WO2024056009A1 - 一种taci抗体及其用途 - Google Patents

一种taci抗体及其用途 Download PDF

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WO2024056009A1
WO2024056009A1 PCT/CN2023/118680 CN2023118680W WO2024056009A1 WO 2024056009 A1 WO2024056009 A1 WO 2024056009A1 CN 2023118680 W CN2023118680 W CN 2023118680W WO 2024056009 A1 WO2024056009 A1 WO 2024056009A1
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seq
antibody
amino acid
acid sequence
antigen
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PCT/CN2023/118680
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English (en)
French (fr)
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姚雪静
贠莎莎
张宝鹏
朱冬雪
胡园
邢玲
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荣昌生物制药(烟台)股份有限公司
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Priority to CN202380014064.4A priority Critical patent/CN118139890A/zh
Publication of WO2024056009A1 publication Critical patent/WO2024056009A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants

Definitions

  • the invention relates to the field of biomedicine, specifically to a human B lymphocyte stimulating factor receptor (TACI) antibody and its use.
  • TACI human B lymphocyte stimulating factor receptor
  • AID Autoimmune diseases
  • AARDA American Autoimmune Related Diseases Association
  • autoimmune diseases include systemic lupus erythematosus (SLE), rheumatoid Rheumatoid Arthritis (RA), Psoriatic Arthritis (PsA), Myasthenia Gravis (MG), Multiple Sclerosis (MS), Sicca Syndrome, SS) etc.
  • SLE systemic lupus erythematosus
  • RA rheumatoid Rheumatoid Arthritis
  • PsA Psoriatic Arthritis
  • MG Myasthenia Gravis
  • MS Multiple Sclerosis
  • Sicca Syndrome SS
  • B lymphocyte stimulators (BLyS) is a new member of the TNF superfamily discovered in 1999. It regulates B lymphocyte maturation, promotes immunoglobulin class switching, helper T cell activation and Participate in autoimmune and other functions.
  • BCMA B cell maturation antigen
  • BAFF-R BAFF receptor, BR3
  • TACI transmembrane activator and calcium modulator and cyclophilin ligand interactor
  • BLyS mainly promotes transitional B cell maturation in mice, extends the lifespan of mouse and human mature B cells, and activates helper T cells through BAFF-R; BCMA has Maintains the survival of plasmablasts and promotes the antigen presentation of mature B cells; and the TACI receptor has a dual effect on the activation of mouse B cells: on the one hand, TACI-deficient mice show an increase in peripheral B cells, increased antigen synthesis, and the occurrence of self- Immunity and even lethal lymphoproliferation phenomenon, suggesting that TACI is an inhibitory receptor of BLyS; on the other hand, TACI is involved in the T cell-independent antigen (TI) immune response.
  • BAFF-R is an inhibitory receptor of BLyS.
  • Proprietary receptors, and TACI and BCMA can also bind to APRIL in addition to BLyS, and the TACI receptor has a strong affinity for both BLyS and APRIL, so drugs developed with TACI receptors (such as TACI -Fc fusion protein) is superior to drugs developed with the other two receptors in blocking BLyS and APRIL, and is the preferred drug structure.
  • TACI tumor necrosis factor
  • CAML-interactor transmembrane activator and CAML-interactor
  • TNF tumor necrosis factor
  • TACI-Fc fusion protein brings expected good therapeutic effects, the mechanism of action of autoimmune diseases is complex.
  • Methods start treatment with conventional doses. If patients do not respond adequately, researchers will upgrade the drug dose. Therefore, if you want to provide more specific and individualized treatment options for patients with autoimmune diseases, you must While gaining a deeper understanding of the manifestations of the disease in different patients, it complements specific companion diagnostic methods. While providing effective treatment options, it can also conduct disease assessment, efficacy monitoring and prognosis judgment on the drug absorption level in the patient's body fluids after treatment. , thereby ensuring that patients are provided with correct, specific, and individualized treatment plans.
  • Companion diagnostics is an in vitro diagnostic technology related to targeted drugs. It mainly measures the expression levels of proteins and mutated genes in the human body to understand the therapeutic response of different patients to specific drugs and screen out the most suitable ones. Targeted and individualized treatments are provided to drug users, thereby improving their treatment prognosis and reducing health care expenses.
  • the U.S. FDA promulgated the "Companion Diagnostic Guidelines” on August 6, 2014. Companion diagnostics can help determine the most likely targets for A patient population that responds to a therapeutic drug promotes the use of the drug in a relatively limited market and improves the drug's effectiveness and safety.
  • CDx is conducive to the design of clinical trial plans with small samples, and during the development process, clearer results can be obtained with less investment.
  • the advantage of companion diagnosis is that it can screen out effective treatment plans for patients, save the time and cost of ineffective treatments, improve patients' compliance with medication, reduce the incidence of adverse reactions, and ensure drug safety and efficacy.
  • the present invention provides an antibody that can target TACI and a method for detecting the content of free TACI in a biological sample using the TACI antibody.
  • the detection antibody uses the TACI content in the patient sample as a detection index, and passes The determination of its content can not only provide a basis for the prognosis of patients with autoimmune diseases, but also effectively provide guidance for the medication and treatment of autoimmune diseases.
  • the present invention provides an antibody or antigen-binding fragment thereof that can specifically bind to TACI.
  • the antibodies or antigen-binding fragments provided by the present invention include:
  • Heavy chain variable region includes the VH-CDR1 amino acid sequence described in SEQ ID NO: 1, the VH-CDR2 amino acid sequence described in SEQ ID NO: 2, SEQ The VH-CDR3 amino acid sequence (Kabat numbering, the same below) described in ID NO:3; or
  • VH Heavy chain variable region
  • the heavy chain variable region includes the VH-CDR1 amino acid sequence described in SEQ ID NO: 4, the VH-CDR2 amino acid sequence described in SEQ ID NO: 5, SEQ The VH-CDR3 amino acid sequence described in ID NO:6; or
  • Heavy chain variable region includes the VH-CDR1 amino acid sequence described in SEQ ID NO:7, the VH-CDR2 amino acid sequence described in SEQ ID NO:8, SEQ The VH-CDR3 amino acid sequence described in ID NO:9; and/or
  • VL Light chain variable region
  • the light chain variable region includes the VL-CDR1 amino acid sequence described in SEQ ID NO: 10, the VL-CDR2 amino acid sequence described in SEQ ID NO: 11, SEQ The VL-CDR3 amino acid sequence described in ID NO:12; or
  • Light chain variable region includes the VL-CDR1 amino acid sequence described in SEQ ID NO: 13, the VL-CDR2 amino acid sequence described in SEQ ID NO: 14, SEQ The VL-CDR3 amino acid sequence described in ID NO:15; or
  • Light chain variable region includes the VL-CDR1 amino acid sequence described in SEQ ID NO: 16, the VL-CDR2 amino acid sequence described in SEQ ID NO: 17, SEQ The VL-CDR3 amino acid sequence described in ID NO:18.
  • the antibody is a mouse antibody.
  • the antibody is a rabbit antibody.
  • the antibody is a mouse-derived antibody.
  • the antibody is a rabbit-derived antibody.
  • the antigen-binding fragment is Fab.
  • the antigen-binding fragment is F(ab')2.
  • the antigen-binding fragment is Fab’.
  • the antigen-binding fragment is Fv.
  • the antigen-binding fragment is ScFv.
  • the antibody or antigen-binding fragment includes:
  • VH Heavy chain variable region
  • VL Light chain variable region
  • the antibody or antigen-binding fragment includes:
  • Heavy chain variable region the heavy chain variable region has at least 95%, 96%, 97%, 98%, 99% or 100% similarity with the amino acid sequence shown in SEQ ID NO:19 Sequence identity; and/or light chain variable region (VL), the light chain variable region has at least 95%, 96%, 97%, 98%, 99% with the amino acid sequence shown in SEQ ID NO:22 % or 100% sequence identity; or
  • Heavy chain variable region the heavy chain variable region has at least 95%, 96%, 97%, 98%, 99% or 100% of the amino acid sequence shown in SEQ ID NO:20 Sequence identity; and/or light chain variable region (VL), the light chain variable region has at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity; or
  • Heavy chain variable region which has at least 95%, 96%, 97%, 98%, 99% or 100% similarity with the amino acid sequence shown in SEQ ID NO:21 Sequence identity; and/or light chain variable region (VL), the light chain variable region has at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity.
  • the antibody or antibody-binding fragment includes: a heavy chain variable region (VH), the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 19; and/ Or light chain variable region (VL), the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 22.
  • VH heavy chain variable region
  • VL light chain variable region
  • the antibody or antibody-binding fragment includes: a heavy chain variable region (VH), the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 20; and/ Or light chain variable region (VL), the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 23.
  • VH heavy chain variable region
  • VL light chain variable region
  • the antibody or antibody-binding fragment includes: a heavy chain variable region (VH), the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 21; and/ Or light chain variable region (VL), the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 24.
  • VH heavy chain variable region
  • VL light chain variable region
  • the antibody or antigen-binding fragment contains an Fc region.
  • the Fc has at least 95%, 96%, 97%, 98%, 99% or 100% similarity with the amino acid sequence shown in SEQ ID NO:25, SEQ ID NO:26 or SEQ ID NO:27. % sequence identity.
  • amino acid sequence of the Fc is shown in SEQ ID NO: 25.
  • amino acid sequence of the Fc is shown in SEQ ID NO: 26.
  • amino acid sequence of the Fc is shown in SEQ ID NO: 27.
  • the antibody or antigen-binding fragment includes:
  • Heavy chain the heavy chain has at least 95%, 96%, 97%, 98%, 99% similarity with the amino acid sequence shown in SEQ ID NO:28, SEQ ID NO:29, or SEQ ID NO:30 % or 100% sequence identity; and/or
  • the light chain has at least 95%, 96%, 97%, 98%, 99% similarity with the amino acid sequence shown in SEQ ID NO:31, SEQ ID NO:32, or SEQ ID NO:33 % or 100% sequence identity.
  • the antibody or antigen-binding fragment includes:
  • Heavy chain the heavy chain has at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 28; and/or light Chain, the light chain has at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 31; or
  • Heavy chain the heavy chain has at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 29; and/or light Chain, the light chain has at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 32; or
  • Heavy chain the heavy chain has at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 30; and/or light chain, the light chain has at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:33.
  • the antibody or antigen-binding fragment includes: a heavy chain, the amino acid sequence of the heavy chain is shown in SEQ ID NO: 28; and/or a light chain, the light chain The amino acid sequence is shown in SEQ ID NO:31.
  • the antibody or antigen-binding fragment includes: a heavy chain, the amino acid sequence of the heavy chain is shown in SEQ ID NO: 29; and/or a light chain, the light chain The amino acid sequence is shown in SEQ ID NO:32.
  • the antibody or antigen-binding fragment includes: a heavy chain, the amino acid sequence of the heavy chain is shown in SEQ ID NO: 30; and/or a light chain, the light chain The amino acid sequence is shown in SEQ ID NO:33.
  • the antibody or antigen-binding fragment can be further coupled with a detectable label.
  • the detectable label is an enzyme.
  • the detectable label is a luminescent label.
  • the detectable label is a radioactive isotope.
  • the detectable label is a chromogenic label.
  • the detectable label is a hapten.
  • the detectable label is a metal complex.
  • the present invention also provides the use of the antibody or antigen-binding fragment described in any of the above for detecting the TACI content in a sample.
  • sample is a biological sample.
  • the sample is a blood sample.
  • samples were derived from subjects overexpressing BLyS and APRIL.
  • sample is derived from subjects suffering from autoimmune diseases.
  • the autoimmune disease is selected from rheumatoid arthritis, rheumatoid arthritis, juvenile rheumatoid arthritis, systemic lupus erythematosus (SLE), lupus nephritis (LN), Wegener's disease, inflammation Sexual enteropathy, idiopathic thrombocytopenic purpura (ITP), thrombotic thrombocytopenic purpura (TTP), autoimmune thrombocytopenia, multiple sclerosis, psoriasis, IgA nephropathy, IgM polyneuropathy, severe disease One or more of myasthenia, vasculitis, diabetes, Reynauld's syndrome, Sjorgen's syndrome, glomerulonephritis, autoimmune hepatitis, autoimmune thyroiditis, neuromyelitis optica, and Sjogren's syndrome.
  • the autoimmune disease is rheumatoid arthritis; in other specific embodiments, the autoimmune disease is rheumatoid arthritis; in other specific embodiments In, the autoimmune disease is juvenile rheumatoid arthritis; in other specific embodiments, the autoimmune disease is systemic lupus erythematosus (SLE); in other specific embodiments, the autoimmune disease is systemic lupus erythematosus (SLE).
  • the autoimmune disease is lupus nephritis (LN); in other specific embodiments, the autoimmune disease is Wegener's disease; in other specific embodiments, the autoimmune disease is inflammation sexual enteropathy; in other specific embodiments, the autoimmune disease is idiopathic thrombocytopenic purpura (ITP); in other specific embodiments, the autoimmune disease is thrombotic platelet disease TTP; in other specific embodiments, the autoimmune disease is autoimmune thrombocytopenia; in other specific embodiments, the autoimmune disease is multiple sclerosis ; In other specific embodiments, the autoimmune disease The disease is psoriasis; in other specific embodiments, the autoimmune disease is IgA nephropathy; in other specific embodiments, the autoimmune disease is IgM polyneuropathy; in other specific embodiments, the autoimmune disease is IgA nephropathy.
  • LN lupus nephritis
  • the autoimmune disease is Wegener's disease
  • the autoimmune disease is
  • the autoimmune disease is myasthenia gravis; in other specific embodiments, the autoimmune disease is vasculitis; in other specific embodiments, the autoimmune disease The disease is diabetes; in other specific embodiments, the autoimmune disease is Reynauld's syndrome; in other specific embodiments, the autoimmune disease is Sjorgen's syndrome; in other specific implementations In the scheme, the autoimmune disease is glomerulonephritis; in other specific embodiments, the autoimmune disease is autoimmune hepatitis; in other specific embodiments, the autoimmune disease The disease is autoimmune thyroiditis; in other specific embodiments, the autoimmune disease is neuromyelitis optica; in other specific embodiments, the autoimmune disease is Sjogren's syndrome.
  • the autoimmune disease described above is an overlapping syndrome of two, three, four or even multiple diseases, that is, the patient manifests multiple specific autoimmune diseases at the same time.
  • the autoimmune disease is an overlapping syndrome of rheumatoid arthritis and systemic lupus erythematosus, or the autoimmune disease is rheumatoid arthritis or systemic lupus erythematosus (SLE). , multiple sclerosis, etc.
  • the subject is a human or a mammal. In a preferred embodiment, the subject is a human.
  • the detection was performed by Western blotting.
  • the present invention also provides a method for detecting TACI content in a sample, which is characterized in that the method includes the following steps: contacting the detection sample with the antibody or antigen-binding fragment described in any of the above, and then detecting the bound Presence of antibodies or antigen-binding fragments.
  • the method for retrieving TACI content in a sample provided by the present invention includes, but is not limited to, the following steps:
  • Coating Coat the TACI antibody or antigen-binding fragment thereof as described in any of the above on a 96-well enzyme plate, and incubate at a specific temperature;
  • Substrate color development Add substrate to develop color
  • the microplate reader reads the difference between 450nm and 570nm.
  • the present invention also provides an isolated polynucleotide encoding the antibody or antigen-binding fragment described in any one of the above.
  • the present invention also provides an expression vector comprising the polynucleotide described above.
  • the present invention also provides the use of the above-mentioned expression vector in preparing any of the above-mentioned antibodies or antigen-binding fragments.
  • the present invention also provides a host cell comprising at least one expression vector as described above.
  • the present invention also provides the use of the antibody or antigen-binding fragment described in any of the above in preparing a kit.
  • the present invention also provides a kit comprising the antibody described in any one of the above.
  • the present invention provides an antibody targeting TACI or an antibody-binding fragment thereof.
  • the TACI antibody can detect the content of TACI in patients with immune diseases, especially the content of free TACI or TACI-Fc fusion protein drugs, and can further detect the content of TACI in patients with immune diseases.
  • the content of the drug in the patient's body is calculated to calculate the patient's drug absorption level, which plays a role in assessing the patient's condition, monitoring efficacy, and judging prognosis.
  • Figure 1 is a butterfly diagram of the consistency analysis of deuterated behavior in the control group B-1 and the interaction group B;
  • Figure 2 is a residue map showing the difference in deuteration between control group B-1 and interaction group B;
  • Figure 3 is a residue map showing the difference in deuteration percentage between control group B-1 and interaction group B;
  • Figure 4 is a butterfly diagram of the consistency analysis of deuterated behavior in the control group A-1 and the interaction group A;
  • Figure 5 is a residue map showing the difference in deuteration between control group A-1 and interaction group A;
  • Figure 6 is a residue map showing the difference in deuteration percentage between control group A-1 and interaction group A;
  • Figure 7 is a standard curve of free Tatasip-OD
  • TACI Transmembrane Activator And Caml Interactor. Normally, wild-type TACI contains a type III membrane protein of 293 amino acid residues (166 extracellular functional domains and 10 transmembrane domains). (117 intracellular functional regions), and no signal peptide at the amino terminus. There are two extracellular cysteine-rich repeat regions (S33-66 and C70-C104), which belong to the TNFR superfamily but lack the "death structure" of the TNF family compared with other TNF receptor superfamily members.
  • antibody is used in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (eg, bispecific antibodies), and antibody fragments.
  • antibody refers to a protein containing at least two heavy chains and two light chains interconnected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (one-fifth or one-fourth of the heavy chain near the N-terminus) and a heavy chain constant region (three-quarters or four-fifths of the heavy chain near the C-terminus). Area).
  • Each light chain contains a light chain variable region (the region near the N-terminal half of the light chain) and a light chain constant region (the region near the C-terminal half of the light chain).
  • the heavy chain variable region and light chain variable region region can also be subdivided into multiple regions with high variability, called complementarity determining regions (CDRs).
  • CDR refers to the hypervariable regions of the heavy and light chains of immunoglobulins, including those determined by Kabat, Chothia, IMGT, AbM and Contact systems. There are three heavy chain CDRs and three light chain CDRs per antibody.
  • the term CDR is used herein to indicate one, or several or even all of these regions, as the case may be, which region contains the majority of the amino acid residues responsible for binding by the affinity of the antibody for the antigen or the epitope it recognizes.
  • antigen-binding fragment refers to an antibody fragment comprising an antibody heavy chain variable region or a light chain variable region sufficient to retain the same binding specificity and sufficient affinity as the antibody from which it is derived.
  • Fab fragments Fab' fragments, F(ab) 2 fragments, F(ab') 2 fragments, Fv fragments, scFv fragments and/or isolated complementarity determining regions.
  • Fab Usually refers to a monovalent fragment consisting of VH, VL, CL and CH1 domains.
  • Fab' generally refers to fragments that differ from Fab by adding a small number of residues (including one or more cysteines from the hinge region of the antibody) to the carboxy terminus of the heavy chain CH1 domain.
  • F(ab') 2 generally refers to a dimer of Fab', a bivalent fragment consisting of two Fab fragments linked by a disulfide bond at the hinge region.
  • Fv generally refers to an Fv fragment consisting of the VL and VH domains of a single arm of an antibody.
  • scFv generally refers to a monovalent molecule formed by pairing VH and VL through a linker.
  • Such scFv molecules can have a general structure: NH 2 -VL-linker-VH-COOH or NH 2- VH-linked Sub-VL-COOH.
  • These antibody fragments can be obtained using conventional techniques known to those skilled in the art, and these fragments can be screened for utility in the same manner as intact antibodies.
  • murine antibody refers to an antibody having heavy and light chains derived exclusively from murine B cells.
  • the antibody thus consists of a murine amino acid sequence, regardless of the origin of the cell in which it was produced.
  • rabbit antibody refers to variable and constant regions or domains that substantially correspond to rabbit germline immunoglobulin sequences known in the art
  • mouse-derived antibody and "rabbit-derived antibody” in the present invention refer to monoclonal antibodies prepared according to the knowledge and skills in the art. During preparation, a test subject is injected with the corresponding antigen, and then hybridomas expressing antibodies with desired sequences or functional properties are isolated.
  • the murine or rabbit antibody or antigen-binding fragment thereof may further comprise the light chain constant region of the murine or rabbit kappa or lambda chain or a variant thereof, or further comprise The heavy chain constant region of mouse or rabbit IgG1, IgG2, IgG3 or variants thereof.
  • Fc region defines the C-terminal region of an immunoglobulin heavy chain and includes, for example, native sequence Fc regions, recombinant Fc regions and variant Fc regions. Although the boundaries of the immunoglobulin heavy chain Fc region may vary, the human IgG heavy chain Fc region is generally defined as extending from the amino acid residues at position Cys226 or from Pro230 to its carboxy terminus.
  • the C-terminal lysine of the Fc region (residue 447 according to the EU numbering system) can be removed, for example, during the production or purification of the antibody, or by recombinant engineering of the nucleic acid encoding the antibody heavy chain.
  • compositions of intact antibodies can include populations of antibodies with all K447 residues removed, populations of antibodies with K447 residues removed, and populations of antibodies containing mixtures of antibodies with and without K447 residues.
  • a functional "Fc region” has the "effector functions" of a native sequence Fc region.
  • Exemplary "effector functions” include Clq binding; CDC; Fc receptor binding; ADCC; phagocytosis; downregulation of cell surface receptors (eg, B cell receptors), etc.
  • effector functions generally require binding of the Fc region to a binding region or domain (eg, an antibody variable region or domain) and can be assessed using a variety of assays known to those skilled in the art.
  • a variant "Fc region” includes a variant resulting from at least one amino acid modification (e.g., substitution, An amino acid sequence that is different from the amino acid sequence of the Fc region of the native sequence due to addition or deletion).
  • the variant Fc region has at least one amino acid substitution compared to the native sequence Fc region or compared to the Fc region of the parent polypeptide, for example, from about 1 to about 10 amino acids in the native sequence Fc region or the Fc region of the parent polypeptide. substitution, or about 1 to about 5 amino acid substitutions.
  • a variant Fc region herein may have at least about 80% homology to a native sequence Fc region and/or to an Fc region of a parent polypeptide, or at least about 90% homology thereto, e.g., at least about 95% homology thereto. Homology.
  • detectable label refers to any component capable of providing a detection signal under detection conditions, including directly and indirectly detectable labels.
  • Detectable labels useful in the methods described herein include any component that can be detected indirectly or directly by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical, chemical or other means.
  • antigen markers e.g. digoxigenin (DIG), fluorescein, dinitrophenol (DNP), etc.
  • biotin for staining with labeled streptavidin conjugates e.g.
  • radioactive labels for example, 125i, 35S, 14C, OR32p
  • enzymes for example, Streptavidin-HRP, peroxidase, alkaline phosphate enzymes, galactosidases and other enzymes commonly used in ELISA
  • fluorescent proteins e.g. green fluorescent protein, red fluorescent protein, yellow fluorescent protein, etc.
  • synthetic polymers that chelate metals (metal complexes), colorimetric Tags etc.
  • the term "enzyme” refers to a molecule that is protein in nature and catalyzes metabolic biochemical reactions present in cells or in the extracellular medium.
  • oxidoreductases such as oxidase, reductase, peroxidase, oxygenase, hydrogenase or dehydrogenase
  • transferases such as kinases; transaminase; mutase
  • hydrolases such as esterase; Peptidase; glycosidase; glucosidase
  • lyase such as decarboxylase, aldolase; dehydratase
  • isomerase such as racemase; epimerase
  • ligase is a peroxidase, more preferably a Streptavidin-HRP enzyme.
  • the term "luminescent mark” refers to a mark that is capable of emitting light when externally stimulated. This can be photoluminescence. Fluorescent labels (which include dye molecules or quantum dots), and luminescent labels (eg, electro or chemiluminescent labels) are different types of luminescent labels.
  • the external excitation is light (photons) for fluorescence, electric current for electroluminescence, and chemical reaction for chemiluminescence. External incentives can be a combination of the above.
  • hapten refers to a partial or incomplete antigen. Haptens are protein-free substances that do not stimulate antibody formation but do react with antibodies.
  • metal complex refers to a metal-containing compound that contains a central metal atom or ion and a surrounding array of associated molecules or ions (ie, ligands).
  • biological sample includes, but is not limited to, samples derived from or originating from a living organism. Any amount of matter. Such living organisms include, but are not limited to, humans, mice, monkeys, rats, rabbits and other animals. Such substances include, but are not limited to, blood, serum, urine, cells, organs, tissues, bones, bone marrow, lymph nodes and skin.
  • blood sample refers to any sample prepared from blood, such as plasma, blood cells separated from blood, and the like.
  • BLyS includes Shu et al., J. Leukocyte Biol., 65: 680 (1999); GenBank registration number AF136293; WO98/18921 published on May 7, 1998; EP86/9180 published on October 7, 1998; WO98/27114 published on June 25, 1998; WO99/12964 published on March 18, 1999; WO99/33980 published on page 7, 8, 1999; Moore et al., Science, 285: 260-263 (1999); Those described in Schneider et al., J. Exp. Med., 189: 1747-1756 (1999); Mukhopadhyay et al., J. Biol. Chem., 274: 15978-15981 (1999).
  • APRIL refers to A proliferation-inducing ligand, which is a proliferation-inducing ligand composed of 184 amino acid residues (NCBI Reference Sequence: NP_003799.1) and belongs to the TNF superfamily.
  • autoimmune disease refers to a disease in which people with healthy immune systems have an abnormal immune response.
  • autoimmune diseases include, but are not limited to, acute disseminated encephalomyelitis, acute necrotizing hemorrhagic leukoencephalitis, Addison's disease, agammaglobulinemia, patchy alopecia, amyloidosis, ankylosing spondylitis Spondylitis, anti-GBM/anti-TBM nephritis, antiphospholipid syndrome, autoimmune angioedema, autoimmune aplastic anemia, autoimmune dysautonomia, autoimmune hepatitis, autoimmune hyperlipidemia, autoimmune immunodeficiency , autoimmune inner ear disease, autoimmune myocarditis, autoimmune ovarianitis, autoimmune pancreatitis, autoimmune retinopathy, autoimmune thrombocytopenic purpura, autoimmune thyroid disease, autoimmune urticaria, axonal or neuronal neuropathy, Bar
  • Elisa refers to a qualitative and quantitative detection method in which soluble antigens or antibodies are bound to solid-phase carriers such as polystyrene, and the specificity of antigen-antibody binding is used to perform immune reactions.
  • Western blot includes not only standard western blots but also various variations such as Far-Western blots, Northwestern blots and Southwestern blots.
  • Western blotting involves the transfer of proteins to a membrane and subsequent detection of the proteins on the membrane.
  • membranes known in the art that are suitable for use as Western blotting membranes, including but not limited to: polyvinylidene fluoride (PVDF) membranes, nitrocellulose membranes, polyamide membranes, polyester membranes, and nylon membranes.
  • PVDF polyvinylidene fluoride
  • Western blotting usually uses transfer buffer.
  • a variety of Western blot transfer buffers are known in the art. usually, The pH of the Western blot transfer buffer is above the isoelectric point of the protein to be transferred. Therefore, when a voltage potential is applied, the protein migrates toward the positive electrode. Alternatively, the pH of the transfer buffer can be below the isoelectric point of the protein to be transferred. In such cases, proteins migrate toward the negative electrode.
  • immunohistochemistry refers to a technique used to detect the presence of an antigen in a histological sample using an antibody capable of specifically binding to said antigen. Detection of antibody-antigen complexes is usually performed by chromogenic reactions using enzyme-labeled antibodies or by fluorescently labeled antibodies.
  • the following examples prepare TACI antibodies by immunizing TACI antigens
  • mice After immunizing mice with human TACI antigen, spleen cells from the immunized mice were fused with mouse myeloma cells to establish a series of hybridoma cell lines.
  • the hybridoma cells were cultured, and the supernatant was taken for enzyme-linked immunosorbent assay (ELISA) detection: BLyS (R&D Systems) was coated on a 96-well enzyme plate, and incubated at 4°C overnight; blocked with 3% BSA-PBST, 300ul/ wells and incubate at 37°C for 3 hours; incubate the supernatant diluted 10 times, 100 times, and 1000 times in PBST with 30 ng/ml Tatacept separately for a certain period of time, then add 100ul/well to the 96-well plate, and incubate at 37°C for 1 hour.
  • ELISA enzyme-linked immunosorbent assay
  • the results are shown in Table 1.
  • the results show that the culture supernatant of three cell lines: 1B11D2-1, 7G12F8-1, and 12C9F3-1 can significantly inhibit the binding of BlyS and TACI-Fc fusion protein, while the inhibitory effect of other cell lines is not good. Therefore, the supernatants of the above three cell lines were collected and purified, and antibodies 1B11D2, 7G12F8, and 12C9F3 were obtained respectively.
  • amino acid sequences of antibodies 1B11D2, 7G12F8, and 12C9F3 are as follows:
  • 3Test product B BLyS (R&D Systems), diluted to 40 ⁇ M with buffer;
  • 4Test product C Tatacept, diluted to 40 ⁇ M with buffer;
  • the enzymatically digested products were analyzed by PAL3 autosampler, LEAP Technologies) Load the sample and pass it through the chromatographic column (ACQUITY UPLC Peptide CSH C18 Column, 1.7 ⁇ m, 1mm Mass spectrometry acquisition time: 15min, detection method: positive ion, precursor ion scanning range: 300-1500m/z. MS resolution: 60,000. Secondary mass spectrometry resolution: 15,000. The detection reaction parameters are shown in Table 3.
  • Figure 1 is the butterfly diagram of the consistency analysis of the deuterated behavior of the control group B-1 and the interaction group B. According to the test results, it can be seen that the overall symmetry of the butterfly diagrams of the two is good, but there are local differences.
  • Figures 2 and 3 are residue diagrams showing the difference in deuteration number (Da) and deuteration percentage on different ordinates according to the number of the peptide segment.
  • Figure 2 shows that there are obvious differences in the deuteration behavior of the control group B-1 and the interaction group B.
  • the horizontal line is the 1Da limit
  • the significant areas of differential peptides are 29-64(#15), 50-68(#19), 67-72(#22), 67-73(#23), 68-99(# 25),94-106(#27),96-106(#28),98-106(#29),193-208(#57),235-252(#78),267-290(#94) area, suggesting significant changes in structure.
  • Figure 3 shows that there are obvious differences in the deuteration behavior of control group B-1 and interaction group B (the horizontal line is the 5% limit).
  • the significant areas of differential peptides are 29-64 (#15), 50-68 (#19), and 67 -72(#22),67-73(#23,68-99(#25),94-106(#27),96-106(#28),98-106(#29),99-106( #30), 100-106(#31), 128-137(#39), 132-138(#40), 156-162(#45)193-208(#57), 234-241(#68) , 234-248(#69), 235-252(#78), 267-290(#94) regions, suggesting significant structural changes.
  • Figure 4 is the butterfly diagram of the consistency analysis of the deuteration behavior of the control group A-1 and the interaction group A. According to the test results, it can be seen that the overall symmetry of the butterfly diagrams of the control group A-1 and the interaction group A is good, but there are local differences.
  • Figures 5 and 6 are residue diagrams showing the difference in deuteration number (Da) and deuteration percentage on different ordinates according to the number of the peptide segment.
  • Figure 5 shows that there are obvious differences in the deuteration behavior of the control group A-1 and the interaction group A. , significant differential peptide regions 29-64(#12), 54-92(#18), 67-73(#19), 68-99(#21), 74-107(#23), 252-262(# 76), 269-290(#90), 271-291(#93) and 297-311(#100) regions, suggesting significant structural changes.
  • Figure 6 shows that there are obvious differences in the deuteration behavior between control group A-1 and interaction group A.
  • differential peptides The significant areas of differential peptides are 7-16 (#6), 29-64 (#12), 54-92 (#18), 67 -73(#19),68-99(#21),74-107(#23),95-106(#24),252-262(#76),269-290(#90),297-311 (#93) and 301-309 (#103) regions, suggesting significant structural changes.
  • the residue diagram of the difference in deuteration and the residue diagram of the difference in deuteration percentage it can be seen that the interaction between the two is at 29-32.
  • the 67-73 and 267-290 regions and their vicinity are the ligand receptor binding epitopes of the interacting group B, among which the interacting group has the highest affinity in the 67-73 region.
  • the residue diagram analysis of the deuteration difference and the residue diagram analysis of the deuteration percentage difference it can be seen that the interaction between the two is at 29-32.
  • the 67-73 and 269-290 regions and their vicinity are the ligand receptor binding epitopes of the interacting group A, among which the interacting group has the highest affinity in the 67-73 region.
  • Regions 29-32, 67-73 and 269-290 are both binding epitopes of interacting group B and interacting group A, indicating that 1B11D2 and BLyS are in a competitive relationship with Tatacept respectively, and the degree of overlap is highly consistent.
  • Octet SA Biosensors (Thermo, 87766) was used to immobilize biotinylated 1B11D2 antibody, with different concentrations of Tatacept as the analyte, and the detection pattern was obtained through kinetic fitting.
  • Standard curve sample Prepare Tatacept to the following concentrations: 1000.0ng/ml, 500.0ng/ml, 400.0ng/ml, 300.0ng/ml, 200.0ng/ml, 120.0ng/ml, 60.0ng/ml, 30.0ng /ml, 15.0ng/ml and 10.0ng/ml;
  • Sample to be tested Add 15ul of clinical blood sample to 105ul of 1% BSA-PBS and mix well, then add 10ul of the mixture to 90ul of 1% BSA-PBS and mix well;
  • Blank control Add 15ul of blank serum to 105ul of 1% BSA-PBS and mix well, then add 10ul of the mixture to 90ul of 1% BSA-PBS and mix well.
  • Coating Coat 1B11D2 antibody (14.2ug/ml) on a 96-well enzyme plate and incubate at 4°C overnight.
  • Blocking wash the plate, block with 3% BSA-PBST, 300ul/well, and incubate at 37°C for 2 hours.
  • Detect antibody binding wash the plate, add standard curve samples, test samples and blank controls at 100ul/well, and incubate at room temperature for 1.5 hours to fully combine the free TACI in each sample with the 1B11D2 antibody.
  • Secondary antibody binding wash the plate, dilute the secondary antibody Goat-anti-human IgG Fc-Biotin 1:5000, then add 100ul/well to the enzyme plate, and incubate at room temperature for 40 minutes.
  • Substrate color development wash the plate and add 100ul of substrate (tetramethylbenzidine) to each well for color development.
  • Termination Add 50ul of stop solution (2M H 2 SO 4 ) to each well to terminate the reaction.
  • the sample to be tested reads 1.977 with a microplate reader.
  • the content of free TACI in the sample to be tested is calculated as 207.275ng/ml according to the following formula.
  • the calculation formula is:
  • y represents the OD value
  • x represents the concentration
  • A is the upper asymptote estimate
  • B is the slope
  • C is the corresponding concentration at half the maximum binding (EC 50 )
  • D is the lower asymptote estimate.
  • the TACI antibody detected the content of TACI in patients treated with immune diseases, especially the content of free TACI or TACI-Fc fusion protein drugs. Based on the content of the drug in the patient's body, the patient's drug absorption level was effectively understood. It plays a role in condition assessment, efficacy monitoring and prognosis judgment of patients with autoimmune diseases.

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Abstract

提供一种与TACI具有高特异性和高亲和力的人B淋巴细胞刺激因子受体(TACI)抗体，以及利用所述TACI抗体进行TACI或TACI-Fc融合蛋白检测的方法，该检测方法可以检测患者体内游离TACI或TACI-Fc融合蛋白类药物的含量，根据患者体内药物的含量，有效了解了患者药物吸收的水平，起到了对自身免疫性疾病患者的病情评估、疗效监测及预后判断情况的作用。

Description

一种TACI抗体及其用途 技术领域
本发明涉及生物医药领域，具体涉及一种人B淋巴细胞刺激因子受体(TACI)抗体及其用途。
背景技术
自身免疫性疾病(autoimmune diseases，AID)是一类由机体对自身抗原失去耐受，引起免疫系统攻击自身组织，最终导致各系统组织和器官损伤的一类疾病。根据近年来大规模流行病学研究显示，自身免疫性疾病在世界范围内的患病率已高达5％～8％，临床需求巨大。根据美国自身免疫性相关疾病学会(American Autoimmune Related Diseases Association，AARDA)统计，目前已发现100多种自身免疫性疾病，常见的自身免疫疾病包括系统性红斑狼疮(systemic lupus erythematosus，SLE)、类风湿性关节炎(Rheumatoid Arthritis，RA)、银屑病关节炎(Psoriatic Arthritis，PsA)、重症肌无力(Myasthenia Gravis，MG)、多发性硬化症(Multiple Sclerosis，MS)、干燥综合征(Sicca Syndrome，SS)等。
BLyS和APRIL的过量表达是多种自身免疫疾病的病因之一，临床研究表明，BLyS的浓度往往与自身免疫疾病的严重程度成正相关，因此，阻断BLyS和APRIL的作用就成为治疗自身免疫疾病的一个有效途径。B淋巴细胞刺激因子(B lymphocyte stimulators，BLyS)是1999年发现的TNF超家族新成员，其通过与相应受体结合发挥其调控B淋巴细胞成熟、促进免疫球蛋白类别转换、辅助T细胞活化和参与自身免疫等功能。目前已知BLyS受体有三个：BCMA(B cell maturation antigen)、BAFF-R(BAFF receptor，BR3)、和TACI(transmembrane activater and calcium modulator and cyclophilin ligand interactor，TACI)。BLyS的三种受体在与BLyS结合时发挥的作用不同，BLyS主要通过BAFF-R实现促进小鼠过渡期B细胞成熟、延长小鼠和人成熟B细胞的寿命以及辅助T细胞活化；BCMA具有维持浆母细胞存活和促进成熟B细胞的抗原呈递作用；而TACI受体对小鼠B细胞的活化具有双重效应：一方面TACI缺陷的小鼠表现为外周B细胞增多、抗原合成增加，出现自身免疫甚至致死性淋巴增殖现象，提示TACI为BLyS的抑制性受体；另一方面TACI又参与T细胞非依赖性抗原(TI)的免疫应答，另外，BAFF-R是BLyS的 专有受体，而TACI和BCMA除与BLyS结合外，还能与APRIL结合，并且TACI受体对BLyS和APRIL这两者均有很强的亲和力，因此以TACI受体开发的药物(如TACI-Fc融合蛋白)阻断BLyS和APRIL的作用较以其他两种受体开发的药物为优，是首选的药物结构。
TACI，即穿膜蛋白活化物(transmambrane activator and CAML-interactor)是肿瘤坏死因子(Tumor Necrosis Factor，TNF)受体超家族成员，主要表达于B细胞，微弱表达于单核细胞、树突状细胞和T细胞系中。目前以TACI受体开发的药物主要为TACI-Fc融合蛋白和TACI抗体两种类型，并以TACI-Fc融合蛋白形式为主。截止到2022年09月02日，全球仅一款TACI-Fc融合蛋白获批上市，另有2款药物处于临床阶段。
尽管TACI-Fc融合蛋白带来了预期良好的治疗效果，但是自身免疫疾病作用机制复杂，在临床实践中，由于缺乏针对性的伴随诊断手段，患者常常会被采用反应性逐步增强、反复试验的方法，从常规剂量开始进行治疗，如果患者并未产生充分反映的话，研究人员就会升级药物剂量，因此，想要实现对自身免疫性疾病患者提供更具体和个体化的治疗方案，就必须在更深入地了解疾病在不同患者体内表现的同时，与特异性伴随诊断方法相辅相成，在提供有效治疗方案的同时对患者治疗用药后体液中的药物吸收水平进行病情评估、疗效监测及预后判断的检测，从而保证为患者提供正确、特异性、且个体化的治疗方案。
伴随诊断(companion diagnostics，CDx)，是一种与靶向药物相关的体外诊断技术，主要通过测量人体内蛋白、变异基因的表达水平，了解不同患者对特定药物的治疗反应，筛选出最合适的用药人群并有针对性地进行个体化治疗，从而改善其治疗预后和降低保健开支。美国FDA于2014年8月6日颁布了《伴随诊断指南》，伴随诊断有助于确定最有可能针对 治疗药物产生响应的患者群体，促进药物在相对有限的市场中使用，提高药物的有效性和安全性。在药物的开发中，CDx有利于设计小样本的临床试验方案，在开发过程中，以更少的投入获得更清楚明确的结果。伴随诊断的优势在于可以为患者筛选出有效的治疗方案，节省无效治疗的时间和费用，提高患者服药的依从性，降低不良反应的发生率，保证了药物安全性和疗效。
然而，目前尚缺乏能够满足市场需要的快速对TACI表达进行有效检测的免疫组化检测抗体以及相关检测方法。
发明内容
为了克服现有技术的缺陷，本发明提供了一种能够靶向TACI的抗体以及利用该TACI抗体检测生物样本中游离TACI的含量的方法，该检测抗体以患者样本中TACI含量作为检测指标，通过对其含量的测定，既可对自身免疫性疾病患者的预后判断提供依据，又可以有效的为自身免疫性疾病的用药治疗提供指导。
一方面，本发明提供了一种能够与TACI特异性结合的抗体或其抗原结合片段。具体的，本发明提供的抗体或抗原结合片段包含：
(1)重链可变区(VH)，所述的重链可变区包含SEQ ID NO:1所述的VH-CDR1氨基酸序列、SEQ ID NO:2所述的VH-CDR2氨基酸序列、SEQ ID NO:3所述的VH-CDR3氨基酸序列(Kabat编号，下同)；或
(2)重链可变区(VH)，所述的重链可变区包含SEQ ID NO:4所述的VH-CDR1氨基酸序列、SEQ ID NO:5所述的VH-CDR2氨基酸序列、SEQ ID NO:6所述的VH-CDR3氨基酸序列；或
(3)重链可变区(VH)，所述的重链可变区包含SEQ ID NO:7所述的VH-CDR1氨基酸序列、SEQ ID NO:8所述的VH-CDR2氨基酸序列、SEQ  ID NO:9所述的VH-CDR3氨基酸序列；和/或
(4)轻链可变区(VL)，所述的轻链可变区包含SEQ ID NO:10所述的VL-CDR1氨基酸序列、SEQ ID NO:11所述的VL-CDR2氨基酸序列、SEQ ID NO:12所述的VL-CDR3氨基酸序列；或
(5)轻链可变区(VL)，所述的轻链可变区包含SEQ ID NO:13所述的VL-CDR1氨基酸序列、SEQ ID NO:14所述的VL-CDR2氨基酸序列、SEQ ID NO:15所述的VL-CDR3氨基酸序列；或
(6)轻链可变区(VL)，所述的轻链可变区包含SEQ ID NO:16所述的VL-CDR1氨基酸序列、SEQ ID NO:17所述的VL-CDR2氨基酸序列、SEQ ID NO:18所述的VL-CDR3氨基酸序列。
进一步的，所述的抗体是鼠抗体。
进一步的，所述的抗体是兔抗体。
进一步的，所述的抗体是鼠源抗体。
进一步的，所述的抗体是兔源抗体。
进一步的，所述的抗原结合片段为Fab。
进一步的，所述的抗原结合片段为F(ab’)2。
进一步的，所述的抗原结合片段为Fab’。
进一步的，所述的抗原结合片段为Fv。
进一步的，所述的抗原结合片段为ScFv。
进一步的，所述的抗体或抗原结合片段包含：
(1)重链可变区(VH)(VH)，所述的重链可变区与SEQ ID NO:19、SEQ ID NO:20、或SEQ ID NO:21所示的氨基酸序列具有至少95％、96％、97％、98％、99％或100％的序列同一性；和/或
(2)轻链可变区(VL)，所述的轻链可变区与SEQ ID NO:22、SEQ ID NO:23、或SEQ ID NO:24所示的氨基酸序列具有至少95％、96％、97％、98％、99％或100％的序列同一性。
更进一步的，所述的抗体或抗原结合片段包含：
(1)重链可变区(VH，所述的重链可变区与SEQ ID NO:19所示的氨基酸序列具有至少95％、96％、97％、98％、99％或100％的序列同一性；和/或轻链可变区(VL)，所述的轻链可变区与SEQ ID NO:22所示的氨基酸序列具有至少95％、96％、97％、98％、99％或100％的序列同一性；或
(2)重链可变区(VH)，所述的重链可变区与SEQ ID NO:20所示的氨基酸序列具有至少95％、96％、97％、98％、99％或100％的序列同一性；和/或轻链可变区(VL)，所述的轻链可变区与SEQ ID NO:23所示的氨基酸序列具有至少95％、96％、97％、98％、99％或100％的序列同一性；或
(3)重链可变区(VH)，所述的重链可变区与SEQ ID NO:21所示的氨基酸序列具有至少95％、96％、97％、98％、99％或100％的序列同一性；和/或轻链可变区(VL)，所述的轻链可变区与SEQ ID NO:24所示的氨基酸序列具有至少95％、96％、97％、98％、99％或100％的序列同一性。
在某些具体的实施方案中，所述的抗体或抗体结合片段包含：重链可变区(VH)，所述的重链可变区的氨基酸序列如SEQ ID NO:19所示；和/或轻链可变区(VL)，所述的轻链可变区的氨基酸序列如SEQ ID NO:22所示。
在某些具体的实施方案中，所述的抗体或抗体结合片段包含：重链可变区(VH)，所述的重链可变区的氨基酸序列如SEQ ID NO:20所示；和/或轻链可变区(VL)，所述的轻链可变区的氨基酸序列如SEQ ID NO:23所示。
在某些具体的实施方案中，所述的抗体或抗体结合片段包含：重链可变区(VH)，所述的重链可变区的氨基酸序列如SEQ ID NO:21所示；和/或轻链可变区(VL)，所述的轻链可变区的氨基酸序列如SEQ ID NO:24所示。
进一步的，所述的抗体或抗原结合片段包含Fc区。
更进一步的，所述的Fc与SEQ ID NO:25、SEQ ID NO:26、或SEQ ID NO:27所示的氨基酸序列具有至少95％、96％、97％、98％、99％或100％的序列同一性。

在某些具体的实施方案中，所述的Fc的氨基酸序列如SEQ ID NO:25所示。
在某些具体的实施方案中，所述的Fc的氨基酸序列如SEQ ID NO:26所示。
在某些具体的实施方案中，所述的Fc的氨基酸序列如SEQ ID NO:27所示。
进一步的，所述的抗体或抗原结合片段包含：
(1)重链，所述的重链与SEQ ID NO:28、SEQ ID NO:29、或SEQ ID NO:30所示的氨基酸序列具有至少95％、96％、97％、98％、99％或100％的序列同一性；和/或

(2)轻链，所述的轻链与SEQ ID NO:31、SEQ ID NO:32、或SEQ ID NO:33所示的氨基酸序列具有至少95％、96％、97％、98％、99％或100％的序列同一性。

更进一步的，所述的抗体或抗原结合片段包含：
(1)重链，所述的重链与SEQ ID NO:28所示的氨基酸序列具有至少95％、96％、97％、98％、99％或100％的序列同一性；和/或轻链，所述的轻链与SEQ ID NO:31所示的氨基酸序列具有至少95％、96％、97％、98％、99％或100％的序列同一性；或
(2)重链，所述的重链与SEQ ID NO:29所示的氨基酸序列具有至少95％、96％、97％、98％、99％或100％的序列同一性；和/或轻链，所述的轻链与SEQ ID NO:32所示的氨基酸序列具有至少95％、96％、97％、98％、99％或100％的序列同一性；或
(3)重链，所述的重链与SEQ ID NO:30所示的氨基酸序列具有至少95％、96％、97％、98％、99％或100％的序列同一性；和/或轻链，所述的轻链与SEQ ID NO:33所示的氨基酸序列具有至少95％、96％、97％、98％、99％或100％的序列同一性。
在某些具体的实施方案中，所述的抗体或抗原结合片段包含：重链，所述的重链的氨基酸序列如SEQ ID NO:28所示；和/或轻链，所述的轻链的氨基酸序列如SEQ ID NO:31所示。
在某些具体的实施方案中，所述的抗体或抗原结合片段包含：重链，所述的重链的氨基酸序列如SEQ ID NO:29所示；和/或轻链，所述的轻链的氨基酸序列如SEQ ID NO:32所示。
在某些具体的实施方案中，所述的抗体或抗原结合片段包含：重链，所述的重链的氨基酸序列如SEQ ID NO:30所示；和/或轻链，所述的轻链的氨基酸序列如SEQ ID NO:33所示。
进一步的，所述的抗体或抗原结合片段可进一步与可检测标记偶联。
更进一步的，所述的可检测标记是酶。
更进一步的，所述的可检测标记是发光标记。
更进一步的，所述的可检测标记是放射性同位素。
更进一步的，所述的可检测标记是显色标记。
更进一步的，所述的可检测标记是半抗原。
更进一步的，所述的可检测标记是金属络合物。
另一方面，本发明还提供了上述任一项所述的抗体或抗原结合片段在检测样品中TACI含量中的用途。
进一步的，所述的样品是生物学样品。
更进一步的，所述的样品为血液样品。
进一步的，所述的样品源自BLyS和APRIL过量表达的对象。
更进一步的，所述的样品源自患有自身免疫疾病的对象。
更进一步的，所述的自身免疫疾病选自风湿性关节炎、类风湿性关节炎、青少年类风湿性关节炎、系统性红斑狼疮(SLE)、狼疮肾炎(LN)、韦格纳病、炎症性肠病、特发性血小板减少性紫癜(ITP)、血栓性血小板减少性紫癜(TTP)、自身免疫性血小板减少症、多发性硬化症、银屑病、IgA肾病、IgM多发性神经病、重症肌无力、脉管炎、糖尿病、Reynauld’s综合征、Sjorgen’s综合征、肾小球肾炎、自身免疫性肝炎、自身免疫性甲状腺炎、视神经脊髓炎、干燥综合征疾病中的一种或几种。
在某些具体的实施方案中，所述的自身免疫疾病为风湿性关节炎；在另一些具体的实施方案中，所述的自身免疫疾病为类风湿性关节炎；在另一些具体的实施方案中，所述的自身免疫疾病为青少年类风湿性关节炎；在另一些具体的实施方案中，所述的自身免疫疾病为系统性红斑狼疮(SLE)；在另一些具体的实施方案中，所述的自身免疫疾病为狼疮肾炎(LN)；在另一些具体的实施方案中，所述的自身免疫疾病为韦格纳病；在另一些具体的实施方案中，所述的自身免疫疾病为炎症性肠病；在另一些具体的实施方案中，所述的自身免疫疾病为特发性血小板减少性紫癜(ITP)；在另一些具体的实施方案中，所述的自身免疫疾病为血栓性血小板减少性紫癜(TTP)；在另一些具体的实施方案中，所述的自身免疫疾病为自身免疫性血小板减少症；在另一些具体的实施方案中，所述的自身免疫疾病为多发性硬化症；在另一些具体的实施方案中，所述的自身免疫疾 病为银屑病；在另一些具体的实施方案中，所述的自身免疫疾病为IgA肾病；在另一些具体的实施方案中，所述的自身免疫疾病为IgM多发性神经病；在另一些具体的实施方案中，所述的自身免疫疾病为重症肌无力；在另一些具体的实施方案中，所述的自身免疫疾病为脉管炎；在另一些具体的实施方案中，所述的自身免疫疾病为糖尿病；在另一些具体的实施方案中，所述的自身免疫疾病为Reynauld’s综合征；在另一些具体的实施方案中，所述的自身免疫疾病为Sjorgen’s综合征；在另一些具体的实施方案中，所述的自身免疫疾病为肾小球肾炎；在另一些具体的实施方案中，所述的自身免疫疾病为自身免疫性肝炎；在另一些具体的实施方案中，所述的自身免疫疾病为自身免疫性甲状腺炎；在另一些具体的实施方案中，所述的自身免疫疾病为视神经脊髓炎；在另一些具体的实施方案中，所述的自身免疫疾病为干燥综合征疾病。
在另一些具体的实施方案中，所述的自身免疫疾病上述两种、三种、四种甚至多种疾病的重叠综合征，即患者同时变现为患者多种具体的自身免疫疾病。非限制性举例，所述的自身免疫疾病表现为类风湿性关节炎和系统性红斑狼疮的重叠综合征，或者所述的自身免疫疾病表现为类风湿性关节炎、系统性红斑狼疮(SLE)、多发性硬化症等。
更进一步的，所述的对象为人或哺乳动物，在优选的实施方案中，所述的对象为人。
进一步的，所述的检测通过Elisa进行。
进一步的，所述的检测通过蛋白质印迹进行。
进一步的，所述的检测通过免疫组织化学(IHC)进行。
另一方面，本发明还提供了检测样品中TACI含量的方法，其特征在于，所述的方法包括如下步骤：用上述任一项所述的抗体或抗原结合片段接触检测样品，之后检测结合的抗体或抗原结合片段的存在。
在某些具体的实施方案中，本发明提供的检索样品中TACI含量的方法，非限制性的包括如下步骤：
(1)包被：将上述任一项所述的TACI抗体或其抗原结合片段包被在96孔酶标板上，特定温度下孵育；
(2)封闭：用封闭液后继续孵育。
(3)与检测样品的接触：加入标准曲线样品、质控样品、检测样品及 空白对照，孵育，使样品中的游离TACI与TACI抗体(1B11D2)充分结合；
(4)二抗结合：加入二抗；
(5)二抗显色：加入显色剂；
(6)底物显色：:加入底物显色；
(7)终止：加入终止液，终止反应；
(8)读数酶标仪读取450nm和570nm差值。
另一方面，本发明还提供了一种分离的多核苷酸，其编码上述任一项所述的抗体或抗原结合片段。
另一方面，本发明还提供了一种表达载体，其包含上述所述的多核苷酸。
另一方面，本发明还提供了一种上述所述的表达载体在制备上述任一项所述的抗体或抗原结合片段中的用途。
另一方面，本发明还提供了一种宿主细胞，其包含至少一种上述所述的表达载体。
另一方面，本发明还提供了上述任一项所述的抗体或抗原结合片段在制备试剂盒中的用途。
另一方面，本发明还提供了一种试剂盒，所述的试剂盒包含上述任一项所述的抗体。
本发明提供了一种靶向TACI的抗体或其抗体结合片段，该TACI抗体能够检测治疗免疫性疾病患者体内TACI的含量，尤其是游离TACI或TACI-Fc融合蛋白类药物的含量，进而可根据患者体内药物的含量计算出患者药物吸收水平，从而起到对患者的病情评估、疗效监测及预后判断情况的作用。
附图说明
图1是对照组B-1和相互作用组B氘代行为一致性分析的蝴蝶图；
图2是对照组B-1和相互作用组B氘代差异的残基图；
图3是对照组B-1和相互作用组B氘代百分比差的残基图；
图4是对照组A-1和相互作用组A氘代行为一致性分析的蝴蝶图；
图5是对照组A-1和相互作用组A氘代差异的残基图；
图6是对照组A-1和相互作用组A氘代百分比差的残基图；
图7是游离泰它西普-OD的标准曲线；
具体实施方式
定义
除非另有定义，本文使用的所有术语具有本领域普通技术人员所理解的相同含义。关于本领域的定义及术语，专业人员具体可参考Current Protocols in Molecular Biology(Ausubel)。氨基酸残基的缩写是本领域中所用的指代20个常用L-氨基酸之一的标准3字母和/或1字母代码。
术语的“TACI”是指穿膜蛋白活化物(Transmembrane Activator And Caml Interactor)，通常情况下，野生型TACI包含293个氨基酸残基的Ⅲ型膜蛋白(胞外功能区166个，穿膜区10个，胞内功能区117个)，氨基端无信号肽。胞外有2个富含半胱氨酸的重复区(S33-66和C70-C104)，属于TNFR超家族但与其它TNF受体超家族成员相比缺少TNF家族的“死亡结构”。
术语“抗体”以最广义使用，并且涵盖各种抗体结构，包括但不限于单克隆抗体、多克隆抗体、多特异性抗体(例如双特异性抗体)、和抗体片段。特别的，本文所使用的“抗体”指包含通过二硫键而互连的至少两条重链和两条轻链的蛋白。每条重链包含一重链可变区(重链上靠近N端的五分之一或四分之一的区域)和一重链恒定区(重链上靠近C端四分之三或五分之四的区域)。每条轻链包含一轻链可变区(轻链上靠近N端二分之一的区域)和一轻链恒定区(轻链上靠近C端二分之一的区域)。重链可变区和轻链可变区区域还可再细分为具有高可变性的多个区，被称为互补决定区(CDR)。所述的“CDR”是指免疫球蛋白的重链和轻链的高变区，包括Kabat、Chothia、IMGT、AbM以及Contact体系确定的。每个抗体存在三个重链CDR和三个轻链CDR。根据情况，本文所用术语CDR是为了指示这些区域之一、或者这些区域的几个或者甚至全部，所述区域包含通过抗体对抗原或其识别表位的亲和力而负责结合的大部分氨基酸残基。
术语“抗原结合片段”是指包含抗体重链可变区或轻链可变区的抗体片段，所述片段足以保留与其来源抗体相同的结合特异性和充分的亲和力。特别是但不限于Fab片段、Fab’片段、F(ab)2片段、F(ab’)2片段、Fv片段、scFv片段和/或分离的互补决定区。在本发明中，术语“Fab” 通常是指由VH、VL、CL和CH1结构域组成的单价片段。术语Fab’通常是指在重链CH1结构域的羧基端添加少量残基(包括一个或多个来自抗体铰链区的半胱氨酸)而不同于Fab的片段。术语“F(ab’)2”通常是指Fab’的二聚体，包含在铰链区处通过二硫键连接的两个Fab片段的双价片段。术语“Fv”通常是指由抗体单臂的VL和VH结构域组成的Fv片段。术语“scFv”通常是指VH和VL通过连接子(linker)连接配对形成的单价分子，此类scFv分子可具有一般结构：NH2-VL-连接子-VH-COOH或NH2-VH-连接子-VL-COOH。这些抗体片段可用本领域技术人员公知的常规技术获得，并用与完整抗体相同的方法对这些片段的实用性进行筛选。
术语“鼠抗体”是指具有仅衍生自鼠B细胞的重链和轻链的抗体。该抗体因此由鼠氨基酸序列组成，无论使其产生的细胞的来源是什么。
术语“兔抗体”是指可变区和恒定区或基本上对应于本领域已知的兔种系免疫球蛋白序列的结构域
术语“鼠源抗体”和“兔源抗体”在本发明中为根据本领域知识和技能制备的单克隆抗体。制备时用对应的抗原注射试验对象，然后分离表达具有所需序列或功能特性的抗体的杂交瘤。在本发明一个优选的实施方案中，所述的鼠源或兔源抗体或其抗原结合片段，可进一步包含鼠源或兔源κ、λ链或其变体的轻链恒定区，或进一步包含鼠源或兔源IgG1、IgG2、IgG3或其变体的重链恒定区。
术语“Fc区”定义了免疫球蛋白重链的C-末端区域，包括例如，天然序列Fc区，重组Fc区域和变体Fc区。虽然免疫球蛋白重链Fc区的边界可能会有所不同，但人IgG重链Fc区通常定义为从Cys226位置的氨基酸残基或从Pro230延伸到其羧基末端。Fc区的C末端赖氨酸(根据EU编号系统的残基447)可以被去除，例如，在抗体的生产或纯化过程中，或通过重组工程改造编码抗体重链的核酸。因此，完整抗体的组合物可包含去除了所有K447残基的抗体群，去除了K447残基的抗体群，和包含具有和不具有K447残基的抗体混合物的抗体群。功能性“Fc区”具有天然序列Fc区的“效应物功能”。示例性的“效应功能”包括C1q结合；CDC；Fc受体结合；ADCC；吞噬作用；下调细胞表面受体(例如，B细胞受体)等。这类效应功能通常需要令Fc区与结合区或结合结构域(例如，抗体可变区或结构域)结合，并且可以使用本领域技术人员已知的各种试验进行评估。变体“Fc区”包含由于至少一个氨基酸修饰(例如，取代、 添加或缺失)而与天然序列Fc区的氨基酸序列不同的氨基酸序列。在某些实施方式中，变体Fc区相比天然序列Fc区或相比亲本多肽的Fc区有至少一处氨基酸取代，例如天然序列Fc区或亲本多肽Fc区中约1至约10处氨基酸取代，或约1至约5处氨基酸取代。本文的变体Fc区可与天然序列Fc区和/或与亲本多肽的Fc区具有至少约80％的同源性，或与其至少约90％的同源性，例如，与其至少约95％的同源性。
术语“可检测标记”是指在检测条件下，能够提供检测信号的任何组分，包括直接和间接可检测的标记。可用于本文所述方法的可检测标记包括可通过光谱，光化学，生物化学，免疫化学，电学，光学，化学或其它手段间接或直接检测的任何组分。例如，抗原标记(例如地高辛素(DIG)，荧光素，二硝基苯酚(DNP)等)，用于用标记的链霉亲和素缀合物染色的生物素，荧光染料(例如荧光素，德士古红，罗丹明，荧光团标记如阿利克斯Fluor标记等)，放射性标记(例如，125i，35S，14C，OR32p)酶(例如Streptavidin-HRP、过氧化物酶，碱性磷酸酶，半乳糖苷酶和通常用于ELISA的其它酶)，荧光蛋白(例如绿色荧光蛋白，红色荧光蛋白，黄色荧光蛋白等)，螯合金属的合成聚合物(金属络合物)，比色标记等。
术语“酶”是指性质是蛋白质的分子，其催化细胞或细胞外介质中存在的代谢的生化反应。具体例如氧化还原酶(诸如氧化酶，还原酶，过氧化物酶，加氧酶，氢化酶或脱氢酶)；转移酶(诸如激酶；转氨酶；变位酶)；水解酶(诸如酯酶；肽酶；糖苷酶；葡糖苷酶)；裂合酶(诸如脱羧酶，醛缩酶；脱水酶)；异构酶(诸如消旋酶；表异构酶)；连接酶。优选，所述酶是过氧化物酶，更优选的是Streptavidin-HRP酶。
术语“发光标记”指能够在外部激励发光时的标记。这可以是光致发光。荧光标记(其包括染料分子或量子点)，和发光标记(例如，电或化学发光标记物)都是不同类型的发光标记。外部激励对于荧光是光(光子)，对于电发光时是电流，而对于化学发光时是化学反应。外部激励可以是上述的组合。
术语“半抗原”是指部分或不完全抗原。半抗原是不能刺激抗体形成但与抗体反应的无蛋白质的物质。
术语“金属络合物”是指含金属的化合物，其包含中心金属原子或离子和结合的分子或离子(即配体)的周围阵列。
术语“生物学样品”包括但不限于来自活体生物或原来是活体生物的 任何量的物质。所述活体生物包括但不限于人、小鼠、猴、大鼠、兔和其它动物。所述物质包括但不限于血液、血清、尿液、细胞、器官、组织、骨、骨髓、淋巴结及皮肤。
术语“血液样品”是指从血液，诸如血浆、从血液分离的血细胞等制备的任何样品。
术语“BLyS”包括Shu等，J.Leukocyte Biol.，65：680(1999)；GenBank登记号AF136293；1998年5月7日公开的WO98/18921；1998年10月7日公开的EP86/9180；1998年6月25日公开的WO98/27114；1999年3月18日公开的WO99/12964；1999年7页8日公开的WO99/33980；Moore等，Science，285：260-263(1999)；Schneider等，J.Exp.Med.，189：1747-1756(1999)；Mukhopadhyay等，J.Biol.Chem.，274：15978-15981(1999)中所述的那些。
术语“APRIL”指A proliferation-inducing ligand,其是增殖诱导配体,由184个胺基酸残基组成(NCBI Reference Sequence:NP_003799.1),属于TNF超家族。
术语“自身免疫疾病”是指其中免疫系统较健康人群具有异常免疫应答的疾病。自身免疫疾病的实施例包括但不限于急性播散性脑脊髓炎、急性坏死性出血性脑白质炎、阿狄森氏病、无γ球蛋白血症、斑形脱发、淀粉样变性、僵直性脊椎炎、抗GBM/抗TBM肾炎、抗磷脂综合征、自体免疫血管性水肿、自体免疫再生障碍性贫血、自体免疫自主神经异常、自体免疫肝炎、自体免疫高脂质血症、自体免疫免疫缺乏、自体免疫内耳病、自体免疫心肌炎、自体免疫卵巢炎、自体免疫胰腺炎、自体免疫视网膜病变、自体免疫血小板减少性紫癜、自体免疫甲状腺疾病、自体免疫荨麻疹、轴突或神经元神经病变、巴洛病、贝塞特氏病、心肌病变、卡斯尔曼病、查加斯病、慢性疲劳综合征、慢性炎症性脱髓鞘多发性神经病变、慢性复发性多灶性骨髓炎(CRMO)、车格-施特劳斯综合征、瘢痕性类天疱疮/良性粘膜性类天疱疮、柯根氏综合征、冷凝集素病、先天性心脏阻滞、柯萨奇心肌炎、CREST病、原发性混合冷球蛋白血症、脱髓鞘神经病变、疱疹样皮炎、皮肤肌炎、视神经脊髓炎、盘状狼疮、德雷斯勒氏综合征、子宫内膜异位、嗜酸粒细胞性食管炎、嗜酸粒细胞性筋膜炎、结节性红斑、实验性过敏性脑脊髓炎、埃文斯综合征、纤维肌痛、纤维性肺泡炎、巨细胞动脉炎、巨细胞心肌炎、肾小球性肾炎、古德帕斯彻氏综合征、肉芽肿病伴多血管炎、格雷福斯病、格林-巴利综合征、溶血性贫血、亨诺克-合 恩来因紫癜、妊娠疱疹、低γ球蛋白血症、特发性血小板减少性紫癜、IgA肾病、IgG4相关的硬化性疾病、免疫调控性脂蛋白疾病、包涵体肌炎、间质性膀胱炎、青少年肌炎、川崎综合征、兰伯特-伊顿综合征、白细胞破裂性血管炎、扁平苔癣、硬化性苔癣、木质性结膜炎、线性IgA疾病、莱姆病、慢性关尼尔氏病、显微性多血管炎、混合结缔组织疾病、慕伦氏溃疡、慕夏-哈伯曼病、多发性硬化症、重症肌无力、肌炎、嗜睡病、嗜中性白细胞减少症、眼瘢痕性类天疱疮、视神经炎、复发性风湿病、副赘生性小脑变性、阵发性夜间血红蛋白尿、帕里龙伯格综合征、帕森尼奇-特纳综合征、扁平部睫状体炎、天疱疮、外周神经病变、静脉周脑脊髓炎、恶性贫血、POEMS综合征、多发性结节性动脉炎、I、II和III型自体免疫多腺性综合征、风湿性多肌痛、多发性肌炎、心肌梗塞后综合征、心包切开术后综合征、孕酮皮炎、原发性胆汁性硬化、原发性硬化性胆管炎、特发性肺纤维化、坏疽性脓皮病、纯红细胞再生障碍、雷诺氏现象、反应性关节炎、反射交感性营养不良、莱特尔氏综合征、复发性多软骨炎、多动腿综合征、腹膜后纤维化、风湿热、风湿性关节炎、类风湿性关节炎、青少年类风湿性关节炎、类肉瘤病、施密特综合征、巩膜炎、硬皮病、精子和睾丸自体免疫性、僵人综合征、亚急性细菌性心内膜炎、苏萨克氏综合征、交感性眼炎、高安氏动脉炎、颞动脉炎/巨细胞动脉炎、托洛萨-亨特综合征、横贯性脊髓炎、未分化结缔组织疾病、葡萄膜炎、血管炎、水疱大疱性皮肤病、白癜风或韦格纳氏肉芽肿病、系统性红斑狼疮(SLE)、狼疮肾炎(LN)、韦格纳病、炎症性肠病、血栓性血小板减少性紫癜(TTP)、自身免疫性血小板减少症、银屑病、IgM多发性神经病、脉管炎、糖尿病、Reynauld’s综合征、Sjorgen’s综合征、肾小球肾炎、自身免疫性肝炎、自身免疫性甲状腺炎、干燥综合征。自身免疫疾病由上述两种、三种、四种甚至多种疾病同时发病的情况，被称为“重叠综合征”(overlap syndrome)。
术语“Elisa”，是指可溶性的抗原或抗体结合到聚苯乙烯等固相载体上，利用抗原抗体结合专一性进行免疫反应的定性和定量检测方法。
术语“蛋白质印迹(western blot)”不仅包括标准的蛋白质印迹，还包括各种变形，例如Far-Western印迹、Northwestern印迹和Southwestern印迹。通常，蛋白质印迹包括将蛋白质转移至膜，并后续检测膜上的蛋白质。本领域已知有多种膜适合用作蛋白质印迹膜，包括但不限于：聚偏二氟乙烯(PVDF)膜，硝酸纤维素膜、聚酰胺膜、聚酯膜和尼龙膜。蛋白质印迹通常用到转移缓冲剂。本领域已知多种蛋白质印迹转移缓冲剂。通常， 蛋白质印迹转移缓冲剂的pH高于待转移蛋白质的等电点。因此，在施加电压电势时，蛋白质会向正极电极迁移。或者，转移缓冲剂的pH可低于待转移蛋白质的等电点。此类情况中，蛋白质会向负极电极迁移。
术语“免疫组织化学”是指用于用能够特异性结合组织学样品中抗原的抗体检测所述抗原的存在的技术。抗体-抗原复合物的检测通常通过利用酶标记的抗体的显色反应或通过荧光标记的抗体进行。
术语“泰它西普”是指Telitacicept，商品名为“泰爱”，其氨基酸序列如SEQ ID NO:34所示：(来源于INN：https://extranet.who.int/soinn/mod/page/view.php？id＝49)
实施例
下面将通过实施例对本发明进行进一步的阐述，需要说明的是，以下实施例是对本发明进行进一步的阐述和解释，而不应被看作是对本发明的限制。
以下实施例通过免疫接种TACI抗原制备TACI抗体
实施例1 TACI抗体制备和筛选
用人TACI抗原对小鼠进行免疫接种后，取免疫小鼠脾细胞与小鼠骨髓瘤细胞进行细胞融合，建立一系列杂交瘤细胞株。
对杂交瘤细胞进行培养，取上清液进行酶联免疫吸附法(ELISA)检测：取BLyS(R&D Systems)包被96孔酶标板，4℃过夜孵育；3％BSA-PBST封闭，300ul/孔，37℃孵育3h；将10倍、100倍、1000倍PBST稀释的上清液分别和30ng/ml的泰它西普单独孵育一定时间后，100ul/孔加入96孔板，37℃孵育1h；Goat-anti-human IgG Fc-HRP 1:5000稀释，100ul/孔，37℃孵育1h；100ul/孔加入底物显色，450/655nm读数，根据450/655nm 吸光值差值筛选高活性抗体。
表1杂交瘤细胞株上清液吸光度读数结果
结果参见表1，结果显示，1B11D2-1、7G12F8-1、12C9F3-1三株细胞株的培养上清可以明显抑制BlyS和TACI-Fc融合蛋白的结合，而其他细胞株的抑制效果不好，因此收集上述三株细胞株上清液纯化，分别得抗体1B11D2、7G12F8、12C9F3。
其中，抗体1B11D2、7G12F8、12C9F3的氨基酸序列分别如下所示：
抗体1B11D2的氨基酸序列：

抗体7G12F8的氨基酸序列：

抗体12C9F3的氨基酸序列：

实施例2：结合表位分析
(1)供试品准备
①缓冲液：20mM PB,75mM NaCl,PH 7.4,H2O；
②供试品A:1B11D2，用缓冲液稀释至40μM；
③供试品B:BLyS(R&D Systems)，用缓冲液稀释至40μM；
④供试品C:泰它西普，用缓冲液稀释至40μM；
⑤相互作用组A：供试品A+供试品C，二者等体积混匀；
⑥相互作用组B：供试品B+供试品C，二者等体积混匀；
⑦对照组A-1：供试品C，加入等体积的缓冲液混匀；
⑧对照组B-1：供试品C，加入等体积的缓冲液混匀；
(2)液质检测方式
将相互作用组A、相互作用组B和对照组A-1、对照组B-1进行胃蛋白酶酶解后，采用UPLC液相系统(Ultimate NCS-3500RSLC pump system,ThermoFisher，进行分离(液相A液为0.1％FA/H2O水溶液，B液为0.1％FA/90％ACN/10％H2O溶液，液相色谱梯度见表2)。
表2液相色谱梯度设置
酶解后的产物由自动进样器PAL3 autosampler,LEAP Technologies) 上样，再经色谱柱(ACQUITY UPLC Peptide CSH C18 Column,1.7μm,1mm X 50mm,ThermoFisher)分离后使用高分辨质谱仪(Orbitrap Fusion,ThermoFisher)进行检测扫描质谱分析。质谱采集时间：15min，检测方式：正离子，母离子扫描范围：300-1500m/z。一级质谱分辨率：60,000。二级质谱分辨率：15,000。检测反应参数见表3。
表3反应参数
(3)检测结果
相互作用组B检测结果：
图1是对照组B-1和相互作用组B氘代行为一致性分析的蝴蝶图，根据检测结果可知二者蝴蝶图整体对称性较好，局部有差异。
图2和图3是根据肽段的编号在不同纵坐标的氘代数(Da)和氘代百分比差的残基图，图2显示对照组B-1和相互作用组B氘代行为有明显差异(横线为1Da限)，差异肽显著区域为29-64(#15),50-68(#19)，67-72(#22)，67-73(#23),68-99(#25),94-106(#27)，96-106(#28)，98-106(#29)，193-208(#57)，235-252(#78)，267-290(#94)区域，提示结构发生显著变化。图3显示对照组B-1和相互作用组B氘代行为有明显差异(横线为5％限),差异肽显著区域为29-64(#15),50-68(#19)，67-72(#22)，67-73(#23,68-99(#25),94-106(#27)，96-106(#28)，98-106(#29)，99-106(#30)，100-106(#31)，128-137(#39)，132-138(#40)，156-162(#45)193-208(#57)，234-241(#68)，234-248(#69)，235-252(#78)，267-290(#94)区域，提示结构发生显著变化。
因此在对照组B-1和相互作用组B蝴蝶图整体对称性较好，局部有差异的情况下，通过对图2和图3不同统计方式的残基图和整体肽段的氘代差异情况综合分析，得出29-32，67-73和267-290区域及附近可能发生抗原抗体的相互作用，其中67-73区域氘代下降差异显著性最高。
相互作用组A检测结果：
图4是对照组A-1和相互作用组A氘代行为一致性分析的蝴蝶图，根据检测结果可知对照组A-1和相互作用组A蝴蝶图整体对称性较好，局部有差异。
图5和图6是根据肽段的编号在不同纵坐标的氘代数(Da)和氘代百分比差的残基图，图5显示对照组A-1和相互作用组A氘代行为有明显差异，差异肽显著区域29-64(#12),54-92(#18),67-73(#19),68-99(#21),74-107(#23)，252-262(#76)，269-290(#90)，271-291(#93)和297-311(#100)区域，提示结构发生显著变化。图6显示对照组A-1和相互作用组A氘代行为有明显差异，差异肽显著区域为7-16(#6)，29-64(#12),54-92(#18),67-73(#19),68-99(#21),74-107(#23)，95-106(#24)，252-262(#76)，269-290(#90),297-311(#93)和301-309(#103)区域，提示结构发生显著变化。
因此在对照组A-1和相互作用组A蝴蝶图整体对称性较好，局部有差异的情况下，通过图5和6不同阈值的残基图和整体肽段的氘代差异情况综合分析，得出29-32，67-73和269-290区域及附近能发生相互作用，其中67-73区域氘代下降差异显著性最高。
(4)结论
根据对照组B-1与相互作用组B的氘代行为一致性分析的蝴蝶图、氘代差异的残基图和氘代百分比差的残基图分析可知二者的相互作用在29-32，67-73和267-290区域及附近为相互作用组B的配体受体结合表位，其中相互作用组在67-73区域的亲和力最高。
根据对照组A-1与相互作用组A的氘代行为一致性分析的蝴蝶图、氘代差异的残基图和氘代百分比差的残基图分析可知二者的相互作用在29-32，67-73和269-290区域及附近为相互作用组A的配体受体结合表位，其中相互作用组在67-73区域的亲和力最高。
29-32，67-73和269-290区域同为相互作用组B和相互作用组A的结合表位，表明1B11D2、BLyS分别与泰它西普属于竞争性关系，且重合度高度一致。
实施例3：与TACI的亲和力
使用Sartorius Octet RH16分子相互作用仪，选取适量1B11D2抗体与EZ-Link Sulfo-NHS-LC-LC-Biotin(Sartorius，18-5019)室温反应 30min后，Zeba脱盐离心柱(Thermo，21338)离心，收集生物素化的1B11D2抗体。
采用Octet SA Biosensors(Thermo，87766)固定生物素化的1B11D2抗体，以不同浓度的泰它西普为分析物，检测图谱通过动力学拟合得出结果。
表4 1B11D2抗体与泰它西普亲和力结果
1B11D2抗体与泰它西普的亲和力结果如表4所示。实验结果表明，1B11D2抗体可以特异性结合泰它西普，二者之间具有抗体抗原的强结合力，采用此方法同样验证了抗体7G12F8和抗体12C9F3同样和泰它西普表现出良好的结合力。
实施例4：游离TACI含量的检测
(1)样品的准备
标准曲线样品：将泰它西普配制成以下浓度1000.0ng/ml、500.0ng/ml、400.0ng/ml、300.0ng/ml、200.0ng/ml、120.0ng/ml、60.0ng/ml、30.0ng/ml、15.0ng/ml以及10.0ng/ml；
待测样品：取15ul临床血液样本加入到105ul 1％BSA-PBS混匀，再取10ul混合液加入到90ul 1％BSA-PBS混匀；
空白对照：取15ul空白血清加入到105ul 1％BSA-PBS混匀，再取10ul混合液加入到90ul 1％BSA-PBS混匀。
(2)游离TACI含量的检测
A.包被：将1B11D2抗体(14.2ug/ml)包被在96孔酶标板上，4℃过夜孵育。
B.封闭：洗板，用3％BSA-PBST封闭，300ul/孔，37℃孵育2h。
C.检测抗体结合：洗板，以100ul/孔加入标准曲线样品、待测样品及空白对照，室温条件下孵育1.5h，使各样品中游离的TACI与1B11D2抗体充分结合。
D.二抗结合：洗板，将二抗Goat-anti-human IgG Fc-Biotin进行1:5000稀释，然后以100ul/孔加入酶标板，室温条件孵育40min。
E.二抗显色：洗板，加入Streptavidin-HRP，室温条件孵育40min。
F.底物显色：洗板，每孔加入100ul底物(四甲基联苯胺)显色。
G.终止：每孔加入50ul终止液(2M H2SO4)，终止反应。
H.读数：采用酶标仪读取450nm和570nm差值。
(3)标准曲线的绘制
标准曲线溶液的OD值参见表5，以标准曲线样品浓度为横坐标，OD值为纵坐标，绘制成标准曲线(见附图7)。
表5 1B11D2抗体体外检测标准曲线
(4)待测样品中游离TACI含量的计算
待测样品经酶标仪读数为1.977，按照如下公式计算出待测样品中游离TACI的含量为207.275ng/ml，计算公式为：
其中，y代表OD值，x代表浓度，A是上渐近线估值，B是斜率，C是最大结合一半时对应浓度(EC50)，D是下渐近线估值。在本研究中，TACI抗体检测了治疗免疫性疾病患者体内TACI的含量，尤其是游离TACI或TACI-Fc融合蛋白类药物的含量，根据患者体内药物的含量，有效了解了患者药物吸收的水平，起到了对自身免疫性疾病患者的病情评估、疗效监测及预后判断情况的作用。
以上描述地仅是优选实施方案，其只作为示例而不限制实施本发明所必需特征的组合。所提供的标题并不意指限制本发明的多种实施方案。术语例如“包含”、“含”和“包括”不意在限制。此外，除非另有说明，没有数词修饰时包括复数形式，以及“或”、“或者”意指“和/或”。除非本文另有定义，本文使用的所有技术和科学术语的意思与本领域技术人员通常理解的相同。
本申请中提及的所有公开物和专利通过引用方式并入本文。不脱离本发明的范围和精神，本发明的所描述的方法和组合物的多种修饰和变体对于本领域技术人员是显而易见的。虽然通过具体的优选实施方式描述了本发明，但是应该理解所要求保护的本发明不应该被不适当地局限于这些具体实施方式。事实上，那些对于相关领域技术人员而言显而易见的用于实施本发明的所描述的模式的多种变体意在包括在随附的权利要求的范围内。

Claims (33)

  1. 一种能够与TACI特异性结合的抗体或其抗原结合片段，其特征在于，所述的抗体或抗原结合片段包含：
    (1)重链可变区(VH)，所述的重链可变区包含SEQ ID NO:1所述的VH-CDR1氨基酸序列、SEQ ID NO:2所述的VH-CDR2氨基酸序列、SEQ ID NO:3所述的VH-CDR3氨基酸序列；或
    (2)重链可变区(VH)，所述的重链可变区包含SEQ ID NO:4所述的VH-CDR1氨基酸序列、SEQ ID NO:5所述的VH-CDR2氨基酸序列、SEQ ID NO:6所述的VH-CDR3氨基酸序列；或
    (3)重链可变区(VH)，所述的重链可变区包含SEQ ID NO:7所述的VH-CDR1氨基酸序列、SEQ ID NO:8所述的VH-CDR2氨基酸序列、SEQ ID NO:9所述的VH-CDR3氨基酸序列；和/或
    (4)轻链可变区(VL)，所述的轻链可变区包含SEQ ID NO:10所述的VL-CDR1氨基酸序列、SEQ ID NO:11所述的VL-CDR2氨基酸序列、SEQ ID NO:12所述的VL-CDR3氨基酸序列；或
    (5)轻链可变区(VL)，所述的轻链可变区包含SEQ ID NO:13所述的VL-CDR1氨基酸序列、SEQ ID NO:14所述的VL-CDR2氨基酸序列、SEQ ID NO:15所述的VL-CDR3氨基酸序列；或
    (6)轻链可变区(VL)，所述的轻链可变区包含SEQ ID NO:16所述的VL-CDR1氨基酸序列、SEQ ID NO:17所述的VL-CDR2氨基酸序列、SEQ ID NO:18所述的VL-CDR3氨基酸序列。
  2. 根据权利要求1所述的抗体或其抗原结合片段，其特征在于，所述的抗体是鼠抗体、兔抗体、鼠源抗体或兔源抗体。
  3. 根据权利要求1所述的抗体或抗原结合片段，其特征在于，所述的抗原结合片段为Fab、F(ab’)2、Fab’、Fv、ScFv。
  4. 根据权利要求1所述的抗体或抗原结合片段，其特征在于，所述的抗体或抗原结合片段包含：
    (1)重链可变区(VH)，所述的重链可变区与SEQ ID NO:19、SEQ ID NO:20、或SEQ ID NO:21所示的氨基酸序列具有至少95％、96％、97％、98％、99％或100％的序列同一性；和/或
    (2)轻链可变区(VL)，所述的轻链可变区与SEQ ID NO:22、SEQ ID NO:23、或SEQ ID NO:24所示的氨基酸序列具有至少95％、96％、97％、 98％、99％或100％的序列同一性。
  5. 根据权利要求4所述的抗体或抗原结合片段，其特征在于，所述的抗体或抗原结合片段包含：
    (1)重链可变区(VH)，所述的重链可变区与SEQ ID NO:19所示的氨基酸序列具有至少95％、96％、97％、98％、99％或100％的序列同一性；和/或轻链可变区(VL)，所述的轻链可变区与SEQ ID NO:22所示的氨基酸序列具有至少95％、96％、97％、98％、99％或100％的序列同一性；或
    (2)重链可变区(VH)，所述的重链可变区与SEQ ID NO:20所示的氨基酸序列具有至少95％、96％、97％、98％、99％或100％的序列同一性；和/或轻链可变区(VL)，所述的轻链可变区与SEQ ID NO:23所示的氨基酸序列具有至少95％、96％、97％、98％、99％或100％的序列同一性；或
    (3)重链可变区(VH)，所述的重链可变区与SEQ ID NO:21所示的氨基酸序列具有至少95％、96％、97％、98％、99％或100％的序列同一性；和/或轻链可变区(VL)，所述的轻链可变区与SEQ ID NO:24所示的氨基酸序列具有至少95％、96％、97％、98％、99％或100％的序列同一性。
  6. 根据权利要求1所述的抗体或抗原结合片段，其特征在于，所述的抗体或抗原结合片段包含Fc区。
  7. 根据权利要求6所述的抗体或抗原结合片段，其特征在于，所述的Fc与SEQ ID NO:25、SEQ ID NO:26、或SEQ ID NO:27所示的氨基酸序列具有至少95％、96％、97％、98％、99％或100％的序列同一性。
  8. 根据权利要求1所述的抗体或抗原结合片段，其特征在于，所述的抗体或抗原结合片段包含：
    (1)重链，所述的重链与SEQ ID NO:28、SEQ ID NO:29、或SEQ ID NO:30所示的氨基酸序列具有至少95％、96％、97％、98％、99％或100％的序列同一性；和/或
    (2)轻链，所述的轻链与SEQ ID NO:31、SEQ ID NO:32、或SEQ ID NO:33所示的氨基酸序列具有至少95％、96％、97％、98％、99％或100％的序列同一性。
  9. 根据权利要求8所述的抗体或抗原结合片段，其特征在于，所述的 抗体或抗原结合片段包含：
    (1)重链，所述的重链与SEQ ID NO:28所示的氨基酸序列具有至少95％、96％、97％、98％、99％或100％的序列同一性；和/或轻链，所述的轻链与SEQ ID NO:31所示的氨基酸序列具有至少95％、96％、97％、98％、99％或100％的序列同一性；或
    (2)重链，所述的重链与SEQ ID NO:29所示的氨基酸序列具有至少95％、96％、97％、98％、99％或100％的序列同一性；和/或轻链，所述的轻链与SEQ ID NO:32所示的氨基酸序列具有至少95％、96％、97％、98％、99％或100％的序列同一性；或
    (3)重链，所述的重链与SEQ ID NO:30所示的氨基酸序列具有至少95％、96％、97％、98％、99％或100％的序列同一性；和/或轻链，所述的轻链与SEQ ID NO:33所示的氨基酸序列具有至少95％、96％、97％、98％、99％或100％的序列同一性。
  10. 根据权利要求1所述的抗体或抗原结合片段，其特征在于，所述的抗体或抗原结合片段可进一步与可检测标记偶联。
  11. 根据权利要求10所述的抗体或抗原结合片段，其特征在于，所述的可检测标记是酶。
  12. 根据权利要求10所述的抗体或抗原结合片段，其特征在于，所述的可检测标记是发光标记。
  13. 根据权利要求10所述的抗体或抗原结合片段，其特征在于，所述的可检测标记是放射性同位素。
  14. 根据权利要求10所述的抗体或抗原结合片段，其特征在于，所述的可检测标记是显色标记。
  15. 根据权利要求10所述的抗体或抗原结合片段，其特征在于，所述的可检测标记是半抗原。
  16. 根据权利要求10所述的抗体或抗原结合片段，其特征在于，所述的可检测标记是金属络合物。
  17. 权利要求1-16任一项所述的抗体或抗原结合片段在检测样品中TACI含量中的用途。
  18. 根据权利要求17所述的用途，其特征在于，所述的样品是生物学样品。
  19. 根据权利要求18所述的用途，其特征在于，所述的样品为血液样品。
  20. 根据权利要求17所述的用途，其特征在于，所述的样品源自BLyS和APRIL过量表达的对象。
  21. 根据权利要求17所述的用途，其特征在于，所述的样品源自患有自身免疫疾病的对象。
  22. 根据权利要求21所述的用途，其特征在于，所述的自身免疫疾病选自类风湿性关节炎、风湿性关节炎、青少年类风湿性关节炎、系统性红斑狼疮(SLE)、狼疮肾炎(LN)、韦格纳病、炎症性肠病、特发性血小板减少性紫癜(ITP)、血栓性血小板减少性紫癜(TTP)、自身免疫性血小板减少症、多发性硬化症、银屑病、IgA肾病、IgM多发性神经病、重症肌无力、脉管炎、糖尿病、Reynauld’s综合征、Sjorgen’s综合征、肾小球肾炎、自身免疫性肝炎、自身免疫性甲状腺炎、视神经脊髓炎、干燥综合征疾病中的一种或几种。
  23. 根据权利要求20-22任一项所述的用途，其特征在于，所述的对象为人。
  24. 根据权利要求17所述的用途，其特征在于，所述的检测通过Elisa进行。
  25. 根据权利要求17所述的用途，其特征在于，所述的检测通过蛋白质印迹进行。
  26. 根据权利要求17所述的用途，其特征在于，所述的检测通过免疫组织化学(IHC)进行。
  27. 检测样品中TACI含量的方法，其特征在于，所述的方法包括如下步骤：用权利要求1-16任一项所述的抗体或抗原结合片段接触检测样品，之后检测结合的抗体或抗原结合片段的存在。
  28. 分离的多核苷酸，其编码权利要求1-16中任一项所述的抗体或抗原结合片段。
  29. 表达载体，其包含权利要求28所述的多核苷酸。
  30. 权利要求29所述的表达载体在制备权利要求1-16任一项所述的抗体或抗原结合片段中的用途。
  31. 宿主细胞，其包含至少一种权利要求29所述的表达载体。
  32. 权利要求1-16任一项所述的抗体或抗原结合片段在制备试剂盒中的用途。
  33. 一种试剂盒，其特征在于，所述的试剂盒包含权利要求1-16任一项所述的抗体。
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