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Definitions

• the invention relates to an anti-adrenomedullin non-neutralizing antibody, its preparation method and application, and belongs to the fields of bioengineering and biotherapy.
• Adrenomedullin is a 6kDa polypeptide composed of 52 amino acids. It was first discovered in pheochromocytoma in 1993 and was later found to be widely secreted and expressed in vascular epithelial cells and vascular smooth muscle cells. The adrenomedullin gene is located on chromosome 11. After translation, it is gradually enzymatically cleaved by the prohormone to form calcitonin gene-related peptide (CLR). The heterodimer composed of CLR and RAMP2 or RAMP3 is the receptor of adrenomedullin. Based on its receptor-mediated endocytosis and the action of protease, its half-life is only 22 minutes.
• Adrenomedullin maintains the integrity of endothelial cells and enhances the barrier. This is achieved in the following two ways: (1) enhancing the activity of GTPase Rac1 and increasing the production of cortical actin and stress fibers; (2) reducing myosin light chain kinase-induced actinogenesis by inhibiting the RhoA/ROCK pathway Globulin shrinks. Adrenomedullin also has vasodilation and blood pressure lowering effects.
• adrenomedullin In healthy people, the concentration of adrenomedullin is extremely low, around 10pg/mL. It can flexibly shuttle inside and outside blood vessels, flexibly regulate vasodilation and maintain endothelial cell barrier function. In patients with septic shock, adrenomedullin concentration is 5-6 times higher than normal, which is directly related to the severity of the disease and prognosis. In patients with septic shock, inflammation-based effects weaken the barrier effect of blood vessels, and vasodilation leads to further reductions in blood pressure.
• Non-neutralizing antibodies to adrenomedullin restrict adrenomedullin to blood vessels, correct intravascular barrier function, weaken extracellular vasodilation, and increase the half-life of adrenomedullin in plasma to achieve therapeutic and Attenuates the shock symptoms of sepsis. Therefore, there is an urgent need to develop non-neutralizing antibodies with higher affinity for adrenomedullin.
• 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 N of human adrenomedullin (ADM).
• ADM anti-adrenomedullin
• the N-terminal amino acid sequence 1-21 of the human ADM is as shown in SEQ ID NO: 2, and the monoclonal antibody or fragment exhibits a KD value for ADM of less than 10 - 10 M affinity.
• an “antibody” or “antibody fragment” of the invention is capable of binding ADM, and is therefore directed against ADM, and may therefore be referred to as an "anti-ADM antibody” or “anti-ADM antibody fragment”.
• the "antibody” or “antibody fragment” of the present invention is a non-neutralizing antibody against adrenomedullin or a fragment thereof.
• antibodies having "non-neutralizing anti-ADM activity” or antibody fragments that, for example, block less than 80% of the ADM biological activity are collectively referred to as “non-neutralizing” anti-ADM antibodies or antibody fragments.
• Antibody fragments are defined as:
• One or more molecules that bind ADM upon addition to a culture of a eukaryotic cell line, reduce the amount of cAMP produced by the cell line by the action of a synthetic human ADM peptide added in parallel, said cell line expressing A functional human recombinant ADM receptor composed of CRLR (calcitonin receptor-like receptor) and RAMP3 (receptor activity modifying protein 3), wherein the added synthetic human ADM is added in such an amount that in the absence of In the case of a non-neutralizing antibody to be analyzed that results in a half-maximal stimulation of cAMP synthesis, binding of said molecule to ADM results in a decrease in cAMP of no more than 80%, even when said non-neutralizing molecule to be analyzed is capable of binding to ADM This is also true when an amount 10 times or more of the amount required to obtain the maximum reduction in cAMP obtained using the non-neutralizing antibody to be analyzed is added.
• Bioactivity is defined as the effect of a substance that, following its interaction, appears as a living organism or tissue or organ or functional unit in vivo or in vitro (e.g., in an assay). In terms of ADM biological activity, this could be the role of ADM in the human recombinant adrenomedullin receptor cAMP functional assay. Therefore, according to the present invention, biological activity is defined by adrenomedullin receptor cAMP functional assay.
• Dose response curves were performed with ADM in the human recombinant adrenomedullin receptor cAMP functional assay.
• the ADM concentration for half-maximal cAMP stimulation can be calculated.
• ADM concentration dose-response curves (up to a final concentration of 100 ⁇ g/ml) were performed at constant half-maximal cAMP stimulation with ADM-stabilizing antibodies or adrenomedullin-stabilizing antibody fragments, respectively.
• a 50% maximum inhibition in the ADM bioassay means that the anti-ADM antibody or the anti-adrenomedullin antibody fragment, respectively, blocks 50% of the biological activity of the baseline value.
• a maximum inhibition of 80% in the ADM bioassay means that the anti-ADM antibody or the anti-adrenomedullin antibody fragment, respectively, blocks 80% of the ADM biological activity. The implication is to block no more than 80% of the biological activity of ADM. This means that approximately 20% of residual ADM biological activity remains.
• blocking the biological activity of ADM should be understood as only reducing the biological activity of ADM, preferably maximally reducing the ADM biological activity from 100% to 20% of the remaining ADM biological activity, preferably reducing the ADM biological activity.
• the activity is reduced from 100% to 50% of the remaining ADM biological activity, but in any case there is still ADM biological activity that can be determined as described above.
• an anti-adrenomedullin (ADM) antibody is an antibody that can specifically bind to ADM
• an anti-adrenomedullin antibody fragment is a fragment of an ADM antibody, wherein the fragment can specifically bind to ADM.
• Specific binding to ADM also allows binding of other antigens. This means that the specificity does not preclude the antibody from cross-reacting with polypeptides other than the polypeptide that elicited the antibody. This also applies to the specificity of the anti-ADM antibodies or fragments thereof of the invention.
• non-neutralizing anti-ADM antibodies or non-neutralizing anti-ADM antibody fragments of the invention provide significant therapeutic advantages over neutralizing anti-ADM antibodies or neutralizing anti-ADM antibody fragments.
• Antibodies of the present invention are proteins, including one or more polypeptides that are substantially encoded by immunoglobulin genes and can specifically bind to antigens. Recognized immunoglobulin genes include kappa, lambda, alpha (IgA), gamma (IgG1, IgG2, IgG3, IgG4), delta (IgD), epsilon (IgE) and mu (IgM) constant region genes as well as countless immunoglobulins Variable region genes.
• Full-length immunoglobulin light chain length is typically about 25 KDa or 214 amino acids. The length of a full-length immunoglobulin heavy chain is typically 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 NH 2 -terminus and a kappa or lambda constant region gene located at the COOH-terminus.
• the heavy chain is also encoded by a variable region gene (approximately 116 amino acids in length) and one of the other constant region genes.
• the basic structural unit of an antibody is usually a tetramer consisting of two pairs of identical immunoglobulin chains, each pair having a light chain and a heavy chain. In each pair, the light and heavy chain variable regions bind antigen, while the constant regions mediate effects or functions.
• Immunoglobulins also exist in a variety of other forms, including, for example, Fv, Fab and F(ab') 2 as well as bifunctional hybrid and single-chain antibodies.
• the immunoglobulin light or heavy chain variable region consists of a framework region interrupted by three hypervariable regions, also known as complementarity determining regions (CDR's). As noted above, CDRs are primarily responsible for binding to the epitope of the antigen.
• Immune complexes are antibodies such as monoclonal antibodies, chimeric antibodies, humanized antibodies, or human antibodies or functional antibody fragments that specifically bind to an antigen.
• CDRs of antibodies can be defined through a variety of methods, such as Kabat definition rules based on sequence variability, Chothia definition rules based on the position of the structural loop region, and concepts based on IMGT ontology (IMGT-ONTOLOGY).
• the amino acid sequences of VL and VH of the anti-ADM antibody are encoded according to Chothia coding rules, and the light chain CDR1-3 (LCDR 1-3) and heavy chain CDR1-3 (HCDR 1-3) of the anti-ADM antibody are 3) As defined by Chothia.
• Chimeric antibodies are antibodies whose light and heavy chain genes have been constructed through genetic engineering from immunoglobulin variable and constant region genes belonging to different species.
• variable segments from mouse monoclonal antibody genes can be linked to human constant segments such as kappa and gamma 1 or gamma 3.
• a therapeutic chimeric antibody is thus a hybrid protein consisting of a variable domain or antigen-binding domain from a mouse antibody and a constant or effector domain from a human antibody, although other mammalian species may be used or by molecular techniques to generate variable regions.
• a “humanized” immunoglobulin is an immunoglobulin that includes human framework regions and one or more CDRs of a non-human (such as mouse, rat, or synthetic) immunoglobulin.
• the non-human immunoglobulin that provides the CDRs is called the "donor” and the human immunoglobulin that provides the framework is called the "acceptor.”
• all CDRs in the humanized immunoglobulin are from the donor immunoglobulin. Constant regions need not be present, but if present, they must be substantially identical to human immunoglobulin constant regions, that is, at least about 85-90% identical, such as about 95% identical or more. Therefore, all portions of the humanized immunoglobulin, except perhaps the CDRs, are substantially identical to the corresponding portions of the native human immunoglobulin sequence.
• a "humanized antibody” is an antibody comprising a humanized light chain and a humanized heavy chain immunoglobulin. Humanized antibodies bind the same antigen as the donor antibody that provided the CDRs.
• the acceptor framework of a humanized immunoglobulin or antibody can have a limited number of substitutions of amino acids from the donor framework. Humanized or other monoclonal antibodies may have other conservative amino acid substitutions that have essentially no effect on antigen binding or other immunoglobulin function.
• Humanized immunoglobulins can be constructed through genetic engineering.
• Human antibodies are antibodies in which the light chain and heavy chain genes are of human origin. Human antibodies can be produced by immortalizing human B cells that secrete the antibody of interest. Immortality can be achieved, for example, by EBV infection or by This is accomplished by fusing human B cells with myeloma or hybridoma cells to produce triple hybridoma cells. Human antibodies can also be produced by phage display methods or selected from human combinatorial monoclonal antibody libraries. Human antibodies can also be produced using transgenic animals carrying human immunoglobulin genes
• anti-ADM antibodies may have forms known in the art. Examples are human antibodies, monoclonal antibodies, humanized antibodies, chimeric antibodies, CDR-grafted antibodies.
• the antibody of the invention is a recombinantly produced antibody such as an IgG, or an antibody fragment containing at least the F-variable domain of the heavy and/or light chain such as a chemically coupled antibody (fragment antigen binding) .
• the anti-adrenomedullin monoclonal antibody fragments of the invention include Fab, Fab', F(ab) 2 , Fv fragment, F(ab') 2 , scFv, di - scFv, VHH and/or dAb.
• the antibody of the invention can be produced as follows:
• the synthesized N-terminal 16 amino acid polypeptides of human ADM, the N-terminal 21 amino acid polypeptides of human ADM, or the N-terminal 19 amino acid polypeptides of mouse ADM (referred to as YY-19, SEQ ID NO: 3) and conjugates were used as Immunize mice with the immunogen. Blood was collected one week after the last immunization, and the serum anti-YY-21 titer was determined by ELISA. After the mice with high serum titers were pulse immunized with the immunogen, spleen cells were harvested for fusion. After spleen cell fusion and hybridoma clone screening, hybridoma clones were obtained. Upon affinity determination, the hybridoma clone exhibited an affinity for ADM with a KD value of less than 10 -10 M.
• KD KD
• KD KD
• KD KD
• kd dissociation rate constant
• kon association rate constant
• the heavy chain variable region of the hybridoma clone contained the following CDR sequence:
• the light chain variable region of the hybridoma clone contains the following CDR sequences:
• the hybridoma clone comprises the heavy chain variable region sequence shown in SEQ ID NO: 9, and the hybridoma clone comprises the light chain variable region sequence shown in SEQ ID NO: 10 sequence.
• Humanization of anti-ADM antibodies can be performed according to the following protocol:
• the human antibody germline genes with the highest homology were selected as the humanization design framework.
• the heavy chain variable region of the hybridoma clone was subjected to CDR transplantation and back mutation to obtain a humanized heavy chain variable region sequence.
• the light chain variable region of the hybridoma clone is subjected to CDR transplantation and back mutation to obtain a humanized light chain variable region sequence.
• the heavy chain variable region of the humanized antibody comprises the following CDR sequences:
• the light chain variable region of the humanized antibody contains the following CDR sequences:
• the humanized antibody comprises the heavy chain variable region sequence shown in SEQ ID NO: 17, and the humanized antibody comprises the heavy chain variable region sequence shown in any of SEQ ID NO: 18 and 19 The light chain variable region sequence.
• the humanized antibody comprises the heavy chain variable region sequence set forth in SEQ ID NO: 17, and the humanized antibody comprises the light chain variable region sequence set forth in SEQ ID NO: 18. Variable region sequence.
• the humanized antibody comprises the heavy chain variable region sequence shown in SEQ ID NO:17, and the humanized antibody comprises the light chain variable region sequence shown in SEQ ID NO:19. Variable region sequence.
• the monoclonal antibody further comprises an antibody light chain constant region, and the light chain constant region is a human kappa chain constant region.
• the human Kappa chain constant region includes the amino acid sequence shown in SEQ ID NO: 20.
• the monoclonal antibody further comprises an antibody heavy chain constant region, which is a human IgGl constant region.
• the human IgG1 constant region includes the amino acid sequence shown in SEQ ID NO: 21.
• the light chain variable region sequence of the monoclonal antibody of the present invention is combined with the human Kappa chain constant region to form an antibody light chain
• the heavy chain variable region sequence of the monoclonal antibody of the present invention is combined with the human IgG1 constant region to form an anti- Weight chain.
• the monoclonal antibody comprises the heavy chain sequence shown in any one of SEQ ID NO:23, 25, and 27, and the monoclonal antibody comprises SEQ ID NO:22, The light chain sequence shown in any one of 24 and 26.
• the monoclonal antibody comprises the heavy chain sequence shown in SEQ ID NO:23, and the monoclonal antibody comprises the light chain sequence shown in SEQ ID NO:22.
• the monoclonal antibody comprises the heavy chain sequence shown in SEQ ID NO:25, and the monoclonal antibody comprises the light chain sequence shown in SEQ ID NO:24.
• the monoclonal antibody comprises the heavy chain sequence set forth in SEQ ID NO:27, and the monoclonal antibody comprises the light chain sequence set forth in SEQ ID NO:26.
• the double plasmids are co-transfected into Expi293F or CHO-K1 cells for transient expression of the antibody.
• the antibody is purified through a Protein A affinity chromatography column. Upon affinity determination, the humanized anti-ADM antibody exhibited an affinity for ADM with a KD value of less than 10 -10 M.
• the present invention can also perform anti-ADM screening by selecting a fully human single-chain phage antibody library. Screening of monoclonal antibodies.
• the heavy chain variable region of the monoclonal antibody includes the following CDR sequence:
• the light chain variable region of the monoclonal antibody includes the following CDR sequence:
• “monoclonal antibody” generally refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies making up the population are identical except for possible naturally occurring mutations and/or translations that may be present in minimal amounts.
• post-modification such as isomerization, amidation
• the anti-adrenomedullin monoclonal antibody contains a post-translational modification (PTM) site for NG.
• PTM post-translational modification
• the post-translational modification (PTM) site of NG contained in the anti-adrenomedullin monoclonal antibody is site-directed mutated into QG to eliminate deamidation isomerization.
• the heavy chain variable region of the monoclonal antibody includes the following CDR sequence:
• the light chain variable region of the monoclonal antibody includes the following CDR sequence:
• the monoclonal antibody comprises the heavy chain variable region sequence shown in either SEQ ID NO: 28 and 29, and the monoclonal antibody comprises the light chain variable region sequence shown in SEQ ID NO: 35. Chain variable region sequence.
• the humanized antibody comprises the heavy chain variable region sequence shown in SEQ ID NO: 28, and the humanized antibody comprises the light chain variable region sequence shown in SEQ ID NO: 35. Variable region sequence.
• the humanized antibody comprises the heavy chain variable region sequence shown in SEQ ID NO:29, and the humanized antibody comprises the light chain variable region sequence shown in SEQ ID NO:35. Variable region sequence.
• the present invention In order to improve the affinity of fully human antibodies with human ADM, the present invention also designed and constructed an affinity mature library of fully human antibodies. Through the screening of fully human antibody affinity mature libraries and the identification of monoclones, the present invention obtained different fully human anti-ADM antibodies, all of which exhibit an affinity for ADM with a KD value of less than 10 -10 M.
• the heavy chain variable region of the monoclonal antibody includes the following CDR sequence:
• the light chain variable region of the monoclonal antibody includes the following CDR sequence:
• the heavy chain variable region of the monoclonal antibody includes the following CDR sequence:
• the light chain variable region of the monoclonal antibody includes the following CDR sequence:
• the heavy chain variable region of the monoclonal antibody includes the following CDR sequence:
• the light chain variable region of the monoclonal antibody includes the following CDR sequence:
• the heavy chain variable region of the monoclonal antibody includes the following CDR sequence:
• the light chain variable region of the monoclonal antibody includes the following CDR sequence:
• the heavy chain variable region of the monoclonal antibody includes the following CDR sequence:
• the light chain variable region of the monoclonal antibody includes the following CDR sequence:
• the heavy chain variable region of the monoclonal antibody includes the following CDR sequence:
• the light chain variable region of the monoclonal antibody includes the following CDR sequence:
• the heavy chain variable region of the monoclonal antibody includes the following CDR sequence:
• the light chain variable region of the monoclonal antibody includes the following CDR sequence:
• the heavy chain variable region of the monoclonal antibody includes the following CDR sequence:
• the light chain variable region of the monoclonal antibody includes the following CDR sequence:
• the heavy chain variable region of the monoclonal antibody includes the following CDR sequence:
• the light chain variable region of the monoclonal antibody includes the following CDR sequence:
• the heavy chain variable region of the monoclonal antibody includes the following CDR sequence:
• the light chain variable region of the monoclonal antibody includes the following CDR sequence:
• the heavy chain variable region of the monoclonal antibody includes the following CDR sequence:
• the light chain variable region of the monoclonal antibody includes the following CDR sequence:
• the anti-adrenomedullin monoclonal antibody or a fragment thereof is provided.
• the heavy chain variable region of the monoclonal antibody includes the following CDR sequence:
• the light chain variable region of the monoclonal antibody includes the following CDR sequence:
• the present invention also obtains the heavy chain variable region sequence and light chain variable region sequence of the antibody with improved affinity.
• the monoclonal antibody comprises the heavy chain shown in any one of SEQ ID NO: 30 to 34 Variable region sequence
• the monoclonal antibody includes the light chain variable region sequence shown in any one of SEQ ID NO: 35 to 42.
• the humanized antibody comprises the heavy chain variable region sequence shown in SEQ ID NO:30, and the humanized antibody comprises the light chain variable region sequence shown in SEQ ID NO:35. Variable region sequence.
• the humanized antibody comprises the heavy chain variable region sequence shown in SEQ ID NO: 31, and the humanized antibody comprises the light chain variable region sequence shown in SEQ ID NO: 36. Variable region sequence.
• the humanized antibody comprises the heavy chain variable region sequence set forth in SEQ ID NO: 31, and the humanized antibody comprises the light chain variable region sequence set forth in SEQ ID NO: 37. Variable region sequence.
• the humanized antibody comprises the heavy chain variable region sequence set forth in SEQ ID NO: 32, and the humanized antibody comprises the light chain variable region sequence set forth in SEQ ID NO: 36. Changing area order List.
• the humanized antibody comprises the heavy chain variable region sequence shown in SEQ ID NO:33, and the humanized antibody comprises the light chain variable region sequence shown in SEQ ID NO:38. Variable region sequence.
• the humanized antibody comprises the heavy chain variable region sequence shown in SEQ ID NO: 31, and the humanized antibody comprises the light chain variable region sequence shown in SEQ ID NO: 38. Variable region sequence.
• the humanized antibody comprises the heavy chain variable region sequence shown in SEQ ID NO: 31, and the humanized antibody comprises the light chain variable region sequence shown in SEQ ID NO: 39. Variable region sequence.
• the humanized antibody comprises the heavy chain variable region sequence shown in SEQ ID NO:34, and the humanized antibody comprises the light chain variable region sequence shown in SEQ ID NO:38. Variable region sequence.
• the humanized antibody comprises the heavy chain variable region sequence shown in SEQ ID NO: 34, and the humanized antibody comprises the light chain variable region sequence shown in SEQ ID NO: 40. Variable region sequence.
• the humanized antibody comprises the heavy chain variable region sequence shown in SEQ ID NO: 33, and the humanized antibody comprises the light chain variable region sequence shown in SEQ ID NO: 41 Variable region sequence.
• the humanized antibody comprises the heavy chain variable region sequence shown in SEQ ID NO: 31, and the humanized antibody comprises the light chain variable region sequence shown in SEQ ID NO: 42. Variable region sequence.
• the monoclonal antibody further comprises an antibody light chain constant region, and the light chain constant region is a human kappa chain constant region.
• the human Kappa chain constant region includes the amino acid sequence shown in SEQ ID NO: 20.
• the monoclonal antibody further comprises an antibody heavy chain constant region,
• the heavy chain constant region is a human IgG1 constant region.
• the human IgG1 constant region includes the amino acid sequence shown in SEQ ID NO: 21.
• the light chain variable region sequence of the monoclonal antibody is combined with the human Kappa chain constant region to form an antibody light chain
• the heavy chain variable region sequence of the monoclonal antibody is combined with the human IgG1 constant region to form an antibody heavy chain .
• the synthesized gene fragment is cloned into the expression vector pcDNA3.4, expressed and purified in CHO cells, and affinity matured monoclonal antibodies can be obtained.
• the anti-ADM antibody exhibits an affinity for ADM with a KD value of less than 10 -10 M as determined by affinity determination.
• the present invention provides a nucleotide sequence encoding the anti-adrenomedullin monoclonal antibody of the present invention or a fragment thereof.
• Another aspect of the present invention provides an expression vector comprising the nucleotide sequence of the present invention, preferably the vector pcDNA3.4.
• Another aspect of the present invention provides a host cell comprising the nucleotide sequence or expression vector of the present invention, which is preferably a CHO cell.
• Another aspect of the present invention provides a pharmaceutical composition, detection reagent or kit containing the anti-adrenomedullin monoclonal antibody or fragment thereof according to the present invention.
• Another aspect of the present invention provides the use of the anti-adrenomedullin monoclonal antibody or fragment thereof according to the present invention in the preparation of drugs for maintaining endothelial cell integrity and enhancing barrier function.
• Another aspect of the present invention provides the use of the anti-adrenomedullin monoclonal antibody or fragment thereof according to the present invention in the preparation of drugs for controlling vasodilation and lowering blood pressure.
• Another aspect of the present invention provides the use of the anti-adrenomedullin monoclonal antibody or fragment thereof according to the present invention in the preparation of medicaments for the treatment or prevention of septic shock or traumatic injury, especially in the late stage of sepsis.
• Another aspect of the present invention provides the use of the anti-adrenomedullin monoclonal antibody or fragment thereof according to the present invention in the preparation of a medicament for reducing the risk of death of a patient with a chronic or acute disease or an acute disease, the chronic or acute disease being or acute illness 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.
• 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,
• Another aspect of the present invention provides the use of the anti-adrenomedullin monoclonal antibody or fragment thereof according to the present invention in the preparation of medicaments for stabilizing the circulation, especially the systemic circulation, of patients suffering from chronic or acute diseases or acute Conditions
• the chronic or acute diseases or acute conditions are 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.
• the anti-adrenomedullin monoclonal antibody or fragment thereof is used in combination with another agent.
• the pharmaceutical agent is selected from the group consisting of vasopressors, intravenous solutions, TNF- ⁇ -antibodies and antibiotics.
• the present invention screens out anti-ADM monoclonal antibodies through hybridoma technology, and obtains fully human anti-ADM monoclonal antibodies through humanized expression of antibodies and panning of a fully human single-chain phage antibody library. On this basis, the present invention screened the affinity maturation library and finally obtained a drug with higher activity of binding to the N-terminus of human ADM. Compared with anti-ADM non-neutralizing antibodies existing in the prior art, the anti-ADM non-neutralizing antibodies of the present invention exhibit an affinity for ADM with a KD value of less than 10 -10 M.
• Figure 1 shows that anti-ADM antibody blocks the production of CHOK1/CRLR/RAMP3 cells induced by hADM.
• Figure 2 is a graph showing the results of anti-ADM antibody survival rate in LPS-induced sepsis in mice.
• Example 1 Screening of anti-ADM monoclones using hybridoma technology
• the synthesized N-terminal 16 amino acid polypeptide of human ADM (referred to as YY-16, SEQ ID NO: 1), the N-terminal 21 amino acid polypeptide of human ADM (referred to as YY-21, SEQ ID NO: 2) or mouse ADM
• the conjugate of the N-terminal 19 amino acid peptide (referred to as YY-19, SEQ ID NO: 3) and KLH (Sigma, H8283) was used as an immunogen to immunize mice.
• the immunogen and Freund's complete adjuvant were emulsified at a ratio of 1:1, and intraperitoneally injected into 6 to 8-week-old female Balb/c mice, SJL mice or SD rats at 100 ⁇ g/mouse; thereafter Booster immunizations were performed every 2 to 3 weeks with 50 ⁇ g of immunogen per animal plus Freund's incomplete adjuvant.
• blood was collected to measure the titer. Blood was collected one week after the last immunization, and the serum anti-YY-21 titer was determined by ELISA. After the mice with high serum titer were pulse immunized by intraperitoneal injection of 50 ⁇ g of immunogen, the spleen cells of the animals were harvested for fusion on the third day.
• mice After the mice were euthanized, the cells were dissected, the spleens were removed, the cells were ground and collected, and the cells were suspended in 5 mL of red blood cell lysis buffer and placed at 4°C for 5 min. The reaction was terminated with DMEM+10% FBS. After centrifugation, resuspend the splenocytes in 40 mL DMEM, let stand for 2 to 3 minutes, and then transfer the supernatant to another 50 mL centrifuge tube.
• hybridoma clone 40E12 was screened, the cell line was cryopreserved, and 50 mL small-scale production was carried out using serum-free medium. The protein A column was purified for subsequent identification.
• Octet RED96e was used to determine candidate antibodies and biotin-labeled human ADM (referred to as human ADM-C-biotin, the human ADM sequence is shown in SEQ ID NO: 4) and mouse ADM (referred to as mouse ADM-C-biotin, mouse The affinity of the ADM sequence (shown in SEQ ID NO:5). Both the antigen and antibody were diluted with 1 ⁇ PBST, the antigen concentration was 2 ⁇ g/ml, and the antibody working concentration was 100 nM.
• the sample was added to a 96-well plate (Greiner bio-one, 655209), and the system was 200 ⁇ L/well. Then set the software parameters, the plate temperature is set to 30°C, and the frequency of collecting standard kinetic signals is 5.0HZ.
• prewet the streptavidin sensor (Fortébio, Cat. No.: 18-5020) with 1 ⁇ PBST for 10 minutes, and then put it on the machine for detection.
• Each cycle includes the following steps: 1) Immerse in the buffer for 180 seconds to stabilize the baseline; 2) Solidify the antigen for 10 seconds to bind the antigen to the sensor, and control the antigen binding amount between 0.5-1.0nm; 3) Immerse the sensor into the buffer for 180 seconds ;4) Antigen and antibody combine. When combined, 180s; 5) Dissociation of antigen and antibody, time 10min.
• VH primers sequences such as SEQ ID NO: 6
• VK primer sequence shown in SEQ ID NO: 7
• polyC primer sequence shown in SEQ ID NO: 8
• the light and heavy chain variable regions were Chothia numbered.
• the amino acid sequences of HCDR1, HCDR2 and HCDR3 of clone 40E12 defined by Chothia are shown in SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13 respectively.
• LCDR1, LCDR2 and The amino acid sequences of LCDR3 are shown in SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16 respectively.
• human antibody germline genes with the highest homology were selected as the humanization design framework.
• the heavy chain variable region is based on the human antibody germline gene sequences IGHV7-4-1*02 and IGHJ6*01 as the framework, and 40E12VH (also referred to as 2004hzVH0) is subjected to CDR transplantation and reverse mutation to obtain the humanized heavy chain variable region sequence.
• 2004hzVH9 amino acid sequence such as SEQ ID NO: 17).
• the light chain variable region uses the human antibody germline gene sequences IGKV2-30*02 and IGKJ2*01 as the framework, and combines 40E12VK (also referred to as 2004hzVK0) humanized light chain variable region sequences 2004hzVK7 (amino acid sequence such as SEQ ID NO: 18) and 2004hzVK9 (amino acid sequence such as SEQ ID NO: 19) subjected to CDR transplantation and backmutation.
• 40E12VK also referred to as 2004hzVK0
• 2004hzVK7 amino acid sequence
• 2004hzVK9 amino acid sequence such as SEQ ID NO: 19
• the 2004hzVK7 and 2004hzVK9 sequences were combined with the human Kappa chain constant region (CL, amino acid sequence SEQ ID NO: 20) to form an antibody light chain, and the 2004hzVH9 sequence was combined with the human IgG1 constant region (CH, amino acid sequence SEQ ID NO: 21) to form an antibody Weight chain.
• 2004hzVH0 and 2004hzVK0 were combined with the human heavy chain constant region and the human light chain constant region respectively and then paired to form the chimeric antibody 2004hz00 (sequences are shown in SEQ ID NO: 22 and 23); 2004hzVH9 and 2004hzVK7 were combined with the human heavy chain constant region and the human light chain constant region respectively.
• the light chain constant regions were combined and paired to form humanized antibody 2004hz97 (sequences are shown in SEQ ID NO: 24 and 25); 2004hzVH9 and 2004hzVK9 were combined with human heavy chain constant regions and human light chain constant regions respectively to form humanized antibodies.
• Antibody 2004hz99 sequences are shown in SEQ ID NO: 26 and 27).
• Octet RED96e (Fortebio) was used to determine the affinity of antibodies 2004hz00, 2004hz97, 2004hz99 and biotin-labeled human and mouse ADM (human ADM product number: 894757, mouse ADM product number: 894758, synthesized by GLBiochem). Both antigens and antibodies were used 1 ⁇ PBST (1 ⁇ PBS: Sangon, B548117-0500; 0.02% Tween 20: sigma, P1379) was diluted, the antibody concentration was 100 nM, and the antigen concentration was 2 ⁇ g/mL.
• Each cycle includes the following steps: 1) Immerse in the buffer for 60 seconds; 2) Detect whether the antigen binds non-specifically to the sensor; 3) Regenerate the 10mM glycine solution at pH 1.7; 4) Immerse in the buffer for 60 seconds; 5) The antigen is solidified on the sensor on, the time is 10s; 6) the sensor is immersed in the buffer for 180s; 7) the antigen and antibody are combined, the time is 180s; 8) the antigen-antibody dissociation, the time is 600s; 9) the sensor is regenerated.
• humanized antibodies 2004hz97 and 2004hz99 have good physical and chemical properties after one-step protein A purification.
• Example 5 Panning of fully human single-chain phage antibody library
• the antigen-specifically bound phages were eluted with 450 ⁇ L of 100 mM hydrochloric acid, and 50 ⁇ L of 1M Tris-HCl at pH 11 was added to neutralize and infect E. coli SS320 in the logarithmic growth phase, and the phages were generated and purified for the next round of screening.
• the screening method was the same as in the first round, except that the amount of antigen was reduced to 4 ⁇ g.
• the enriched phages after two rounds of screening were used to identify the enrichment status using enzyme-linked immunoassay (ELISA). The results showed that the phages were significantly enriched after two rounds of panning.
• adrenomedullin bioassay The effect of selected anti-ADM antibodies on ADM biological activity was tested in a human recombinant adrenomedullin receptor cAMP functional assay (adrenomedullin bioassay).
• the anti-ADM antibody was diluted to 1600 ⁇ g/mL with Stimulation Buffer 1 (Cisbio, 64SB1FDD), with a working concentration of 400 ⁇ g/mL, followed by 3-fold gradient dilution with Stimulation Buffer 1 (8 ⁇ L + 16 ⁇ L Stimulation Buffer 1), in which the antagonist human ADM (22-52) (Alfa Aesar, Cat. No.: 159899-65-7) was diluted to 12000 ⁇ g/mL, with a working concentration of 3000 ⁇ g/mL, and then 2.5 ⁇ L of anti-ADM antibody was added to the corresponding well in the experimental plate. Dilute human ADM JMB2004 YY-52 protein (Gill Biochemical, Cat.
• CHO-K1/CRLR/RAMP3 human recombinant adrenomedullin receptor
• the cAMP content was detected using the HTRF kit (Cisbio, 62AM4PEB). That is, 5 ⁇ L of cAMP-d2 reagent working solution was added to the experimental well, and then 5 ⁇ L of cAMP Eu-Cryptate antibody working solution was added. After incubation at room temperature for 1 hour, the HTRF value was detected by a microplate reader.
• the fully human antibody 1F12 obtained through screening was affinity matured to improve its affinity with human ADM.
• the heavy chain antigen-binding determinant 2 (CDR_H2) of 1F12 contains the post-translational modification (PTM) site of NG, site-directed mutation into QG eliminates deamidation isomerization, and the mutated antibody is named 1F12PTM ⁇ (variable region amino acid Sequences are shown in SEQ ID NO: 29 and 35).
• coding was performed according to Chothia rules, the CDR region was defined according to Chothia, and the amino acids at the HCDR-3 and LCDR-3 sites were randomly mutated to construct a mutation library.
• NNK mutation primers were designed to perform polymerase chain reaction (PCR) to amplify the HCDR-3 and LCDR-3 mutation library gene fragments.
• PCR polymerase chain reaction
• the amplified VH and VL gene fragments were recovered and electroporated together with the yeast display plasmid into the Saccharomyces cerevisiae strain EBY100 ( Purchased from ATCC), the VH and VL genes were inserted into the yeast display plasmid through homologous recombination of Saccharomyces cerevisiae, thereby realizing a Fab mutation library that displays antibodies on the yeast cell wall surface.
• the library was named JYYDL196-197.
• the library JYYDL196-197 was cultured in 250 mL of SD-Trp-Leu liquid medium (Clontech, Cat. No.: 630316) at 30°C overnight; 1.0 ⁇ 10 9 bacteria were taken from each and resuspended in 200 mL of YPGP induction medium (2% Galactose, 2% peptone, 1% yeast extract, 0.54% Na 2 HPO 4 , 0.86% NaH 2 PO 4 ⁇ H 2 O), cultured at 20°C for 24h, and placed at 4°C until use.
• the sequence of the parent 1F12PTM ⁇ was displayed on the yeast surface and used as a parental control.
• Ab2004.Am01 continues to undergo affinity maturation to improve its affinity with human and mouse ADM.
• the amino acids of HCDR-1, HCDR-2, LCDRL-1, LCDR-2, and LCDR-3 of Ab2004.Am01 were selected for mutation library construction, and the library number was JYYDL208-212.
• the library JYYDL208-212 was cultured and induced before use.
• the Fab sequence of Ab2004.Am01 was displayed on the yeast surface and used as a parental control for this round of affinity maturation.
• JYYDL208-209 the second and third round screening products of JYYDL211, selected several single clones from each to construct the light and heavy chain mutation combinatorial library JYYDL227.
• .Am32 (the amino acid sequence of the variable region is shown in SEQ ID NO: 31 and 37), Ab2004.Am33 (the amino acid sequence of the variable region is shown in SEQ ID NO: 32 and 36), Ab2004.Am34 (the amino acid sequence of the variable region is shown in SEQ ID NO: 32 and 36) As shown in SEQ ID NO:33 and 38), Ab2004.Am35 (the amino acid sequence of the variable region is as shown in SEQ ID NO:31 and 38), Ab2004.Am36 (the amino acid sequence of the variable region is as shown in SEQ ID NO:31 and 39) shown), Ab2004.Am37 (the variable region amino acid sequence is shown in SEQ ID NO: 34 and 38), Ab2004.Am38 (the amino acid sequence of the variable region is shown in SEQ ID NO:34 and 40), Ab2004.Am39 (the amino acid sequence of the variable region is shown in SEQ ID NO:33 and 41) and Ab2004.Am40 (the amino acid sequence of the variable region is shown in SEQ ID NO:33 and 41) The sequences are shown in
• Example 2.2 the VH and VK sequences of each clone were combined with the human Kappa chain constant region (CL, amino acid sequence SEQ ID NO: 20) and the human IgG1 constant region (CH, amino acid sequence SEQ ID NO: 21) respectively to form an antibody.
• the light chain and heavy chain were loaded into the expression vector pcDNA3.4 (Life Technologies), entrusted Nanjing GenScript Biotechnology Co., Ltd. to perform transient CHO cell expression and purification, and finally obtained the affinity mature candidate antibodies as shown in Table 9.
• Example 3 continue to measure the affinity of the affinity mature antibodies Ab2004.Am31-Ab2004.Am40 and the parent antibody Ab2004.Am01 to human and mouse ADM respectively.
• the results are shown in Table 10.
• Table 10 the affinity of Ab2004.Am31, Ab2004.Am34, and Ab2004.Am39 to human ADM is the most improved compared to the parent antibody Ab2004.Am01, and the affinity to mouse ADM is comparable to the positive control antibody Enibarcimab.
• Example 14 Evaluation of physicochemical properties of candidate antibodies
• Example 15 Pharmacodynamic study of ADM antibody in LPS-induced C57BL/6J mouse sepsis model
• C57BL/6J mice were purchased from Beijing Vitong Lihua Experimental Animal Technology Co., Ltd., 10-11 weeks old, male, 50 in total.

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