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Definitions

• the invention belongs to the field of genetic engineering medicine, and particularly relates to a novel B cell activating factor (BAFF) antagonist, a preparation method thereof and use thereof.
• BAFF B cell activating factor
• RA rheumatoid arthritis
• SLE systemic lupus erythematosus
• Sjggren's syndrome Sjogren's syndrome, Sjogren's Syndrome
• the body's B cells or plasma cells are closely related to excessive proliferation and humoral immune activation.
• BAFF B cell activating factor
• BLyS also known as BLyS, TALL-1, THANK, zT F4 or TNFSF-13B
• BAFF has the same subtype as the other three ligands (APRIL, EDA and TWEAK) and has similar functional and structural characteristics [2].
• BAFF is a type II transmembrane protein, which exists in both membrane-bound and soluble forms. The former consists of 285 amino acids. The proteolytic enzyme can separate the polypeptide chain from R133 and A134 to form a 152 amino acid.
• Soluble protein this process is regulated by cells and factors [3]. Under normal physiological conditions, soluble BAFF exists in the form of a trimer and is biologically active [4]. BAFF is mainly expressed by peripheral blood mononuclear cells (PBMNCs), including macrophages, monocytes and dendritic cells in the spleen and lymph nodes [5].
• PBMNCs peripheral blood mononuclear cells
• BCMA B cell maturation antigen
• TACI transmembrane activator and CAML interactor
• BAFF-R BAFF receptor, Br3
• BCMA B cell maturation antigen
• TACI transmembrane activator and CAML interactor
• BAFF-R BAFF receptor, Br3
• Both BAFF and APRIL bind TACI and BCMA with high affinity, but BAFF can also bind to BAFF-R.
• the extracellular domain of the T F receptor contains a plurality of cysteine-rich domains (CRD), each of which forms three disulfide bonds from six cysteine residues.
• BCMA has a single CRD.
• TACI contains two typical CRDs: CRD1 and CRD2, and only CRD2 is involved in ligand binding (TACI (aa.70-104): see SEQ ID No. 8) [6].
• Br3 contains only one CRD consisting of four cysteine residues (Br3 (aa.18-35): see SEQ ID No. 9), and the binding domain to BAFF is reduced to 26 amino acids [7].
• BAFF In addition to promoting the survival of B cells, BAFF has an important regulatory role in maintaining germinal center response, homotypic switching, and activation of T cells. The effect of BAFF on T cell activation may play an important role in the pathogenesis of autoimmune diseases. Therefore, BAFF and its receptors have received wide attention as new targets for the treatment of autoimmune diseases.
• BAFF-specific antagonists including soluble receptors TACI-Fc, Br3-Fc or anti-BAFF antibodies
• RA rheumatoid arthritis
• Sj0gren syndrome systemic lupus erythematosus
• the technical problem to be solved by the present invention is to find a new and effective choice for the prevention and treatment of autoimmune diseases, especially by antagonizing BAFF to prevent and treat autoimmune diseases.
• the technical solution of the present invention to solve the technical problem is to provide a novel B cell activating factor antagonist.
• the B cell activating factor antagonist is a protein.
• the structure of the B cell activating factor antagonist is:
• a fusion protein comprising: a domain obtained by fusion of a CRD2 domain which binds to BAFF in a TACI receptor and a CRD domain which binds to BAFF in a Br3 receptor;
• Or (2) a protein having the same or similar function as (1) the defined fusion protein obtained by substituting and/or deleting and/or adding at least one amino acid in the amino acid sequence of the (1) defined fusion protein.
• This B cell activator antagonist contains this domain as the primary B cell activating factor binding.
• the above B cell activating factor antagonist is:
• amino acid sequence thereof is a protein in which the amino acid sequence of the Fc fragment of human immunoglobulin is ligated to the C-terminus of the amino acid sequence shown in SEQ ID No. 1.
• Or (2) a protein having the same or similar function as (1) obtained by substituting and/or deleting and/or adding at least one amino acid in the amino acid sequence of the (1) defined protein.
• the above B cell activating factor antagonist is a protein having the amino acid sequence of SEQ ID No. 2;
• the function of (1) the protein represented by SEQ ID No. 2 is identical or similar to that obtained by substituting and/or deleting and/or adding at least one amino acid in the amino acid sequence of the protein represented by SEQ ID No. 2. protein. Due to the addition of the Fc fragment of human immunoglobulin, the genetically engineered fusion protein will exist as a dimer.
• the N-terminus of the above B cell activator antagonist protein is linked to a signal peptide.
• the above B cell activating factor antagonist is a protein having the amino acid sequence of SEQ ID No. 3; or: substituted and/or deleted in the amino acid sequence of the protein represented by SEQ ID No. 3 and/or Or a protein obtained by adding at least one amino acid to have the same or similar function as the protein represented by (1) SEQ ID ⁇ 2.
• the present invention provides a nucleotide sequence encoding the above-described ⁇ cell activating factor antagonist while providing the above-described ⁇ cell activating factor antagonist which is a protein.
• nucleotide sequence encoding the sputum cell activating factor antagonist is:
• nucleotide sequence derived by substituting, deleting or adding at least one nucleotide in the nucleotide sequence defined by (1), and having the same function as the nucleotide sequence of SEQ ID NO. Or a similar protein.
• nucleotide sequence encoding the B cell activating factor antagonist is:
• nucleotide sequence derived by substituting, deleting or adding at least one nucleotide in the nucleotide sequence defined by (1), and having the same function as the nucleotide sequence of SEQ ID N0.4 Or a similar protein.
• nucleotide sequence encoding the B cell activating factor antagonist is:
• nucleotide sequence derived by substituting, deleting or adding at least one nucleotide in the nucleotide sequence defined by (1), and having the same function as the nucleotide sequence of SEQ ID N0.5 Or a similar protein.
• the present invention also provides a gene vector comprising the above nucleotide sequence.
• the above gene vector is an expression vector capable of expressing the above nucleotide sequence.
• the invention also provides a host cell comprising the above gene vector.
• the present invention also provides the use of the above B cell activating factor antagonist, or the above nucleotide sequence, or the above gene vector for the preparation of a medicament for treating an autoimmune disease.
• the present invention also provides the above-mentioned B cell activating factor antagonist, or the above nucleotide sequence, or the above gene carrier as a main active ingredient for preventing and treating an autoimmune disease.
• the above autoimmune diseases mainly refer to rheumatoid arthritis, systemic lupus erythematosus, and dry syndrome (Sj0gren's syndrome).
• the present invention also provides a method for preparing the above B cell activating factor antagonist. Law.
• the method comprises the steps of: operably loading a gene encoding a B cell activating factor antagonist into an expression vector, transferring the expression vector into a host, and separating and purifying the host and/or its culture supernatant after the host culture is propagated to obtain B. Cell activating factor antagonist.
• the expression vector described in the above method for producing a B cell activating factor antagonist is a eukaryotic plasmid expression vector, an adenovirus vector or an adeno-associated virus vector.
• the host described in the above method for producing a B cell activating factor antagonist is a eukaryotic cell.
• the method for separating and purifying the above-mentioned B cell activating factor antagonist is to purify the supernatant of the large-scale culture host by using a gel Mab-Select column of GE, USA, and then performing SP column chromatography. B cell activating factor antagonist.
• the expression vector in the above method can use a common eukaryotic expression vector, a host cell commonly used in various genetic engineering, and the isolation and purification method can also refer to the use of the existing common methods to obtain a relatively pure B cell activating factor antagonist of the present invention.
• Agent Whereas a gene encoding a B cell activating factor antagonist is operably loaded into an expression vector, and the expression vector is transferred into a host, the description can be made with reference to various genetic engineering manuals and specific vectors and host cell instructions.
• the present invention designs and constructs a BAFF antagonist fragment in which the TACI receptor binds to BAFF-binding domain 2 (CRD2) and the Br3 receptor binds to the BAFF-binding domain (CRD) (the amino acid sequence is represented by SEQ ID No. 1, which may be In order to increase its in vivo stability and prolong the half-life, the nucleotide sequence shown by SEQ ID No. 10 can be fused with an Fc fragment of an immunoglobulin to obtain a novel fusion protein molecule.
• BAFF Trap amino acid sequence is shown in SEQ ID No. 2
• SEQ ID No. 4 amino acid sequence is shown in SEQ ID No. 4
• experiments have demonstrated the function of a B cell activating factor antagonist.
• the B cell activating factor antagonist of the present invention is a protein
• the main preparation method is to carry out fermentation production by using an existing genetic engineering method.
• nucleotide sequence of various commonly used secretion signal peptides such as the human IL-2 signal peptide
• the nucleotide sequence is encoded (the amino acid sequence of the human IL-2 signal peptide is SEQ ID No. 3 and can be encoded by the nucleotide sequence shown in SEQ ID No. 5).
• a gene sequence similar to "the nucleotide sequence in SEQ ID NO: 1 is substituted, deleted or added with at least one nucleotide-derived sequence" generally means that the coding has the coding of SEQ ID NO: 1.
• the degenerate sequence refers to a sequence produced by the substitution of one or more codons in the sequence by degenerate codons encoding the same amino acid. Due to the degeneracy of the codon, a degenerate sequence having a homology of less than about 89% to SEQ ID NO: 1 can also encode the sequence set forth in SEQ ID NO: 1.
• nucleotide sequence of SEQ ID NO: 1 by substitution, deletion or addition of at least one nucleotide-derived sequence also includes SEQ ID NO: under moderately stringent conditions, more preferably under highly stringent conditions. a nucleotide sequence in which a nucleotide sequence hybridizes. The term also includes at least 80%, preferably at least 89%, more preferably at least 90% homology to the nucleotide sequence of SEQ ID NO: 1. Optimum at least 95% of the nucleotide sequence. Having the same function in the present invention means having an effect of binding to BAFF and thereby antagonizing the biological activity of BAFF.
• nucleotide sequence in SEQ ID NO: 1 The substituted, deleted or added at least one nucleotide-derived sequence "also includes variant forms encoding the open reading frame sequence of SEQ ID NO: 1 having the same function as the protein encoded by SEQ ID NO: 1. These variant forms Including (but not limited to): a plurality of (usually 1 to 90, preferably 1 to 60, more preferably 1 to 20, optimally 1 to 10) nucleotide deletions, insertions, and / or replace, and add a few at the 5' and / or 3' end (usually less than 60, preferably Within 30, more preferably 10, optimally 5 or less nucleotides.
• amino acid sequence derived from amino acid derivatization includes, but is not limited to, several (typically 1-50, preferably 1-30, more preferably 1-20, optimally 1-10) amino acids. Deletions, insertions and/or substitutions, and the addition of one or several (usually within 20, preferably within 10, more preferably within 5) amino acids at the C-terminus and/or N-terminus.
• amino acids typically 1-50, preferably 1-30, more preferably 1-20, optimally 1-10) amino acids.
• the function of the protein is usually not changed.
• the addition of one or several amino acids at the C-terminus and/or the N-terminus generally does not change the function of the protein.
• the term also encompasses active fragments and active derivatives of the protein.
• the same function in the present invention refers to the effect of binding to
• the amino acid sequence obtained by substituting, deleting or adding at least one amino acid in the amino acid sequence includes, but is not limited to, having up to 10 (ie one or several), preferably up to 8, More preferably, at most 5 amino acids are replaced by amino acids of similar or similar nature to form a polypeptide, ie a conservative variant polypeptide.
• conservative variant polypeptides are preferably produced by substitution according to Table 1.
• the invention also includes analogs of the claimed proteins or polypeptides.
• the difference between these analogs and the original protein may be a difference in amino acid sequence, a difference in the modification form which does not affect the sequence, or a combination thereof.
• These proteins include natural or induced genetic variants. Induced variants can be obtained by a variety of techniques, such as random mutagenesis by irradiation or exposure to a mutagen, or by site-directed mutagenesis or other techniques known to molecular biology.
• Analogs also include analogs having residues other than the native L-amino acid (e.g., D-amino acids), as well as analogs having non-naturally occurring or synthetic amino acids (e.g., beta, ⁇ -amino acids). It will be understood that the proteins or peptides of the invention are not limited to the representative proteins or polypeptides exemplified above.
• Modifications include: chemically derived forms of the polypeptide, such as acetylation or carboxylation, in vivo or in vitro. Modifications also include glycosylation, such as those produced by glycosylation modifications in the synthesis and processing of the polypeptide or in further processing steps. Such modification can be accomplished by exposing the polypeptide to an enzyme that performs glycosylation, such as a mammalian glycosylation enzyme or a deglycosylation enzyme. Modified forms also include sequences having phosphorylated amino acid residues such as phosphotyrosine, phosphoserine, phosphothreonine. Also included are polypeptides modified to increase their resistance to proteolytic properties or to optimize solubility properties.
• “Operably linked to” as used in the present invention means that certain portions of a linear DNA sequence are capable of affecting the activity of other portions of the same linear DNA sequence. For example, if the signal peptide DNA is expressed as a precursor and is involved in the secretion of the polypeptide, then the signal peptide (secretion leader sequence) DNA is operably linked to the polypeptide DNA; if the promoter controls the transcription of the sequence, then it is operably linked to A coding sequence; if the ribosome binding site is placed at a position that enables translation, then it is operably linked to the coding sequence.
• “operably linked” means adjacent, and for secretory leader sequences means adjacent in the reading frame.
• the present invention has the beneficial effects of:
• the present invention designs and constructs a fragment in which the TACI receptor binds to BAFF-binding domain 2 (CRD2) and the Br3 receptor binds to the BAFF-binding domain (CRD), in order to improve its stability in vivo,
• the half-life is extended and can be fused to the Fc fragment of an immunoglobulin, for example, it can be fused to an Fc fragment of IgG1, IgG2 or IgG4.
• a signal peptide can be ligated at its N-terminus, which results in a series of new fusion protein molecules.
• the novel B cell activating factor antagonist of the present invention can greatly improve the binding effect with BAFF, reduce the therapeutic dose, and improve the effect of treating autoimmune diseases. It provides a new and effective choice for the prevention and treatment of autoimmune diseases.
• FIG. 1 Schematic diagram of the interaction of BAFF with its receptor, from Nature Review Immunology, 2002, 2: 465-475.
• FIG. 2 Schematic diagram of the BAFF Trap structure. Due to the addition of the Fc fragment, the fusion protein is easily present as a dimer.
• Figure 3 RT-PCR results of IgGl Fc (1% Agarose electrophoresis). M: lOObp DNA Ladder (Invitrogen); 1: RT-PCR product.
• Figure 4 Schematic diagram of the construction process of recombinant plasmid pEF-BT.
• Figure 5 is a plasmid map of the eukaryotic expression vector pEFl/V5-His A.
• M lkb DN A Ladder (Invitrogen); 11, 15, 16, 26, 27 represent different clones, respectively.
• DNA Ladder (Invitrogen); 1, 2, 5, 6, 7, 10 represent different clones, respectively.
• Figure 8 Electropherogram of purified protein (12% SDS-PAGE). M: protein molecular weight standard; 1 : non-reducing conditions; 2: reducing conditions.
• Figure 9 Changes in body weight and clinical scores in a rheumatoid arthritis model.
• A is the change in body weight;
• B is the change in clinical score.
• Figure 10 HE diagram of the ankle joint in the rheumatoid arthritis model (magnification: 40) and pathological score analysis.
• A is a HE map, wherein: a is a normal rat, b is a modeling group, c is a hlgG group, d is a BT group; B is a pathological score analysis.
• Figure 11 B220 flow detection diagram A: normal rat group; B: saline group; H: hlgG group; B: BT group.
• BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail by way of specific embodiments with reference to the accompanying drawings. In the following examples, where no specific experimental conditions are indicated, they are in accordance with conventional conditions well known to those skilled in the art, such as Sambrook J, Russell DW, 2001, Molecular Cloning: A laboratory manual (3 rd ed), Spring Harbor. The conditions described in the Laboratory Press, or in accordance with the conditions recommended by the manufacturer.
• the cDNA fragment of TACI domain 2 and Br3 domain was obtained by whole-genome synthesis, and the human 11-2 signal peptide sequence was included at the 5' end.
• a BamH I site was designed in the 5' primer and a Pme I site was designed in the 3' primer.
• the IgG1 Fc fragment was amplified by RT-PCR using the total RNA of human lymphocytes as a template.
• the reaction conditions were as follows:
• Reverse transcription reaction denaturation at 94 ° C for 30 seconds; annealing at 55 ° C for 30 seconds; extension at 68 ° C for 1 minute. The reaction was carried out for 10 cycles.
• PCR reaction denaturation at 94 ° C for 30 seconds; annealing at 60 ° C for 30 seconds; extension at 68 ° C for 1 minute. The reaction was carried out for 25 cycles. Then extend at 68 °C for another 12 minutes.
• the nucleotide and protein sequences of the full-length BAFF Trap are shown in SEQ ID No. 5 and SEQ ID No. 3, respectively. Due to the addition of the Fc fragment, the fusion protein exists as a dimer through a disulfide bond, and the structure of the BAFF Trap is as follows. Figure 2 shows.
• the Kpn I restriction site was designed at the 5' end of the whole gene synthesis fragment (TACI-Br3), and the BamH I restriction site was designed at the 3' end, so that the cDNA fragment of TACI-Br3 can be directly Inserted into the multiple cloning site Kpn l/BamH I of the eukaryotic expression vector pEF1/V5-HisA (purchased from Invitrogen, USA, see Figure 5), the IgGl Fc fragment was inserted into the multiple cloning site BamH I/Pme l , you can get the BAFF Trap fusion fragment.
• the construction process is shown in Figure 4.
• the whole gene synthesis fragment TACI-Br3 was inserted into the Kpn I/BamH I site of pEF1/V5-HisA, and the ligation product pEF-TACI-Br3 was transformed into E. coli JM109, and 28 clones were randomly selected for screening.
• the clones of clones 11, 15, 16, 26 and 27 were all cut out with Kpn I/BamH I, and the results of restriction enzyme digestion of recombinant plasmid pEF-TACI-Br3 are shown in Fig. 6.
• the clone No. 26 was selected for sequencing and the sequence was correct, and the fusion gene with IgG1 Fc was further constructed.
• plasmid pEF-BT 5 g of the above-prepared recombinant plasmid pEF-BT was prepared, and CHO-K1 cells (purchased from ATCC, USA) were transfected with liposome Lipofectamine 2000. After two days, the cells were passaged at a ratio of 1:5, and 0.4 mg/ml of G418 was added. Screening was observed in 10 days from the screening of Invitrogen, USA. Randomly digested 96 clones with distinctly separated, well-celled cells were seeded in 6 24-well plates (first round of screening).
• the expression of the fusion protein was detected by ELISA, and 24 clones positively selected were inoculated into one 24-well plate (second round screening). After 2 days of culture, the supernatant was taken for ELISA.
• the expression of the fusion protein was selected from the six clones with higher expression: 1-B2, 1-B8, 1-D7, 1-E1, 2-Dl, 2-F6 were further screened by limiting dilution method.
• 1-B8-1, l-D7-7, 1-E1-7 were inoculated into 96-well plates (1 cell/? ⁇ /200 ⁇ 1) (fourth round of screening), after the cells were full (about 10 days later), The culture supernatant was used to detect the expression of the fusion protein by ELISA. All the three clones were homogenous clones. The highest expression was amplified and preserved, and named CHO-BTCl-B8-i;>, CHO-BT ( ; i-D7-7;), CH0-BT (l-El-7), pick CH0-BT (l-El-7) for further expression and purification. 4.
• BIAcore T100 was used to detect the binding ability of BAFF Trap and BAFF under in vitro conditions, and the binding constant was analyzed by BIAcore T100 Evaluation Soft worn.
• Standards BAFF R-Fc and TACI-Fc were used as controls at the same time. Proceed as follows:
• BAFF standard is diluted with HEPES solution, ie 1800nm, 600nm,
• the KD value is about 12.7 nM, and the binding constants of TACI-Fc and Br3-Fc to BAFF are 14.8 nM and 34 nM, respectively.
• Raji cells (purchased from ATCC, USA) are human B cell lines that highly express BAFF receptors (TACI and Br3), We used to detect the binding of BT to competitively antagonize BAFF.
• Harvest Raji cells in logarithmic growth phase add different concentrations of BT protein and anti-TACI antibody/anti-Br3 antibody, incubate for 1 hour at room temperature, add FITC-labeled secondary antibody after washing, incubate at room temperature for half an hour in the dark, wash and then flow up
• Table 4 Which hlgG contains a human IgGl Fc fragment as an unrelated protein group.
• BT competitively inhibits the binding of antibodies to the BAFF receptors on the Raji cells, TACI and Br3, and exhibits a dose-dependent relationship. It is indicated that in vitro, BT can act as an antagonist to inhibit the binding of BAFF to its cell membrane surface receptor.
• mice 6-8 weeks old female Lewis rats were injected intradermally with ⁇ incomplete Freund's adjuvant containing 5 mg/ml inactivated BCG, and the control mice were not treated. After the onset, they were randomly divided into 3 groups: saline group, hlgG group, and fusion protein BT group, 10 rats in each group, intraperitoneally administered 100 ⁇ ⁇ /100 ⁇ l/only, twice a week.
• the hlgG group containing the human IgGl Fc fragment, as an unrelated treatment group.
• the body weight was weighed daily, and the limbs were clinically scored in a double-blind manner. Blood was taken from the orbital vein once a week, serum was separated, and stored in a negative 80 degree refrigerator.
• mice were sacrificed 45 days after treatment, and the ankle joints were taken and HE stained. Lymphocytes were isolated from the spleen and spleen B cells were detected by flow cytometry (B220 antibody detection).
• the rate of decrease in the BAFF Trap group was significantly higher than that of the other two groups (P ⁇ 0.05), and returned to normal around the 37th day. At this time, the score of the AIA group was 10 ⁇ 1, and that of the hlgG group was 6 ⁇ 1. .
• Fig. 10A The HE staining results of the joints in each group are shown in Fig. 10A.
• AIA group and the hlgG group lymphocyte infiltration, bone destruction, and synovial inflammation (bone fibrosis and vasospasm can also be found in the AIA group); It can be seen that the synovial membrane is intact, lymphocytes are regressed, and damaged bone is repaired, similar to normal rat joints.
• the pathological scores are shown in Figure 10B).
• the pathological scores of the AIA group and the hlgG group were as high as 4 points, while in the BT group, the pathological scores were significantly lower (P ⁇ 0.05) and tend to normalize.

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