Anti-s100a8 / a9 monoclonal antibody for treating severe acute pancreatitis and use thereof

Abstract

Provided are an antibody binding to a dimer of S100A8 and S100A9 or an antigen-binding fragment thereof, and use thereof in treating pancreatitis.

Description

An anti-S100A8 / A9 monoclonal antibody for the treatment of severe acute pancreatitis and its application.

[0001] Cross-references to related applications

[0002] This application claims priority to Chinese Patent Application No. 2024117085617, filed with the China National Intellectual Property Administration on November 26, 2024, and to Chinese Patent Application No. 2025102626624, filed with the China National Intellectual Property Administration on March 6, 2025, the entire contents of which are incorporated herein by reference for all purposes. Technical Field

[0003] This application relates to the field of biomedical technology, specifically to an anti-S100A8 / A9 monoclonal antibody for the treatment of severe acute pancreatitis and its application. Background Technology

[0004] Acute pancreatitis (AP) is a disease that can cause a syndrome of local or systemic inflammatory response. The severity varies significantly among individuals, with an overall mortality rate of approximately 10%-15%. Most AP patients recover within a week, but nearly 20% progress to severe acute pancreatitis (SAP). SAP patients may experience persistent organ failure within two weeks of diagnosis, resulting in a mortality rate exceeding 40%. Currently, there are no drugs that can effectively reduce the mortality rate (in-hospital mortality or mortality within 6 months) of SAP patients. Therefore, early intervention and preventing AP from progressing to SAP are key treatment approaches for the prevention and treatment of SAP.

[0005] S100A8 / A9, also known as myeloid-related protein 8 / 14 (MRP8 / 14), belongs to the S100 family of Ca 2+S100A8 / A9 is a binding protein widely expressed in neutrophils and monocytes. Previous reports have indicated that S100A8 / A9 can further induce neutrophil chemotaxis and amplify the inflammatory cascade through signal transduction via toll-like receptor-4 (TLR-4) or receptor for advanced glycation end products (RAGE). Treatment of endothelial cells with exogenous S100A8 / A9 increases their adhesion molecule expression, chemokine secretion, and cell permeability. Therefore, both play important roles in inflammatory and immune responses and have become biomarkers of the degree of inflammation in infectious and autoimmune diseases. Previous studies have shown elevated S100A9 levels in the ductal fluid of patients with pancreatic ductal adenocarcinoma (PDAC), indicating a poor prognosis; significantly elevated S100A9 levels were also observed in the plasma of mice and patients with acute pancreatitis, confirming S100A9's important regulatory role in pancreatic duct injury. In summary, S100A8 / A9 has significant clinical implications; however, to date, there are no drugs that can specifically block S100A8 / A9. Paquinimod, an orally active small molecule inhibitor, has been investigated for the treatment of diseases such as idiopathic pulmonary fibrosis and COVID-19, but its targeting is poor. It does not directly bind to S100A8 / A9, but rather exerts its effect by inhibiting the activity of S100A9, thereby reducing its binding to TLR-4. Therefore, there is an urgent need to develop a highly specific and affinity humanized S100A8 / A9 monoclonal antibody that can precisely target diseased cells or molecules, thereby reducing damage to normal cells.

[0006] Nanobodies (Nb) are a novel type of antibody comprising four conserved framework regions (FRs) and three complementarity-determining regions (CDRs). The CDRs of Nb generally have a longer amino acid count than those of traditional human or mouse CDR3s, allowing for the formation of convex ring structures and numerous CDR rings exposed to the solvent. Compared to traditional antibodies (which typically have concave or flat antigen-binding sites), Nb exhibits better specificity and affinity for antigen binding. Furthermore, due to their smaller molecular weight, Nb can more easily bind to some difficult-to-reach antigenic epitopes. In addition, high-quality Nb can overcome the risk of Fc-related antibody-dependent enhancement, making it a promising candidate for neutralizing antibody therapy. Nanobodies have high solubility and can be formulated into various dosage forms, including injections, inhalers, ophthalmic preparations, and oral medications. Compared to traditional macromolecular antibodies, nanobodies have a relatively short half-life, averaging about 20 hours. By linking nanobodies to albumin, the half-life can be extended to approximately two weeks. These advantages have made nanobodies a hot topic in the development of next-generation therapeutic antibodies. Therefore, nanobodies have enormous development potential and application prospects.

[0007] It should be noted that the methods described in this section are not necessarily methods that had been previously conceived or adopted. Unless otherwise specified, no method described in this section should be assumed to be prior art simply because it is included in this section. Similarly, unless otherwise specified, the issues mentioned in this section should not be considered to be accepted in any prior art. Summary of the Invention

[0008] To address the aforementioned issues, this application provides a nanobody or antigen-binding fragment of a dimeric antibody combining S100A8 and S100A9, and demonstrates its effectiveness in treating acute pancreatitis, particularly severe acute pancreatitis. Furthermore, this application also provides the use of an antibody or antigen-binding fragment of a dimeric antibody combining S100A8 and S100A9 in the treatment of pancreatitis.

[0009] According to one embodiment of this application, this application provides a nanobody or antigen-binding fragment thereof that binds a dimer of S100A8 and S100A9, wherein the nanobody or antigen-binding fragment thereof includes a heavy chain variable region, and the heavy chain variable region contains at least the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3.

[0010] According to one embodiment of this application, a polynucleotide is also provided, said polynucleotide encoding the nanobody or its antigen-binding fragment described in this application.

[0011] According to one embodiment of this application, a recombinant vector is also provided, the recombinant vector comprising the polynucleotide described in this application.

[0012] According to one embodiment of this application, a host cell is also provided, the host cell comprising the polynucleotides described in this application and / or the recombinant vectors described in this application.

[0013] According to one embodiment of this application, an antibody is also provided, the antibody comprising the nanobody or antigen-binding fragment described in this application.

[0014] According to one embodiment of this application, a chimeric antigen receptor is also provided, the chimeric antigen receptor comprising the nanobody or its antigen-binding fragment described in this application.

[0015] According to one embodiment of this application, an antibody conjugate is also provided, the antibody conjugate comprising the nanobody or its antigen-binding fragment described in this application, and further comprising a cytotoxin or a radioisotope.

[0016] According to one embodiment of this application, a pharmaceutical composition is also provided, the pharmaceutical composition comprising the nanobody or antigen-binding fragment described in this application.

[0017] According to one embodiment of this application, a kit is also provided, the kit comprising the nanobody or antigen-binding fragment thereof described in this application.

[0018] According to one embodiment of this application, the use of the nanobody or antigen-binding fragment thereof described in this application, the antibody described in this application, the chimeric antigen receptor described in this application, the antibody conjugate described in this application, the pharmaceutical composition described in this application, and / or the kit described in this application in the preparation of a medicament, pharmaceutical composition, or kit for detecting, preventing, alleviating, or treating a disease is also provided.

[0019] According to one embodiment of this application, a detection method is also provided, the method comprising using the nanobody or its antigen-binding fragment described in this application to detect the dimer expression level of S100A8 and S100A9 in a sample.

[0020] According to one embodiment of this application, a method for preventing, alleviating, or treating a disease is also provided, the method comprising administering to a subject in need the nanobody or antigen-binding fragment described in this application, the antibody described in this application, the chimeric antigen receptor described in this application, the antibody-drug conjugate described in this application, the pharmaceutical composition described in this application, and / or the kit described in this application.

[0021] According to one embodiment of this application, an antibody or antigen-binding fragment thereof that binds to a dimer of S100A8 and S100A9 is also provided for use in the preparation of a medicament, pharmaceutical composition or kit for detecting, preventing, alleviating or treating pancreatitis or pancreatic tissue damage, wherein the heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 of the antibody or antigen-binding fragment are the amino acid sequences shown in SEQ ID NO: 19, 20, 21, 22, 23, and 24, respectively.

[0022] According to one embodiment of this application, a method for preventing, alleviating, or treating pancreatitis or pancreatic tissue damage is also provided, the method comprising administering to a subject in need an antibody or antigen-binding fragment thereof that binds to a dimer of S100A8 and S100A9, wherein the heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 of the antibody or antigen-binding fragment are the amino acid sequences shown in SEQ ID NO: 19, 20, 21, 22, 23, and 24, respectively.

[0023] It should be understood that the description in this section is not intended to identify key or essential features of the embodiments of this application, nor is it intended to limit the scope of this application. Other features of this application will become readily apparent from the following description. Attached Figure Description

[0024] The accompanying drawings exemplify embodiments and form part of the specification, serving together with the textual description to explain exemplary implementations of the embodiments. The illustrated embodiments are for illustrative purposes only and do not limit the scope of the claims. Throughout the drawings, the same reference numerals refer to similar but not necessarily identical elements.

[0025] Figure 1 is a flowchart of the preparation process of the anti-S100A8 / A9 nanobody provided in one embodiment of this application.

[0026] Figure 2 shows the results of biological activity assays for the anti-S100A8 / A9 nanobody in Example 2. Figure 2A shows the results of the antigen binding assay; Figure 2B shows the results of the cellular activity assay.

[0027] Figure 3 shows the therapeutic effect of the anti-S100A8 / A9 nanobody on a severe acute pancreatitis animal model in Example 4. Specifically, Figure 3A shows HE staining to assess pancreatic tissue damage; Figure 3B shows IHC (F4 / 80) staining to assess macrophage infiltration in pancreatic tissue; Figure 3C shows IHC (Ly6G) staining to assess neutrophil infiltration in pancreatic tissue; and Figure 3D shows ELISA detection of serum inflammatory factors (IL-1beta, IL-6).

[0028] Figure 4 shows a transmission electron microscope (TEM) image of the submicroscopic structure of pancreatic tissue in Example 4. Figure 4A shows pancreatic tissue from a mouse model of severe acute pancreatitis; Figure 4B shows pancreatic tissue from mice in the monoclonal antibody treatment group.

[0029] Figure 5 shows the HE staining results and statistical chart of pancreatic tissue in Example 6. Figure 5A shows the HE staining results of pancreatic tissue from a mouse model of severe acute pancreatitis; Figure 5B shows the statistical chart of HE staining results obtained according to Figure 5A and the pathological tissue scoring criteria in Table 5. Detailed Implementation

[0030] Unless otherwise stated, all figures used in this specification and claims to represent content, concentration, proportion, mass, volume, time, temperature, thickness, technical effect, etc., should in any instance be understood to be modified by the terms “about” or “approximately”. Therefore, unless indicated to the contrary, the numerical parameters listed in the following specification and appended claims are approximate values. They can vary for those skilled in the art depending on the desired properties and effects sought through this disclosure, and each numerical parameter should be interpreted according to the number of significant figures and conventional rounding methods or in a manner understood by those skilled in the art.

[0031] Although the numerical ranges and parameters described in this disclosure are approximate, the values ​​presented in the specific embodiments are provided as precisely as possible. However, any numerical value will inherently contain some errors, which are necessarily caused by the standard deviation found in its corresponding test measurements. Each numerical range given in this specification will include every narrower numerical range falling within that wider range, as if these narrower numerical ranges were explicitly stated herein.

[0032] Unless otherwise stated or contradicted by the context, the terms or expressions used herein should be read in the context of the document and as understood by one of ordinary skill in the art. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0033] When used in this document, the expression “A and / or B” includes three cases: (1) A; (2) B; and (3) A and B. The expression “A, B and / or C” includes seven cases: (1) A; (2) B; (3) C; (4) A and B; (5) A and C; (6) B and C; and (7) A, B and C. The meanings of similar expressions can be deduced by analogy.

[0034] When used in this document, the terms “include,” “contain,” and “comprising” mean that other elements are not excluded in addition to the listed elements.

[0035] The terms “nucleic acid” and “polynucleotide” used in this application are used interchangeably to refer to a polymeric form of nucleotides of any length, including deoxyribonucleotides, ribonucleotides, combinations thereof, and analogues.

[0036] In this application, the terms "polypeptide" and "peptide" are used interchangeably and refer to a polymer of amino acids of any length. Therefore, polypeptides, oligopeptides, proteins, antibodies, and enzymes are all included in the definition of polypeptide.

[0037] The term "identity" as used in this application refers to the degree of similarity between a pair of sequences (nucleotides or amino acids). Identity is determined by dividing the number of identical residues by the total number of residues and multiplying the quotient by 100 to obtain a percentage. Gap is not considered when evaluating identity. Therefore, two copies of an identical sequence have 100% identity, but sequences with deletions, additions, or substitutions may have a lower degree of identity. Those skilled in the art are aware of computer programs that can be used to determine sequence identity, such as those employing algorithms like BLAST. BLAST nucleotide searches are performed using the NBLAST program, and BLAST protein searches are performed using the BLASTP program, with the default parameters of each program used.

[0038] The terms “S100A8 / A9 dimer,” “S100A8 / S100A9 dimer,” “S100A8 / S100A9 heterodimer,” “S100A8 / S100A9 polypeptide,” “S100A8 / S100A9 protein,” “S100A8 and S100A9 dimer,” or “S100A8 and S100A9 heterodimer” used in this application have the same meaning and can be used interchangeably. They refer to a protein complex in the form of a heterodimer formed by calcium-binding protein A8 (S100A8) and calcium-binding protein A9 (S100A9) in the presence of calcium and zinc ions.

[0039] The term "antibody" as used in this application refers to a specific immunoglobulin targeting an antigenic site. Antibodies can be manufactured according to methods known in the art. Antibodies can take the form of polyclonal or monoclonal antibodies, antibody fragments (e.g., Fab, Fab', F(ab')2, and Fv fragments), single-chain Fv (scFv) antibodies, multispecific antibodies (e.g., bispecific antibodies), monospecific antibodies, monovalent antibodies, chimeric antibodies, fully human antibodies, human antibodies, fusion proteins containing an antigen-binding site, and any other modified immunoglobulin molecule containing an antigen-binding site, provided that the antibody exhibits the desired biobinding activity.

[0040] The term "antigen-binding fragment" as used in this application refers to one or more portions of an antibody that retain the specificity for binding to a target antigen. Antigen-binding fragments include, but are not limited to, VHH fragments and fragments containing CDRs.

[0041] The term "specific binding" as used in this application refers to the non-covalent interaction between an antibody or its antigen-binding fragment and the antigen. The strength or affinity of this interaction can be expressed by the equilibrium dissociation constant (KD or Kd) of the antigen and the corresponding antibody: the smaller the KD value, the stronger the binding strength between the epitope and the antibody. The equilibrium dissociation constant (KD) is calculated as the ratio of Koff / kon, where the "binding rate constant (Kon)" and the "dissociation rate constant (Koff)" can be determined by calculating the concentration and the actual binding and dissociation rates (see Nature 361:186-87 (1993)).

[0042] The precise amino acid sequence boundaries of a given complementarity-determining region (CDR) or backbone region (FR) can be readily determined using many numbering schemes well-known in the art. These schemes include: Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5th edition, Public Health Service, National Institutes of Health, Bethesda, Maryland (“Kabat” numbering scheme); Al-Lazikani et al. (1997) JMB 273, 927-948 (“Chothia” numbering scheme); MacCallum et al., J. Mol. Biol. 262: 732-745 (1996), “Antibody-antigen interactions: Contact analysis and binding sitetopography,” J. Mol. Biol. 262, 732-745 (“Contact” numbering scheme); Lefranc MP et al., “IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains,” Dev Comp Immunol, January 2003; 27(1):55-77 (“IMGT” numbering scheme); Honegger A and Plückthun A, “Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool,” JMol Biol, June 8, 2001; 309(3):657-70 (“Aho” numbering scheme); and Martin et al., “Modeling antibody hypervariable loops: a combined algorithm,” PNAS, 1989, 86(23):9268-9272 (“AbM” numbering scheme).

[0043] The boundaries of a given CDR or FR can vary depending on the protocol used for identification. For example, the Kabat protocol is based on structure alignment, while the Chothia protocol is based on structural information. Both the Kabat and Chothia protocols number antibodies based on the length of the most common antibody region sequences, where insertions are indicated by insert letters (e.g., "30a") and deletions occur in some antibodies. These two protocols place certain insertions and deletions (indels) in different positions, resulting in different numbering schemes. The Contact protocol is based on the analysis of complex crystal structures and is similar to the Chothia numbering scheme in many ways. The AbM protocol is a compromise between the Kabat and Chothia definitions, based on the scheme used by the Oxford Molecular AbM antibody modeling software.

[0044] Therefore, unless otherwise specified, it should be understood that the “CDR” of a given antibody or its region (such as its variable region) encompasses the CDRs defined by any of the above-described schemes or other known schemes. For example, in specifying that a particular CDR (e.g., CDR3) contains a given amino acid sequence, it should be understood that such a CDR may also have the sequence of the corresponding CDR (e.g., CDR3) as defined by any of the above-described schemes or other known schemes. Similarly, unless otherwise specified, it should be understood that the FR of a given antibody or its region (such as its variable region) encompasses the FRs defined by any of the above-described schemes or other known schemes. Unless specifically indicated, the numbering scheme used herein to delineate the boundaries between CDRs and FRs adopts the Kabat scheme.

[0045] The terms "exogenous" and "heterogeneous" used in this application are used interchangeably and refer to sources different from the native (original) organism, such as organisms derived from another species. The terms "heterogeneous gene" or "exogenous gene" used in this application refer to genes that do not naturally exist in the host organism and are introduced into the host organism through gene transfer.

[0046] The term "vector" as used in this application refers to a self-replicating DNA molecule that transfers a foreign target gene into a host organism, and is often in the form of a circular double-stranded DNA molecule. Typical vectors include plasmids, viruses, bacteriophages, kinases, and mini-chromosomes. Among these, plasmids are the most common form of vector, referring to circular double-stranded DNA that can accept foreign nucleic acid fragments and replicate in prokaryotic or eukaryotic cells.

[0047] In this application, the terms "expression vector" and "recombinant vector" are used interchangeably, referring to a vector containing a foreign gene and also including regulatory elements for expression in a specified host organism. Introducing the expression vector into a suitable host organism enables it to express the inserted target gene.

[0048] In this application, "transformation" refers to the transfer of a foreign gene into a host organism, such as a host cell, resulting in stable genetic inheritance. The transformed gene can be in plasmid form retained in the host organism or integrated into the host organism's genome. A host organism containing the transformed gene is referred to as a "transgenic," "recombinant," "transformed," or "engineered" organism. Transformation of the host organism using an expression vector can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryote, competent cells capable of uptake DNA can be harvested after the exponential growth phase and treated with the CaCl2 method, the steps of which are well known in the art. If necessary, methods such as microinjection, electroporation, or liposome packaging can also be used. These are well-known techniques in the art and will not be described in detail here.

[0049] The term "chimeric antigen receptor" or "CAR" as used in this application refers to a group of polypeptides, and in some non-limiting embodiments, two polypeptides; when in immune effector cells, it provides the cell with specificity for a target cell (e.g., for cancer cells) and provides intracellular signaling. In some embodiments, the CAR includes at least an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signal transduction domain. In some embodiments, the extracellular antigen-binding domain comprises the nanobody provided in this application.

[0050] The terms "relief" and "treatment" as used in this application, and their synonyms, refer to the improvement of a disease, symptom, and / or condition. "Relief" and "treatment" can be an improvement in at least one measurable physical parameter, which is not necessarily identifiable by the patient. "Relief" and "treatment" can also be the physical (e.g., stabilizing identifiable symptoms), physiological (e.g., stabilizing physical parameters), or both, suppression of the progression of a disease, symptom, and / or condition. "Relief" and "treatment" can also be the slowing of or reversal of the progression of a disease, symptom, and / or condition.

[0051] The term “prevention” as used in this application and its synonyms refer to delaying the onset of a particular disease, condition and / or symptom or related symptoms of such disease, condition and / or symptom or reducing the risk of acquiring such disease, condition and / or symptom.

[0052] To make the above-mentioned objectives, features and advantages of this application more apparent and understandable, the specific embodiments of this application are described in detail below.

[0053] Nanobodies or their antigen-binding fragments

[0054] According to one embodiment of this application, this application provides a nanobody or antigen-binding fragment thereof that binds a dimer of S100A8 and S100A9, wherein the nanobody or antigen-binding fragment thereof includes a heavy chain variable region, and the heavy chain variable region contains at least the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3.

[0055] In some embodiments, the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3 are the sites where the nanobody or its antigen-binding fragment specifically binds to the dimers of S100A8 and S100A9. Therefore, those skilled in the art, based on the disclosure of this application, will understand that antibodies or their antigen-binding fragments containing the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3 can effectively bind to the dimers of S100A8 and S100A9.

[0056] In some embodiments, the heavy chain variable region includes a complementarity-determining region (CDR). In some embodiments, the amino acid sequence of the complementarity-determining region is defined according to the KABAT system, and the complementarity-determining region contains the following amino acid sequences: the amino acid sequence of CDR1 is shown in SEQ ID NO:1; the amino acid sequence of CDR2 is shown in SEQ ID NO:2; and the amino acid sequence of CDR3 is shown in SEQ ID NO:3.

[0057] In some embodiments, the heavy chain variable region further includes a backbone region (FR). Suitable FR sequences are known in the art, and any suitable FR sequence can be used in this application. In some embodiments, the amino acid sequence of the backbone region is defined according to the KABAT system, and the backbone region contains the following amino acid sequences: the amino acid sequence of FR1 is shown in SEQ ID NO:4; the amino acid sequence of FR2 is shown in SEQ ID NO:5; the amino acid sequence of FR3 is shown in SEQ ID NO:6; and the amino acid sequence of FR4 is shown in SEQ ID NO:7.

[0058] In some embodiments, the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:8, or comprises an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence shown in SEQ ID NO:8.

[0059] In some embodiments, the nanobody or its antigen-binding fragment further includes a constant region. Suitable constant region sequences are known in the art, and any suitable constant region sequence can be used in this application. In some preferred embodiments, the constant region comprises an amino acid sequence as shown in SEQ ID NO:9.

[0060] In some embodiments, the nanobody or its antigen-binding fragment comprises an amino acid sequence as shown in SEQ ID NO:10, or comprises an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence shown in SEQ ID NO:10.

[0061] Polynucleotides, recombinant vectors, host cells

[0062] According to one embodiment of this application, a polynucleotide is also provided, which encodes the nanobody or its antigen-binding fragment described in this application. Due to the degeneracy of codons, those skilled in the art will understand that there are various nucleic acid sequences capable of encoding the nanobody provided in this application, and no limitation is made here. Those skilled in the art can perform appropriate codon optimization and select suitable nucleic acid sequences for expressing the nanobody provided in this application, depending on the expression purpose or host.

[0063] According to one embodiment of this application, a recombinant vector comprising the polynucleotide described in this application is also provided. Recombinant vectors suitable for expressing the nanobodies described in this application are well known in the art and are not limited thereto.

[0064] In some embodiments, the recombinant vector includes cloning vectors and expression vectors. Both viral and non-viral expression vectors can be used to generate antibodies in mammalian host cells. Non-viral vectors and systems include plasmids, augmentative vectors (typically having expression cassettes for expressing proteins or RNA), and human artificial chromosomes (see, for example, Harrington et al., Nat Genet. [Nature Genetics] 15:345, 1997, the contents of which are hereby incorporated by reference). Viral vectors include, but are not limited to, lentiviral vectors, retroviral vectors, adenovirus vectors, adeno-associated virus vectors, poxvirus vectors, and herpesvirus vectors.

[0065] In some embodiments, the recombinant vector further comprises a promoter. The promoter can be any suitable promoter sequence, i.e., a nucleic acid sequence that can be recognized by a host cell expressing a nucleic acid sequence. The promoter sequence contains a transcriptional regulatory sequence that mediates the expression of the antibody. The promoter can be any nucleic acid sequence that is transcriptionally active in the selected host cell, including mutated, truncated, and heterozygous promoters, and can be derived from genes encoding extracellular or intracellular proteins or polypeptides that are homologous or heterologous to those of the host cell.

[0066] The recombinant vector of this application can be constructed using methods known in the art. For example, appropriate restriction enzyme sites can be added to both ends of the polynucleotide of this application according to the restriction enzyme sites contained in the backbone vector used, and then the polynucleotide can be inserted into the backbone vector.

[0067] According to one embodiment of this application, a host cell is also provided, the host cell comprising the polynucleotide described in this application and / or the recombinant vector described in this application. The host cell can be selected according to the type of expression vector. The polynucleotide and / or the expression vector can be delivered into the host cell using any suitable method known in the art, without limitation herein.

[0068] In some embodiments, mammalian host cells are used to express and produce the nanobodies or antigen-binding fragments of the present application. For example, these may be hybridoma cell lines expressing endogenous immunoglobulin genes or mammalian cell lines containing exogenous expression vectors. These include any normal non-immortalized or normal or abnormal immortalized animal or human cells. For example, many suitable host cell lines capable of secreting intact immunoglobulins have been developed, including CHO cell lines, various COS cell lines, HeLa cells, myeloma cell lines, transformed B cells, and hybridomas. Exemplary host cells include, but are not limited to, Chinese hamster ovary (CHO) cells, human embryonic kidney (HEK) cells (e.g., HEK293, HEK293T, HEK293F), monkey kidney (COS) cells (e.g., COS-1, COS-7), young hamster kidney (BHK) cells (e.g., BHK-21), African green monkey kidney cells (e.g., BSC-1), HeLa cells, human hepatocellular carcinoma cells (e.g., Hep G2), myeloma cells (e.g., NS0, 653, SP2 / 0), lymphoma cells, oocytes, and cells from transgenic animals (e.g., mammary epithelial cells) or any derived, immortalized, or transformed cells thereof.

[0069] Antibodies, chimeric antigen receptors, antibody-drug conjugates

[0070] According to one embodiment of this application, an antibody is also provided, the antibody comprising the nanobody or antigen-binding fragment described in this application.

[0071] In some embodiments, the antibody is a multispecific antibody comprising the nanobody or its antigen-binding fragment provided in this application, and further comprising one or more second antibodies or their antigen-binding fragments that specifically bind to other antigens. In some embodiments, the second antibody or its antigen-binding fragment is selected from full-length antibodies, Fab, Fab', (Fab')2, Fv, scFv, scFv-scFv, microantibodies, biantibodies, or sdAbs. Those skilled in the art can select a suitable second antibody or its antigen-binding fragment as needed and use methods known in the art to conjugate the nanobody provided in this application with the second antibody or its antigen-binding fragment to form a multispecific antibody.

[0072] According to one embodiment of this application, a chimeric antigen receptor is also provided, comprising the nanobody or its antigen-binding fragment described in this application. In some embodiments, the chimeric antigen receptor comprises an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signal transduction domain. In some embodiments, the extracellular antigen-binding domain comprises the nanobody or its antigen-binding fragment provided in this application. The chimeric antigen receptor can be constructed by conjugating the nanobody or its antigen-binding fragment provided in this application with other portions using methods known in the art.

[0073] According to one embodiment of this application, an antibody conjugate is also provided, comprising the nanobody or its antigen-binding fragment described in this application, and further comprising a cytotoxin or a radioactive isotope. Suitable radioactive isotopes and cytotoxins are known in the art, and those skilled in the art can select appropriate types as needed and use methods known in the art to conjugate the nanobody provided in this application with a cytotoxin or radioactive isotope.

[0074] In some non-limiting embodiments, the antibody conjugate comprises the nanobody and cytotoxicant provided in this application to form an antibody-drug conjugate (ADC). Examples of cytotoxicants in some embodiments include, but are not limited to, methotrexate, aminopterin, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil, dacarbazine, nitrogen mustard, thiotepa, chlorambucil, melphalan, carmustine, lomustine, 1-methylnitrosourea, cyclophosphamide, nitrogen mustard, busulfan, dibromomannitol, streptozoline, mitomycin, cis-dichlorodiamineplatin, cisplatin, carboplatin, zorubicin, doxorubicin, detoxoplasmosis, carminoxetine, idarubicin, epirubicin, and mitoxantrone. Actinomycin D, bleomycin, cephalosporin, sclerosomycin, atrazomycin, vincristine, vinblastine, paclitaxel, ricin, pseudomonadine exotoxin, gemcitabine, cytochalasin B, bacitracin D, ethidium bromide, emetine, etoposide, teniposide, colchicine, dihydroxyanthradinone, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, procarbazine, hydroxyurea, asparaginase, corticosteroids, mitotane, interferon, and combinations thereof.

[0075] In some non-limiting embodiments, the antibody conjugate comprises the nanobodies and radioisotopes provided in this application to form radionuclide drug conjugates (RDCs). Examples of radioisotopes that can be used in this application include, but are not limited to, At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32, Pb212, 99mTc, 123I, 18F, and 68Ga.

[0076] In addition to the antibodies, chimeric antigen receptors, and antibody-drug conjugates disclosed above, the nanobodies or their antigen-binding fragments disclosed herein can also be conjugated with other factors using chemical methods or through genetic engineering. These factors provide the ability to target the antibody to desired functional sites or to enhance or provide other properties to the antibody.

[0077] Drug compositions, reagent kits

[0078] According to one embodiment of this application, a pharmaceutical composition is also provided, the pharmaceutical composition comprising the nanobody or antigen-binding fragment described in this application.

[0079] In some embodiments, the pharmaceutical composition further includes a pharmaceutically or physiologically acceptable carrier. The carrier may be any compatible, physiologically acceptable, non-toxic substance suitable for delivering the polypeptide, polynucleotide, or recombinant vector provided in this application into a mammal (e.g., a human).

[0080] "Pharmaceutically acceptable carrier" refers to a carrier, diluent, or adjuvant used in the formulation or administration of the polypeptide, polynucleotide, or recombinant carrier provided in this application, which is not an essential active ingredient and does not cause excessive toxicity after administration. Suitable pharmaceutically acceptable carriers are well known to those skilled in the art.

[0081] "Physiologically acceptable carrier" refers to a carrier, diluent, or adjuvant that does not cause significant irritation to an organism and does not eliminate the pharmaceutical activity and properties of the peptide, polynucleotide, or recombinant carrier provided in this application. Suitable physiologically acceptable carriers are also well known to those skilled in the art.

[0082] In some embodiments, the pharmaceutical composition further includes pharmaceutically acceptable excipients. In some embodiments, the excipients include at least one selected from solubilizers, disintegrants, wetting agents, stabilizers, thickeners, diluents, buffers, and flavoring agents.

[0083] In some non-limiting embodiments, the carrier and / or excipients used in the pharmaceutical compositions of this application may comprise, for example, liquid, gel or solid carriers, aqueous mediators, non-aqueous mediators, antimicrobial agents, isotonic agents, buffers, antioxidants, suspending agents, dispersants, chelating agents, diluents, adjuvants, excipients or non-toxic excipients, other components known in the art, or various combinations thereof.

[0084] According to one embodiment of this application, a kit is also provided, the kit comprising the nanobody or antigen-binding fragment thereof described in this application.

[0085] Uses and methods

[0086] According to one embodiment of this application, the use of the nanobody or antigen-binding fragment thereof described in this application, the antibody described in this application, the chimeric antigen receptor described in this application, the antibody conjugate described in this application, the pharmaceutical composition described in this application, and / or the kit described in this application in the preparation of a medicament, pharmaceutical composition, or kit for detecting, preventing, alleviating, or treating a disease is also provided.

[0087] According to one embodiment of this application, a method for preventing, alleviating, or treating a disease is also provided, the method comprising administering to a subject in need the nanobody or antigen-binding fragment described in this application, the antibody described in this application, the chimeric antigen receptor described in this application, the antibody-drug conjugate described in this application, the pharmaceutical composition described in this application, and / or the kit described in this application.

[0088] In some embodiments, the disease includes pancreatitis. In some embodiments, the pancreatitis includes acute pancreatitis. In some embodiments, the pancreatitis includes severe acute pancreatitis.

[0089] In the uses described in this application, the dosage of the nanobody provided can depend on several factors, including the severity and responsiveness of symptoms, the route of administration, the duration of treatment (from days to months to years), and the time to symptom improvement. Those skilled in the art can adjust the dosage regimen to provide a therapeutic response based on the patient's specific circumstances. For example, a single dose can be administered, several separate doses can be administered over a predetermined time period, or the dose can be reduced or increased as indicated by the treatment outcome. The dosage specification is determined by the specific therapeutic effect to be achieved. The dosage value can also vary depending on the type and severity of the condition to be alleviated. For any particular subject, the specific dosage regimen can be adjusted over time according to individual needs and the professional judgment of the treating clinician.

[0090] Detection methods

[0091] According to one embodiment of this application, a detection method is also provided, the method comprising using the nanobody or its antigen-binding fragment described in this application to detect the dimer expression level of S100A8 and S100A9 in a sample.

[0092] In some embodiments, the detection method is a non-diagnostic method. In some embodiments, the detection method is used for diagnostic purposes.

[0093] In some embodiments, the detection method includes the following steps: mixing the nanobody or its antigen-binding fragment with the sample; and detecting the dimer expression levels of S100A8 and S100A9 in the sample.

[0094] The nanobodies or antigen-binding fragments disclosed herein can be chemically labeled or genetically engineered to provide detectable nanobodies. Detectable antibodies include detectable components. Detectable components include, but are not limited to, enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron-emitting metals, and non-radioactive paramagnetic metal ions.

[0095] In some embodiments, the methods for detecting the dimer expression levels of S100A8 and S100A9 in the sample include enzyme-linked immunosorbent assay (ELISA), Western blotting, flow cytometry, and / or immunostaining. Those skilled in the art can select appropriate methods as needed; the implementation steps of these detection methods are known in the art and are not limited herein.

[0096] In some embodiments, the sample may refer to a biological sample, including blood and other bodily fluids (e.g., peripheral blood, serum, plasma, urine, and saliva); it may also include solid tissue samples (e.g., biopsy specimens). In some preferred embodiments, blood-containing samples (e.g., serum or plasma) are the most preferred sample type in this application.

[0097] Antibody or its antigen-binding fragment

[0098] According to one embodiment of this application, an antibody or antigen-binding fragment thereof that binds to a dimer of S100A8 and S100A9 is also provided for use in the preparation of a medicament, pharmaceutical composition or kit for detecting, preventing, alleviating or treating pancreatitis or pancreatic tissue damage, wherein the heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 of the antibody or antigen-binding fragment are the amino acid sequences shown in SEQ ID NO: 19, 20, 21, 22, 23, and 24, respectively.

[0099] According to one embodiment of this application, a method for preventing, alleviating, or treating pancreatitis or pancreatic tissue damage is also provided, the method comprising administering to a subject in need an antibody or antigen-binding fragment thereof that binds to a dimer of S100A8 and S100A9, wherein the heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 of the antibody or antigen-binding fragment are the amino acid sequences shown in SEQ ID NO: 19, 20, 21, 22, 23, and 24, respectively.

[0100] In some embodiments, the pancreatitis includes acute pancreatitis. In some embodiments, the pancreatitis includes severe acute pancreatitis.

[0101] In some embodiments, the pancreatic tissue injury includes at least one of the following: (1) reducing pancreatic tissue edema; (2) reducing pancreatic tissue hemorrhage and fat necrosis; (3) reducing pancreatic tissue inflammatory cell infiltration; and / or (4) reducing pancreatic tissue acinar necrosis.

[0102] In some embodiments, the pancreatic tissue damage is caused by or exacerbated by pancreatitis.

[0103] In some embodiments, the antibody or its antigen-binding fragment includes a heavy chain variable region and a light chain variable region.

[0104] In some embodiments, the heavy chain variable region and the light chain variable region include complementarity-determining regions (CDRs), the amino acid sequences of which are defined according to the KABAT system, and the complementarity-determining regions contain the following amino acid sequences: heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 are the amino acid sequences shown in SEQ ID NO: 19, 20, 21, 22, 23, and 24, respectively.

[0105] In some embodiments, the heavy chain variable region and the light chain variable region further include a backbone region (FR). Suitable FR region sequences are known in the art, and any suitable FR region sequence can be applied in this application.

[0106] In some embodiments, the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:25, or comprises an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence shown in SEQ ID NO:25.

[0107] In some embodiments, the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:26, or comprises an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence shown in SEQ ID NO:26.

[0108] In some embodiments, the antibody or its antigen-binding fragment further includes a heavy chain constant region and a light chain constant region. Suitable constant region sequences are known in the art, and any suitable constant region sequence can be used in this application. In some embodiments, the antibody or its antigen-binding fragment further includes a heavy chain constant region as shown in SEQ ID NO:27 and a light chain constant region as shown in SEQ ID NO:28.

[0109] In some embodiments, the antibody or its antigen-binding fragment comprises a heavy chain and a light chain.

[0110] In some embodiments, the heavy chain comprises an amino acid sequence as shown in SEQ ID NO:29, or comprises an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence shown in SEQ ID NO:29.

[0111] In some embodiments, the light chain comprises an amino acid sequence as shown in SEQ ID NO:30, or comprises an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence shown in SEQ ID NO:30.

[0112] The various embodiments and preferences disclosed above can be combined with each other (as long as they are not inherently contradictory), and all embodiments formed by such combinations are considered as part of the disclosure of this application.

[0113] The exemplary embodiments of this application will now be described with reference to the accompanying drawings, including various details of the embodiments to aid understanding. It should be understood that these are merely exemplary and are in no way intended to limit the scope of protection of this application. The scope of protection of this application is defined only by the claims. Therefore, those skilled in the art will recognize that various changes and modifications can be made to the embodiments described herein without departing from the scope of this application. Similarly, for clarity and brevity, descriptions of well-known functions and structures are omitted in the following description.

[0114] Example

[0115] Unless otherwise specified in this embodiment, the techniques or conditions described in the literature in this field or in accordance with the product instructions shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0116] I. Laboratory Animals

[0117] Severe acute pancreatitis model: Humanized S100A8 / A9 mice (C57BL / 6J, hereinafter referred to as hS100A8 / A9) were bred and bred by Cyagen Biosciences Co., Ltd. All animals were housed and bred in the SPF-grade environmental animal facility of the Beijing Institute of Cardiovascular and Pulmonary Diseases. hS100A8 / A9 mice were required to be approximately 8 weeks old and weighing approximately 20–22 g (male). All experimental procedures were performed according to the NIH's 1996 Guidelines for the Management and Use of Laboratory Animals and the experimental procedures stipulated by the Laboratory Animal Management Committee of Capital Medical University. All experimental animals were randomly assigned to groups.

[0118] II. Experimental Instruments and Reagents

[0119] Table 1 Biochemical reagents and kits

[0120] Table 2 Experimental Instruments

[0121] Example 1: Preparation of anti-S100A8 / A9 nanobodies

[0122] As shown in Figure 1, the preparation process of alpaca-derived anti-S100A8 / A9 nanobodies is as follows:

[0123] 1. Preparation of antigen (recombinant protein)

[0124] Human S100A8 (Uniprot: P05109, amino acid sequence shown in SEQ ID No: 11, encoding nucleic acid sequence shown in SEQ ID No: 12) and S100A9 (Uniprot: P06702, amino acid sequence shown in SEQ ID No: 13, encoding nucleic acid sequence shown in SEQ ID No: 14) gene fragments were synthesized and their sequences were optimized for protein expression in *E. coli*. The synthesized gene fragments were cloned into the pET21a vector (Novagen) digested with NdeI and BamHI to construct the pET21-S100A8-S100A9 co-expression vector, with S100A8 carrying a His-tag. Recombinant proteins from different species, including human S100A8 / A9 (hS100A8 / A9) and mouse S100A8 / A9 (mS100A8 / A9), were constructed and prepared using similar methods. A His-tag was designed at the C-terminus of the recombinant proteins for purification. E. coli BL21(DE3)T7 expression cells were transformed with the plasmid to be expressed. Colonies were inoculated in 20 mL LB flasks containing 100 μg / mL ampicillin and cultured at 37°C for 2 h. 20 mL of the pre-culture was inoculated into 800 mL LB flasks containing 100 μg / mL ampicillin. When the cell density reached an OD600 of 0.5, 0.5 mM isopropyl β-d-1-thiogalactoside (IPTG) was added, and the cells were induced for 3 h. E. coli were harvested by centrifugation, and the cells were washed with 0.15 M NaCl. The cells were resuspended in 80 mL of 50 mM Tris-HCl buffer (pH 7.5) containing 50 mM NaCl and 5 mM MgSO4, and the cells were sonicated on ice. S100A8 and S100A9 were expressed separately in E. coli, forming a dimer. The supernatant collected after sonication and centrifugation was purified using Ni-NTA chromatography to obtain the His-tag-tagged recombinant protein. The purified protein was then dialyzed to replace the PBS solution, and aliquoted and stored at -80°C. Pyrogen-free recombinant protein was further depyrogenated according to different experimental requirements, and then aliquoted and stored at -80°C.

[0125] 2. Alpaca Immunization (Antigen Immunization)

[0126] (1) Antigen preparation: The antigen dose is about 0.5 mg / 1.5 mL / time. Before immunization, the antigen and adjuvant are emulsified in a 1:1 ratio to form a homogeneous mixture.

[0127] (2) Alpaca Immunization: Each time, administer a subcutaneous injection near the lymph nodes in the alpaca's neck, dividing the injection site into two points on each side. Inject approximately 0.4 mL of emulsified antigen at each point. Observe the alpaca for half an hour after immunization to ensure it is in good condition and shows no signs of discomfort. Immunize every two weeks, for a minimum of four immunizations.

[0128] (3) Serum separation: Blood was collected before each antigen immunization for immune evaluation, with 5 mL of blood collected each time. The blood was centrifuged at 4000 rpm for 10 min using a pre-cooled 25°C centrifuge on the same day, and the upper serum was separated and frozen for subsequent antibody titer detection.

[0129] (4) Blood collection: 50 mL of blood was collected from the alpaca’s jugular vein 5-7 days after the fourth immunization.

[0130] (5) Lymphocyte separation: Add 15 mL of cell separation medium to a 50 mL centrifuge tube, then slowly add 15 mL of blood. Add the blood carefully and slowly to prevent mixing with the separation medium. After centrifuging at 25°C, centrifuge at 400×g for 30 min. Observe the blood separation in the centrifuge tube. Store the upper serum layer in a new centrifuge tube at -80°C. Carefully pipette the middle cotton-like upper layer of immune cells into a new 50 mL centrifuge tube. Add 10 mL of room-temperature PBS buffer to each tube and centrifuge at 25°C, 400×g for 20 min. Remove the supernatant, add 5 mL of room-temperature PBS buffer to each tube, mix gently, count the cell number using a hemocytometer, and then centrifuge at 25°C, 400×g for 20 min. Remove the supernatant, and lyse the separated lymphocytes using RNAiso Plus according to the cell number to obtain 10... 7 / mL cell lysate, store at -80℃.

[0131] 3. Constructing phage libraries

[0132] (1) RNA extraction: Peripheral blood lymphocytes preserved with Trizol were thawed on ice and transferred to 1.5 mL centrifuge tubes. 1 / 5 volume of chloroform was added and the mixture was shaken and mixed. After standing at room temperature for 5 min, the mixture was centrifuged at 12000 g for 15 min at 4 °C. The supernatant after centrifugation was transferred to a new centrifuge tube. An equal volume of isopropanol was added to the new centrifuge tube. The mixture was inverted and mixed. After standing at room temperature for 10 min, the mixture was centrifuged at 12000 g for 10 min at 4 °C. The supernatant was discarded and the precipitate was washed with 75% ethanol. After centrifugation at 7500 g for 5 min at 4 °C, the supernatant was discarded. The precipitate was dried at room temperature and then dissolved in an appropriate amount of RNase-free water.

[0133] (2) Obtaining cDNA via reverse transcription: Follow the instructions of the Prime Script reverse transcription kit. TMThe instructions for the II 1st Strand cDNA Synthesis Kit (catalog number 6210A) state that the RNA obtained in the previous step should be reverse transcribed into cDNA, using OligodT and random primers for the reverse transcription.

[0134] (3) Antibody fragment amplification: Specific antibody fragments were amplified from the reverse-transcribed cDNA using PCR amplification with 2×Pfu PCR premixed reagent. The PCR reaction system consisted of: 2 μL cDNA template, 2 μL Alpa001-F primer (sequence shown in SEQ ID No: 15), 2 μL Alpa001-R primer (sequence shown in SEQ ID No: 16), 2×Pfu PCR premixed reagent, and ddH2O to a final volume of 50 μL. The PCR reaction conditions were: 98℃ for 3 min; 95℃ for 30 s, 57℃ for 30 s, 72℃ for 40 s, 25 cycles; 72℃ for 5 min. The obtained PCR amplification product was subjected to 1% agarose gel electrophoresis, which revealed a PCR band of approximately 0.4 kb. The 0.4 kb band was excised from the gel and recovered using a DNA purification and recovery kit according to the manufacturer's instructions.

[0135] (4) Cloning to phage plasmid: The diverse antibody gene sequences and phage vectors obtained in the previous step are spliced ​​together using recombination. The splicing products are recovered using a DNA purification and recovery kit according to the instructions and dissolved in ultrapure water.

[0136] (5) Transformation of TG1: Pre-chill the sterile electroporation cuvette on ice. After thawing 100 μL of TG1 competent cells, add 100 ng of the recovered ligation product. Transfer the mixed competent cells and ligation product to the pre-chilled electroporation cuvette and electroporate using the Bacteria transformation program preset on the electroporator. Immediately after electroporation, add 1 mL of SOC medium to the cuvette. Perform at least 20 electroporations. After thawing the cells at 37°C for 60 min, spread them on LB culture plates containing ampicillin for overnight growth. Wash and scrape the cells from the culture plate after overnight growth with 2×YT medium and a spreader. Add 20% glycerol, measure the OD600 nm value, and store at -80°C to form the bacterial bank.

[0137] (6) Amplification and purification of the phage library: After thoroughly mixing the bacterial cells scraped off in the previous step, the number of phage libraries should be approximately 10. 9The bacteria were transferred to 100 mL of 2×YT culture medium pre-added with ampicillin and cultured at 37°C and 220 rpm until the OD600 nm reached 0.65. Helper phage was added at a ratio of 50:1 (helper phage to bacterial cells), and the culture was incubated at room temperature for 30 min. Kanamycin was added to a final concentration of 50 μg / mL, and the culture was incubated overnight at 30°C on a shaker. The overnight cultured bacteria were centrifuged at 8000 rpm for 20 min at 4°C. The supernatant was transferred to a new centrifuge tube, and 1 / 4 volume of pre-chilled 5×PEG 8000 / NaCl was added. The tube was incubated on ice for at least 60 min. After centrifugation at 10000 rpm for 35 min at 4°C, the supernatant was removed, the liquid was drained, and 1-2 mL of PBS buffer was added to dissolve the precipitate. The phage library was filtered through a 0.45 μm filter and stored long-term at -80°C, or short-term (1-2 weeks) at -20°C.

[0138] 4. Antibody screening and identification

[0139] The antigen was coated onto the immunotubes at a concentration of 20 μg / mL and incubated overnight at 4°C. The next day, the tubes were washed four times with PBS for 3 min each time, and then blocked with 1.5 mL of 2% BSA at 37°C for 2 h. The phage library was blocked with blocking buffer (2% BSA, 0.1% Tween-20) at 37°C for 30 min before being added to the immunotubes, with a total volume of 10 μg / mL. 9 -10 12 / tube, incubated at 37℃ for 1 hour. After thorough washing with PBST and PBS, 1 mL of 0.2 mol / L glycine-hydrochloric acid (pH 2.2) was added for elution, and the mixture was shaken at room temperature for 10 min. Immediately after aspiration, the phage was neutralized to approximately pH 7.0 with 1 mol / L Tris at pH 8.8. The neutralized phage was then used to infect logarithmically growing XL1-Blue activated bacteria. The mixture was incubated at 37℃ for 20-30 min, and then shaken on a shaker for 30 min at 37℃ and 150 rpm. 1% and 0.1% of the bacterial suspension were then plated for counting. The remaining bacterial suspension was centrifuged at 4000 rpm for 10 min, the supernatant was discarded, and the bacterial cells were plated on 2 YTCG (2×YT medium containing chloramphenicol (C) and glucose (G)) solid plates and incubated overnight at 37℃. The following day, bacterial cells were collected from Petri dishes and inoculated into 2YTCG liquid medium. The culture was then incubated with shaking until the logarithmic growth phase, followed by infection with M13KO7. The culture was allowed to stand at room temperature for 15-30 minutes, then incubated at 37℃ / 150rpm for 1 hour. Finally, 50μg / mL kanamycin was added, and the culture was incubated overnight at 30℃. The following day, the phages were purified by precipitation using PEG8000 / NaCl. These phages were then ready for the next round of screening. A total of three rounds of phage library enrichment and screening were performed.

[0140] 5. ELISA identification

[0141] Single colonies obtained after three rounds of phage selection were inoculated into 200 μL of 2YTCTG medium and cultured at 37°C and 220 rpm until the logarithmic growth phase. Then, 10 μL of phage spores were added to each well. 8 Infect each well with helper phage M13KO7 at room temperature for 20 min, then incubate at 37°C and 150 rpm for 1 h. Add an equal volume of 2×YTCTKI (kanamycin 50 μg / mL, IPTG 0.2 mmol / L), and incubate overnight at 30°C and 200 rpm. The next day, centrifuge and collect the supernatant. Add BSA to a final concentration of 2%, add Tween-20 to a final concentration of 0.1%, and incubate at 37°C for 15 min for ELISA identification. Coat 96-well ELISA plates with hS100A8 / A9 antigen (200 ng / well) at 4°C overnight. The next day, block the ELISA plates with PBST-4% milk (250 μL / well) at 37°C for 2 h. Discard the blocking buffer, add the blocked phage antibody, and incubate at 37°C for 1 h. Discard the liquid and wash three times with PBST. Dilute Anti-M13 antibody with blocking buffer and add to the ELISA plates, then incubate at 37°C for 30 min. Wash three times with PBST, add OPD substrate chromogenic solution, incubate at room temperature for about 10 minutes, and terminate the chromogenic process with 2M H2SO4. Measure the absorbance using a microplate reader.

[0142] 6. Expression and purification of nanobodies (monoclonal antibodies)

[0143] Full-length antibodies were prepared from the heavy chain antibodies obtained after screening. The VH variable region gene was amplified using primers VHH-F (sequence shown in SEQ ID No: 17) and VHH-R (sequence shown in SEQ ID No: 18). VH was cloned into the IgG1 vector using homologous recombination. After transformation into *E. coli* Top10, single clones were picked, and the recombinant plasmid was sequenced for identification to select the correct antibody expression vector. After plasmid extraction, the heavy chain was transfected into 293-T cells for transient antibody expression. The expression supernatant was purified by Protein A affinity chromatography, and the purified antibody was identified by electrophoresis, aliquoted, and stored for subsequent experimental identification. Pyrogens were removed from the antibodies according to the requirements of different experiments.

[0144] The sequences of the obtained nanobodies are shown in Table 3 below:

[0145] Table 3 Antibody-related sequences

[0146] Example 2: Bioactivity assay of anti-S100A8 / A9 nanobodies

[0147] The anti-S100A8 / A9 nanobody prepared in Example 1 was subjected to binding activity and cytological activity detection according to the following methods.

[0148] 1. Combined with activity experiments

[0149] The experimental steps are briefly described below:

[0150] (1) Coating antigen: The S100A8 / A9 recombinant fusion protein was diluted with PBS to 5 μg / mL, 50 μL / well, and placed in a 4°C refrigerator overnight;

[0151] (2) Blocking: Discard the liquid in the plate, add 4% skim milk to block, 250 μL / well, and incubate at 37°C for 2 h;

[0152] (3) Add antibody: Discard the liquid in the plate, dilute the nanobody (20 μg / ml) into 8 gradients, 50 μL / well, and incubate at 37℃ for 1 h;

[0153] (4) Washing the plate: Discard the liquid in the plate and wash the plate three times with a plate washing machine;

[0154] (5) Add secondary antibody: horseradish enzyme-labeled goat anti-human IgG (H+L), 50 μL / well, add to the plate and incubate at 37℃ for 30 min;

[0155] (6) Washing the plate: After incubation, discard the liquid in the plate and wash the plate three times;

[0156] (7) Color development: Mix color development solution A and color development solution B at a ratio of 1:1 and add them to the plate, 100 μL / well; develop color at room temperature for 3-10 min.

[0157] (8) Stop solution: Add stop solution immediately after color development, 100 μL / well;

[0158] (9) Reading: Use a multi-functional microplate detector to read the OD value and plot the dose-effect curve.

[0159] As shown in Figure 2A, the OD value increased with increasing monoclonal antibody concentration, indicating that the nanobody prepared in Example 1 exhibited good binding activity with S100A8 / A9.

[0160] 2. Cellular activity assay

[0161] The experimental procedure is briefly described as follows: The monoclonal antibody (400 μg / mL) was diluted into seven gradients and added to each well of a 96-well plate (50 μL per well). The S100A8 / A9 recombinant fusion protein was prepared at a concentration of 80 μg / mL and added to each well (50 μL per well) of the same 96-well plate. The pyrogens used in this experiment were both less than 2 EU / mL. The ligand protein was mixed with equal volumes of the gradient-diluted antibody and incubated at room temperature for 30 min. 100 μL of 1.0 × 10⁻⁶ mol / L 10⁻⁶ mol / L of the fusion protein was added to each well. 6Human peripheral blood mononuclear cells (PBMCs, catalog number PB010C) were seeded into the appropriate wells of a 96-well plate and mixed thoroughly with antibodies. A blank control group (PBMCs only) and a positive control group (PBMCs + S100A8 / A9) were also set up. The culture medium volume in the control group was replenished to make up the difference. The cell plate was placed in a 37°C CO2 incubator and incubated for approximately 20 hours before subsequent assays. All antibody and antigen dilutions and cell density adjustments were performed using RPMI 1640 medium (catalog number A1049101) containing 10% fetal bovine serum (FBS, catalog number 10099141C). After incubation, the cell supernatant was collected and diluted. Cytokine detection was performed according to the instructions of the Human IL-6 ELISA kit (catalog number 1110603), and OD450 was read to plot a dose-response curve.

[0162] As shown in Figure 2B, the IL-6 level decreased with increasing antibody concentration, indicating that the nanobody prepared in Example 1 exhibited good cellular activity.

[0163] Example 3: Construction of an animal model of severe acute pancreatitis

[0164] Mechanism of establishing animal models of severe acute pancreatitis (SAP): Ceruletide, an analogue of cholecystokinin, stimulates excessive secretion from pancreatic acinar cells, impairing the separation of trypsinogen and lysosomal hydrolases within the cells. Following cathepsin B-dependent activation, this further activates zymogens, leading to a series of enhanced protease activities and ultimately resulting in pancreatic autodigestion. Ceruletide-induced AP is mostly edematous and is primarily used for studies of mild AP and the transformation of mild AP into SAP. Lipopolysaccharide (LPS) is often combined with ceruletide to construct SAP models. LPS, as an endotoxin, activates monocytes to release cytokines, initiating a systemic inflammatory response and further exacerbating pancreatic tissue inflammation and damage.

[0165] Rain frog peptide (MedChemExpress, catalog number HY-A0190) is a decapeptide containing 14 amino acid residues. It can play a role similar to CCK and has a strong effect on stimulating gallbladder contraction and pancreatic enzyme secretion.

[0166] LPS (MedChemExpress, catalog number HY-D1056) can activate pathogen-associated molecular patterns (PAMPs) of the immune system and TLR-4 of immune cells; at the same time, it promotes the release of inflammatory mediators, thereby causing local pancreatitis to progress to a systemic inflammatory response and aggravating pancreatitis on the basis of hyoscyamine induction.

[0167] Eight-week-old hS100A8 / A9 mice were used. Rain frog peptide and LPS were dissolved in physiological saline at doses of 50 μg / kg and 10 mg / kg, respectively. Rain frog peptide was administered intraperitoneally once every hour for 10 consecutive hours, with LPS administered concurrently with the last injection.

[0168] Two hours after the last injection, mice were anesthetized by intraperitoneal injection of pentobarbital (100 mg / kg). One carotid artery was dissected and severed, and serum was collected. The heart was perfused with heparinized saline to remove residual blood. The pancreatic tissue was dissected and separated; a portion was fixed in tissue fixative, embedded in paraffin, and sectioned for hematoxylin-eosin (HE) staining and immunohistochemical (IHC) staining; another portion was fixed in electron microscopy fixative for transmission electron microscopy sample preparation.

[0169] Example 4: Therapeutic effect of S100A8 / A9 monoclonal antibody on an animal model of severe acute pancreatitis

[0170] The anti-S100A8 / A9 nanobody prepared in Example 1 and the severe acute pancreatitis animal model constructed in Example 3 were used in the experiment. First, hS100A8 / A9 mice were randomly divided into 3 groups:

[0171] (1) The mAb group is the anti-S100A8 / A9 nanobody therapy group prepared in Example 1: 0.5h before the first injection of the frog peptide, the anti-S100A8 / A9 monoclonal antibody (1mg / kg) was injected intraperitoneally.

[0172] (2) The IgG group was the positive control group: the control IgG antibody (1 mg / kg) was injected intraperitoneally at the same time as the mAb group;

[0173] (3) The Saline group was the negative control group (non-disease group): except that the antibody was not given in advance, the same dosing frequency (rain frog peptide combined with lipopolysaccharide) as the mAb and IgG groups was given, and the same volume of physiological saline was injected intraperitoneally.

[0174] Subsequent experiments included: histopathological assessment of pancreatic tissue damage (HE and IHC staining); detection of serum inflammatory factors (IL-1 beta and IL-6) using ELISA; and evaluation of submicroscopic structural changes in pancreatitis tissue using transmission electron microscopy. The steps and results are briefly described below:

[0175] 1. Preparation of paraffin sections of tissue

[0176] Two hours after the last injection, mice were anesthetized by intraperitoneal injection of pentobarbital (100 mg / kg), and their hearts were perfused with heparinized saline to remove residual blood. The pancreatic tissue of the mice was dissected and separated, and fixed in tissue fixative. After at least 24 hours, the tissue was removed, dehydrated, and embedded in paraffin. Serial sections with a thickness of 5 μm were prepared and mounted on poly-L-lysine-coated glass slides.

[0177] 2. Histopathological evaluation

[0178] (1) HE staining to assess pancreatic tissue damage

[0179] The specific steps are as follows:

[0180] Xylene dewaxing: Paraffin sections were dewaxed and hydrated by alcohol of different concentrations: Xylene I 10 min → Xylene II 10 min → Xylene III 10 min → Anhydrous ethanol 10 min → 95% alcohol 5 min → 80% alcohol 5 min → double distilled water to remove alcohol.

[0181] Stain with hematoxylin for 3-5 minutes, wash with double-distilled water, differentiate with 1% hydrochloric acid alcohol for a few seconds, rinse with double-distilled water, restore blue with 0.6% ammonia water, and rinse with running water.

[0182] Stain with eosin solution for 2-3 minutes, then rinse with double-distilled water to remove excess stain.

[0183] The slices were sequentially immersed in 80% ethanol I for 5 min → 95% ethanol II for 5 min → anhydrous ethanol I for 5 min → xylene I for 5 min → xylene II for 5 min to dehydrate and clear them;

[0184] Slices were dried in a fume hood and then mounted with neutral resin.

[0185] Using a Nikon ECLIPSE 90i microscope, the cell nuclei were stained a bright blue by hematoxylin; the cytoplasm was stained with eosin in varying shades of pink to peach.

[0186] (2) IHC staining to assess the infiltration of inflammatory cells in pancreatic tissue

[0187] Xylene dewaxing: Paraffin sections were dewaxed and hydrated by alcohol of different concentrations: Xylene I 10 min → Xylene II 10 min → Xylene III 10 min → Anhydrous ethanol 10 min → 95% alcohol 5 min → 80% alcohol 5 min → double distilled water to remove alcohol.

[0188] Antigen retrieval: Heat the citric acid antigen retrieval solution on high for 10 minutes, then cool to room temperature.

[0189] Treat with endogenous peroxidase inhibitor for 15 min;

[0190] Primary antibody incubated overnight at 4°C;

[0191] Rewarm to 37℃, then incubate with the secondary antibody at room temperature for 1 hour;

[0192] Develop DAB for 10 minutes and monitor the staining degree under a microscope. Wash with double-distilled water to stop the staining.

[0193] Hematoxylin redyeing, followed by rinsing with running water;

[0194] The slices were placed in 80% alcohol for 5 minutes, 95% alcohol for 5 minutes, anhydrous ethanol for 5 minutes, xylene for 5 minutes, and xylene for 5 minutes in sequence to dehydrate and clear the slices;

[0195] Slices were dried in a fume hood and then mounted with neutral resin.

[0196] Observation was performed using a Nikon ECLIPSE 90i microscope.

[0197] 3. ELISA detection of serum inflammatory factors (IL-1beta, IL-6) expression.

[0198] Prepare all reagents, samples, and standards according to the instructions;

[0199] Add 100 μL of standard or sample to each well and incubate at room temperature for 2.5 h;

[0200] Aspirate the liquid from each well, wash 2-3 times with washing buffer, then add 100 μL of antibody to each well and incubate at room temperature for 1 hour;

[0201] Aspirate the liquid from each well, wash 2-3 times with washing buffer, then add 100 μL of Streptavidin solution to each well and incubate at room temperature for 45 min;

[0202] Aspirate the liquid from each well, wash 2-3 times with washing buffer, then add 100 μL of TMB One-Step substrate to each well and incubate at room temperature for 30 min;

[0203] Add 50 μL of stop solution to each well and immediately read the values ​​at 450 nm using a microplate reader.

[0204] Figure 3 shows the results:

[0205] (1) The pancreatic damage of mice injected with anti-S100A8 / A9 monoclonal antibody (mAb group) was significantly improved compared with that of mice in the positive control group (IgG group) (Fig. 3A), the infiltration of inflammatory cells was significantly reduced (Fig. 3B, C), and the serum inflammatory factors were significantly reduced (Fig. 3D).

[0206] (2) The pancreatic tissue of mice in the anti-S100A8 / A9 monoclonal antibody group showed only mild loose edema and partial fat-like vacuolar degeneration. At the same time, the infiltration of inflammatory cells (neutrophils and macrophages) was significantly reduced compared with the IgG group, and the serum inflammatory factors (IL-1beta and IL-6) levels were significantly reduced.

[0207] 4. Transmission electron microscopy observation of the submicroscopic structure of mouse tissues affected by anti-S100A8 / A9 monoclonal antibody in severe acute pancreatitis.

[0208] Sampling and fixation: Perform pancreatitis tissue sampling as described above, minimizing mechanical damage such as traction, contusion, and compression. Sampling should be performed within 1-3 minutes, with each sample being 1 mm thick. 3 Size. Before sampling, prepare a culture dish containing electron microscopy fixative. Immediately after removing the small tissue block from the body, place it into the culture dish and cut it into 1mm pieces using a scalpel in the fixative. 3 Small tissue blocks were then transferred to EP tubes containing fresh electron microscopy fixative for further fixation. The tissue blocks were stored and transported at 4°C. The tissue blocks were rinsed three times with 0.1M phosphate-buffered saline (PBS) (pH 7.4), 15 min each time.

[0209] Post-fixation: 1% osmium tetroxide was prepared in 0.1M phosphate buffer PB (pH 7.4) and fixed at room temperature in the dark for 2 hours. Washed 3 times with 0.1M phosphate buffer PBS (pH 7.4), 15 min each time.

[0210] Dehydration at room temperature: Tissues were sequentially immersed in 30%-50%-70%-80%-95%-100%-100% alcohol for upward dehydration, 20 minutes each time, followed by 100% acetone twice, 15 minutes each time.

[0211] Infiltration embedding: Acetone: 812 embedding agent = 1:1, 37℃, 2-4h; Acetone: 812 embedding agent = 1:2, infiltration overnight at 37℃; Pure 812 embedding agent, 37℃, 5-8h. Pour pure 812 embedding agent into the embedding plate, insert the sample into the embedding plate, and incubate overnight at 37℃.

[0212] Polymerization: The embedding plate was placed in a 60℃ oven for polymerization for 48 hours, and the resin block was removed for later use.

[0213] Positioning: The resin block was sectioned into 1.5μm sections using a microtome, stained with toluidine blue, and positioned under a light microscope.

[0214] Ultrathin sectioning: Resin blocks are used to make ultrathin sections of 60-80nm using an ultrathin microtome, and the sections are retrieved using a 150-mesh copper screen.

[0215] Staining: Copper mesh was stained with 2% uranium acetate saturated alcohol solution in the dark for 8 min; washed 3 times with 70% alcohol; washed 3 times with ultrapure water; stained with 2.6% lead citrate solution in the dark for 8 min; washed 3 times with ultrapure water; and slightly blotted dry with filter paper. The copper mesh sections were placed in a copper mesh box and dried at room temperature overnight.

[0216] Observe under a transmission electron microscope and collect and analyze images.

[0217] Figure 4 shows that the number and size of autolysosomes containing undegraded tissue increased in the pancreatic tissue of mice with severe acute pancreatitis, while mitochondria showed swelling and cristae breakage (Figure 4A); while the number of autolysosomes in the pancreatic tissue of the monoclonal antibody treatment group was less and the mitochondrial structure was normal (Figure 4B).

[0218] In summary, the results of Example 4 show that the anti-S100A8 / A9 nanobody prepared in Example 1 can significantly improve the symptoms of mice with severe acute pancreatitis and has a good therapeutic effect.

[0219] Example 5: Construction and bioactivity assay of anti-S100A8 / A9 antibody

[0220] The preparation process and bioactivity assay of the fully human anti-S100A8 / A9 monoclonal antibody are detailed in the published patent application CN119192371A, and its amino acid sequence is shown in Table 4 below:

[0221] Table 4 Antibody-related sequences

[0222] Example 6: Therapeutic effect of anti-S100A8 / A9 antibody on an animal model of severe acute pancreatitis

[0223] The anti-S100A8 / A9 antibody prepared in Example 5 and the severe acute pancreatitis animal model constructed in Example 3 were used in the experiment. First, hS100A8 / A9 mice were randomly divided into 3 groups:

[0224] (1) The experimental group was the anti-S100A8 / A9 nanobody treatment group prepared in Example 1: 0.5 h before the first injection of rain frog peptide, low-dose (2.5 mg / kg), medium-dose (5 mg / kg) and high-dose (10 mg / kg) anti-S100A8 / A9 antibodies were injected intraperitoneally.

[0225] (2) The IgG group was the positive control group: control IgG antibody (5 mg / kg) was injected intraperitoneally at the same time as the experimental group;

[0226] (3) The Ctrl group was the negative control group (non-disease group): except that the antibody was not given in advance, the same frequency of administration (rain frog peptide combined with lipopolysaccharide) as the experimental group and the IgG group was given, and the same volume of physiological saline was injected intraperitoneally.

[0227] Subsequent experiments included: HE staining to assess pancreatic tissue damage, following the same procedure as in Example 4. The HE staining pathological tissue scoring criteria are shown in Table 5 below.

[0228] Table 5. HE staining pathological tissue scoring criteria

[0229] Figure 5 shows the results:

[0230] (1) The pancreatic damage in mice injected with medium and high doses of nanobodies (medium dose group and high dose group) was significantly improved compared with that in the positive control group (IgG group) (Figure 5A).

[0231] (2) The pancreatic tissue of mice in the medium-dose and high-dose groups showed only mild loose edema and partial fat-like vacuolar degeneration, while the low-dose group still had patchy acinar necrosis. The pathological tissue score showed that the score decreased with the increase of anti-S100A8 / A9 antibody dose, indicating that the pancreatic tissue damage of mice in the medium-dose and high-dose groups was significantly improved compared with other groups (Figure 5B).

[0232] In summary, the results of Example 6 show that the anti-S100A8 / A9 nanobody prepared in Example 5 can significantly improve the symptoms of mice with severe acute pancreatitis and has a good therapeutic effect.

[0233] It should be noted that the above are merely preferred embodiments of this application and are not intended to limit the application. Various modifications and variations are possible for those skilled in the art. Although specific embodiments have been described, alternatives, modifications, variations, improvements, and substantial equivalents of the above embodiments may exist or be unforeseeable to the applicant or other those skilled in the art. Therefore, the appended claims and any possible amendments to the claims are intended to cover all such alternatives, modifications, variations, improvements, and substantial equivalents. Importantly, as technology evolves, many elements described herein can be replaced by equivalent elements appearing after this application.

Claims

1. A nanobody or antigen-binding fragment thereof that combines a dimer of S100A8 and S100A9, characterized in that, The nanobody or its antigen-binding fragment includes a heavy chain variable region, which contains at least the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:

3.

2. The nanobody or its antigen-binding fragment according to claim 1, wherein the heavy chain variable region includes a complementarity-determining region (CDR), the complementarity-determining region comprising the following amino acid sequence: The amino acid sequence of CDR1 is shown in SEQ ID NO:1; The amino acid sequence of CDR2 is shown in SEQ ID NO:2; and The amino acid sequence of CDR3 is shown in SEQ ID NO:

3.

3. The nanobody or its antigen-binding fragment according to claim 2, wherein the heavy chain variable region further comprises a backbone region (FR), the backbone region comprising the following amino acid sequence: The amino acid sequence of FR1 is shown in SEQ ID NO:4; The amino acid sequence of FR2 is shown in SEQ ID NO:5; The amino acid sequence of FR3 is shown in SEQ ID NO:6; and The amino acid sequence of FR4 is shown in SEQ ID NO:

7.

4. The nanobody or its antigen-binding fragment according to claim 3, wherein the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:8, or comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity with the amino acid sequence shown in SEQ ID NO:

8.

5. The nanobody or antigen-binding fragment thereof according to claim 1, wherein the nanobody or antigen-binding fragment thereof further comprises a constant region, the constant region comprising an amino acid sequence as shown in SEQ ID NO:

9.

6. The nanobody or antigen-binding fragment of claim 1, wherein the nanobody or antigen-binding fragment comprises an amino acid sequence as shown in SEQ ID NO:10, or comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity with the amino acid sequence shown in SEQ ID NO:

10.

7. A polynucleotide, characterized in that, The polynucleotide encodes the nanobody or its antigen-binding fragment as described in any one of claims 1-6.

8. A recombinant vector, characterized in that, The recombinant vector comprises the polynucleotide of claim 7.

9. A host cell, characterized in that, The host cell comprises the polynucleotide of claim 7 and / or the recombinant vector of claim 8.

10. An antibody, characterized in that, The antibody comprises the nanobody or antigen-binding fragment of any one of claims 1-6.

11. A chimeric antigen receptor, characterized in that, The chimeric antigen receptor comprises the nanobody or antigen-binding fragment thereof as described in any one of claims 1-6.

12. An antibody conjugate, characterized in that, The antibody conjugate comprises any one of the nanobodies or antigen-binding fragments of claims 1-6, and further comprises cytotoxins or radioisotopes.

13. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises any one of the nanobodies or antigen-binding fragments thereof according to claims 1-6.

14. A reagent kit, characterized in that, The kit comprises any one of the nanobodies or antigen-binding fragments thereof according to claims 1-6.

15. Use of the nanobody or antigen-binding fragment thereof of any one of claims 1-6, the antibody of claim 10, the chimeric antigen receptor of claim 11, the antibody-drug conjugate of claim 12, the pharmaceutical composition of claim 13, and / or the kit of claim 14 in the preparation of a medicament, pharmaceutical composition, or kit for detecting, preventing, alleviating, or treating a disease.

16. The use according to claim 15, wherein the disease includes pancreatitis; preferably, the pancreatitis includes acute pancreatitis.

17. A detection method, characterized in that, The method includes detecting the dimer expression levels of S100A8 and S100A9 in a sample using a nanobody or its antigen-binding fragment as described in any one of claims 1-6.

18. A method for preventing, alleviating, or treating a disease, characterized in that, The method comprises administering to a subject in need the nanobody or antigen-binding fragment of any one of claims 1-6, the antibody of claim 10, the chimeric antigen receptor of claim 11, the antibody-drug conjugate of claim 12, the pharmaceutical composition of claim 13, and / or the kit of claim 14.

19. The method of claim 18, wherein the disease includes pancreatitis; preferably, the pancreatitis includes acute pancreatitis.

20. The use of an antibody or antigen-binding fragment thereof that binds a dimer of S100A8 and S100A9 in the preparation of a medicament, pharmaceutical composition, or kit for detecting, preventing, alleviating, or treating pancreatitis or pancreatic tissue damage, characterized in that, The heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 of the antibody or its antigen-binding fragment are the amino acid sequences shown in SEQ ID NO: 19, 20, 21, 22, 23, and 24, respectively.

21. The use according to claim 20, wherein: The heavy chain variable region of the antibody or its antigen-binding fragment comprises an amino acid sequence as shown in SEQ ID NO:25, or comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity with the amino acid sequence shown in SEQ ID NO:25; and The light chain variable region of the antibody or its antigen-binding fragment contains an amino acid sequence as shown in SEQ ID NO:26, or contains an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity with the amino acid sequence shown in SEQ ID NO:

26.

22. The use according to claim 21, wherein: The antibody or its antigen-binding fragment comprises a heavy chain as shown in SEQ ID NO:29; and The antibody or its antigen-binding fragment comprises a light chain as shown in SEQ ID NO:

30.

23. The use according to any one of claims 20-22, wherein the pancreatitis includes acute pancreatitis.

24. The use according to any one of claims 20-22, wherein the pancreatic tissue injury comprises at least one of the following: (1) Reduce pancreatic tissue edema; (2) Reduce pancreatic tissue hemorrhage and fat necrosis; (3) Reduce inflammatory cell infiltration in pancreatic tissue; and / or (4) Reduce pancreatic tissue acinar necrosis.

25. The use according to claim 24, wherein the pancreatic tissue damage is caused by or aggravated by pancreatitis.

26. A method for preventing, alleviating, or treating pancreatitis or pancreatic tissue damage, the method comprising administering to a subject in need an antibody or antigen-binding fragment thereof that binds to a dimer of S100A8 and S100A9, wherein the heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 of the antibody or antigen-binding fragment are the amino acid sequences shown in SEQ ID NO: 19, 20, 21, 22, 23, and 24, respectively.

27. The method according to claim 26, wherein: The heavy chain variable region of the antibody or its antigen-binding fragment comprises an amino acid sequence as shown in SEQ ID NO:25, or comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity with the amino acid sequence shown in SEQ ID NO:25; and The light chain variable region of the antibody or its antigen-binding fragment contains an amino acid sequence as shown in SEQ ID NO:26, or contains an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity with the amino acid sequence shown in SEQ ID NO:

26.

28. The method of claim 27, wherein: The antibody or its antigen-binding fragment comprises a heavy chain as shown in SEQ ID NO:29; and The antibody or its antigen-binding fragment comprises a light chain as shown in SEQ ID NO:

30.

29. The method according to any one of claims 26-28, wherein the pancreatitis includes acute pancreatitis.

30. The method according to any one of claims 26-28, wherein the pancreatic tissue injury comprises at least one of the following: (1) Reduce pancreatic tissue edema; (2) Reduce pancreatic tissue hemorrhage and fat necrosis; (3) Reduce inflammatory cell infiltration in pancreatic tissue; and / or (4) Reduce pancreatic tissue acinar necrosis.

31. The method of claim 30, wherein the pancreatic tissue damage is caused by or aggravated by pancreatitis.

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