary temperature 25°C, sample chamber temperature 15°C, and detection wavelength 220 nm. The corrected peak area percentage of the main peak was calculated.

[0402] As shown in Table 5, the humanized antibodies 2004hz97 and 2004hz99 have good physicochemical properties after one-step protein A purification.

[0403] Table 5 Analysis results of physicochemical properties of candidate antibodies

[0404] Example 5: Panning of a fully human single-chain phage antibody library

[0405] Take 150 μL Streptavidin Magnetic Beads (Thermo fisher, product number: 88817) and pre-bind with 2 mL of fully human single-chain phage antibody library, incubate at room temperature for 90 minutes to remove non-specific binding. Add 10 μg human ADM (product number NT-H-2, synthesized by GenScript) and 150 μL Streptavidin Magnetic Beads to the library phage after background removal, incubate at room temperature for 15 minutes, and wash 14 times with PBST (PBS containing 0.05% Tween-20) to wash away unbound phage. Use 450 μL 100mM hydrochloric acid to elute the antigen-specifically bound phage, add 50 μL 1M Tris-HCl at pH 11 to neutralize and infect Escherichia coli SS320 in the logarithmic growth phase, produce and purify phage for the next round of screening. The screening method is the same as the first round, except that the amount of antigen is reduced to 4 μg. The enriched phages after two rounds of screening were identified by enzyme-linked immunosorbent assay (ELISA). The results showed that the phages were significantly enriched after two rounds of panning.

[0406] After two rounds of panning, 10 μL of phage eluted from the panning pool was diluted 10,000-fold and added to 90 μL of Escherichia coli SS320 in logarithmic growth phase. The cells were inoculated for 30 minutes before being plated on a resistance plate and incubated overnight at 37°C. The next day, single colonies were isolated from the resistance plate and plated into a 96-well plate containing ampicillin / IPTG / 2YT and incubated overnight at 37°C. The supernatant was centrifuged at 4000 g for 10 minutes, and the supernatant was collected. Binding activity was assessed by enzyme-linked immunosorbent assay (ELISA), and clone 1F12 (amino acid sequences of the heavy and light chain variable regions are shown in SEQ ID NOs: 28 and 35, respectively) was selected.

[0407] Example 6: Effect of anti-ADM antibodies on the biological activity of ADM

[0408] The effects of selected anti-ADM antibodies on ADM bioactivity were tested in a human recombinant adrenomedullin receptor cAMP functional assay (Adrenomedullin bioassay).

[0409] Anti-ADM antibody was diluted to 1600 μg / mL with Stimulation Buffer 1 (Cisbio, 64SB1FDD) for a working concentration of 400 μg / mL, followed by a 3-fold serial dilution with Stimulation Buffer 1 (8 μL + 16 μL Stimulation Buffer 1), where the antagonist human ADM (22-52) (Alfa Aesar, Cat. No.: 159899-65-7) was diluted to 12000 μg / mL for a working concentration of 3000 μg / mL. 2.5 μL of anti-ADM antibody was then added to the corresponding wells of the experimental plate. Human ADM JMB2004 YY-52 protein (Gill Biochemical, Catalog No. 196191) was diluted to 0.6 μg / mL with Stimulation Buffer 1, and 2.5 μL was added to the corresponding wells, i.e., the working concentration of human ADM JMB2004 YY-52 protein was 0.15 μg / mL. The experimental plate was then incubated at room temperature for 60 min. CHO-K1 cells expressing human recombinant adrenomedullin receptor (hereinafter referred to as CHO-K1 / CRLR / RAMP3, where the gene accession number of CRLR is U17473 and the gene accession number of RAMP3 is AJ001016) were digested and isolated using TrypLE Express (gibco, Catalog No. 12604-021), collected by centrifugation and resuspended in Stimulation Buffer 1, and the cell density was adjusted to 4 × 10 6 / mL, 5 μL CHO-K1 / CRLR / RAMP3 cells (2×10 4The experimental plate was then incubated in a 37°C cell culture incubator for 90 min, and the cAMP content was detected using an HTRF kit (Cisbio, 62AM4PEB). That is, 5 μL of cAMP-d2 reagent working solution was added to the experimental well, followed by 5 μL of cAMP Eu-Cryptate antibody working solution. After incubation at room temperature for 1 h, the HTRF value was detected by a microplate reader.

[0410] The experimental results are shown in FIG1 . As can be seen from FIG1 , antibody 1F12 has no blocking activity.

[0411] Example 7: Design and Construction of Affinity Maturation Library for the Fully Human Antibody 1F12

[0412] The fully human antibody 1F12 obtained through screening was affinity matured to improve its affinity for human ADM. Furthermore, since the heavy chain antigen-binding determinant cluster 2 (CDR_H2) of 1F12 contains a post-translational modification (PTM) site of NG, a site-directed mutagenesis to QG was performed to eliminate deamidation. The resulting mutant was named 1F12 PTMΔ (the amino acid sequences of the heavy and light chain variable regions are shown in SEQ ID NOs: 29 and 35, respectively). Using the mutant 1F12 PTMΔ as the parent, encoding was performed according to the Chothia rules, and the CDR regions were defined according to Chothia. Random amino acid mutations were performed at the HCDR-3 and LCDR-3 positions to construct a mutation library. NNK mutation primers were designed to perform polymerase chain reaction (PCR) amplification of HCDR-3 and LCDR-3 mutation library gene fragments. The amplified VH and VL gene fragments were recovered and co-transformed with the yeast display plasmid into the cerevisiae strain EBY100 (purchased from ATCC). The VH and VL genes were inserted into the yeast display plasmid through homologous recombination of Saccharomyces cerevisiae, thereby realizing the Fab mutation library of antibodies displayed on the yeast cell wall surface. The library was named JYYDL196-197. After electroporation, the library JYYDL196-197 was cultured in 250 mL of SD-Trp-Leu liquid medium (Clontech, catalog number: 630316) at 30°C overnight; 1.0×10 9 The cells were resuspended in 200 mL of YPGP induction medium (2% galactose, 2% peptone, 1% yeast extract, 0.54% Na2HPO4, 0.86% NaH2PO4·H2O), cultured at 20°C for 24 hours, and stored at 4°C until use. The parental 1F12 PTMΔ sequence was also displayed on the yeast surface as a parental control.

[0413] Example 8: Affinity maturation library screening and monoclonal identification of the fully human antibody 1F12

[0414] After induction of JYYDL196-197 library, measure the OD of the bacterial solution 600 , 1OD is 1.0×10 7 The cell number was calculated as 1.0×10 9 Cells were enriched using a magnetic bead sorting system in the first round: washed once with 50 mL 1×PBSA (1×PBS + 1% BSA), centrifuged and discarded the supernatant; incubated with 5 mL 1×PBSA containing 100 nM biotin-labeled human ADM (hADM-Biotin, Product No. NT-H-2, synthesized by GenScript) at room temperature for 30 minutes; after washing, anti-biotin magnetic beads (miltenyi, Product No.: 130-090-485) were added and mixed and incubated for 10 minutes, and positive cells were collected by magnetic column (Quadro MACS Starting Kit). After the positive cells were cultured and induced again, 3.0×10 7 The cells were subjected to a second round of flow cytometry sorting: 1 mL of 1×PBSA was used, the supernatant was discarded after centrifugation; the cells were incubated on ice for 30 min with 1 mL of 1×PBSA containing 10 nM hADM-Biotin and mouse anti-V5 antibody (Invitrogen, cat. no. 2156578, 1:1000 dilution); the cells were centrifuged and the supernatant was discarded, and 1 mL of 1×PBSA was added for washing once; 500 μL of 1×PBSA containing fluorescent antibodies (SA-PE manufacturer eBioscience, cat. no.: 12-4317-8, 1:200 dilution; goat anti-mouse-647 manufacturer Invitrogen, cat. no.: A21235, 1:400 dilution) was added and incubated on ice in the dark for 20 min; after washing, 2 mL of 1×PBSA was added to resuspend the cells, and the cell population with strong 647 and PE fluorescent signals was collected using a flow cytometer. After the second round of flow cytometry sorting, the cells were cultured and induced again, and 3.0×10 7 After incubation with 3 nM hADM-Biotin, the cells were subjected to a third round of flow cytometry sorting. After sorting, a portion of the cells were spread on SD-Trp-Leu solid medium (Clontech, catalog number: 630317) plates and cultured at 30°C for 3 days.

[0415] The third round of screening products of JYYDL196-197 were selected, and 92 single clones were selected for sequencing analysis. Finally, the yeast monoclonal colonies with unique sequences were obtained for flow cytometry analysis. 1×10 6 The cells were stained and evaluated. Based on the staining results of each clone and the similarity of the clone sequences, the fully human antibody Ab2004.Am01 (the amino acid sequences of the heavy and light chain variable regions are shown in SEQ ID NOs: 30 and 35, respectively) was finally selected for expression.

[0416] Example 9: Design and Construction of Ab2004.Am01 Affinity Maturation Library

[0417] Ab2004.Am01 was further affinity matured to improve its affinity for human and mouse ADM. Referring to Example 7, amino acids in the HCDR-1, HCDR-2, LCDRL-1, LCDR-2, and LCDR-3 of Ab2004.Am01 were selected for construction of a mutation library, designated JYYDL208-212. Library JYYDL208-212 was cultured and induced before use. Simultaneously, the Fab sequence of Ab2004.Am01 was displayed on the surface of yeast cells as a parental control for this round of affinity maturation.

[0418] Example 10: Ab2004.Am01 affinity maturation library screening

[0419] After induction of JYYDL208-212 library, 1.0×10 9 The cells were enriched in the first round using a magnetic bead sorting system. After magnetic bead screening, the positive cells were cultured and induced again, and 3.0×10 7 The cells were sorted by flow cytometry for the second round, and the cell population with strong 647 and PE fluorescence signals was collected by flow cytometry. After the cell population was cultured and induced, the third round of flow cytometry screening was performed. The second round screening products of JYYDL208-209 were taken as 3.0×10 7 The cells were sorted for the third round under the condition of 10 nM mADM-Biotin; 3.0×10 7 The cells were sorted for the third round of flow cytometry under the condition of 100 nM mADM-Biotin. After the second and third rounds of sorting, the cells were spread on SD-Trp-Leu solid medium plates and cultured at 30°C for 3 days.

[0420] Example 11: Construction and screening of light and heavy chain mutant combinatorial libraries

[0421] From the second and third round screening products of JYYDL208-209 and JYYDL211, several single clones were picked to construct the light and heavy chain mutant combinatorial library JYYDL227.

[0422] JYYDL227 takes 2.0×10 7 The cells were sorted by flow cytometry in the first round using 3 nM hADM-Biotin and 1.2 nM mADM-Biotin, respectively. After sorting, the cells were spread on SD-Trp-Leu solid culture medium and cultured at 30°C for 3 days.

[0423] The first round of screening products of JYYDL227 were selected and 46 single clones were sequenced. Finally, the yeast monoclonal colonies with unique sequences were obtained for flow cytometry staining EC 50 Identification. Take 1×10 5 1. Evaluate the binding level of each clone to human ADM at different antigen concentrations and calculate the EC value of each clone to human ADM. 50 The smaller the value, the stronger the affinity; 2. Evaluate the binding level of each clone to mouse ADM at different antigen concentrations. Similarly, the affinity of each clone to mouse ADM can be obtained.

[0424] According to the staining results of each clone (as shown in Table 6), the similarity of each clone sequence was comprehensively considered and the expression antibodies Ab2004.Am31 (the amino acid sequences of the heavy chain and light chain variable regions are shown in SEQ ID NOs: 31 and 36, respectively), Ab2004.Am32 (the amino acid sequences of the heavy chain and light chain variable regions are shown in SEQ ID NOs: 31 and 37, respectively), Ab2004.Am33 (the amino acid sequences of the heavy chain and light chain variable regions are shown in SEQ ID NOs: 32 and 36, respectively), Ab2004.Am34 (the amino acid sequences of the heavy chain and light chain variable regions are shown in SEQ ID NOs: 33 and 38, respectively), Ab2004.Am35 (the amino acid sequences of the heavy chain and light chain variable regions are shown in SEQ ID NOs: 31 and 38, respectively), Ab2004.Am36 (the amino acid sequences of the heavy chain and light chain variable regions are shown in SEQ ID NOs: 31 and 39, respectively), and Ab2004.Am37 (the amino acid sequences of the heavy chain and light chain variable regions are shown in SEQ ID NOs: 31 and 39, respectively). The monoclonal antibodies of the candidate antibodies are Ab2004.Am38 (the amino acid sequences of the heavy and light chain variable regions are shown in SEQ ID NOs: 34 and 40, respectively), Ab2004.Am39 (the amino acid sequences of the heavy and light chain variable regions are shown in SEQ ID NOs: 33 and 41, respectively), and Ab2004.Am40 (the amino acid sequences of the heavy and light chain variable regions are shown in SEQ ID NOs: 31 and 42, respectively). The Chothia numbered CDR region amino acid sequences of the candidate antibodies are shown in Table 7, and the amino acid sequences of the light and heavy chain variable regions of the candidate antibodies are shown in Table 8.

[0425] Table 6 Flow cytometry staining results of yeast monoclonal colonies

[0426] Table 7 Chothia numbered CDR sequences of candidate antibodies

[0427] Table 8 Variable region sequences of candidate antibodies

[0428] Example 12: Expression of candidate antibodies

[0429] As in Example 2.2, the VH and VK sequences of each clone were combined with the light chain constant region (CL, amino acid sequence SEQ ID NO: 20) and the heavy chain constant region (CH, amino acid sequence SEQ ID NO: 21) to form the antibody light chain (amino acid sequence SEQ ID NO: 69-76) and heavy chain (amino acid sequence SEQ ID NO: 64-68), respectively. The antibodies were loaded into the expression vector pcDNA3.4 (Life Technologies) and entrusted to Nanjing GenScript Biotechnology Co., Ltd. for transient expression in CHO cells and purification. The affinity matured candidate antibodies were finally obtained, as shown in Table 9.

[0430] Table 9 Affinity maturation candidate antibody expression and purification data

[0431] Example 13: Affinity determination of candidate antibodies

[0432] Similar to Example 3, the affinities of affinity-matured antibodies Ab2004.Am31-Ab2004.Am40 and parental antibody Ab2004.Am01 for human and mouse ADM, respectively, were determined. The results are shown in Table 10. As shown in Table 10, Ab2004.Am31, Ab2004.Am34, and Ab2004.Am39 exhibited the greatest improvements in affinity for human ADM relative to parental antibody Ab2004.Am01, and their affinities for mouse ADM were comparable to those of the positive control antibody, Enibarcimab.

[0433] Table 10 Affinity determination of candidate antibodies to human and mouse ADM

[0434] To further assess the binding of Ab2004.Am34 to human, mouse, and rat ADM, the affinity of the candidate antibody to human ADM (Gill Biochem, Catalog No. 196191), mouse ADM (Gill Biochem, Catalog No. 772653), and rat ADM (GlpBio, Catalog No. GC34230) was determined using a Biacore T200 (Cytiva). A multi-cycle kinetics approach was employed, with software parameters set to 25°C. Antigen and antibody were diluted in 1× HBS-EP+ buffer (10× HBS-EP+: Cytiva, Catalog No. BR100669). Human, mouse, and rat ADM were prepared starting at 25 nM and serially diluted in 2-fold increments. An S-series sensor chip, Protein A (Cytiva, Catalog No. 29127556), and 1× HBS-EP+ buffer were placed, primed, and placed in standby mode before testing. Each cycle consisted of the following steps: (1) Capture: Antibody flowed into channels 2, 3, and 4, and the antibody was added at a flow rate of 10 μL / min for 60 s. (2) Analyte: Analyte antigen was injected into channels 1, 2, 3, and 4 at a flow rate of 30 μL / min, with an association time of 180 s and a dissociation time of 600 s. (3) Regeneration: 10 mM glycine-HCl, pH 1.5, was injected into channels 1, 2, 3, and 4 at a flow rate of 30 μL / min for 30 s. The data were fitted using Biacore T200 Evaluation 3.2.1 software using a 1:1 binding model. The results are shown in Tables 11-13.

[0435] Table 11 Affinity determination of candidate antibody Ab2004.Am34 and human ADM

[0436] Table 12 Affinity determination of candidate antibody Ab2004.Am34 and mouse ADM

[0437] Table 13 Affinity determination of candidate antibody Ab2004.Am34 and rat ADM

[0438] The results showed that the affinity of candidate antibody Ab2004.Am34 to human ADM was approximately 60.4 times higher than that of Enibarcimab, and its affinity to mouse and rat ADM was comparable to that of Enibarcimab.

[0439] Example 14: Evaluation of Physicochemical Properties of Candidate Antibodies

[0440] The expression levels and affinities of candidate antibodies Ab2004.Am31, Ab2004.Am34, and Ab2004.Am39 were ideal, and further physicochemical drugability evaluation was performed, with the results summarized in Table 14. As shown in Table 14, Ab2004.Am31, Ab2004.Am34, and Ab2004.Am39 met drugability criteria in terms of purity, thermal stability, hydrophilicity, and charge isoforms, using the same method as in Example 4.

[0441] Table 14 Analysis results of physicochemical properties of affinity matured antibodies

[0442] Example 15: Pharmacodynamic Study of ADM Antibody in LPS-Induced C57BL / 6J Mouse Sepsis Model

[0443] Fifty male C57BL / 6J mice, aged 10-11 weeks, were purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd. The animals were randomly divided into the following five groups (n=10) according to body weight: Group G1 was a model group and administered with the isotype control RSV-IgG1 (2 mg / kg, IV, single dose); Group G2 was a positive control group and administered with Enibarcimab (2 mg / kg, IV, single dose); and Groups G3, G4, and G5 were administered with the antibodies of the present invention, 2004hz97 (2 mg / kg, IV, single dose), 2004hz99 (2 mg / kg, IV, single dose), and Ab2004.Am34 (2 mg / kg, IV, single dose), respectively. Mice were treated with the corresponding antibodies intravenously 5 minutes before LPS-induced sepsis. Five minutes later, mice were given a single IP dose of 20 mg / kg LPS (E. coli 055:B5; Sigma) to induce sepsis. Animal mortality was observed twice daily for 7 consecutive days. Data were plotted using GraphPad Prism 8 software, and statistically analyzed using the Log-rank (Mantel-Cox) test.

[0444] After 7 days of LPS treatment, the survival rates of animals in each group were: RSV-IgG1 (40%), Enibarcimab (50%, P = 0.6713 vs RSV-IgG1), 2004hz97 (90%, P < 0.05 vs RSV-IgG1), 2004hz99 (80%, P = 0.1001 vs RSV-IgG1) and Am34 (80%, P < 0.05 vs RSV-IgG1). The results showed that treatment with the antibodies of the present invention can significantly improve the survival rate of animals and improve the symptoms of sepsis (see Table 15, Figure 2).

[0445] Table 15: Effects of candidate antibodies on the survival rate of mice with LPS-induced sepsis

[0446] Example 16: Pharmacodynamic study of candidate antibodies in mouse ischemic stroke model

[0447] 16.1 Experimental Grouping and Methods

[0448] Thirty-two ICR mice were adaptively housed and divided equally into four groups of eight animals each based on body weight: G1 served as a model control group and received a single IV dose of PBS; G2 served as a positive control group and received a single IV dose of edaravone and dextroborneol (Xanbixin, 20 mg / kg); and G3 (preventive) and G4 (therapeutic) groups received a single IV dose of the antibody JMB2004.Am34 (2 mg / kg). Specific group information is shown in Table 16. Mice were fasted for 12 hours preoperatively with free access to water. Before the experiment, mice were anesthetized with isoflurane and maintained in spontaneous breathing. They were fixed in the supine position on a mouse board, the midline neck was depilated, and the animals were disinfected with 75% alcohol. A midline surgical incision was made on the ventral side of the neck. The muscles and fascia were separated along the medial border of the sternocleidomastoid muscle, and the right common carotid artery, external carotid artery, and internal carotid artery were isolated. A small beveled incision was made in the common carotid artery approximately 0.5 cm proximal to the ligature using vascular scissors. The proximal end of the external carotid artery was retracted until it was aligned with the internal carotid artery. A nylon suture was slowly advanced through the incision in the right external carotid artery trunk toward the cranial entrance of the internal carotid artery, using the common carotid artery bifurcation as a marker. When slight resistance was felt during advancement, the middle cerebral artery was occluded. The suture was removed 1 hour after the infarction, completing the cerebral ischemia-reperfusion injury model. During the operation, an incandescent lamp was used to maintain rectal temperature at 36.5°C to 37.0°C. The G3 preventive medication group was administered 0.5 hour before the infarction, while the other groups were administered 0.5 hour after the infarction. After 48 h of ischemia-reperfusion, the experimental mice in each group were euthanized, and the brain tissues were collected for serial coronal sections, TTC (2,3,5-triphenyltetrazolium chloride) staining, and photographed. Image Pro Plus 6.0 was used to measure the infarct area, calculate the total infarct volume, and calculate the percentage of the infarct volume to the total brain volume.

[0449] Infarct volume ratio = infarct volume ÷ total brain volume × 100%;

[0450] Infarction improvement rate = (infarction volume ratio of model group - infarction volume ratio of drug-treated group) ÷ infarction volume ratio of model group × 100%

[0451] 16.2 TTC staining

[0452] Collect mouse brain tissue and place it in a graduated trough. Cut four slices every 2 mm. Place the slices in a 1% TTC stain solution, wrap the bottle in tin foil, and stain in a 37°C incubator for 15-30 minutes. Stir the slices occasionally to ensure even contact with the stain solution. After staining, remove the slices, observe, and photograph them.

[0453] 16.3 Data Processing and Analysis

[0454] Experimental data are presented as mean ± SEM. Homogeneity of variance was tested across groups. If homogeneity of variance was observed, one-way ANOVA was performed. If heterogeneity was observed, a t-test was used for follow-up analysis. P < 0.05 was considered a significant difference, and P < 0.01 was considered an extremely significant difference. Graphs were plotted using GraphPad Prism 8.

[0455] Table 16 Animal grouping and dosing

[0456] The results are shown in Figure 3. The cerebral infarction volume ratio of mice in the PBS group was 47.72±7.16%; the cerebral infarction volume ratios of mice in the Xianbixin group, JMB2004.Am34 preventive administration group and JMB2004.Am34 therapeutic administration group were 26.72±10.17%, 36.83±5.48% and 28.75±13.52%, respectively. The infarction improvement rates were 44.00%, 22.82% and 39.72%, respectively. This shows that the antibody of the present invention has a certain protective and improving effect on the mouse ischemic stroke model through preventive administration or therapeutic administration.

[0457] Example 17: Therapeutic Effects of Candidate Antibodies on Ischemic Stroke Model in Mice

[0458] To further verify the therapeutic effect of the candidate antibody JMB2004.Am34 on the mouse ischemic stroke model, the experimental groups in this example were: G1 was a model control group, treated with PBS (IV, single dose); G2 (therapeutic administration) group was treated with antibody JMB2004.Am34 (2 mg / kg, IV, single dose); G3 was a positive control group treated with a concentrated solution of edaravone and dextromethorphan for injection (Xanbixin, 20 mg / kg, IV, single dose); G4 was a drug combination control group treated with a concentrated solution of edaravone and dextromethorphan for injection and antibody JMB2004.Am34 (antibody JMB2004.Am34, 2 mg / kg; Xianbixin, 20 mg / kg, IV, single dose); and G5 was a positive control group treated with enebarcimab (2 mg / kg, IV, single dose). Specific group information is shown in Table 17. The specific experimental and data analysis procedures are the same as in Example 16.

[0459] Table 17 Animal grouping and dosing

[0460] The experimental results are shown in Table 18 and Figure 4. As shown above, the cerebral infarction area ratio of mice in the PBS group was 44.30±7.52%; the cerebral infarction area ratios of mice in the JMB2004.Am34 treatment group, the Cerebrospinal fluid group, and the Enibarcimab group were 30.42±14.74%, 26.59±16.05%, and 30.98%±9.00%, respectively, with infarction improvement rates of 31.32%, 39.97%, and 30.07%, respectively. Furthermore, the cerebral infarction area ratio of mice in the drug combination group was 25.72%±7.48%, with an infarction improvement rate of 41.95%, indicating that the combination of the present antibody JMB2004.Am34 and Cerebrospinal fluid has no synergistic effect, and that the effect of using the antibody JMB2004.Am34 alone can achieve a certain improvement and protective effect.

[0461] Table 18 Experimental results of different groups

[0462] *p<0.05, **p<0.01 vs. model group, N=6 / group for endpoint analysis.

[0463] Example 18: Safety Verification of Candidate Antibodies

[0464] To further verify the in vivo safety of the candidate antibodies, this example set up relevant toxicology studies. Specific experiments included:

[0465] (1) Toxicity study of a single intravenous infusion of JMB2004 DS in Sprague-Dawley rats and cynomolgus monkeys (in compliance with GLP regulations);

[0466] (2) Toxicity study in Sprague-Dawley rats and cynomolgus monkeys with repeated intravenous infusion of JMB2004 DS once a week for 5 times for 4 consecutive weeks (in compliance with GLP regulations, including safety pharmacology and local tolerance studies);

[0467] (3) In vitro hemolysis test of New Zealand rabbit erythrocytes by JMB2004 DS.

[0468] Sprague-Dawley rats received a single intravenous infusion of 0 (JMB2004 placebo), 125, 250, and 500 mg / kg JMB2004 DS at a rate of 1 mL / min, with a maximum tolerated dose (MTD) of 500 mg / kg. Sprague-Dawley rats received repeated intravenous infusions of 0 (JMB2004 placebo), 100, 200, and 400 mg / kg JMB2004 DS at a rate of 1 mL / min once weekly for five consecutive weeks. The no-observed adverse effect level (NOAEL) was 400 mg / kg. No central nervous system effects related to JMB2004 DS were found, and no vascular and muscle irritation effects related to JMB2004 DS were observed at the administration site. Cynomolgus monkeys received single intravenous infusions of 0 (JMB2004 placebo), 30, 100, and 300 mg / kg at a rate of 1 mL / min, resulting in a maximum tolerated dose (MTD) of 300 mg / kg. Cynomolgus monkeys received repeated intravenous infusions of 0 (JMB2004 placebo), 25, 50, and 100 mg / kg of JMB2004 DS at a rate of 1 mL / min once weekly for five consecutive weeks. The no-observed-adverse-effect level (NOAEL) was 100 mg / kg. No JMB2004 DS-related cardiovascular, respiratory, or central nervous system effects were observed. No vascular or muscle irritation related to JMB2004 DS was observed at the administration site. Furthermore, JMB2004 DS at a concentration of 50.2 mg / mL did not cause hemolysis or erythrocyte aggregation in vitro.

[0469] The above description is only a preferred embodiment of the present invention, and the present invention should not be limited to the contents disclosed in the embodiment and the accompanying drawings. Any equivalent or modification completed without departing from the spirit disclosed in the present invention shall fall within the scope of protection of the present invention.

[0470] Table 19 Sequence Listing

Claims

1. Use of an anti - adrenomedullin (anti - ADM) antibody or a fragment thereof in the preparation of a medicament for preventing or treating stroke, wherein, The antibody or its fragment specifically binds to the amino acid sequence of the first 1-21 amino acids at the N-terminus of human adrenomedullin (ADM). The amino acid sequence of the first 1-21 amino acids at the N-terminus of human ADM is shown in SEQ ID NO: 2, and the monoclonal antibody or fragment exhibits a KD value for ADM of less than 10- 10 M affinity.

2. Use according to claim 1, wherein the fragment comprises Fab, Fab’, F(ab)2, Fv fragment, F(ab’)2, scFv, di-scFv, VHH and / or dAb.

3. Use according to claim 1 or 2, wherein (a) the heavy chain variable region of the antibody comprises the following CDR sequences: (i) GYTFTX1Y, wherein X1 is selected from H, S or Q, (ii) SX2YX3GX4, wherein X2 is selected from A or P, X3 is selected from Q, N, S or T, and X4 is selected from N or K, (iii) EGRX5GGSFX6I, wherein X5 is selected from W or S, and X6 is selected from N or D; and (b) the light chain variable region of the antibody comprises the following CDR sequences: (i)RAX7X8GIX9X 10 YLA, where X7 is selected from S or A, X8 is selected from Q or E, X9 is selected from S or G, and X 10 is selected from S or E, (ii) DX 11 SX 12 X 13 X 14 X 15 where X 11 selects V, A or T, X 12 is selected from I, N or D, X 13 is selected from L or V, X 14 is selected from D or E, X 15 is selected from A or T (iii)QQYDX 16 LX 17 LX 18 , wherein X 16 is selected from N or D, X 17 is selected from P or D, X 18 is selected from T or S.

4. Use according to claim 3, wherein (a) the heavy chain variable region of the antibody comprises HCDR1, HCDR2 and HCDR3 sequences, wherein the HCDR1 comprises the amino acid sequence shown in any one of SEQ ID NO:55, 43 and 51, the HCDR2 comprises the amino acid sequence shown in any one of SEQ ID NO:49, 44, 54 and 58, and the HCDR3 comprises the amino acid sequence shown in any one of SEQ ID NO:50 and 45; and (b) the light chain variable region of the antibody comprises LCDR1, LCDR2 and LCDR3 sequences, wherein the LCDR1 comprises the amino acid sequence shown in any one of SEQ ID NO:46, 52 and 53, the LCDR2 comprises the amino acid sequence shown in any one of SEQ ID NO:56, 47, 57 and 59, and the LCDR3 comprises the amino acid sequence shown in any one of SEQ ID NO:48, 60 and 61.

5. The use according to claim 4, wherein, The antibody is selected from any one of the following combinations: (1) the heavy chain variable region of the antibody comprises: (i) a CDR1 sequence having the following formula: GYTFTHY (shown in SEQ ID NO:55), (ii) a CDR2 sequence having the following formula: SAYQGN (shown in SEQ ID NO:49), (iii) a CDR3 sequence having the following formula: EGRWGGSFNI (shown in SEQ ID NO:50); and the light chain variable region of the antibody comprises: (i) a CDR1 sequence having the following formula: RASQGISSYLA (shown in SEQ ID NO:46), (ii) a CDR2 sequence having the following formula: DVSILDA (shown in SEQ ID NO:56), (iii) a CDR3 sequence having the following formula: QQYDNLPLT (shown in SEQ ID NO:48); or (2) the heavy chain variable region of the antibody comprises: (i) a CDR1 sequence having the following formula: GYTFTSY (shown in SEQ ID NO:43), (ii) a CDR2 sequence having the following formula: SAYNGN (shown in SEQ ID NO:44), (iii) A CDR3 sequence having the following formula: EGRSGGSFDI (as shown in SEQ ID NO: 45); and The variable region of the light chain of the antibody comprises: (i) A CDR1 sequence having the following formula: RASQGISSYLA (as shown in SEQ ID NO: 46), (ii) A CDR2 sequence having the following formula: DASNLET (as shown in SEQ ID NO: 47), (iii) A CDR3 sequence having the following formula: QQYDNLPLT (as shown in SEQ ID NO: 48); or (3) The variable region of the heavy chain of the antibody comprises: (i) A CDR1 sequence having the following formula: GYTFTSY (as shown in SEQ ID NO: 43), (ii) A CDR2 sequence having the following formula: SAYQGN (as shown in SEQ ID NO: 49), (iii) A CDR3 sequence having the following formula: EGRSGGSFDI (as shown in SEQ ID NO: 45); and The variable region of the light chain of the antibody comprises: (i) A CDR1 sequence having the following formula: RASQGISSYLA (as shown in SEQ ID NO: 46), (ii) A CDR2 sequence having the following formula: DASNLET (as shown in SEQ ID NO: 47), (iii) A CDR3 sequence having the following formula: QQYDNLPLT (as shown in SEQ ID NO: 48); or (4) The variable region of the heavy chain of the antibody comprises: (i) A CDR1 sequence having the following formula: GYTFTSY (as shown in SEQ ID NO: 43), (ii) A CDR2 sequence having the following formula: SAYQGN (as shown in SEQ ID NO: 49), (iii) A CDR3 sequence having the following formula: EGRWGGSFNI (as shown in SEQ ID NO: 50); and The variable region of the light chain of the antibody comprises: (i) A CDR1 sequence having the following formula: RASQGISSYLA (as shown in SEQ ID NO: 46), (ii) A CDR2 sequence having the following formula: DASNLET (as shown in SEQ ID NO: 47), (iii) A CDR3 sequence having the following formula: QQYDNLPLT (as shown in SEQ ID NO: 48); or (5) The variable region of the heavy chain of the antibody comprises: (i) A CDR1 sequence having the following formula: GYTFTQY (as shown in SEQ ID NO: 51), (ii) A CDR2 sequence having the following formula: SAYQGN (as shown in SEQ ID NO: 49), (iii) A CDR3 sequence having the following formula: EGRWGGSFNI (as shown in SEQ ID NO: 50); and The variable region of the light chain of the antibody comprises: (i) A CDR1 sequence having the following formula: RASEGISEYLA (as shown in SEQ ID NO: 52), (ii) A CDR2 sequence having the formula: DASNLET (as shown in SEQ ID NO: 47), (iii) A CDR3 sequence having the formula: QQYDNLPLT (as shown in SEQ ID NO: 48); or (6) The heavy chain variable region of the antibody comprises: (i) A CDR1 sequence having the formula: GYTFTQY (as shown in SEQ ID NO: 51), (ii) A CDR2 sequence having the formula: SAYQGN (as shown in SEQ ID NO: 49), (iii) A CDR3 sequence having the formula: EGRWGGSFNI (as shown in SEQ ID NO: 50), and The light chain variable region of the antibody comprises: (i) A CDR1 sequence having the formula: RAAEGIGSYLA (as shown in SEQ ID NO: 53), (ii) A CDR2 sequence having the formula: DASNLET (as shown in SEQ ID NO: 47), (iii) A CDR3 sequence having the formula: QQYDNLPLT (as shown in SEQ ID NO: 48); or (7) The heavy chain variable region of the antibody comprises: (i) A CDR1 sequence having the formula: GYTFTSY (as shown in SEQ ID NO: 43), (ii) A CDR2 sequence having the formula: SPYSGN (as shown in SEQ ID NO: 54), (iii) A CDR3 sequence having the formula: EGRWGGSFNI (as shown in SEQ ID NO: 50); and The light chain variable region of the antibody comprises: (i) A CDR1 sequence having the formula: RASEGISEYLA (as shown in SEQ ID NO: 52), (ii) A CDR2 sequence having the formula: DASNLET (as shown in SEQ ID NO: 47), (iii) A CDR3 sequence having the formula: QQYDNLPLT (as shown in SEQ ID NO: 48); or (8) The heavy chain variable region of the antibody comprises: (i) A CDR1 sequence having the formula: GYTFTQY (as shown in SEQ ID NO: 51), (ii) A CDR2 sequence having the formula: SAYQGN (as shown in SEQ ID NO: 49), (iii) A CDR3 sequence having the formula: EGRWGGSFNI (as shown in SEQ ID NO: 50); and The light chain variable region of the antibody comprises: (i) A CDR1 sequence having the formula: RASQGISSYLA (as shown in SEQ ID NO: 46), (ii) A CDR2 sequence having the formula: DVSILDA (as shown in SEQ ID NO: 56), (iii) A CDR3 sequence having the formula: QQYDNLPLT (as shown in SEQ ID NO: 48); or (9) The heavy chain variable region of the antibody comprises: (i) A CDR1 sequence having the formula: GYTFTQY (as shown in SEQ ID NO: 51), (ii) a CDR2 sequence having the formula: SAYQGN (as shown in SEQ ID NO: 49), (iii) a CDR3 sequence having the formula: EGRWGGSFNI (as shown in SEQ ID NO: 50); and The variable light chain region of the antibody comprises: (i) a CDR1 sequence having the formula: RASQGISSYLA (as shown in SEQ ID NO: 46), (ii) a CDR2 sequence having the formula: DASNVDT (as shown in SEQ ID NO: 57), (iii) a CDR3 sequence having the formula: QQYDNLPLT (as shown in SEQ ID NO: 48); or (10) The variable heavy chain region of the antibody comprises: (i) a CDR1 sequence having the formula: GYTFTSY (as shown in SEQ ID NO: 43), (ii) a CDR2 sequence having the formula: SPYTGK (as shown in SEQ ID NO: 58), (iii) a CDR3 sequence having the formula: EGRWGGSFNI (as shown in SEQ ID NO: 50); and The variable light chain region of the antibody comprises: (i) a CDR1 sequence having the formula: RASQGISSYLA (as shown in SEQ ID NO: 46), (ii) a CDR2 sequence having the formula: DVSILDA (as shown in SEQ ID NO: 56), (iii) a CDR3 sequence having the formula: QQYDNLPLT (as shown in SEQ ID NO: 48); or (11) The variable heavy chain region of the antibody comprises: (i) a CDR1 sequence having the formula: GYTFTSY (as shown in SEQ ID NO: 43), (ii) a CDR2 sequence having the formula: SPYTGK (as shown in SEQ ID NO: 58), (iii) a CDR3 sequence having the formula: EGRWGGSFNI (as shown in SEQ ID NO: 50); and The variable light chain region of the antibody comprises: (i) a CDR1 sequence having the formula: RASQGISSYLA (as shown in SEQ ID NO: 46), (ii) a CDR2 sequence having the formula: DTSDLDT (as shown in SEQ ID NO: 59), (iii) a CDR3 sequence having the formula: QQYDNLPLT (as shown in SEQ ID NO: 48); or (12) The variable heavy chain region of the antibody comprises: (i) a CDR1 sequence having the formula: GYTFTHY (as shown in SEQ ID NO: 55), (ii) a CDR2 sequence having the formula: SAYQGN (as shown in SEQ ID NO: 49), (iii) a CDR3 sequence having the formula: EGRWGGSFNI (as shown in SEQ ID NO: 50); and The variable light chain region of the antibody comprises: (i) A CDR1 sequence having the formula: RASQGISSYLA (as shown in SEQ ID NO: 46), (ii) A CDR2 sequence having the formula: DASNLET (as shown in SEQ ID NO: 47), (iii) A CDR3 sequence having the formula: QQYDDLDLT (as shown in SEQ ID NO: 60); or (13) The heavy chain variable region of the antibody comprises: (i) A CDR1 sequence having the formula: GYTFTQY (as shown in SEQ ID NO: 51), (ii) A CDR2 sequence having the formula: SAYQGN (as shown in SEQ ID NO: 49), (iii) A CDR3 sequence having the formula: EGRWGGSFNI (as shown in SEQ ID NO: 50); and The light chain variable region of the antibody comprises: (i) A CDR1 sequence having the formula: RASQGISSYLA (as shown in SEQ ID NO: 46), (ii) A CDR2 sequence having the formula: DASNLET (as shown in SEQ ID NO: 47), (iii) A CDR3 sequence having the formula: QQYDDLPLS (as shown in SEQ ID NO: 61).

6. Use according to any one of claims 3 - 5, wherein, The antibody comprises a heavy chain variable region sequence shown in any one of SEQ ID NO: 28 - 34, and the antibody comprises a light chain variable region sequence shown in any one of SEQ ID NO: 35 - 42; Preferably, the antibody is selected from any one of the following combinations: (1) Comprising the heavy chain variable region sequence shown in SEQ ID NO: 33 and the light chain variable region sequence shown in SEQ ID NO: 38; or (2) Comprising the heavy chain variable region sequence shown in SEQ ID NO: 28 and the light chain variable region sequence shown in SEQ ID NO: 35; or (3) Comprising the heavy chain variable region sequence shown in SEQ ID NO: 29 and the light chain variable region sequence shown in SEQ ID NO: 35; or (4) Comprising the heavy chain variable region sequence shown in SEQ ID NO: 30 and the light chain variable region sequence shown in SEQ ID NO: 35; or (5) Comprising the heavy chain variable region sequence shown in SEQ ID NO: 31 and the light chain variable region sequence shown in SEQ ID NO: 36; or (6) Comprising the heavy chain variable region sequence shown in SEQ ID NO: 31 and the light chain variable region sequence shown in SEQ ID NO: 37; or (7) Comprising the heavy chain variable region sequence shown in SEQ ID NO: 32 and the light chain variable region sequence shown in SEQ ID NO: 36; or (8) Comprising the heavy chain variable region sequence shown in SEQ ID NO: 31 and the light chain variable region sequence shown in SEQ ID NO: 38; or (9) Comprising the heavy chain variable region sequence shown in SEQ ID NO: 31 and the light chain variable region sequence shown in SEQ ID NO: 39; or (10) Comprising a heavy chain variable region sequence as shown in SEQ ID NO:34 and a light chain variable region sequence as shown in SEQ ID NO:38; or (11) Comprising a heavy chain variable region sequence as shown in SEQ ID NO:34 and a light chain variable region sequence as shown in SEQ ID NO:40; or (12) Comprising a heavy chain variable region sequence as shown in SEQ ID NO:33 and a light chain variable region sequence as shown in SEQ ID NO:41; or (13) Comprising a heavy chain variable region sequence as shown in SEQ ID NO:31 and a light chain variable region sequence as shown in SEQ ID NO:

42.

7. The use according to claim 6, wherein the antibody comprises a heavy chain amino acid sequence as shown in any one of SEQ ID NO:62-68, and the antibody comprises a light chain amino acid sequence as shown in any one of SEQ ID NO:69-76; Preferably, the antibody is selected from any one of the following combinations: (1) Comprising a heavy chain amino acid sequence as shown in SEQ ID NO:67 and a light chain amino acid sequence as shown in SEQ ID NO:72; or (2) Comprising a heavy chain amino acid sequence as shown in SEQ ID NO:62 and a light chain amino acid sequence as shown in SEQ ID NO:69; or (3) Comprising a heavy chain amino acid sequence as shown in SEQ ID NO:63 and a light chain amino acid sequence as shown in SEQ ID NO:69; or (4) Comprising a heavy chain amino acid sequence as shown in SEQ ID NO:64 and a light chain amino acid sequence as shown in SEQ ID NO:69; or (5) Comprising a heavy chain amino acid sequence as shown in SEQ ID NO:65 and a light chain amino acid sequence as shown in SEQ ID NO:70; or (6) Comprising a heavy chain amino acid sequence as shown in SEQ ID NO:65 and a light chain amino acid sequence as shown in SEQ ID NO:71; or (7) Comprising a heavy chain amino acid sequence as shown in SEQ ID NO:66 and a light chain amino acid sequence as shown in SEQ ID NO:70; or (8) Comprising a heavy chain amino acid sequence as shown in SEQ ID NO:65 and a light chain amino acid sequence as shown in SEQ ID NO:72; or (9) Comprising a heavy chain amino acid sequence as shown in SEQ ID NO:65 and a light chain amino acid sequence as shown in SEQ ID NO:73; or (10) Comprising a heavy chain amino acid sequence as shown in SEQ ID NO:68 and a light chain amino acid sequence as shown in SEQ ID NO:72; or (11) Comprising a heavy chain amino acid sequence as shown in SEQ ID NO:68 and a light chain amino acid sequence as shown in SEQ ID NO:74; or (12) Comprising a heavy chain amino acid sequence as shown in SEQ ID NO:67 and a light chain amino acid sequence as shown in SEQ ID NO:75; or (13) comprising the heavy chain amino acid sequence shown in SEQ ID NO: 65 and the light chain amino acid sequence shown in SEQ ID NO:

76.

8. The use according to claim 1 or 2, wherein (a) the heavy chain variable region of the antibody comprises the following CDR sequences: (i) GYAFTTF (as shown in SEQ ID NO: 11), (ii) NTYSRV (as shown in SEQ ID NO: 12) (iii) GYGGEGGLGF (as shown in SEQ ID NO: 13); and (b) the light chain variable region of the antibody comprises the following CDR sequences: (i) RSSQSIIDSDGNTYLE (as shown in SEQ ID NO: 14), (ii) KVSNRFS (as shown in SEQ ID NO: 15), (iii) FQGSHFPYT (as shown in SEQ ID NO: 16).

9. The use according to claim 8, wherein The antibody comprises the heavy chain variable region sequence shown in any one of SEQ ID NOs: 9 and 17, and the antibody comprises the light chain variable region sequence shown in any one of SEQ ID NOs: 10, 18 and 19; Preferably, the antibody is selected from any one of the following combinations: (a) comprising the heavy chain variable region sequence shown in SEQ ID NO: 9 and the light chain variable region sequence shown in SEQ ID NO: 10; or (b) comprising the heavy chain variable region sequence shown in SEQ ID NO: 17 and the light chain variable region sequence shown in SEQ ID NO: 18; or (c) comprising the heavy chain variable region sequence shown in SEQ ID NO: 17 and the light chain variable region sequence shown in SEQ ID NO:

19.

10. The use according to claim 9, wherein, The antibody is selected from any one of the following combinations: (a) comprising the heavy chain sequence shown in SEQ ID NO: 23 and the light chain sequence shown in SEQ ID NO: 22; or (b) comprising the heavy chain sequence shown in SEQ ID NO: 25 and the light chain sequence shown in SEQ ID NO: 24; or (c) comprising the heavy chain sequence shown in SEQ ID NO: 27 and the light chain sequence shown in SEQ ID NO:

26.

11. Use according to any one of claims 1-10, wherein, The type of the antibody includes IgG, IgA, IgM, IgD or IgE.

12. Use according to any one of claims 1-11, wherein, The stroke includes ischemic stroke or hemorrhagic stroke.

13. An anti - adrenomedullin (anti - ADM) antibody or a fragment thereof, wherein the antibody comprises the heavy chain amino acid sequence shown in any one of SEQ ID NOs: 62 - 68, and the antibody comprises the light chain amino acid sequence shown in any one of SEQ ID NOs: 69 - 76; Preferably, the antibody is selected from any one of the following combinations: (1) comprising the heavy chain amino acid sequence shown in SEQ ID NO: 67 and the light chain amino acid sequence shown in SEQ ID NO: 72; or (2) comprising the heavy chain amino acid sequence shown in SEQ ID NO: 62 and the light chain amino acid sequence shown in SEQ ID NO: 69; or (3) comprising the heavy chain amino acid sequence shown in SEQ ID NO: 63 and the light chain amino acid sequence shown in SEQ ID NO: 69; or (4) comprising the heavy chain amino acid sequence shown in SEQ ID NO: 64 and the light chain amino acid sequence shown in SEQ ID NO: 69; or (5) comprising the heavy chain amino acid sequence shown in SEQ ID NO: 65 and the light chain amino acid sequence shown in SEQ ID NO: 70; or (6) comprising the heavy chain amino acid sequence shown in SEQ ID NO: 65 and the light chain amino acid sequence shown in SEQ ID NO: 71; or (7) comprising the heavy chain amino acid sequence shown in SEQ ID NO: 66 and the light chain amino acid sequence shown in SEQ ID NO: 70; or (8) comprising the heavy chain amino acid sequence shown in SEQ ID NO: 65 and the light chain amino acid sequence shown in SEQ ID NO: 72; or (9) comprising the heavy chain amino acid sequence shown in SEQ ID NO: 65 and the light chain amino acid sequence shown in SEQ ID NO: 73; or (10) comprising the heavy chain amino acid sequence shown in SEQ ID NO: 68 and the light chain amino acid sequence shown in SEQ ID NO: 72; or (11) comprising the heavy chain amino acid sequence shown in SEQ ID NO: 68 and the light chain amino acid sequence shown in SEQ ID NO: 74; or (12) comprising the heavy chain amino acid sequence shown in SEQ ID NO: 67 and the light chain amino acid sequence shown in SEQ ID NO: 75; or (13) comprising the heavy chain amino acid sequence shown in SEQ ID NO: 65 and the light chain amino acid sequence shown in SEQ ID NO:

76.

14. The anti - adrenomedullin (anti - ADM) antibody or fragment thereof according to claim 13, wherein, The types of the antibody include IgG, IgA, IgM, IgD or IgE.

15. A nucleic acid molecule encoding the anti - adrenomedullin (anti - ADM) antibody or its fragment according to claim 13 or 14.

16. An expression vector comprising the nucleic acid molecule according to claim 15; Preferably, the vector is selected from plasmid expression vectors, lentiviral expression vectors, adenoviral expression vectors, adeno - associated viral expression vectors or transposon vectors; The plasmid vector is further preferably pcDNA3.

4.

17. A host cell comprising the nucleic acid molecule according to claim 15 or the expression vector according to claim 16, wherein the host cell is selected from hamster cells, human cells or mouse cells; Preferably, the hamster cells are selected from CHO cells or BHK cells; Preferably, the human cells are selected from Expi293f cells, HEK293 cells, HT - 1080 cells, PER.C6 cells, CAP cells, HKB - 11 cells or HuH - 7 cells; Preferably, the mouse cells are selected from NS0 cells or Sp2 / 0 cells.

18. A method for preparing the anti - adrenomedullin (anti - ADM) antibody or its fragment according to claim 13 or 14, comprising culturing the host cell according to claim 17, and then isolating the anti - adrenomedullin (anti - ADM) antibody or its fragment.

19. A pharmaceutical composition, comprising the anti - adrenomedullin (anti - ADM) antibody or its fragment according to claim 13 or 14, or the anti - adrenomedullin (anti - ADM) antibody or its fragment prepared by the preparation method according to claim 18.

20. The pharmaceutical composition according to claim 19, wherein the anti - adrenomedullin (anti - ADM) antibody or its fragment is used in combination with at least one other pharmaceutical component.

21. The pharmaceutical composition according to claim 20, wherein the other pharmaceutical component is selected from a neurotransmitter release regulator, a neuroreceptor ligand or agonist or antagonist, a GLP - 1R agonist, a calcium channel agent, an acid ion channel agent, an immunomodulator, an anti - platelet drug, an anticoagulant drug, an anti - atherosclerotic drug, a thrombolytic drug, a neuroprotective drug, a vasopressor, a TNF - α - antibody, an antibiotic or a separate pharmaceutical dosage form of other central nervous system - reactive antibodies; Preferably, the thrombolytic drug is alteplase; Preferably, the neuroprotective drug is edaravone.

22. The pharmaceutical composition according to any one of claims 19 - 21, wherein the anti - adrenomedullin (anti - ADM) antibody or its fragment is used in combination with edaravone.

23. The pharmaceutical composition according to any one of claims 19 - 21, wherein the anti - adrenomedullin (anti - ADM) antibody or its fragment is used in combination with alteplase.

24. A reagent or kit, comprising the anti - adrenomedullin (anti - ADM) antibody or its fragment according to claim 13 or 14, the anti - adrenomedullin (anti - ADM) antibody or its fragment prepared by the preparation method according to claim 18, or the pharmaceutical composition according to any one of claims 19 - 23.

25. The reagent or kit according to claim 24, wherein The dosage form of the reagent includes an injection or a powder; Preferably, the injection includes a sterile or sterilized solution, an aqueous injection, an oily injection or a powder injection; Preferably, the powder includes a freeze - dried powder.

26. The reagent or kit according to claim 24 or 25, wherein The administration mode of the reagent is intravenous injection, subcutaneous injection, intraperitoneal injection or intramuscular injection; Preferably, the administration mode of the reagent is intravenous injection.

27. The reagent or kit according to any one of claims 24-26, wherein, The administration dose of the reagent is 0.25 mg / kg - 10 mg / kg; Preferably, the administration dose of the reagent is 0.5 mg / kg - 8 mg / kg.

28. A method for preventing or treating stroke, wherein, The method includes administering a therapeutically effective amount of the anti - adrenomedullin (anti - ADM) antibody or its fragment according to claim 13 or 14, the anti - adrenomedullin (anti - ADM) antibody or its fragment prepared by the preparation method according to claim 18, the pharmaceutical composition according to any one of claims 19 - 23 or the reagent or kit according to any one of claims 24 - 27 to a subject.

29. The anti - adrenomedullin (anti - ADM) antibody or its fragment as described in claim 13 or 14, the anti - adrenomedullin (anti - ADM) antibody or its fragment prepared by the preparation method described in claim 18, the pharmaceutical composition as described in any one of claims 19 - 23, or the reagent or kit as described in any one of claims 24 - 27, which is used for preventing or treating stroke.

30. Use of the anti - adrenomedullin (anti - ADM) antibody or its fragment as described in claim 13 or 14, the anti - adrenomedullin (anti - ADM) antibody or its fragment prepared by the preparation method described in claim 18, the pharmaceutical composition as described in any one of claims 19 - 23, or the reagent or kit as described in any one of claims 24 - 27 in the preparation of a medicament for preventing or treating stroke.

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