Anti-human copeptidin monoclonal antibody and application thereof

Abstract

The invention provides an anti-human copeptidin monoclonal antibody and an application thereof. The antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a heavy chain CDR1, a heavy chain CDR2 and a heavy chain CDR3 in the heavy chain variable region as shown in SEQ ID NO.8; the light chain variable region comprises a light chain CDR1, a light chain CDR2 and a light chain CDR3 in the light chain variable region as shown in SEQ ID NO. 9. The antibody of the invention can effectively bind to peptin and has been verified to be suitable for use in an ELISA detection platform. The antibody provided by the invention has wide application prospect and practical value.

Description

Technical Field

[0001] This invention belongs to the field of antibodies, specifically relating to an anti-human and anti-peptide monoclonal antibody and its applications. Background Technology

[0002] Copeptin is the C-terminal fragment produced by the breakdown of arginine vasopressin (AVP) precursor (ProAVP), and is released into the bloodstream in a 1:1 ratio with AVP. Due to its longer half-life (approximately 7 days), copeptin is more stable in vivo than AVP, making it a stable biomarker for assessing acute events. It has broad clinical application value, particularly in cardiovascular disease, prognostic assessment of acute and critical illnesses, and diagnosis of multisystem diseases. In the field of cardiovascular disease, it can be used for the early diagnosis and exclusion of acute coronary syndromes (ACS). The combined detection of copeptin and troponin forms a dual-marker strategy (DMS), which can significantly improve diagnostic sensitivity, especially suitable for non-ST-segment elevation ACS (NSTE-ACS). It has significant kinetic advantages: copeptin rapidly increases in the early stages of acute myocardial infarction (AMI), forming a "reverse release kinetic" with troponin, compensating for the insufficient early sensitivity of troponin and shortening the diagnostic time. The 2020 ESC guidelines list it as a Category IIa B recommendation. Furthermore, elevated tropin levels are significantly associated with mortality risk in ACS patients, particularly showing independent predictive value for both short-term and long-term prognosis: patients with tropin levels >10 pmol / L have a nearly 3-fold increased all-cause mortality rate, and when combined with elevated high-sensitivity troponin T (hs-cTnT), the 30-day mortality rate increases 10-fold. tropin is of significant value in pulmonary embolism (PE) risk stratification; normotensive PE patients with tropin levels ≥24 pmol / L have a 6.3-fold increased risk of adverse outcomes and a 7.6-fold increased risk of death, with predictive power comparable to NT-proBNP. Additionally, in patients with acute ischemic stroke, elevated tropin levels are an independent predictor of poor prognosis, associated with the risk of death, hemiplegia, and recurrent stroke. Plasma tropin levels in patients with cerebral hemorrhage are positively correlated with mortality. Moreover, in respiratory diseases, severe pneumonia, and ventilator-associated pneumonia (VAP) patients, tropin levels increase with disease severity, with significantly higher levels in deceased patients than in survivors. In patients with acute exacerbations of chronic obstructive pulmonary disease (AECOPD), the combination of hydroxyvitamin and other inflammatory markers can improve diagnostic and prognostic efficacy. In sepsis and infectious diseases, hydroxyvitamin levels are positively correlated with sepsis severity and mortality; combining it with procalcitonin (PCT) can improve predictive accuracy. Therefore, hydroxyvitamin, with its high stability and convenient testing, demonstrates significant value in the early diagnosis of cardiovascular diseases, prognostic assessment of acute and critical illnesses, and management of multi-system diseases.

[0003] As a key diagnostic and prognostic biomarker for various critical diseases, the widespread and important clinical applications of phytokinin directly determine the sustainability and scale of market demand. Developing a high-performance antibody is of great significance for improving the sensitivity, specificity, and accessibility of phytokinin detection. Summary of the Invention

[0004] In view of this, in order to overcome the shortcomings of the prior art, the present invention is proposed.

[0005] The first aspect of the present invention provides an anti-peptidase antibody or an antigen-binding fragment thereof, the anti-peptidase antibody or the antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 as shown in SEQ ID NO.8; the light chain variable region comprising light chain CDR1, light chain CDR2, and light chain CDR3 as shown in SEQ ID NO.9.

[0006] In some embodiments, the amino acid sequences of the heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 are shown in SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4, respectively, and the amino acid sequences of the light chain CDR1, light chain CDR2 and light chain CDR3 are shown in SEQ ID NO.5, SEQ ID NO.6 and SEQ ID NO.7, respectively.

[0007] In some embodiments, the heavy chain variable region has an amino acid sequence as shown in SEQ ID NO. 8 or an amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO. 8; the light chain variable region has an amino acid sequence as shown in SEQ ID NO. 9 or an amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO. 9.

[0008] In some embodiments, the heavy chain variable region has an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence shown in SEQ ID NO. 8; the light chain variable region has an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence shown in SEQ ID NO. 9.

[0009] In some embodiments, the heavy chain variable region has an amino acid sequence as shown in SEQ ID NO. 8; and the light chain variable region has an amino acid sequence as shown in SEQ ID NO. 9.

[0010] As used in this invention, the term "CDR," also known as "complementarity-determining region," "CDR region," or "hypervariant region," refers to a region in the antibody variable region that is highly variable in sequence and forms a structurally defined loop ("hypervariant loop") and / or contains antigen contact residues ("antigen contact sites"). The CDR is primarily responsible for binding to antigen epitopes.

[0011] Based on the amino acid sequences of the heavy chain variable region and light chain variable region contained in the anti-peptide antibody or its antigen-binding fragment provided in this invention, those skilled in the art can determine the CDRs contained therein using conventional methods in the art. CDR sequences determined by different methods all fall within the scope of protection of this application. For example, the IMGT method, Kabat method, AbM method, Chothia method, or Contact method can be used to define the CDRs in the variable region amino acid sequence.

[0012] When referring to antibodies defined by a specific CDR sequence as defined in this invention, the scope of said antibody also includes antibodies whose variable region sequence contains the specific CDR sequence, but whose claimed CDR boundaries differ from those defined in this invention due to the application of different schemes (e.g., different assignment system rules or combinations).

[0013] The boundaries of the CDR of the antibody of the present invention can be determined artificially according to any method or combination thereof in the art. Unless otherwise stated, in this invention, the term "CDR" or "CDR sequence" covers the CDR sequence determined in any of the foregoing methods.

[0014] In some embodiments, functional variants of the anti- and peptidoglycan antibody or its antigen-binding fragment described in this invention are also included within the scope of protection of this invention. The term "functional variant" refers to a protein that has significant or marked sequence identity or similarity to the parent antibody, and that retains the biological activity of the parent antibody. Functional variants encompass, for example, the following variants of the anti- and peptidoglycan antibody or its antigen-binding fragment (parent antibody) described herein, which retain the ability to recognize target antigens to a similar, equal, or greater extent than the parent antibody. Referring to the parent antibody, the functional variant may, for example, have at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher identity with the parent antibody in terms of amino acid sequence.

[0015] In some embodiments, the functional variant may, for example, comprise the amino acid sequence of a parent antibody having at least one conserved amino acid substitution. Alternatively or supplementally, the functional variant may comprise the amino acid sequence of a parent antibody having at least one non-conserved amino acid substitution. In this case, the non-conserved amino acid substitution preferably does not interfere with or inhibit the biological activity of the functional variant. The non-conserved amino acid substitution can enhance the biological activity of the functional variant, resulting in increased biological activity of the functional variant compared to the parent antibody.

[0016] In some embodiments, conservative amino acid substitution is known in the art and includes the substitution of one amino acid having a particular physical and / or chemical property with another amino acid having the same or similar chemical or physical property.

[0017] Those skilled in the art can readily mutate the nucleotide sequence corresponding to the antibody described in this invention using known methods, such as directed evolution and point mutation. Artificially modified nucleotides that have 90% or more homology to the nucleotide sequence corresponding to the anti-peptide antibody or its antigen-binding fragment described in this invention, as long as they encode the aforementioned anti-peptide antibody or its antigen-binding fragment, are all derived from and equivalent to the sequence of this invention, and are also included within the scope of protection of this invention.

[0018] In this invention, the antibody includes monoclonal antibody, polyclonal antibody, and recombinant antibody.

[0019] As used herein, a "monoclonal antibody" refers to an antibody molecule or antibody formulation that shares a common heavy chain amino acid sequence and a common light chain amino acid sequence, as opposed to a "polyclonal" antibody formulation containing a mixture of antibodies with different amino acid sequences. Monoclonal antibodies can be produced using several known techniques such as phage, bacterial, yeast, or ribosome display, as well as the classic method exemplified by antibodies derived from hybridomas. Therefore, the term "monoclonal" is used to refer to all antibodies derived from a single nucleic acid clone.

[0020] As used herein, the term "polyclonal antibody" refers to a mixture of antibodies produced by multiple B cell clones following immunization in mammals (such as humans, rabbits, sheep, mice, horses, and rabbits). Because the antigen contains multiple determinants, it stimulates different B cells to produce antibodies against multiple epitopes.

[0021] As used herein, the term “recombinant antibody” refers to an antibody manufactured, expressed, produced, or isolated by recombinant methods, such as antibodies expressed using a recombinant expression vector transfected into a host cell; antibodies isolated from a recombinant antibody library; or antibodies manufactured, expressed, produced, or isolated by any other means, such as antibodies manufactured, expressed, produced, or isolated by assembling a specific immunoglobulin gene sequence (such as the human immunoglobulin gene sequence) with other DNA sequences.

[0022] In this invention, the antibodies include chimeric antibodies, modified antibodies, SDR transplantation antibodies, antigen epitope-directed selection antibodies, surface remodeling antibodies, framework region replacement antibodies, and fully humanized antibodies.

[0023] As used herein, a “chimeric antibody” refers to an antibody whose variable domain sequence, derived from a non-human mammalian species (e.g., mouse or rabbit), is transplanted onto a constant domain sequence derived from a human antibody.

[0024] As used herein, the “modified antibody” refers to the transplantation of the complementarity-determining region (CDR) of an antibody variable region derived from a non-human mammalian species (e.g., mouse or rabbit) into the backbone region (FR) of a human antibody variable region.

[0025] "Modified antibodies" include antibodies with complete CDR transplantation and antibodies with partial CDR transplantation.

[0026] Complete CDR transplantation antibody: Six CDRs from a non-human antibody are cloned into the corresponding FRs of a human antibody using methods such as PCR to construct a new antibody. Compared with chimeric antibodies, CDR transplantation further reduces the content of heterologous sequences in the antibody and reduces antibody heterology.

[0027] Partial CDR transplantation antibodies: The properties of six CDRs on non-human antibodies are studied to identify the CDRs necessary for binding antigens in the antibody. These CDRs are then transplanted onto human antibody FR, resulting in lower immunogenicity than fully CDR transplanted antibodies.

[0028] SDR transfer antibodies: Based on partial CDR transplantation, a few residues from specific regions (SDRs) of the CDR that are specifically recognized and bound to the antigen are transplanted to the corresponding positions on the human antibody. These mainly include: SDR residues exposed to the outside of the CDR that are closely related to antigen binding; residues related to maintaining the 4th-order structure of the antibody's V region; amino acid position 71 of the light and heavy chains; and residues at the N-terminus of the antibody.

[0029] Antigen epitope-directed antibody selection: Antigen epitope-directed selection is a humanization strategy developed based on phage antibody library technology. Taking non-human antibodies derived from mice as an example, antigen epitope-directed selection uses the variable regions of light and heavy chain genes as basic units to construct human heavy chain mouse light chain and human light chain mouse heavy chain hybrid antibody libraries. Using a phage surface selection system, human heavy chain mouse light chain genes and human light chain mouse heavy chain genes are screened separately. Then, the human light and heavy chain genes are mixed and screened to obtain fully humanized antibody light and heavy chain genes that recognize the same epitopes as the parent mouse monoclonal antibody.

[0030] Surface-remodeled antibodies: Surface remodeling is a humanization technique directly based on the three-dimensional structure of antibodies. Taking non-human antibodies derived from mice as an example, the immunogenicity of the V region of mouse monoclonal antibodies largely stems from the motility and solution accessibility of surface residues in this region (solution-accessible residues (SAR): amino acid residues that can come into contact with the solvent when the antibody dissolves). Amino acids with high solution accessibility may have strong antigenicity. Surface remodeling emphasizes using the precise or modeled structure of antigen-antibody interactions as a basis, replacing highly surface-accessible amino acid residues in the variable regions of the light and heavy chains of mouse-derived antibodies with corresponding human amino acid residues, while retaining the CDR region and embedded amino acid residues of the mouse-derived antibody. Surface remodeling can maximize the preservation of antibody binding activity while improving its humanization degree, making it a relatively ideal humanization method.

[0031] Frame region replacement antibodies: Unlike CDR transplantation antibodies, the frame region replacement method does not search for human antibody frame regions similar to those from murine sources. Instead, it establishes a human antibody frame region library based on human germline genes, and then transplants the six CDRs (three from the light chain and three from the heavy chain) of the heterologous antibody into the frame regions of the human germline genes. The corresponding antibody combination library can then be screened using antigens. This method can not only be used for humanized antibodies but is also a more general antibody modification method. Through screening, it may even be possible to find human antibodies with stronger affinity than parental murine antibodies.

[0032] In some implementations, the non-human antibody is derived from rabbits.

[0033] In this invention, the antibodies include monospecific antibodies and multispecific antibodies.

[0034] In this invention, the multispecific antibody is a type of antibody that can simultaneously bind to two or more different antigenic epitopes or antigens.

[0035] In some implementations, the multispecific antibody is bispecific, trispecific, quadrispecific, or even more specific.

[0036] In some implementations, the multispecific antibody is bispecific.

[0037] In this invention, the antigen-binding fragments include Fab, Fab′, F(ab′)2, single-chain antibody scFv, and nanobody VHH.

[0038] Fab: Fab is a functional unit of a monoclonal antibody molecule, which contains the heavy chain variable region VH, the heavy chain constant region CH1, the light chain variable region VL, and the light chain constant region CL. It does not contain the Fc fragment and has a molecular weight of approximately 50 kDa.

[0039] scFv: scFv is the variable region of the monoclonal antibody molecule, namely VH+VL, with a molecular weight of approximately 25 kDa.

[0040] VHH: Unlike Fab and scFv, VHH originates from heavy chain antibodies (a type of antibody that naturally lacks a light chain). VHH is the variable region of the heavy chain in a heavy chain antibody, with a molecular weight of approximately 15 kDa. The molecular weight of VHH is only one-tenth that of conventional antibodies, and its size is in the nanometer range, hence it is also called a nanobody (Nano-antibody). Because it has a single structural domain, it is also called a single-domain antibody (SdAbs).

[0041] In some embodiments, the antibody further includes a constant region, and according to the structure of the constant region, the antibody includes IgG antibodies, IgA antibodies, IgM antibodies, IgE antibodies, and IgD antibodies.

[0042] In some implementations, the constant region is preferably the human IgG constant region, which is divided into four isotypes: IgG1, IgG2, IgG3 and IgG4.

[0043] A second aspect of the present invention provides a polynucleotide molecule that encodes the anti-peptide antibody or its antigen-binding fragment as described in the first aspect of the present invention.

[0044] The term "polynucleotide," synonymously referred to as "nucleic acid molecule," "nucleotide," or "nucleic acid," refers to any polynucleotide or polydeoxynucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. "Polynucleotide" includes, but is not limited to, single-stranded and double-stranded DNA, DNA consisting of a mixture of single-stranded and double-stranded regions, single-stranded and double-stranded RNA, and RNA consisting of a mixture of single-stranded and double-stranded regions, and hybrid molecules containing DNA and RNA that may be single-stranded or more typically double-stranded, or a mixture of single-stranded and double-stranded regions. Additionally, "polynucleotide" refers to a triple-stranded region containing RNA or DNA, or both RNA and DNA. The term polynucleotide also includes DNA or RNA containing one or more modified bases, and DNA or RNA with a backbone modified for stability or other reasons.

[0045] In this invention, the polynucleotide molecule may comprise natural, non-natural, or modified nucleotides; and it may comprise natural, non-natural, or modified internucleotide linkages, such as aminophosphate linkages or thiophosphate linkages, instead of phosphodiester linkages present between the nucleotides of the unmodified oligonucleotide. In some embodiments, the nucleotides do not contain any insertions, deletions, inversions, and / or substitutions. However, in some cases, it may be suitable for a nucleotide to contain one or more insertions, deletions, inversions, and / or substitutions, and therefore, nucleotides formed by these insertions, deletions, inversions, and / or substitutions are also within the scope of this invention.

[0046] When applied to polynucleotide molecules, the term "encoding" refers to a polynucleotide that, if in its natural state or when manipulated by methods known to those skilled in the art, can be transcribed and / or translated to produce an mRNA containing a polypeptide and / or fragments thereof, is called "encoding" the polypeptide. The antisense strand is the complement of this nucleic acid, and the coding sequence can be deduced from it.

[0047] A third aspect of the present invention provides a carrier comprising the polynucleotide molecule described in the second aspect of the present invention.

[0048] In some embodiments, examples of vectors that can be used in this invention include, but are not limited to, plasmids, phage particles, granules, artificial chromosomes, and virus-derived vectors.

[0049] Various vectors known in the art can be used, such as commercially available vectors, and then a polynucleotide encoding the antibody or its antigen-binding fragment can be operatively linked to the expression regulatory sequence to form an expression vector. In some embodiments, the virus-derived vectors include, but are not limited to: lentiviral vectors, retroviral vectors, adenovirus vectors, adeno-associated virus vectors, poxvirus vectors, herpesvirus vectors, baculovirus vectors, papillomavirus vectors, and papillomavirus vectors.

[0050] In some embodiments, the expression vector may contain expression regulatory sequences, such as transcription and translation start and stop codons, which are specific to the type of host cell (e.g., bacteria, fungi, plants, or animals) into which the vector is to be introduced, depending on the circumstances and whether the vector is DNA-based or RNA-based. Recombinant expression vectors may contain restriction sites to facilitate cloning.

[0051] In some embodiments, the vector may also contain one or more marker genes that allow selection of host cells for transformation or transfection. Marker genes include biocidal resistance (e.g., resistance to antibiotics, heavy metals, etc.); prototrophic complementation in auxotrophic hosts, etc. Suitable marker genes for the expression vector of the present invention include, for example, neomycin / G418 resistance genes, hygromycin resistance genes, histidine resistance genes, tetracycline resistance genes, ampicillin resistance genes, kanamycin resistance genes, and puromycin resistance genes.

[0052] In some embodiments, the vector includes vectors selected from plasmid vectors.

[0053] In some implementations, the plasmid vector is selected from expression vectors.

[0054] In some embodiments, the expression vectors include, but are not limited to, pET28a-sumo, pET28a-sumo-TEV / EK, pET28a-GST-TEV / EK, pET28a-MBP-TEV / EK, pET32a-Trx-TEV / EK, pcDNA3.1, pcDNA3.4, PTT5, and pEF1 / myc-His A.

[0055] In some embodiments, the expression vector is selected from pcDNA3.4.

[0056] A fourth aspect of the present invention provides a modified host cell or a population of host cells comprising the same, said modified host cell comprising the anti-peptidase antibody or antigen-binding fragment thereof as described in the first aspect of the present invention, or the polynucleotide molecule as described in the second aspect of the present invention, or the vector as described in the third aspect of the present invention.

[0057] In some implementations, the host cell population may also include host cells other than the modified host cells.

[0058] In some implementations, the prokaryotic cells include, but are not limited to, bacteria, actinomycetes, cyanobacteria, mycoplasma, chlamydia, and rickettsia.

[0059] In some embodiments, the bacteria include, but are not limited to, Escherichia coli, Bacillus subtilis, Salmonella typhimurium, Pseudomonas, Streptomyces, and Staphylococcus.

[0060] In some embodiments, the eukaryotic cells include, but are not limited to, mammalian cells, insect cells, plant cells, and yeast cells.

[0061] In some embodiments, the mammalian cells include, but are not limited to, immune cells, CHO cells, HEK293T cells, and HEK293F cells.

[0062] In some embodiments, the mammalian cells are selected from 293F cells.

[0063] The fifth aspect of the present invention provides an antibody derivative of an anti-peptide antibody or an antigen-binding fragment thereof, said antibody derivative comprising a complex formed by directly or indirectly coupling the anti-peptide antibody or an antigen-binding fragment thereof to a detectable marker as described in the first aspect of the present invention.

[0064] In this invention, the term "detectable marker" refers to a reagent that is detectable, for example, by spectroscopic, photochemical, biochemical, immunochemical, or chemical means.

[0065] In some implementations, the detectable markers include, but are not limited to, radionuclides, chemiluminescent agents, bioluminescent agents, paramagnetic ions, enzymes, and photosensitizing diagnostic agents.

[0066] In some embodiments, the radionuclides include, but are not limited to, those mentioned above. 18 F, 52 Fe、 62 Cu、 64 Cu、 67 Cu、 86 Y、 90 Y、 89 Zr、 120 I, 123 I, 124 I, 125 I, 131 I, 13 N、 15 O、 186 Re、 188 Re、 51 Mn, 55 Co、 72 As.

[0067] In some embodiments, the chemiluminescent agent includes, but is not limited to, luminol, isoluminol, aromatic acridine esters, imidazole, acridine salts, and oxalates.

[0068] In some embodiments, the bioluminescent agent includes, but is not limited to, luciferin, luciferase, and jellyfish luminescent protein.

[0069] In some embodiments, the paramagnetic ions include, but are not limited to, chromium (III), manganese (II), iron (III), iron (II), cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III), holmium (III), and erbium (III).

[0070] In some embodiments, the enzyme includes, but is not limited to, horseradish peroxidase, alkaline phosphatase, glucose oxidase, β-D-galactosidase, urease, catalase, or glucosylamylase.

[0071] In some embodiments, the photosensitive diagnostic agent includes, but is not limited to, dihydroxysilyl phthalocyanine, methylene blue, protoporphyrin, hematoporphyrin, and photoporphyrin.

[0072] The sixth aspect of the present invention provides a product for detecting and peptidin protein or a functional fragment thereof or a polypeptide containing and peptidin, the product comprising an anti-and peptidin antibody or an antigen-binding fragment thereof as described in the first aspect of the present invention, a polynucleotide molecule as described in the second aspect of the present invention, a carrier as described in the third aspect of the present invention, and / or a host cell or a population of host cells containing the present invention as described in the fourth aspect of the present invention.

[0073] In some implementations, the products include, but are not limited to, reagent kits, test strips, reagents, and chips.

[0074] In some embodiments, the kit may also include, but is not limited to, a container for holding anti- and peptidoglycan antibodies or antigen-binding fragments thereof when not in use, instructions for use, anti- and peptidoglycan antibodies or antigen-binding fragments thereof attached to a solid support, known standards, and reference samples, or one or more of these.

[0075] The term "known standard" can refer to a solution containing a known amount or concentration of anti-peptide, wherein such solution can be a naturally occurring solution; or such solution can be a synthetic solution, such as a buffered aqueous solution in which a known amount of anti-peptide is diluted. Known standards described herein may include anti-peptide isolated from subjects, recombinant or purified anti-peptide proteins, or anti-peptide associated with disease symptoms.

[0076] The term "reference sample" is a sample that can be compared with another sample, such as a test sample, to characterize the sample being compared. The reference sample will have certain characteristic properties as the basis for comparison with the test sample.

[0077] In some implementations, the kit includes, but is not limited to, ELISA detection kits, immunofluorescence detection kits, FACS kits, and IHC detection kits.

[0078] In some implementations, the kit is selected from ELISA detection kits.

[0079] In some embodiments, the product is detected by an ELISA method using peptidin proteins or functional fragments thereof, or polypeptides containing peptidins.

[0080] In some embodiments, the polypeptide containing arginine vasopressin is an arginine vasopressin precursor.

[0081] The seventh aspect of the present invention provides any of the following methods:

[0082] (1) A method for preparing the anti-peptide antibody or antigen-binding fragment thereof as described in the first aspect of the present invention, the method comprising the following steps: culturing the modified host cells or host cell populations containing the modified host cells as described in the third aspect of the present invention; (2) A method for detecting KIR3DL2 or a fragment thereof in a test sample, the method comprising the following steps: contacting the test sample with the anti-peptidase antibody or its antigen-binding fragment as described in the first aspect of the present invention, or contacting the test sample with an antibody derivative of the anti-peptidase antibody or its antigen-binding fragment as described in the fifth aspect of the present invention, or contacting the test sample with a product for detecting peptidase protein or its functional fragment or a polypeptide containing peptidase as described in the sixth aspect of the present invention, and detecting the formation of a complex of the anti-peptidase antibody or its antigen-binding fragment or the antibody derivative of the anti-peptidase antibody or its antigen-binding fragment with peptidase protein or its functional fragment or a polypeptide containing peptidase; In some embodiments, the polypeptide containing arginine vasopressin is an arginine vasopressin precursor; (3) A method for preparing the modified host cell or host cell population containing the present invention according to the fourth aspect of the present invention, the method comprising the following steps: introducing the polynucleotide molecule according to the second aspect of the present invention or the vector according to the third aspect of the present invention into the host cell.

[0083] In some implementations, the vector is introduced into the host cell by means including but not limited to physical methods, chemical methods, and biological methods.

[0084] In some implementations, the physical methods include, but are not limited to, the use of calcium phosphate precipitation, lipid transfection, particle bombardment, microinjection, and electroporation.

[0085] In some implementations, the chemical method includes, but is not limited to, the use of colloidal dispersion systems and lipid-based systems.

[0086] In some implementations, the biological method includes, but is not limited to, the use of DNA vectors, lentiviral vectors, poxvirus vectors, herpes simplex virus vectors, adenovirus vectors, and adeno-associated virus vectors.

[0087] In some embodiments, the term "sample" refers to similar fluids, cells, or tissues separated from the subject (e.g., surgically removed tumor tissue, biopsy sections, including fine-needle aspirated tissue) and collections of fluids, cells, or tissues present in the subject. In some embodiments, the sample is a biological fluid. Biological fluids are generally liquids at physiological temperatures and may include naturally occurring fluids present in, extracted from, expressed from, or otherwise extracted from the subject or biological source. Some biological fluids originate from a specific tissue, organ, or localized area, and some other biological fluids may be more systemic or present in the subject or biological source. Examples of biological fluids include blood, serum, and serous fluid, plasma, lymph, urine, saliva, cystic fluid, tears, feces, sputum, mucosal secretions of secretory tissues and organs, vaginal secretions, ascites such as those associated with non-solid tumors, fluids from the pleura, pericardium, peritoneum, abdomen, and other body cavities, fluids collected via bronchial lavage, etc. Biofluids may also include liquid solutions that come into contact with the subject or biological sources, such as cell and organ culture media, including cell or organ conditioned media, lavage solutions, etc. As used herein, the term "sample" encompasses substances taken from or present in the subject.

[0088] In some implementations, the subject may be human or non-human. Furthermore, the subject may preferably be a mammal (e.g., human or non-human). Examples of mammals include, but are not limited to, any member of the mammalian class: humans, non-human primates (e.g., chimpanzees) and other apes and monkeys; livestock such as cattle, horses, sheep, goats, and pigs; domestic animals such as rabbits, dogs, and cats; and laboratory animals including rodents such as rats, mice, and guinea pigs.

[0089] The eighth aspect of the present invention provides any of the following applications: (1) The use of the anti-peptidase antibody or its antigen-binding fragment as described in the first aspect of the present invention, and the antibody derivative of the anti-peptidase antibody or its antigen-binding fragment as described in the fifth aspect of the present invention, in the preparation of products for detecting peptidase protein or its functional fragment or peptides containing peptidase. (2) The use of the anti-peptidase antibody or its antigen-binding fragment as described in the first aspect of the present invention, the antibody derivative of the anti-peptidase antibody or its antigen-binding fragment as described in the fifth aspect of the present invention, and the product for detecting peptidase protein or its functional fragment or peptide containing peptidase as described in the sixth aspect of the present invention in the preparation of a product for diagnosing whether a subject has a peptidase-related disease or condition. (3) The use of the anti-peptidase antibody or antigen-binding fragment thereof described in the first aspect of the present invention, the antibody derivative of the anti-peptidase antibody or antigen-binding fragment thereof described in the fifth aspect of the present invention, and the product for detecting peptidase protein or functional fragment thereof or containing peptidase peptides described in the sixth aspect of the present invention in the preparation of products for predicting the prognosis of peptidase-related diseases or conditions.

[0090] In some implementations, diseases or conditions related to peptides include, but are not limited to, cardiovascular diseases, respiratory diseases, and infectious diseases.

[0091] In some implementations, the cardiovascular disease includes, but is not limited to, coronary syndrome, myocardial infarction, and stroke.

[0092] In some implementations, the coronary syndrome is acute coronary syndrome.

[0093] In some implementations, the myocardial infarction is an acute myocardial infarction.

[0094] In some implementations, the stroke is an acute stroke.

[0095] In some implementations, the respiratory diseases include, but are not limited to, pulmonary embolism, pneumonia, and obstructive pulmonary disease.

[0096] In some implementations, the pneumonia includes, but is not limited to, chronic obstructive pulmonary disease (COPD) and severe pneumonia.

[0097] In some implementations, the obstructive pulmonary disease includes, but is not limited to, chronic obstructive pulmonary disease.

[0098] In some implementations, the infectious disease includes, but is not limited to, sepsis.

[0099] The ninth aspect of the present invention provides a method for diagnosing whether a subject suffers from a peptidin-related disease or condition, characterized in that the method includes contacting the subject to be tested with an anti-peptidin antibody or its antigen-binding fragment as described in the first aspect of the present invention, or contacting the subject to be tested with an antibody derivative of an anti-peptidin antibody or its antigen-binding fragment as described in the fifth aspect of the present invention, or contacting the subject to be tested with a product for detecting peptidin protein or its functional fragment or a polypeptide containing peptidin as described in the sixth aspect of the present invention, detecting the formation of a complex of the anti-peptidin antibody or its antigen-binding fragment or its antibody derivative with peptidin protein or its functional fragment or a polypeptide containing peptidin; if the formation of the complex is detected, the subject is diagnosed with a peptidin-related disease or condition.

[0100] The advantages and beneficial effects of this invention are as follows: This invention provides an anti-human and anti-peptide monoclonal antibody and its applications. The antibody comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region includes heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 as shown in SEQ ID NO. 8; the light chain variable region includes light chain CDR1, light chain CDR2, and light chain CDR3 as shown in SEQ ID NO. 9. The antibody of this invention can effectively bind to and against peptides, and its suitability for ELISA detection platforms has been verified. The antibody of this invention has broad application prospects and practical value. Attached Figure Description

[0101] Figure 1 This is the result of antigenicity prediction; Figure 2 This is a graph showing the ELISA titer of rabbit serum 8 weeks after immunization. Figure 3 This is a titer graph of the ELISA detection in the supernatant after B cell sorting. Detailed Implementation

[0102] The present invention will be further described below with reference to embodiments. The following description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any other way. Any person skilled in the art may make equivalent modifications to the disclosed technical content to create equivalent embodiments. Any simple modifications or equivalent changes made to the following embodiments based on the technical essence of the present invention without departing from the scope of the invention are all within the protection scope of the present invention.

[0103] Example: Preparation and functional testing of antibodies I. Experimental Methods Immunization was performed using KLH-conjugated human Copeptin antigen peptide and Bio-Long Quick Antibody™ adjuvant. The KLH-conjugated peptide and rabbit Quick Antibody™-8-week rapid adjuvant were mixed thoroughly and injected intramuscularly into the thigh of rabbits. A total of three immunizations were administered over eight weeks. After the immunization cycle, ELISA plates were coated with bovine serum albumin (BSA)-conjugated human Copeptin peptide for detection. The titer of anti-Copeptin antibodies in rabbit serum was detected by ELISA. Rabbits that passed the test were treated with air injection, and lymphocytes were obtained from the spleen of the immunized rabbits. Subsequently, specific single B cell sorting and enrichment were performed using biotin-labeled bovine serum albumin (BSA)-conjugated Copeptin peptide. Rabbit B cells that recognize the antigen Copeptin were cultured using rabbit single B cell culture technology. After 7-10 days of culture, the antibody supernatant of the cultured single B cells in a 96-well plate was detected by ELISA. The titer of the antibody against the recombinant human HSA-Copeptin fusion protein was detected by ELISA, and rabbit B cell clones that recognize human Copeptin were obtained.

[0104] The steps for screening positive B cells using an ELISA assay are as follows: 1. Coating antigen: Bovine serum albumin (BSA) conjugated with Copeptin peptide at a concentration of 2 μg / mL, 100 μL / well, is coated onto the microplate and incubated overnight at 4°C. 2. Cleaning: Use a plate washer to clean the ELISA plate 4-5 times; 3. Blocking: Prepare 0.5% BSA, add 300 μL of blocking solution to each well, and block at room temperature for 2 hours; 3. Cleaning: Use a plate washer to clean the ELISA plate 4-5 times; 4. Add sample: Add 100 μL of sample to each well and incubate at 37℃ for 1 h; 5. Cleaning: Use a plate washer to clean the ELISA plate 4-5 times; 6. Add secondary antibody: Prepare goat anti-rabbit HRP secondary antibody (Anti-Rabbit IgG (H+L), HRPConjugate, W4011, Promega) at a ratio of 1:8000. Add 100 μL of secondary antibody solution to each well, place on a shaker, adjust the speed to medium, and incubate in the dark for 40 min. 7. Cleaning: Use a plate washer to clean the ELISA plate 4-5 times; 8. Color development: Add 100 μL of TMB color development solution to each well, incubate in the dark, check the color development at any time, incubate for no more than 5 minutes, and stop the incubation immediately when the color turns dark blue; 9. Stop color development: Add 100 μL of stop solution to each well; 10. Reading: After turning on the microplate, read the OD452 value and save the data.

[0105] By extracting RNA from B cells and reverse transcribing cDNA, the full-length genes of the variable regions of the light and heavy chains in the B cell clones were amplified using specific primers. The nucleotides expressing the antibody were then constructed into the pcDNA3.4 expression vector containing the constant regions of the rabbit IgG heavy chain Kapa chain and light chain. After in vitro recombinant expression in HEK293 suspension cells, a high concentration of recombinant rabbit monoclonal antibody was obtained, and its titer was re-detected by ELISA.

[0106] The steps for detecting Copeptin using the serial dilution method via ELISA are as follows: 1. Coating antigen: Human serum albumin HAS-Copeptin fusion protein recombinantly expressed in 293 suspension cells was serially diluted at a concentration of mol / L, 100 μL / well, and coated onto the microplate at 4°C overnight; 2. Cleaning: Use a plate washer to clean the ELISA plate 4-5 times; 3. Blocking: Prepare 0.5% BSA, add 300 μL of blocking solution to each well, and block at room temperature for 2 hours; 3. Cleaning: Use a plate washer to clean the ELISA plate 4-5 times; 4. Add purified antibody: Add 100 μL of purified antibody diluted 1:3000 to each well and incubate at 37°C for 1 hour; 5. Cleaning: Use a plate washer to clean the ELISA plate 4-5 times; 6. Add secondary antibody: Prepare goat anti-rabbit HRP secondary antibody at a ratio of 1:8000. Add 100 μL of secondary antibody solution to each well, place on a shaker, adjust the speed to medium, and incubate in the dark for 40 min. 7. Cleaning: Use a plate washer to clean the ELISA plate 4-5 times; 8. Color development: Add 100 μL of TMB color development solution to each well, incubate in the dark, check the color development at any time, incubate for no more than 5 minutes, and stop the incubation immediately when the color turns dark blue; 9. Stop color development: Add 100 μL of stop solution to each well; 10. Reading: After turning on the microplate, read the OD452 value and save the data.

[0107] The HAS-Copeptin fusion protein and the control HAS-His fusion protein were detected by serial dilution. The purified antibody was expressed at a concentration of 1 mg / ml and coated with 5 μg / ml HAS-Copeptin fusion protein and 5 μg / ml HAS-His fusion protein, respectively. 2A7 was diluted at 1:1000, 1:3000, 1:9000, 1:27000, 1:81000, 1:243000, and 1:729000 to serve as primary antibodies. In addition, control wells without antibody were prepared for ELISA experiments.

[0108] II. Experimental Results The KLH-conjugated antigen polypeptide sequence and the amino acid sequence of the Copeptin polypeptide antigen are SEQ ID NO.1: ASDRSNATQLDGPAGALLLRLVQLAGAPEPFEPAQPDAY. Antigenicity prediction revealed that AGAPEPFEPAQPDAY has strong antigenicity, scoring 16 points. A score greater than 3 points can be considered an excellent antigen. Figure 1 ).

[0109] Figure 2 This is a graph showing the ELISA titer of rabbit serum 8 weeks after immunization in this invention.

[0110] Figure 3 The image shows the titer of the supernatant after B cell sorting in this invention by ELISA. Among them, the cells with OD values ​​greater than 2.8 are 1A2, 2E5, 2A7, 2E7, 2E11, 2F7, 2F9, 3C11, and 3A11.

[0111] The amino acid sequences of the antibodies, after sequencing, are shown in Table 1 below: Table 1 Antibody Sequences

[0112] Table 2 shows the results of detecting coated human Copeptin using a serial dilution method after expressing and purifying the antibody from monoclonal antibody strain 2A7. The concentration of the purified antibody after expression was 1 mg / ml, and the results were obtained by detecting the purified antibody at a dilution of 1:3000.

[0113] Table 2 Antibody binding activity

[0114] Table 3 shows the experimental results of detecting the coated HSA-CPP fusion protein and the control HSA-His fusion protein using a serial dilution method after expressing purified antibody from monoclonal antibody strain 2A7 at a concentration of 1 mg / ml. The antibody obtained in this invention can specifically bind to the Copeptin antigen without binding to HSA.

[0115] Table 3 Antibody Specificity

[0116] The above description of the embodiments is only for understanding the method and core ideas of the present invention. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from the principles of the invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims

1. An anti-peptide antibody or its antigen-binding fragment, characterized in that, The anti-peptide antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 as shown in SEQ ID NO.8; and the light chain variable region comprises light chain CDR1, light chain CDR2, and light chain CDR3 as shown in SEQ ID NO.

9. Preferably, the amino acid sequences of the heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 are as shown in SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4, respectively, and the amino acid sequences of the light chain CDR1, light chain CDR2 and light chain CDR3 are as shown in SEQ ID NO.5, SEQ ID NO.6 and SEQ ID NO.7, respectively.

2. The anti-peptide antibody or its antigen-binding fragment as described in claim 1, characterized in that, The heavy chain variable region has an amino acid sequence as shown in SEQ ID NO.8 or an amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO.8; the light chain variable region has an amino acid sequence as shown in SEQ ID NO.9 or an amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO.

9.

3. A polynucleotide molecule, characterized in that, The polynucleotide molecule encodes the anti-peptide antibody or its antigen-binding fragment as described in any one of claims 1-2.

4. A carrier, characterized in that, The carrier comprises the polynucleotide molecule of claim 3; Preferably, the vector comprises a selection from plasmid vectors; Preferably, the plasmid vector is selected from expression vectors; Preferably, the expression vector is selected from pcDNA3.

4.

5. A modified host cell or a population of host cells containing the same, characterized in that, The modified host cell comprises the anti-peptidase antibody or its antigen-binding fragment as described in any one of claims 1-2, or the polynucleotide molecule as described in claim 3, or the vector as described in claim 4; Preferably, the host cell is selected from eukaryotic cells; Preferably, the eukaryotic cells are selected from mammalian cells; Preferably, the mammalian cells include HEK293F cells, HEK293T cells, and CHO cells; Preferably, the mammalian cells are selected from HEK293F cells.

6. An antibody derivative of an anti-peptide antibody or its antigen-binding fragment, characterized in that, The antibody derivative comprises a complex formed by directly or indirectly conjugating the anti-peptide antibody or its antigen-binding fragment as described in any one of claims 1-2 to a detectable marker. Preferably, the detectable markers include radionuclides, chemiluminescent agents, bioluminescent agents, paramagnetic ions, enzymes, and photosensitizing diagnostic agents; Preferably, the radionuclide includes 18 F, 52 Fe、 62 Cu、 64 Cu、 67 Cu、 86 Y、 90 Y、 89 Zr、 120 I, 123 I, 124 I, 125 I, 131 I, 13 N、 15 O、 186 Re、 188 Re、 51 Mn, 55 Co、 72 As; Preferably, the chemiluminescent agent includes luminol, isoluminol, aromatic acridine ester, imidazole, acridine salt, and oxalate ester; Preferably, the bioluminescent agent includes luciferin, luciferase, and jellyfish luminescent protein; Preferably, the paramagnetic ions include chromium (III), manganese (II), iron (III), iron (II), cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III), holmium (III), and erbium (III); Preferably, the enzyme includes horseradish peroxidase, alkaline phosphatase, glucose oxidase, β-D-galactosidase, urease, catalase, or glucosylamylase. Preferably, the photosensitive diagnostic agent includes dihydroxysilylphthalocyanine, methylene blue, protoporphyrin, hematoporphyrin, and photoporphyrin.

7. A product for detecting and peptidin proteins or functional fragments thereof or polypeptides containing and peptidins, characterized in that, The product comprises the anti-peptide antibody or its antigen-binding fragment as described in any one of claims 1-2, the polynucleotide molecule as described in claim 3, the vector as described in claim 4, and / or the host cell or host cell population containing the present as described in claim 5. Preferably, the product includes a reagent kit, test strips, reagents, and a chip; Preferably, the kit includes an ELISA detection kit; Preferably, the polypeptide containing arginine vasopressin is an arginine vasopressin precursor.

8. The product for detecting and peptidin proteins or functional fragments thereof or polypeptides containing and peptidins according to claim 7, characterized in that, The product is detected by ELISA using peptide proteins or functional fragments thereof, or polypeptides containing peptides.

9. Any one of the following methods: (1) A method for preparing the anti-peptide antibody or its antigen-binding fragment as described in any one of claims 1-2, characterized in that, The method includes the following steps: culturing the modified host cells of claim 4 or a population of host cells containing them; (2) A method for detecting anti- and peptidoglycan or its fragments in a test sample, characterized in that the method comprises the following steps: contacting the test sample with the anti- and peptidoglycan antibody or its antigen-binding fragment as described in any one of claims 1-2, or contacting the test sample with an antibody derivative of the anti- and peptidoglycan antibody or its antigen-binding fragment as described in claim 6, or contacting the test sample with a product for detecting peptidoglycan protein or its functional fragments or a polypeptide containing peptidoglycan as described in any one of claims 7-8, and detecting the formation of a complex of the anti- and peptidoglycan antibody or its antigen-binding fragments or its antibody derivatives with peptidoglycan protein or its functional fragments or a polypeptide containing peptidoglycan; Preferably, the polypeptide containing arginine vasopressin is an arginine vasopressin precursor; (3) A method for preparing the modified host cell or host cell population containing the present invention as described in claim 5, characterized in that the method comprises the following steps: introducing the polynucleotide molecule of claim 3 or the vector of claim 4 into the host cell.

10. Any of the following applications: (1) The use of the anti-peptidase antibody or antigen-binding fragment thereof as described in any one of claims 1-2, or the antibody derivative thereof as described in claim 6, in the preparation of products for detecting peptidase protein or its functional fragments or peptides containing peptidase; (2) The use of the anti-peptidase antibody or antigen-binding fragment thereof as described in any one of claims 1-2, the antibody derivative of the anti-peptidase antibody or antigen-binding fragment thereof as described in claim 6, and the product for detecting peptidase protein or functional fragment thereof or peptide containing peptidase as described in any one of claims 7-8 in the preparation of a product for diagnosing whether a subject has a peptidase-related disease or condition. (3) The use of the anti-peptidase antibody or antigen-binding fragment thereof as described in any one of claims 1-2, the antibody derivative of the anti-peptidase antibody or antigen-binding fragment thereof as described in claim 6, and the product for detecting peptidase protein or functional fragment thereof or peptide containing peptidase as described in any one of claims 7-8 in the preparation of products for predicting the prognosis of peptidase-related diseases or conditions. Preferably, diseases or conditions related to peptides include cardiovascular diseases, respiratory diseases, and infectious diseases; Preferably, the cardiovascular disease includes coronary syndrome, myocardial infarction, and stroke; Preferably, the respiratory diseases include pulmonary embolism, pneumonia, and obstructive pulmonary disease; Preferably, the infectious disease includes sepsis.

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