Anti-adenomedullin non-neutralizing antibody, method for producing the same, and use thereof

Abstract

The present invention belongs to the fields of biotechnology and biotherapy, and discloses an anti-ADM monoclonal antibody or a fragment thereof, and a pharmaceutical composition, a detection reagent or a kit containing the monoclonal antibody or the fragment thereof. The present invention further discloses the use of the monoclonal antibody or the fragment thereof in the manufacture of a drug.

Description

Technical Field

[0001] This application claims the priority of a prior application filed with the China National Intellectual Property Administration on July 20, 2022, with the patent application number 202210863441.9 and the title "Anti-Adrenomedullin Non-Neutralizing Antibody, Its Production Method and Use". The full text of the prior application is incorporated herein by reference.

[0002] The present invention relates to an anti-adrenomedullin non-neutralizing antibody, its production method and use, and belongs to the fields of biotechnology and biotherapy.

Background Art

[0003] Adrenomedullin (ADM) is a 6 kDa polypeptide consisting of 52 amino acids. It was first discovered in pheochromocytoma in 1993, and later it was discovered that it is widely secreted and expressed in both vascular endothelial cells and vascular smooth muscle cells. The gene of adrenomedullin is located on chromosome 11. After translation, it is gradually enzymatically cleaved by prohormone and formed as calcitonin gene-related peptide (CLR). The heterodimer consisting of CLR and RAMP2 or RAMP3 is the receptor of adrenomedullin, and based on its receptor-mediated endocytosis and the action of protease, its half-life is only 22 min.

[0004] Adrenomedullin has the effect of maintaining the integrity of endothelial cells and enhancing the barrier. This is achieved by two means: (1) enhancing the activity of GTPase Rac1 to increase the production of cortical actin and stress fibers; (2) inhibiting the RhoA / ROCK pathway to reduce the contraction of actomyosin induced by myosin light chain kinase. Adrenomedullin also has the effects of vasodilation and blood pressure reduction. This is realized by two aspects: (1) through the PI3K / Akt pathway, releasing NO and activating cyclic guanosine monophosphate (cGMP) / activated protein kinase K (PKG); (2) by binding to vascular smooth muscle, leading to an increase in the concentration of cyclic adenosine monophosphate (cAMP) / activated protein kinase A (PKA), and causing the relaxation of smooth muscle cells by the action of phosphorylation.

[0005] In healthy people, the concentration of adrenomedullin is extremely low, about 10 pg / mL, and it can freely reciprocate inside and outside blood vessels, flexibly regulate vasodilation, and maintain the barrier function of endothelial cells. In patients with septic shock, the concentration of adrenomedullin is 5 to 6 times that of normal cases, and it is directly related to the severity and prognosis of the disease. In patients with septic shock, the barrier function of blood vessels is weakened by the action based on inflammation, and the blood pressure further decreases due to vasodilation.

[0006] The adrenomedullin non-neutralizing antibody restricts adrenomedullin within blood vessels, corrects the intravascular barrier function, weakens the extracellular vasodilatory effect, and increases the half-life of adrenomedullin in plasma, thereby achieving the treatment and alleviation of the shock symptoms of sepsis. Therefore, at present, it is urgently necessary to develop a non-neutralizing antibody with relatively high affinity for adrenomedullin. Summary of the Invention

[0007] In view of the above technical problems, one aspect of the present invention provides an anti - adrenomedullin (anti - ADM) monoclonal antibody or a fragment thereof, wherein the monoclonal antibody or fragment specifically binds to the amino acid sequence of positions 1 to 21 at the N - terminus of human adrenomedullin (ADM), the amino acid sequence of positions 1 to 21 at the N - terminus of the human ADM is shown in SEQ ID NO: 2, and the monoclonal antibody or fragment has a KD value for ADM of less than 10 -10 M, indicating an affinity.

[0008] Throughout the specification of the present invention, the "antibody" or "antibody fragment" of the present invention can bind to ADM, and thus targets ADM, and may thereby be referred to as an "anti - ADM antibody" or "anti - ADM antibody fragment".

[0009] The "antibody" or "antibody fragment" described in the present invention is a non - neutralizing antibody against adrenomedullin or a fragment thereof.

[0010] In this context, for the purpose of simplification, an antibody or antibody fragment having "non - neutralizing anti - ADM activity", collectively referred to as a "non - neutralizing" anti - ADM antibody or antibody fragment (e.g., blocking 80% or less of ADM biological activity), is defined as follows: After adding one or more molecules that bind to ADM to a culture of a eukaryotic cell line, the amount of cAMP produced by the cell line is decreased by the action of a synthetic human ADM peptide added in parallel. The cell line expresses a functional human recombinant ADM receptor composed of CRLR (calcitonin receptor - like receptor) and RAMP3 (receptor activity - modifying protein 3). Among them, the added synthetic human ADM is added in an amount that provides a half - maximal stimulation of cAMP synthesis in the absence of the non - neutralizing antibody awaiting analysis. Among them, the binding of the molecule to ADM results in a decrease of 80% or less of cAMP, and the same is true even when the non - neutralizing molecule awaiting analysis that can bind to ADM is added in an amount 10 times or more of the above amount (required to achieve the maximum decrease in cAMP obtained using the non - neutralizing antibody awaiting analysis).

[0011] The same definition applies to other ranges such as 95%, 90%, 50%, etc.

[0012] Bioactivity is defined as the action that a substance exhibits as a living organism, tissue, organ, or functional unit in vivo or in vitro (e.g., in an assay) after interaction. For ADM bioactivity, this may be the action of ADM in a human recombinant adrenomedullin receptor cAMP functional assay. Thus, according to the present invention, bioactivity is defined by the adrenomedullin receptor cAMP functional assay.

[0013] To measure ADM bioactivity in such an assay, the following steps can be performed: In the above human recombinant adrenomedullin receptor cAMP functional assay, perform a dose-response curve using ADM.

[0014] The ADM concentration for half-maximal cAMP stimulation can be calculated.

[0015] At a constant half-maximal cAMP stimulation, perform an ADM concentration dose-response curve (the final concentration reaches 100 μg / mL) with an ADM stabilizing antibody or an adrenomedullin stabilizing antibody fragment, respectively.

[0016] In the above ADM bioassay, 50% maximal inhibition indicates that the anti-ADM antibody or the anti-adrenomedullin antibody fragment blocks 50% of the bioactivity of the baseline value, respectively. In the above ADM bioassay, 80% maximal inhibition indicates that the anti-ADM antibody or the anti-adrenomedullin antibody fragment blocks 80% of the ADM bioactivity, respectively. The meaning is to block 80% or less of the ADM bioactivity. This means that there is about 20% residual ADM bioactivity.

[0017] However, throughout this specification and in the above context, in accordance with the anti-ADM antibodies and anti-ADM antibody fragments disclosed herein, the expression "blocking the biological activity of ADM" should be understood to merely reduce the biological activity of ADM, preferably, the remaining ADM biological activity with the ADM biological activity reduced from 100% to 20% at most, preferably, the remaining ADM biological activity with the ADM biological activity reduced from 100% to 50%, but in any case, there is ADM biological activity that can be measured as described above.

[0018] In this specification, 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, among which, the above fragment can specifically bind to ADM. Specifically binding to ADM also allows binding to other antigens. This means that the specificity does not exclude the ability of the antibody to cross-react with polypeptides other than the polypeptide that excites the antibody. This also applies to the specificity of the anti-ADM antibody or its fragment of the present invention.

[0019] The non-neutralizing anti-ADM antibody or non-neutralizing anti-ADM antibody fragment of the present invention provides therapeutic advantages that significantly exceed those of neutralizing anti-ADM antibodies or neutralizing anti-ADM antibody fragments.

[0020] The antibody of the present invention is a protein comprising one or more polypeptides substantially encoded by immunoglobulin genes and capable of specifically binding to an antigen. Widely recognized immunoglobulin genes include κ, λ, α (IgA), γ (IgG1, IgG2, IgG3, IgG4), δ (IgD), ε (IgE) and μ (IgM) constant region genes as well as numerous immunoglobulin variable region genes. The length of the full-length immunoglobulin light chain is usually about 25 KDa or 214 amino acids. The length of the full-length immunoglobulin heavy chain is usually about 50 KDa or 446 amino acids. The light chain is encoded by a variable region gene (about 110 amino acids in length) located at the NH2-terminus and a κ or λ constant region gene located at the COOH-terminus. The heavy chain is similarly encoded by a variable region gene (about 116 amino acids in length) and one of the other constant region genes.

[0021] The basic structural unit of an antibody is usually a tetramer consisting of two pairs of identical immunoglobulin chains, each pair having one light chain and one heavy chain. In each pair, the light chain and heavy chain variable regions bind to the antigen, and the constant regions mediate the effect or function. Immunoglobulins further exist in a plurality of other forms, including, for example, Fv, Fab and F(ab')2, and bifunctional hybrid antibodies and single-chain antibodies. The immunoglobulin light chain or heavy chain variable region contains a framework region interrupted by three hypervariable regions, which are also called complementarity-determining regions (CDRs). As described above, the CDRs mainly play a role in binding to the epitope of the antigen. An immune complex is one in which an antibody such as a monoclonal antibody, a chimeric antibody, a humanized antibody, or a human antibody or a functional antibody fragment specifically binds to an antigen.

[0022] In the art, the CDRs of antibodies can be defined by various methods such as the Kabat definition rules based on sequence variability, the Chothia definition rules based on the positions of structural loop regions, and the concept based on the IMGT ontology (IMGT-ONTOLOGY). In the present invention, the amino acid sequences of the VL and VH of the anti-ADM antibody are encoded according to the Chothia coding rules, and the light-chain CDRs 1-3 (LCDR1-3) and heavy-chain CDRs 1-3 (HCDR1-3) of the anti-ADM antibody are defined according to Chothia.

[0023] A chimeric antibody is an antibody whose light-chain and heavy-chain genes are constructed by genetic engineering from immunoglobulin variable-region and constant-region genes belonging to different species. For example, variable segments from a mouse monoclonal antibody gene can be ligated to human constant segments such as κ and γ1 or γ3. In one example, a therapeutic chimeric antibody is a hybrid protein consisting of a variable domain or antigen-binding domain derived from a mouse antibody and a constant or effector domain derived from a human antibody, although other mammalian species can also be used or variable regions can be generated by molecular techniques.

[0024] A "humanized" immunoglobulin is an immunoglobulin that contains a human framework region and one or more CDRs of a non-human (mouse, rat, synthetic, etc.) immunoglobulin. The non-human immunoglobulin that provides the CDR is called the "donor", and the human immunoglobulin that provides the framework is called the "acceptor".

[0025] In one embodiment, all CDRs in a humanized immunoglobulin are derived from a donor immunoglobulin. The presence of a constant region is not necessary, but if present, it must be substantially identical to a human immunoglobulin constant region, i.e., at least about 85 - 90% identical, such as about 95% identical, or more. Thus, all parts of the humanized immunoglobulin, probably except for the CDRs, are substantially identical to the corresponding parts of the native human immunoglobulin sequence. A "humanized antibody" is an antibody that includes a humanized light chain and a humanized heavy chain immunoglobulin. A humanized antibody can bind to the same antigen as the donor antibody that provides the CDRs. The acceptor framework of a humanized immunoglobulin or antibody can have a limited number of amino acid substitutions from the donor framework. A humanized or other monoclonal antibody can have other conservative amino acid substitutions that do not substantially affect antigen binding or other immunoglobulin functions. A humanized immunoglobulin can be constructed by means of genetic engineering.

[0026] A human antibody is an antibody in which the genes of the light chain and the heavy chain are of human origin. Human antibodies can be produced by immortalizing human B cells that secrete the antibody of interest. Immortalization can be achieved, for example, by EBV infection or by fusing human B cells with myeloma or hybridoma cells to produce trioma cells. Human antibodies can also be produced by phage display methods or selected from a human combinatorial monoclonal antibody library. Human antibodies can also be manufactured using transgenic animals that carry human immunoglobulin genes.

[0027] Accordingly, anti-ADM antibodies can have forms known in the art. Examples are human antibodies, monoclonal antibodies, humanized antibodies, chimeric antibodies, CDR-grafted antibodies.

[0028] In a preferred embodiment, the antibody of the present invention is a recombinantly produced antibody, such as IgG, or an antibody fragment containing at least the heavy chain and / or the F-variable domain of the light chain, such as a chemically coupled antibody (a fragment that binds to an antigen).

[0029] Therefore, in a preferred embodiment of the present invention, the anti - adrenomedullin monoclonal antibody fragment described in the present invention includes Fab, Fab', F(ab)2, Fv fragment, F(ab')2, scFv, di - scFv, VHH and / or dAb.

[0030] In a preferred embodiment of the present invention, the antibody of the present invention can be produced as follows: Respectively, a polypeptide of 16 amino acids at the N - terminus of synthetic human ADM, a polypeptide of 21 amino acids at the N - terminus of human ADM, or a polypeptide of 19 amino acids at the N - terminus of mouse ADM (abbreviated as YY - 19, SEQ ID NO: 3) and a conjugate are used as immunogens to immunize mice. One week after the last immunization, blood is collected, and the titer of serum anti - YY - 21 is measured by ELISA. After immunizing the mice with high serum titers with an immunogen for an immunization shock, spleen cells are collected and fused. After the fusion of spleen cells and the screening of hybridoma clones, hybridoma clones are obtained. By affinity measurement, the hybridoma clones show an affinity with a KD value for ADM of less than 10 -10 M.

[0031] In the present invention, the term "K D ", "K D " or "KD" may be used interchangeably and generally refers to the equilibrium dissociation constant of the antibody - antigen interaction. "KD" used in the present invention is the ratio of the dissociation rate constant (kdis, also called "dissociation rate (off - rate) (koff)" or "kd") to the association rate constant (kon, also called "association rate (kon)" or "ka").

[0032] By sequencing, the heavy - chain variable region of the above - mentioned hybridoma clones is (i) GYAFTTF (shown in SEQ ID NO: 11), (ii) NTYSRV (shown in SEQ ID NO: 12), (iii) GYGGEGGLGF (shown in SEQ ID NO: 13), and includes the CDR sequences, and the light chain variable region of the above hybridoma clone is (i) RSSQSIIDSDGNTYLE (shown in SEQ ID NO: 14), (ii) KVSNRFS (shown in SEQ ID NO: 15), (iii) FQGSHFPYT (shown in SEQ ID NO: 16), and includes the CDR sequences.

[0033] In some embodiments of the present invention, the above hybridoma clone includes the heavy chain variable region sequence shown in SEQ ID NO: 9, and the above hybridoma clone includes the light chain variable region sequence shown in SEQ ID NO: 10.

[0034] Humanization of the anti-ADM antibody can be performed according to the following protocol.

[0035] By sequence alignment, the human antibody germline gene with the highest homology is selected as the humanized design framework. CDR transplantation and back mutations are performed on the heavy chain variable region of the hybridoma clone to obtain the humanized heavy chain variable region sequence. CDR transplantation and back mutations are performed on the light chain variable region of the hybridoma clone to obtain the humanized light chain variable region sequence.

[0036] Thus, in some embodiments of the present invention, the heavy chain variable region of the above humanized antibody is (i) GYAFTTF (shown in SEQ ID NO: 11), (ii) NTYSRV (shown in SEQ ID NO: 12), (iii) GYGGEGGLGF (shown in SEQ ID NO: 13), and includes the CDR sequences, and the light chain variable region of the above humanized antibody is (i) RSSQSIIDSDGNTYLE (shown in SEQ ID NO: 14), (ii) KVSNRFS (shown in SEQ ID NO: 15), (iii) It contains a CDR sequence of FQGSHFPYT (shown in SEQ ID NO: 16).

[0037] In some embodiments of the present invention, the humanized antibody contains a heavy chain variable region sequence shown in SEQ ID NO: 17, and the humanized antibody contains a light chain variable region sequence shown in either SEQ ID NO: 18 or 19.

[0038] In some embodiments of the present invention, the humanized antibody contains a heavy chain variable region sequence shown in SEQ ID NO: 17, and the humanized antibody contains a light chain variable region sequence shown in SEQ ID NO: 18.

[0039] In some embodiments of the present invention, the humanized antibody contains a heavy chain variable region sequence shown in SEQ ID NO: 17, and the humanized antibody contains a light chain variable region sequence shown in SEQ ID NO: 19.

[0040] In some embodiments of the present invention, the monoclonal antibody further contains an antibody light chain constant region, and the light chain constant region is a human Kappa chain constant region. In the present invention, the human Kappa chain constant region contains the amino acid sequence shown in SEQ ID NO: 20.

[0041] In some embodiments of the present invention, the monoclonal antibody further contains an antibody heavy chain constant region, and the heavy chain constant region is a human IgG1 constant region. In the present invention, the human IgG1 constant region contains the amino acid sequence shown in SEQ ID NO: 21.

[0042] Combine the light chain variable region sequence of the monoclonal antibody described in the present invention and the human Kappa chain constant region as the antibody light chain, and combine the heavy chain variable region sequence of the monoclonal antibody described in the present invention and the human IgG1 constant region as the antibody heavy chain.

[0043] Thereby, in some embodiments of the present invention, the monoclonal antibody contains a heavy chain sequence shown in any one of SEQ ID NO: 23, 25, 27, and the monoclonal antibody contains a light chain sequence shown in any one of SEQ ID NO: 22, 24, 26.

[0044] In some embodiments of the present invention, the monoclonal antibody comprises a heavy chain sequence shown in SEQ ID NO: 23, and the monoclonal antibody comprises a light chain sequence shown in SEQ ID NO: 22.

[0045] In some embodiments of the present invention, the monoclonal antibody comprises a heavy chain sequence shown in SEQ ID NO: 25, and the monoclonal antibody comprises a light chain sequence shown in SEQ ID NO: 24.

[0046] In some embodiments of the present invention, the monoclonal antibody comprises a heavy chain sequence shown in SEQ ID NO: 27, and the monoclonal antibody comprises a light chain sequence shown in SEQ ID NO: 26.

[0047] After codon optimization, gene synthesis was performed, and the synthesized gene fragment was cloned into an expression vector. After amplification of the expression plasmid and extraction of the plasmid, the double plasmid was co-transfected into Expi293F or CHO-K1 cells for transient expression of the antibody, and after expression, it was purified by a Protein A affinity chromatography column. By affinity measurement, the humanized anti-ADM antibody showed an affinity with a KD value for ADM of less than 10 -10 M.

[0048] The present invention can further screen for anti-ADM monoclonal antibodies by panning a fully human single-chain phage antibody library.

[0049] Thus, in a preferred embodiment of the present invention, in the above anti-adrenomedullin monoclonal antibody or a fragment thereof, (a) the heavy chain variable region of the monoclonal antibody is (i) GYTFTSY (shown in SEQ ID NO: 43), (ii) SAYNGN (shown in SEQ ID NO: 44), (iii) EGRSGGSFDI (shown in SEQ ID NO: 45), and contains the CDR sequences of (b) the light chain variable region of the monoclonal antibody is (i) RASQGISSYLA (shown in SEQ ID NO: 46), (ii) DASNLET (shown in SEQ ID NO: 47), (iii) QQYDNLPLT (shown in SEQ ID NO: 48), and includes the CDR sequences.

[0050] In the present application, the "monoclonal antibody" generally refers to an antibody obtained from a substantially homogeneous population of antibodies, that is, each antibody constituting the population is identical except for the presence of extremely small amounts of naturally occurring mutations and / or post-translational modifications (e.g., isomerization, amidation). For example, in some embodiments of the present invention, the anti-adrenomedullin monoclonal antibody includes a post-translational modification (PTM) site of NG.

[0051] In some embodiments of the present invention, the post-translational modification (PTM) site of NG contained in the anti-adrenomedullin monoclonal antibody is site-specifically mutated to QG to remove the deamidation isomerization effect.

[0052] Therefore, in some embodiments of the present invention, in the above anti-adrenomedullin monoclonal antibody or a fragment thereof, (a) The heavy chain variable region of the monoclonal antibody is (i) GYTFTSY (shown in SEQ ID NO: 43), (ii) SAYQGN (shown in SEQ ID NO: 49), (iii) EGRSGGSFDI (shown in SEQ ID NO: 45), and includes the CDR sequences, and (b) The light chain variable region of the monoclonal antibody is (i) RASQGISSYLA (shown in SEQ ID NO: 46), (ii) DASNLET (shown in SEQ ID NO: 47), (iii) QQYDNLPLT (shown in SEQ ID NO: 48), and includes the CDR sequences.

[0053] In some embodiments of the present invention, the monoclonal antibody comprises a heavy chain variable region sequence shown in any one of SEQ ID NOs: 28 and 29, and the monoclonal antibody comprises a light chain variable region sequence shown in SEQ ID NO: 35.

[0054] In some embodiments of the present invention, the humanized antibody comprises a heavy chain variable region sequence shown in SEQ ID NO: 28, and the humanized antibody comprises a light chain variable region sequence shown in SEQ ID NO: 35.

[0055] In some embodiments of the present invention, the humanized antibody comprises a heavy chain variable region sequence shown in SEQ ID NO: 29, and the humanized antibody comprises a light chain variable region sequence shown in SEQ ID NO: 35.

[0056] In order to improve the affinity between the fully human antibody and human ADM, the present invention further designs and constructs an affinity maturation library of the fully human antibody. The present invention obtains different fully human anti-ADM antibodies, all of which show a KD value of less than 10 -10 M against ADM by screening the fully human antibody affinity maturation library and identifying monoclonal antibodies.

[0057] Therefore, in a preferred embodiment of the present invention, in the above-mentioned anti-adrenomedullin monoclonal antibody or its fragment, (a) the heavy chain variable region of the monoclonal antibody is (i) GYTFTX1Y, wherein X1 is selected from S, Q or H, (ii) SX2YX3GX4, wherein X2 is selected from A or P, X3 is selected from N, Q, S or T, X4 is selected from N or K, (iii) EGRX5GGSFX6I, wherein X5 is selected from S or W, X6 is selected from D or N, and contains the following CDR sequences, and (b) the light chain variable region of the monoclonal antibody is (i) RAX7X8GIX9X 10 YLA, wherein X7 is selected from S or A, X8 is selected from Q or E, X9 is selected from S or G, X 10 is selected from S or E, (ii) DX 11 SX 12 X 13 X 14 X 15 Among them, X 11 is selected from A, V or T, and X 12 is selected from N, I or D, and X 13 is selected from L or V, and X 14 is selected from E or D, and X 15 is selected from T or A, (iii) QQYDX 16 LX 17 LX 18 Among them, X 16 is selected from N or D, and X 17 is selected from P or D, and X 18 is selected from T or S, and includes a CDR sequence of

[0058] In a more preferred embodiment of the present invention, in the above anti - adrenomedullin monoclonal antibody or its fragment, (a) The heavy - chain variable region of the above monoclonal antibody is (i) GYTFTSY (shown in SEQ ID NO: 43), (ii) SAYQGN (shown in SEQ ID NO: 49), (iii) EGRWGGSFNI (shown in SEQ ID NO: 50), and includes a CDR sequence of (b) The light - chain variable region of the above monoclonal antibody is (i) RASQGISSYLA (shown in SEQ ID NO: 46), (ii) DASNLET (shown in SEQ ID NO: 47), (iii) QQYDNLPLT (shown in SEQ ID NO: 48), and includes a CDR sequence of

[0059] In a more preferred embodiment of the present invention, in the above anti - adrenomedullin monoclonal antibody or its fragment, (a) The heavy - chain variable region of the above monoclonal antibody is (i) GYTFTQY (shown in SEQ ID NO: 51), (ii) SAYQGN (shown in SEQ ID NO: 49), (iii) It contains the CDR sequence of EGRWGGSFNI (shown in SEQ ID NO: 50), and (b) The variable region of the light chain of the monoclonal antibody is (i) RASEGISEYLA (shown in SEQ ID NO: 52), (ii) DASNLET (shown in SEQ ID NO: 47), (iii) It contains the CDR sequence of QQYDNLPLT (shown in SEQ ID NO: 48).

[0060] In a more preferred embodiment of the present invention, in the above anti - adrenomedullin monoclonal antibody or its fragment, (a) The variable region of the heavy chain of the monoclonal antibody is (i) GYTFTQY (shown in SEQ ID NO: 51), (ii) SAYQGN (shown in SEQ ID NO: 49), (iii) It contains the CDR sequence of EGRWGGSFNI (shown in SEQ ID NO: 50), and (b) The variable region of the light chain of the monoclonal antibody is (i) RAAEGIGSYLA (shown in SEQ ID NO: 53), (ii) DASNLET (shown in SEQ ID NO: 47), (iii) It contains the CDR sequence of QQYDNLPLT (shown in SEQ ID NO: 48).

[0061] In a more preferred embodiment of the present invention, in the above anti - adrenomedullin monoclonal antibody or its fragment, (a) The variable region of the heavy chain of the monoclonal antibody is (i) GYTFTSY (shown in SEQ ID NO: 43), (ii) SPYSGN (shown in SEQ ID NO: 54), (iii) It contains the CDR sequence of EGRWGGSFNI (shown in SEQ ID NO: 50), and (b) The variable region of the light chain of the monoclonal antibody is (i) RASEGISEYLA (shown in SEQ ID NO: 52), (ii) DASNLET (shown in SEQ ID NO: 47), (iii) It contains a CDR sequence of QQYDNLPLT (shown in SEQ ID NO: 48).

[0062] In a more preferred embodiment of the present invention, in the above-mentioned anti - adrenomedullin monoclonal antibody or its fragment, (a) The heavy - chain variable region of the above - mentioned monoclonal antibody is (i) GYTFTHY (shown in SEQ ID NO: 55), (ii) SAYQGN (shown in SEQ ID NO: 49), (iii) It contains a CDR sequence of EGRWGGSFNI (shown in SEQ ID NO: 50), and (b) The light - chain variable region of the above - mentioned monoclonal antibody is (i) RASQGISSYLA (shown in SEQ ID NO: 46), (ii) DVSILDA (shown in SEQ ID NO: 56), (iii) It contains a CDR sequence of QQYDNLPLT (shown in SEQ ID NO: 48).

[0063] In a more preferred embodiment of the present invention, in the above - mentioned anti - adrenomedullin monoclonal antibody or its fragment, (a) The heavy - chain variable region of the above - mentioned monoclonal antibody is (i) GYTFTQY (shown in SEQ ID NO: 51), (ii) SAYQGN (shown in SEQ ID NO: 49), (iii) It contains a CDR sequence of EGRWGGSFNI (shown in SEQ ID NO: 50), and (b) The light - chain variable region of the above - mentioned monoclonal antibody is (i) RASQGISSYLA (shown in SEQ ID NO: 46), (ii) DVSILDA (shown in SEQ ID NO: 56), (iii) It contains a CDR sequence of QQYDNLPLT (shown in SEQ ID NO: 48).

[0064] In a more preferred embodiment of the present invention, in the above - mentioned anti - adrenomedullin monoclonal antibody or its fragment, (a) The heavy - chain variable region of the above - mentioned monoclonal antibody is (i) GYTFTQY (set forth in SEQ ID NO:51); (ii) SAYQGN (set forth in SEQ ID NO: 49); (iii) comprises the CDR sequence EGRWGGSFNI (set forth in SEQ ID NO:50); and (b) The light chain variable region of the monoclonal antibody is (i) RASQGISSYLA (set forth in SEQ ID NO:46); (ii) DASNVDT (set forth in SEQ ID NO:57); (iii) contains the CDR sequence QQYDNLPLT (set forth in SEQ ID NO:48).

[0065] In a further preferred embodiment of the present invention, the anti-adrenomedullin monoclonal antibody or a fragment thereof is (a) The heavy chain variable region of the monoclonal antibody is (i) GYTFTSY (shown in SEQ ID NO: 43); (ii) SPYTGK (set forth in SEQ ID NO:58); (iii) comprises the CDR sequence EGRWGGSFNI (set forth in SEQ ID NO:50); and (b) The light chain variable region of the monoclonal antibody is (i) RASQGISSYLA (set forth in SEQ ID NO:46); (ii) DVSILDA (set forth in SEQ ID NO:56); (iii) contains the CDR sequence QQYDNLPLT (set forth in SEQ ID NO:48).

[0066] In a further preferred embodiment of the present invention, the anti-adrenomedullin monoclonal antibody or a fragment thereof is (a) The heavy chain variable region of the monoclonal antibody is (i) GYTFTSY (shown in SEQ ID NO: 43); (ii) SPYTGK (set forth in SEQ ID NO:58); (iii) comprises the CDR sequence EGRWGGSFNI (set forth in SEQ ID NO:50); and (b) The light chain variable region of the monoclonal antibody is (i) RASQGISSYLA (shown in SEQ ID NO: 46), (ii) DTSDLDT (shown in SEQ ID NO: 59), (iii) QQYDNLPLT (shown in SEQ ID NO: 48), and includes a CDR sequence.

[0067] In a more preferred embodiment of the present invention, in the above anti - adrenomedullin monoclonal antibody or its fragment, (a) The heavy - chain variable region of the above monoclonal antibody (i) GYTFTHY (shown in SEQ ID NO: 55), (ii) SAYQGN (shown in SEQ ID NO: 49), (iii) EGRWGGSFNI (shown in SEQ ID NO: 50), and includes a CDR sequence, and (b) The light - chain variable region of the above monoclonal antibody (i) RASQGISSYLA (shown in SEQ ID NO: 46), (ii) DASNLET (shown in SEQ ID NO: 47), (iii) QQYDDLDLT (shown in SEQ ID NO: 60), and includes a CDR sequence.

[0068] In a more preferred embodiment of the present invention, in the above anti - adrenomedullin monoclonal antibody or its fragment, (a) The heavy - chain variable region of the above monoclonal antibody (i) GYTFTQY (shown in SEQ ID NO: 51), (ii) SAYQGN (shown in SEQ ID NO: 49), (iii) EGRWGGSFNI (shown in SEQ ID NO: 50), and includes a CDR sequence, and (b) The light - chain variable region of the above monoclonal antibody (i) RASQGISSYLA (shown in SEQ ID NO: 46), (ii) DASNLET (shown in SEQ ID NO: 47), (iii) QQYDDLPLS (shown in SEQ ID NO: 61), and includes a CDR sequence.

[0069] Furthermore, the present invention further obtains heavy and light chain variable region sequences of an antibody with improved affinity.

[0070] Thus, in a preferred embodiment of the present invention, in the above-mentioned anti-adrenomedullin monoclonal antibody or its fragment, the monoclonal antibody contains a heavy chain variable region sequence shown in any one of SEQ ID NOs: 30 to 34, and the monoclonal antibody contains a light chain variable region sequence shown in any one of SEQ ID NOs: 35 to 42.

[0071] In some embodiments of the present invention, the humanized antibody contains the heavy chain variable region sequence shown in SEQ ID NO: 30, and the humanized antibody contains the light chain variable region sequence shown in SEQ ID NO: 35.

[0072] In some embodiments of the present invention, the humanized antibody contains the heavy chain variable region sequence shown in SEQ ID NO: 31, and the humanized antibody contains the light chain variable region sequence shown in SEQ ID NO: 36.

[0073] In some embodiments of the present invention, the humanized antibody contains the heavy chain variable region sequence shown in SEQ ID NO: 31, and the humanized antibody contains the light chain variable region sequence shown in SEQ ID NO: 37.

[0074] In some embodiments of the present invention, the humanized antibody contains the heavy chain variable region sequence shown in SEQ ID NO: 32, and the humanized antibody contains the light chain variable region sequence shown in SEQ ID NO: 36.

[0075] In some embodiments of the present invention, the humanized antibody contains the heavy chain variable region sequence shown in SEQ ID NO: 33, and the humanized antibody contains the light chain variable region sequence shown in SEQ ID NO: 38.

[0076] In some embodiments of the present invention, the humanized antibody contains the heavy chain variable region sequence shown in SEQ ID NO: 31, and the humanized antibody contains the light chain variable region sequence shown in SEQ ID NO: 38.

[0077] In some embodiments of the present invention, the humanized antibody comprises a heavy chain variable region sequence shown in SEQ ID NO: 31, and the humanized antibody comprises a light chain variable region sequence shown in SEQ ID NO: 39.

[0078] In some embodiments of the present invention, the humanized antibody comprises a heavy chain variable region sequence shown in SEQ ID NO: 34, and the humanized antibody comprises a light chain variable region sequence shown in SEQ ID NO: 38.

[0079] In some embodiments of the present invention, the humanized antibody comprises a heavy chain variable region sequence shown in SEQ ID NO: 34, and the humanized antibody comprises a light chain variable region sequence shown in SEQ ID NO: 40.

[0080] In some embodiments of the present invention, the humanized antibody comprises a heavy chain variable region sequence shown in SEQ ID NO: 33, and the humanized antibody comprises a light chain variable region sequence shown in SEQ ID NO: 41.

[0081] In some embodiments of the present invention, the humanized antibody comprises a heavy chain variable region sequence shown in SEQ ID NO: 31, and the humanized antibody comprises a light chain variable region sequence shown in SEQ ID NO: 42.

[0082] In some embodiments of the present invention, the monoclonal antibody further comprises an antibody light chain constant region, and the light chain constant region is a human Kappa chain constant region. In the present invention, the human Kappa chain constant region comprises the amino acid sequence shown in SEQ ID NO: 20.

[0083] In some embodiments of the present invention, the monoclonal antibody further comprises an antibody heavy chain constant region, and the heavy chain constant region is a human IgG1 constant region. In the present invention, the human IgG1 constant region comprises the amino acid sequence shown in SEQ ID NO: 21.

[0084] The light chain variable region sequence of the monoclonal antibody and the human Kappa chain constant region are combined as an antibody light chain, and the heavy chain variable region sequence of the monoclonal antibody and the human IgG1 constant region are combined as an antibody heavy chain.

[0085] After codon optimization, gene synthesis is performed, and the synthesized gene fragment is cloned into the expression vector pcDNA3.4, and then expressed and purified in CHO cells to obtain an affinity-matured monoclonal antibody. By affinity measurement, the KD value of the anti-ADM antibody against ADM is 10 -10 less than M, indicating an affinity.

[0086] Yet another aspect of the present invention provides a nucleotide sequence encoding the anti-adrenomedullin monoclonal antibody or a fragment thereof described in the present invention.

[0087] Yet another aspect of the present invention provides an expression vector comprising the nucleotide sequence described in the present invention, preferably the vector pcDNA3.4.

[0088] Yet another aspect of the present invention provides a host cell comprising the nucleotide sequence or expression vector described in the present invention, preferably a CHO cell.

[0089] Yet another aspect of the present invention provides a pharmaceutical composition, detection reagent or kit comprising the anti-adrenomedullin monoclonal antibody or a fragment thereof described in the present invention.

[0090] Yet another aspect of the present invention provides the use of the anti-adrenomedullin monoclonal antibody or a fragment thereof described in the present invention in the manufacture of a drug for maintaining endothelial cell integrity and enhancing the barrier function.

[0091] Yet another aspect of the present invention provides the use of the anti-adrenomedullin monoclonal antibody or a fragment thereof described in the present invention in the manufacture of a drug for controlling vasodilation and reducing blood pressure.

[0092] Yet another aspect of the present invention provides the use of the anti-adrenomedullin monoclonal antibody or a fragment thereof described in the present invention in the manufacture of a drug for treating or preventing septic shock or traumatic injury, particularly the end stage of sepsis.

[0093] Another aspect of the present invention is the use of the anti - adrenomedullin monoclonal antibody or a fragment thereof according to the present invention in the manufacture of a medicament for reducing the risk of death from chronic or acute diseases or acute illnesses of a patient, wherein the chronic or acute disease or acute illness is selected from severe infections such as meningitis, systemic inflammatory response syndrome (SIRS), sepsis, other diseases such as diabetes, cancer, acute and chronic vascular diseases such as heart failure, myocardial infarction, stroke, atherosclerosis, edema, shock such as septic shock, and organ dysfunction such as renal dysfunction, liver dysfunction, burns, surgery, trauma, poisoning.

[0094] Another aspect of the present invention is the use of the anti - adrenomedullin monoclonal antibody or a fragment thereof according to the present invention in the manufacture of a medicament for stabilizing the circulation, particularly the systemic circulation, of a patient suffering from a chronic or acute disease or acute illness, wherein the chronic or acute disease or acute illness is selected from severe infections such as meningitis, systemic inflammatory response syndrome (SIRS), sepsis, other diseases such as diabetes, cancer, acute and chronic vascular diseases such as heart failure, myocardial infarction, stroke, atherosclerosis, edema, shock such as septic shock and organ dysfunction such as renal dysfunction, liver dysfunction, burns, surgery, trauma, poisoning.

[0095] In a preferred embodiment of the present invention, the anti - adrenomedullin monoclonal antibody or a fragment thereof is used in combination with another agent.

[0096] In a more preferred embodiment of the present invention, the agent is selected from vasopressors, intravenous injection solutions, TNF - α - antibodies, and antibiotics.

Advantages of the Invention

[0097] The present invention screens anti-ADM monoclonal antibodies by hybridoma technology, and obtains fully human anti-ADM monoclonal antibodies by humanization expression of the antibodies and panning of a fully human single-chain phage antibody library. Based on this, the present invention screens an affinity maturation library to finally obtain those with relatively high binding activity to the N-terminus of human ADM. Compared with the anti-ADM non-neutralizing antibodies existing in the prior art, the anti-ADM non-neutralizing antibodies of the present invention have a KD value for ADM of 10 -10 less than M and show an affinity.

[0098] Animal tests show that treatment with the anti-ADM non-neutralizing antibody of the present invention can significantly improve the symptoms of sepsis and improve the survival rate of animals.

Brief Description of the Drawings

[0099]

Figure 1

Figure 2

Modes for Carrying Out the Invention

[0100] To further explain the objectives and advantages of this method, the specific implementation content of the present invention will be described in more detail by combining the drawings and specific examples.

[0101] Example 1, Screening of Anti-ADM Monoclonal by Hybridoma Technology 1.1 Immunization of Mice The conjugates of the N-terminal 16-amino acid polypeptide of synthetic human ADM (abbreviated as YY-16, SEQ ID NO: 1), the N-terminal 21-amino acid polypeptide of human ADM (abbreviated as YY-21, SEQ ID NO: 2), or the N-terminal 19-amino acid polypeptide of mouse ADM (abbreviated as YY-19, SEQ ID NO: 3) with KLH (Sigma, H8283) were used as immunogens to immunize mice. At the first immunization, the immunogen and Freund's complete adjuvant were emulsified at a ratio of 1:1 and intraperitoneally injected into female Balb / c mice, SJL mice or SD rats aged 6-8 weeks at 100 μg per mouse. Subsequently, booster immunizations were performed at intervals of 2-3 weeks. 50 μg of the immunogen and Freund's incomplete adjuvant were added to each animal, and after 3-4 immunizations, blood was collected to measure the titer. Blood was collected 1 week after the last immunization, and the serum titer of anti-YY-21 was measured by ELISA. Mice with high serum titers were intraperitoneally injected with 50 μg of the immunogen to induce an immune shock, and on the 3rd day, the spleen cells of the animals were collected and fused.

[0102] 1.2 Spleen cell fusion After euthanizing the mice, they were dissected, the spleens were collected, crushed to collect cells, the cells were suspended in 5 mL of erythrocyte lysate, left at 4°C for 5 min, and the reaction was stopped with DMEM + 10% FBS. After centrifugation, the spleen cells were resuspended in 40 mL of DMEM, left standing for 2-3 min, and then the supernatant was transferred to another 50 mL centrifuge tube. SP2 / 0: spleen cells were mixed at a ratio of 1:2, centrifuged, and after thoroughly aspirating the supernatant, the mixed cells were precipitated, washed twice with DMEM, resuspended in electrofusion buffer, and added to the electrofusion chamber. After the electrofusion program was completed, the fused cells were first left standing for 5 min and then added to DMEM + 10% FBS + 1×HAT screening medium. The above cell suspension was added into a 96-well cell culture plate and cultured in an incubator at 37°C, 75% humidity, and 5% CO2 for 7-9 days.

[0103] 1.3 Screening of hybridoma clones YY-21 or YY-19 was diluted to 1.0 μg / mL with PBS, added to a 96-well plate (Corning, 9018) at 100 μL / well, and incubated overnight at 4°C for coating. The next day, the ELISA plate was washed three times with a washing buffer (PBS + 0.05% Tween20) using an automatic plate washer. 300 μL of blocking buffer (PBS + 0.05% Tween20 + 1% BSA) was added to each well and blocked at room temperature for 1 h. Then, it was washed three times with the washing buffer using an automatic plate washer. The hybridoma supernatant was added to each well of the ELISA plate, incubated at room temperature for 1 h, and then the plate was washed three times according to the above method. Goat Anti-mouse IgG Fc-HRP (Sigma, A0168) or Goat Anti-rat-IgG-HRP (Sigma, A5795) was diluted 1:5000 with the blocking buffer, 100 μL was added to each well, and incubated at room temperature for 1 h. Then, the plate was washed three times according to the above method. The TMB substrate solution was added at 100 μL / well and incubated at room temperature for 10 min. Then, 50 μL of 1.0 M hydrochloric acid was added to each well to stop the reaction, and the plate was read at OD 450 nm using a microplate reader. Positive cells were selected and subcloned and subcloning screening was performed until a stable hybridoma cell line that could secrete monoclonal antibodies binding to YY-21 and YY-19 was obtained. Through the binding experiment, hybridoma clone 40E12 was screened, the cell line was cryopreserved, small-scale production of 50 mL was carried out using a serum-free medium, and after purification by a protein A column, subsequent identification was performed.

[0104] 1.4 Measurement of the affinity of hybridoma clone 40E12 The affinity of the candidate antibody with biotinylated human ADM (abbreviated as human ADM-C-biotin, and the human ADM sequence is shown in SEQ ID NO: 4) and mouse ADM (abbreviated as mouse ADM-C-biotin, and the mouse ADM sequence is shown in SEQ ID NO: 5) was measured using Octet RED96e (Fortebio). Both the antigen and the antibody were diluted with 1×PBST. The use concentration of the antigen was 2 μg / mL, and the working concentration of the antibody was 100 nM.

[0105] First, the samples were added to a 96-well plate (Greiner bio-one, 655209), and the reaction system was 200 μL / well. Next, the software parameters were set, the plate temperature was set at 30°C, and the frequency of collecting the standard kinetic signal was 5.0 HZ. Subsequently, after pre-wetting the streptavidin sensor (Fortebio, part number 18-5020) with 1×PBST for 10 min, it was loaded and detected. Each cycle included the following steps: 1) Immersed in the buffer for 180 s to stabilize the baseline; 2) The antigen was cured for 10 s to bind the antigen to the sensor, and the amount of antigen bound was controlled between 0.5 and 1.0 nm; 3) The sensor was immersed in the buffer for 180 s; 4) The antigen and the antibody were allowed to bind for 180 s; 5) The antigen-antibody complex was dissociated for 10 min.

[0106] 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 thereby calculate the equilibrium dissociation constant (KD) of the antibody. The results are shown in Table 1.

[0107]

Table 1

[0108] 1.5 Sequencing of Hybridoma Clone 40E12 The hybridoma monoclonal cell line was cultured, and 5×10 6Individual hybridoma cells were collected by centrifugation, total RNA was extracted by the Trizol method, and after performing a G addition reaction on the cDNA obtained after reverse transcription reaction using terminal transferase, a DNA containing a variable region sequence was amplified using a VH primer (the sequence is shown in SEQ ID NO: 6), a VK primer (the sequence is shown in SEQ ID NO: 7), and a polyC primer (the sequence is shown in SEQ ID NO: 8), TA cloning was performed, and after sequencing, the sequences of the heavy chain variable region (40E12VH) and the light chain variable region (40E12VK) of the mouse-derived hybridoma clone 40E12 obtained were shown in SEQ ID NO: 9 and SEQ ID NO: 10, respectively. The light chain and heavy chain variable regions were numbered according to Chothia, and the amino acid sequences of HCDR1, HCDR2, and HCDR3 of clone 40E12 defined by Chothia were 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 were shown in SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16, respectively.

[0109] Example 2, Humanization and Expression of Anti-ADM Antibody 2.1 Humanization of Anti-ADM Antibody By sequence alignment, the human antibody germline gene with the highest homology was selected as the humanized design framework.

[0110] For the heavy chain variable region, using the human antibody germline gene sequences IGHV7-4-1*02 and IGHJ6*01 as the framework, CDR transplantation and back mutations were performed on 40E12VH (also abbreviated as 2004hzVH0), and the humanized heavy chain variable region sequence 2004hzVH9 (the amino acid sequence is shown in SEQ ID NO: 17) was obtained. For the light chain variable region, using the human antibody germline gene sequences IGKV2-30*02 and IGKJ2*01 as the framework, CDR transplantation and back mutations were performed on 40E12VK (also abbreviated as 2004hzVK0), and the humanized light chain variable region sequences 2004hzVK7 (the amino acid sequence is shown in SEQ ID NO: 18) and 2004hzVK9 (the amino acid sequence is shown in SEQ ID NO: 19) were obtained.

[0111] 2.2 Expression of anti-ADM antibody The 2004hzVK7 and 2004hzVK9 sequences were combined with the human kappa chain constant region (CL, amino acid sequence shown in SEQ ID NO: 20) as the antibody light chain, and the 2004hzVH9 sequence was combined with the human IgG1 constant region (CH, amino acid sequence shown in SEQ ID NO: 21) as the antibody heavy chain. After combining the 2004hzVH0 and 2004hzVK0 with the human heavy chain constant region and the human light chain constant region respectively, they paired to form the chimeric antibody 2004hz00 (sequences shown in SEQ ID NO: 22 and 23). After combining the 2004hzVH9 and 2004hzVK7 with the human heavy chain constant region and the human light chain constant region respectively, they paired to form the humanized antibody 2004hz97 (sequences shown in SEQ ID NO: 24 and 25). After combining the 2004hzVH9 and 2004hzVK9 with the human heavy chain constant region and the human light chain constant region respectively, they paired to form the humanized antibody 2004hz99 (sequences shown in SEQ ID NO: 26 and 27).

[0112] After codon optimization, gene synthesis was performed, and the synthesized gene fragment was cloned into the expression vector pcDNA3.4 (Life Technologies). After amplification of the expression plasmid and extraction of the plasmid (Qiagen, EndoFree® Plasmid Maxi Kit, Cat. No. 12362), the double plasmid was co-transfected into Expi293F (ThermoFisher Scientific, A14527) or CHO-K1 cells (ECACC catalogue no. 85051005), and transient expression of the antibody was performed by the supplier's Expi293F or CHO-K1 expression system method. After expression, it was purified by a protein A affinity chromatography column, eluted with citrate buffer (pH 3.4), the absorbance value at 280 nm was read using a NanoDrop instrument, and the antibody was collected by dialysis for use.

[0113] The results of antibody expression are shown in Table 2, indicating that anti-ADM antibody can be expressed using the above method.

[0114]

Table 2

[0115] Example 3, Measurement of Affinity of Anti-ADM Antibody Using Octet RED96e (Fortebio), the affinities of antibodies 2004hz00, 2004hz97, 2004hz99 with human and mouse ADM labeled with biotin (human ADM product number 894757, mouse ADM product number 894758, synthesized by GLBiochem) were measured. Both the antigen and the antibody were diluted with 1×PBST (1×PBS, Sangon Biotech, B548117 - 0500, 0.02% Tween20, sigma, P1379). The antibody concentration used was 100 nM and the antigen concentration used was 2 μg / mL. The candidate antibody samples were added to a 96-well plate (Greiner bio-one, 655209) at 200 μL / well. The software parameters were set with the temperature at 30°C and the frequency of collecting the standard kinetic signal at 5.0 Hz. After pre-wetting the SA sensor (Fortebio, product number 18 - 5020) with 1×PBST for 10 min, it was loaded and detected.

[0116] Each cycle includes the following steps: 1) Immerse in the buffer for 60 s, 2) Detect whether the antigen binds non-specifically to the sensor, 3) Regenerate with a 10 mM glycine solution at pH 1.7, 4) Immerse in the buffer for 60 s, 5) Immobilize the antigen on the sensor for 10 s, 6) Immerse the sensor in the buffer for 180 s, 7) Bind the antigen and the antibody for 180 s, 8) Dissociate the antigen-antibody complex for 600 s, 9) Regenerate the sensor.

[0117] Using Fortebio's Data Analysis 12.0 software, the association rate (Kon) and dissociation rate (Koff) of the antigen-antibody were measured in a 1:1 binding mode, and thereby the equilibrium dissociation constant (KD) of the antibody was calculated. The results are shown in Table 3 and Table 4 respectively. As can be seen from Table 3 and Table 4, the affinities of the humanized antibodies 2004hz97, 2004hz99 with human and mouse ADM are comparable to the affinity of the chimeric antibody 2004hz00.

[0118]

Table 3

[0119]

Table 4

[0120] Example 4, Evaluation of Physicochemical Properties of Humanized Antibodies The expression levels and affinities of antibodies 2004hz97 and 2004hz99 are relatively ideal, and the evaluation of physicochemical drug-likeness as candidate molecules continues, specifically as follows.

[0121] 4.1 SEC-HPLC Purity Analysis The sample concentration was adjusted to 1 mg / mL, and the supernatant was taken by centrifugation and transferred to a sample bottle and placed in an HPLC sample tray. The chromatography conditions were set as follows: chromatography column TSK G3000SWxl, detection wavelength 280 nm, column temperature 25°C, sample chamber temperature 5°C, and flow rate 0.5 mL / min. After equilibrating the chromatography column with the mobile phase (200 mM phosphate buffer, pH 6.8), the sample was injected for analysis, and data analysis was performed using chromatography software. The peak area percentage of each peak was calculated by the peak area normalization method, indicating that the higher the percentage, the higher the antibody purity.

[0122] 4.2 HIC-HPLC Analysis The sample concentration was adjusted to 1 mg / mL, and the supernatant was taken by centrifugation in preparation for measurement. The chromatography conditions were: chromatography column MAbPac TMHIC-10 was set at a detection wavelength of 214 nm, a column temperature of 30 °C, a sample chamber temperature of 5 °C, and a flow rate of 0.8 mL / min. Gradient elution was performed using mobile phase A (50 mM phosphate buffer / 1 M ammonium sulfate, pH 7.0) and mobile phase B (50 mM phosphate buffer, pH 7.0), and the retention time of the main peak was recorded. The shorter the peak appearance time, the higher the hydrophilicity of the antibody.

[0123] 4.3 Melting temperature (Tm) value analysis Protein Thermal Shift TM According to the Starter Kit instructions, 13 μL of the sample solution was taken and added into a PCR tube, 5 μL of Protein Thermal shift TM Buffer was added, 2 μL of 10× staining solution was added, the reaction volume was made up to 20 μL, and after mixing uniformly, it was centrifuged at 12000 rpm for 5 min to remove bubbles. The detection sample was placed in a PCR instrument for sample analysis, the Tm value of the sample was recorded, and the higher the Tm value, the higher the thermal stability of the antibody.

[0124] 4.4 Isoelectric focusing capillary electrophoresis (iCIEF) analysis The sample solution was taken and added to a system that had already been thoroughly and uniformly mixed, consisting of 70 μL of 1% methyl cellulose (MC), 80 μL of 5 M urea, 8 μL of amphoteric electrolyte Pharmalyte with a pH range of 3 - 10, 2 μL each of pI markers 5.5 and 9.5. An appropriate volume of ultrapure water was added to make up to 200 μL and mixed uniformly. The supernatant was taken by centrifugation and the sample was injected for analysis. After the analysis was completed, the result file was imported into ChromPerfect software for spectral integration processing, and the isoelectric point and percentage of each peak were calculated to analyze the distribution of charge isomers of the candidate antibody.

[0125] 4.5 nrCE-SDS The sample was diluted to 4 mg / mL with ultrapure water. 25 μL was taken, 75 μL of SDS sample buffer and 5 μL of 0.25 mol.L-1 iodoacetamide (IAM) were added, and after mixing uniformly, it was heated at 70 °C for 10 min. Then 90 μL of the supernatant was taken and put into a sample bottle for loading and analysis. The Beckman PA800 Plus capillary electrophoresis system was adopted for detection with an uncoated capillary (total length 31 cm, effective length 21 cm). The detection conditions were: separation voltage 15 KV, capillary temperature 25 °C, sample chamber temperature 15 °C, and detection wavelength 220 nm. The percentage of the corrected peak area of the main peak was calculated.

[0126] As can be seen from Table 5, the humanized antibodies 2004hz97 and 2004hz99 already had good physicochemical properties by one-step protein A purification.

[0127]

Table 5

[0128] Example 5, Panning of a fully human single-chain phage antibody library Take 150 μL of Streptavidin Magnetic Beads (Thermo Fisher, product number 88817), pre-bind it with 2 mL of a complete human single-chain phage antibody library, incubate at room temperature for 90 min, and remove non-specific binding. After removing the background, add the library phage to 10 μg of human ADM (product number NT-H-2, synthesized by GeneScript), 150 μL of Streptavidin Magnetic Beads, incubate at room temperature for 15 min, wash 14 times with PBST (containing 0.05% Tween-20 in PBS) to wash away unbound phage. Elute the phage specifically bound to the antigen with 450 μL of 100 mM hydrochloric acid, add 50 μL of 1M Tris-HCl at pH 11 to neutralize it, infect Escherichia coli SS320 in the logarithmic growth phase, generate phage for the next round of screening, and purify it. The screening method is the same as the first round, only reducing the antigen dosage to 4 μg. Take the phage enriched after two rounds of screening, use enzyme-linked immunosorbent assay (ELISA) to identify the enrichment situation, and the results showed that the phage was significantly enriched after two rounds of panning.

[0129] Take 10 μL of the phage eluted after two rounds of panning, serially dilute it 10,000-fold, add 90 μL of Escherichia coli SS320 in the logarithmic growth phase, let it stand for 30 min to infect, then spread on a resistant plate and culture overnight at 37°C. The next day, select monoclonal plaques from the resistant plate, put them into a 96-deep well plate with ampicillin / IPTG / 2YT added, and culture overnight at 37°C. The next day, centrifuge at 4000 g for 10 min to collect the supernatant, and use enzyme-linked immunosorbent assay (ELISA) to identify the monoclonal binding ability, and screen for monoclonal 1F12 (the variable region amino acid sequences are shown in SEQ ID NO: 28 and 35).

[0130] Example 6, Effect of Anti-ADM Antibody on ADM Biological Activity In a human recombinant adrenomedullin receptor cAMP function assay (bioassay of adrenomedullin), the effect of a selected anti-ADM antibody on ADM bioactivity was detected.

[0131] The anti-ADM antibody was diluted to 1600 μg / mL with Stimulation Buffer 1 (Cisbio, 64SB1FDD), and the working concentration was set to 400 μg / mL. Then, it was serially diluted 3-fold with Stimulation Buffer 1 (8 μL + 16 μL of Stimulation Buffer 1). Among them, the antagonist human ADM (22 - 52) (Alfa Aesar, product number 159899-65-7) was diluted to 12000 μg / mL, and the working concentration was set to 3000 μg / mL. Then, 2.5 μL of the anti-ADM antibody was added to the corresponding wells of the experimental plate. Human ADM JMB2004 YY-52 protein (GLBiochem, product number 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 set to 0.15 μg / mL. Then, the experimental plate was incubated at room temperature for 60 min. CHO-K1 cells expressing the human recombinant adrenomedullin receptor (hereinafter abbreviated as CHO-K1 / CRLR / RAMP3, among which the gene accession number of CRLR is U17473 and the gene accession number of RAMP3 is AJ001016) were digested and separated with TrypLE Express (gibco, product number 12604-021), collected by centrifugation and resuspended in Stimulation Buffer 1, and the cell density was adjusted to 4×10 6 / mL, and 5 μL of CHO-K1 / CRLR / RAMP3 cells (2×10 4(Cells / well) were added. Subsequently, the experimental plate was incubated in a 37°C cell incubator for 90 min, and then the cAMP content was detected using the HTRF kit (Cisbio, 62AM4PEB). That is, after adding 5 μL of the cAMP-d2 reagent working solution to the experimental well, 5 μL of the cAMP Eu-Cryptate antibody working solution was added, incubated at room temperature for 1 h, and then the HTRF value was detected using a microplate reader.

[0132] The experimental results are shown in Figure 1, and it was found from Figure 1 that antibody 1F12 has no blocking activity.

[0133] Example 7, Design and Construction of an Affinity Maturation Library of the Fully Human Antibody 1F12 Affinity maturation was performed on the fully human antibody 1F12 obtained by screening to improve its affinity for human ADM. At the same time, since the heavy-chain antigen-binding determinant 2 (CDR_H2) of 1F12 contains an NG post-translational modification (PTM) site, it was site-specifically mutated to QG to remove the deamidation isomerization effect, and the mutated antibody was named 1F12 PTMΔ (the variable-region amino acid sequences are shown in SEQ ID NOs: 29 and 35). Using the mutant 1F12 PTMΔ as the parent, it was encoded according to the Chothia rules, the CDR regions were defined according to Chothia, and 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) to amplify the HCDR-3 and LCDR-3 mutant library gene fragments. After recovering the amplified VH and VL gene fragments, they were electrotransformed into the budding yeast strain EBY100 (purchased from ATCC) together with the yeast display plasmid, and the VH and VL genes were inserted into the yeast display plasmid by homologous recombination of budding yeast, and further realized the display of the Fab mutant library of the antibody on the yeast cell wall surface, and the library was named JYYDL196-197. After electrotransformation, the library JYYDL196-197 was cultured overnight at 30 °C in 250 mL of SD-Trp-Leu liquid medium (Clontech, product number 630316), and the bacterial amounts of 1.0×10 9 were taken and resuspended in 200 mL of YPGP induction medium (2% galactose, 2% peptone, 1% yeast extract, 0.54% Na2HPO4, 0.86% NaH2PO4·H2O), cultured at 20 °C for 24 h, 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 parental control.

[0134] Example 8, Screening and Monoclonal Identification of the Affinity Maturation Library of the Fully Human Antibody 1F12 For the bacterial solution after induction of the JYYDL196-197 library, the OD 600 of the bacterial solution was measured and calculated to be 1.0×10 7 cells per OD, and 1.0×10 9Take the cells and perform the first round of enrichment using a magnetic bead sorting system. Wash once with 50 mL of 1×PBSA (1×PBS + 1% BSA), discard the supernatant by centrifugation, and incubate at room temperature for 30 min with 5 mL of 1×PBSA containing 100 nM biotin-labeled human ADM (abbreviated as hADM-Biotin, product number NT-H-2, synthesized by GeneScript). After washing, add anti-biotin magnetic beads (Miltenyi, product number 130-090-485), mix uniformly, incubate for 10 min, and collect the positive cells through a magnetic column (Quadro MACS Starting Kit). After culturing and inducing the positive cells again, 3.0×10 7 Take the cells and perform the second round of fluorescence-activated cell sorting (FACS). Centrifuge with 1 mL of 1×PBSA and discard the supernatant. Incubate on ice for 30 min with 1 mL of 1×PBSA containing 10 nM hADM-Biotin and mouse anti-V5 antibody (Invitrogen, product number 2156578, diluted 1:1000). Centrifuge and discard the supernatant, add 1 mL of 1×PBSA and wash once. Add 500 μL of 1×PBSA containing fluorescent antibodies (eBioscience for SA-PE, product number 12-4317-8, diluted 1:200; Invitrogen for donkey anti-mouse-647, product number A21235, diluted 1:400), incubate in the dark on ice for 20 min. After washing, add 2 mL of 1×PBSA to resuspend the cells, and collect the cell population with strong 647 fluorescence signal and PE fluorescence signal using a FACS device. After the second round of FACS, after culturing and inducing the cells again, 3.0×10 7 Take the cells, incubate with 3 nM hADM-Biotin, and then perform the third round of FACS. After sorting, take some of the cells and spread them on an SD-Trp-Leu solid medium (Clontech, product number 630317) plate, and statically culture at 30 °C for 3 days.

[0135] For the screening products in the third round of JYYDL196 - 197, 92 monoclonal antibodies were selected for sequencing analysis respectively. Finally, yeast monoclonal colonies with unique sequences were obtained and flow cytometry staining analysis was performed. 1×10 6 cells were taken for staining evaluation. Based on the staining results of each clone and in accordance with the similarity of each clone sequence, finally, the fully human antibody Ab2004.Am01 (the variable region amino acid sequences are shown in SEQ ID NO: 30 and 35) was selected and expressed.

[0136] Example 9, Design and Construction of the Affinity Maturation Library of Ab2004.Am01 Subsequent affinity maturation of Ab2004.Am01 was carried out to improve its affinity with 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 to construct a mutant library, and the library numbers were JYYDL208 - 212. After culturing and inducing the library JYYDL208 - 212, it was prepared for use. At the same time, the Fab sequence of Ab2004.Am01 was displayed on the yeast surface and used as the parental control for this round of affinity maturation.

[0137] Example 10, Screening of the Affinity Maturation Library of Ab2004.Am01 For the bacterial solution after induction of the JYYDL208 - 212 library, 1.0×10 9 cells were taken and enriched in the first round using a magnetic bead sorting system. The positive cells after magnetic bead screening were cultured and induced again, and 3.0×10 7 cells were taken for the second round of flow sorting. A cell population with strong 647 fluorescence signal and PE fluorescence signal was collected by a flow sorting device. After culturing and inducing the cell population, the third round of flow sorting was performed. For the screening products in the second round of JYYDL208 - 209, 3.0×10 7Take cells and perform the third round of fluorescence-activated cell sorting (FACS) under the condition of 10 nM mADM-Biotin, with 3.0×10 7 Take cells and perform the third round of FACS under the condition of 100 nM mADM-Biotin. After the second and third rounds of screening, spread the cells on SD-Trp-Leu solid medium plates and statically culture them at 30 °C for 3 days.

[0138] Example 11: Construction and Screening of Light- and Heavy-Chain Mutant Combinatorial Libraries For the second-round screening products and third-round screening products of JYYDL208-209 and JYYDL211, several monoclonal antibodies were selected respectively to construct a light- and heavy-chain mutant combinatorial library JYYDL227.

[0139] For JYYDL227, take 2.0×10 7 Take cells and perform the first round of FACS using 3 nM hADM-Biotin and 1.2 nM mADM-Biotin respectively. After screening, take the cells and spread them on SD-Trp-Leu solid medium, and statically culture them at 30 °C for 3 days.

[0140] For the first-round screening products of JYYDL227, 46 monoclonal antibodies were selected respectively for sequencing, and finally yeast monoclonal colonies with unique sequences were obtained for EC 50 Identification was performed by flow cytometry staining. Take 1×10 5 cells each for staining evaluation. 1. Evaluate the binding level of each clone to human ADM at different antigen concentrations, calculate the EC 50 value of each clone and human ADM. The smaller the value, the higher the affinity. 2. Evaluate the binding level of each clone to mouse ADM at different antigen concentrations, and similarly, the strength of the affinity between each clone and mouse ADM can be obtained.

[0141] Based on the staining results of each clone (shown in Table 6), in accordance with the similarity of each clone sequence, finally, monoclonal antibodies of expressed antibody Ab2004.Am31 (the variable region amino acid sequences are shown in SEQ ID NO: 31 and 36), Ab2004.Am32 (the variable region amino acid sequences are shown in SEQ ID NO: 31 and 37), Ab2004.Am33 (the variable region amino acid sequences are shown in SEQ ID NO: 32 and 36), Ab2004.Am34 (the variable region amino acid sequences are shown in SEQ ID NO: 33 and 38), Ab2004.Am35 (the variable region amino acid sequences are shown in SEQ ID NO: 31 and 38), Ab2004.Am36 (the variable region amino acid sequences are shown in SEQ ID NO: 31 and 39), Ab2004.Am37 (the variable region amino acid sequences are shown in SEQ ID NO: 34 and 38), Ab2004.Am38 (the variable region amino acid sequences are shown in SEQ ID NO: 34 and 40), Ab2004.Am39 (the variable region amino acid sequences are shown in SEQ ID NO: 33 and 41), and Ab2004.Am40 (the variable region amino acid sequences are shown in SEQ ID NO: 31 and 42) were selected. The amino acid sequences of the CDR regions with Chothia numbers 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.

[0142]

Table 6

[0143]

Table 7

[0144]

Table 8

[0145] Example 12, Expression of Candidate Antibodies Similar to Example 2.2, the VH and VK sequences of each clone were combined with the human Kappa chain constant region (CL, amino acid sequence shown in SEQ ID NO: 20) and the human IgG1 constant region (CH, amino acid sequence shown in SEQ ID NO: 21) with the antibody light chain and heavy chain, respectively, inserted into the expression vector pcDNA3.4 (Life Technologies), and transient expression and purification of CHO cells were performed by entrusting Nanjing GeneScript Biotechnology Co., Ltd. The finally obtained affinity maturation candidate antibodies are shown in Table 9.

[0146]

Table 9

[0147] Example 13, Measurement of Affinity of Candidate Antibodies Similar to Example 3, the affinities of the affinity maturation antibodies Ab2004.Am31 - Ab2004.Am40 and the parental antibody Ab2004.Am01 with human ADM and mouse ADM were continuously measured, and the results are shown in Table 10. As can be seen from Table 10, the affinities of Ab2004.Am31, Ab2004.Am34, and Ab2004.Am39 with human ADM were most highly improved compared to the parental antibody Ab2004.Am01, and the affinities with mouse ADM were equivalent to those of enibacumab as a positive control antibody.

[0148]

Table 10

[0149] Example 14, Evaluation of Physicochemical Properties of Candidate Antibodies The expression levels and affinities of the candidate antibodies Ab2004.Am31, Ab2004.Am34, and Ab2004.Am39 were relatively ideal, and the evaluation of physicochemical drug - making properties was continuously carried out, and the results were summarized as shown in Table 11. As can be seen from Table 11, Ab2004.Am31, Ab2004.Am34, and Ab2004.Am39 all met the drug - making criteria in terms of purity, thermal stability, hydrophilicity, charge isomers, etc. The method referred to Example 4.

[0150]

Table 11

[0151] Example 15, Pharmacodynamic Study of ADM Antibody in a C57BL / 6J Mouse Sepsis Model Induced by LPS Male C57BL / 6J mice at 10 - 11 weeks of age were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., with a total of 50 mice. The animals were randomly divided into the following 5 groups (n = 10) according to body weight. Group G1 was the model group, which was administered isotype control RSV-IgG1 (2 mg / kg, IV, Single). Group G2 was the positive control group, which was administered enibasumab (2 mg / kg, IV, Single). Groups G3, G4, and G5 were respectively administered the antibody 2004hz97 (2 mg / kg, IV, Single), 2004hz99 (2 mg / kg, IV, Single), and Ab2004.Am34 (2 mg / kg, IV, Single) of the present invention. 5 minutes before LPS induced sepsis, the corresponding antibody was administered by single IV injection for treatment. 5 minutes later, 20 mg / kg of LPS (E. coli 055:B5, Sigma) was administered by single IP injection to induce mouse sepsis. After modeling, the death status of the animals was observed twice a day for 7 consecutive days. Data were graphed using GraphPad Prism 8 software, and statistical analysis of the data was performed using the Log-rank (Mantel-Cox) test method.

[0152] After treatment with LPS for 7 days, the animal survival rates of each group were RSV-IgG1 (40%), enibasumab (50%, P = 0.6713 vs RSV-IgG1), 2004hz97 (90%, P < 0.05 vs RSV-IgG1), 2004hz99 (80%, P = 0.1001 vs RSV-IgG1), Am34 (80%, P < 0.05 vs RSV-IgG1), respectively. From the results, it was shown that after treatment with the antibody of the present invention, the animal survival rate was significantly improved and the symptoms of sepsis could be alleviated (see Table 12 and Figure 2).

[0153]

Table 12

[0154] The above are only preferred embodiments of the present invention, and the present invention should not be limited to the content disclosed in this embodiment and the drawings. All equivalent or modified implementations made without departing from the spirit disclosed in the present invention are included in the claims of the present invention.

[0155] Sequence Listing

[0156]

Table 13

Claims

1. An anti-adrenomedullin (anti-ADM) monoclonal antibody or a fragment thereof, wherein the monoclonal antibody or fragment specifically binds to the amino acid sequence of amino acids 1 to 21 at the N-terminus of human adrenomedullin (ADM), the amino acid sequence of amino acids 1 to 21 at the N-terminus of human ADM being set forth in SEQ ID NO: 2, and the KD value of the monoclonal antibody or fragment for ADM is 10 -10 exhibiting an affinity of less than M Anti-adrenomedullin (anti-ADM) monoclonal antibody or a fragment thereof.

2. The fragments include Fab, Fab', F(ab)2, Fv fragment, F(ab')2, scFv, di-scFv, VHH and / or dAb. The anti-adrenomedullin monoclonal antibody or a fragment thereof according to claim 1.

3. (a) The heavy chain variable region of the monoclonal antibody comprises: (i) GYTFTHY (set forth in SEQ ID NO: 55); (ii) SAYQGN (set forth in SEQ ID NO: 49); (iii) comprising the CDR sequence EGRWGGSFNI (set forth in SEQ ID NO: 50); and (b) the light chain variable region of the monoclonal antibody comprises: (i) RASQGISSYLA (set forth in SEQ ID NO: 46); (ii) DVSILDA (set forth in SEQ ID NO: 56); (iii) comprising the CDR sequence QQYDNLPLT (set forth in SEQ ID NO: 48); or (a) the heavy chain variable region of the monoclonal antibody comprises: (i) GYTFTSY (set forth in SEQ ID NO: 43), (ii) SAYQGN (set forth in SEQ ID NO: 49); (iii) comprising the CDR sequence EGRWGGSFNI (set forth in SEQ ID NO: 50); and (b) the light chain variable region of the monoclonal antibody comprises: (i) RASQGISSYLA (set forth in SEQ ID NO: 46); (ii) DASNLET (set forth in SEQ ID NO: 47); (iii) comprising the CDR sequence QQYDNLPLT (set forth in SEQ ID NO: 48); or (a) the heavy chain variable region of the monoclonal antibody comprises: (i) GYTFTQY (set forth in SEQ ID NO: 51); (ii) SAYQGN (set forth in SEQ ID NO: 49); (iii) comprising the CDR sequence EGRWGGSFNI (set forth in SEQ ID NO: 50); and (b) the light chain variable region of the monoclonal antibody comprises: (i) RASEGISEYLA (set forth in SEQ ID NO: 52); (ii) DASNLET (set forth in SEQ ID NO: 47); (iii) comprising the CDR sequence QQYDNLPLT (set forth in SEQ ID NO: 48); or (a) the heavy chain variable region of the monoclonal antibody comprises: (i) GYTFTQY (set forth in SEQ ID NO: 51); (ii) SAYQGN (set forth in SEQ ID NO: 49); (iii) comprising the CDR sequence EGRWGGSFNI (set forth in SEQ ID NO: 50); and (b) the light chain variable region of the monoclonal antibody comprises: (i) RAAEGIGSYLA (set forth in SEQ ID NO: 53); (ii) DASNLET (set forth in SEQ ID NO: 47); (iii) comprising the CDR sequence QQYDNLPLT (set forth in SEQ ID NO: 48); or (a) the heavy chain variable region of the monoclonal antibody comprises: (i) GYTFTSY (set forth in SEQ ID NO: 43), (ii) SPYSGN (set forth in SEQ ID NO: 54); (iii) comprising the CDR sequence EGRWGGSFNI (set forth in SEQ ID NO: 50); and (b) the light chain variable region of the monoclonal antibody comprises: (i) RASEGISEYLA (set forth in SEQ ID NO: 52); (ii) DASNLET (set forth in SEQ ID NO: 47); (iii) comprising the CDR sequence QQYDNLPLT (set forth in SEQ ID NO: 48); or (a) the heavy chain variable region of the monoclonal antibody comprises: (i) GYTFTQY (set forth in SEQ ID NO: 51); (ii) SAYQGN (set forth in SEQ ID NO: 49); (iii) comprising the CDR sequence EGRWGGSFNI (set forth in SEQ ID NO: 50); and (b) the light chain variable region of the monoclonal antibody comprises: (i) RASQGISSYLA (set forth in SEQ ID NO: 46); (ii) DVSILDA (set forth in SEQ ID NO: 56); (iii) comprising the CDR sequence QQYDNLPLT (set forth in SEQ ID NO: 48); or (a) the heavy chain variable region of the monoclonal antibody comprises: (i) GYTFTQY (set forth in SEQ ID NO: 51); (ii) SAYQGN (set forth in SEQ ID NO: 49); (iii) comprising the CDR sequence EGRWGGSFNI (set forth in SEQ ID NO: 50); and (b) the light chain variable region of the monoclonal antibody comprises: (i) RASQGISSYLA (set forth in SEQ ID NO: 46); (ii) DASNVDT (set forth in SEQ ID NO: 57); (iii) comprising the CDR sequence QQYDNLPLT (set forth in SEQ ID NO: 48); or (a) the heavy chain variable region of the monoclonal antibody comprises: (i) GYTFTSY (set forth in SEQ ID NO: 43), (ii) SPYTGK (set forth in SEQ ID NO: 58); (iii) comprising the CDR sequence EGRWGGSFNI (set forth in SEQ ID NO: 50); and (b) the light chain variable region of the monoclonal antibody comprises: (i) RASQGISSYLA (set forth in SEQ ID NO: 46); (ii) DVSILDA (set forth in SEQ ID NO: 56); (iii) comprising the CDR sequence QQYDNLPLT (set forth in SEQ ID NO: 48); or (a) the heavy chain variable region of the monoclonal antibody comprises: (i) GYTFTSY (set forth in SEQ ID NO: 43), (ii) SPYTGK (set forth in SEQ ID NO: 58); (iii) comprising the CDR sequence EGRWGGSFNI (set forth in SEQ ID NO: 50); and (b) the light chain variable region of the monoclonal antibody comprises: (i) RASQGISSYLA (set forth in SEQ ID NO: 46); (ii) DTSDLDT (set forth in SEQ ID NO: 59); (iii) comprising the CDR sequence QQYDNLPLT (set forth in SEQ ID NO: 48); or (a) the heavy chain variable region of the monoclonal antibody comprises: (i) GYTFTHY (set forth in SEQ ID NO: 55); (ii) SAYQGN (set forth in SEQ ID NO: 49); (iii) comprising the CDR sequence EGRWGGSFNI (set forth in SEQ ID NO: 50); and (b) the light chain variable region of the monoclonal antibody comprises: (i) RASQGISSYLA (set forth in SEQ ID NO: 46); (ii) DASNLET (set forth in SEQ ID NO: 47); (iii) comprising the CDR sequence QQYDDLDLT (set forth in SEQ ID NO: 60); or (a) the heavy chain variable region of the monoclonal antibody comprises: (i) GYTFTQY (set forth in SEQ ID NO: 51); (ii) SAYQGN (set forth in SEQ ID NO: 49); (iii) comprising the CDR sequence EGRWGGSFNI (set forth in SEQ ID NO: 50); and (b) the light chain variable region of the monoclonal antibody comprises: (i) RASQGISSYLA (set forth in SEQ ID NO: 46); (ii) DASNLET (set forth in SEQ ID NO: 47); (iii) QQYDDLPLS (shown in SEQ ID NO: 61), The anti-adrenomedullin monoclonal antibody or a fragment thereof according to claim 1.

4. The monoclonal antibody comprises a heavy chain variable region sequence shown in any one of SEQ ID NOs: 30 to 34, and the monoclonal antibody comprises a light chain variable region sequence shown in any one of SEQ ID NOs: 35 to 42. The anti-adrenomedullin monoclonal antibody or a fragment thereof according to claim 3.

5. The monoclonal antibody comprises a heavy chain variable region sequence shown in SEQ ID NO: 33 and a light chain variable region sequence shown in SEQ ID NO: 38; the monoclonal antibody comprises a heavy chain variable region sequence shown in SEQ ID NO: 30 and a light chain variable region sequence shown in SEQ ID NO: 35; the monoclonal antibody comprises a heavy chain variable region sequence shown in SEQ ID NO: 31 and a light chain variable region sequence shown in SEQ ID NO: 36; the monoclonal antibody comprises a heavy chain variable region sequence shown in SEQ ID NO: 31 and a light chain variable region sequence shown in SEQ ID NO: 37; the monoclonal antibody comprises a heavy chain variable region sequence shown in SEQ ID NO: 32 and a light chain variable region sequence shown in SEQ ID NO: 36; the monoclonal antibody comprises a heavy chain variable region sequence shown in SEQ ID NO: 31 and a light chain variable region sequence shown in SEQ ID NO: 38 The anti-adrenomedullin monoclonal antibody or a fragment thereof according to claim 4, wherein the monoclonal antibody comprises a heavy chain variable region sequence shown in SEQ ID NO: 31 and a light chain variable region sequence shown in SEQ ID NO: 39; the monoclonal antibody comprises a heavy chain variable region sequence shown in SEQ ID NO: 34 and a light chain variable region sequence shown in SEQ ID NO: 38; the monoclonal antibody comprises a heavy chain variable region sequence shown in SEQ ID NO: 34 and a light chain variable region sequence shown in SEQ ID NO: 40; the monoclonal antibody comprises a heavy chain variable region sequence shown in SEQ ID NO: 33 and a light chain variable region sequence shown in SEQ ID NO: 41; or the monoclonal antibody comprises a heavy chain variable region sequence shown in SEQ ID NO: 31 and a light chain variable region sequence shown in SEQ ID NO:

42.

6. (a) the heavy chain variable region of the monoclonal antibody comprises: (i) GYAFTTF (set forth in SEQ ID NO: 11); (ii) NTYSRV (set forth in SEQ ID NO: 12); (iii) GYGGEGGLGF (shown in SEQ ID NO: 13), and (b) the light chain variable region of the monoclonal antibody comprises: (i) RSSQSIIDSDGNTYLE (set forth in SEQ ID NO: 14), (ii) KVSNRFS (set forth in SEQ ID NO: 15); (iii) FQGSHFPYT (shown in SEQ ID NO: 16), comprising the CDR sequence The anti-adrenomedullin monoclonal antibody or a fragment thereof according to claim 1.

7. The monoclonal antibody comprises a heavy chain variable region sequence shown in any one of SEQ ID NOs: 9 and 17, and the monoclonal antibody comprises a light chain variable region sequence shown in any one of SEQ ID NOs: 10, 18, and 19, wherein the monoclonal antibody comprises a heavy chain variable region sequence shown in SEQ ID NO: 9 and a light chain variable region sequence shown in SEQ ID NO: 10, the monoclonal antibody comprises a heavy chain variable region sequence shown in SEQ ID NO: 17 and a light chain variable region sequence shown in SEQ ID NO: 18, and the monoclonal antibody comprises a heavy chain variable region sequence shown in SEQ ID NO: 17 and a light chain variable region sequence shown in SEQ ID NO: 19, The anti-adrenomedullin monoclonal antibody or a fragment thereof according to claim 6.

8. The monoclonal antibody comprises a heavy chain sequence shown in SEQ ID NO: 23 and a light chain sequence shown in SEQ ID NO:

22. the monoclonal antibody comprises a heavy chain sequence shown in SEQ ID NO: 25 and a light chain sequence shown in SEQ ID NO: 24; or The monoclonal antibody comprises a heavy chain sequence shown in SEQ ID NO: 27 and a light chain sequence shown in SEQ ID NO:

26. The anti-adrenomedullin monoclonal antibody or a fragment thereof according to claim 6.

9. (a) the heavy chain variable region of the monoclonal antibody comprises: (i) GYTFTX 1 Y, of which X 1 is selected from S, Q or H; (ii) SX 2 YX 3 GX 4 , among which, X 2 is selected from A or P, and 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, among them, 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 monoclonal antibody comprises: (i) RAX 7 X 8 GIX 9 X 10 YLA, among which X 7 is selected from S or A, and X 8 is selected from Q or E, and X 9 is selected from S or G, and X 10 is selected from S or E, (ii) Digital Transformation 11 SX 12 X 13 X 14 X 15 , among which, X 11 is selected from A, V or T, and X 12 is selected from N, I or D, and X 13 is selected from L or V, and X 14 is selected from E or D, and X 15 is selected from T or A, (iii) QQYDX 16 LX 17 LX 18 , among which, X 16 is selected from N or D, and X 17 is selected from P or D, and X 18 is selected from T or S, The anti-adrenomedullin monoclonal antibody or a fragment thereof according to claim 1.

10. (a) the heavy chain variable region of the monoclonal antibody comprises HCDR1, HCDR2, and HCDR3 sequences, wherein the HCDR1 comprises the amino acid sequence set forth in any one of SEQ ID NOs: 43, 51, and 55, the HCDR2 comprises the amino acid sequence set forth in any one of SEQ ID NOs: 44, 49, 54, and 58, and the HCDR3 comprises the amino acid sequence set forth in any one of SEQ ID NOs: 45 and 50; and (b) the light chain variable region of the monoclonal antibody comprises LCDR1, LCDR2, and LCDR3 sequences, wherein the LCDR1 comprises the amino acid sequence set forth in any one of SEQ ID NOs: 46, 52, and 53, the LCDR2 comprises the amino acid sequence set forth in any one of SEQ ID NOs: 47, 56, 57, and 59, and the LCDR3 comprises the amino acid sequence set forth in any one of SEQ ID NOs: 48, 60, and 61; The anti-adrenomedullin monoclonal antibody or a fragment thereof according to claim 9.

11. (a) the heavy chain variable region of the monoclonal antibody comprises: (i) GYTFTSY (set forth in SEQ ID NO: 43), (ii) SAYNGN (set forth in SEQ ID NO: 44); (iii) EGRSGGSFDI (shown in SEQ ID NO: 45), and (b) the light chain variable region of the monoclonal antibody comprises: (i) RASQGISSYLA (set forth in SEQ ID NO: 46); (ii) DASNLET (set forth in SEQ ID NO: 47); (iii) QQYDNLPLT (shown in SEQ ID NO: 48), which contains the CDR sequence The anti-adrenomedullin monoclonal antibody or a fragment thereof according to claim 10.

12. (a) the heavy chain variable region of the monoclonal antibody comprises: (i) GYTFTSY (set forth in SEQ ID NO: 43), (ii) SAYQGN (set forth in SEQ ID NO: 49); (iii) EGRSGGSFDI (shown in SEQ ID NO: 45), and (b) the light chain variable region of the monoclonal antibody comprises: (i) RASQGISSYLA (set forth in SEQ ID NO: 46); (ii) DASNLET (set forth in SEQ ID NO: 47); (iii) QQYDNLPLT (shown in SEQ ID NO: 48), which contains the CDR sequence The anti-adrenomedullin monoclonal antibody or a fragment thereof according to claim 10.

13. The monoclonal antibody comprises a heavy chain variable region sequence shown in SEQ ID NO: 28 or 29, and the monoclonal antibody comprises a light chain variable region sequence shown in SEQ ID NO:

35. The anti-adrenomedullin monoclonal antibody or a fragment thereof according to claim 9.

14. The monoclonal antibody comprises a heavy chain variable region sequence set forth in SEQ ID NO: 28 and a light chain variable region sequence set forth in SEQ ID NO: 35; or The monoclonal antibody comprises a heavy chain variable region sequence shown in SEQ ID NO: 29 and a light chain variable region sequence shown in SEQ ID NO:

35. The anti-adrenomedullin monoclonal antibody or a fragment thereof according to claim 13.

15. A nucleotide sequence encoding the anti-adrenomedullin monoclonal antibody or a fragment thereof according to any one of claims 1 to 14.

16. An expression vector comprising the nucleotide sequence of claim 15.

17. The expression vector described in Claim 16, wherein the expression vector is pcDNA3.

4.

18. 16. A host cell comprising the nucleotide sequence of claim 15 or an expression vector comprising said nucleotide sequence.

19. The host cell described in claim 18, wherein the host cell is a CHO cell.

20. A pharmaceutical composition, detection reagent or kit comprising the anti-adrenomedullin monoclonal antibody or a fragment thereof according to any one of claims 1 to 14.

21. Use of the anti-adrenomedullin monoclonal antibody or a fragment thereof described in any one of claims 1 to 14 in the manufacture of a drug for maintaining the integrity of endothelial cells and enhancing barrier function.

22. Use of the anti-adrenomedullin monoclonal antibody or a fragment thereof described in any one of claims 1 to 14 in the manufacture of a drug for controlling vasodilation and lowering blood pressure.

23. Use of the anti-adrenomedullin monoclonal antibody or a fragment thereof described in any one of claims 1 to 14 in the manufacture of a drug for treating or preventing septic shock or traumatic injury, particularly the end stage of sepsis.

24. Use of an anti-adrenomedullin monoclonal antibody or a fragment thereof described in any one of claims 1 to 14 in the manufacture of a drug for reducing the risk of death from a chronic or acute disease or acute illness in a patient, wherein the chronic or acute disease or acute illness is selected from severe infectious diseases such as meningitis, systemic inflammatory response syndrome (SIRS), sepsis, other diseases such as diabetes, cancer, acute and chronic vascular diseases such as heart failure, myocardial infarction, stroke, atherosclerosis, edema, shock such as septic shock and organ dysfunction such as renal dysfunction, liver dysfunction, burns, surgery, trauma, and poisoning.

25. Use of an anti-adrenomedullin monoclonal antibody or a fragment thereof described in any one of claims 1 to 14 in the manufacture of a drug for stabilizing circulation, particularly systemic circulation, in a patient, wherein the patient is suffering from a chronic or acute disease or acute illness, and the chronic or acute disease or acute illness is selected from severe infections, such as meningitis, systemic inflammatory response syndrome (SIRS), sepsis, other diseases, such as diabetes, cancer, acute and chronic vascular diseases, such as heart failure, myocardial infarction, stroke, atherosclerosis, edema, shock, such as septic shock, and organ dysfunction, such as renal dysfunction, liver dysfunction, burns, surgery, trauma, and poisoning.

26. The anti-adrenomedullin monoclonal antibody or a fragment thereof is used in combination with another drug.

26. The use according to claim 25.

27. The drug is selected from the group consisting of a hypertensive drug, an intravenous fluid, a TNF-α antibody, and an antibiotic.

27. The use according to claim 26.

28. A pharmaceutical composition comprising an anti-adrenomedullin monoclonal antibody or a fragment thereof described in any one of claims 1 to 14, for use in a method for maintaining the integrity of endothelial cells and enhancing barrier function.

29. A pharmaceutical composition comprising an anti-adrenomedullin monoclonal antibody or a fragment thereof described in any one of claims 1 to 14, for use in a method for controlling vasodilation and lowering blood pressure.

30. A pharmaceutical composition comprising an anti-adrenomedullin monoclonal antibody or a fragment thereof described in any one of claims 1 to 14, for use in the treatment or prevention of septic shock or traumatic injury, particularly end-stage sepsis.

31. A pharmaceutical composition comprising an anti-adrenomedullin monoclonal antibody or a fragment thereof described in any one of claims 1 to 14, for use in a method for reducing the risk of death from a chronic or acute disease or acute illness in a patient, wherein the chronic or acute disease or acute illness is selected from severe infectious diseases, such as meningitis, systemic inflammatory response syndrome (SIRS), sepsis, other diseases, such as diabetes, cancer, acute and chronic vascular diseases, such as heart failure, myocardial infarction, stroke, atherosclerosis, edema, shock, such as septic shock, and organ dysfunction, such as renal dysfunction, hepatic dysfunction, burns, surgery, trauma, and poisoning.

32. A pharmaceutical composition comprising an anti-adrenomedullin monoclonal antibody or a fragment thereof described in any one of claims 1 to 14, for use as a drug for stabilizing circulation, particularly systemic circulation, in a patient, wherein the patient is suffering from a chronic or acute disease or acute illness, and the chronic or acute disease or acute illness is selected from severe infections, such as meningitis, systemic inflammatory response syndrome (SIRS), sepsis, other diseases, such as diabetes, cancer, acute and chronic vascular diseases, such as heart failure, myocardial infarction, stroke, atherosclerosis, edema, shock, such as septic shock, and organ dysfunction, such as renal dysfunction, hepatic dysfunction, burns, surgery, trauma, and poisoning.

33. The anti-adrenomedullin monoclonal antibody or a fragment thereof is used in combination with another drug.

33. The pharmaceutical composition of claim 32.

34. The drug is selected from the group consisting of a hypertensive drug, an intravenous fluid, a TNF-α antibody, and an antibiotic.

34. The pharmaceutical composition of claim 33.

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