WO2025152915A1 - Use of anti-adrenomedullin antibody in prevention or treatment of cerebral stroke - Google Patents

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

CROSS-REFERENCE TO RELATED APPLICATIONS

The present 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

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

Adrenomedullin (ADM) is a 6kDa polypeptide composed of 52 amino acids. It was first discovered in pheochromocytoma in 1993 and was later found to be widely secreted and expressed in vascular epithelial cells and vascular smooth muscle cells. The gene of adrenomedullin is located on chromosome 11. After translation, it is gradually cleaved by preprohormone to form a calcitonin gene-related peptide receptor-like receptor (CRLR). The heterodimer composed of CRLR and RAMP2 or RAMP3 is the receptor of adrenomedullin. Based on its receptor-mediated endocytosis and the action of proteases, its half-life is only 22 minutes.

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

In healthy people, the concentration of adrenomedullin is extremely low, at around 10 pg/mL, and it can flexibly shuttle in and out of blood vessels, flexibly regulating vasodilation and maintaining endothelial cell barrier function. In patients with septic shock, the concentration of adrenomedullin is 5 to 6 times that of normal, which is directly related to the severity of the disease and prognosis. In patients with septic shock, the barrier function of blood vessels is weakened by inflammation, and vasodilation leads to a further decrease in blood pressure.

"Cerebral stroke" is also known as "cerebral vascular accident" (CVA). It is an acute cerebrovascular disease, a group of diseases caused by sudden rupture of brain blood vessels or blood vessel blockage that prevents blood from flowing into the brain, causing brain tissue damage. Adrenomedullin non-neutralizing antibodies restrict adrenomedullin to blood vessels, correct the intravascular barrier function, weaken the extracellular vasodilation effect, and increase the half-life of adrenomedullin in plasma, which is expected to be used to develop drugs for stroke indications. Therefore, there is an urgent need to develop non-neutralizing antibodies with high affinity to adrenomedullin, and then try to develop effective drugs for the prevention or treatment of stroke.

Summary of the invention

In order to address the deficiencies of the prior art, the purpose of the present disclosure is 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 prevent or treat stroke, and be used for drug development.

In order to achieve the above-mentioned object, 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 the fragment thereof specifically binds to the 1st to 21st amino acid sequence of the N-terminus of human adrenomedullin (ADM), the 1st to 21st amino acid sequence of the N-terminus of human ADM is as shown in SEQ ID NO: 2, and the monoclonal antibody or fragment exhibits an affinity for ADM with a KD value of less than ^10-10 M.

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

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

In an alternative embodiment, the antibody comprises,

(a) The heavy chain variable region of the antibody comprises the following CDR sequence:

(i) _GYTFTX1Y , wherein _X1 is selected from S, Q or H,

(ii) SX ₂ YX ₃ GX ₄ , wherein X ₂ is selected from A or P, X ₃ is selected from N, Q, S or T, and X ₄ is selected from N or K,

(iii) EGRX ₅ GGSFX ₆ I, wherein X ₅ is selected from S or W, and X ₆ is selected from D or N; and,

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

(i) RAX ₇ X ₈ GIX ₉ X ₁₀ YLA, wherein X ₇ is selected from S or A, X ₈ is selected from Q or E, X ₉ 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 , wherein X ₁₁ is selected from A, V or T, X ₁₂ is selected from N, I or D, X ₁₃ is selected from L or V, X ₁₄ is selected from E or D, and X ₁₅ is selected from T or A,

(iii) QQYDX ₁₆ LX ₁₇ LX ₁₈ , wherein X ₁₆ is selected from N or D, X ₁₇ is selected from P or D, and X ₁₈ is selected from T or S.

In another alternative embodiment, the antibody comprises,

(a) The heavy chain variable region of the antibody comprises the following CDR sequence:

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

(ii) NTYSRV (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 sequence:

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

(ii) KVSNRFS (shown in SEQ ID NO: 15),

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

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;

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) 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.

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.

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

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 transposition vector.

More preferably, the plasmid vector is pcDNA3.4.

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.

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.

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.

In a seventh aspect, the present disclosure provides a pharmaceutical composition comprising the anti-adrenomedullin (anti-ADM) antibody or a fragment thereof as 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.

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

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.

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.

In an eighth aspect, the present disclosure provides a reagent or a kit of parts, 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.

In the ninth aspect, the present disclosure provides a method for preventing or treating stroke, the method 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.

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.

Beneficial technical effects of the present disclosure:

The anti-ADM non-neutralizing antibody disclosed in the present invention exhibits high affinity to ADM and can be used in the development of drugs for the prevention or treatment of stroke.

Furthermore, animal experiments have shown that the anti-ADM monoclonal antibody provided by the present invention has a preventive and therapeutic effect on stroke mice that is comparable to that of the existing drug Cerebrospinal Fluoride, and can significantly improve the survival status of stroke mice.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a schematic diagram showing that anti-ADM antibodies block the production of cAMP in CHOK1/CRLR/RAMP3 cells induced by hADM;

FIG2 is a graph showing the effect of anti-ADM antibody on the survival rate of mice induced by LPS sepsis;

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

FIG. 4 shows the effects of different drug administration groups on the cerebral infarction area in MCAO mice.

DETAILED DESCRIPTION

I. Definitions

In the present disclosure, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, chemistry-related terms and laboratory procedures used herein are terms and conventional procedures widely used in the corresponding fields. At the same time, in order to better understand the present disclosure, the definitions and explanations of the relevant terms are provided below.

For purposes of clarity and concise description, 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.

The term "antibody" or "antibody fragment" refers to an anti-adrenomedullin non-neutralizing antibody or 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".

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:

One or more molecules that bind to ADM, after being added 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, 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 maximum reduction in cAMP that can be obtained with the non-neutralizing antibodies to be analyzed.

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

Biological activity is defined as the effect that a substance exhibits as a living organism or tissue or organ or functional unit in vivo or in vitro (e.g., in an assay) following its interaction. In the case of ADM biological activity, this may 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 an adrenomedullin receptor cAMP functional assay.

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

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

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

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

A maximum inhibition of 50% in the ADM bioassay means that the anti-ADM antibody or the anti-adrenomedullin antibody fragment, respectively, blocks 50% of the bioactivity of the baseline value. A maximum inhibition of 80% in the ADM bioassay means that the anti-ADM antibody or the anti-adrenomedullin antibody fragment, respectively, blocks 80% of the ADM bioactivity. This means that no more than 80% of the ADM bioactivity is blocked. This means that there is still about 20% residual ADM bioactivity.

However, through the present 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.

Herein, an anti-adrenomedullin (ADM) antibody is an antibody that can specifically bind to ADM, and an anti-adrenomedullin antibody fragment is a fragment of an ADM antibody, wherein the fragment can specifically bind to ADM. Specific binding to ADM also allows binding to other antigens. This means that the specificity does not exclude that the antibody can cross-react with polypeptides other than the polypeptide that stimulated the antibody. This also applies to the specificity of the anti-ADM antibodies or fragments thereof disclosed herein.

The non-neutralizing anti-ADM antibodies or non-neutralizing anti-ADM antibody fragments of the present disclosure provide significant therapeutic advantages over neutralizing anti-ADM antibodies or neutralizing anti-ADM antibody fragments.

The antibodies disclosed herein are proteins, including one or more polypeptides that can specifically bind to an antigen, which are substantially encoded by immunoglobulin genes. Recognized immunoglobulin genes include κ, λ, α (IgA), γ (IgG1, IgG2, IgG3, IgG4), δ (IgD), ε (IgE) and μ (IgM) constant region genes and countless immunoglobulin variable region genes. The length of a full-length immunoglobulin light chain is generally about 25KDa or 214 amino acids. The length of a full-length immunoglobulin heavy chain is generally about 50KDa or 446 amino acids. The light chain is encoded by a variable region gene (about 110 amino acids in length) at the NH2-terminus and a κ or λ 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.

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 and heavy chain variable regions bind to the antigen, while the constant region mediates the effect 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, which are also referred to as complementary determining regions (CDR's). As noted above, CDRs are primarily responsible for binding to the epitope of the antigen. An immune complex is an antibody such as a monoclonal antibody, a chimeric antibody, a humanized antibody or a human antibody or a functional antibody fragment that specifically binds to the antigen.

In the art, the CDR of an antibody can be defined by a variety of methods, such as the Kabat definition rules based on sequence variability, the Chothia definition rules based on the position of the structural loop region, and the concept based on the IMGT ontology (IMGT-ONTOLOGY). In the present disclosure, the amino acid sequences of the VL and VH of the anti-ADM antibody are encoded according to the Chothia encoding rules, and the light chain CDR1 to 3 (LCDR 1 to 3) and heavy chain CDR1 to 3 (HCDR 1 to 3) of the anti-ADM antibody are defined according to Chothia.

Chimeric antibodies are antibodies whose 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 connected to human constant segments such as κ and γ1 or γ3. In one example, therapeutic chimeric antibodies are thus hybrid proteins composed of variable domains or antigen-binding domains from mouse antibodies and constant or effector domains from human antibodies, although variable regions can be produced using other mammalian species or by molecular techniques.

A "humanized" immunoglobulin is an immunoglobulin that includes a human framework region and one or more CDRs of 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."

In one embodiment, all CDRs in the humanized immunoglobulin are from donor immunoglobulins. Constant regions are not required to be present, but if present, they must be substantially consistent with human immunoglobulin constant regions, i.e., at least about 85% to 90%, such as about 95% consistent or more consistent. Therefore, all parts of the humanized immunoglobulin, except for the CDR, are substantially consistent with the corresponding parts of the natural human immunoglobulin sequence. "Humanized antibodies" are antibodies comprising humanized light chain and humanized heavy chain immunoglobulins. Humanized antibodies can bind to the same antigen as the donor antibody providing the CDR. The acceptor framework of a humanized immunoglobulin or antibody may have a limited number of substitutions of amino acids 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 means of genetic engineering.

Human antibodies are antibodies in which the light and heavy chain genes are 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 prepared using transgenic animals carrying human immunoglobulin genes.

Therefore, the anti-ADM antibody may have a form known in the art, such as a human antibody, a monoclonal antibody, a humanized antibody, a chimeric antibody, or a CDR-grafted antibody.

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.

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.

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

The synthetic N-terminal 16 amino acid polypeptide of human ADM, the N-terminal 21 amino acid polypeptide of human ADM or the N-terminal 19 amino acid polypeptide of mouse ADM (abbreviated as YY-19, SEQ ID NO: 3) and the conjugate were used as immunogens to immunize mice. 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 titer were immunized with immunogen shock, spleen cells were taken for fusion. After spleen cell fusion and hybridoma clone screening, hybridoma clones were obtained. After affinity determination, the hybridoma clones showed an affinity for ADM with a KD value of less than ^10-10M .

In the present 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 the present 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 "binding rate (kon)" or "ka").

In this disclosure, the term "specific binding" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and its antigen. The strength or affinity of a specific binding interaction can be expressed as the equilibrium dissociation constant (KD) or half-maximal effect concentration ( _EC50 ) of the interaction.

The specific binding properties between two molecules can be determined using methods known in the art. One method involves measuring the speed 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 by concentration and the actual rate 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 by any effective method. In certain embodiments, bioluminescence interferometry (e.g., ForteBio Octet method) can be used to measure the dissociation constant. In addition, surface plasmon resonance technology (e.g., Biacore) or Kinexa can be used to measure the dissociation constant.

In the present disclosure, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector. The vector can be introduced into a host cell by 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 (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC); bacteriophages such as lambda phage or M13 phage and animal viruses, etc. 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, papillomaviruses (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, the vector may also contain a replication initiation site.

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 contains 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).

In the present disclosure, the term "host cell" refers to cells 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.

In the present disclosure, the term "pharmaceutically acceptable" means that when the molecular entity, molecular fragment or composition is properly administered to an animal or a human, they will not produce adverse, allergic or other adverse reactions. Specific examples of some substances that can be used as pharmaceutically acceptable carriers or their components include sugars (such as lactose), starch, cellulose and its derivatives, vegetable oils, gelatin, polyols (such as propylene glycol), alginic acid, etc.

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 disease recurrence, etc., whether detectable or undetectable. In addition, "treatment" can also refer to prolonging survival compared to the expected survival (if not receiving treatment).

In the present 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 disorder related to coagulation or thromboembolism, or has a risk of suffering from the above disease.

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 associated with coagulation or thromboembolism) refers to an amount sufficient to prevent, prevent, or delay the occurrence of a disease (e.g., a disease or condition associated with coagulation or thromboembolism); an effective amount for treating a disease refers to an amount sufficient to cure or at least partially prevent 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 effective amount 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 gender, the mode of administration of the drug, and other treatments administered simultaneously, etc.

In the present disclosure, the term "stroke" is well known in the art. Stroke can be occlusive (due to vascular closure) or hemorrhagic (due to vascular bleeding). The term "ischemia" 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 at the distal end of the abnormal stenosis (narrowing) of the blood vessels. Although most occlusive strokes are caused by atherosclerosis and thrombosis, and most hemorrhagic strokes are associated with hypertension or aneurysms, any type of stroke may occur at any age due to a variety of reasons, including heart disease, trauma, infection, tumors, blood dyscrasias, vascular malformations, immune disorders, and exogenous toxins.

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, stroke can cause irreversible tissue damage due to the death of brain cells. The symptoms of stroke are well known in the art. For example, stroke symptoms include sudden numbness or weakness of the face, arms or legs (especially on one side of the body), sudden confusion, difficulty speaking or understanding, sudden loss of vision in one or both eyes, and sudden difficulty walking, dizziness, loss of balance or coordination. Ischemic stroke may be caused by atherosclerotic thrombosis or cerebral aorta embolism, by coagulation disorders or non-tumor vascular diseases, or by cardiac ischemia causing a decrease in total blood flow. For example, atherosclerotic thrombotic stroke, cardiogenic stroke and lacunar stroke, atrial fibrillation can also cause cardiogenic stroke (also commonly referred to as embolic or thromboembolic stroke).

In the present disclosure, the term "Enibarcimab", namely Anti-Adrenomedullin, is a humanized mouse monoclonal immunoglobulin G1 (IgG1) antibody targeting the vascular protective peptide adrenomedullin, with a molecular weight of 145.5 KD.

II. Embodiment

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 1st to 21st amino acid sequence of the N-terminus of human adrenomedullin (ADM), the 1st to 21st amino acid sequence of the N-terminus of human ADM is as shown in SEQ ID NO: 2, and the monoclonal antibody or fragment exhibits an affinity for ADM with a KD value of less than ^10-10 M.

On the other hand, the present disclosure provides a method for preventing or treating stroke, comprising administering a therapeutically effective amount of an anti-adrenomedullin (anti-ADM) antibody or a fragment thereof to a subject, 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 an affinity for ADM with a KD value of less than ^10-10 M.

On the other hand, 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 is as shown in SEQ ID NO: 2, and the monoclonal antibody or fragment exhibits an affinity for ADM with a KD value of less than ^10-10 M.

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

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

In certain embodiments, hybridoma clones are prepared and screened by mouse immunization, wherein the heavy chain variable region of the hybridoma clones comprises:

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

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

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

The light chain variable region of the hybridoma clone comprises:

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

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

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

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.

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

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 transplantation 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 transplantation and back mutation to obtain the humanized light chain variable region sequence.

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

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

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

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

The light chain variable region of the humanized antibody comprises:

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

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

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

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.

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.

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.

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

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

The light chain variable region sequence of the monoclonal antibody described in the present disclosure is combined with the light chain constant region to form an antibody light chain, and the heavy chain variable region sequence of the monoclonal antibody described in the present disclosure is combined with the heavy chain constant region to form an antibody heavy chain.

Thus, in certain embodiments of the present disclosure, the monoclonal antibody comprises a heavy chain sequence as shown in any one of SEQ ID NO: 23, 25, and 27, and the monoclonal antibody comprises a light chain sequence as shown in any one of SEQ ID NO: 22, 24, and 26.

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.

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.

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.

After codon optimization, gene synthesis was performed, and the synthesized gene fragment was cloned into the expression vector. After expression plasmid amplification and plasmid extraction, the double plasmids were co-transfected into Expi293F or CHO-K1 cells for transient antibody expression, and after expression, the antibody was purified by Protein A affinity chromatography column. According to affinity determination, the humanized anti-ADM antibody showed an affinity for ADM with a KD value of less than 10 ^-10 M.

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

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

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

(a) 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: SAYNGN (as shown in SEQ ID NO:44),

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

(b) 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 following formula: QQYDNLPLT (as shown in SEQ ID NO:48).

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 isomerism.

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

(a) 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: SAYQGN (as shown in SEQ ID NO:49),

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

(b) 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 following formula: QQYDNLPLT (as shown in SEQ ID NO:48).

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

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.

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.

In order to improve the affinity of fully human antibodies to human ADM, the present disclosure also designs and constructs an affinity maturation library of fully human antibodies. Through screening of the fully human antibody affinity maturation library and identification of monoclonal clones, the present disclosure obtains different fully human anti-ADM antibodies, all of which exhibit an affinity for ADM with a KD value of less than 10 ^-10 M.

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

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

(i) a CDR1 sequence having the formula: _GYTFTX1Y , wherein _X1 is selected from S, Q or H,

(ii) a CDR2 sequence having the formula: SX ₂ YX ₃ GX ₄ , wherein X ₂ is selected from A or P, X ₃ is selected from N, Q, S or T, and X ₄ is selected from N or K,

(iii) a CDR3 sequence having the formula: EGRX ₅ GGSFX ₆ I, wherein X ₅ is selected from S or W, and X ₆ is selected from D or N; and,

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

(i) a CDR1 sequence having the formula: RAX ₇ X ₈ GIX ₉ X ₁₀ YLA, wherein X ₇ is selected from S or A, X ₈ is selected from Q or E, X ₉ is selected from S or G, X ₁₀ is selected from S or E,

(ii) _{a CDR2} sequence having the formula: _{DX11SX12X13X14X15} , wherein _X11 is selected from A, V or T, _X12 is selected from N, _I or D, _X13 is selected from _L or V, _X14 is selected from E or D, _X15 is selected from T or A,

(iii) a CDR3 sequence having the following formula: QQYDX ₁₆ LX ₁₇ LX ₁₈ , wherein X ₁₆ is selected from N or D, X ₁₇ is selected from P or D, and X ₁₈ is selected from T or S.

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

(1) 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: SAYNGN (as shown in SEQ ID NO:44),

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

(2) 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 following formula: QQYDNLPLT (as shown in SEQ ID NO:48).

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

(1) 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: 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

(2) 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 following formula: QQYDNLPLT (as shown in SEQ ID NO:48).

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

(a) 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: SAYQGN (as shown in SEQ ID NO:49),

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

(b) 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 following formula: QQYDNLPLT (as shown in SEQ ID NO:48).

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

(a) 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

(b) 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 following formula: QQYDNLPLT (as shown in SEQ ID NO:48).

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

(a) 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

(b) 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 following formula: QQYDNLPLT (as shown in SEQ ID NO:48).

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

(a) 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

(b) 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 following formula: QQYDNLPLT (as shown in SEQ ID NO:48).

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

(a) The heavy chain variable 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

(b) 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 following formula: QQYDNLPLT (as shown in SEQ ID NO:48).

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

(a) The heavy chain variable region of the monoclonal 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

(b) 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 following formula: QQYDNLPLT (as shown in SEQ ID NO:48).

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

(a) 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

(b) 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: DASNVDT (as shown in SEQ ID NO:57),

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

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

(a) 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: SPYTGK (as shown in SEQ ID NO:58),

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

(b) 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 following formula: QQYDNLPLT (as shown in SEQ ID NO:48).

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

(a) 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: SPYTGK (as shown in SEQ ID NO:58),

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

(b) 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: DTSDLDT (as shown in SEQ ID NO:59),

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

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

(a) The heavy chain variable 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

(b) 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 following formula: QQYDDLDLT (as shown in SEQ ID NO:60).

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

(a) 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

(b) 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 following formula: QQYDDLPLS (as shown in SEQ ID NO:61).

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

Therefore, in a preferred embodiment of the present disclosure, in the anti-adrenomedullin antibody or its fragment, the antibody comprises a heavy chain variable region sequence as shown in any one of SEQ ID NO: 28 to 34, and the antibody comprises a light chain variable region sequence as shown in any one of SEQ ID NO: 35 to 42.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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

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

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

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;

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;

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;

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;

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;

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;

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;

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;

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;

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;

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;

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;

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;

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.

After codon optimization, gene synthesis is performed, and the synthesized gene fragment is cloned into the expression vector pcDNA3.4, expressed in CHO cells, and purified to obtain affinity-matured monoclonal antibodies. According to affinity measurement, the anti-ADM antibody exhibits an affinity for ADM with a KD value of less than 10-10M.

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. The stroke includes ischemic stroke or hemorrhagic stroke.

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;

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;

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;

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;

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;

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;

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;

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;

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;

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;

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;

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;

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;

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.

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

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

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.

In yet 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.

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

In yet another aspect, the present disclosure provides a pharmaceutical composition comprising the anti-adrenomedullin antibody or fragment thereof described in the present disclosure.

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.

In further preferred embodiments of the present disclosure, the 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 antiatherosclerotic 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, Dibutylphthalide, Troxerutin, Citicoline, Piracetam, Aspirin, Alteplase, Atorvastatin or a GLP-1R agonist, that is, the anti-adrenomedullin antibody or a fragment thereof is used in combination with Enibarcimab, Cefixime, Edaravone, Dibutylphthalide, Troxerutin, Citicoline, Piracetam, Aspirin, Alteplase, Atorvastatin or a GLP-1R agonist.

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

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

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.

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 a fragment thereof, the anti-adrenomedullin (anti-ADM) antibody or a fragment thereof prepared by the aforementioned preparation method, the aforementioned pharmaceutical composition, or the aforementioned reagent.

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.

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.

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, ocular, topical, parenteral, rectal, intrathecal, intracytoplasmic reticulum, inguinal, intravesical, topical (e.g., powder, ointment or drops), or nasal route. However, for many therapeutic uses, the preferred route of administration/mode is parenteral administration (e.g., intravenous injection, subcutaneous injection, intraperitoneal injection, intramuscular injection). Technicians should understand that the route of administration and/or mode will change according to the intended purpose. In a preferred embodiment, the antibody or its antigen-binding fragment, pharmaceutical composition of the present invention is administered by intravenous infusion or injection.

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.

The present disclosure is described in more detail below with reference to specific examples, however, the examples are for illustrative purposes only and have no limiting effect on the present disclosure. The reagents and biological materials described in the following examples, unless otherwise specified, can all be obtained from commercial sources.

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

1.1 Immunization of mice

The synthetic N-terminal 16 amino acid peptide of human ADM (referred to as YY-16, SEQ ID NO: 1), the N-terminal 21 amino acid peptide of human ADM (referred to as YY-21, SEQ ID NO: 2) or the N-terminal 19 amino acid peptide of mouse ADM (referred to as YY-19, SEQ ID NO: 3) and KLH (Sigma, H8283) were used as immunogens to immunize mice. In the first immunization, the immunogen was emulsified with Freund's complete adjuvant at a ratio of 1:1 and intraperitoneally injected into 6-8 week old female Balb/c mice, SJL mice or SD rats, 100 μg/mouse; after that, booster immunization was performed every 2-3 weeks, with 50 μg of immunogen plus Freund's incomplete adjuvant per animal, and blood was collected after 3-4 immunizations to measure the titer. Blood was collected one week after the last immunization, and the titer of serum anti-YY-21 was measured by ELISA. 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.

1.2 Spleen cell fusion

After the mouse was euthanized, the spleen was dissected, the cells were collected by grinding and 5 mL of red blood cell lysis buffer was used to suspend the cells, and the cells were placed at 4 ° C for 5 minutes, and the reaction was terminated with DMEM + 10% FBS. After centrifugation, the spleen cells were resuspended with 40 mL of DMEM, and the supernatant was transferred to another 50 mL centrifuge tube after standing for 2 to 3 minutes. Mix them according to the ratio of SP2/0: spleen cells = 1:2, centrifuge, and fully absorb the supernatant. The mixed cell pellets were washed twice with DMEM, and the cells were resuspended with electrofusion buffer and added to the electrofusion tank. After the electrofusion procedure is completed, the fused cells were first allowed to stand for 5 minutes, and then added to DMEM + 10% FBS + 1×HAT screening medium. The above cell suspension was added to a 96-well cell culture plate and cultured in a 37 ° C, 75% humidity, 5% CO ₂ incubator for 7 to 9 days.

1.3 Screening of hybridoma clones

Dilute YY-21 or YY-19 to 1.0 μg/mL with PBS, add to 96-well plate (Corning, 9018), 100 μL/well, and coat at 4°C overnight. The next day, wash the ELISA plate 3 times with washing buffer (PBS + 0.05% Tween 20) on an automatic plate washer. Add 300 μL of blocking buffer (PBS + 0.05% Tween 20 + 1% BSA) to each well and block at room temperature for 1 hour. Then wash 3 times with washing buffer on an automatic plate washer, add hybridoma supernatant to each well of the ELISA plate, incubate at room temperature for 1 hour, and then wash the plate 3 times according to the above method. Dilute Goat Anti-mouse IgG Fc-HRP (Sigma, A0168) or Goat Anti-rat-IgG-HRP (Sigma, A5795) 1:5000 in blocking buffer, add 100 μL to each well, and incubate at room temperature for 1 hour. Then wash the plate three 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, and read the plate at OD ₄₅₀ nm using a microplate reader. Pick positive cells for subcloning and subclone screening until a stable hybridoma cell line that can secrete monoclonal antibodies that bind to YY-21 and YY-19 is obtained. Through the binding experiment, the hybridoma clone 40E12 was screened, the cell line was frozen, and a 50mL small-scale production was carried out using serum-free medium, and subsequent identification was performed after protein A column purification.

1.4 Affinity determination of hybridoma clone 40E12

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

First, add the sample to a 96-well plate (Greiner bio-one, 655209) with a system of 200 μL/well. Then set the software parameters, set the plate temperature to 30°C, and collect the standard kinetic signal at a frequency of 5.0 Hz. Next, pre-wet the streptavidin sensor (Fortébio, catalog number: 18-5020) with 1×PBST for 10 min, and then test it on the machine. Each cycle contains the following steps: 1) immerse in buffer for 180 s to stabilize the baseline; 2) antigen solidification for 10 s to allow the antigen to bind to the sensor, and the antigen binding amount is controlled between 0.5-1.0 nm; 3) the sensor is immersed in buffer for 180 s; 4) antigen binds to the antibody, and the binding time is 180 s; 5) antigen-antibody dissociation, time 10 min.

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. The results are shown in Table 1.

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

1.5 Sequencing of hybridoma clone 40E12

The hybridoma monoclonal cell line was cultured, and 5×10 ⁶ hybridoma cells were collected by centrifugation. Total RNA was extracted by the Trizol method, and the cDNA obtained after reverse transcription was subjected to a G reaction using terminal transferase. The DNA containing the variable region sequence was then amplified using a VH primer (amino acid sequence as shown in SEQ ID NO: 6), a VK primer (amino acid sequence as shown in SEQ ID NO: 7), and a polyC primer (amino acid sequence as shown in SEQ ID NO: 8). TA cloning was performed, and after sequencing, the sequences of the mouse hybridoma clone 40E12 heavy chain variable region (40E12VH) and 40E12 light chain variable region (40E12VK) were 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 defined using Chothia are shown in SEQ ID NO:11, SEQ ID NO:12 and SEQ ID NO:13, respectively, and 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.

Example 2: Humanization and expression of anti-ADM antibodies

2.1 Humanization of anti-ADM antibodies

Through sequence alignment, the human antibody germline genes with the highest homology were selected as the humanization design framework.

The heavy chain variable region was based on the human antibody germline gene sequences IGHV7-4-1*02 and IGHJ6*01, and 40E12VH (also referred to as 2004hzVH0) was subjected to CDR transplantation and back mutation to obtain the humanized heavy chain variable region sequence 2004hzVH9 (amino acid sequence such as SEQ ID NO: 17). The light chain variable region was based on the human antibody germline gene sequences IGKV2-30*02 and IGKJ2*01, and 40E12VK (also referred to as 2004hzVK0) was subjected to CDR transplantation and back mutation to obtain the humanized light chain variable region sequences 2004hzVK7 (amino acid sequence such as SEQ ID NO: 18) and 2004hzVK9 (amino acid sequence such as SEQ ID NO: 19).

2.2 Expression of anti-ADM antibodies

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

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, After the double plasmids were co-transfected into Expi293F (ThermoFisher Scientific, A14527) or CHO-K1 cells (ECACC catalogue no.85051005) using the DNA sequencing technology of the DNA sequencing company Plasmid Maxi Kit, Cat. No. 12362, the antibodies were transiently expressed according to the supplier's Expi293F or CHO-K1 expression system method, and after expression, they were purified by Protein A affinity chromatography column, eluted with citrate buffer (PH 3.4), and the absorbance at 280 nm was read using a NanoDrop instrument; the antibodies were dialyzed and collected for later use.

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

Table 2 Expression levels of candidate antibodies

Example 3: Affinity determination of anti-ADM antibodies

Octet RED96e (Fortebio) was used to measure the affinity of antibodies 2004hz00, 2004hz97, and 2004hz99 to biotin-labeled 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: Biochem, 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 samples were added into a 96-well plate (Greiner bio-one, 655209) at 200 μL/well, and 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 min and then tested on the machine.

Each cycle includes the following steps: 1) immersion in buffer for 60 seconds; 2) detection of non-specific binding of antigen to the sensor; 3) regeneration with 10 mM glycine solution at pH 1.7; 4) immersion in buffer for 60 seconds; 5) antigen solidification 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.

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, and the equilibrium dissociation constant (KD) of the antibody was calculated. The results are shown in Tables 3 and 4. As shown in Tables 3 and 4, the affinity of humanized antibodies 2004hz97 and 2004hz99 to human and mouse ADM is comparable to that of chimeric antibody 2004hz00.

Table 3 Affinity of candidate antibodies to human ADM

Table 4 Affinity of candidate antibodies to mouse ADM

Example 4: Evaluation of physicochemical properties of humanized antibodies

The expression levels and affinities of antibodies 2004hz97 and 2004hz99 were ideal, and they were further evaluated for physicochemical drugability as candidate molecules, as follows.

4.1 SEC-HPLC purity analysis

The sample concentration was adjusted to 1 mg/mL, and the supernatant was transferred to a sample bottle by centrifugation and placed in an HPLC sample plate. The chromatographic conditions were set as follows: chromatographic column, TSK G3000SWxl; detection wavelength, 280 nm; column temperature, 25°C; sample chamber temperature, 5°C; flow rate, 0.5 mL/min. After the chromatographic column was equilibrated with the mobile phase (200 mM phosphate buffer, pH 6.8), the sample was injected for analysis, and the data was analyzed using the chromatographic software. The peak area normalization method was used to calculate the peak area percentage of each peak. The higher the percentage, the higher the purity of the antibody.

4.2 HIC-HPLC analysis

The sample concentration was adjusted to 1 mg/mL, and the supernatant was collected for testing after centrifugation. 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. 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) were used for gradient elution, and the retention time of the main peak was recorded. The shorter the peak time, the stronger the hydrophilicity of the antibody.

4.3 Melting temperature (Tm) value analysis

According to the instructions of Protein Thermal Shift ^TM Starter Kit, add 13μL of the test solution to the PCR tube, add 5μL Protein Thermal shift TM Buffer, add 2μL 10× staining solution, make the reaction volume 20μL, mix well, and centrifuge at 12000rpm for 5min to remove bubbles. Place the test sample in the PCR instrument, analyze the sample, and record the Tm value of the sample. The higher the Tm value, the better the thermal stability of the antibody.

4.4 Isoelectric focusing (iCIEF) analysis

Take the sample solution and add it to the following system that has been fully mixed: 70μL of 1% methylcellulose (MC), 80μL of 5M urea, 2μL of ampholyte Pharmalyte pH 3-108μL, and 2μL of pI marker 5.5 and 9.5. Add an appropriate volume of ultrapure water to 200μL and mix well. Centrifuge and take the supernatant for sample analysis. After the analysis, import the result file into ChromPerfect software for spectral integration processing and calculate the isoelectric point and percentage of each peak to analyze the charge isomer distribution of the candidate antibody.

4.5 nrCE-SDS

Dilute the sample to 4 mg/mL with ultrapure water, take 25 μL, add 75 μL of SDS sample buffer, 5 μL of 0.25 mol.L-1 iodoacetamide (IAM), mix well, heat at 70℃ for 10 min, take 90 μL of supernatant and put it into a sample bottle for analysis. Use Beckman PA800 Plus capillary electrophoresis system, uncoated capillary (total length 31 cm, effective length 21 cm) for detection; detection conditions: separation voltage 15KV, capillary temperature 25℃, sample chamber temperature 15℃, detection wavelength 220nm. Calculate the main peak corrected peak area percentage.

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

Table 5 Results of analysis of physicochemical properties of candidate antibodies

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

Take 150μL Streptavidin Magnetic Beads (Thermo fisher, catalog number: 88817) and pre-bind with 2mL of fully human single-chain phage antibody library, incubate at room temperature for 90min to remove non-specific binding. Add 10μg human ADM (catalog number NT-H-2, synthesized by GenScript) and 150μL Streptavidin Magnetic Beads to the library phage after background removal, incubate at room temperature for 15min, wash 14 times with PBST (PBS containing 0.05% Tween-20) to wash away unbound phage. Elute the antigen-specifically bound phage with 450μL 100mM hydrochloric acid, add 50μL 1M Tris-HCl at pH11 to neutralize and infect Escherichia coli SS320 in the logarithmic growth phase, and 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 4μg. 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 selection.

Take 10 μL of phage eluted after two rounds of panning, dilute 10,000 times, add 90 μL of Escherichia coli SS320 in the logarithmic growth phase, let it stand for 30 minutes, then spread it on the resistance plate and culture it at 37°C overnight. The next day, pick monoclonal plaques from the resistance plate and put them into a 96-deep-well plate with ampicillin/IPTG/2YT added, and culture it at 37°C overnight. The next day, centrifuge at 4000g for 10 minutes to take the supernatant, and identify the monoclonal binding ability through enzyme-linked immunosorbent assay (ELISA) to screen out the monoclonal 1F12 (the amino acid sequences of the heavy chain and light chain variable regions are shown in SEQ ID NO: 28 and 35, respectively).

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

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

Anti-ADM antibody was diluted to 1600 μg/mL with Stimulation Buffer 1 (Cisbio, 64SB1FDD) with a working concentration of 400 μg/mL, followed by 3-fold gradient dilution with Stimulation Buffer 1 (8 μL+16 μL Stimulation Buffer 1), where antagonist human ADM (22-52) (Alfa Aesar, Catalog No.: 159899-65-7) was diluted to 12000 μg/mL with a working concentration of 3000 μg/mL, and then 2.5 μL of anti-ADM antibody was added to the corresponding wells in 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, that is, the working concentration of human ADM JMB2004 YY-52 protein was 0.15 μg/mL, and then the experimental plate was incubated at room temperature for 60 minutes. 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 separated using TrypLE Express (gibco, catalog number: 12604-021), collected by centrifugation and resuspended in Stimulation Buffer 1, the cell density was adjusted to 4×10 ⁶ /mL, and 5μL CHO-K1/CRLR/RAMP3 cells (2×10 ⁴ cells/well) were added to each well. After the experimental plate was incubated in a cell culture incubator at 37°C for 90 minutes, the cAMP content was detected using the HTRF kit (Cisbio, 62AM4PEB), that is, 5μL cAMP-d2 reagent working solution was added to the experimental well, and then 5μL cAMP Eu-Cryptate antibody working solution was added. After incubation at room temperature for 1 hour, the HTRF value was detected by a microplate reader.

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

Example 7: Design and construction of affinity maturation library for fully human antibody 1F12

The fully human antibody 1F12 obtained by screening was affinity matured to improve its affinity with human ADM. At the same time, since the heavy chain antigen binding determinant cluster 2 (CDR_H2) of 1F12 contains a post-translational modification (PTM) site of NG, the site-directed mutation to QG removes the deamidation isomerization effect, and the mutant antibody is named 1F12 PTMΔ (the amino acid sequences of the heavy chain and light chain variable regions are shown in SEQ ID NO: 29 and 35, respectively). The mutant 1F12 PTMΔ was used as the parent, encoded according to the Chothia rule, and the CDR region was defined according to Chothia. The amino acids at the HCDR-3 and LCDR-3 sites were randomly mutated to construct a mutant 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 electroporated together 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 display of the antibody Fab mutation library 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 bacteria were taken and resuspended in 200 mL of YPGP induction medium (2% galactose, 2% peptone, 1% yeast extract, 0.54% Na ₂ HPO ₄ , 0.86% NaH ₂ PO ₄ ·H ₂ O), cultured at 20°C for 24 hours, and placed at 4°C for use. At the same time, the sequence of the parent 1F12 PTMΔ was displayed on the yeast surface and used as a parent control.

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

After the JYYDL196-197 library was induced, the _OD600 of the bacterial solution was measured. Based on the calculation that 1OD was 1.0× ¹⁰⁷ cells, 1.0× ¹⁰⁹ cells were taken and the first round of enrichment was performed using a magnetic bead sorting system: wash once with 50mL 1×PBSA (1×PBS+1% BSA), centrifuge and discard the supernatant; incubate with 5mL 1×PBSA containing 100nM biotin-labeled human ADM (hADM-Biotin, NT-H-2, synthesized by GenScript) at room temperature for 30min; after washing, add anti-biotin magnetic beads (miltenyi, 130-090-485), mix and incubate for 10min, and collect positive cells through a magnetic column (Quadro MACS Starting Kit). After the positive cells were cultured and induced again, 3.0×10 ⁷ cells were taken for the second round of flow cytometry: 1 mL 1×PBSA was used, and the supernatant was discarded after centrifugation; incubated on ice for 30 min with 1 mL 1×PBSA containing 10 nM hADM-Biotin and mouse anti-V5 antibody (Invitrogen, catalog number 2156578, diluted 1:1000); centrifuged and the supernatant was discarded, and 1 mL 1×PBSA was added to wash once; 500 μL 1×PBSA containing fluorescent antibody (SA-PE manufacturer eBioscience, catalog number: 12-4317-8, diluted 1:200; goat anti-mouse-647 manufacturer Invitrogen, catalog number: A21235, diluted 1:400) was added, and incubated on ice in the dark for 20 min; after washing, 2 mL 1×PBSA was added to resuspend the cells, and the cell population with strong 647 fluorescence signal and PE fluorescence signal was collected by flow cytometry. After the second round of flow cytometric sorting, the cells were cultured and induced again. The same method was used in the second round. 3.0×10 ⁷ cells were incubated with 3 nM hADM-Biotin for the third round of flow cytometric sorting. After sorting, some cells were spread on SD-Trp-Leu solid culture medium (Clontech, catalog number: 630317) plates and cultured at 30°C for 3 days.

For the third round of screening products of JYYDL196-197, 92 monoclones were picked for sequencing analysis, and finally yeast monoclonal colonies with unique sequences were obtained for flow cytometry analysis. 1×10 ⁶ cells were taken for staining evaluation. According to the staining results of each clone and the similarity of each clone sequence, 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 picked for expression.

Example 9: Design and construction of Ab2004.Am01 affinity maturation library

Ab2004.Am01 was further subjected to affinity maturation to improve its affinity to human and mouse ADM. Referring to Example 7, the amino acids of HCDR-1, HCDR-2, LCDRL-1, LCDR-2, and LCDR-3 of Ab2004.Am01 were selected for mutation library construction, and the library number was JYYDL208-212. Library JYYDL208-212 was cultured and induced for standby use. At the same time, the Fab sequence of Ab2004.Am01 was displayed on the yeast surface and used as a parent control for this round of affinity maturation.

Example 10: Ab2004.Am01 affinity maturation library screening

After the induction of JYYDL208-212 library, 1.0×10 ⁹ cells were taken from the bacterial liquid and the magnetic bead sorting system was used for the first round of enrichment. After the magnetic bead screening, the positive cells were cultured and induced again, and 3.0×10 ⁷ cells were taken for the second round of flow sorting. The cell population with strong 647 fluorescence signal and PE fluorescence signal was collected by the flow sorting instrument. After the cell population was cultured and induced, the third round of flow screening was carried out. For the second round of screening products of JYYDL208-209, 3.0×10 ⁷ cells were taken for the third round of flow sorting under the condition of 10nM mADM-Biotin; for JYYDL210-212, 3.0×10 ⁷ cells were taken for the third round of flow sorting under the condition of 100nM mADM-Biotin. After the second and third rounds of sorting, the cells were coated on SD-Trp-Leu solid culture medium plates and cultured at 30℃ for 3 days.

Example 11: Construction and screening of light and heavy chain mutation combinatorial libraries

From the second and third round screening products of JYYDL208-209 and JYYDL211, several single clones were selected to construct the light and heavy chain mutation combinatorial library JYYDL227.

2.0×10 ⁷ cells of JYYDL227 were taken and 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.

For the first round of screening products of JYYDL227, 46 monoclones were selected for sequencing, and finally the yeast monoclonal colonies with unique sequences were obtained for flow staining EC ₅₀ identification. 1×10 ⁵ cells were taken for staining evaluation: 1. The binding level of each clone with human ADM at different antigen concentrations was evaluated, and the EC ₅₀ value of each clone with human ADM was calculated. The smaller the value, the stronger the affinity; 2. The binding level of each clone with mouse ADM at different antigen concentrations was evaluated. Similarly, the affinity of each clone with mouse ADM can be obtained.

According to the staining results of each clone (as shown in Table 6), and considering the similarity of each clone sequence, we finally selected the antibodies expressing 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), and Ab2004.Am35 (the amino acid sequences of the heavy chain and light chain variable regions are shown in SEQ ID NOs: 34 and 35, respectively). The amino acid sequences of the heavy and light chain variable regions are shown in SEQ ID NOs: 31 and 38, respectively), Ab2004.Am36 (the amino acid sequences of the heavy and light chain variable regions are shown in SEQ ID NOs: 31 and 39, respectively), Ab2004.Am37 (the amino acid sequences of the heavy and light chain variable regions are shown in SEQ ID NOs: 34 and 38, respectively), 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.

Table 6 Flow cytometry staining results of yeast monoclonal colonies

Table 7 Chothia numbered CDR sequences of candidate antibodies

Table 8 Variable region sequences of candidate antibodies

Example 12: Expression of candidate antibodies

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 antibody light chain (amino acid sequence SEQ ID NO: 69-76) and heavy chain (amino acid sequence SEQ ID NO: 64-68), loaded into the expression vector pcDNA3.4 (Life Technologies), and entrusted to Nanjing GenScript Biotechnology Co., Ltd. for transient CHO cell expression and purification, and finally the affinity matured candidate antibodies were obtained (see Table 9).

Table 9 Affinity maturation candidate antibody expression and purification data

Example 13: Affinity determination of candidate antibodies

Similar to Example 3, the affinity of affinity matured antibodies Ab2004.Am31-Ab2004.Am40 and parent antibody Ab2004.Am01 to human and mouse ADM was determined, and the results are shown in Table 10. As shown in Table 10, the affinity of Ab2004.Am31, Ab2004.Am34, and Ab2004.Am39 to human ADM was the most improved relative to the parent antibody Ab2004.Am01, and the affinity to mouse ADM was comparable to that of the positive control antibody Enibarcimab.

Table 10 Affinity determination of candidate antibodies with human and mouse ADM

In addition, in order to once again determine the binding of antibody Ab2004.Am34 to human, mouse and rat ADM, the affinity of the candidate antibody to human ADM (Gill Biochemical, No.: 196191), mouse ADM (Gill Biochemical, No.: 772653) and rat ADM (GlpBio, Catalog No.: GC34230) was determined using Biacore T200 (cytiva). The multi-cycle kinetic method was used, the software parameters were set, the temperature was 25°C, and the antigen and antibody were diluted with 1×HBS-EP+ buffer (10×HBS-EP+: Cytiva, Catalog No.: BR100669). The concentration of human, mouse and rat ADM was configured to start at 25nM and was diluted 2 times. First, the S series sensor chip Protein A (Cytiva, Catalog No.: 29127556) and 1×HBS-EP+ buffer were placed, primed and entered the standby state, and then tested on the machine. Each cycle includes the following steps: (1) Capture: Antibody flows into channels 2, 3, and 4, antibody is added, flow rate is 10 μL/min, and time is 60 s. (2) Analyte: Inject analyte antigen, flow into channels 1, 2, 3, and 4, flow rate is 30 μL/min, binding time is 180 s, and dissociation time is 600 s. (3) Regeneration: Inject 10 mM glycine-HCL, pH 1.5, sample flows into channels 1, 2, 3, and 4, flow rate is 30 μL/min, and time is 30 s. The data obtained from the experiment were fitted with the 1:1 binding model using Biacore T200 Evaluation 3.2.1 software. The results are shown in Tables 11-13.

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

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

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

The results showed that the affinity of candidate antibody Ab2004.Am34 to human ADM was increased by about 60.4 times relative to Enibarcimab, and its affinity to mouse and rat ADM was comparable to that of Enibarcimab.

Example 14: Evaluation of Physicochemical Properties of Candidate Antibodies

The expression levels and affinities of candidate antibodies Ab2004.Am31, Ab2004.Am34, and Ab2004.Am39 were relatively ideal, and the physicochemical drugability evaluation was continued, and the results are summarized in Table 14. As can be seen from Table 14, Ab2004.Am31, Ab2004.Am34, and Ab2004.Am39 all meet the drugability standards in terms of purity, thermal stability, hydrophilicity, and charge isomers, and the method is referred to Example 4.

Table 14 Analysis results of physicochemical properties of affinity matured antibodies

Example 15: Pharmacodynamic study of ADM antibody in LPS-induced sepsis model in C57BL/6J mice

C57BL/6J mice were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd., 10-11 weeks old, male, a total of 50. The animals were randomly divided into the following five groups (n=10) according to body weight: G1 group was a model group given isotype control RSV-IgG1 (2 mg/kg, IV, Single), G2 group was a positive control group given Enibarcimab (2 mg/kg, IV, Single), G3, G4, G5 groups were given the antibody of the present invention 2004hz97 (2 mg/kg, IV, Single), 2004hz99 (2 mg/kg, IV, Single) and Ab2004.Am34 (2 mg/kg, IV, Single), respectively. The corresponding antibody treatment was given IV once 5 minutes before LPS induced sepsis. 20 mg/kg LPS (E. coli 055:B5; Sigma) was given IP once 5 minutes later to induce sepsis in mice. After modeling, the death of animals was observed twice a day for 7 consecutive days. The data were plotted using GraphPad Prism 8 software, and the data were statistically analyzed using the Log-rank (Mantel-Cox) test method.

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

Table 15 Effect of candidate antibodies on survival rate of mice with LPS-induced sepsis

Example 16: Pharmacodynamic study of candidate antibodies on mouse ischemic stroke model

16.1 Experimental Grouping and Methods

32 ICR mice were adaptively raised and divided into 4 groups according to their body weight, with 8 animals in each group, namely: G1 was a model control group, treated with PBS (IV, single time), G2 was a positive control group, treated with edaravone and dextroborneol concentrated solution for injection (Xenbixin, 20 mg/kg, IV, single time), G3 (preventive administration) and G4 (therapeutic administration) groups were treated with antibody JMB2004.Am34 (2 mg/kg, IV, single time), and the specific grouping information is shown in Table 16. The mice were fasted for 12 hours before surgery and had free access to water. Before the experiment, the mice were anesthetized with isoflurane, and spontaneous breathing was retained. They were fixed on a mouse board in the supine position, the hair was removed in the middle of the neck, and disinfected with 75% alcohol. A surgical incision was made in the middle of the ventral neck of the mouse, and the muscles and fascia were separated along the inner edge of the sternocleidomastoid muscle, and the right common carotid artery, external carotid artery, and internal carotid artery were separated. A small beveled incision was made with vascular scissors about 0.5 cm from the proximal end of the common carotid artery to the ligature, and the proximal end of the external carotid artery was pulled to form a straight line with the internal carotid artery. The nylon suture was slowly pushed into the direction of the internal carotid artery entering the skull through the incision of the right external carotid artery trunk, with the common carotid artery bifurcation as a mark. When slight resistance was felt during the advancement, the middle cerebral artery was blocked. The suture was removed 1 hour after infarction to complete the cerebral ischemia-reperfusion injury model. During the operation, an incandescent lamp was used to heat the rectal temperature to maintain 36.5℃～37.0℃. The G3 preventive medication group was administered 0.5h before infarction, and the other groups were administered 0.5h after the animal infarction. After 48 hours 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.

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

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

16.2 TTC staining

Collect the mouse brain tissue, place the brain tissue in a brain groove with scales, make brain slices every 2mm, and cut four slices in total. Place the slices in a 1% TTC dye solution, wrap the bottle with tin foil, and place it in a 37℃ constant temperature box for dyeing for 15 to 30 minutes. During this period, turn the brain slices from time to time to make them evenly contact the dye solution. After the dyeing is completed, take out the slices for observation and photography.

16.3 Data Processing and Analysis

The experimental data were expressed as Mean ± SEM. The variance of different groups was tested for homogeneity. If the variance was homogeneous, one-way ANOVA was performed. If the variance was unequal, T test was used for follow-up analysis. P < 0.05 was considered to be significantly different, and P < 0.01 was considered to be extremely significant. GraphPad Prism 8 was used for plotting.

Table 16 Animal grouping and dosing schedule

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 Xinbixin 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, and the infarction improvement rates were 44.00%, 22.82% and 39.72%, respectively, indicating 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.

Example 17: Therapeutic effect of candidate antibodies on mouse ischemic stroke model

In order to further verify the therapeutic effect of the candidate antibody JMB2004.Am34 on the mouse ischemic stroke model, the experimental groups in this embodiment are: G1 is a model control group, treated with PBS (IV, single), G2 (treatment administration) group is treated with antibody JMB2004.Am34 (2 mg/kg, IV, single), G3 is a positive control group given edaravone and dexamethasone injection concentrated solution (Xenbixin, 20 mg/kg, IV, single), G4 is a drug combination control group given edaravone and dexamethasone injection concentrated solution and antibody JMB2004.Am34 (antibody JMB2004.Am34, 2 mg/kg; Xianbixin, 20 mg/kg, IV, single), G5 is a positive control group given Enibarcimab (2 mg/kg, IV, single), and the specific grouping information is shown in Table 17. The specific experimental and data analysis process is the same as in Example 16.

Table 17 Animal grouping and dosing schedule

The experimental results are shown in Table 18 and Figure 4. From the above results, it can be seen that 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 Enibarcimab group and the Enibarcimab group were 30.42±14.74%, 26.59±16.05% and 30.98%±9.00%, respectively, and the infarction improvement rates were 31.32%, 39.97% and 30.07%, respectively; in addition, the cerebral infarction area ratio of mice in the drug combination group was 25.72%±7.48%, and the infarction improvement rate was 41.95%, indicating that the combination of the antibody JMB2004.Am34 of the present invention and Enibarcimab has no synergistic effect, and the effect of the antibody JMB2004.Am34 alone can achieve a certain improvement and protection effect.

Table 18 Experimental results of different groups

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

Example 18: Safety Verification of Candidate Antibodies

In order to further verify the in vivo safety of the candidate antibodies, this example sets up relevant toxicology studies. The specific experiments include:

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

(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);

(3) JMB2004 DS in vitro hemolysis test on New Zealand rabbit erythrocytes.

Sprague-Dawley rats were given a single intravenous infusion of 0 (JMB2004 placebo), 125, 250, and 500 mg/kg JMB2004 DS at a rate of 1 mL/min, and the maximum tolerated dose (MTD) was 500 mg/kg. Sprague-Dawley rats were repeatedly infused with 0 (JMB2004 placebo), 100, 200, and 400 mg/kg JMB2004 DS at a rate of 1 mL/min, once a week for a total of 5 times for 4 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 stimulatory effects related to JMB2004 DS were observed at the administration site. The maximum tolerated dose (MTD) of cynomolgus monkeys was 300 mg/kg after a single intravenous infusion of 0 (JMB2004 placebo), 30, 100 and 300 mg/kg at a rate of 1 mL/min. The cynomolgus monkeys were repeatedly intravenously infused with 0 (JMB2004 placebo), 25, 50, 100 mg/kg JMB2004 DS at a rate of 1 mL/min once a week for 5 times for 4 consecutive weeks. The no-observed-adverse-effect level (NOAEL) was 100 mg/kg. No effects on the cardiovascular, respiratory and central nervous systems related to JMB2004 DS were observed. No vascular and muscle irritation effects related to JMB2004 DS were observed at the site of administration. In addition, JMB2004 DS at a concentration of 50.2 mg/mL did not cause hemolysis and erythrocyte aggregation in vitro.

The above 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 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.

Table 19 Sequence Listing

Claims (30)

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 a fragment thereof specifically binds to the 1st to 21st amino acid sequence of the N-terminus of human adrenomedullin (ADM), the 1st to 21st amino acid sequence of the N-terminus of human ADM is as shown in SEQ ID NO: 2, and the monoclonal antibody or fragment exhibits an affinity for ADM with a KD value of less than ^10-10 M. The 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. The use according to claim 1 or 2, wherein (a) The heavy chain variable region of the antibody comprises the following CDR sequence: (i) _GYTFTX1Y , wherein _X1 is selected from H, S or Q, (ii) SX ₂ YX ₃ GX ₄ , wherein X ₂ is selected from A or P, X ₃ is selected from Q, N, S or T, and X ₄ is selected from N or K, (iii) EGRX ₅ GGSFX ₆ I, wherein X ₅ is selected from W or S, and X ₆ is selected from N or D; and, (b) The light chain variable region of the antibody comprises the following CDR sequence: (i) RAX ₇ X ₈ GIX ₉ X ₁₀ YLA, wherein X ₇ is selected from S or A, X ₈ is selected from Q or E, X ₉ 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 , wherein X ₁₁ is selected from 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, and 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, and X ₁₈ is selected from T or S. The 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 NOs: 55, 43 and 51, the HCDR2 comprises the amino acid sequence shown in any one of SEQ ID NOs: 49, 44, 54 and 58, and the HCDR3 comprises the amino acid sequence shown in any one of SEQ ID NOs: 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. 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 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 following 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 following formula: QQYDNLPLT (as shown in SEQ ID NO:48); or (2) 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: SAYNGN (as shown in SEQ ID NO:44), (iii) a CDR3 sequence having the following formula: EGRSGGSFDI (as shown in SEQ ID NO:45); 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 following formula: QQYDNLPLT (as shown in SEQ ID NO:48); or (3) 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: 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 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 following formula: QQYDNLPLT (as shown in SEQ ID NO:48); or (4) 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: 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 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 following formula: QQYDNLPLT (as shown in SEQ ID NO:48); or (5) 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 following 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 following 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 following 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 following 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 following 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 following 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 following 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 following 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: DASNVDT (as shown in SEQ ID NO:57), (iii) a CDR3 sequence having the following formula: QQYDNLPLT (as shown in SEQ ID NO:48); or (10) 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: SPYTGK (as shown in SEQ ID NO:58), (iii) a CDR3 sequence having the following 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 following formula: QQYDNLPLT (as shown in SEQ ID NO:48); or (11) 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: SPYTGK (as shown in SEQ ID NO:58), (iii) a CDR3 sequence having the following 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: DTSDLDT (as shown in SEQ ID NO:59), (iii) a CDR3 sequence having the following formula: QQYDNLPLT (as shown in SEQ ID NO:48); or (12) The heavy chain variable 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 following 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 following 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 following 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 following formula: QQYDDLPLS (as shown in SEQ ID NO:61). The use according to any one of claims 3 to 5, wherein 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; Preferably, the antibody is selected from any one of the following combinations: (1) 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:38; or (2) comprising a heavy chain variable region sequence as shown in SEQ ID NO:28 and a light chain variable region sequence as shown in SEQ ID NO:35; or (3) comprising a heavy chain variable region sequence as shown in SEQ ID NO:29 and a light chain variable region sequence as shown in SEQ ID NO:35; or (4) comprising a heavy chain variable region sequence as shown in SEQ ID NO:30 and a light chain variable region sequence as shown in SEQ ID NO:35; or (5) 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:36; or (6) 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:37; or (7) comprising a heavy chain variable region sequence as shown in SEQ ID NO:32 and a light chain variable region sequence as shown in SEQ ID NO:36; or (8) 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:38; or (9) 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: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) comprises 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. The use according to claim 6, 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; 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) 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. The use according to claim 1 or 2, wherein (a) The heavy chain variable region of the antibody comprises the following CDR sequence: (i)GYAFTTF (as shown in SEQ ID NO:11), (ii) NTYSRV (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 sequence: (i)RSSQSIIDSDGNTYLE (as shown in SEQ ID NO:14), (ii) KVSNRFS (shown in SEQ ID NO: 15), (iii)FQGSHFPYT (as shown in SEQ ID NO:16). The use according to claim 8, wherein the antibody comprises a heavy chain variable region sequence as shown in any one of SEQ ID NOs: 9 and 17, and the antibody comprises a light chain variable region sequence as 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 a heavy chain variable region sequence as shown in SEQ ID NO:9 and a light chain variable region sequence as shown in SEQ ID NO:10; or (b) comprising a heavy chain variable region sequence as shown in SEQ ID NO: 17 and a light chain variable region sequence as shown in SEQ ID NO: 18; or (c) comprises a heavy chain variable region sequence as shown in SEQ ID NO:17 and a light chain variable region sequence as shown in SEQ ID NO:19. The use according to claim 9, wherein the antibody is selected from any one of the following combinations: (a) comprising a heavy chain sequence as shown in SEQ ID NO:23 and a light chain sequence as shown in SEQ ID NO:22; or (b) comprising a heavy chain sequence as shown in SEQ ID NO:25 and a light chain sequence as shown in SEQ ID NO:24; or (c) comprises a heavy chain sequence as shown in SEQ ID NO:27 and a light chain sequence as shown in SEQ ID NO:26. The use according to any one of claims 1 to 10, wherein the type of the antibody comprises IgG, IgA, IgM, IgD or IgE. The use according to any one of claims 1 to 11, wherein the stroke includes ischemic stroke or hemorrhagic stroke. 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; 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) 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. The anti-adrenomedullin (anti-ADM) antibody or fragment thereof according to claim 13, wherein the type of the antibody comprises IgG, IgA, IgM, IgD or IgE. A nucleic acid molecule encoding the anti-adrenomedullin (anti-ADM) antibody or a fragment thereof according to claim 13 or 14. An expression vector comprising the nucleic acid molecule of claim 15; 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 transposition vector; The plasmid vector is further preferably pcDNA3.4. A host cell comprising the nucleic acid molecule of claim 15 or the expression vector of claim 16, wherein the host cell is selected from a hamster cell, a human cell or a mouse cell; Preferably, the hamster cell is 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. A method for preparing the anti-adrenomedullin (anti-ADM) antibody or fragment thereof according to claim 13 or 14, comprising culturing the host cell according to claim 17, and then isolating and obtaining the anti-adrenomedullin (anti-ADM) antibody or fragment thereof. A pharmaceutical composition comprising the anti-adrenomedullin (anti-ADM) antibody or a fragment thereof according to claim 13 or 14, or the anti-adrenomedullin (anti-ADM) antibody or a fragment thereof prepared by the preparation method according to claim 18. The pharmaceutical composition according to claim 19, wherein the anti-adrenomedullin (anti-ADM) antibody or fragment thereof is used in combination with at least one other pharmaceutical component. The pharmaceutical composition according to claim 20, wherein the other pharmaceutical components are selected from the group consisting of neurotransmitter release regulators, neuroreceptor ligands or agonists or antagonists, GLP-1R agonists, calcium channel agents, acid ion channel agents, immunomodulators, antiplatelet drugs, anticoagulants, anti-atherosclerotic drugs, thrombolytic drugs, neuroprotective drugs, vasopressors, TNF-α-antibodies, antibiotics or other central nervous system reactive antibodies in separate pharmaceutical dosage forms; Preferably, the thrombolytic drug is alteplase; Preferably, the neuroprotective drug is edaravone. The pharmaceutical composition according to any one of claims 19 to 21, wherein the anti-adrenomedullin (anti-ADM) antibody or a fragment thereof is used in combination with edaravone. The pharmaceutical composition according to any one of claims 19 to 21, wherein the anti-adrenomedullin (anti-ADM) antibody or a fragment thereof is used in combination with alteplase. A reagent or a kit comprising the anti-adrenomedullin (anti-ADM) antibody or a fragment thereof according to claim 13 or 14, the anti-adrenomedullin (anti-ADM) antibody or a fragment thereof prepared by the preparation method according to claim 18, or the pharmaceutical composition according to any one of claims 19 to 23. The reagent or kit according to claim 24, wherein the dosage form of the reagent comprises an injection or a powder; Preferably, the injection comprises a sterile or sterilized solution, water injection, oil injection or powder injection; Preferably, the powder comprises a lyophilized powder. The reagent or kit according to claim 24 or 25, wherein the reagent is administered by intravenous injection, subcutaneous injection, intraperitoneal injection or intramuscular injection; Preferably, the agent is administered by intravenous injection. The reagent or kit according to any one of claims 24 to 26, wherein the dosage of the reagent is 0.25 mg/kg to 10 mg/kg; Preferably, the dosage of the agent is 0.5 mg/kg-8 mg/kg. A method for preventing or treating stroke, comprising administering to a subject a therapeutically effective amount of the anti-adrenomedullin (anti-ADM) antibody or fragment thereof according to claim 13 or 14, the anti-adrenomedullin (anti-ADM) antibody or fragment thereof prepared by the preparation method according to claim 18, the pharmaceutical composition according to any one of claims 19 to 23, or the reagent or kit according to any one of claims 24 to 27. The anti-adrenomedullin (anti-ADM) antibody or fragment thereof according to claim 13 or 14, the anti-adrenomedullin (anti-ADM) antibody or fragment thereof prepared by the preparation method according to claim 18, the pharmaceutical composition according to any one of claims 19 to 23, or the reagent or kit according to any one of claims 24 to 27, which is used for preventing or treating stroke. Use of the anti-adrenomedullin (anti-ADM) antibody or fragment thereof according to claim 13 or 14, the anti-adrenomedullin (anti-ADM) antibody or fragment thereof prepared by the preparation method according to claim 18, the pharmaceutical composition according to any one of claims 19 to 23, or the reagent or kit according to any one of claims 24 to 27 in the preparation of a drug for preventing or treating stroke.