Antibodies that specifically bind ssb and uses thereof

Abstract

The present disclosure relates to the field of antibodies, and particularly relates to antibodies specifically binding to SSB and applications thereof. The present disclosure provides an antibody or an antigen-binding fragment thereof specifically binding to SSB of an animal, a coding nucleic acid, an expression vector, a cell, a composition comprising the antibody or the antigen-binding fragment thereof, and a method and an application for preparing the antibody or the antigen-binding fragment thereof. The antibody specifically binding to SSB of the present disclosure has good binding capacity, high specificity, and good thermal stability and freeze-thaw stability to SSB protein, and provides raw material guarantee for downstream development of high-quality diagnostic reagents.

Description

Technical Field

[0001] This invention relates to the field of antibodies, and more particularly to an antibody or antigen-binding fragment thereof that specifically binds to SSB, and its applications. Background Technology

[0002] SSB (single-stranded nucleic acid-binding protein), also known as La protein, is an RNA-binding protein that plays a crucial role in RNA polymerase III transcription and transcript maturation during the cell cycle. It participates in regulating cell proliferation, tumor development, and interfering with RNA virus invasion, and is widely distributed in the cell nucleus and cytoplasm. Anti-SSB antibodies are autoantibodies targeting La / SSB proteins. Serological positivity for these antibodies is typically associated with autoimmune diseases such as connective tissue disorders, including primary Sjögren's syndrome (pSS), systemic lupus erythematosus (SLE), vasculitis, and scleroderma.

[0003] The detection rate of anti-SSB antibodies in the serum of patients with primary Sjögren's syndrome (pSS) is very high, and it is one of the serum diagnostic markers for pSS. The main characteristic of pSS is T-lymphocyte infiltration of exocrine glands (primarily affecting the salivary and lacrimal glands), leading to damage to the ductal epithelium and parenchymal structure, causing tissue damage and glandular dysfunction, with dry eyes and dry mouth as the main symptoms. However, pSS is clinically heterogeneous, affecting multiple tissues and organs. Therefore, patients with Sjögren's syndrome have a high probability of developing respiratory diseases, especially interstitial lung diseases such as interstitial lung disease (ILD) and pulmonary arterial hypertension (PAH). Interstitial lung disease is a heterogeneous group of non-neoplastic and non-infectious lung diseases with inflammation of the alveolar units and interstitial fibrosis as the basic pathological changes. It can be divided into connective tissue disease-associated ILD and idiopathic ILD of unknown etiology. Its most prominent feature is its insidious onset, difficulty in early detection, progressive worsening, and eventual cardiopulmonary failure. Therefore, detecting autoantibody profiles, including anti-SSB antibodies, plays an important role in the typing, early diagnosis, treatment, and control of ILD.

[0004] Most commercially available anti-SSB antibodies are animal-derived antibodies, such as mouse or rabbit antibodies, produced by repeated immunization of mice or rabbits with recombinant SSB antigens. These antibodies are obtained through methods such as hybridoma technology, phage display technology, yeast display technology, and single-cell B-cell technology, and exhibit high affinity for the SSB antigen. However, antibodies produced by immunizing animals are incompatible with anti-human secondary antibodies in chemiluminescence detection systems, necessitating humanization of these animal-derived antibodies. Antibody humanization generally employs the following methods: 1) replacing the Fc fragment of animal-derived antibodies with the Fc fragment of human monoclonal antibodies; 2) transplanting the CDR region of animal antibodies onto the framework of human monoclonal antibodies; 3) producing fully human antibodies from transgenic mice. While these techniques can achieve the goal of converting animal-derived antibodies into human-derived antibodies, altering the antibody Fc fragment or framework may change the antibody conformation, leading to decreased antibody affinity and stability. This requires further optimization and modification of the humanized antibodies, increasing development time and costs. Therefore, there is a need to develop human-derived antibodies that specifically bind to SSB. Summary of the Invention

[0005] This disclosure provides an antibody or antigen-binding fragment thereof that specifically binds to SSB, a composition including the antibody or antigen-binding fragment thereof, a method of preparation and application.

[0006] According to a first aspect of this disclosure, an antibody or antigen-binding fragment thereof that specifically binds to SSB is provided, said antibody or antigen-binding fragment comprising: a1) The amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 in the heavy chain variable region as shown in SEQ ID NO: 3; and the amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 in the light chain variable region as shown in SEQ ID NO: 5; or a2) Compared with CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 described in a1), at least one CDR contains one or more amino acid substitutions, deletions or additions; The CDR is defined by IMGT, Kabat, Chothia, or the Contact numbering system.

[0007] In some embodiments, the antibody or its antigen-binding fragment comprises: b1) A heavy chain variable region comprising the amino acid sequences CDR-H1, CDR-H2, and CDR-H3 as shown in SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9, respectively; and a light chain variable region comprising the amino acid sequences CDR-L1, CDR-L2, and CDR-L3 as shown in SEQ ID NO: 18, GAS, and SEQ ID NO: 19, respectively; wherein the CDRs are defined by the IMGT numbering system, or The heavy chain variable region comprises amino acid sequences CDR-H1, CDR-H2, and CDR-H3 as shown in SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, respectively; and the light chain variable region comprises amino acid sequences CDR-L1, CDR-L2, and CDR-L3 as shown in SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22, respectively; wherein the CDRs are defined by the Kabat numbering system, or The heavy chain variable region comprises the amino acid sequences CDR-H1, CDR-H2, and CDR-H3 as shown in SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 12, respectively; and the light chain variable region comprises the amino acid sequences CDR-L1, CDR-L2, and CDR-L3 as shown in SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22, respectively; wherein the CDRs are defined by the Chothia numbering system, or The heavy chain variable region comprises the amino acid sequences CDR-H1, CDR-H2, and CDR-H3 as shown in SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17, respectively; and the light chain variable region comprises the amino acid sequences CDR-L1, CDR-L2, and CDR-L3 as shown in SEQ ID NO: 23, SEQ ID NO: 24, and SEQ ID NO: 25, respectively; wherein the CDRs are defined by the Contact numbering system, or b2) Heavy chain variable region; and, light chain variable region, wherein, compared with the heavy chain variable region and / or light chain variable region of b1), at least one CDR contains a substitution, deletion or addition of one or more amino acids.

[0008] In some embodiments, the heavy chain variable region of the antibody or its antigen-binding fragment further includes a framework region of the heavy chain variable region, and / or the light chain variable region of the antibody or its antigen-binding fragment further includes a framework region of the light chain variable region.

[0009] In some embodiments, the frame region of the heavy chain variable region includes the frame region of the heavy chain variable region or a variant thereof derived from immunoglobulins of humans, mice, primates, cattle, horses, pigs, sheep, goats, dogs, cats, rabbits, camels, donkeys, deer, minks, chickens, ducks, or geese; and further includes the frame region of the heavy chain variable region or a variant thereof derived from human immunoglobulins.

[0010] In some embodiments, the framework region of the light chain variable region includes the framework region of the heavy chain variable region or a variant thereof derived from immunoglobulins of humans, mice, primates, cattle, horses, pigs, sheep, goats, dogs, cats, rabbits, camels, donkeys, deer, minks, chickens, ducks, or geese; and further includes the framework region of the light chain variable region or a variant thereof derived from human immunoglobulins.

[0011] In some embodiments, the antibody or its antigen-binding fragment comprises: The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 3, or an amino acid sequence having at least 80%, at least 85%, 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% sequence identity with it; and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 5, or an amino acid sequence having at least 80%, at least 85%, 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% sequence identity with it.

[0012] In some embodiments, the antibody or its antigen-binding fragment further includes a heavy chain constant region and / or a light chain constant region.

[0013] In some embodiments, the heavy chain constant region includes at least a portion of the heavy chain constant region or a variant thereof derived from immunoglobulins of humans, mice, primates, cattle, horses, pigs, sheep, goats, dogs, cats, rabbits, camels, donkeys, deer, minks, chickens, ducks, or geese.

[0014] In some embodiments, the light chain constant region includes at least a portion of the light chain constant region or a variant thereof derived from immunoglobulins of humans, mice, primates, cattle, horses, pigs, sheep, goats, dogs, cats, rabbits, camels, donkeys, deer, minks, chickens, ducks, or geese.

[0015] In some embodiments, the antibody may be a monoclonal antibody, a monospecific antibody, a bispecific antibody, or a multispecific antibody.

[0016] In some embodiments, the antibody may be murine, porcine, porcine-derived, chimeric, humanized, or fully human.

[0017] In some embodiments, the antibody may be of type IgA, IgD, IgE, IgG, or IgM. In some specific embodiments, the antibody may be of type IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2.

[0018] In some embodiments, the antigen-binding fragment may be one known in the art. In specific embodiments, the antigen-binding fragment may include scFv, Fab, Fab', (Fab')2, Fv fragment, Fd, dsFv.

[0019] According to another aspect of this disclosure, a nucleic acid molecule is provided that encodes the antibody or antigen-binding fragment thereof described in this disclosure.

[0020] According to another aspect of this disclosure, a carrier is provided that includes the nucleic acid molecules described herein.

[0021] In some embodiments, the vector includes prokaryotic expression vectors and eukaryotic expression vectors. In some embodiments, the eukaryotic expression vector includes, for example, but not limited to, yeast expression vectors, mammalian expression vectors, and insect expression vectors. For example, the expression vector may include, but is not limited to, plasmids, retroviral vectors, lentiviral vectors, bacteriophage vectors, adenovirus vectors, adeno-associated vectors, or herpes simplex vectors.

[0022] According to another aspect of this disclosure, a cell is provided that includes the antibody or antigen-binding fragment thereof described in this disclosure, the nucleic acid molecule, or the vector described in this disclosure.

[0023] In some embodiments, the cells are conventional host cells in the art, as long as the expression vector stably expresses the carried nucleic acid molecules as the antibody or its antigen-binding fragment described in this invention. In some embodiments, the host cells can be prokaryotic cells and / or eukaryotic cells. Prokaryotic cells may include, for example, *Escherichia coli*, and eukaryotic cells may include, for example, CHO cells, HEK293 cells, BHK cells, NSO cells, SP2 / 0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells, HeLa cells, Vero cells, Expi293 cells, hybridoma cells, yeast cells, and insect cells. Transforming the expression vector into the host cells yields the host cells of this invention. The transformation method is a conventional transformation method in the art, preferably a chemical transformation, heat shock, or electroporation.

[0024] According to another aspect of this disclosure, a conjugate is provided, comprising: an antibody or an antigen-binding fragment thereof as described in this disclosure; and a conjugation portion, wherein the conjugation portion is selected from detectable substances, small molecule drugs, and / or proteins.

[0025] In some embodiments, the protein may include, but is not limited to, cytokines, antibodies, antibody Fc fragments, antibody scFv fragments, enzymes, and viral capsid proteins.

[0026] In some embodiments, the detectable substance can be any substance conventionally used in the art, such as, but not limited to, fluorescent substances, luminescent markers, or radioactive substances. In a preferred embodiment, the detectable substance can be selected from any one of acridine esters, acridine sulfonamides, luminol, isoluminol, horseradish peroxidase, alkaline phosphatase, and isotopes.

[0027] According to another aspect of this disclosure, a method for preparing the antibody or antigen-binding fragment thereof described in this disclosure is provided.

[0028] In some embodiments, the method includes culturing the cells described in this disclosure and isolating the antibody under conditions that allow expression of the antibody or its antigen-binding fragment.

[0029] According to another aspect of this disclosure, a pharmaceutical composition is provided comprising: the antibody or antigen-binding fragment thereof described in this disclosure, the nucleic acid molecule, the carrier, the cell, or the conjugate.

[0030] In some embodiments, the pharmaceutical composition may also contain a pharmaceutically acceptable carrier.

[0031] In some embodiments, the pharmaceutically acceptable carrier may be a carrier conventionally used in the art.

[0032] In some embodiments, the route of administration of the pharmaceutical composition may be parenteral, injection, oral, or topical. The pharmaceutical composition may be formulated into a form suitable for administration, such as a solid, semi-solid, or liquid form, and may be an aqueous solution, non-aqueous solution, or suspension, or in the form of powder, tablet, capsule, granules, injection, or infusion.

[0033] According to another aspect of this disclosure, applications are provided for the antibodies or antigen-binding fragments thereof described herein, the nucleic acid molecules, the vectors, the cells, the conjugates, or the pharmaceutical compositions described herein, the applications including one or more of the following: d1) Detect the presence or level of SSB in the sample; d2) Diagnosis or prognostic assessment of SSB-related diseases; d3) Prevention and / or treatment of SSB-related diseases; d4) Prepare products for detecting the presence or level of SSB in samples; d5) Prepare products for the diagnosis or prognostic assessment of SSB-related diseases; d6) Prepare medicines for the treatment and / or prevention of SSB-related diseases.

[0034] In some embodiments, the SSB-related diseases are not limited to autoimmune diseases, such as lupus erythematosus, ankylosing spondylitis, primary Sjögren's syndrome, vasculitis, scleroderma, interstitial lung disease, primary biliary cholangitis, idiopathic inflammatory myopathy, and rheumatoid arthritis.

[0035] In some embodiments, the sample includes whole blood, red blood cell concentrate, platelet concentrate, white blood cell concentrate, tissue, bone marrow aspirate, plasma, serum, cerebrospinal fluid, feces, urine, cultured cells, saliva, oral secretions and / or nasal secretions from the subject.

[0036] In some implementations, the subject may include a human, a mouse, a primate, a cow, a horse, a pig, a sheep, a goat, a dog, a cat, a rabbit, a camel, a donkey, a deer, a mink, a chicken, a duck, or a goose.

[0037] In some embodiments, a method for detecting SSB in a sample is provided, the method comprising the step of contacting the sample with an antibody or antigen-binding fragment thereof as described in the first aspect of this disclosure.

[0038] In some embodiments, a method is provided for diagnosing or prognostically assessing SSB-related diseases using the antibodies or antigen-binding fragments described in this disclosure. In some embodiments, the method includes the step of contacting a sample of the subject with the antibody or antigen-binding tablet described in this disclosure.

[0039] According to another aspect of this disclosure, the antibodies or antigen-binding fragments thereof described herein, the nucleic acid molecules, the vectors, the cells, the conjugates, or the pharmaceutical compositions described herein are provided for the treatment and / or prevention of SSB-related diseases.

[0040] In some embodiments, this disclosure provides a method for treating and / or preventing SSB-related diseases, the method comprising: administering to a subject in need an effective amount of the antibody or antigen-binding fragment thereof described in this disclosure, the nucleic acid molecule, the carrier, the cell, the conjugate, or the pharmaceutical composition.

[0041] The beneficial effects of this disclosure are: The antibody that specifically binds to SSB provided in this disclosure has excellent binding ability to SSB protein, high specificity, no cross-activity with other autoimmune antigens, and good thermal stability and freeze-thaw stability, providing a raw material guarantee for the downstream development of high-quality diagnostic reagents. Attached Figure Description

[0042] Figure 1 An SDS-PAGE image of the SSB protein is shown.

[0043] Figure 2 The image shows an electrophoresis diagram of gene amplification in the variable region of B cell antibodies.

[0044] Figure 3 The SDS-PAGE results of the purified antibody 2H2 are shown. Detailed Implementation

[0045] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention in any way. Furthermore, descriptions of well-known structures and techniques are omitted in the following description to avoid unnecessarily obscuring the concepts of this disclosure. Such structures and techniques have also been described in many publications.

[0046] definition Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly used in the field to which this invention pertains. For the purposes of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural forms, and vice versa.

[0047] Unless the context clearly indicates otherwise, the terms “a” and “an” as used herein include plural references. For example, reference to “a cell” includes multiple such cells and equivalents known to those skilled in the art, etc.

[0048] As used herein, the term "about" indicates a range of ±20% of the following value. In some embodiments, the term "about" indicates a range of ±10% of the following value. In some embodiments, the term "about" indicates a range of ±5% of the following value.

[0049] The term "antibody" in this document encompasses a wide range of antibody structures, referring to an immunoglobulin capable of specifically binding to a target antigen via at least one of its antigen-binding domains. In this document, the term "antibody" includes, but is not limited to, monoclonal antibodies, polyclonal antibodies, monospecific and multispecific antibodies (e.g., bispecific or trispecific antibodies), single-chain molecules, and antibody fragments, as long as they exhibit the desired antigen-binding activity.

[0050] Antibodies can be classified into different types based on the amino acid sequence of their constant heavy chain regions. There are five main types of immunoglobulins: IgG, IgA, IgM, IgD, and IgE, with heavy chains of γ, α, μ, δ, and ε, respectively. Antibodies can also be further classified into subtypes, such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The light chains of antibodies, based on the amino acid sequence of their constant structural domains, can be divided into two types: κ and λ.

[0051] The term "variable region" or "variable domain" used in this article refers to the domain of the antibody heavy or light chain in an antigen-binding molecule that participates in antigen binding. The variable domains (VH and VL, respectively) of the heavy and light chains of natural antibodies typically have similar structures, with each domain containing four conserved frame regions (FRs) and three hypervariable regions (HVRs). A single VH or VL domain is sufficient to confer antigen-binding specificity. The HVRs in each chain are tightly held together by the FR regions and, together with the HVRs of other chains, contribute to the formation of the antibody's antigen-binding site. Constant regions do not directly participate in antibody-antigen binding but have other effector functions, such as participating in antibody-dependent cytotoxicity.

[0052] The term "hypervariant region" or "HVR" in this article refers to a region in the variable domain region of an antibody that is highly variable in sequence and / or forms a structurally defined loop ("hypervariant loop"). Typically, a natural tetrachain antibody contains six HVRs: three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). HVRs typically contain amino acid residues from the hypervariant loop and / or from the "complementarity-determining region (CDR)," the amino acid residues from the "CDR" having the highest sequence variability and / or being involved in antigen recognition.

[0053] The term "framework" or "FR" in this article refers to the variable domain residues other than the hypervariable region (HVR) residues. A variable domain FR typically consists of four FR domains: FR1, FR2, FR3, and FR4. Therefore, the HVR and FR sequences typically appear in the VH (or VL) in the following sequence: FR-H1(L1)-FR-H2(L2)-FR-H3(L3)-FR4.

[0054] The term "substitution" or "replacement" used in this article to describe amino acids can refer to the substitution of conserved amino acids, in which an amino acid residue is replaced by an amino acid residue with a similar side chain. Families of amino acid residues with similar side chains have been defined in the art, including basic side chains (e.g., lysine (L), arginine (R), histidine (H)), acidic side chains (e.g., aspartic acid (D), glutamic acid (E)), uncharged polar side chains (e.g., glycine (G), asparagine (N), glutamine (Q), serine (S), threonine (T), tyrosine (Y), cysteine ​​(C)), nonpolar side chains (e.g., alanine (A), valine (V), leucine (L), isoleucine (I), proline (P), phenylalanine (F), methionine (M), tryptophan (W)), β-branched side chains (e.g., threonine (T), valine (V), isoleucine (I)), and aromatic side chains (e.g., tyrosine (Y), phenylalanine (F), tryptophan (W), histidine (H)). Therefore, if an amino acid in a polypeptide is replaced by another amino acid from the same side chain family, such substitution is considered conserved. In another respect, an amino acid chain can be conservatively replaced by a structurally similar amino acid chain that differs in the order and / or composition of its side chain family members.

[0055] The term "nucleic acid molecule" used in this article refers to one of the most important biological macromolecules in organisms. It is the material basis for carrying and transmitting genetic information and includes two major categories: deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).

[0056] The term "antibody fragment" or "antigen-binding fragment" used in this article refers to a portion of a complete antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv, bisomatic antibodies, trisomatic antibodies, tetrasomatic antibodies, cross-Fab fragments, linear antibodies, single-chain antibody molecules (e.g., scFv), single-domain antibodies (single-domain antibodies), etc.

[0057] After obtaining the DNA fragments encoding the VH and / or VL of the antibody, these DNA fragments can be further manipulated using recombinant DNA techniques, such as converting variable region genes into full-length antibody chain genes, Fab fragment genes, or scFv genes. In these operations, the DNA fragment encoding the VH and / or VL is operatively ligated to another DNA fragment encoding a different protein, such as the antibody constant region or a flexible linker. As used herein, the term "operatively ligated" means that two DNA fragments are joined together in such a way that the amino acid sequences encoded by both DNA fragments remain within the reading frame.

[0058] By operatively linking the DNA encoding VH to another DNA molecule encoding the heavy chain constant region (CH1, CH2, and CH3), isolated DNA encoding the VH region can be converted into a full-length heavy chain gene. Similarly, by operatively linking the DNA encoding VL to another DNA molecule encoding the light chain constant region CL, isolated DNA encoding the VL region can be converted into a full-length light chain gene (and a Fab light chain gene).

[0059] The term "antigen-binding domain" or "antigen-binding site" of an antibody, as used herein, refers to the portion of an antibody or its antigen-binding fragment that specifically binds to an antigenic determinant. More specifically, the term "antigen-binding domain" refers to a region of an antibody that specifically binds to and is complementary to a portion or all of an antigen. In cases where the antigen molecule is large, the antibody may bind only to a specific portion of the antigen, called an epitope. The antigen-binding domain may be provided by, for example, one or more variable domains (also called variable regions). Preferably, the antigen-binding domain comprises a variable region (VL) of the antibody light chain and a variable region (VH) of the antibody heavy chain. In some embodiments, the antigen-binding domain is capable of binding its antigen and blocking or partially blocking the function of said antigen.

[0060] The term "antigenic determinant" used herein is interchangeable with "antigen" and "epitope" and refers to a site on a polypeptide molecule (e.g., a continuous amino acid sequence or a conformation composed of discontinuous amino acids) to which an antigen-binding moiety binds, thereby forming an antigen-binding moiety-antigen complex. Unless otherwise stated, the protein used as an antigen in this disclosure can be any naturally occurring protein of any vertebrate origin, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), dogs, cats, etc.

[0061] The terms "vector" or "expression vector" and "expression construct" used herein are used interchangeably to describe a DNA molecule to which a specific gene, operatively linked, is introduced into a target cell and directed for expression. The vector includes a vector as a self-replicating nucleic acid structure and a vector incorporated into the genome of the host cell into which it has been introduced. The expression vectors disclosed herein contain expression cassettes. Expression vectors can perform transcription of large amounts of stable mRNA. Once the expression vector is within the target cell, the cellular transcription and / or translation mechanisms generate a ribonucleic acid molecule or protein encoded by the gene. The term "expression cassette" herein refers to a recombinant or synthetically produced polynucleotide having a set of nucleic acid elements that allow transcription of a specific nucleic acid in the target cell.

[0062] The term "pharmaceutical composition" herein refers to a mixture containing an antibody or antibody- or antigen-binding fragment thereof, biological material, or conjugate of the present disclosure, and other chemical components, such as physiological / pharmaceutical-grade carriers or excipients. The purpose of a pharmaceutical composition is to facilitate administration to a living organism, thereby promoting the absorption of the active ingredient and the exertment of its biological activity.

[0063] The term "treatment" herein refers to the administration of an oral or topical therapeutic agent, such as a composition comprising any antibody of the present disclosure or an antibody thereof or an antigen-binding fragment thereof, or a nucleic acid molecule encoding an antibody thereof or an antibody thereof or an antigen-binding fragment thereof, to a subject who has one or more diseases or symptoms, and the therapeutic agent has a therapeutic effect on these diseases or symptoms. Typically, the therapeutic agent is administered in a treated patient or population in an amount that effectively relieves one or more diseases or symptoms, to induce regression of such symptoms or to inhibit the development of such symptoms to any clinically measurable extent.

[0064] The "sequence identity percentage" or "identity percentage" between two polynucleotide or polypeptide sequences refers to the number of identical matching positions shared by sequences within a comparison window, taking into account additions or deletions (i.e., vacancies) that must be introduced for optimal alignment of the two sequences. A matching position is any location where the same nucleotide or amino acid is present in both the target and reference sequences. Vacancies are not nucleotides or amino acids and are not counted in the target sequence. Similarly, vacancies in the reference sequence are not counted because nucleotides or amino acids from the target sequence are counted, but those from the reference sequence are not.

[0065] The percentage of sequence identity can be calculated as follows: determine the number of positions in both sequences where the same amino acid residue or nucleic acid base appears (the number of matching positions), divide the number of matching positions by the total number of positions in the comparison window, and multiply the result by 100 to obtain the percentage of sequence identity. Sequence comparison and determination of the percentage of sequence identity between two sequences can be accomplished using software that is readily available online and downloadable. Suitable software programs are available from various sources for protein and nucleotide sequence alignment. A suitable program for determining the percentage of sequence identity is bl2seq, which is part of the BLAST program suite available from the National Center for Biotechnology Information (NCBI) website (blast.ncbi.nlm.nih.gov). Bl2seq uses either the BLASTN or BLASTP algorithm for comparing two sequences. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. Other suitable programs are, for example, Needle, Stretcher, Water, or Matcher, which are part of the EMBOSS suite of bioinformatics programs and are also available from the European Institute of Bioinformatics (EBI) at www.ebi.ac.uk / Tools / psa.

[0066] The following embodiments and accompanying drawings are provided to aid in understanding the present invention. However, it should be understood that these embodiments and drawings are for illustrative purposes only and do not constitute any limitation. The actual scope of protection of the present invention is set forth in the claims. It should be understood that any modifications and changes can be made without departing from the spirit of the invention. The reagents and / or kits used in the following embodiments are commercially available or can be synthesized by known methods.

[0067] It should be noted that, unless specific conditions are specified in the examples, experimental conditions should be performed according to standard conditions, manufacturer recommendations, or publicly reported experimental conditions. Reagents or instruments whose manufacturers are not specified are all commercially available, standard products. For reagents whose manufacturers are specified, similar products from other manufacturers are substitutes.

[0068] Unless otherwise specified, the quantitative experiments in the following examples were all repeated three times, and the results were averaged.

[0069] Example Example 1 Expression and purification of SSB protein The SSB protein amino acid sequence was obtained from the Uniport database (UniPort ID: P05455). The full-length amino acid sequence of 1-408 amino acids (SEQ ID NO: 1, nucleotide sequence SEQ ID NO: 2) was selected as the antigen protein for expression. Six HIS tags were added to the C-terminus of the protein. The expression sequence was codon-optimized for the prokaryotic expression system by Beijing Qingke Biotechnology Co., Ltd., and the gene was synthesized. The synthesized full-length SSB gene sequence was ligated into the PET-28a vector using a seamless cloning method. The SSB protein expression plasmid was transformed into BL21 competent cells, and expression was induced overnight at 18°C ​​with 0.2 mM IPTG. The bacterial culture was collected by centrifugation in 300 ml of bacterial culture, resuspended in 20 ml of PBS, and 1 mM PMSF was added. The bacterial cells were then disrupted using an ultrasonic disruptor (output power 60%, working for 4 seconds, stopping for 7 seconds as one cycle, total time 15 min). The bacteria were collected by centrifugation at 12000g for 30 min. The supernatant was purified by affinity chromatography using Ni-NTA affinity resin (Invitrogen™, R90115). First, 1 ml of Ni-NTA was equilibrated with 10 ml Native Binding Buffer (10 mM imidazole). Then, the bacterial supernatant was slowly loaded through the Ni-NTA. Impurities bound to Ni-NTA were washed with 10 ml Native Wash Buffer (20 mM imidazole). Finally, the target protein was eluted with 10 ml Native Elution Buffer (250 mM imidazole). The eluted target protein was concentrated by ultrafiltration to replace the PBS buffer, and then identified by SDS-PAGE. The electrophoresis image is shown below. Figure 1 The molecular weight of SSB protein is approximately 47 kDa, and its purity is >90%.

[0070] SSB protein amino acid sequence: MAENGDNEKMAALEAKICHQIEYYFGDFNLPRDKFLKEQIKLDEGWVPLEIMIKFNRLNRLTTDFNVIVEALSKSKAELMEISEDKTKIRRSPSKPLPEVTDEYKNDVKNRSVYIKGFPTDATLDDIKEWLEDKGQVLNIQMRRTLHKAFKGSIFVVFDSIESAKKFVETPGQKYKETDLLILFKDDYFAKKNEERKQNKVEAKLRAKQEQEAKQKLEEDAEMKSLEEKIGCLLKFSGDLDDQTCREDLHILFSNHGEIKWIDFVRGAKEGIILFKEKAKEALGKAKDANNGNLQLRNKEVTWEVLEGEVEKEALKKIIEDQQESLNKWKSKGRRFKGKGKGNKAAQPGSGKGKVQFQGKKTKFASDDEHDEHDENGATGPVKRAREETDKEEPASKQQKTENGAGDQ (SEQ ID NO: 1), Nucleotide sequence of the SSB protein:

[0071] Example 2: Obtaining Monoclonal Antibodies 1) Biotinylation of SSB protein and conjugation with fluorescein Biotin can be used for simple and effective biotinylation of antibodies, proteins, and other macromolecules containing primary amines in solution. Biotin (Thermo Fisher) was dissolved in dimethyl sulfoxide (DMSO) to prepare a 10 mM biotin solution. 100 μl of the 1 mg / mL SSB protein prepared in Example 1 was taken, and 20 times the excess biotin was added at a molar ratio. The mixture was incubated on ice for 2 h, and excess biotin was removed by passing it through a desalting column. Following a streptavidin:biotin binding molar ratio of 1:4, the biotinylated SSB protein was conjugated with streptavidin-conjugated fluorescein (BD Biosciences). Incubation on ice for 30 min completed the SSB-fluorescein conjugation, yielding fluorescein-labeled SSB.

[0072] 2) Sorting of B cells Peripheral blood was collected from 10 ml of patients with Sjögren's syndrome complicated with interstitial pneumonia. Peripheral blood mononuclear cells (PBMCs) were isolated using human lymphocyte separation medium and analyzed at a concentration of 5 × 10⁻⁶ cells / ml. 6 Each cell / tube was cryopreserved in liquid nitrogen for later use. After cryopreservation, PBMCs were rapidly thawed in a 37°C water bath. The thawed PBMCs were washed and resuspended in FACS buffer (10 mM PBS, 1 mM EDTA, 2% FBS). 100 μL of the resuspended cells (5 × 10⁶ cells / tube) was then collected. 6 PBMCs were sequentially added with the following fluorescent markers: DAPI (biolegend, 422801), CD3-BV241 (BD Biosciences, 562426), CD19-PE / cy7 (BD Biosciences, 555413), CD27-APC / cy7 (BD Biosciences, 560222), IgG-FITC (BD Biosciences, 550931), and SSB-PE (prepared in step 1). The cells were incubated on ice for 30 min. PBMCs were sorted using a BD FACSAria III flow cytometer with a flow cytometry gate of DAPI- / CD3- / CD19+ / CD27+ / IgG+ / SSB+ to obtain an SSB-specific memory B cell population. A 96-well plate was sorted, and excluding 8 cell-free negative control wells, a total of 88 cells were obtained.

[0073] 3) Amplify the antibody variable region gene of a single B cell. Referring to Smith, K., et al. (Rapid generation of fully human monoclonal antibodies specific to a vaccinating antigen. Nat Protoc 4, 372–384 (2009). https: / / doi.org / 10.1038 / nprot.2009.3), antibody variable region genes were obtained from single B cells. In simple terms, B cells were lysed, RNA was extracted, and RT-PCR was performed to amplify the heavy chain Igγ, light chain Igκ, or Igλ variable region gene products. RT-PCR was performed using a mixture of primers from the heavy chain, Kappa chain, and Lambda chain, as shown in Table 1.

[0074] Table 1 Antibody Amplification Primers

[0075] To cover all possible antibody variable region (V) gene families, nested PCR was used to enrich the variable region genes, amplifying DNA to a level sufficient to obtain heavy and light chain V gene sequences. Cloning PCR was performed using highly specific primers for each V gene family, designed to integrate cloning restriction sites and place the VDJ heavy chain or VJ light chain gene sequences within the same open reading frame as the signal peptide sequences and constant region genes in their respective cloning vectors. Gene amplification results are shown below. Figure 2 As shown, heavy chains 59 / 88 (67%), Kappa chains (60 / 88, 68%), and Lambda chains (26 / 88, 29%).

[0076] 4) Antibody expression plasmid construction First, the gamma, kappa, or lambda cloning vectors were double-digested using restriction endonucleases Age I & Sal I, Age I & BsiWI, and Age I & Xho I, respectively. The cloning vectors were referenced in Smith K., et al., and digestion was performed at 37°C for 2 hours. The variable region gene products of the gamma, kappa, or lambda chains amplified in step 3) were purified and recovered using a standard DNA purification kit. The double-digested cloning vector samples were subjected to 1% agarose gel electrophoresis; the vector length was approximately 5700 bp. Homologous recombination of the variable region gene fragment and the vector fragment was performed using the ClonExpress II One Step Cloning Kit at 37°C for 30 minutes. The recombinant product was transformed into competent *E. coli* cells and evenly plated on antibiotic-containing plates, incubated overnight at 37°C. The next day, single colonies from three plates were picked using a pipette tip and placed in 500 μL of antibiotic-containing LB medium, incubated at 37°C for 4–6 hours. PCR was performed using 2 × Taq Master Mix to verify whether the variable region was successfully inserted into the vector. Positive clones were then sequenced to confirm the accuracy of the variable region sequence.

[0077] 5) Small-batch expression of antibodies The recombinant antibody plasmid from step 4) was transfected into 293T cells for low-level expression. 24 hours before transfection, 293T cells were seeded into 96-well plates to maintain 90% confluence. Before transfection, 30 ml of DMEM medium was thoroughly mixed with 500 μl of PEI (1 mg / ml), and 300 μl was added to each well of a new, clean, sterile 96-well plate. Then, 10 ng of each of the corresponding light and heavy chain expression plasmids were added to the same 96-well plate, and the plates were incubated at room temperature for 15 min. The complete culture medium (DMEM + 10% FBS) from the 96-well plates containing the cells was gently aspirated. The DMEM mixture containing the light / heavy chain and transfection reagents was added to the 96-well plates, and the plates were cultured at 37°C and 5% CO2 for 3 days. The cell supernatant was then collected.

[0078] 6) Antibody ELISA specific screening Dilute the SSB protein antigen obtained in Example 1 to 2 μg / ml with carbonate coating buffer, add 100 μL per well to an ELISA plate, and incubate overnight at 4°C. Discard the antigen from the plate and wash for 10 seconds. Add 200 μL of 5% skim milk powder to each well and block at 37°C for 1 hour. Wash the plate three times with 300 μL PBST for 3 minutes each time, adding 100 μL of cell supernatant obtained in step 5) to each well, and incubate at 37°C for 40 minutes. Wash the plate three times with 300 μL PBST for 3 minutes each time. Add 100 μL of goat anti-human IgG secondary antibody (abcam, ab97225) (diluted 1:10000 with 5% skim milk) to each well and block at 37°C for 40 minutes. Wash the plate three times with 300 μL PBST for 3 minutes each time, then add 100 μL of TMB substrate to each well in the dark and incubate at room temperature for 10 minutes. Add 50 μl of 2M sulfuric acid to each well to stop the colorimetric reaction, then place the well on a microplate reader to detect the OD450 value. The results are shown in Table 2. Positive clone 2H2 was screened from 88 samples, and the sequences of its heavy chain variable region and light chain variable region are shown in Table 2.

[0079] Table 2

[0080] 7) Specific antibody expression The selected specific antibody 2H2 was expressed in small batches using 293F cells. The 293F cells were passaged one day before transfection, and on the day of transfection, cell viability was required to be >90%, and the cell density to be 3-4 × 10⁶ cells / year. 6 Cells / mL. Adjust cell density to 1.0 × 10⁶ cells / mL during transfection. 6 Cells / mL were transfected with PEI (1 mg / mL) and 80 μg plasmid DNA (light chain plasmid: heavy chain plasmid = 1:1) and incubated at room temperature for 10 min to form a cationic polymer (PEI:DNA = 3:1, mass ratio). This polymer was slowly added to 293F cells, with each antibody transfecting 80 mL of cells. After 6 days, the cell supernatant was collected for purification. The cell supernatant after expression was purified using ATProtein A Diamond (Bestchrom, AA0272). The supernatant was washed with 10 mL of equilibration buffer (20 mM Na2HPO4, 0.15 M NaCl, pH = 7.0), followed by antibody elution with 5 mL of 0.1 M glycine. The eluted antibody solution was immediately neutralized with 500 μl of 1 M Tris-HCl and dialyzed against 10 mM PBS at 4 °C. The dialysate was changed every 2 h (10 mM PBS), with at least one overnight dialyze. The dialysis was repeated 3 times to obtain the purified antibody 2H2. The purity and concentration of the antibody were identified by SDS-PAGE and ultraviolet absorption spectrometry, and the results are as follows: Figure 3 As shown.

[0081] Example 3: Monoclonal Antibody Performance Detection 1) Detection of 2H2 antibody specificity and sensitivity The monoclonal antibody 2H2 obtained in Example 2 was used to test its specificity and sensitivity. SSB antigen and other autoimmune antigens SmD1 (UniProt: P62314), SmD2 (UniProt: P62316), SmD3 (UniProt: P62318), RNPC (UniProt: P09234), CENPB (UniProt: P07199), JO-1 (UniProt: P12081), Sc1-70 (UniProt: P11387), RNPA (UniProt: P09012), RNP70 (UniProt: P08621), SSA (UniProt: P10155), Ro52 (UniProt: P19474), and PCNA (UniProt: P12004) were diluted to 1 μg / ml with carbonate coating buffer and added to 100 μL per well of the ELISA plate. The plates were incubated overnight at 4°C. After removing the antigen coating solution, blot the ELISA plate dry, then add 200 μL of 5% skim milk powder and block at 37°C for 1 h. Remove the blocking solution and wash the plate three times with 300 μL of PBST, 3 min each time. Dilute the monoclonal antibody 2H2 with 5% skim milk powder to concentrations of 1 μg / mL, 0.1 μg / mL, 0.01 μg / mL, and 0.001 μg / mL, respectively. Add 100 μL of each concentration to the corresponding ELISA plate and incubate at 37°C for 40 min. After discarding the antibody diluent, wash the plate twice with PBST for 3 min each time; add HRP-labeled goat anti-human IgG secondary antibody (1:10000 diluted to 5% skim milk powder), incubate at 37℃ for 40 min, then wash the plate four times with PBST for 3 min each time, add 100 μL of TMB chromogenic solution to each well, react in the dark for 10 min, add 50 μL of 2M sulfuric acid stop solution to each well, and immediately measure the absorbance at 450 nm using a microplate reader. The results are shown in Tables 3 and 4.

[0082] Table 3. Specificity detection of anti-SSB antibodies

[0083] Table 4. Sensitivity detection of anti-SSB antibodies

[0084] The results showed that the SSB monoclonal antibody 2H2 had excellent specificity, did not cross-react with other irrelevant autoimmune antigens such as SmD1, and specifically bound only to the SSB antigen. Furthermore, the 2H2 antibody could still be detected at OD450 when diluted to 0.001 μg / ml, indicating high sensitivity and excellent SSB antigen binding ability.

[0085] 2) Accelerated and freeze-thaw stability tests of 2H2 antibody The 2H2 monoclonal antibody was diluted to two different concentrations: 1 μg / ml and 0.01 μg / ml, and aliquoted into several tubes, 300 μl per tube. Samples for accelerated stability testing were placed in a 37°C incubator and subjected to ELISA testing on days 1, 3, and 7 of accelerated stability. Freeze-thaw stability samples were placed at -80°C. After the antibody was completely frozen, it was removed and thawed at room temperature for 1 hour, then returned to -80°C. This process was repeated once, twice, and three times before ELISA testing. The control antibodies for accelerated and freeze-thaw stability testing were untreated 2H2 antibodies aliquoted and stored at 2–8°C.

[0086] The specific ELISA detection method is as follows: Dilute the SSB antigen to 1 μg / ml with carbonate coating buffer, add 100 μL to each well of the ELISA plate, and incubate overnight at 4°C. After discarding the antigen coating buffer, blot the ELISA plate dry, add 200 μL of 5% skim milk powder, and block at 37°C for 1 h. Discard the blocking buffer, wash the plate three times with 300 μL PBST, 3 min each time. Add 100 μL each of accelerated, freeze-thawed, and untreated 1 μg / mL and 0.01 μg / mL 2H2 solutions to the corresponding wells of the ELISA plate, and incubate at 37°C for 40 min. After discarding the antibody diluent, wash the plate twice with PBST for 3 min each time; add HRP-labeled goat anti-human IgG secondary antibody (1:10000 diluted to 5% skim milk powder), incubate at 37℃ for 40 min, then wash the plate four times with PBST for 3 min each time, add 100 μL of TMB chromogenic solution to each well, react in the dark for 10 min, add 50 μL of 2M sulfuric acid stop solution to each well, and immediately measure the absorbance at 450 nm using a microplate reader.

[0087] The coefficient of variation (CV) of the OD450 of the accelerated and freeze-thaw treated samples was <15% compared with that of the untreated samples, which is considered to indicate that the antibody can maintain stable functional activity under these conditions.

[0088] Table 5. SSB antibody stability test

[0089] As shown in Table 5, whether it is high concentration (1 μg / ml) or low concentration (0.01 μg / ml) SSB antibody 2H2, after 1 day, 3 days or 7 days of accelerated treatment at 37℃, or after 1, 2 or 3 freeze-thaw cycles, the CV relative to the untreated control sample does not exceed 10%, indicating that the 2H2 antibody has good accelerated stability and freeze-thaw stability.

[0090] The technical solutions of the present invention are not limited to the specific embodiments described above. Any technical modifications made in accordance with the technical solutions of the present invention fall within the protection scope of the present invention.

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to SSB, characterized in that, The antibody or its antigen-binding fragment comprises: The heavy chain variable region comprises the amino acid sequences CDR-H1, CDR-H2, and CDR-H3 as shown in SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9, respectively; and the light chain variable region comprises the amino acid sequences CDR-L1, CDR-L2, and CDR-L3 as shown in SEQ ID NO: 18, GAS, and SEQ ID NO: 19, respectively; wherein the CDRs are defined by the IMGT numbering system, or The heavy chain variable region comprises amino acid sequences CDR-H1, CDR-H2, and CDR-H3 as shown in SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, respectively; and the light chain variable region comprises amino acid sequences CDR-L1, CDR-L2, and CDR-L3 as shown in SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22, respectively; wherein the CDRs are defined by the Kabat numbering system, or The heavy chain variable region comprises the amino acid sequences CDR-H1, CDR-H2, and CDR-H3 as shown in SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 12, respectively; and the light chain variable region comprises the amino acid sequences CDR-L1, CDR-L2, and CDR-L3 as shown in SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22, respectively; wherein the CDRs are defined by the Chothia numbering system, or The heavy chain variable region comprises amino acid sequences CDR-H1, CDR-H2, and CDR-H3 as shown in SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17, respectively; and the light chain variable region comprises amino acid sequences CDR-L1, CDR-L2, and CDR-L3 as shown in SEQ ID NO: 23, SEQ ID NO: 24, and SEQ ID NO: 25, respectively; wherein the CDRs are defined by the Contact numbering system.

2. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The heavy chain variable region of the antibody or its antigen-binding fragment further includes a framework region of the heavy chain variable region, and / or the light chain variable region of the antibody or its antigen-binding fragment further includes a framework region of the light chain variable region.

3. The antibody or its antigen-binding fragment according to claim 2, characterized in that, The framework region of the heavy chain variable region includes the framework region of the heavy chain variable region derived from immunoglobulins from mice, primates, cattle, horses, pigs, sheep, goats, dogs, cats, rabbits, camels, donkeys, deer, minks, chickens, ducks, or geese.

4. The antibody or its antigen-binding fragment according to claim 3, characterized in that, The framework region of the heavy chain variable region includes the framework region of the heavy chain variable region derived from human immunoglobulins.

5. The antibody or its antigen-binding fragment according to claim 2, characterized in that, The framework region of the light chain variable region includes the framework region of the heavy chain variable region of immunoglobulins derived from mice, primates, cattle, horses, pigs, sheep, goats, dogs, cats, rabbits, camels, donkeys, deer, minks, chickens, ducks, or geese.

6. The antibody or its antigen-binding fragment according to claim 5, characterized in that, The framework region of the light chain variable region includes the framework region of the light chain variable region derived from human immunoglobulins.

7. The antibody or its antigen-binding fragment according to claim 2, characterized in that, The antibody or its antigen-binding fragment includes: The heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 3, or an amino acid sequence having at least 80%, at least 85%, 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% sequence identity with it; and the light chain variable region has the amino acid sequence shown in SEQ ID NO: 5, or an amino acid sequence having at least 80%, at least 85%, 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% sequence identity with it.

8. The antibody or antigen-binding fragment thereof according to any one of claims 1-7, characterized in that, The antibody or its antigen-binding fragment further includes a heavy chain constant region and / or a light chain constant region.

9. The antibody or its antigen-binding fragment according to claim 8, characterized in that, The heavy chain constant region includes at least a portion of the heavy chain constant region derived from immunoglobulins from mice, primates, cattle, horses, pigs, sheep, goats, dogs, cats, rabbits, camels, donkeys, deer, minks, chickens, ducks, or geese.

10. The antibody or its antigen-binding fragment according to claim 9, characterized in that, The heavy chain constant region includes at least a portion of the heavy chain constant region derived from humans.

11. The antibody or its antigen-binding fragment according to claim 8, characterized in that, The light chain constant region includes at least a portion of the light chain constant region derived from immunoglobulins from mice, primates, cattle, horses, pigs, sheep, goats, dogs, cats, rabbits, camels, donkeys, deer, minks, chickens, ducks, or geese.

12. The antibody or its antigen-binding fragment according to claim 11, characterized in that, The light chain constant region includes at least a portion of the light chain constant region derived from humans.

13. The antibody or its antigen-binding fragment according to claim 8, characterized in that, The antibody is a monoclonal antibody, a monospecific antibody, a bispecific antibody, or a multispecific antibody.

14. The antibody or its antigen-binding fragment according to claim 8, characterized in that, The antibody is a murine, porcine, porcine, chimeric, humanized, or fully human antibody.

15. The antibody or its antigen-binding fragment according to claim 8, characterized in that, The antibody is of type IgA, IgD, IgE, IgG, or IgM.

16. The antibody or its antigen-binding fragment according to claim 15, characterized in that, The antibody is of type IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2.

17. The antibody or antigen-binding fragment thereof according to claim 8, characterized in that, The antigen-binding fragments include scFv, Fab, Fab', (Fab')2, Fv fragments, or dsFv.

18. A nucleic acid molecule, characterized in that, It encodes the antibody or antigen-binding fragment thereof as described in any one of claims 1-17.

19. A carrier, characterized in that, It includes the nucleic acid molecule as described in claim 18.

20. A cell, characterized in that, It includes the antibody or antigen-binding fragment thereof as described in any one of claims 1-17, the nucleic acid molecule as described in claim 18, or the vector as described in claim 19.

21. A coupling, characterized in that, It includes: The antibody or antigen-binding fragment thereof as described in any one of claims 1-17; And, a coupling portion, wherein the coupling portion is a detectable substance.

22. The coupling according to claim 21, characterized in that, The detectable substance is a luminescent marker or a radioactive substance.

23. The coupling according to claim 21, characterized in that, The detectable substance is a fluorescent substance.

24. The coupling according to claim 21, characterized in that, The detectable substance is selected from any one of acridine ester, acridine sulfonamide, luminol, isoluminol, horseradish peroxidase, alkaline phosphatase, and isotopes.

25. A method for preparing the antibody or antigen-binding fragment thereof according to any one of claims 1-17, characterized in that, The method includes culturing the cells of claim 20 and isolating the antibody under conditions that allow expression of the antibody or its antigen-binding fragment.

26. A pharmaceutical composition, characterized in that, It includes: The antibody or antigen-binding fragment thereof according to any one of claims 1-17, the nucleic acid molecule according to claim 18, the vector according to claim 19, the cell according to claim 20, or the conjugate according to any one of claims 21-24; and a pharmaceutically acceptable vector.

27. The pharmaceutical composition of claim 26, wherein the route of administration of the pharmaceutical composition includes parenteral, injection, oral, or topical administration.

28. The pharmaceutical composition according to claim 26, wherein the pharmaceutical composition is in solid, semi-solid or liquid form.

29. The use of the antibody or antigen-binding fragment thereof according to any one of claims 1-17, the nucleic acid molecule according to claim 18, the carrier according to claim 19, the cell according to claim 20, the conjugate according to any one of claims 21-24, or the pharmaceutical composition according to any one of claims 26-28, characterized in that, The application includes one or more of the following: d1) Prepare a product for detecting the presence or level of SSB in a sample; d2) Prepare products for the diagnosis or prognostic assessment of SSB-related diseases.

30. The application according to claim 29, characterized in that, The SSB-related diseases are selected from one or more of the following: lupus erythematosus, ankylosing spondylitis, primary Sjögren's syndrome, vasculitis, scleroderma, interstitial lung disease, primary biliary cholangitis, idiopathic inflammatory myopathy, and rheumatoid arthritis.

31. The application according to claim 29, characterized in that, The samples include whole blood, red blood cell concentrate, platelet concentrate, white blood cell concentrate, tissue, bone marrow aspirate, plasma, serum, cerebrospinal fluid, feces, urine, cultured cells, saliva, oral secretions and / or nasal secretions from the subject.

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