WO2022223028A1 - 抗BLyS抗体、其药物组合物及其用途 - Google Patents

抗BLyS抗体、其药物组合物及其用途 Download PDF

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WO2022223028A1
WO2022223028A1 PCT/CN2022/088507 CN2022088507W WO2022223028A1 WO 2022223028 A1 WO2022223028 A1 WO 2022223028A1 CN 2022088507 W CN2022088507 W CN 2022088507W WO 2022223028 A1 WO2022223028 A1 WO 2022223028A1
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seq
variable region
amino acid
acid sequence
chain variable
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PCT/CN2022/088507
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English (en)
French (fr)
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王莹
文珺
周岳华
向丹丹
开仁冬
李薇
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上海君实生物医药科技股份有限公司
苏州君盟生物医药科技有限公司
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Publication of WO2022223028A1 publication Critical patent/WO2022223028A1/zh

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Definitions

  • the invention belongs to the field of biomedicine, and relates to an anti-BLyS antibody, its pharmaceutical composition and use thereof.
  • Systemic lupus erythematosus is an autoimmune autoimmune disease that affects multiple systems in the body, such as skin, joints, heart, lungs, kidneys, blood, and brain. disease.
  • Systemic lupus erythematosus mainly affects Afro-Caribbean, Asian and Hispanic people, and to a lesser extent Caucasian (Caucasian) people.
  • Caucasian Caucasian
  • systemic lupus erythematosus The causes of systemic lupus erythematosus are complex and unclear, not caused by a single factor, but may be related to multiple factors such as heredity, environment, sex hormones and immunity.
  • the cause of systemic lupus erythematosus recognized by the world scientific community is that at the cellular level, self-reactive B cells exist in peripheral tissues for too long, producing human self-antigens and causing autoimmunity. Therefore, if the growth and proliferation of early B cells can be inhibited, the lupus erythematosus disease can be treated.
  • B Lymphocyte Stimulator also known as Tall-1 (TNF and Apol related leukocyte expressed ligand 1), BAFF (B cell activating factor belonging to the TNF family), THANK (TNF homologues that activate apoptosis, NF- ⁇ B and JNK), belonging to the tumor necrosis factor (TNF) family
  • B Lymphocyte Stimulator also known as Tall-1 (TNF and Apol related leukocyte expressed ligand 1)
  • BAFF B cell activating factor belonging to the TNF family
  • THANK TNF homologues that activate apoptosis, NF- ⁇ B and JNK
  • TNF tumor necrosis factor
  • BLyS induces massive proliferation and secretion of large amounts of IgM and IgA after preactivation of B cells with IgM, but this stimulation is not evident for B cells during resting period (3). Further research showed that BLyS mainly acts on pre-B lymphocytes, immature B lymphocytes, and activated lymphocytes, but has no effect on plasma cells and lymphoid pluripotent stem cells. Like most cytokines, BLyS stimulates downstream signaling primarily through B cell surface receptors.
  • B cell activating factor receptor BR3, BLyS receptor 3 or BAFF-R
  • TACI transmembrane activator-1 and calcium modulator and cyclophilin ligand-interactor
  • BCMA B cell maturation Antigen
  • Therapeutic antibodies against BLyS have been shown to effectively inhibit B cell growth, IgA and IgM secretion in vivo and in vitro to achieve the effect of treating systemic lupus erythematosus (Edwards BM et al., The remarkable flexibility of the human antibody repertoire; isolation of over one thousand different antibodies to a single protein, BLyS.J Mol Biol. 2003 Nov 14; 334(1): 103-18; Baker KP et al., Generation and characterization of LymphoStat-B, a human monoclonal antibody that antagonizes the bioactivities of B lymphocyte stimulator. Arthritis Rheum. 2003 Nov;48(11):3253-65).
  • Benlysta an anti-BLyS antibody developed by Human Genome Corporation, has become the world's first new drug for the treatment of lupus erythematosus in the past 60 years. Benlysta only targets BLyS-stimulated B cells, which greatly reduces the side effects during treatment compared with chemotherapy drugs, thus providing a safe and effective treatment for systemic lupus erythematosus patients. In recent years, the research and clinical application of targeted therapy for BLyS has developed rapidly. Except for the American Human Genome Company, other companies use fusion proteins modified on the basis of BLyS or its receptor. Genentech of the United States developed BR3-FC drugs, Zymogenetics developed TACI-FC drugs, and AMGEN developed peptide-FC drugs. Compared with Benlysta, they have poor specificity, weak binding, relatively poor efficacy and strong toxicity. All three drugs were discontinued or terminated in Phase II clinical trials. Therefore, anti-BLyS antibody drugs are an effective drug method for this target.
  • the anti-BLyS antibody provided by the present invention has excellent binding ability of human BLyS protein and the ability to competitively inhibit the binding of human BLyS to its receptor human BR3.
  • the pharmaceutical composition is a highly stable pharmaceutical composition containing an anti-BLyS antibody.
  • the present invention finds that the humanized anti-BLyS antibody is in a histidine buffer system and L-arginine, L-arginine.
  • An unexpected feature in the combination of hydrochloride or methionine is high stability.
  • the present invention provides a humanized anti-BLyS antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein:
  • amino acid sequences of LCDR1, LCDR2 and LCDR3 of the humanized anti-BLyS antibody or its antigen-binding fragment are respectively shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3;
  • amino acid sequences of HCDR1, HCDR2 and HCDR3 of the humanized anti-BLyS antibody or its antigen-binding fragment are shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, respectively;
  • the FR1 of the light chain variable region of the humanized anti-BLyS antibody or its antigen-binding fragment is selected from the FR1 described in any one of SEQ ID NO: 7, 9 and 13, and the FR2 is the FR2 described in SEQ ID NO: 7 , FR3 is selected from the FR3 described in any one of SEQ ID NO: 7, 9 and 11, and FR4 is the FR4 described in SEQ ID NO: 7;
  • the FR1 of the heavy chain variable region of the humanized anti-BLyS antibody or antigen-binding fragment thereof is selected from FR1 described in any one of SEQ ID NOs: 8, 10, 12 and 15, and FR2 is selected from SEQ ID NO: 8, The FR2 of any one of 10 and 15, the FR3 is selected from the FR3 of any one of SEQ ID NOs: 8, 12 and 14, and the FR4 is the FR4 of SEQ ID NO: 8 or 10; and
  • the humanized anti-BLyS antibody or its antigen-binding fragment does not include: the variable region of the light chain is the amino acid sequence shown in SEQ ID NO: 7, and the variable region of the heavy chain is the amino acid sequence shown in SEQ ID NO: 8. Humanized anti-BLyS antibodies or antigen-binding fragments thereof.
  • the above-mentioned humanized anti-BLyS antibody or antigen-binding fragment thereof is a humanized anti-BLyS antibody or antigen-binding fragment thereof:
  • the FR1 of the light chain variable region is selected from the FR1 described in any one of SEQ ID NO:7, 9 and 13, the FR2 is the FR2 described in SEQ ID NO:7, and the FR3 is selected from the FR1 described in SEQ ID NO:7, 9 and 11 FR3 described in any one, FR4 is FR4 described in SEQ ID NO:7; FR1-FR4 of heavy chain variable region are respectively FR1-FR4 of SEQ ID NO:15; Or
  • the FR1 of the light chain variable region is selected from the FR1 described in any one of SEQ ID NO:7, 9 and 13, the FR2 is the FR2 described in SEQ ID NO:7, and the FR3 is selected from the FR1 described in SEQ ID NO:7, 9 and 11 FR3 described in any one, FR4 is FR4 described in SEQ ID NO:7; FR1-FR4 of heavy chain variable region are respectively FR1-FR4 of SEQ ID NO:10; Or
  • the FR1 of the light chain variable region is selected from the FR1 described in any one of SEQ ID NO:7, 9 and 13, the FR2 is the FR2 described in SEQ ID NO:7, and the FR3 is selected from the FR1 described in SEQ ID NO:7, 9 and 11
  • the FR3 described in any one, FR4 is the FR4 described in SEQ ID NO:7;
  • the FR1-FR4 of the heavy chain variable region are respectively the FR1-FR4 of SEQ ID NO:12; or
  • FR1 of the light chain variable region is FR1 described in SEQ ID NO: 11 or 13
  • FR2 is FR2 described in SEQ ID NO: 11
  • FR3 is FR3 described in SEQ ID NO: 11 or 13
  • FR4 is SEQ ID NO: 11
  • the FR4 described in NO:11; the FR1-FR4 of the heavy chain variable region are respectively the FR1-FR4 of SEQ ID NO:14.
  • the humanized anti-BLyS antibody or antigen-binding fragment thereof described above comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises, for example, SEQ ID NO: 7, SEQ ID NO : 9, the amino acid sequence shown in SEQ ID NO: 11 or SEQ ID NO: 13, the heavy chain variable region comprises such as SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO : 14 or the amino acid sequence shown in SEQ ID NO: 15.
  • the above-mentioned humanized anti-BLyS antibody or antigen-binding fragment thereof comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region and heavy chain variable region are selected from:
  • the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:7
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14 or the amino acid sequence shown in SEQ ID NO: 15; or
  • the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:9
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12 , the amino acid sequence shown in SEQ ID NO: 14 or SEQ ID NO: 15; or
  • the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 11
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12 , the amino acid sequence shown in SEQ ID NO: 14 or SEQ ID NO: 15; or
  • the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 13
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12 , SEQ ID NO: 14 or the amino acid sequence shown in SEQ ID NO: 15;
  • the light chain variable region and the heavy chain variable region are selected from:
  • the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 9
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 10;
  • the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 11
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 12;
  • the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 13
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 14;
  • the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 7
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 15;
  • the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 9
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 15;
  • the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 11
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 15;
  • the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 13
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 15.
  • the humanized anti-BLyS antibody of the present invention further contains a human light chain constant region and a heavy chain constant region, and the light chain variable region and heavy chain variable region are connected to the human light chain constant region and heavy chain constant region, respectively . That is, the humanized anti-BLyS antibody contains a complete light chain and a complete heavy chain, wherein the complete light chain is formed by connecting the light chain variable region contained in the anti-BLyS antibody and the human light chain constant region, so The complete heavy chain is composed of the variable region of the heavy chain contained in the anti-BLyS antibody and the constant region of the human heavy chain.
  • the human light chain constant region is a human light chain kappa constant region.
  • the human heavy chain constant region is a human heavy chain Fc fragment.
  • the human light chain kappa constant region and the human heavy chain Fc fragment are both derived from healthy human B lymphocytes.
  • the variable region and constant region were connected by overlapping extension PCR to obtain the light chain and heavy chain of the complete humanized anti-BLyS antibody.
  • the humanized anti-BLyS antibody or antigen-binding fragment thereof described above comprises a light chain and a heavy chain, wherein the light chain comprises, for example, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20 or The amino acid sequence set forth in SEQ ID NO: 22, the heavy chain comprising the amino acids set forth in SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23 or SEQ ID NO: 24 sequence.
  • the humanized anti-BLyS antibody or antigen-binding fragment thereof described above comprises a light chain and a heavy chain, wherein:
  • the light chain comprises the amino acid sequence as shown in SEQ ID NO: 16
  • the heavy chain comprises as SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23 or SEQ ID NO: 24 the amino acid sequence shown; or
  • the light chain comprises the amino acid sequence shown in SEQ ID NO: 18, and the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23 or the amino acid sequence shown in SEQ ID NO: 24; or
  • the light chain comprises the amino acid sequence shown in SEQ ID NO: 20
  • the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23 or the amino acid sequence shown in SEQ ID NO: 24; or
  • the light chain comprises the amino acid sequence shown in SEQ ID NO: 22, and the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23 Or the amino acid sequence shown in SEQ ID NO: 24;
  • the light chain comprises the amino acid sequence shown in SEQ ID NO: 18, and the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 19; or
  • the light chain comprises the amino acid sequence shown in SEQ ID NO: 20
  • the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 21;
  • the light chain comprises the amino acid sequence shown in SEQ ID NO: 22, and the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 23; or
  • the light chain comprises the amino acid sequence shown in SEQ ID NO: 16
  • the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 24;
  • the light chain comprises the amino acid sequence shown in SEQ ID NO: 18, and the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 24; or
  • the light chain comprises the amino acid sequence shown in SEQ ID NO: 20
  • the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 24;
  • the light chain comprises the amino acid sequence shown in SEQ ID NO:22, and the heavy chain comprises the amino acid sequence shown in SEQ ID NO:24.
  • the antigen-binding fragments of the invention are selected from Fab, Fab', Fab'-SH, Fv, scFv, F(ab')2, sdAb, or diabodies.
  • the present invention provides a polynucleotide molecule selected from the group consisting of: a polynucleotide molecule encoding a humanized anti-BLyS antibody or antigen-binding fragment thereof as described in any one of the schemes herein, or its complement.
  • the present invention provides an expression vector comprising a polynucleotide molecule as described herein, preferably, the expression vector is a eukaryotic expression vector.
  • the present invention provides a host cell comprising a polynucleotide molecule or expression vector as described herein, preferably the host cell is a eukaryotic cell, more preferably a mammalian cell.
  • the present invention provides a method of making a humanized anti-BLyS antibody or antigen-binding fragment thereof as described in any one of the schemes herein, the method comprising expressing a humanized anti-BLyS antibody or antigen-binding fragment thereof suitable for expression of the antibody or antigen-binding fragment thereof.
  • a host cell as described herein is cultured under conditions such that it expresses the antibody or antigen-binding fragment thereof, and the expressed antibody or antigen-binding fragment thereof is recovered.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a humanized anti-BLyS antibody or antigen-binding fragment thereof as in any one of the schemes herein, a polynucleotide as described herein, an expression vector as described herein or a host cell as described herein, and a pharmaceutically acceptable carrier or excipient.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising: (1) a buffer; (2) a humanized anti-BLyS antibody or antigen-binding fragment thereof, the humanized anti-BLyS antibody or antigen-binding fragment thereof
  • the fragments have the amino acid sequences of LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, respectively, and the amino acid sequences as SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:3 respectively HCDR1, HCDR2 and HCDR3 shown in ID NO:6.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising: (1) a buffer; (2) a humanized anti-BLyS antibody or an antigen-binding fragment thereof; the humanized anti-BLyS antibody or an antigen-binding fragment thereof Fragments are humanized anti-BLyS antibodies or antigen-binding fragments thereof as described in any of the schemes herein.
  • the concentration of the humanized anti-BLyS antibody or antigen-binding fragment thereof in the above pharmaceutical composition is about 1-300 mg/mL, preferably about 10-300 mg/mL, more preferably about 20-280 mg/mL, More preferably about 30-150 mg/mL, more preferably about 80-120 mg/mL, more preferably about 180-220 mg/mL.
  • the aforementioned buffer is selected from one or more of acetate buffer, citrate buffer, succinate buffer, phosphate buffer, and histidine buffer.
  • the concentration of the aforementioned buffer is about 1-200 mM, preferably about 1-100 mM, preferably about 5-50 mM, preferably about 10-40 mM, preferably about 20-40 mM.
  • the pH of the above buffer is about 5.0-6.5, preferably about 5.0-6.0, preferably about 5.5-6.0.
  • the above-described pharmaceutical composition further includes a stabilizer.
  • the stabilizer is selected from the group consisting of arginine, arginine hydrochloride, methionine, proline, glycine, sodium chloride, mannitol, sorbitol, sucrose, maltose, xylitol, and One or more of trehalose.
  • the aforementioned stabilizer is selected from one or more of L-arginine, L-arginine hydrochloride, sucrose, methionine, and sodium chloride.
  • the concentration of the aforementioned stabilizer is about 10 mM to 400 mM, preferably about 50 mM to 300 mM, more preferably about 100 mM to 300 mM, more preferably about 100 mM to 200 mM.
  • the above stabilizer is a combination of L-arginine hydrochloride at a concentration of about 10-30 mM and sodium chloride at a concentration of about 50-200 mM; or the stabilizer is L-arginine at a concentration of about 10-30 mM in combination with about 50-200 mM sodium chloride; or the stabilizer is a combination of L-arginine at a concentration of about 10-30 mM and sucrose at a concentration of about 80-220 mM; or the stabilizer is at a concentration of about 10-30 mM combination of L-arginine hydrochloride and about 80-220 mM sucrose; or the stabilizer is a combination of about 30-90 mM methionine and about 50-200 mM sodium chloride; or the stabilizer or the stabilizer is about 10-120mM methionine; preferably, the stabilizer is L-arginine at a concentration of about 10-30mM The hydrochloride salt in combination with about 80-120 mM sodium chloride; or the
  • the above-mentioned pharmaceutical composition further includes a surfactant, preferably, the surfactant is selected from one or more of polysorbate 80, polysorbate 20, and poloxamer 188.
  • the above-mentioned surfactant concentration is about 0.001%-0.1%, preferably about 0.01%-0.1%, preferably about 0.01%-0.05%, on a w/v basis.
  • the above pharmaceutical composition further includes water for injection.
  • the pharmaceutical composition is a liquid formulation or a lyophilized formulation.
  • the pharmaceutical composition is a liquid formulation.
  • the present invention provides an injection, which contains the pharmaceutical composition described in any one of the schemes herein and a sodium chloride solution; preferably, the concentration of the sodium chloride solution is 0.85-0.9%; preferably, in the injection, the concentration of the humanized anti-BLyS antibody is 3-100 mg/mL, more preferably about 3-60 mg/mL; preferably, the pH of the injection is 5.5-6.0.
  • the pharmaceutical composition or injection is administered by subcutaneous injection.
  • the present invention provides a humanized anti-BLyS antibody or antigen-binding fragment thereof as described in any one of the schemes herein, a polynucleotide molecule described herein, an expression vector described herein, a Use of the host cell of the present invention, the pharmaceutical composition described herein, or the injection described herein in the preparation of a medicament for preventing and/or treating a disease caused by excessive B cell proliferation.
  • the present invention provides a humanized anti-BLyS antibody or antigen-binding fragment thereof as described in any one of the schemes herein, a polynucleotide molecule described herein, an expression vector described herein, a
  • the host cells, the pharmaceutical compositions described herein, or the injections described herein are used to prevent and/or treat diseases caused by excessive B cell proliferation.
  • the present invention provides a method of preventing and/or treating a disease caused by excessive B cell proliferation, comprising administering to a subject in need thereof a humanized antibody as described in any one of the schemes herein A BLyS antibody or antigen-binding fragment thereof, a polynucleotide molecule described herein, an expression vector described herein, a host cell described herein, a pharmaceutical composition described herein, or an injection described herein.
  • the above-mentioned disease caused by excessive proliferation of B cells is systemic lupus erythematosus, rheumatoid arthritis, ankylosing arthritis or B cell lymphoma.
  • the present invention provides a pharmaceutical combination comprising a humanized anti-BLyS antibody or antigen-binding fragment thereof as described in any one of the schemes herein, a polynucleotide molecule as described herein, a The expression vector described herein, the host cell described herein, the pharmaceutical composition described herein, or the injectable formulation described herein, and one or more additional therapeutic agents.
  • the present invention provides a kit comprising a humanized anti-BLyS antibody or antigen-binding fragment thereof as described in any one of the schemes herein, a polynucleotide molecule described herein, a The expression vector described herein, the host cell described herein, the pharmaceutical composition described herein, or the injection described herein, preferably it further comprises an administration device.
  • the present invention provides a histidine buffer and one or more stabilizers selected from the group consisting of L-arginine, L-arginine hydrochloride, methionine and sodium chloride and Use of an optional surfactant (preferably polysorbate 80) in improving the stability of a pharmaceutical formulation of a humanized anti-BLyS antibody or antigen-binding fragment thereof, or in the preparation of a humanized anti-BLyS antibody with improved stability or the application of the antigen-binding fragment thereof in the pharmaceutical preparation.
  • an optional surfactant preferably polysorbate 80
  • the histidine buffer, stabilizer and surfactant and the amounts thereof are as described in any of the embodiments herein; the improved stability is as described in any of the embodiments herein; the humanized anti-BLyS The antibody or antigen-binding fragment thereof is as described in any of the embodiments herein.
  • composition refers to a mixture comprising one or more of the antibodies described herein in admixture with other components, such as physiologically acceptable carriers and excipients.
  • the purpose of the pharmaceutical composition is to facilitate the administration to the organism, facilitate the absorption of the active ingredient and then exert the biological activity.
  • liquid formulation refers to a formulation in a liquid state and is not intended to refer to a resuspended lyophilized formulation.
  • the liquid formulations of the present invention are stable on storage, and their stability is independent of lyophilization (or other state-changing methods, such as spray drying).
  • aqueous liquid formulation refers to a liquid formulation using water as a solvent.
  • aqueous liquid formulations are formulations that do not require lyophilization, spray drying, and/or freezing to maintain stability (eg, chemical and/or physical stability and/or biological activity).
  • excipient refers to an agent that can be added to a formulation to provide desired properties (eg, consistency, increased stability) and/or to adjust osmotic pressure.
  • excipients include, but are not limited to, sugars, polyols, amino acids, surfactants, and polymers.
  • buffer pH of about 5.0-6.5 refers to an agent that, through the action of its acid/base conjugate component, renders a solution containing the agent resistant to pH changes.
  • the buffer used in the formulations of the present invention may have a pH in the range of about 5.0 to about 6.5, or a pH in the range of about 5.5 to about 6.5, or a pH in the range of about 5.0 to about 6.0.
  • buffers that control pH within this range include succinic acid, succinate (eg, sodium succinate), gluconic acid, histidine, histidine hydrochloride, methionine, Citric acid, citrate, phosphoric acid, phosphate, citrate/phosphate, imidazole, acetic acid, acetate, combinations thereof and other organic acid buffers.
  • Hetidine buffer is a buffer containing histidine ions.
  • histidine buffers include histidine and histidine salts, such as histidine hydrochloride, histidine acetate, histidine phosphate, histidine sulfate, and the like, such as histidine-containing Histidine buffers of acid and histidine hydrochloride; histidine buffers of the present invention also include histidine buffers containing histidine and acetate salts (eg, sodium or potassium salts).
  • citrate buffer is a buffer that includes citrate ions.
  • citrate buffers include citrate-sodium citrate, citrate-potassium citrate, citrate-calcium citrate, citrate-magnesium citrate, and the like.
  • the citrate buffer is a citric acid-sodium citrate buffer.
  • Acetate buffer is a buffer that includes acetate ions.
  • acetate buffers include acetate-sodium acetate, acetate-potassium acetate, acetate-calcium acetate, acetate-magnesium acetate, and the like.
  • the acetate buffer is acetic acid-sodium acetate buffer.
  • succinate buffer is a buffer that includes succinate ions.
  • succinate buffers include succinate-sodium succinate, succinate-potassium succinate, succinate-calcium succinate, succinate-magnesium succinate, and the like.
  • succinate buffer is a succinate-sodium succinate buffer.
  • a “phosphate buffer” is a buffer that includes phosphate ions.
  • phosphate buffers include disodium hydrogen phosphate-sodium dihydrogen phosphate, dipotassium hydrogen phosphate-potassium dihydrogen phosphate, and the like.
  • the phosphate buffer is disodium hydrogen phosphate-sodium hydrogen phosphate.
  • stabilizer means a pharmaceutically acceptable excipient which protects the active pharmaceutical ingredient and/or formulation from chemical and/or physical degradation during manufacture, storage and application.
  • Stabilizers include, but are not limited to, sugars, amino acids, salts, polyols and their metabolites as defined below, such as sodium chloride, calcium chloride, magnesium chloride, mannitol, sorbitol, sucrose, trehalose, methionine, L-arginine or its salts (such as L-arginine hydrochloride), glycine, alanine ( ⁇ -alanine, ⁇ -alanine), betaine, leucine, lysine, Glutamic acid, aspartic acid, proline, 4-hydroxyproline, sarcosine, gamma-aminobutyric acid (GABA), opines, alanine, octopine, glycine Strombine) and trimethylamine N-oxide (TMAO), human serum albumin (hsa), bo
  • the stabilizer specifically used in the present invention is selected from one or more of polyols, amino acids, salts, and sugars.
  • the sugars are sucrose and trehalose, preferably the polyol is mannitol.
  • Preferred amino acids are L-arginine or a salt thereof (eg L-arginine hydrochloride), methionine, glycine, proline.
  • Preferred stabilizers are sodium chloride, mannitol, sorbitol, sucrose, trehalose, methionine, L-arginine, L-arginine hydrochloride, glycine, proline, sodium chloride - L-arginine, sodium chloride-L-arginine hydrochloride, sodium chloride-methionine, sodium chloride-sorbitol, sodium chloride-mannitol, sodium chloride-sucrose, chloride Sodium-trehalose, L-arginine hydrochloride-mannitol, L-arginine hydrochloride-sucrose, more preferably L-arginine hydrochloride, L-arginine, methionine , sodium chloride-L-arginine, sodium chloride-L-arginine hydrochloride, sodium chloride-methionine, more preferably sodium chloride-L-arginine, sodium chloride- L-arginine hydrochloride.
  • the stabilizer used sodium chloride,
  • surfactant generally includes agents that protect proteins, such as antibodies, from air/solution interface induced stress, solution/surface induced stress to reduce aggregation of the antibody or to minimize the formation of particulate matter in the formulation.
  • exemplary surfactants include, but are not limited to, nonionic surfactants such as polyoxyethylene sorbitan fatty acid esters (eg, polysorbate 20 and polysorbate 80), polyethylene-polypropylene copolymers, polysorbate Ethylene-polypropylene glycol, polyoxyethylene-stearate, polyoxyethylene alkyl ethers, such as polyoxyethylene monolauryl ether, alkylphenyl polyoxyethylene ether (Triton-X), polyoxyethylene- Polyoxypropylene copolymer (Poloxamer, Pluronic), sodium dodecyl sulfate (SDS).
  • the surfactant used in the present invention is polysorbate 80.
  • isotonic means that the formulation has substantially the same osmotic pressure as human blood.
  • Isotonic formulations generally have an osmolarity of about 250 to 350 mOsm. Isotonicity can be measured using a vapor pressure or freezing point depression osmometer.
  • stable formulation is one in which the antibody substantially retains its physical and/or chemical stability and/or biological activity during the manufacturing process and/or upon storage.
  • a pharmaceutical formulation may be stable even if the contained antibody fails to retain 100% of its chemical structure or biological function after a certain period of storage.
  • an antibody that maintains about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% of its structure or function after storage for a certain period of time may also be considered “" stable”.
  • Various analytical techniques for measuring protein stability are available in the art and are reviewed in Peptide and Protein Drug Delivery 247-301, edited by Vincent Lee, Marcel Dekker, Inc. ., New York, N.Y., Pubs. (1991)), and Jones, A. (1993) Adv. Drug Delivery Rev. 10:29-90 (both incorporated by reference).
  • the stability of a formulation can be measured by determining the percentage (among other methods) of native antibody remaining in a formulation after storage at a given temperature for a given period of time.
  • the percentage of native antibody can be measured by size exclusion chromatography (eg, size exclusion high performance liquid chromatography [SEC-HPLC]), with "native" meaning unaggregated and undegraded.
  • protein stability is determined as the percentage of monomeric protein in solution with a low percentage of degraded (eg, fragmented) and/or aggregated protein.
  • the formulations are stable for storage at room temperature, about 25-30°C, or 40°C for at least 2 weeks, at least 28 days, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 months, at least 24 months, or Longer, up to about 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% of the antibody in aggregated form.
  • Stability can be measured by determining (among other methods) the percentage of antibody (“acidic form") that migrates in the more acidic fraction of this antibody main fraction ("predominantly charged form”) during ion exchange, where stability is related to The percentage of antibody in the acidic form is inversely proportional.
  • the percentage of "acidified” antibody can be measured by ion exchange chromatography (eg, cation exchange high performance liquid chromatography [CEX-HPLC]).
  • an acceptable degree of stability means that upon storage of the formulation at a certain temperature and for a certain period of time, the detectable acidic form of the antibody therein does not exceed at most about 49%, 45%, 40%, 35% %, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, 0.5% or 0.1%.
  • the certain period of storage prior to measuring stability can be at least 2 weeks, at least 28 days, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 months, at least 24 months, or longer.
  • the temperature at which the pharmaceutical formulation is allowed to be stored can be any temperature in the range of about -80°C to about 45°C, eg, at about -80°C, about -30°C, about -20°C, about 0°C , about 2-8°C, about 5°C, about 25°C, or about 40°C.
  • the antibody shows substantially no evidence of, for example, aggregation, precipitation and/or denaturation upon visual inspection of color and/or clarity or as measured by UV light scattering or by size exclusion chromatography, the antibody is in the drug combination "maintain its physical stability". Aggregation is the process by which individual molecules or complexes associate covalently or non-covalently to form aggregates. Aggregation can proceed to the point where a visible precipitate is formed.
  • the stability of the formulation can be assessed by methods well known in the art, including measuring the apparent extinction (absorbance or optical density) of the sample. Such extinction measurements correlate to the turbidity of the formulation.
  • the turbidity of a formulation is, in part, an inherent property of proteins dissolved in solution, and is typically measured by nephelometric methods and measured in nephelometric turbidity units (NTU).
  • the level of turbidity as a function of, for example, the concentration of one or more components in solution is also referred to as the "opaque” or "opaque appearance" of the formulation.
  • Turbidity levels can be calculated with reference to a standard curve generated using suspensions of known turbidity.
  • Reference standards for determining the turbidity level of pharmaceutical compositions can be based on the European Pharmacopoeia standards (European Pharmacopoeia, 4th edition, "Directorate for the Quality of Medicine”). of the Council of Europe) (EDQM), France).
  • a clear solution is defined as a solution with a turbidity lower than or equal to that of a reference suspension according to the European Pharmacopoeia standard of about 3.
  • Nephelometric turbidity measurements can detect Rayleigh scattering in the absence of associative or non-ideal effects, which typically vary linearly with concentration. Other methods for assessing physical stability are known in the art.
  • Chemical stability can be assessed by, for example, detecting or quantifying chemically altered forms of the antibody.
  • Chemical changes can include size changes (eg, clipping), which can be assessed using, for example, size exclusion chromatography, SDS-PAGE, and/or matrix-assisted laser desorption ionization/time-of-flight mass spectrometry (MALDI/TOF MS).
  • size changes eg, clipping
  • MALDI/TOF MS matrix-assisted laser desorption ionization/time-of-flight mass spectrometry
  • charge changes eg occur as a result of deamidation or oxidation
  • An antibody in a pharmaceutical composition "retains its biological activity" in a pharmaceutical composition if it is biologically active for its intended purpose. For example, if the formulation is stored at a temperature such as 5°C, 25°C, 45°C, etc. for a certain period of time (eg, 1 to 12 months), the humanized monoclonal antibody contained in the formulation binds to COVID-19 with the stated affinity
  • a formulation of the invention is considered stable if the binding affinity of the antibody is at least 90%, 95% or more prior to storage. Binding affinity can also be determined using techniques such as ELISA or plasmon resonance.
  • a “therapeutically effective amount” or “effective amount” of an antibody in the pharmacological sense refers to an amount effective in the prevention or treatment or alleviation of the symptoms of the disorder that the antibody can effectively treat.
  • a “therapeutically effective amount” or “therapeutically effective dose” of a drug is any amount of drug that, when used alone or in combination with another therapeutic agent, protects a subject from disease onset or promotes disease regression, so The disease regression is evidenced by a reduction in the severity of disease symptoms, an increase in the frequency and duration of asymptomatic periods of the disease, or prevention of injury or disability caused by the suffering of the disease.
  • a pharmacologically effective amount includes a "prophylactically effective amount,” that is, any amount that inhibits the progression or recurrence of a disease when administered to a subject at risk for the disease or to a subject relapsed with the disease, alone or as in combination with other therapeutic agents medicine.
  • subject or “patient” are intended to include mammalian organisms.
  • subjects/patients include humans and non-human mammals, such as non-human primates, dogs, cows, horses, pigs, sheep, goats, cats, mice, rabbits, rats, and transgenic non-human animals.
  • the subject is a human.
  • administering refers to introducing a composition comprising a therapeutic agent into a subject using any of a variety of methods or delivery systems known to those skilled in the art.
  • Routes of administration of humanized anti-BLyS antibodies or antigen-binding fragments thereof include intravenous, intramuscular, subcutaneous, peritoneal, spinal or other parenteral routes of administration, such as injection or infusion.
  • Parenter administration refers to modes of administration other than enteral or topical administration, usually by injection, including, but not limited to, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular , intra-frame, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subepidermal, intraarticular, subcapsular, subarachnoid, intraspinal, intradural, and intrasternal injection and infusion and in vivo electroporation.
  • antibody as used herein should be understood to include whole antibody molecules and antigen-binding fragments thereof.
  • antigen-binding portion or “antigen-binding fragment” of an antibody (or simply “antibody portion” or “antibody fragment”) refers to one or more fragments of an antibody that retain the ability to specifically bind BLyS.
  • full-length antibody or "intact antibody molecule” as used herein refers to an immunoglobulin molecule comprising four peptide chains, two heavy (H) chains (about 50-70 kDa in full length) and two light (L) chains (about 25 kDa in full length) are interconnected by disulfide bonds.
  • Each heavy chain (herein abbreviated as HC) consists of a heavy chain variable region (herein abbreviated as VH) and a heavy chain constant region (herein abbreviated as CH).
  • the heavy chain constant region consists of three domains, CH1, CH2 and CH3.
  • Each light chain (abbreviated herein as LC) consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region.
  • the light chain constant region consists of one domain, CL.
  • the VH and VL regions can be further subdivided into highly variable complementarity determining regions (CDRs) and spaced by more conserved regions called framework regions (FRs).
  • CDRs highly variable complementarity determining regions
  • FRs framework regions
  • Each VH or VL region consists of 3 CDRs and 4 FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from amino terminus to carboxy terminus.
  • the variable regions of the heavy and light chains contain binding domains that interact with the antigen.
  • the constant regions of the antibodies mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq)
  • CDR refers to the complementarity determining regions within antibody variable sequences. There are three CDRs in each variable region of the heavy and light chains, which are designated HCDR1, HCDR2 and HCDR3 or LCDR1, LCDR2 and LCDR3 for each heavy and light chain variable region. The exact boundaries of these CDRs are defined differently by different systems.
  • variable region CDRs of the antibodies of the invention can be determined using any of a number of well-known protocols, including those described by Kabat et al. (1991), "Sequences of Proteins of Immunological Interest, p. 5 ed. Public Health Service, National Institutes of Health, Bethesda, MD ("Kabat” numbering scheme) described the Kabat protocol and Lefranc M.-P. et al. described the IMGT protocol (1999 Nucleic Acids Research, 27, 209-212).
  • an "antigen-binding fragment” includes a fragment of an antibody or derivative thereof, typically including at least a fragment of the antigen-binding or variable region (eg, one or more CDRs) of the parent antibody, which retains at least some of the parent antibody binding specificity.
  • antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2 and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules such as sc-Fv; nanobodies formed from antibody fragments and multispecific antibodies.
  • the binding fragment or derivative thereof retains at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more of the antigen binding affinity of the parent antibody.
  • antigen-binding fragments of antibodies may include conservative or non-conservative amino acid substitutions that do not significantly alter their biological activity (referred to as “conservative variants” or “functionally conservative variants” of an antibody).
  • Specific binding when referring to a ligand/receptor, antibody/antigen, or other binding pair refers to determining the presence or absence of a protein and/or other biological agent in a heterogeneous population.
  • the monoclonal antibody of the present invention binds to the BLyS protein.
  • a specific ligand/antigen binds to a specific receptor/antibody, and does not bind to other proteins present in the sample in significant amounts.
  • the humanized monoclonal antibodies or antigen-binding fragments thereof described herein include any one of the anti-BLyS antibodies described in Application No. CN201210160474.3, the entire disclosures of which are incorporated herein by reference.
  • the CDR sequences of the antibodies used in the methods and compositions of the invention include CDR sequences from the antibody BLyS-IV described in CN201210160474.3.
  • the non-limiting, exemplary antibodies used in the examples herein are selected from the humanized anti-BLyS antibody BLyS-IV described in CN201210160474.3.
  • the non-limiting, exemplary humanized anti-BLyS antibodies used in the Examples herein have the light chain amino acid sequence set forth in SEQ ID NO: 18, and the amino acid sequence set forth in SEQ ID NO: 19
  • the heavy chain amino acid sequence of ; the humanized anti-BLyS antibody was named H482L472.
  • the present invention provides a humanized anti-BLyS antibody or antigen-binding fragment thereof capable of binding to B lymphocyte stimulating factor and capable of inhibiting the binding of B lymphocyte stimulating factor to its receptor BR3-Fc, having Excellent binding ability of human BLyS protein and the ability to competitively inhibit the binding of human BLyS to its receptor human BR3.
  • an expression vector can be constructed by referring to the methods shown in Example 7 and Example 8 in Application No. CN201210160474.3, and 293F cells are used for transient expression in a 100 mL system. , after one-step affinity purification.
  • the present invention provides a humanized anti-BLyS antibody or an antigen-binding fragment thereof, the amino acid sequences of LCDR1, LCDR2 and LCDR3 of the humanized anti-BLyS antibody or antigen-binding fragment thereof are respectively as SEQ ID NO: 1.
  • SEQ ID NO:2 and SEQ ID NO:3 Shown in SEQ ID NO:2 and SEQ ID NO:3, the amino acid sequences of HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6 respectively; light chain variable
  • the FR1 of the region is selected from the FR1 described in any one of SEQ ID NO:7, 9 and 13, the FR2 is the FR2 described in SEQ ID NO:7, and the FR3 is selected from any one of SEQ ID NO:7, 9 and 11.
  • FR3, FR4 are FR4 described in SEQ ID NO:7; FR1 of heavy chain variable region is selected from FR1 described in any one of SEQ ID NO:8, 10, 12 and 15, FR2 is selected from SEQ ID NO FR2 described in any one of: 8, 10 and 15, FR3 is selected from the FR3 described in any one of SEQ ID NO: 8, 12 and 14, and FR4 is the FR4 described in SEQ ID NO: 8 or 10.
  • the humanized anti-BLyS antibody or antigen-binding fragment thereof of the present invention does not include: the light chain variable region is the amino acid sequence shown in SEQ ID NO: 7, and the heavy chain variable region is SEQ ID NO: 7 The humanized anti-BLyS antibody or antigen-binding fragment thereof of the amino acid sequence shown in ID NO: 8.
  • the humanized anti-BLyS antibody or antigen-binding fragment thereof of the present invention does not include: the light chain is the amino acid sequence shown in SEQ ID NO: 16, while the heavy chain is shown in SEQ ID NO: 17 The amino acid sequence of a humanized anti-BLyS antibody or antigen-binding fragment thereof.
  • the amino acid sequences of LCDR1, LCDR2 and LCDR3 are as set forth in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, respectively As shown, the amino acid sequences of HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 respectively; FR1 of the light chain variable region is selected from SEQ ID NO: 7, 9 and 13 FR1 described in any one of, FR2 is FR2 described in SEQ ID NO:7, FR3 is selected from FR3 described in any one of SEQ ID NO:7, 9 and 11, FR4 is described in SEQ ID NO:7 FR4; FR1-FR4 of the heavy chain variable region are FR1-FR4 of SEQ ID NO: 15, respectively.
  • the amino acid sequences of LCDR1, LCDR2 and LCDR3 are as set forth in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, respectively As shown, the amino acid sequences of HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 respectively; FR1 of the light chain variable region is selected from SEQ ID NO: 7, 9 and 13 FR1 described in any one of, FR2 is FR2 described in SEQ ID NO:7, FR3 is selected from FR3 described in any one of SEQ ID NO:7, 9 and 11, FR4 is described in SEQ ID NO:7 FR4; FR1-FR4 of the heavy chain variable region are FR1-FR4 of SEQ ID NO: 10, respectively.
  • the amino acid sequences of LCDR1, LCDR2 and LCDR3 are as set forth in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, respectively As shown, the amino acid sequences of HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 respectively; FR1 of the light chain variable region is selected from SEQ ID NO: 7, 9 and 13 FR1 described in any one of, FR2 is FR2 described in SEQ ID NO:7, FR3 is selected from FR3 described in any one of SEQ ID NO:7, 9 and 11, FR4 is described in SEQ ID NO:7 FR4; FR1-FR4 of the heavy chain variable region are FR1-FR4 of SEQ ID NO: 12, respectively.
  • the amino acid sequences of LCDR1, LCDR2 and LCDR3 are as set forth in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, respectively shown, the amino acid sequences of HCDR1, HCDR2 and HCDR3 are respectively shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6; FR1 of the light chain variable region is described in SEQ ID NO:11 or 13 FR1, FR2 are FR2 described in SEQ ID NO: 11, FR3 are FR3 described in SEQ ID NO: 11 or 13, FR4 are FR4 described in SEQ ID NO: 11; FR1-FR4 of the heavy chain variable region are respectively are FR1-FR4 of SEQ ID NO: 14.
  • the invention provides a humanized anti-BLyS antibody or antigen-binding fragment thereof comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises as SEQ ID The amino acid sequence shown in NO: 7, SEQ ID NO: 9, SEQ ID NO: 11 or SEQ ID NO: 13, the heavy chain variable region comprises as SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID The amino acid sequence shown in NO: 12, SEQ ID NO: 14 or SEQ ID NO: 15.
  • the above-described humanized anti-BLyS antibody or antigen-binding fragment thereof comprises a light chain variable region and a heavy chain variable region, wherein:
  • the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:7
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14 or the amino acid sequence shown in SEQ ID NO: 15; or
  • the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:9
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12 , the amino acid sequence shown in SEQ ID NO: 14 or SEQ ID NO: 15; or
  • the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 11
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12 , the amino acid sequence shown in SEQ ID NO: 14 or SEQ ID NO: 15; or
  • the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 13
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12 , SEQ ID NO: 14 or the amino acid sequence shown in SEQ ID NO: 15.
  • the light chain variable region and the heavy chain variable region are selected from one of the following groups:
  • the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 9
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 10;
  • the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 11
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 12;
  • the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 13
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 14;
  • the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 7
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 15;
  • the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 9
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 15;
  • the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 11
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 15;
  • the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 13
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 15.
  • the above-described humanized anti-BLyS antibody or antigen-binding fragment thereof comprises a light chain variable region and a heavy chain variable region, wherein:
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:8, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:13;
  • the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 10
  • the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 7, 9, 11 or 13;
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 12
  • the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 7, 9, 11 or 13;
  • the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 14, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 11 or 13; or
  • the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 15
  • the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 7, 9, 11 or 13.
  • the humanized anti-BLyS antibody of the present invention further contains a human light chain constant region and a heavy chain constant region, and the light chain variable region and heavy chain variable region are connected to the human light chain constant region and heavy chain constant region, respectively . That is, the humanized anti-BLyS antibody contains a complete light chain and a complete heavy chain, wherein the complete light chain is formed by connecting the light chain variable region contained in the anti-BLyS antibody and the human light chain constant region, so The complete heavy chain is composed of the variable region of the heavy chain contained in the anti-BLyS antibody and the constant region of the human heavy chain.
  • the human light chain constant region is a human light chain kappa constant region.
  • the human heavy chain constant region is a human heavy chain Fc fragment.
  • the human light chain kappa constant region and the human heavy chain Fc fragment are both derived from healthy human B lymphocytes.
  • overlapping extension PCR to connect the variable region and constant region to obtain the complete light chain and heavy chain of humanized anti-BLyS antibody.
  • the above-mentioned humanized anti-BLyS antibody or antigen-binding fragment thereof comprises a light chain and a heavy chain, wherein the light chain comprises, for example, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20 or the amino acid sequence shown in SEQ ID NO: 22, the heavy chain comprising the amino acid sequence shown in SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23 or SEQ ID NO: 24 amino acid sequence.
  • the humanized anti-BLyS antibody or antigen-binding fragment thereof described above comprises a light chain and a heavy chain, wherein:
  • the light chain comprises the amino acid sequence as shown in SEQ ID NO: 16
  • the heavy chain comprises as SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23 or SEQ ID NO: 24 the amino acid sequence shown; or
  • the light chain comprises the amino acid sequence shown in SEQ ID NO: 18, and the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23 or the amino acid sequence shown in SEQ ID NO: 24; or
  • the light chain comprises the amino acid sequence shown in SEQ ID NO: 20
  • the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23 or the amino acid sequence shown in SEQ ID NO: 24; or
  • the light chain comprises the amino acid sequence shown in SEQ ID NO: 22, and the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23 or the amino acid sequence shown in SEQ ID NO: 24; or
  • the light and heavy chains are selected from one of the following groups:
  • the light chain comprises the amino acid sequence shown in SEQ ID NO: 18, and the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 19; or
  • the light chain comprises the amino acid sequence shown in SEQ ID NO: 20
  • the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 21;
  • the light chain comprises the amino acid sequence shown in SEQ ID NO: 22, and the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 23; or
  • the light chain comprises the amino acid sequence shown in SEQ ID NO: 16
  • the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 24;
  • the light chain comprises the amino acid sequence shown in SEQ ID NO: 18, and the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 24; or
  • the light chain comprises the amino acid sequence shown in SEQ ID NO: 20
  • the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 24;
  • the light chain comprises the amino acid sequence shown in SEQ ID NO:22, and the heavy chain comprises the amino acid sequence shown in SEQ ID NO:24.
  • the humanized anti-BLyS antibody or antigen-binding fragment thereof described above comprises a light chain and a heavy chain, wherein:
  • the heavy chain comprises the amino acid sequence set forth in SEQ ID NO:17, and the light chain comprises the amino acid sequence set forth in SEQ ID NO:22;
  • the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 19 and the light chain comprises the amino acid sequence set forth in SEQ ID NO: 16, 18, 20 or 22;
  • the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 21, and the light chain comprises the amino acid sequence set forth in SEQ ID NO: 16, 18, 20 or 22;
  • the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 23, and the light chain comprises the amino acid sequence set forth in SEQ ID NO: 20 or 22; or
  • the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 24, and the light chain comprises the amino acid sequence set forth in SEQ ID NO: 16, 18, 20 or 22.
  • the pharmaceutical composition of the present invention is a highly stable pharmaceutical composition containing an anti-BLyS antibody.
  • the anti-BLyS antibody has an unexpected feature in the combination of histidine buffer system and L-arginine, L-arginine hydrochloride or methionine, namely high stability .
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a humanized anti-BLyS antibody or antigen-binding fragment thereof described herein, a polynucleotide described herein, an expression vector described herein, or a host cell described herein, and a pharmaceutically acceptable carrier or excipient.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising: (1) a buffer; (2) a humanized anti-BLyS antibody or antigen-binding fragment thereof, the humanized anti-BLyS antibody or antigen-binding fragment thereof
  • the fragments have the amino acid sequences of LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, respectively, and the amino acid sequences as SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:3 respectively HCDR1, HCDR2 and HCDR3 shown in ID NO:6.
  • the humanized anti-BLyS antibody or antigen-binding fragment thereof in the pharmaceutical composition of the present invention is as described in any one of the embodiments in the "Antibody” section of the present application; or the humanized anti-BLyS antibody or antigen-binding fragment thereof comprises A light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:7, and the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:8 The amino acid sequence shown; or the humanized anti-BLyS antibody or antigen-binding fragment thereof comprises a light chain and a heavy chain, wherein the light chain comprises the amino acid sequence shown in SEQ ID NO: 16, and the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 16 The amino acid sequence shown in SEQ ID NO: 17.
  • the humanized anti-BLyS antibody or antigen-binding fragment thereof described above comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises as in SEQ ID NO: 7 , SEQ ID NO: 9, SEQ ID NO: 11 or the amino acid sequence shown in SEQ ID NO: 13, the heavy chain variable region comprises such as SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12 , SEQ ID NO: 14 or the amino acid sequence shown in SEQ ID NO: 15.
  • the above-described humanized anti-BLyS antibody, or antigen-binding fragment thereof comprises a light chain variable region and a heavy chain variable region, wherein:
  • the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:7
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12 , the amino acid sequence shown in SEQ ID NO: 14 or SEQ ID NO: 15; or
  • the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:9
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12 , the amino acid sequence shown in SEQ ID NO: 14 or SEQ ID NO: 15; or
  • the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 11
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12 , the amino acid sequence shown in SEQ ID NO: 14 or SEQ ID NO: 15; or
  • the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 13
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12 , the amino acid sequence shown in SEQ ID NO: 14 or SEQ ID NO: 15; or
  • the light chain variable region and the heavy chain variable region are selected from one of the following groups:
  • the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 9
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 10;
  • the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 11
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 12;
  • the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 13
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 14;
  • the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 7
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 15;
  • the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 9
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 15;
  • the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 11
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 15;
  • the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 13
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 15;
  • the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:7, and the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:8.
  • the humanized anti-BLyS antibody of the present invention further contains a human light chain constant region and a heavy chain constant region, and the light chain variable region and heavy chain variable region are connected to the human light chain constant region and heavy chain constant region, respectively . That is, the humanized anti-BLyS antibody contains a complete light chain and a complete heavy chain, wherein the complete light chain is formed by connecting the light chain variable region contained in the anti-BLyS antibody and the human light chain constant region, so The complete heavy chain is composed of the variable region of the heavy chain contained in the anti-BLyS antibody and the constant region of the human heavy chain.
  • the human light chain constant region is a human light chain kappa constant region.
  • the human heavy chain constant region is a human heavy chain Fc fragment.
  • the human light chain kappa constant region and the human heavy chain Fc fragment are both derived from healthy human B lymphocytes.
  • overlapping extension PCR to connect the variable region and constant region to obtain the complete light chain and heavy chain of humanized anti-BLyS antibody.
  • the above-mentioned humanized anti-BLyS antibody or antigen-binding fragment thereof comprises a light chain and a heavy chain, wherein the light chain comprises, for example, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20 or the amino acid sequence shown in SEQ ID NO: 22, the heavy chain comprising the amino acid sequence shown in SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23 or SEQ ID NO: 24 amino acid sequence.
  • the above-described humanized anti-BLyS antibody, or antigen-binding fragment thereof, comprising a light chain and a heavy chain, wherein:
  • the light chain comprises the amino acid sequence shown in SEQ ID NO: 16
  • the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23 or the amino acid sequence shown in SEQ ID NO: 24; or
  • the light chain comprises the amino acid sequence shown in SEQ ID NO: 18, and the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23 or the amino acid sequence shown in SEQ ID NO: 24; or
  • the light chain comprises the amino acid sequence shown in SEQ ID NO: 20
  • the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23 or the amino acid sequence shown in SEQ ID NO: 24; or
  • the light chain comprises the amino acid sequence shown in SEQ ID NO: 22, and the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23 or the amino acid sequence shown in SEQ ID NO: 24; or
  • the light and heavy chains are selected from one of the following groups:
  • the light chain comprises the amino acid sequence shown in SEQ ID NO: 18, and the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 19; or
  • the light chain comprises the amino acid sequence shown in SEQ ID NO: 20
  • the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 21;
  • the light chain comprises the amino acid sequence shown in SEQ ID NO: 22, and the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 23; or
  • the light chain comprises the amino acid sequence shown in SEQ ID NO: 16
  • the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 24;
  • the light chain comprises the amino acid sequence shown in SEQ ID NO: 18, and the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 24; or
  • the light chain comprises the amino acid sequence shown in SEQ ID NO: 20
  • the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 24;
  • the light chain comprises the amino acid sequence shown in SEQ ID NO: 22, and the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 24; or
  • the light chain comprises the amino acid sequence shown in SEQ ID NO: 16
  • the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 17.
  • the antigen-binding fragments of the invention are selected from Fab, Fab', Fab'-SH, Fv, scFv, F(ab')2, sdAb, or diabodies.
  • the concentration of the humanized anti-BLyS antibody or antigen-binding fragment thereof in the pharmaceutical composition is about 1-300 mg/mL, preferably about 10-300 mg/mL, more preferably about 20-280 mg/mL , more preferably about 30-150mg/mL, more preferably about 80-120mg/mL, more preferably about 180-220mg/mL; more preferably, the concentration of the above-mentioned humanized monoclonal antibody or its antigen-binding fragment is about 5mg/mL, 10mg/mL, 15mg/mL, 20mg/mL, 25mg/mL, 30mg/mL, 35mg/mL, 40mg/mL, 45mg/mL, 50mg/mL, 60mg/mL, 70mg/mL, 80mg/mL mL, 90 mg/mL, 100 mg/mL, 110 mg/mL, 120 mg/mL, 130 mg/mL, 140 mg
  • the aforementioned buffer is selected from one or more of acetate buffer, citrate buffer, succinate buffer, phosphate buffer, and histidine buffer.
  • the concentration of the above buffer is about 1-200 mM, preferably about 1-100 mM, preferably about 5-50 mM, preferably about 10-40 mM, preferably about 20-40 mM; the above buffer concentration is not limiting Typical examples are about 5mM, 10mM, 15mM, 20mM, 25mM, 30mM, 35mM, 40mM, 45mM, 50mM, 60mM, 70mM, 80mM, 90mM, 100mM, 105mM, 110mM, 115mM, 120mM, 130mM, 140mM, 150mM, 160mM , 170 mM or 180 mM or a range formed by any two values within these ranges as endpoints, preferably 10 mM, 15 mM, 20 mM, 25 mM, 30 mM or 35 mM.
  • the pH of the above buffer is about 5.0-6.5, preferably about 5.0-6.0, preferably about 5.5-6.0, non-limiting examples of pH of the above buffer are about 5.0, 5.1, 5.2 , 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, preferably about 5.5, 5.8 or 6.0.
  • the above buffer is histidine buffer, preferably, the histidine buffer is selected from histidine-hydrochloride buffer or histidine-acetate buffer, preferably histidine acid-hydrochloride buffer.
  • the histidine-hydrochloride buffers described above are made from histidine and histidine hydrochloride, preferably L-histidine and L-histidine monohydrochloride.
  • the histidine buffer is made from 5-40 mM L-histidine and 5-40 mM L-histidine monohydrochloride.
  • the histidine buffer is made from histidine and histidine hydrochloride in a molar ratio of 1:1 to 1:4.
  • the histidine buffer is made from histidine and histidine hydrochloride in a molar ratio of 1:2 to 1:2.5.
  • the histidine buffer is made of histidine and histidine hydrochloride in a molar ratio of 1:2.3.
  • the histidine buffer is: histidine buffer at pH 5.8 made from about 10.0-11.0 mM L-histidine and about 24.0-25.0 mM L-histidine monohydrochloride liquid.
  • 35 mM histidine buffer at pH 5.8 is prepared by first preparing 35 mM L-histidine and L-histidine monohydrochloride solutions separately, and then mixing the two to pH 5.8 That's it.
  • the histidine buffer is: histidine buffer at pH 5.8 made from about 10.0 mM histidine and about 25.0 mM histidine hydrochloride.
  • the above buffer is acetate buffer, preferably, the acetate buffer is acetic acid-sodium acetate buffer or acetic acid-potassium acetate buffer, preferably acetic acid-sodium acetate buffer.
  • the above buffer is a citrate buffer, preferably, the citrate buffer is a citric acid-sodium citrate buffer.
  • the above buffer is succinate buffer, preferably, the succinate buffer is succinate-sodium succinate buffer.
  • the above buffer is a phosphate buffer, preferably, the phosphate buffer is a sodium dihydrogen phosphate-disodium hydrogen phosphate buffer or a potassium dihydrogen phosphate-dipotassium hydrogen phosphate buffer.
  • the above-described pharmaceutical composition further includes a stabilizer.
  • the stabilizer is selected from the group consisting of arginine, arginine hydrochloride, methionine, proline, glycine, sodium chloride, mannitol, sorbitol, sucrose, maltose, xylitol, and One or more of trehalose.
  • the aforementioned stabilizer is selected from one or more of L-arginine, L-arginine hydrochloride, sucrose, methionine, and sodium chloride.
  • the concentration of the aforementioned stabilizer is about 10 mM to 400 mM, preferably about 50 mM to 300 mM, more preferably about 100 mM to 300 mM, more preferably about 100 mM to 200 mM.
  • the above stabilizer is a combination of L-arginine hydrochloride at a concentration of about 10-30 mM and sodium chloride at a concentration of about 50-200 mM; or the stabilizer is L-arginine at a concentration of about 10-30 mM in combination with about 50-200 mM sodium chloride; or the stabilizer is a combination of L-arginine at a concentration of about 10-30 mM and sucrose at a concentration of about 80-220 mM; or the stabilizer is at a concentration of about 10-30 mM combination of L-arginine hydrochloride and about 80-220 mM sucrose; or the stabilizer is a combination of about 30-90 mM methionine and about 50-200 mM sodium chloride; or the stabilizer or the stabilizer is about 10-120mM methionine; preferably, the stabilizer is L-arginine at a concentration of about 10-30mM The hydrochloride salt in combination with about 80-120 mM sodium chloride; or the
  • the above-mentioned stabilizer consists of sodium chloride at a concentration of about 50-200 mM and L-arginine at a concentration of about 10-30 mM; preferably, the above-mentioned stabilizer consists of sodium chloride at a concentration of about 80-120 mM and a concentration of consisting of about 10-30 mM L-arginine; non-limiting examples of the above stabilizers are chlorides at concentrations of about 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 95 mM, 100 mM, 105 mM, 110 mM, 115 mM, 120 mM or 130 mM Sodium and L-arginine at concentrations of about 10mM, 15mM, 20mM, 25mM, 30mM.
  • the above-mentioned stabilizer consists of sodium chloride at a concentration of about 50-200 mM and L-arginine hydrochloride at a concentration of about 10-30 mM; preferably, the above-mentioned stabilizer is made of chloride at a concentration of about 80-120 mM Sodium and L-arginine hydrochloride at a concentration of about 10-30 mM; non-limiting examples of the above stabilizers are at a concentration of about 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 95 mM, 100 mM, 105 mM, 110 mM, 115 mM , 120mM or 130mM sodium chloride and concentrations of about 10mM, 15mM, 20mM, 25mM, 30mM L-arginine hydrochloride composition.
  • the above-mentioned stabilizer consists of sodium chloride at a concentration of about 50-200 mM and methionine at a concentration of about 30-90 mM; preferably, the above-mentioned stabilizer consists of sodium chloride at a concentration of about 80-120 mM and 50-70 mM methionine; non-limiting examples of the above stabilizers are sodium chloride at concentrations of about 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 95 mM, 100 mM, 105 mM, 110 mM, 115 mM, 120 mM or 130 mM and methionine at concentrations of approximately 30 mM, 40 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM or 80 mM.
  • the above stabilizer consists of L-arginine hydrochloride at a concentration of about 10-30 mM and sucrose at a concentration of about 80-220 mM; -30 mM L-arginine hydrochloride; non-limiting examples of the above stabilizers are composed of concentrations of about 80 mM, 90 mM, 100 mM, 110 mM, 120 Mm, 125 Mm, 130 Mm, 135 Mm, 140 Mm, 145 Mm, 150 Mm, 155 Mm, 160mM or 170mM sucrose and L-arginine hydrochloride at concentrations of about 10mM, 15mM, 20mM, 25mM, 30mM.
  • the aforementioned stabilizer is L-arginine.
  • the above-mentioned stabilizer is L-arginine at a concentration of about 10-100 mM, and the above-mentioned L-arginine concentration is preferably about 15-50 mM, preferably about 20-30 mM, and the above-mentioned L-arginine concentration
  • Non-limiting examples of are about 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, preferably 25 mM or 30 mM.
  • the aforementioned stabilizer is methionine.
  • the above-mentioned stabilizer is methionine at a concentration of about 20-200 mM, preferably the concentration of the above-mentioned methionine is about 20-180 mM, preferably about 40-150 mM, preferably about 45-100 mM, preferably about 50-90 mM, non-limiting examples of the above methionine concentrations are about 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 85 mM , 90mM, 100mM, preferably 60mM or 65mM.
  • the above stabilizer is sodium chloride.
  • the stabilizer is sodium chloride at a concentration of about 30-200 mM, preferably at a concentration of about 50-190 mM, preferably about 60-180 mM, preferably about 70-150 mM, preferably about 80 -120mM, non-limiting examples of the above sodium chloride concentrations are about 60mM, 70mM, 80mM, 85mM, 90mM, 95mM, 100mM, 105mM, 110mM, 115mM, 120mM, 130mM, 140mM, 150mM, 160mM, 170mM, 180mM, 190mM, 200mM, preferably 100mM or 105mM.
  • the aforementioned stabilizer is L-arginine hydrochloride.
  • the above-mentioned stabilizer is L-arginine hydrochloride at a concentration of about 30-200 mM, preferably the concentration of the above-mentioned L-arginine hydrochloride is about 50-190 mM, preferably about 100-180 mM, preferably About 120-170 mM, preferably about 130-150 mM, non-limiting examples of the above L-arginine hydrochloride concentrations are about 100 mM, 110 mM, 120 mM, 125 mM, 130 mM, 135 mM, 140 mM, 145 mM, 150 mM, 155 mM , 160mM, 170mM, 180mM, 190mM, 200mM, preferably 135mM or 140mM.
  • the above-mentioned pharmaceutical composition further comprises a surfactant selected from one or more of polysorbate 80, polysorbate 20, and poloxamer 188.
  • the above-mentioned surfactant is selected from polysorbate 80.
  • the above-mentioned surfactant is selected from polysorbate 20.
  • the above-mentioned surfactant concentration is about 0.001%-0.1%, preferably about 0.01%-0.1%, preferably about 0.01%-0.05%; as a non-limiting example, the above-mentioned surfactant concentration
  • the concentration of surfactant is about 0.01%, 0.02%, 0.03%, 0.04%, 0.05%. 0.06%, 0.07%, 0.09% or 0.08%.
  • the above pharmaceutical composition further includes water for injection.
  • the above pharmaceutical compositions contain: (a) about 20 mg/mL-280 mg/mL of the humanized anti-BLyS antibody or antigen-binding fragment thereof described in any one of the herein; (b) about 5-50 mM of Histidine buffer or about 5-50 mM acetate buffer, pH about 5.0-6.5; (c) about 10-30 mM L-arginine or about 10-30 mM L-arginine hydrochloride; (d) about 80-220 mM sucrose; (e) and about 0.01%-0.1% polysorbate 80.
  • the pharmaceutical compositions described above contain: (a) about 20 mg/mL-280 mg/mL of the humanized anti-BLyS antibody or antigen-binding fragment thereof described in any of the embodiments herein; (b) about 10-40 mM Histidine buffer or about 10-40mM acetate buffer, pH about 5.0-6.5; (c) about 10-30mM L-arginine or about 10-30mM L-arginine hydrochloride ; (d) about 130-170 mM sucrose; (e) and about 0.01%-0.1% polysorbate 80.
  • the pharmaceutical compositions described above contain: (a) about 30 mg/mL-150 mg/mL of the humanized anti-BLyS antibody or antigen-binding fragment thereof of any of the embodiments herein; (b) about 5-50 mM Histidine buffer or about 5-50mM acetate buffer, pH about 5.0-6.5; (c) about 10-30mM L-arginine, about 10-30mM L-arginine hydrochloride or about 30-90 mM methionine; (d) about 50-200 mM sodium chloride; (e) about 0.01%-0.1% polysorbate 80.
  • the above pharmaceutical composition comprises: (a) about 30 mg/mL-150 mg/mL of the humanized anti-BLyS antibody or antigen-binding fragment thereof of any of the embodiments herein; (b) about 5- 50 mM histidine buffer, pH about 5.0-6.5; (c) about 10-30 mM L-arginine, about 10-30 mM L-arginine hydrochloride, or about 30-90 mM methylsulfide amino acid; (d) sodium chloride at about 50-200 mM; (e) and polysorbate 80 at about 0.01%-0.1%.
  • the above pharmaceutical composition comprises: (a) about 30 mg/mL-150 mg/mL of the humanized anti-BLyS antibody or antigen-binding fragment thereof of any of the embodiments herein; (b) about 5- 50 mM acetate buffer, pH about 5.0-6.5; (c) about 10-30 mM L-arginine, about 10-30 mM L-arginine hydrochloride, or about 30-90 mM methionine ; (d) sodium chloride at about 50-200 mM; (e) and polysorbate 80 at about 0.01%-0.1%.
  • the pharmaceutical compositions described above contain: (a) about 80 mg/mL-120 mg/mL of the humanized anti-BLyS antibody or antigen-binding fragment thereof of any of the embodiments herein; (b) about 20-40 mM of histidine buffer or about 20-40 mM acetate buffer, pH about 5.5-6.0; (c) about 10-30 mM L-arginine, about 50-70 mM methionine or about 10- 30 mM L-arginine hydrochloride; (d) about 80-120 mM sodium chloride; (e) and about 0.01%-0.05% polysorbate 80.
  • the pharmaceutical compositions described above contain: (a) about 80 mg/mL-120 mg/mL of the humanized anti-BLyS antibody or antigen-binding fragment thereof of any of the embodiments herein; (b) about 20-40 mM Histidine buffer or, pH about 5.5-6.0; (c) about 10-30 mM L-arginine, about 50-70 mM methionine or about 10-30 mM L-arginine salt salt; (d) sodium chloride at about 80-120 mM; (e) and polysorbate 80 at about 0.01%-0.05%.
  • the pharmaceutical compositions described above contain: (a) about 80 mg/mL-120 mg/mL of the humanized anti-BLyS antibody or antigen-binding fragment thereof of any of the embodiments herein; (b) about 20-40 mM (c) about 10-30 mM L-arginine, about 50-70 mM methionine or about 10-30 mM L-arginine hydrochloride; (d) sodium chloride at about 80-120 mM; (e) and polysorbate 80 at about 0.01%-0.05%.
  • the above pharmaceutical compositions contain: (a) about 180 mg/mL-220 mg/mL of the humanized anti-BLyS antibody or antigen-binding fragment thereof described in any one of the herein; (b) about 20-40 mM of Histidine buffer or about 20-40 mM acetate buffer, pH about 5.5-6.0; (c) about 10-30 mM L-arginine or about 10-30 mM L-arginine hydrochloride; (d) about 130-170 mM sucrose; (e) and about 0.01%-0.05% polysorbate 80; or
  • the pharmaceutical composition described above comprises: (a) about 200 mg/mL of the humanized anti-BLyS antibody or antigen-binding fragment thereof of any of the embodiments herein; (b) about 25 mM histidine buffer, About 25 mM acetate buffer or about 35-36 mM histidine buffer, pH about 5.5-6.0; (c) about 15 mM L-arginine or about 15 mM L-arginine hydrochloride; (d) 150 mM sucrose; (e) and about 0.02% polysorbate 80.
  • the pharmaceutical composition described above comprises: (a) about 100 mg/mL of the humanized anti-BLyS antibody or antigen-binding fragment thereof of any of the embodiments herein; (b) about 25 mM histidine buffer, pH about 5.5-6.0; (c) about 25 mM L-arginine or about 25 mM L-arginine hydrochloride; (d) about 100 mM sodium chloride; (e) and about 0.02% poly Sorbitan Ester 80.
  • the pharmaceutical composition described above comprises: (a) about 100 mg/mL of the humanized anti-BLyS antibody or antigen-binding fragment thereof of any of the embodiments herein; (b) about 25 mM acetate buffer, pH about (c) about 60 mM methionine; (d) about 100 mM sodium chloride; (e) and about 0.02% polysorbate 80.
  • the pharmaceutical compositions described above contain: (a) about 100 mg/mL of the humanized anti-BLyS antibody or antigen-binding fragment thereof of any of the embodiments herein; (b) about 35-36 mM histidine buffer solution, pH about 5.5-6.0; (c) about 15 mM L-arginine hydrochloride or about 15 mM L-arginine; (d) about 100 mM sodium chloride; (e) and about 0.02% of polysorbate 80.
  • the above-mentioned pharmaceutical composition comprises the components shown in any one of the following (1)-(20):
  • (9) (a) about 80 mg/mL-120 mg/mL of a humanized anti-BLyS antibody or an antigen-binding fragment thereof comprising a light chain variable region and a heavy chain variable region or an antigen-binding fragment thereof variable region, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 9, and the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 10; (b) about 20-40mM Histidine buffer, pH about 5.5-6.0; (c) about 10-30 mM L-arginine hydrochloride; (d) about 80-120 mM sodium chloride; (e) and about 0.01%- 0.05% polysorbate 80; or
  • (10) (a) about 80 mg/mL-120 mg/mL of a humanized anti-BLyS antibody or an antigen-binding fragment thereof comprising a light chain variable region and a heavy chain variable region or an antigen-binding fragment thereof variable region, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 9, and the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 10; (b) about 20-40mM Acetate buffer, pH about 5.5-6.0; (c) about 50-70 mM methionine; (d) about 80-120 mM sodium chloride; (e) and about 0.01%-0.05% polysorbate Ester 80; or
  • (11) (a) about 180 mg/mL-220 mg/mL of a humanized anti-BLyS antibody or an antigen-binding fragment thereof comprising a light chain variable region and a heavy chain variable region or an antigen-binding fragment thereof variable region, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 9, and the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 10; (b) about 20-40mM Histidine buffer, pH about 5.5-6.0; (c) about 10-30 mM L-arginine or about 10-30 mM L-arginine hydrochloride; (d) about 130-170 mM sucrose ; (e) and about 0.01%-0.05% polysorbate 80; or
  • (16) (a) about 100 mg/mL of a humanized anti-BLyS antibody or antigen-binding fragment thereof comprising a light chain variable region and a heavy chain variable region, wherein The light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 9, and the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 10; (b) about 25mM histidine buffer, (c) about 25 mM L-arginine hydrochloride; (d) about 100 mM sodium chloride; (e) about 0.02% polysorbate 80; or
  • the light chain amino acid sequence of the humanized anti-BLyS antibody is shown in SEQ ID NO: 18, and the heavy chain amino acid sequence is shown in SEQ ID NO: 19.
  • the pharmaceutical composition is a liquid formulation or a lyophilized formulation.
  • the pharmaceutical composition is a liquid formulation.
  • the above liquid formulation or lyophilized formulation is stable at 2-8°C for at least 3 months, at least 6 months, at least 12 months, at least 18 months or at least 24 months.
  • aqueous or lyophilized formulations described above are stable at 40°C for at least 7 days, at least 14 days or at least 28 days.
  • the present invention provides a humanized anti-BLyS antibody or antigen-binding fragment thereof as described in any one of the schemes herein, a polynucleotide described herein, an expression vector described herein, a host cell described herein, a Use of the pharmaceutical composition or the injection described herein in the preparation of a medicament for preventing and/or treating a disease caused by excessive B cell proliferation.
  • the present invention provides a humanized anti-BLyS antibody or antigen-binding fragment thereof as described in any one of the schemes herein, a polynucleotide described herein, an expression vector described herein, a host cell described herein, a The pharmaceutical composition described herein, or the injection described herein, is used for preventing and/or treating diseases caused by excessive B cell proliferation.
  • the present invention provides a method of preventing and/or treating a disease caused by excessive B cell proliferation, comprising administering to a subject in need thereof a humanized anti-BLyS antibody or an antigen thereof as described in any one of the schemes herein Binding fragments, polynucleotides described herein, expression vectors described herein, host cells described herein, pharmaceutical compositions described herein, or injectables described herein.
  • diseases caused by excessive proliferation of B cells include systemic lupus erythematosus, rheumatoid arthritis, septic arthritis or B cell lymphoma.
  • SEC-HPLC Size exclusion chromatography
  • CEX-HPLC cation exchange chromatography
  • Stability was assessed by the following parameters: (1) visual appearance and visible foreign matter; (2) UV spectrophotometric determination of protein content; (3) SEC-HPLC measurement of antibody monomer, aggregate or fragment content; (4) CEX -HPLC to measure the main charge, acidic charge or basic charge of the antibody; (5) NR-CE-SDS method to detect the molecular weight of the antibody; (6) R-CE-SDS method to detect the molecular weight of the antibody; (7) ELISA method to detect the antibody binding activity.
  • the protein concentration was detected using a UV spectrophotometer (Thermo, model: Biomate 3S).
  • the percent extinction coefficient (E1%) was set at 1.47 (g/mL)-1 cm-1.
  • BIO MATE 3S instrument wash the cuvette with ultrapure water three times, then add 150 ⁇ L of ultrapure water to the cuvette, click to measure, and use ultrapure water for blank correction. Two solutions were measured in parallel for each sample, and each solution was repeated three times; before each solution was measured, the cuvette was rinsed twice with 150 ⁇ L of solution, and then 150 ⁇ L of solution was taken for measurement. The concentration of the corresponding sample was calculated from the extinction coefficient and OD value.
  • K0 is the peak with the highest peak area percentage
  • K1 is the first peak after K0
  • the retention time difference between K2 and K1 value is about 2.5min
  • the sum of the peak area percentages of the three peaks K0, K1 and K2 is the lysine variant content (that is, the sample purity)
  • the sum of all peak area percentages before the K0 peak is the acidic peak content
  • the K0 peak, K1 The sum of the area percentages of all peaks remaining after the K2 peak is the basic peak content. See Table 2 below for details:
  • test product Place the test product at the edge of the visor, gently rotate and flip the container to suspend visible foreign matter that may exist in the liquid (do not make the liquid bubble), at about 25 cm, on black and white backgrounds, respectively Observe, check the time limit for 20 seconds.
  • the framework region ( FR) was mutated, and the following amino acid sequences were screened out: the FR region of the light chain L47 (SEQ ID NO: 16) was mutated to generate L472 (SEQ ID NO: 18), L473 (SEQ ID NO: 20) and L474 ( SEQ ID NO: 22) three sequences, the FR region of heavy chain H48 (SEQ ID NO: 17) was mutated to generate H482 (SEQ ID NO: 19), H483 (SEQ ID NO: 21), H484 (SEQ ID NO: 21) NO: 23) and H485 (SEQ ID NO: 24) four sequences.
  • Example 2 Binding ELISA method to determine the binding ability of antibody to human BLyS protein
  • Antibody name EC50(ng/mL) H48L47 twenty one H48L472 22.78 H48L473 22.76 H48L474 17.26 H482L47 14.67 H482L472 17.54 H482L473 11.9 H482L474 11.55 H483L47 11.86 H483L472 13.82 H483L473 14.43 H483L474 15.68 H484L47 19.02
  • H484L472 24.7 H484L473 15.37 H484L474 10.81 H485L47 7.588 H485L472 7.25 H485L473 11.73 H485L474 7.685
  • Example 3 Blocking ELISA method to determine the ability of antibodies to competitively inhibit the binding of human BLyS to human BR3
  • Binding ELISA show that the EC50 of the antibody modified by the framework region (FR) sequence is smaller than the EC50 of the unmodified antibody, so only the combination with modified light and heavy chains, namely H482L472, H483L473 and H484L474, was selected for Blocking ELISA. And compared with the marketed positive control antibody Benlysta. The test results are shown in Table 6 below.
  • Example 4 Reporter gene assay for antibody competitive inhibition of human BLyS binding to TACI-stimulated downstream NF- ⁇ B signaling
  • Antibody name IC50 (ng/mL) H482L472 37.56 H483L473 29.27 H484L474 31.52 Benlysta 51.68
  • the buffer system and pH closely affect the stability of the antibody, and each antibody with unique physicochemical properties has the most suitable buffer type and pH.
  • the purpose of this example is to screen an optimal buffer system and pH so that the antibody disclosed in the present invention has the best stability for clinical application.
  • the antibody in this example is H482L472.
  • Centrifugal ultrafiltration was performed with a 15 mL centrifugal ultrafiltration tube (Millipore, UCF803096) (5000 g ⁇ 15 min). Each group of samples 1mL protein, with 2mL buffer exchange 3 times. After the liquid exchange was completed, the concentration was measured with a spectrophotometer, and the final concentration of the preparation was adjusted to an antibody concentration of about 30 mg/mL.
  • the content of histidine buffer is 35mM; the content of acetate buffer is 25mM, the antibody in this example is H482L472, the final concentration of the antibody in each formulation is 100mg/mL, and the concentration of sodium chloride in each formulation is 100mM.
  • the sterilized and filtered samples were incubated in a 37°C constant temperature incubator for 7 days. Detect SEC, CEX-HPLC.
  • the pH of the formulation has a significant effect on stability regardless of histidine or acetic acid.
  • the purity first increased and then decreased, and adding L-arginine or methionine into the formulation could effectively reduce the production of aggregates.
  • two groups of formulations were screened out: 35mM histidine+15mM L-arginine+100mM sodium chloride, pH5.8; 25mM acetic acid+60mM methionine+100mM sodium chloride, pH5.8 .
  • Embodiment 8 Compatibility stability research experiment
  • 500 mL of 0.9% physiological saline was prepared with ultrapure water, stirred with a magnetic stirrer until completely dissolved, and used for later use.
  • 500 mL of 0.9% physiological saline was prepared with ultrapure water, stirred with a magnetic stirrer until completely dissolved, and used for later use.
  • the preparation sample (preparation formula: 100 mg/mL antibody + 35 mM histidine buffer + 15 mM L-arginine hydrochloride + 100 mM sodium chloride + 0.02% Tween- 80, pH 5.8) to the corresponding final concentration (60, 30, 10, 3 mg/mL), the volume of the diluted sample is about 25 mL, and the sample is fully shaken for later use; the antibody in this example is H482L472.
  • the diluted samples were sterilized and filtered in the ultra-clean workbench, and then aseptically packaged into 6 mL vials. Each concentration sample was packaged into 5 vials, each with a volume of 5 mL, and then capped with a capping machine.
  • samples of each group were incubated at 25°C and 30°C for 0, 2, and 4 hours, respectively, and the samples were stored at -80°C. After being frozen at -80°C for 5 days, the samples were sent for inspection, and the samples were tested for SEC purity, biological activity (blocking ELISA) and insoluble particles.
  • the compatibility stability experiment showed that the final formulation was diluted with 0.9% sodium chloride solution, and the properties of the sample remained stable within 4 hours at room temperature within the range of protein concentration of 3-60 mg/ml.
  • the optimal formulation was finally determined by investigating different buffer systems, different pH conditions, different antibody concentrations and different excipient compositions. Histidine buffer was used to adjust pH, arginine hydrochloride and sodium chloride to adjust formulation osmotic pressure, and polysorbate 20 was added to increase formulation solubility.
  • Example 9 Inhibitory effect of UBP1213sc preparation on human BLyS-induced changes in Balb/c mice physiological indicators
  • Day1 and Day3 administration a total of 2 administrations.
  • 0.3 mg/kg of human BLyS was administered subcutaneously on Day 1 to Day 4. During the experiment, the body weight of the animals was checked every day.
  • the results of the Vehicle group showed that 0.3 mg/kg of human BLyS (subcutaneous injection) successfully induced a significant increase in the wet weight of the spleen/spleen organ coefficient, the ratio of spleen B220+/ThB+ cells, and the level of serum IgA in Balb/c mice.
  • it can significantly inhibit the increase of various physiological indicators in model mice; 1.5mg/kg subcutaneous UBP1213 can significantly inhibit the increase of various physiological indicators in model mice; 0.5, 1.5 and 5 mg/kg UBP1213sc can significantly inhibit model mice. The increase of various physiological indicators.
  • Subcutaneous injection of human BLyS can induce significant increase in spleen wet weight/spleen organ coefficient, spleen B220+/ThB+ cell ratio and serum IgA level in Balb/c mice.
  • Subcutaneous injection of different doses (0.15, 0.5, 1.5, 5 mg/kg) of anti-BLyS monoclonal antibody UBP1213sc, 1.5 and 5 mg/kg UBP1213sc can significantly inhibit the increase of spleen wet weight/spleen organ coefficient in mice, 0.5, 1.5 And 5mg/kg UBP1213sc could significantly inhibit the increase of B220+/ThB+ cell ratio and serum IgA level, and showed a good dose-response relationship. There was no significant change in the body weight of animals in each administration group.

Abstract

提供抗BLyS抗体、其药物组合物及其用途。抗BLyS抗体或其抗原结合片段包含重链可变区和轻链可变区，且其LCDR1、LCDR2和LCDR3的氨基酸序列分别如SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3所示，HCDR1、HCDR2和HCDR3的氨基酸序列分别如SEQ ID NO:4、SEQ ID NO:5和SEQ ID NO:6所示。抗BLyS抗体或其抗原结合片段具有优异的人BLyS蛋白的结合能力和竞争抑制人BLyS与其受体人BR3结合的能力。药物组合物是一种含有抗BLyS抗体和缓冲液的高稳定性药物组合物，还可含有至少一种稳定剂和任选的表面活性剂。

Description

抗BLyS抗体、其药物组合物及其用途 技术领域
本发明属于生物医药领域，涉及一种抗BLyS抗体、其药物组合物及其用途，所述抗BLyS抗体具有优异的人BLyS蛋白的结合能力和竞争抑制人BLyS与其受体人BR3结合的能力。
背景技术
系统性红斑狼疮(systemic lupus erythematosus，SLE)是一种累及全身多个系统如皮肤、关节、心脏、肺、肾、血液以及大脑的弥慢性进行性、常以缓解和复发交替出现的自身免疫性疾病。系统性红斑狼疮主要影响非洲-加勒比人、亚洲人和西班牙裔人，而高加索人(白种人)受其影响的程度则较小。美国狼疮基金会认为，保守预测系统性红斑狼疮在美国影响到30万人。而Datamonitor公司估计，仅在中国、印度和墨西哥三国就有220万系统性红斑狼疮患者，其中中国系统性红斑狼疮患者超过100万，为全球之最。由于系统性红斑狼疮初期症状比较隐蔽，因此实际病患人数可能远大于目前估计。由此可见临床在系统性红斑狼疮的诊断和治疗方面均存在极大的客观需求。
系统性红斑狼疮的成因复杂且不明确，并非单一因素引起，可能与遗传、环境、性激素及免疫等多种因素有关。目前，世界科学界公认的系统性红斑狼疮的病因是由于在细胞水平上，自反应B细胞在外周组织中存在时间过长，产生人体自身抗原而造成自身免疫。所以如果可以抑制早期B细胞的生长增殖，则可以治疗红斑狼疮疾病。
B淋巴细胞刺激因子(B Lymphocyte Stimulator，BLyS),又称Tall-1(TNF and Apol related leukocyte expressed ligand 1)、BAFF(B cell activating factor belonging to the TNF family)、THANK(TNF homologues that activate apoptosis,NF-κB and JNK)，属肿瘤坏死因子(TNF)家族，是由舒红兵等人于1999年首先发现并克隆的一种新的细胞因子。BLyS作为一种B淋巴细胞的共刺激因子，在anti-IgM和IL-4存在下，它可以专一刺激B细胞增殖和分化，在体液免疫中起着极其重要作用；而其在体内的过量表达又与自身免疫性疾病密切相关。
体外实验表明，在用IgM预活化B细胞后，BLyS能诱导其大量增殖并分泌大量IgM和IgA，但是，对于静息期间B细胞，这种刺激作用却不明显(3)。进一步的研究表明，BLyS主要作用于前B淋巴细胞、未成熟B淋巴细胞、激活淋巴细胞，而对浆细胞、淋巴 多功能干细胞没有作用。同大多数细胞因子相同，BLyS主要是通过B细胞表面受体而刺激下游信号传导。多个研究小组证实与BLyS结合的受体是：B细胞活化因子受体(BR3、BLyS receptor 3或BAFF-R)、穿膜蛋白活化物(transmembrane activator–1 and calcium modulator and cyclophilin ligand–interactor，TACI)和B细胞成熟抗原(B cell maturation Antigen，BCMA)。这种特异性决定了BLyS是针对B细胞抗体介导的自身免疫疾病和淋巴癌中一个很好的标靶。
抗BLyS的治疗性抗体在体内和体外已经被证明可以有效抑制B细胞生长、IgA和IgM分泌从而达到治疗系统性红斑狼疮的效果(Edwards BM等，The remarkable flexibility of the human antibody repertoire；isolation of over one thousand different antibodies to a single protein,BLyS.J Mol Biol.2003 Nov 14；334(1):103-18；Baker KP等，Generation and characterization of LymphoStat-B,a human monoclonal antibody that antagonizes the bioactivities of B lymphocyte stimulator.Arthritis Rheum.2003 Nov；48(11):3253-65)。美国人类基因组公司开发的抗BLyS抗体Benlysta成为过去60年来世界首个治疗红斑狼疮的新型药物。Benlysta只针对BLyS刺激的B细胞，与化疗药物相比，大大降低了治疗过程中的副作用，因此为系统性红斑狼疮患者提供了一个安全有效的治疗方法。近年来针对BLyS的靶向治疗研究与临床应用迅速发展，除美国人类基因组公司外，其他公司均是采用在BLyS或其受体基础上改造的融合蛋白。美国基因泰克(Genentech)公司开发了BR3-FC药物、Zymogenetics公司开发了TACI-FC药物、安晋(AMGEN)公司开发了多肽-FC药物。与Benlysta相比，它们的特异性差，结合力弱，疗效相对较差，毒性较强。这三种药物均停止或终止在临床实验二期。所以，抗BLyS的抗体药物才是针对此靶点的有效药物方法。
发明内容
本发明提供的抗BLyS抗体具有优异的人BLyS蛋白的结合能力和竞争抑制人BLyS与其受体人BR3结合的能力。药物组合物是一种含有抗BLyS抗体的高稳定性药物组合物，特别地，本发明发现该人源化抗BLyS抗体在组氨酸缓冲液体系和L-精氨酸、L-精氨酸盐酸盐或甲硫氨酸的组合中具有出人意料的特征，即具有高稳定性。
本发明提供了一种人源化抗BLyS抗体或其抗原结合片段，其包含重链可变区和轻链可变区，其中：
该人源化抗BLyS抗体或其抗原结合片段的LCDR1、LCDR2和LCDR3的氨基酸序列分别如SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3所示；
该人源化抗BLyS抗体或其抗原结合片段的HCDR1、HCDR2和HCDR3的氨基酸序列分别如SEQ ID NO:4、SEQ ID NO:5和SEQ ID NO:6所示；
该人源化抗BLyS抗体或其抗原结合片段的轻链可变区的FR1选自SEQ ID NO:7、9和13中任一所述的FR1，FR2为SEQ ID NO:7所述的FR2，FR3选自SEQ ID NO:7、9和11中任一所述的FR3，FR4为SEQ ID NO:7所述的FR4；
该人源化抗BLyS抗体或其抗原结合片段的重链可变区的FR1选自SEQ ID NO:8、10、12和15中任一所述的FR1，FR2选自SEQ ID NO:8、10和15中任一所述的FR2，FR3选自SEQ ID NO:8、12和14中任一所述的FR3，FR4为SEQ ID NO:8或10所述的FR4；且
该人源化抗BLyS抗体或其抗原结合片段不包括：轻链可变区为SEQ ID NO:7所示的氨基酸序列，同时重链可变区为SEQ ID NO:8所示的氨基酸序列的人源化抗BLyS抗体或其抗原结合片段。
在一些方案中，上述人源化抗BLyS抗体或其抗原结合片段中：
轻链可变区的FR1选自SEQ ID NO:7、9和13中任一所述的FR1，FR2为SEQ ID NO:7所述的FR2，FR3选自SEQ ID NO:7、9和11中任一所述的FR3，FR4为SEQ ID NO:7所述的FR4；重链可变区的FR1-FR4分别为SEQ ID NO:15的FR1-FR4；或
轻链可变区的FR1选自SEQ ID NO:7、9和13中任一所述的FR1，FR2为SEQ ID NO:7所述的FR2，FR3选自SEQ ID NO:7、9和11中任一所述的FR3，FR4为SEQ ID NO:7所述的FR4；重链可变区的FR1-FR4分别为SEQ ID NO:10的FR1-FR4；或
轻链可变区的FR1选自SEQ ID NO:7、9和13中任一所述的FR1，FR2为SEQ ID NO:7所述的FR2，FR3选自SEQ ID NO:7、9和11中任一所述的FR3，FR4为SEQ ID NO:7所述的FR4；重链可变区的FR1-FR4分别为SEQ ID NO:12的FR1-FR4；或
轻链可变区的FR1为SEQ ID NO:11或13所述的FR1，FR2为SEQ ID NO:11所述的FR2，FR3为SEQ ID NO:11或13所述的FR3，FR4为SEQ ID NO:11所述的FR4；重链可变区的FR1-FR4分别为SEQ ID NO:14的FR1-FR4。
在一些方案中，上述人源化抗BLyS抗体或其抗原结合片段包含轻链可变区和重链可变区，其中，所述轻链可变区包含如SEQ ID NO：7、SEQ ID NO：9、SEQ ID NO：11或SEQ ID NO：13所示的氨基酸序列，所述重链可变区包含如SEQ ID NO：8、SEQ ID NO：10、SEQ ID NO：12、SEQ ID NO：14或SEQ ID NO：15所示的氨基酸序列。
在一些方案中，上述人源化抗BLyS抗体或其抗原结合片段包含轻链可变区和重链可变区，其中，所述轻链可变区和重链可变区选自：
(1)所述轻链可变区包含如SEQ ID NO：7所示的氨基酸序列，和所述重链可变区包含如SEQ ID NO：10、SEQ ID NO：12、SEQ ID NO：14或SEQ ID NO：15所示的氨基酸序列；或
(2)所述轻链可变区包含如SEQ ID NO：9所示的氨基酸序列，和所述重链可变区包含如SEQ ID NO：8、SEQ ID NO：10、SEQ ID NO：12、SEQ ID NO：14或SEQ ID NO：15所示的氨基酸序列；或
(3)所述轻链可变区包含如SEQ ID NO：11所示的氨基酸序列，和所述重链可变区包含如SEQ ID NO：8、SEQ ID NO：10、SEQ ID NO：12、SEQ ID NO：14或SEQ ID NO：15所示的氨基酸序列；或
(4)所述轻链可变区包含如SEQ ID NO：13所示的氨基酸序列，和所述重链可变区包含如SEQ ID NO：8、SEQ ID NO：10、SEQ ID NO：12、SEQ ID NO：14或SEQ ID NO：15所示的氨基酸序列；
优选地，所述轻链可变区和重链可变区选自：
(5)所述轻链可变区包含如SEQ ID NO：9所示的氨基酸序列，和所述重链可变区包含如SEQ ID NO：10所示的氨基酸序列；或
(6)所述轻链可变区包含如SEQ ID NO：11所示的氨基酸序列，和所述重链可变区包含如SEQ ID NO：12所示的氨基酸序列；或
(7)所述轻链可变区包含如SEQ ID NO：13所示的氨基酸序列，和所述重链可变区包含如SEQ ID NO：14所示的氨基酸序列；或
(8)所述轻链可变区包含如SEQ ID NO：7所示的氨基酸序列，和所述重链可变区包含如SEQ ID NO：15所示的氨基酸序列；或
(9)所述轻链可变区包含如SEQ ID NO：9所示的氨基酸序列，和所述重链可变区包含如SEQ ID NO：15所示的氨基酸序列；或
(10)所述轻链可变区包含如SEQ ID NO：11所示的氨基酸序列，和所述重链可变区包含如SEQ ID NO：15所示的氨基酸序列；或
(11)所述轻链可变区包含如SEQ ID NO：13所示的氨基酸序列，和所述重链可变区包含如SEQ ID NO：15所示的氨基酸序列。
本发明所述人源化抗BLyS抗体还含有人类轻链恒定区和重链恒定区，且所述轻链可变区及重链可变区分别连接至人类轻链恒定区和重链恒定区。即所述人源化抗BLyS抗体含有完整的轻链和完整的重链，其中所述完整的轻链由抗BLyS抗体所含有的轻链可变区与人类轻链恒定区连接而成，所述完整的重链由抗BLyS抗体所含有的重链可变区与人类重链恒定区连接而成。
在一些方案中，所述人类轻链恒定区为人类轻链κ恒定区。
在一些方案中，所述人类重链恒定区为人类重链Fc片段。
其中，所述人类轻链κ恒定区和人类重链Fc片段，均来源于健康人B淋巴细胞。通 过基因工程技术，重叠延伸PCR连接可变区和恒定区得到完整的人源化抗BLyS抗体的轻链和重链。
在一些方案中，上述人源化抗BLyS抗体或其抗原结合片段包含轻链和重链，其中，所述轻链包含如SEQ ID NO：16、SEQ ID NO：18、SEQ ID NO：20或SEQ ID NO：22所示的氨基酸序列，所述重链包含如SEQ ID NO：17、SEQ ID NO：19、SEQ ID NO：21、SEQ ID NO：23或SEQ ID NO：24所示的氨基酸序列。
在一些方案中，上述人源化抗BLyS抗体或其抗原结合片段包含轻链和重链，其中：
(1)所述轻链包含如SEQ ID NO：16所示的氨基酸序列，和所述重链包含如SEQ ID NO：19、SEQ ID NO：21、SEQ ID NO：23或SEQ ID NO：24所示的氨基酸序列；或
(2)所述轻链包含如SEQ ID NO：18所示的氨基酸序列，和所述重链包含如SEQ ID NO：17、SEQ ID NO：19、SEQ ID NO：21、SEQ ID NO：23或SEQ ID NO：24所示的氨基酸序列；或
(3)所述轻链包含如SEQ ID NO：20所示的氨基酸序列，和所述重链包含如SEQ ID NO：17、SEQ ID NO：19、SEQ ID NO：21、SEQ ID NO：23或SEQ ID NO：24所示的氨基酸序列；或
(4)所述轻链包含如SEQ ID NO：22所示的氨基酸序列，和所述重链包含如SEQ ID NO：17、SEQ ID NO：19、SEQ ID NO：21、SEQ ID NO：23或SEQ ID NO：24所示的氨基酸序列；
优选地：
(5)所述轻链包含如SEQ ID NO：18所示的氨基酸序列，和所述重链包含如SEQ ID NO：19所示的氨基酸序列；或
(6)所述轻链包含如SEQ ID NO：20所示的氨基酸序列，和所述重链包含如SEQ ID NO：21所示的氨基酸序列；或
(7)所述轻链包含如SEQ ID NO：22所示的氨基酸序列，和所述重链包含如SEQ ID NO：23所示的氨基酸序列；或
(8)所述轻链包含如SEQ ID NO：16所示的氨基酸序列，和所述重链包含如SEQ ID NO：24所示的氨基酸序列；或
(9)所述轻链包含如SEQ ID NO：18所示的氨基酸序列，和所述重链包含如SEQ ID NO：24所示的氨基酸序列；或
(10)所述轻链包含如SEQ ID NO：20所示的氨基酸序列，和所述重链包含如SEQ ID NO：24所示的氨基酸序列；或
(11)所述轻链包含如SEQ ID NO：22所示的氨基酸序列，和所述重链包含如SEQ ID NO：24所示的氨基酸序列。
在一些方案中，本发明所述抗原结合片段选自Fab、Fab'、Fab'-SH、Fv、scFv、F(ab')2、sdAb或双抗体。
在又一个方面，本发明提供了多核苷酸分子，选自：编码如本文任一项方案中所述的人源化抗BLyS抗体或其抗原结合片段的多核苷酸分子或其互补序列。
在又一个方面，本发明提供了表达载体，其包含如本文所述的多核苷酸分子，优选地，所述表达载体为真核表达载体。
在又一个方面，本发明提供了宿主细胞，其包含如本文所述的多核苷酸分子或表达载体，优选地，所述宿主细胞是真核细胞，更优选是哺乳动物细胞。
在又一个方面，本发明提供了制备如本文任一项方案中所述的人源化抗BLyS抗体或其抗原结合片段的方法，所述方法包括在适合于所述抗体或其抗原结合片段表达的条件下培养如本文所述的宿主细胞，以使其表达所述抗体或其抗原结合片段，并回收所表达的抗体或其抗原结合片段。
在又一个方面，本发明提供了一种药物组合物，其包含如本文任一项方案中人源化抗BLyS抗体或其抗原结合片段、本文所述的多核苷酸、本文所述的表达载体或本文所述的宿主细胞，和药学上可接受的载体或赋形剂。
在一些方案中，本发明提供了一种药物组合物，包含：(1)缓冲液；(2)人源化抗BLyS抗体或其抗原结合片段，所述人源化抗BLyS抗体或其抗原结合片段具有氨基酸序列分别如SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3所示的LCDR1、LCDR2和LCDR3，和氨基酸序列分别如SEQ ID NO:4、SEQ ID NO:5和SEQ ID NO:6所示的HCDR1、HCDR2和HCDR3。
在一些方案中，本发明提供了一种药物组合物，包含：(1)缓冲液；(2)人源化抗BLyS抗体或其抗原结合片段；所述人源化抗BLyS抗体或其抗原结合片段如本文任一项方案中所述的人源化抗BLyS抗体或其抗原结合片段。
在一些方案中，上述药物组合物中人源化抗BLyS抗体或其抗原结合片段的浓度约为1-300mg/mL，优选约为10-300mg/mL，更优选约为20-280mg/mL，更优选约为30-150mg/mL，更优选约为80-120mg/mL，更优选约为180-220mg/mL。
在一些方案中，上述缓冲液选自醋酸缓冲液、柠檬酸缓冲液、琥珀酸缓冲液、磷酸缓冲液和组氨酸缓冲液中的一种或多种。
在一些方案中，上述缓冲液的浓度约为1-200mM，优选约为1-100mM，优选约为5-50mM，优选约为10-40mM，优选约为20-40mM。
在一些方案中，上述缓冲液的pH约为5.0-6.5，优选约为5.0-6.0，优选约为优选约为5.5-6.0。
在一些方案中，上述的药物组合物还包括稳定剂。
在一些方案中，所述稳定剂选自精氨酸、精氨酸盐酸盐、甲硫氨酸、脯氨酸、甘氨酸、氯化钠、甘露醇、山梨醇、蔗糖、麦芽糖、木糖醇和海藻糖中的一种或多种。
在一些方案中，上述稳定剂选自L-精氨酸、L-精氨酸盐酸盐、蔗糖、甲硫氨酸和氯化钠中的一种或多种。
在一些方案中，上述稳定剂的浓度为约10mM～400mM，优选约50mM～300mM，更优选约100mM～300mM，更优选约100mM～200mM。
在一些方案中，上述稳定剂为浓度约10-30mM的L-精氨酸盐酸盐与约50-200mM的氯化钠的组合；或所述稳定剂为浓度约10-30mML-精氨酸与约50-200mM的氯化钠的组合；或所述稳定剂为浓度约10-30mM的L-精氨酸与约80-220mM的蔗糖的组合；或所述稳定剂为浓度约10-30mM的L-精氨酸盐酸盐与约80-220mM的蔗糖的组合；或所述稳定剂为约30-90mM的甲硫氨酸与约50-200mM的氯化钠的组合；或所述稳定剂为约10-120mM的L-精氨酸盐酸盐；或所述稳定剂为约10-120mM的甲硫氨酸；优选地，所述稳定剂为浓度约10-30mM的L-精氨酸盐酸盐与约80-120mM的氯化钠的组合；或所述稳定剂为浓度约10-30mM的L-精氨酸与约80-120mM的氯化钠的组合；或所述稳定剂为约50-70mM的甲硫氨酸与约80-120mM的氯化钠的组合；或所述稳定剂为浓度约10-30mM的L-精氨酸与约130-170mM的蔗糖的组合；或所述稳定剂为浓度约10-30mM的L-精氨酸盐酸盐与约130-170mM的蔗糖的组合。
在一些方案中，上述药物组合物还包括表面活性剂，优选地，所述表面活性剂选自聚山梨醇酯80、聚山梨醇酯20、泊洛沙姆188中的一种或多种。
在一些方案中，以w/v计算，上述表面活性剂浓度约为0.001％-0.1％，优选约为0.01％-0.1％，优选约为0.01％-0.05％。
在一些方案中，上述药物组合物还包括注射用水。
在一些方案中，所述药物组合物为液体制剂或冻干制剂。
在一些方案中，所述药物组合物为液体制剂。
在又一个方面，本发明提供了一种注射剂，其含有本文任一项方案中所述的药物组合物与氯化钠溶液；优选地，所述氯化钠溶液浓度为0.85～0.9％；优选地，所述注射剂中，所述人源化抗BLyS抗体的浓度为3～100mg/mL，更优选为约3～60mg/mL；优选地，所述注射剂的pH为5.5～6.0。
在一些方案中，所述的药物组合物或注射剂，其经皮下注射施用。
在又一个方面，本发明提供了如本文任一项方案中所述的人源化抗BLyS抗体或其抗原结合片段、本文所述的多核苷酸分子、本文所述的表达载体、本文所述的宿主细胞、本文所述的药物组合物、或本文所述的注射剂在制备预防和/或治疗B细胞增殖过量引起的疾病的药物中的应用。
在又一个方面，本发明提供了如本文任一项方案中所述的人源化抗BLyS抗体或其抗原结合片段、本文所述的多核苷酸分子、本文所述的表达载体、本文所述的宿主细胞、本文所述的药物组合物、或本文所述的注射剂，其用于预防和/或治疗B细胞增殖过量引起的疾病。
在又一个方面，本发明提供了一种预防和/或治疗B细胞增殖过量引起的疾病的方法，其包括向有需要的受试者施用如本文任一项方案中所述的人源化抗BLyS抗体或其抗原结合片段、本文所述的多核苷酸分子、本文所述的表达载体、本文所述的宿主细胞、本文所述的药物组合物、或本文所述的注射剂。
优选地，上述B细胞增殖过量引起的疾病为系统性红斑狼疮、类风湿性关节炎、强制性关节炎或B细胞淋巴癌。
在又一个方面，本发明提供了一种药物组合，其包含如本文任一项方案中所述的人源化抗BLyS抗体或其抗原结合片段、本文所述的多核苷酸分子、本文所述的表达载体、本文所述的宿主细胞、本文所述的药物组合物、或本文所述的注射剂，以及一种或多种另外的治疗剂。
在又一个方面，本发明提供了一种试剂盒，其包括如本文任一项方案中所述的人源化抗BLyS抗体或其抗原结合片段、本文所述的多核苷酸分子、本文所述的表达载体、本文所述的宿主细胞、本文所述的药物组合物、或本文所述的注射剂，优选其进一步包括给药装置。
在又一个方面，本发明提供了组氨酸缓冲液和选自L-精氨酸、L-精氨酸盐酸盐、甲硫氨酸和氯化钠中的一种或多种稳定剂以及任选的表面活性剂(优选聚山梨醇酯80)在提高人源化抗BLyS抗体或其抗原结合片段的药物制剂的稳定性中的应用，或在制备稳定性提高的人源化抗BLyS抗体或其抗原结合片段的药物制剂中的应用。优选地，所述组氨酸缓冲液、稳定剂以及表面活性剂及其用量如本文任一实施方案所述；所述稳定性提高如本文任一实施方案所述；所述人源化抗BLyS抗体或其抗原结合片段如本文任一实施方案所述。
具体实施方式
定义和说明
为了更容易理解本发明，以下具体定义了某些技术和科学术语。除非在本文中另有明确定义，本文使用的所有其它技术和科学术语都具有本发明所属领域的一般技术人员通常理解的含义。应理解本发明不限于具体的方法、试剂、化合物、组合物或生物系统，当然可以对以上进行变化。还应理解本申请所用术语仅为了描述具体的实施方式，并不旨在进行限制。
除非该内容被另外明确说明，否则本说明书以及所附权利要求中所用的单数形式"一个"、"一种"和"该"包括复数指代。因此，例如，提及"一种多肽"包括了两种或更多种多肽等的组合。
术语"药物组合物"或“制剂”表示含有一种或多种本文所述抗体与其他组分的混合物，所述其他组分例如生理学可药用的载体和赋形剂。药物组合物的目的是促进对生物体的给药，利于活性成分的吸收进而发挥生物活性。
术语“液体制剂”是指处于液体状态下的制剂，且不意图指称重悬浮的冻干制剂。本发明的液体制剂在储存时稳定，并且其稳定性不依赖于冻干(或其他状态改变方法，例如喷雾干燥)。
术语“水性液体制剂”是指使用水作为溶剂的液体制剂。在一些方案中，水性液体制剂是不需冻干、喷雾干燥和/或冷冻来维持稳定性(例如化学和/或物理稳定性和/或生物活性)的制剂。
术语“赋形剂”是指可以向制剂添加以提供所需特性(例如稠度、提高的稳定性)和/或调节渗透压的试剂。常用赋形剂的实例包括但不限于糖类、多元醇、氨基酸、表面活性剂和聚合物。
本申请所用的"约"在指代可测量数值(如量、持续时间等)时意在涵盖相对于具体数值±20％或±10％的变化，包括±5％、±1％和±0.1％，因为这些变化适于进行所公开的方法。
术语"缓冲液pH约为5.0-6.5"是指这样的试剂，通过其酸/碱共轭组分的作用使得包含该试剂的溶液能抵抗pH变化。本发明的制剂中使用的缓冲液可具有约5.0至约6.5范围内的pH、或约5.5至约6.5范围内的pH、或约5.0至约6.0范围内的pH。
在本文中，将pH控制在该范围内的“缓冲液”实例包括琥珀酸、琥珀酸盐(例如琥珀酸钠)、葡萄糖酸、组氨酸、组氨酸盐酸盐、甲硫氨酸、柠檬酸、柠檬酸盐、磷酸、磷酸盐、柠檬酸盐/磷酸盐、咪唑、醋酸、醋酸盐、其组合和其他有机酸缓冲剂。
"组氨酸缓冲液"为包含组氨酸离子的缓冲液。组氨酸缓冲液的实例包括组氨酸和组氨酸的盐，如组氨酸盐酸盐、组氨酸乙酸盐、组氨酸磷酸盐和组氨酸硫酸盐等，如含有组氨酸与组氨酸盐酸盐的组氨酸缓冲液；本发明的组氨酸缓冲液也包括含有组氨酸和醋酸盐(如钠盐或钾盐)的组氨酸缓冲液。
“柠檬酸缓冲液”是包括柠檬酸根离子的缓冲液。柠檬酸盐缓冲液的实例包括柠檬酸-柠檬酸纳、柠檬酸-柠檬酸钾、柠檬酸-柠檬酸钙、柠檬酸-柠檬酸镁等。优选地柠檬酸盐缓冲液为柠檬酸-柠檬酸纳缓冲液。
“醋酸缓冲液”是包括醋酸根离子的缓冲液。醋酸盐缓冲液的实例包括醋酸-醋酸纳、醋酸-醋酸钾、醋酸-醋酸钙、醋酸-醋酸镁等。优选地醋酸盐缓冲液为醋酸-醋酸纳缓冲液。
“琥珀酸缓冲液”是包括琥珀酸根离子的缓冲液。琥珀盐缓冲液的实例包括琥珀酸-琥珀酸纳、琥珀-琥珀酸钾、琥珀酸-琥珀酸钙、琥珀酸-琥珀酸镁等。优选地琥珀酸盐缓冲液为琥珀-琥珀酸纳缓冲液。
“磷酸缓冲液”是包括磷酸根离子的缓冲液。磷酸盐缓冲液的实例包括磷酸氢二钠-磷酸二氢钠、磷酸氢二钾-磷酸二氢钾等。优选地磷酸盐缓冲液为磷酸氢二钠-磷酸二氢钠。
术语“稳定剂”表示药学上可接受的赋形剂，其在制造，储存和应用过程中保护活性药物成分和/或制剂免受化学和/或物理降解。稳定剂包括但不限于如以下定义的糖，氨基酸，盐，多元醇和他们的代谢产物，例如氯化钠、氯化钙、氯化镁、甘露醇、山梨醇、蔗糖、海藻糖、甲硫氨酸、L-精氨酸或其盐(如L-精氨酸盐酸盐)、甘氨酸、丙氨酸(α-丙氨酸、β-丙氨酸)、甜菜碱、亮氨酸、赖氨酸、谷氨酸、天冬氨酸、脯氨酸、4-羟基脯氨酸、肌氨酸、γ-氨基丁酸(GABA)、奥品类(opines)、丙氨奥品、章鱼碱、甘氨奥品(strombine))和三甲胺的N-氧化物(TMAO)、人血清白蛋白(hsa)、牛血清白蛋白(bsa)、α-酪蛋白、球蛋白、α-乳白蛋白、LDH、溶菌酶、肌红蛋白、卵清蛋白和RNAaseA。部分稳定剂，如氯化钠、氯化钙、氯化镁、甘露醇、山梨醇、蔗糖等也可起到控制渗透压的作用。在本发明中具体地使用的稳定剂选自多元醇、氨基酸、盐、糖中的一种或多种。优选地糖为蔗糖和海藻糖，优选地多元醇为甘露醇。优选地氨基酸为L-精氨酸或其盐(如L-精氨酸盐酸盐)、甲硫氨酸、甘氨酸、脯氨酸。优选地稳定剂为氯化钠、甘露醇、山梨醇、蔗糖、海藻糖、甲硫氨酸、L-精氨酸、L-精氨酸盐酸盐、甘氨酸、脯氨酸、氯化钠-L-精氨酸、氯化钠-L-精氨酸盐酸盐、氯化钠-甲硫氨酸、氯化钠-山梨醇、氯化钠-甘露醇、氯化钠-蔗糖、氯化钠-海藻糖、L-精氨酸盐酸盐-甘露醇、L-精氨酸盐酸盐-蔗糖，更优选为L-精氨酸盐酸盐、L-精氨酸、甲硫氨酸、氯化钠-L-精氨酸、氯化钠-L-精氨酸盐酸盐、氯化钠-甲硫氨酸，更优选为氯化钠-L-精氨酸、氯化钠-L-精氨酸盐酸盐。在特别优选地实施方案中，本发明使用的稳定剂选自甲硫氨酸、L-精氨酸、L-精氨酸盐酸盐和氯化钠中的一种或多种。
术语“表面活性剂”一般包括保护蛋白质例如抗体免受空气/溶液界面诱导的应力、溶液/表面诱导的应力的影响以减少抗体的聚集或使制剂中颗粒物的形成最小化的试剂。示例性的表面活性剂包括但不限于非离子型表面活性剂例如聚氧乙烯脱水山梨醇脂肪酸酯(如聚山梨醇酯20和聚山梨醇酯80)、聚乙烯-聚丙烯共聚物、聚乙烯-聚丙烯二醇、聚氧 乙烯-硬脂酸酯、聚氧乙烯烷基醚、例如聚氧乙烯单月桂基醚、烷基苯基聚氧乙烯醚(Triton-X)、聚氧乙烯-聚氧丙烯共聚物(泊洛沙姆，Pluronic)、十二烷基硫酸钠(SDS)。在特别优选地实施方案中，本发明使用的表面活性剂为聚山梨醇酯80。
术语"等渗"是指该制剂具有与人血液基本相同的渗透压。等渗制剂一般具有约250至350mOsm的渗透压。可使用蒸汽压或冰点下降式的渗透压计测量等渗性。
术语“稳定的”制剂是其中的抗体在制造过程期间和/或储存时基本上保持其物理稳定性和/或化学稳定性和/或生物活性的制剂。即使所含的抗体在经过一定时间储存之后未能保持其100％的化学结构或生物功能，医药制剂也可以是稳定的。在某些情况下，在经过一定时间储存之后，能维持约90％、约95％、约96％、约97％、约98％或约99％的抗体结构或功能，也可被认为是“稳定的”。用于测量蛋白质稳定性的各种分析技术在本技术领域中是可得的，并综述在《肽和蛋白质药物递送》(Peptide and Protein Drug Delivery)247-301，Vincent Lee主编，Marcel Dekker，Inc.，New York，N.Y.，Pubs.(1991))，和Jones，A.(1993)Adv.Drug Delivery Rev.10：29-90中(二者引入作为参考)。
制剂在一定温度下经过一定时间的储存之后，通过测定其中剩余的天然抗体的百分比(及其它方法)，可以测量其稳定性。除其它方法外，天然抗体的百分比可以通过尺寸排阻色谱法(例如尺寸排阻高效液相色谱法[SEC-HPLC])来测量，“天然的”指未聚集的和未降解的。在一些方案中，蛋白质的稳定性按照具有低百分比的降解(例如片段化)和/或聚集蛋白质的溶液中单体蛋白质的百分数来确定。在一些方案中，制剂可以在室温、约25-30℃或40℃下稳定储存至少2周、至少28天、至少1个月、至少2个月、至少3个月、至少4个月、至少5个月、至少6个月、至少7个月、至少8个月、至少9个月、至少10个月、至少11个月、至少12个月、至少18个月、至少24个月，或更长，最多不超过约6％、5％、4％、3％、2％、1％、0.5％，或0.1％聚集形式的抗体。
通过测定在离子交换期间在此抗体主馏分(“主要荷电形式”)较为酸性的馏分中迁移的抗体(“酸性形式”)的百分比(及其它方法)，可以测量稳定性，其中稳定性与酸性形式抗体的百分比成反比。除其它方法外，“酸化”抗体的百分比可以通过离子交换色谱法(例如阳离子交换高效液相色谱法[CEX-HPLC])来测量。在一些实施方式中，可接受程度的稳定性意为当制剂在一定温度下经过一定时间的储存之后，其中可检测出的酸性形式的抗体最多不超过约49％、45％、40％、35％、30％、25％、20％、15％、10％、5％、4％、3％、2％、1％、0.5％或0.1％。在测量稳定性之前储存的一定时间可以是至少2周、至少28天、至少1个月、至少2个月、至少3个月、至少4个月、至少5个月、至少6个月、至少7个月、至少8个月、至少9个月、至少10个月、至少11个月、至少12个月、至少18个月、至少24个月，或更长。当评估稳定性时，容许储存医药制剂的一定温度可以是约 -80℃至约45℃范围内的任何温度，例如储存于约-80℃、约-30℃、约-20℃、约0℃、约2-8℃、约5℃、约25℃，或约40℃。
如果抗体在颜色和/或澄清度目测检查时或通过UV光散射或通过孔径排阻层析测量时基本上不显示出例如聚集、沉淀和/或变性的迹象，则所述抗体在该药物组合物中“保持其物理稳定性”。聚集是单个分子或复合物共价或非共价缔合以形成聚集体的过程。聚集可以进行到形成可见沉淀物的程度。
制剂的稳定性例如物理稳定性可以通过本技术领域中公知的方法来评估，包括测量样品的表观消光度(吸光度或光密度)。这样的消光测量与制剂的浊度相关。制剂的浊度部分地是溶解在溶液中的蛋白质的固有性质，并且通常通过比浊法来测量，并用比浊法浊度单位(NTU)来量度。
随着例如溶液中一种或多种组分的浓度(例如蛋白质和/或盐浓度)而变化的浊度水平也被称为制剂的“乳浊”或“乳浊外观”。浊度水平可以参照使用已知浊度的悬液产生的标准曲线来计算。用于测定药物组合物的浊度水平的参比标准品可以基于《欧洲药典》标准(《欧洲药典》(European Pharmacopoeia)，第四版，“欧洲药品质量委员会指令”(Directorate for the Quality of Medicine of the Council of Europe)(EDQM)，Strasbourg，France)。根据《欧洲药典》标准，澄清溶液被定义为浊度低于或等于按照《欧洲药典》标准具有约3的参比悬液的浊度的溶液。比浊法的浊度测量可以检测在不存在缔合或非理想效应的情况下的瑞利散射，其通常随浓度线性变化。用于评估物理稳定性的其他方法在本技术领域中是公知的。
如果抗体在给定时间点的化学稳定性使得抗体被认为仍保持如下文中所定义的其生物活性，则所述抗体在药物组合物中“保持其化学稳定性”。可以通过例如检测或定量抗体的化学改变的形式来评估化学稳定性。化学改变可以包括尺寸改变(例如剪短)，其可以使用例如孔径排阻层析、SDS-PAGE和/或基质辅助的激光解吸电离/飞行时间质谱(MALDI/TOF MS)来评估。其他类型的化学改变包括电荷改变(例如作为脱酰胺或氧化的结果而发生)，其可以通过例如离子交换层析来评估。
如果药物组合物中的抗体对于其预期目的来说是生物活性的，则所述抗体在药物组合物中“保持其生物活性”。例如，如果制剂于例如5℃、25℃、45℃等温度下储存一定时间(例如1至12个月)之后，该制剂所含人源化单克隆抗体与COVID-19结合的亲和力为所述储存之前抗体结合亲和力的至少90％、95％或以上，则可认为本发明之制剂是稳定的。结合亲和力也可用例如ELISA或等离子共振技术测定。
在本发明的情形中，在药理学意义上，抗体的“治疗有效量”或“有效量”是指在抗体可以有效治疗的障碍的症状的预防或治疗或减轻方面有效的量。本发明中，药物的“治疗有 效量”或“治疗有效剂量”是当单独使用或与另一种治疗剂组合使用时保护受试者免于疾病发作或促进疾病消退的任何量的药物，所述疾病消退通过疾病症状的严重性的降低，疾病无症状期的频率和持续时间的增加，或由疾病痛苦引起的损伤或失能的预防来证明。药物促进疾病消退的能力可以使用本领域技术人员已知的多种方法来评价，比如在临床试验期间的人受试者中，在预测人类功效的动物模型系统中，或通过在体外测定法中测定所述药剂的活性。药物治疗有效量包括“预防有效量”，即当单独或如与其它治疗药物组合给与处于患病风险的受试者或患病复发的受试者时，抑制疾病的发展或复发的任何量的药物。
术语“受试者”或“患者”意图包括哺乳动物生物体。受试者/患者的实例包括人类和非人类哺乳动物，例如非人类灵长动物、狗、奶牛、马、猪、绵羊、山羊、猫、小鼠、兔、大鼠和转基因非人类动物。在本发明的特定实施方式中，受试者是人类。
术语“施用”、“给与”及“处理”是指采用本领域技术人员已知的各种方法或递送系统中的任意一种将包含治疗剂的组合物引入受试者。人源化抗BLyS抗体或其抗原结合片段的给药途径包括静脉内、肌内、皮下、腹膜、脊髓或其他胃肠外给药途径，比如注射或输注。“胃肠外给药”是指除了肠内或局部给药以外的通常通过注射的给药方式，包括但不限于静脉内、肌内、动脉内、鞘内、淋巴内、损伤内、囊内、框内、心内、皮内、腹膜内、经气管、皮下、表皮下、关节内、囊下、蛛网膜下、脊柱内、硬膜内和胸骨内注射和输注以及经体内电穿孔。
抗体
本文所用的术语“抗体”应被理解为包括完整抗体分子及其抗原结合片段。本文所用的术语抗体的“抗原结合部分”或“抗原结合片段”(或简称为“抗体部分”或“抗体片段”)是指抗体中保持了与BLyS特异性结合能力的一个或多个片段。
本文所用的术语“全长抗体”或“完整抗体分子”指包含四条肽链的免疫球蛋白分子，两条重(H)链(全长时约50-70kDa)和两条轻(L)链(全长时约25kDa)通过二硫键互相连接。每一条重链(在本文中缩写为HC)由重链可变区(在本文中缩写为VH)和重链恒定区(在本文中缩写为CH)组成。重链恒定区由3个结构域CH1、CH2和CH3组成。每一条轻链(在本文中缩写为LC)由轻链可变区(在本文中缩写为VL)和轻链恒定区组成。轻链恒定区由一个结构域CL组成。VH和VL区可被进一步细分为具有高可变性的互补决定区(CDR)和其间隔以更保守的称为框架区(FR)的区域。每一个VH或VL区由按下列顺序：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4从氨基末端至羧基末端排列的3个CDR和4个FR组成。重链和轻链的可变区含有与抗原相互作用的结合结构域。抗体的恒定区可介导免疫球蛋白对宿主组织或因子(包括免疫系统的各种细胞(例如，效应细胞)和经典补体 系统的第一组分(Clq))的结合。
当在本文中使用时，术语“CDR”是指抗体可变序列内的互补决定区。在重链和轻链的各个可变区中存在3个CDR，其对于各个重链和轻链可变区被命名为HCDR1、HCDR2和HCDR3或LCDR1、LCDR2和LCDR3。这些CDR的准确边界按照不同的系统有不同的定义。
本发明的所述抗体的可变区CDR的精确氨基酸序列边界可使用许多公知的方案中的任何一种方案来确定，包括由Kabat等人(1991)，“Sequences of Proteins of Immunological Interest，第5版.Public Health Service，National Institutes of Health，Bethesda，MD(“Kabat”编号方案)描述的Kabat方案和Lefranc M.-P.等人描述的IMGT方案(1999 Nucleic Acids Research,27,209-212)。
本文所使用的“抗原结合片段”包括抗体的片段或其衍生物，通常包括亲代抗体的抗原结合区或可变区(例如一个或多个CDR)的至少一个片段，其保持亲代抗体的至少一些结合特异性。抗原结合片段的实例包括但不限于Fab，Fab'，F(ab')2和Fv片段；双抗体；线性抗体；单链抗体分子，例如sc-Fv；由抗体片段形成的纳米抗体(nanobody)和多特异性抗体。当抗体的结合活性在摩尔浓度基础上表示时，结合片段或其衍生物通常保持亲代抗体抗原结合活性的至少10％。优选结合片段或其衍生物保持亲代抗体的抗原结合亲和力的至少20％、50％、70％、80％、90％、95％或100％或更高。还预期抗体的抗原结合片段可包括不明显改变其生物活性的保守或非保守氨基酸取代(称为抗体的“保守变体”或“功能保守变体”)。
当提及配体/受体、抗体/抗原或其它结合对时，“特异性”结合是指在蛋白和/或其它生物试剂的异质群体中确定是否存在所述蛋白。例如本发明的单克隆抗体与BLyS蛋白的结合反应。因此，在所指定的条件下，特定的配体/抗原与特定的受体/抗体结合，并且并不以显著量与样品中存在的其它蛋白结合。
本文所述的人源化单克隆抗体或其抗原结合片段包括申请号为CN201210160474.3中描述的任意一个抗BLyS抗体，本文将其所公开的全部内容以引入的方式纳入本文。在一些方案中，在本发明的方法和组合物中使用的抗体的CDR序列包括来自于CN201210160474.3中描述的抗体BLyS-IV的CDR序列。在一些方案中，在本文实施例中所用的非限制性、示范性抗体选自CN201210160474.3中描述的人源化抗BLyS抗体BLyS-IV。
在一些方案中，在本文实施例中所用的非限制性、示范性人源化抗BLyS抗体，其具有如SEQ ID NO:18所示的轻链氨基酸序列，和如SEQ ID NO:19所示的重链氨基酸序列；该人源化抗BLyS抗体命名为H482L472。
在一些方案中，本发明提供了一种人源化抗BLyS抗体或其抗原结合片段，其能够与B淋巴细胞刺激因子结合并且能够抑制B淋巴细胞刺激因子与其受体BR3-Fc的结合，具有优异的人BLyS蛋白的结合能力和竞争抑制人BLyS与其受体人BR3结合的能力。本发明提供的人源化抗BLyS抗体或其抗原结合片段可以参照申请号为CN201210160474.3中实施例7和实施例8所示的方法，构建表达载体，使用293F细胞进行瞬转表达，100mL体系、经过一步亲和纯化得到。
在一些实施方案中，本发明提供一种人源化抗BLyS抗体或其抗原结合片段，该人源化抗BLyS抗体或其抗原结合片段的LCDR1、LCDR2和LCDR3的氨基酸序列分别如SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3所示，HCDR1、HCDR2和HCDR3的氨基酸序列分别如SEQ ID NO:4、SEQ ID NO:5和SEQ ID NO:6所示；轻链可变区的FR1选自SEQ ID NO:7、9和13中任一所述的FR1，FR2为SEQ ID NO:7所述的FR2，FR3选自SEQ ID NO:7、9和11中任一所述的FR3，FR4为SEQ ID NO:7所述的FR4；重链可变区的FR1选自SEQ ID NO:8、10、12和15中任一所述的FR1，FR2选自SEQ ID NO:8、10和15中任一所述的FR2，FR3选自SEQ ID NO:8、12和14中任一所述的FR3，FR4为SEQ ID NO:8或10所述的FR4。
在一些实施方案中，本发明所述的人源化抗BLyS抗体或其抗原结合片段不包括：轻链可变区为SEQ ID NO:7所示的氨基酸序列，同时重链可变区为SEQ ID NO:8所示的氨基酸序列的人源化抗BLyS抗体或其抗原结合片段。在一些实施方案中，本发明所述的人源化抗BLyS抗体或其抗原结合片段不包括：轻链为SEQ ID NO:16所示的氨基酸序列，同时重链为SEQ ID NO:17所示的氨基酸序列的人源化抗BLyS抗体或其抗原结合片段。
在一些实施方案中，本发明的人源化抗BLyS抗体或其抗原结合片段中，LCDR1、LCDR2和LCDR3的氨基酸序列分别如SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3所示，HCDR1、HCDR2和HCDR3的氨基酸序列分别如SEQ ID NO:4、SEQ ID NO:5和SEQ ID NO:6所示；轻链可变区的FR1选自SEQ ID NO:7、9和13中任一所述的FR1，FR2为SEQ ID NO:7所述的FR2，FR3选自SEQ ID NO:7、9和11中任一所述的FR3，FR4为SEQ ID NO:7所述的FR4；重链可变区的FR1-FR4分别为SEQ ID NO:15的FR1-FR4。
在一些实施方案中，本发明的人源化抗BLyS抗体或其抗原结合片段中，LCDR1、LCDR2和LCDR3的氨基酸序列分别如SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3所示，HCDR1、HCDR2和HCDR3的氨基酸序列分别如SEQ ID NO:4、SEQ ID NO:5和SEQ ID NO:6所示；轻链可变区的FR1选自SEQ ID NO:7、9和13中任一所述的FR1，FR2为SEQ ID NO:7所述的FR2，FR3选自SEQ ID NO:7、9和11中任一所述的FR3，FR4为SEQ ID NO:7所述的FR4；重链可变区的FR1-FR4分别为SEQ ID NO:10的FR1-FR4。
在一些实施方案中，本发明的人源化抗BLyS抗体或其抗原结合片段中，LCDR1、LCDR2和LCDR3的氨基酸序列分别如SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3所示，HCDR1、HCDR2和HCDR3的氨基酸序列分别如SEQ ID NO:4、SEQ ID NO:5和SEQ ID NO:6所示；轻链可变区的FR1选自SEQ ID NO:7、9和13中任一所述的FR1，FR2为SEQ ID NO:7所述的FR2，FR3选自SEQ ID NO:7、9和11中任一所述的FR3，FR4为SEQ ID NO:7所述的FR4；重链可变区的FR1-FR4分别为SEQ ID NO:12的FR1-FR4。
在一些实施方案中，本发明的人源化抗BLyS抗体或其抗原结合片段中，LCDR1、LCDR2和LCDR3的氨基酸序列分别如SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3所示，HCDR1、HCDR2和HCDR3的氨基酸序列分别如SEQ ID NO:4、SEQ ID NO:5和SEQ ID NO:6所示；轻链可变区的FR1为SEQ ID NO:11或13所述的FR1，FR2为SEQ ID NO:11所述的FR2，FR3为SEQ ID NO:11或13所述的FR3，FR4为SEQ ID NO:11所述的FR4；重链可变区的FR1-FR4分别为SEQ ID NO:14的FR1-FR4。
在一些方案中，本发明提供了一种人源化抗BLyS抗体或其抗原结合片段，其包含轻链可变区和重链可变区，其中，所述轻链可变区包含如SEQ ID NO：7、SEQ ID NO：9、SEQ ID NO：11或SEQ ID NO：13所示的氨基酸序列，所述重链可变区包含如SEQ ID NO：8、SEQ ID NO：10、SEQ ID NO：12、SEQ ID NO：14或SEQ ID NO：15所示的氨基酸序列。
在一些方案中，上述人源化抗BLyS抗体或其抗原结合片段包含轻链可变区和重链可变区，其中：
(1)所述轻链可变区包含如SEQ ID NO：7所示的氨基酸序列，和所述重链可变区包含如SEQ ID NO：10、SEQ ID NO：12、SEQ ID NO：14或SEQ ID NO：15所示的氨基酸序列；或
(2)所述轻链可变区包含如SEQ ID NO：9所示的氨基酸序列，和所述重链可变区包含如SEQ ID NO：8、SEQ ID NO：10、SEQ ID NO：12、SEQ ID NO：14或SEQ ID NO：15所示的氨基酸序列；或
(3)所述轻链可变区包含如SEQ ID NO：11所示的氨基酸序列，和所述重链可变区包含如SEQ ID NO：8、SEQ ID NO：10、SEQ ID NO：12、SEQ ID NO：14或SEQ ID NO：15所示的氨基酸序列；或
(4)所述轻链可变区包含如SEQ ID NO：13所示的氨基酸序列，和所述重链可变区包含如SEQ ID NO：8、SEQ ID NO：10、SEQ ID NO：12、SEQ ID NO：14或SEQ ID NO：15所示的氨基酸序列。
优选地，所述轻链可变区和重链可变区选自以下各组中的一组：
(5)所述轻链可变区包含如SEQ ID NO：9所示的氨基酸序列，和所述重链可变区包含如SEQ ID NO：10所示的氨基酸序列；或
(6)所述轻链可变区包含如SEQ ID NO：11所示的氨基酸序列，和所述重链可变区包含如SEQ ID NO：12所示的氨基酸序列；或
(7)所述轻链可变区包含如SEQ ID NO：13所示的氨基酸序列，和所述重链可变区包含如SEQ ID NO：14所示的氨基酸序列；或
(8)所述轻链可变区包含如SEQ ID NO：7所示的氨基酸序列，和所述重链可变区包含如SEQ ID NO：15所示的氨基酸序列；或
(9)所述轻链可变区包含如SEQ ID NO：9所示的氨基酸序列，和所述重链可变区包含如SEQ ID NO：15所示的氨基酸序列；或
(10)所述轻链可变区包含如SEQ ID NO：11所示的氨基酸序列，和所述重链可变区包含如SEQ ID NO：15所示的氨基酸序列；或
(11)所述轻链可变区包含如SEQ ID NO：13所示的氨基酸序列，和所述重链可变区包含如SEQ ID NO：15所示的氨基酸序列。
在一些方案中，上述人源化抗BLyS抗体或其抗原结合片段包含轻链可变区和重链可变区，其中：
(a)所述重链可变区包含如SEQ ID NO：8所示的氨基酸序列，和所述轻链可变区包含如SEQ ID NO：13所示的氨基酸序列；
(b)所述重链可变区包含如SEQ ID NO：10所示的氨基酸序列，和所述轻链可变区包含如SEQ ID NO：7、9、11或13所示的氨基酸序列；
(c)所述重链可变区包含如SEQ ID NO：12所示的氨基酸序列，和所述轻链可变区包含如SEQ ID NO：7、9、11或13所示的氨基酸序列；
(d)所述重链可变区包含如SEQ ID NO：14所示的氨基酸序列，和所述轻链可变区包含如SEQ ID NO：11或13所示的氨基酸序列；或
(e)所述重链可变区包含如SEQ ID NO：15所示的氨基酸序列，和所述轻链可变区包含如SEQ ID NO：7、9、11或13所示的氨基酸序列。
本发明所述人源化抗BLyS抗体还含有人类轻链恒定区和重链恒定区，且所述轻链可变区及重链可变区分别连接至人类轻链恒定区和重链恒定区。即所述人源化抗BLyS抗体含有完整的轻链和完整的重链，其中所述完整的轻链由抗BLyS抗体所含有的轻链可变区与人类轻链恒定区连接而成，所述完整的重链由抗BLyS抗体所含有的重链可变区与人类重链恒定区连接而成。
在一些方案中，所述人类轻链恒定区为人类轻链κ恒定区。
在一些方案中，所述人类重链恒定区为人类重链Fc片段。
其中，所述人类轻链κ恒定区和人类重链Fc片段，均来源于健康人B淋巴细胞。通过基因工程技术，重叠延伸PCR连接可变区和恒定区得到完整的人源化抗BLyS抗体的轻链和重链。
在一些方案中，上述人源化抗BLyS抗体或其抗原结合片段，其包含轻链和重链，其中，所述轻链包含如SEQ ID NO：16、SEQ ID NO：18、SEQ ID NO：20或SEQ ID NO：22所示的氨基酸序列，所述重链包含如SEQ ID NO：17、SEQ ID NO：19、SEQ ID NO：21、SEQ ID NO：23或SEQ ID NO：24所示的氨基酸序列。
在一些方案中，上述人源化抗BLyS抗体或其抗原结合片段包含轻链和重链，其中：
(1)所述轻链包含如SEQ ID NO：16所示的氨基酸序列，和所述重链包含如SEQ ID NO：19、SEQ ID NO：21、SEQ ID NO：23或SEQ ID NO：24所示的氨基酸序列；或
(2)所述轻链包含如SEQ ID NO：18所示的氨基酸序列，和所述重链包含如SEQ ID NO：17、SEQ ID NO：19、SEQ ID NO：21、SEQ ID NO：23或SEQ ID NO：24所示的氨基酸序列；或
(3)所述轻链包含如SEQ ID NO：20所示的氨基酸序列，和所述重链包含如SEQ ID NO：17、SEQ ID NO：19、SEQ ID NO：21、SEQ ID NO：23或SEQ ID NO：24所示的氨基酸序列；或
(4)所述轻链包含如SEQ ID NO：22所示的氨基酸序列，和所述重链包含如SEQ ID NO：17、SEQ ID NO：19、SEQ ID NO：21、SEQ ID NO：23或SEQ ID NO：24所示的氨基酸序列；或
优选地，所述轻链和重链选自以下各组中的一组：
(5)所述轻链包含如SEQ ID NO：18所示的氨基酸序列，和所述重链包含如SEQ ID NO：19所示的氨基酸序列；或
(6)所述轻链包含如SEQ ID NO：20所示的氨基酸序列，和所述重链包含如SEQ ID NO：21所示的氨基酸序列；或
(7)所述轻链包含如SEQ ID NO：22所示的氨基酸序列，和所述重链包含如SEQ ID NO：23所示的氨基酸序列；或
(8)所述轻链包含如SEQ ID NO：16所示的氨基酸序列，和所述重链包含如SEQ ID NO：24所示的氨基酸序列；或
(9)所述轻链包含如SEQ ID NO：18所示的氨基酸序列，和所述重链包含如SEQ ID NO：24所示的氨基酸序列；或
(10)所述轻链包含如SEQ ID NO：20所示的氨基酸序列，和所述重链包含如SEQ ID NO：24所示的氨基酸序列；或
(11)所述轻链包含如SEQ ID NO：22所示的氨基酸序列，和所述重链包含如SEQ ID NO：24所示的氨基酸序列。
在一些方案中，上述人源化抗BLyS抗体或其抗原结合片段包含轻链和重链，其中：
(a)所述重链包含如SEQ ID NO：17所示的氨基酸序列，和所述轻链包含如SEQ ID NO：22所示的氨基酸序列；
(b)所述重链包含如SEQ ID NO：19所示的氨基酸序列，和所述轻链包含如SEQ ID NO：16、18、20或22所示的氨基酸序列；
(c)所述重链包含如SEQ ID NO：21所示的氨基酸序列，和所述轻链包含如SEQ ID NO：16、18、20或22所示的氨基酸序列；
(d)所述重链包含如SEQ ID NO：23所示的氨基酸序列，和所述轻链包含如SEQ ID NO：20或22所示的氨基酸序列；或
(e)所述重链包含如SEQ ID NO：24所示的氨基酸序列，和所述轻链包含如SEQ ID NO：16、18、20或22所示的氨基酸序列。
医药制剂
本发明所述的药物组合物是一种含有抗BLyS抗体的高稳定性药物组合物。特别地，本发明发现该抗BLyS抗体在组氨酸缓冲液体系和L-精氨酸、L-精氨酸盐酸盐或甲硫氨酸的组合中具有出人意料的特征，即具有高稳定性。
本发明提供了一种药物组合物，其包含本文所述的人源化抗BLyS抗体或其抗原结合片段、本文所述的多核苷酸、本文所述的表达载体或本文所述的宿主细胞，和药学上可接受的载体或赋形剂。
在一些方案中，本发明提供了一种药物组合物，包含：(1)缓冲液；(2)人源化抗BLyS抗体或其抗原结合片段，所述人源化抗BLyS抗体或其抗原结合片段具有氨基酸序列分别如SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3所示的LCDR1、LCDR2和LCDR3，和氨基酸序列分别如SEQ ID NO:4、SEQ ID NO:5和SEQ ID NO:6所示的HCDR1、HCDR2和HCDR3。
优选地，本发明所述药物组合物中的人源化抗BLyS抗体或其抗原结合片段如本申请“抗体”部分任一实施方案所述；或人源化抗BLyS抗体或其抗原结合片段包含轻链可变区和重链可变区，其中，所述轻链可变区包含如SEQ ID NO：7所示的氨基酸序列，和所述重链可变区包含如SEQ ID NO：8所示的氨基酸序列；或人源化抗BLyS抗体或其抗原结 合片段包含轻链和重链，其中，所述轻链包含如SEQ ID NO：16所示的氨基酸序列，和所述重链包含如SEQ ID NO：17所示的氨基酸序列。
例如，在一些方案中，上述人源化抗BLyS抗体或其抗原结合片段，其包含轻链可变区和重链可变区，其中，所述轻链可变区包含如SEQ ID NO：7、SEQ ID NO：9、SEQ ID NO：11或SEQ ID NO：13所示的氨基酸序列，所述重链可变区包含如SEQ ID NO：8、SEQ ID NO：10、SEQ ID NO：12、SEQ ID NO：14或SEQ ID NO：15所示的氨基酸序列。
在一些方案中，上述人源化抗BLyS抗体或其抗原结合片段，其包含轻链可变区和重链可变区，其中：
(1)所述轻链可变区包含如SEQ ID NO：7所示的氨基酸序列，和所述重链可变区包含如SEQ ID NO：8、SEQ ID NO：10、SEQ ID NO：12、SEQ ID NO：14或SEQ ID NO：15所示的氨基酸序列；或
(2)所述轻链可变区包含如SEQ ID NO：9所示的氨基酸序列，和所述重链可变区包含如SEQ ID NO：8、SEQ ID NO：10、SEQ ID NO：12、SEQ ID NO：14或SEQ ID NO：15所示的氨基酸序列；或
(3)所述轻链可变区包含如SEQ ID NO：11所示的氨基酸序列，和所述重链可变区包含如SEQ ID NO：8、SEQ ID NO：10、SEQ ID NO：12、SEQ ID NO：14或SEQ ID NO：15所示的氨基酸序列；或
(4)所述轻链可变区包含如SEQ ID NO：13所示的氨基酸序列，和所述重链可变区包含如SEQ ID NO：8、SEQ ID NO：10、SEQ ID NO：12、SEQ ID NO：14或SEQ ID NO：15所示的氨基酸序列；或
优选地，所述轻链可变区和重链可变区选自以下各组中的一组：
(5)所述轻链可变区包含如SEQ ID NO：9所示的氨基酸序列，和所述重链可变区包含如SEQ ID NO：10所示的氨基酸序列；或
(6)所述轻链可变区包含如SEQ ID NO：11所示的氨基酸序列，和所述重链可变区包含如SEQ ID NO：12所示的氨基酸序列；或
(7)所述轻链可变区包含如SEQ ID NO：13所示的氨基酸序列，和所述重链可变区包含如SEQ ID NO：14所示的氨基酸序列；或
(8)所述轻链可变区包含如SEQ ID NO：7所示的氨基酸序列，和所述重链可变区包含如SEQ ID NO：15所示的氨基酸序列；或
(9)所述轻链可变区包含如SEQ ID NO：9所示的氨基酸序列，和所述重链可变区包含如SEQ ID NO：15所示的氨基酸序列；或
(10)所述轻链可变区包含如SEQ ID NO：11所示的氨基酸序列，和所述重链可变区包含如SEQ ID NO：15所示的氨基酸序列；或
(11)所述轻链可变区包含如SEQ ID NO：13所示的氨基酸序列，和所述重链可变区包含如SEQ ID NO：15所示的氨基酸序列；或
(12)所述轻链可变区包含如SEQ ID NO：7所示的氨基酸序列，和所述重链可变区包含如SEQ ID NO：8所示的氨基酸序列。
本发明所述人源化抗BLyS抗体还含有人类轻链恒定区和重链恒定区，且所述轻链可变区及重链可变区分别连接至人类轻链恒定区和重链恒定区。即所述人源化抗BLyS抗体含有完整的轻链和完整的重链，其中所述完整的轻链由抗BLyS抗体所含有的轻链可变区与人类轻链恒定区连接而成，所述完整的重链由抗BLyS抗体所含有的重链可变区与人类重链恒定区连接而成。
在一些方案中，所述人类轻链恒定区为人类轻链κ恒定区。
在一些方案中，所述人类重链恒定区为人类重链Fc片段。
其中，所述人类轻链κ恒定区和人类重链Fc片段，均来源于健康人B淋巴细胞。通过基因工程技术，重叠延伸PCR连接可变区和恒定区得到完整的人源化抗BLyS抗体的轻链和重链。
在一些方案中，上述人源化抗BLyS抗体或其抗原结合片段，其包含轻链和重链，其中，所述轻链包含如SEQ ID NO：16、SEQ ID NO：18、SEQ ID NO：20或SEQ ID NO：22所示的氨基酸序列，所述重链包含如SEQ ID NO：17、SEQ ID NO：19、SEQ ID NO：21、SEQ ID NO：23或SEQ ID NO：24所示的氨基酸序列。
在一些方案中，上述人源化抗BLyS抗体或其抗原结合片段，其包含轻链和重链，其中：
(1)所述轻链包含如SEQ ID NO：16所示的氨基酸序列，和所述重链包含如SEQ ID NO：17、SEQ ID NO：19、SEQ ID NO：21、SEQ ID NO：23或SEQ ID NO：24所示的氨基酸序列；或
(2)所述轻链包含如SEQ ID NO：18所示的氨基酸序列，和所述重链包含如SEQ ID NO：17、SEQ ID NO：19、SEQ ID NO：21、SEQ ID NO：23或SEQ ID NO：24所示的氨基酸序列；或
(3)所述轻链包含如SEQ ID NO：20所示的氨基酸序列，和所述重链包含如SEQ ID NO：17、SEQ ID NO：19、SEQ ID NO：21、SEQ ID NO：23或SEQ ID NO：24所示的氨基酸序列；或
(4)所述轻链包含如SEQ ID NO：22所示的氨基酸序列，和所述重链包含如SEQ ID NO：17、SEQ ID NO：19、SEQ ID NO：21、SEQ ID NO：23或SEQ ID NO：24所示的氨基酸序列；或
优选地，所述轻链和重链选自以下各组中的一组：
(5)所述轻链包含如SEQ ID NO：18所示的氨基酸序列，和所述重链包含如SEQ ID NO：19所示的氨基酸序列；或
(6)所述轻链包含如SEQ ID NO：20所示的氨基酸序列，和所述重链包含如SEQ ID NO：21所示的氨基酸序列；或
(7)所述轻链包含如SEQ ID NO：22所示的氨基酸序列，和所述重链包含如SEQ ID NO：23所示的氨基酸序列；或
(8)所述轻链包含如SEQ ID NO：16所示的氨基酸序列，和所述重链包含如SEQ ID NO：24所示的氨基酸序列；或
(9)所述轻链包含如SEQ ID NO：18所示的氨基酸序列，和所述重链包含如SEQ ID NO：24所示的氨基酸序列；或
(10)所述轻链包含如SEQ ID NO：20所示的氨基酸序列，和所述重链包含如SEQ ID NO：24所示的氨基酸序列；或
(11)所述轻链包含如SEQ ID NO：22所示的氨基酸序列，和所述重链包含如SEQ ID NO：24所示的氨基酸序列；或
(12)所述轻链包含如SEQ ID NO：16所示的氨基酸序列，和所述重链包含如SEQ ID NO：17所示的氨基酸序列。
在一些方案中，本发明所述抗原结合片段选自Fab、Fab'、Fab'-SH、Fv、scFv、F(ab')2、sdAb或双抗体。
在一些方案中，所述药物组合物中人源化抗BLyS抗体或其抗原结合片段的浓度约为1-300mg/mL，优选约为10-300mg/mL，更优选约为20-280mg/mL，更优选约为30-150mg/mL，更优选约为80-120mg/mL，更优选约为180-220mg/mL；更优选地，上述人源化单克隆抗体或其抗原结合片段浓度约为5mg/mL，10mg/mL，15mg/mL，20mg/mL，25mg/mL，30mg/mL，35mg/mL，40mg/mL，45mg/mL，50mg/mL，60mg/mL，70mg/mL，80mg/mL，90mg/mL，100mg/mL，110mg/mL，120mg/mL，130mg/mL，140mg/mL，150mg/mL，160mg/mL，170mg/mL，180mg/mL或200mg/mL，优选约为40mg/mL，60mg/mL，70mg/mL，80mg/mL，90mg/mL，95mg/mL，100mg/mL，105mg/mL，110mg/mL，120mg/mL，150mg/mL，160mg/mL，170mg/mL，180mg/mL，190mg/mL，195mg/mL，200mg/mL，205mg/mL，210mg/mL，220mg/mL。
在一些方案中，上述缓冲液选自醋酸缓冲液、柠檬酸缓冲液、琥珀酸缓冲液、磷酸缓冲液和组氨酸缓冲液中的一种或多种。
在一些方案中，上述缓冲液的浓度约为1-200mM，优选约为1-100mM，优选约为5-50mM，优选约为10-40mM，优选约为20-40mM；上述缓冲液浓度非限制性实施例约为5mM，10mM，15mM，20mM，25mM，30mM，35mM，40mM，45mM，50mM，60mM，70mM，80mM，90mM，100mM，105mM，110mM，115mM，120mM，130mM，140mM，150mM，160mM，170mM或180mM或这些范围内任意两个数值作为端点形成的范围，优选为10mM，15mM，20mM，25mM，30mM或35mM。
在一些方案中，上述缓冲液的pH约为5.0-6.5，优选约为5.0-6.0，优选约为优选约为5.5-6.0，上述缓冲液的pH非限制性实施例约为5.0，5.1，5.2，5.3，5.4，5.5，5.6，5.7，5.8，5.9，6.0，6.1，6.2，6.3，6.4，6.5，优选约为5.5、5.8或6.0。
在一些方案中，上述缓冲液为组氨酸缓冲液，优选地，所述组氨酸缓冲液选自组氨酸-盐酸盐缓冲液或组氨酸-醋酸盐缓冲液，优选组氨酸-盐酸盐缓冲液。
在一些方案中，上述组氨酸-盐酸盐缓冲液由组氨酸和组氨酸盐酸盐制成，优选L-组氨酸和L-组氨酸单盐酸盐。在一些方案中，组氨酸缓冲液由5-40mM的L-组氨酸和5-40mM的L-组氨酸单盐酸盐制成。在一些方案中，组氨酸缓冲液由摩尔比为1:1到1:4的组氨酸和组氨酸盐酸盐制成。在一些方案中，组氨酸缓冲液由摩尔比为1:2到1:2.5的组氨酸和组氨酸盐酸盐制成。在一些方案中，组氨酸缓冲液由摩尔比为1:2.3组氨酸和组氨酸盐酸盐制成。在一些方案中，组氨酸缓冲液为：由约10.0-11.0mM的L-组氨酸和约24.0-25.0mM的L-组氨酸单盐酸盐制成的pH为5.8的组氨酸缓冲液。在一些方案中，pH为5.8的35mM组氨酸缓冲液制备方法为：先分别配置35mM的L-组氨酸和L-组氨酸单盐酸盐溶液，再将两者混合到pH为5.8即得。在一些方案中，组氨酸缓冲液为：由约10.0mM的组氨酸和约25.0mM的组氨酸盐酸盐制成的pH为5.8的组氨酸缓冲液。
在一些方案中，上述缓冲液为醋酸缓冲液，优选地，所述醋酸缓冲液为醋酸-醋酸钠缓冲液或醋酸-醋酸钾缓冲液，优选醋酸-醋酸钠缓冲液。
在一些方案中，上述缓冲液为柠檬酸缓冲液，优选地，所述柠檬酸缓冲液为柠檬酸-柠檬酸钠缓冲液。
在一些方案中，上述缓冲液为琥珀酸缓冲液，优选地，所述琥珀酸缓冲液为琥珀酸-琥珀酸钠缓冲液。
在一些方案中，上述缓冲液为磷酸缓冲液，优选地，所述磷酸缓冲液为磷酸二氢钠-磷酸氢二钠缓冲液或磷酸二氢钾-磷酸氢二钾缓冲液。
在一些方案中，上述的药物组合物还包括稳定剂。
在一些方案中，所述稳定剂选自精氨酸、精氨酸盐酸盐、甲硫氨酸、脯氨酸、甘氨酸、氯化钠、甘露醇、山梨醇、蔗糖、麦芽糖、木糖醇和海藻糖中的一种或多种。
在一些方案中，上述稳定剂选自L-精氨酸、L-精氨酸盐酸盐、蔗糖、甲硫氨酸和氯化钠中的一种或多种。
在一些方案中，上述稳定剂的浓度为约10mM～400mM，优选约50mM～300mM，更优选约100mM～300mM，更优选约100mM～200mM。
在一些方案中，上述稳定剂为浓度约10-30mM的L-精氨酸盐酸盐与约50-200mM的氯化钠的组合；或所述稳定剂为浓度约10-30mML-精氨酸与约50-200mM的氯化钠的组合；或所述稳定剂为浓度约10-30mM的L-精氨酸与约80-220mM的蔗糖的组合；或所述稳定剂为浓度约10-30mM的L-精氨酸盐酸盐与约80-220mM的蔗糖的组合；或所述稳定剂为约30-90mM的甲硫氨酸与约50-200mM的氯化钠的组合；或所述稳定剂为约10-120mM的L-精氨酸盐酸盐；或所述稳定剂为约10-120mM的甲硫氨酸；优选地，所述稳定剂为浓度约10-30mM的L-精氨酸盐酸盐与约80-120mM的氯化钠的组合；或所述稳定剂为浓度约10-30mM的L-精氨酸与约80-120mM的氯化钠的组合；或所述稳定剂为约50-70mM的甲硫氨酸与约80-120mM的氯化钠的组合；或所述稳定剂为浓度约10-30mM的L-精氨酸与约130-170mM的蔗糖的组合；或所述稳定剂为浓度约10-30mM的L-精氨酸盐酸盐与约130-170mM的蔗糖的组合。
在一些方案中，上述稳定剂由浓度约50-200mM的氯化钠和浓度约10-30mM的L-精氨酸组成；优选地，上述稳定剂由浓度约80-120mM的氯化钠和浓度约10-30mM的L-精氨酸组成；上述稳定剂的非限制性实施例为由浓度约50mM，60mM，70mM，80mM，90mM，95mM，100mM，105mM，110mM，115mM，120Mm或130mM的氯化钠和浓度约10mM，15mM，20mM，25mM，30mM的L-精氨酸组成。
在一些方案中，上述稳定剂由浓度约50-200mM的氯化钠和浓度约10-30mM的L-精氨酸盐酸盐组成；优选地，上述稳定剂由浓度约80-120mM的氯化钠和浓度约10-30mM的L-精氨酸盐酸盐组成；上述稳定剂的非限制性实施例为由浓度约50mM，60mM，70mM，80mM，90mM，95mM，100mM，105mM，110mM，115mM，120Mm或130mM的氯化钠和浓度约10mM，15mM，20mM，25mM，30mM的L-精氨酸盐酸盐组成。
在一些方案中，上述稳定剂由浓度约50-200mM的氯化钠和浓度约30-90mM的甲硫氨酸组成；优选地，上述稳定剂由浓度约80-120mM的氯化钠和浓度约50-70mM的甲硫氨酸组成；上述稳定剂的非限制性实施例为由浓度约50mM，60mM，70mM，80mM，90mM，95mM，100mM，105mM，110mM，115mM，120Mm或130mM的氯化钠和浓度约30mM，40mM，50mM，55mM，60mM，65mM，70mM，75Mm或80mM的甲硫氨酸组成。
在一些方案中，上述稳定剂由浓度约10-30mM的L-精氨酸盐酸盐与约80-220mM的蔗糖组成；优选地，上述稳定剂由浓度约130-170mM的蔗糖和浓度约10-30mM的L-精氨酸盐酸盐组成；上述稳定剂的非限制性实施例为由浓度约80mM，90mM，100mM，110mM，120Mm，125Mm，130Mm，135Mm，140Mm，145Mm，150Mm，155Mm，160Mm或170mM的蔗糖和浓度约10mM，15mM，20mM，25mM，30mM的L-精氨酸盐酸盐组成。
在一些方案中，上述稳定剂为L-精氨酸。在一些方案中，上述稳定剂为浓度约10-100mM的L-精氨酸，上述L-精氨酸的浓度优选约为15-50mM，优选约为20-30mM，上述L-精氨酸浓度的非限制性实施例为约10mM，15mM，20mM，25mM，30mM，35mM，40mM，45mM，50mM，55mM，60mM，70mM，80mM，90mM，100mM，优选25mM或30mM。
在一些方案中，上述稳定剂为甲硫氨酸。在一些方案中，上述稳定剂为浓度约20-200mM的甲硫氨酸，上述甲硫氨酸的浓度优选约为20-180mM，优选约为40-150mM，优选约为45-100mM，优选约为50-90mM，上述甲硫氨酸浓度的非限制性实施例为约10mM，15mM，20mM，25mM，30mM，35mM，40mM，45mM，50mM，55mM，60mM，65mM，70mM，75mM，80mM，85mM，90mM，100mM，优选60mM或65mM。
在一些方案中，上述稳定剂为氯化钠。在一些方案中，上述稳定剂为浓度约30-200mM的氯化钠，上述氯化钠的浓度优选约为50-190mM，优选约为60-180mM，优选约为70-150mM，优选约为80-120mM，上述氯化钠浓度的非限制性实施例为约60mM，70mM，80mM，85mM，90mM，95mM，100mM，105mM，110mM，115mM，120mM，130mM，140mM，150mM，160mM，170mM，180mM，190mM，200mM，优选100mM或105mM。
在一些方案中，上述稳定剂为L-精氨酸盐酸盐。在一些方案中，上述稳定剂为浓度约30-200mM的L-精氨酸盐酸盐，上述L-精氨酸盐酸盐的浓度优选约为50-190mM，优选约为100-180mM，优选约为120-170mM，优选约为130-150mM，上述L-精氨酸盐酸盐浓度的非限制性实施例为约100mM，110mM，120mM，125mM，130mM，135mM，140mM，145mM，150mM，155mM，160mM，170mM，180mM，190mM，200mM，优选135mM或140mM。
在一些方案中，上述药物组合物还包括表面活性剂，所述表面活性剂选自聚山梨醇酯80、聚山梨醇酯20、泊洛沙姆188中的一种或多种。
在一些方案中，上述表面活性剂选自聚山梨醇酯80。
在一些方案中，上述表面活性剂选自聚山梨醇酯20。
在一些方案中，以w/v计算，上述表面活性剂浓度约为0.001％-0.1％，优选约为0.01％-0.1％，优选约为0.01％-0.05％；作为非限制性实施例，上述表面活性剂的浓度约为0.01％，0.02％，0.03％，0.04％，0.05％。0.06％，0.07％，0.09％或0.08％。
在一些方案中，上述药物组合物还包括注射用水。
在一些方案中，上述药物组合物含有：(a)约20mg/mL-280mg/mL的本文任一项所述的人源化抗BLyS抗体或其抗原结合片段；(b)约5-50mM的组氨酸缓冲液或约5-50mM的醋酸缓冲液，pH约为5.0-6.5；(c)约10-30mM的L-精氨酸或约10-30mM的L-精氨酸盐酸盐；(d)约80-220mM的蔗糖；(e)以及约0.01％-0.1％的聚山梨醇酯80。
在一些方案中，上述药物组合物含有：(a)约20mg/mL-280mg/mL的本文任一实施方案所述的人源化抗BLyS抗体或其抗原结合片段；(b)约10-40mM的组氨酸缓冲液或约10-40mM的醋酸缓冲液，pH约为5.0-6.5；(c)约10-30mM的L-精氨酸或约10-30mM的L-精氨酸盐酸盐；(d)约130-170mM的蔗糖；(e)以及约0.01％-0.1％的聚山梨醇酯80。
在一些方案中，上述药物组合物含有：(a)约30mg/mL-150mg/mL的本文任一实施方案所述的人源化抗BLyS抗体或其抗原结合片段；(b)约5-50mM的组氨酸缓冲液或约5-50mM的醋酸缓冲液，pH约为5.0-6.5；(c)约10-30mM的L-精氨酸、约10-30mM的L-精氨酸盐酸盐或约30-90mM的甲硫氨酸；(d)约50-200mM的氯化钠；(e)以及约0.01％-0.1％的聚山梨醇酯80。
在一些实施方案中，上述药物组合物含有：(a)约30mg/mL-150mg/mL的本文任一实施方案所述的人源化抗BLyS抗体或其抗原结合片段；(b)约5-50mM的组氨酸缓冲液，pH约为5.0-6.5；(c)约10-30mM的L-精氨酸、约10-30mM的L-精氨酸盐酸盐或约30-90mM的甲硫氨酸；(d)约50-200mM的氯化钠；(e)以及约0.01％-0.1％的聚山梨醇酯80。
在一些实施方案中，上述药物组合物含有：(a)约30mg/mL-150mg/mL的本文任一实施方案所述的人源化抗BLyS抗体或其抗原结合片段；(b)约5-50mM的醋酸缓冲液，pH约为5.0-6.5；(c)约10-30mM的L-精氨酸、约10-30mM的L-精氨酸盐酸盐或约30-90mM的甲硫氨酸；(d)约50-200mM的氯化钠；(e)以及约0.01％-0.1％的聚山梨醇酯80。
在一些方案中，上述药物组合物含有：(a)约80mg/mL-120mg/mL的本文任一实施方案所述的人源化抗BLyS抗体或其抗原结合片段；(b)约20-40mM的组氨酸缓冲液或约20-40mM的醋酸缓冲液，pH约为5.5-6.0；(c)约10-30mM的L-精氨酸、约50-70mM的甲硫氨酸或约10-30mM的L-精氨酸盐酸盐；(d)约80-120mM的氯化钠；(e)以及 约0.01％-0.05％的聚山梨醇酯80。
在一些方案中，上述药物组合物含有：(a)约80mg/mL-120mg/mL的本文任一实施方案所述的人源化抗BLyS抗体或其抗原结合片段；(b)约20-40mM的组氨酸缓冲液或，pH约为5.5-6.0；(c)约10-30mM的L-精氨酸、约50-70mM的甲硫氨酸或约10-30mM的L-精氨酸盐酸盐；(d)约80-120mM的氯化钠；(e)以及约0.01％-0.05％的聚山梨醇酯80。
在一些方案中，上述药物组合物含有：(a)约80mg/mL-120mg/mL的本文任一实施方案所述的人源化抗BLyS抗体或其抗原结合片段；(b)约20-40mM的醋酸缓冲液，pH约为5.5-6.0；(c)约10-30mM的L-精氨酸、约50-70mM的甲硫氨酸或约10-30mM的L-精氨酸盐酸盐；(d)约80-120mM的氯化钠；(e)以及约0.01％-0.05％的聚山梨醇酯80。
在一些方案中，上述药物组合物含有：(a)约180mg/mL-220mg/mL的本文任一项所述的人源化抗BLyS抗体或其抗原结合片段；(b)约20-40mM的组氨酸缓冲液或约20-40mM的醋酸缓冲液，pH约为5.5-6.0；(c)约10-30mM的L-精氨酸或约10-30mM的L-精氨酸盐酸盐；(d)约130-170mM的蔗糖；(e)以及约0.01％-0.05％的聚山梨醇酯80；或
在一些方案中，上述药物组合物含有：(a)约200mg/mL的本文任一实施方案所述的人源化抗BLyS抗体或其抗原结合片段；(b)约25mM组氨酸缓冲液、约25mM醋酸缓冲液或约35-36mM组氨酸缓冲液，pH约为5.5-6.0；(c)约15mM的L-精氨酸或约15mM的L-精氨酸盐酸盐；(d)150mM的蔗糖；(e)以及约0.02％的聚山梨醇酯80。
在一些方案中，上述药物组合物含有：(a)约100mg/mL的本文任一实施方案所述的人源化抗BLyS抗体或其抗原结合片段；(b)约25mM组氨酸缓冲液，pH约为5.5-6.0；(c)约25mM的L-精氨酸或约25mM的L-精氨酸盐酸盐；(d)约100mM的氯化钠；(e)以及约0.02％的聚山梨醇酯80。
在一些方案中，上述药物组合物含有：(a)约100mg/mL的本文任一实施方案所述的人源化抗BLyS抗体或其抗原结合片段；(b)约25mM醋酸缓冲液，pH约为5.5-6.0；(c)约60mM的甲硫氨酸；(d)约100mM的氯化钠；(e)以及约0.02％的聚山梨醇酯80。
在一些方案中，上述药物组合物含有：(a)约100mg/mL的本文任一实施方案所述的人源化抗BLyS抗体或其抗原结合片段；(b)约35-36mM组氨酸缓冲液，pH约为5.5-6.0；(c)约15mM的L-精氨酸盐酸盐或约15mM的L-精氨酸；(d)约100mM的氯化钠；(e)以及约0.02％的聚山梨醇酯80。
在一些方案中，上述药物组合物包含如下(1)-(20)任一项所示的组分：
(1)(a)约20mg/mL-280mg/mL的人源化抗BLyS抗体或其抗原结合片段，所述人源化抗BLyS抗体或其抗原结合片段包含轻链可变区和重链可变区，所述轻链可变区包含如SEQ ID NO：9所示的氨基酸序列，所述重链可变区包含如SEQ ID NO：8、SEQ ID NO：10、SEQ ID NO：12、SEQ ID NO：14或SEQ ID NO：15所示的氨基酸序列；(b)约5-50mM的组氨酸缓冲液，pH约为5.0-6.5；(c)约10-30mM的L-精氨酸或约10-30mM的L-精氨酸盐酸盐；(d)约80-220mM的蔗糖；(e)以及约0.01％-0.1％的聚山梨醇酯80；或
(2)(a)约30mg/mL-150mg/mL的人源化抗BLyS抗体或其抗原结合片段，所述人源化抗BLyS抗体或其抗原结合片段包含轻链可变区和重链可变区，所述轻链可变区包含如SEQ ID NO：9所示的氨基酸序列，所述重链可变区包含如SEQ ID NO：8、SEQ ID NO：10、SEQ ID NO：12、SEQ ID NO：14或SEQ ID NO：15所示的氨基酸序列；(b)约5-50mM组氨酸缓冲液，pH约为5.0-6.5；(c)约10-30mM的L-精氨酸；(d)约50-200mM的氯化钠；(e)以及约0.01％-0.1％的聚山梨醇酯80；或
(3)(a)约30mg/mL-150mg/mL的人源化抗BLyS抗体或其抗原结合片段，所述人源化抗BLyS抗体或其抗原结合片段包含轻链可变区和重链可变区，所述轻链可变区包含如SEQ ID NO：9所示的氨基酸序列，所述重链可变区包含如SEQ ID NO：8、SEQ ID NO：10、SEQ ID NO：12、SEQ ID NO：14或SEQ ID NO：15所示的氨基酸序列；(b)约5-50mM组氨酸缓冲液，pH约为5.0-6.5；(c)约10-30mM的L-精氨酸盐酸盐；(d)约50-200mM的氯化钠；(e)以及约0.01％-0.1％的聚山梨醇酯80；或
(4)(a)约30mg/mL-150mg/mL的人源化抗BLyS抗体或其抗原结合片段，所述人源化抗BLyS抗体或其抗原结合片段包含轻链可变区和重链可变区，所述轻链可变区包含如SEQ ID NO：9所示的氨基酸序列，所述重链可变区包含如SEQ ID NO：8、SEQ ID NO：10、SEQ ID NO：12、SEQ ID NO：14或SEQ ID NO：15所示的氨基酸序列；(b)约5-50mM组氨酸缓冲液，pH约为5.0-6.5；(c)约30-90mM的甲硫氨酸；(d)约50-200mM的氯化钠；(e)以及约0.01％-0.1％的聚山梨醇酯80；或
(5)(a)约30mg/mL-150mg/mL的人源化抗BLyS抗体或其抗原结合片段，所述人源化抗BLyS抗体或其抗原结合片段包含轻链可变区和重链可变区，所述轻链可变区包含如SEQ ID NO：9所示的氨基酸序列，所述重链可变区包含如SEQ ID NO：8、SEQ ID NO：10、SEQ ID NO：12、SEQ ID NO：14或SEQ ID NO：15所示的氨基酸序列；(b)约5-50mM醋酸缓冲液，pH约为5.0-6.5；(c)约10-30mM的L-精氨酸；(d)约50-200mM的氯化钠；(e)以及约0.01％-0.1％的聚山梨醇酯80；或
(6)(a)约30mg/mL-150mg/mL的人源化抗BLyS抗体或其抗原结合片段，所述人源化抗BLyS抗体或其抗原结合片段包含轻链可变区和重链可变区，所述轻链可变区包含如SEQ ID NO：9所示的氨基酸序列，所述重链可变区包含如SEQ ID NO：8、SEQ ID NO：10、SEQ ID NO：12、SEQ ID NO：14或SEQ ID NO：15所示的氨基酸序列；(b)约5-50mM醋酸缓冲液，pH约为5.0-6.5；(c)约10-30mM的L-精氨酸盐酸盐；(d)约50-200mM的氯化钠；(e)以及约0.01％-0.1％的聚山梨醇酯80；或
(7)(a)约30mg/mL-150mg/mL的人源化抗BLyS抗体或其抗原结合片段，所述人源化抗BLyS抗体或其抗原结合片段包含轻链可变区和重链可变区，所述轻链可变区包含如SEQ ID NO：9所示的氨基酸序列，所述重链可变区包含如SEQ ID NO：8、SEQ ID NO：10、SEQ ID NO：12、SEQ ID NO：14或SEQ ID NO：15所示的氨基酸序列；(b)约5-50mM醋酸缓冲液，pH约为5.0-6.5；(c)约30-90mM的甲硫氨酸；(d)约50-200mM的氯化钠；(e)以及约0.01％-0.1％的聚山梨醇酯80；或
(8)(a)约80mg/mL-120mg/mL的人源化抗BLyS抗体或其抗原结合片段，所述人源化抗BLyS抗体或其抗原结合片段包含轻链可变区和重链可变区，所述轻链可变区包含如SEQ ID NO：9所示的氨基酸序列，所述重链可变区包含如SEQ ID NO：10所示的氨基酸序列；(b)约20-40mM组氨酸缓冲液，pH约为5.5-6.0；(c)约10-30mM的L-精氨酸；(d)约80-120mM的氯化钠；(e)以及约0.01％-0.05％的聚山梨醇酯80；或
(9)(a)约80mg/mL-120mg/mL的人源化抗BLyS抗体或其抗原结合片段，所述人源化抗BLyS抗体或其抗原结合片段包含轻链可变区和重链可变区，所述轻链可变区包含如SEQ ID NO：9所示的氨基酸序列，所述重链可变区包含如SEQ ID NO：10所示的氨基酸序列；(b)约20-40mM组氨酸缓冲液，pH约为5.5-6.0；(c)约10-30mM的L-精氨酸盐酸盐；(d)约80-120mM的氯化钠；(e)以及约0.01％-0.05％的聚山梨醇酯80；或
(10)(a)约80mg/mL-120mg/mL的人源化抗BLyS抗体或其抗原结合片段，所述人源化抗BLyS抗体或其抗原结合片段包含轻链可变区和重链可变区，所述轻链可变区包含如SEQ ID NO：9所示的氨基酸序列，所述重链可变区包含如SEQ ID NO：10所示的氨基酸序列；(b)约20-40mM醋酸缓冲液，pH约为5.5-6.0；(c)约50-70mM的甲硫氨酸；(d)约80-120mM的氯化钠；(e)以及约0.01％-0.05％的聚山梨醇酯80；或
(11)(a)约180mg/mL-220mg/mL的人源化抗BLyS抗体或其抗原结合片段，所述人源化抗BLyS抗体或其抗原结合片段包含轻链可变区和重链可变区，所述轻链可变区包含如SEQ ID NO：9所示的氨基酸序列，所述重链可变区包含如SEQ ID NO：10所示的氨基酸序列；(b)约20-40mM组氨酸缓冲液，pH约为5.5-6.0；(c)约10-30mM的L- 精氨酸或约10-30mM的L-精氨酸盐酸盐；(d)约130-170mM的蔗糖；(e)以及约0.01％-0.05％的聚山梨醇酯80；或
(12)(a)约200mg/mL的人源化抗BLyS抗体或其抗原结合片段，所述人源化抗BLyS抗体或其抗原结合片段包含轻链可变区和重链可变区，所述轻链可变区包含如SEQ ID NO：9所示的氨基酸序列，所述重链可变区包含如SEQ ID NO：10所示的氨基酸序列；(b)约35-36mM组氨酸缓冲液，pH约为5.5-6.0；(c)约15mM的L-精氨酸盐酸盐；(d)约150mM的蔗糖；(e)以及约0.02％的聚山梨醇酯80；或
(13)(a)约200mg/mL的人源化抗BLyS抗体或其抗原结合片段，所述人源化抗BLyS抗体或其抗原结合片段包含轻链可变区和重链可变区，所述轻链可变区包含如SEQ ID NO：9所示的氨基酸序列，所述重链可变区包含如SEQ ID NO：10所示的氨基酸序列；(b)约35-36mM组氨酸缓冲液，pH约为5.5-6.0；(c)约15mM的L-精氨酸；(d)约150mM的蔗糖；(e)以及约0.02％的聚山梨醇酯80；或
(14)(a)约200mg/mL的人源化抗BLyS抗体或其抗原结合片段，所述人源化抗BLyS抗体或其抗原结合片段包含轻链可变区和重链可变区，所述轻链可变区包含如SEQ ID NO：9所示的氨基酸序列，所述重链可变区包含如SEQ ID NO：10所示的氨基酸序列；(b)约25mM组氨酸缓冲液，pH约为5.5-6.0；(c)约15mM的L-精氨酸盐酸盐；(d)约150mM的蔗糖；(e)以及约0.02％的聚山梨醇酯80；或
(15)(a)约100mg/mL的人源化抗BLyS抗体或其抗原结合片段，所述人源化抗BLyS抗体或其抗原结合片段包含轻链可变区和重链可变区，所述轻链可变区包含如SEQ ID NO：9所示的氨基酸序列，所述重链可变区包含如SEQ ID NO：10所示的氨基酸序列；(b)约25mM组氨酸缓冲液，pH约为5.5-6.0；(c)约25mM的L-精氨酸；(d)约100mM的氯化钠；(e)以及约0.02％的聚山梨醇酯80；或
(16)(a)约100mg/mL的人源化抗BLyS抗体或其抗原结合片段，所述人源化抗BLyS抗体或其抗原结合片段包含轻链可变区和重链可变区，所述轻链可变区包含如SEQ ID NO：9所示的氨基酸序列，所述重链可变区包含如SEQ ID NO：10所示的氨基酸序列；(b)约25mM组氨酸缓冲液，pH约为5.5-6.0；(c)约25mM的L-精氨酸盐酸盐；(d)约100mM的氯化钠；(e)以及约0.02％的聚山梨醇酯80；或
(17)(a)约100mg/mL的人源化抗BLyS抗体或其抗原结合片段，所述人源化抗BLyS抗体或其抗原结合片段包含轻链可变区和重链可变区，所述轻链可变区包含如SEQ ID NO：9所示的氨基酸序列，所述重链可变区包含如SEQ ID NO：10所示的氨基酸序列；(b)约25mM醋酸缓冲液，pH约为5.5-6.0；(c)约60mM的甲硫氨酸；(d)约100mM的氯化钠；(e)以及约0.02％的聚山梨醇酯80；或
(18)(a)约100mg/mL的人源化抗BLyS抗体或其抗原结合片段，所述人源化抗BLyS抗体或其抗原结合片段包含轻链可变区和重链可变区，所述轻链可变区包含如SEQ ID NO：9所示的氨基酸序列，所述重链可变区包含如SEQ ID NO：10所示的氨基酸序列；(b)约35-36mM组氨酸缓冲液，pH约为5.5-6.0；(c)约15mM的L-精氨酸盐酸盐；(d)约100mM的氯化钠；(e)以及约0.02％的聚山梨醇酯80；或
(19)(a)约100mg/mL的人源化抗BLyS抗体或其抗原结合片段，所述人源化抗BLyS抗体或其抗原结合片段包含轻链可变区和重链可变区，所述轻链可变区包含如SEQ ID NO：9所示的氨基酸序列，所述重链可变区包含如SEQ ID NO：10所示的氨基酸序列；(b)约35-36mM组氨酸缓冲液，pH约为5.5-6.0；(c)约15mM的L-精氨酸；(d)约100mM的氯化钠；(e)以及约0.02％的聚山梨醇酯80；或
(20)(a)约100mg/mL人源化抗BLyS抗体或其抗原结合片段，所述人源化抗BLyS抗体或其抗原结合片段包含轻链可变区和重链可变区，所述轻链可变区包含如SEQ ID NO：9所示的氨基酸序列，所述重链可变区包含如SEQ ID NO：10所示的氨基酸序列；(b)约35mM组氨酸缓冲液，pH约为5.8；(c)约15mM的L-精氨酸盐酸盐；(d)约100mM的氯化钠；(e)以及约0.02％的聚山梨醇酯80；
优选地，上述各药物组合物中，所述人源化抗BLyS抗体的轻链氨基酸序列如SEQ ID NO:18所示，重链氨基酸序列如SEQ ID NO:19所示。
在一些方案中，所述药物组合物为液体制剂或冻干制剂。
在一些方案中，所述药物组合物为液体制剂。
上述液体制剂或冻干制剂于2-8℃稳定至少3个月，至少6个月，至少12个月，至少18个月或至少24个月。
上述水溶液或冻干制剂于40℃稳定至少7天，至少14天或至少28天。
医药用途和方法
本发明提供了如本文任一项方案中所述的人源化抗BLyS抗体或其抗原结合片段、本文所述的多核苷酸、本文所述的表达载体、本文所述的宿主细胞、本文所述的药物组合物、或本文所述的注射剂在制备预防和/或治疗B细胞增殖过量引起的疾病的药物中的应用。
本发明提供了如本文任一项方案中所述的人源化抗BLyS抗体或其抗原结合片段、本文所述的多核苷酸、本文所述的表达载体、本文所述的宿主细胞、本文所述的药物组合物、或本文所述的注射剂，其用于预防和/或治疗B细胞增殖过量引起的疾病。
本发明提供了一种预防和/或治疗B细胞增殖过量引起的疾病的方法，其包括向有需要的受试者施用如本文任一项方案中所述的人源化抗BLyS抗体或其抗原结合片段、本文 所述的多核苷酸、本文所述的表达载体、本文所述的宿主细胞、本文所述的药物组合物、或本文所述的注射剂。
本发明中，B细胞增殖过量引起的疾病包括系统性红斑狼疮、类风湿性关节炎、强制性关节炎或B细胞淋巴癌。
下文将以具体实施例的方式阐述本发明。应理解，这些实施例仅仅是阐述性的，并非意图限制本发明的范围。实施例中所用到的方法和材料，除非另有说明，否则为本领域的常规方法和材料。
检测方法
蛋白降解的主要途径是聚集物、裂解产品和带电变体的形成。采用尺寸排阻色谱法(SEC-HPLC)测定天然形式(蛋白单体)与聚集形式所占的百分比，采用阳离子交换色谱法(CEX-HPLC)测定酸性与碱性形式抗体所占的百分比。
通过以下参数评估稳定性：(1)目视外观和可见异物；(2)紫外分光光度法测定蛋白含量；(3)SEC-HPLC测量抗体单体、聚体或片段的含量；(4)CEX-HPLC测量抗体主要电荷、酸性电荷或碱性电荷含量；(5)NR-CE-SDS法检测抗体的分子量；(6)R-CE-SDS法检测抗体的分子量；(7)ELISA法检测抗体结合活性。
(1)蛋白含量
蛋白浓度使用紫外分光光度计(Thermo，型号：Biomate 3S)来检测。将百分比消光系数(E1％)设定在1.47(g/mL)-1cm-1。使用BIO MATE 3S仪器，用超纯水清洗比色皿三次再向皿中加入150μL超纯水，点击测量，以超纯水做空白校正。每个样品平行测定2份溶液，每份溶液重复测定3次；每份溶液测定之前均需先用150μL溶液润洗比色皿2次，然后取150μL溶液进行测定。通过消光系数及OD值计算出对应检品的浓度。
(2)SEC-HPLC纯度
SEC纯度采用安装了SEC色谱柱(TOSOH G4000sxl SEC 7.8mm ID x 30cm,5μm)的HPLC(Ultimate 3000,DIONEX仪器)进行检测。流动相组成为50mM PB/300mM NaCl pH7.0±0.2。采用面积归一化法计算主峰，高聚物和低聚物的相对百分比。详细见下表1：
表1：SEC-HPLC纯度检测方法
色谱条件 色谱参数
检测器波长 280nm
自动进样器温度 25±3℃
柱温 25±2℃
流速 1mL/min
进样体积 20μl
运行模式 等度模式
运行时间 20.00min
(3)CEX-HPLC纯度
采用峰面积归一化对结果进行定量分析，计算K0、K1、K2的峰面积百分比(K0为峰面积百分比最高的那个峰，K1为K0之后的第一个高峰，K2与K1的保留时间差值约为2.5min)。K0、K1、K2三个峰的峰面积百分比之和为赖氨酸变体含量(即为样品纯度)，K0峰前的所有峰峰面积百分比之和为酸性峰含量，K0峰后除去K1、K2峰后余下的所有峰峰面积百分比之和为碱性峰含量。详细见下表2：
表2：CEX-HPLC纯度检测方法
Figure PCTCN2022088507-appb-000001
(4)不溶性微粒
打开仪器，调节照度旋钮使照度为1200lx。
取供试品20支，去除容器标签，擦拭容器外壁使容器干净透明，室温静置12小时；
把供试品置于遮光板边缘处，轻轻旋转和翻转容器，使药液中可能存在的可见异物悬浮(不要使药液产生气泡)，在约25厘米处，分别在黑色和白色背景下观察，检查时限20秒。
实施例1：抗体制备
抗体序列设计：
以抗体H48L47(轻链如SEQ ID NO：16所示和重链如SEQ ID NO：17所示)起始，出于降低抗体的免疫原性，提高稳定性等目的，对抗体的框架区(FR)进行突变，筛选出以下多种氨基酸序列：轻链L47(SEQ ID NO：16)的FR区进行突变，产生L472(SEQ ID NO：18)、L473(SEQ ID NO：20)和L474(SEQ ID NO：22)三个序列，将重链H48(SEQ ID NO：17)的FR区进行突变，产生H482(SEQ ID NO：19)、H483(SEQ ID NO：21)、H484(SEQ ID NO：23)和H485(SEQ ID NO：24)四个序列。
将上述轻重链两两配对组合，构建表达载体，抗体表达轻重链组合如下表3所示，使用293F细胞进行瞬转表达，100ml体系、经过一步亲和纯化得到蛋白，得到的二十个人源化抗体如下表4所示。
表3：抗体表达轻重链组合表
  H48 H482 H483 H484 H485
L47 H48L47 H482L47 H483L47 H484L47 H485L47
L472 H48L472 H482L472 H483L472 H484L472 H485L472
L473 H48L473 H482L473 H483L473 H484L473 H485L473
L474 H48L474 H482L474 H483L474 H484L474 H485L474
表4：抗体的可变区氨基酸序列和全长序列
抗体名称 VL VH LC HC
H48L47 SEQ ID NO：7 SEQ ID NO：8 SEQ ID NO：16 SEQ ID NO：17
H48L472 SEQ ID NO：9 SEQ ID NO：8 SEQ ID NO：18 SEQ ID NO：17
H48L473 SEQ ID NO：11 SEQ ID NO：8 SEQ ID NO：20 SEQ ID NO：17
H48L474 SEQ ID NO：13 SEQ ID NO：8 SEQ ID NO：22 SEQ ID NO：17
H482L47 SEQ ID NO：7 SEQ ID NO：10 SEQ ID NO：16 SEQ ID NO：19
H482L472 SEQ ID NO：9 SEQ ID NO：10 SEQ ID NO：18 SEQ ID NO：19
H482L473 SEQ ID NO：11 SEQ ID NO：10 SEQ ID NO：20 SEQ ID NO：19
H482L474 SEQ ID NO：13 SEQ ID NO：10 SEQ ID NO：22 SEQ ID NO：19
H483L47 SEQ ID NO：7 SEQ ID NO：12 SEQ ID NO：16 SEQ ID NO：21
H483L472 SEQ ID NO：9 SEQ ID NO：12 SEQ ID NO：18 SEQ ID NO：21
H483L473 SEQ ID NO：11 SEQ ID NO：12 SEQ ID NO：20 SEQ ID NO：21
H483L474 SEQ ID NO：13 SEQ ID NO：12 SEQ ID NO：22 SEQ ID NO：21
H484L47 SEQ ID NO：7 SEQ ID NO：14 SEQ ID NO：16 SEQ ID NO：23
H484L472 SEQ ID NO：9 SEQ ID NO：14 SEQ ID NO：18 SEQ ID NO：23
H484L473 SEQ ID NO：11 SEQ ID NO：14 SEQ ID NO：20 SEQ ID NO：23
H484L474 SEQ ID NO：13 SEQ ID NO：14 SEQ ID NO：22 SEQ ID NO：23
H485L47 SEQ ID NO：7 SEQ ID NO：15 SEQ ID NO：16 SEQ ID NO：24
H485L472 SEQ ID NO：9 SEQ ID NO：15 SEQ ID NO：18 SEQ ID NO：24
H485L473 SEQ ID NO：11 SEQ ID NO：15 SEQ ID NO：20 SEQ ID NO：24
H485L474 SEQ ID NO：13 SEQ ID NO：15 SEQ ID NO：22 SEQ ID NO：24
实施例2：Binding ELISA法测定抗体与人BLyS蛋白的结合能力
将人BLyS包被在疏水酶标板上，用Binding ELISA法测定抗体与人BLyS蛋白的结合能力，拟合曲线算出EC50。测试结果如下表5所示。
表5：抗体与人BLyS蛋白的结合能力
抗体名称 EC50(ng/mL)
H48L47 21
H48L472 22.78
H48L473 22.76
H48L474 17.26
H482L47 14.67
H482L472 17.54
H482L473 11.9
H482L474 11.55
H483L47 11.86
H483L472 13.82
H483L473 14.43
H483L474 15.68
H484L47 19.02
H484L472 24.7
H484L473 15.37
H484L474 10.81
H485L47 7.588
H485L472 7.25
H485L473 11.73
H485L474 7.685
结果显示，本发明20个抗体与重组人BLyS蛋白均有结合，经过框架区(FR)序列改造的抗体，其EC50较H48L47的EC50更小或相当。
实施例3：Blocking ELISA法测定抗体竞争抑制人BLyS与人BR3结合的能力
将人BLyS包被在疏水酶标板上，用Blocking ELISA法测定经过框架区(FR)序列改造的抗体竞争抑制人BLyS与人BR3结合的能力，拟合曲线算出IC50。Binding ELISA结果显示经过框架区(FR)序列改造的抗体的EC50比未经过改造的抗体的EC50更小，因此只选择轻重链都经过改造的组合，即H482L472、H483L473和H484L474进行Blocking ELISA法测定，并且与已上市阳性对照抗体Benlysta进行比较。测试结果如下表6所示。
阳性对照：Benlysta，Human Genome Sciences，专利参见CN1279055C、CN106661111A。
表6：抗体竞争抑制人BLyS与人BR3结合的能力
抗体名称 IC50(μg/mL)
H482L472 1.238
H483L473 0.8576
H484L474 1.275
Benlysta 4.714
由测试结果可见，经过框架区(FR)序列改造的抗体和阳性药Benlysta均可抑制BLyS和其受体BR3结合，抗体H482L472，H483L473和H484L474的IC50显著优于对照Benlysta。
实施例4：报告基因法测定抗体竞争抑制人BLyS结合TACI刺激的下游NF-κB信号
人可溶性BLyS与TACI结合，刺激下游NF-κB信号通路，下游的荧光素酶也随之释放信号被检测到，抗体阻断BLyS和TACI结合，则下游的NF-κB信号将被抑制，用荧光素酶检测试剂盒可以检测其信号值的变化，拟合曲线算出IC50。同样选择轻重链都经过改造的组合，即H482L472、H483L473和H484L474，进行细胞生物学测定，并且与已上市阳性对照抗体Benlysta进行比较，测试结果如下表7所示。
阳性对照：Benlysta，Human Genome Sciences，专利参见CN1279055C、CN106661111A。
表7：抗体竞争抑制人BLyS结合TACI刺激的下游NF-κB信号
抗体名称 IC 50(ng/mL)
H482L472 37.56
H483L473 29.27
H484L474 31.52
Benlysta 51.68
由测试结果可见，经过框架区(FR)序列改造的抗体和阳性药Benlysta均可抑制人BLyS结合TACI刺激的下游NF-κB信号，抗体H482L472，H483L473和H484L474的IC50显著优于对照Benlysta。
实施例5：缓冲液体系、pH筛选实验
液体型药物组合物中，缓冲液体系、pH密切影响抗体的稳定性，每种具有独特理化性质的抗体都具有最适宜的缓冲液的种类、pH。本实施例旨在筛选一种最佳缓冲液体系和pH，使本发明公开的抗体具有最佳的稳定性以适宜临床应用。本实施例中的抗体为H482L472。
1.1缓冲液配制
分别配制25mM的醋酸钠(pH5.0,5.5,6.0)、25mM的磷酸钠(pH6.0,6.5,7.0)、35mM的组氨酸(pH5.5,6.0,6.5)、25mM的琥珀酸缓冲液(pH5.5,6.0,6.5)，每种溶液各配制50mL。
1.2换液
用15mL离心超滤管(Millipore，UCF803096)进行离心超滤换液(5000g×15min)。每组样品1mL蛋白，用2mL缓冲液换液3次。换液完成后，用分光光度计测定浓度，将制剂终浓度调节至抗体浓度为约30mg/mL。
1.3除菌过滤及放样
在超净台中用针头滤器将换液后的样品除菌过滤于1.5mL冻存管中，每支约1mL。于37℃孵育7d，然后检测SEC、CEX-HPLC。
实验方案如下表8所示。
表8：缓冲液体系和pH筛选方案
Figure PCTCN2022088507-appb-000002
Figure PCTCN2022088507-appb-000003
检测结果如下表9所示。
表9：缓冲液体系和pH筛选检测结果
Figure PCTCN2022088507-appb-000004
从实验结果看出，在37℃放置6d后，醋酸盐缓冲液和组氨酸盐缓冲液的SEC和CEX纯度下降幅度比较小，优于磷酸盐和琥珀酸盐缓冲液。因此选择醋酸盐和组氨酸盐缓冲液进行下一轮的筛选。
实施例6：稳定剂筛选实验
根据文献报道，当单抗制剂中蛋白浓度较高时，往往会导致蛋白凝集、高聚物含量增加等不利影响。本实验在第一轮筛选出的制剂缓冲体系的基础上，添加了几种不同氨基酸(L-精氨酸、甲硫氨酸)，考察这些辅料对制剂稳定性的影响。
2.1缓冲液配制
分别按表10和11中的缓冲液配方进行配制。其中组氨酸盐缓冲液含量为35mM；醋酸盐缓冲液含量为25mM，本实施例中的抗体为H482L472，各制剂处方中抗体终浓度为100mg/mL，各制剂处方中氯化钠浓度为100mM。
表10：组氨酸缓冲液体系下稳定剂筛选实验方案
Figure PCTCN2022088507-appb-000005
表11：醋酸缓冲液体系下稳定剂筛选实验方案
Figure PCTCN2022088507-appb-000006
Figure PCTCN2022088507-appb-000007
2.2换液
用50mL离心超滤管(Millipore，UFC903096)进行超滤浓缩换液(5000g×25min)。每组样品3.5mL，超滤浓缩至1mL后加对应的缓冲液4mL换液3次。换液完成后定容至1mL备用，样品中抗体终浓度约为100mg/mL。
2.3除菌过滤
在超净台中将换液后的样品无菌过滤后，分装至1mL预灌封注射器中，每支约1mL。
2.4放样和检测
除菌过滤后的样品置于37℃恒温培养箱内孵育7d。检测SEC、CEX-HPLC。
实验结果如下表12和表13所示。
表12：组氨酸缓冲液体系下稳定剂筛选实验结果
Figure PCTCN2022088507-appb-000008
表13：醋酸缓冲液体系下稳定剂筛选实验结果
Figure PCTCN2022088507-appb-000009
Figure PCTCN2022088507-appb-000010
根据实验结果，无论组氨酸或醋酸，制剂的pH对稳定性都具有显著影响。随着pH的升高，纯度呈现先增大后减小的趋势，而在制剂处方中加入L-精氨酸或甲硫氨酸能有效降低聚体的产生。根据以上结果，筛选出两组制剂处方：35mM组氨酸+15mM的L-精氨酸+100mM氯化钠，pH5.8；25mM醋酸+60mM甲硫氨酸+100mM氯化钠，pH5.8。
实施例7：强制降解实验
对筛选出的处方做进一步的强制降解实验，以考察处方在各种筛选压力下的稳定性。本实施例中的抗体为H482L472，实验方案如下表14所示。
表14：强制降解实验方案
Figure PCTCN2022088507-appb-000011
3.1缓冲液配制
按下表15的配方分别配制实验所需的制剂缓冲液各500mL，用0.2μm滤器过滤后备用。
表15：制剂缓冲液配方
Figure PCTCN2022088507-appb-000012
3.2浓缩换液
用50mL离心超滤管(Millipore，UFC903096)进行超滤浓缩换液(5000g×25min)。每管加原液4.5mL，超滤浓缩至约1.5mL后加对应的缓冲液4mL换液3次。换液完成后混匀测蛋白浓度，调节样品终浓度约为100mg/mL。
3.3配制吐温-80母液
配制含2％吐温-80的水溶液10mL，震荡混匀。向换液后的样品中加入适量体积的配制好的吐温-80母液，调节样品中的吐温-80含量为0.02％。
3.4除菌过滤和分装
在超净台中将配制好的样品用0.2μm除菌滤器过滤后，分装至1mL预灌封注射器中，每支约1mL。
3.5放样和检测
按表14中的方案放样，并对样品进行检测。
实验结果如下表16-20所示。
表16：振摇实验结果
Figure PCTCN2022088507-appb-000013
表17：冻融实验结果
Figure PCTCN2022088507-appb-000014
Figure PCTCN2022088507-appb-000015
表18：紫外照射实验结果
Figure PCTCN2022088507-appb-000016
表19：SEC/CEX纯度高温加速实验结果
Figure PCTCN2022088507-appb-000017
表20：外观和可见异物高温加速试验结果
Figure PCTCN2022088507-appb-000018
Figure PCTCN2022088507-appb-000019
从实验结果可知，对F1和F2处方振摇和冻融后未见明显纯度下降，但紫外照射8h后两种处方制剂的SEC和CEX纯度均有下降趋势，提示制剂处方对紫外光较敏感，需要避光保存；高温加速实验显示样品在高温条件下放置4W和8W后外观均没有明显变化，但酸性峰和聚体含量均有明显上升，其中，处方F1上升的趋势要略低于F2。在后续按处方F1进行生产时，因L-精氨酸碱性略高需要用盐酸调节pH，后将原处方F1中L-精氨酸替换为L-精氨酸盐酸盐(CAS号：1119-34-2)，对更换后的处方F3(制剂处方：100mg/mL抗体+35mM组氨酸缓冲液+15mM的L-精氨酸盐酸盐+100mM氯化钠+0.02％吐温-80，pH5.8)进行稳定性考察，在4℃和37℃条件下放置4W后，检测SEC和CEX纯度。检测结果见下表21。
表21：更换盐酸精氨酸后的稳定性检测结果
Figure PCTCN2022088507-appb-000020
Figure PCTCN2022088507-appb-000021
从实验结果可以看出，将精氨酸更换为精氨酸盐酸盐后，SEC和CEX的下降趋势与使用精氨酸的情况基本一致，本发明药物组合物分别添加上述两种稳定剂均可使处方制剂保持较佳的稳定性。
实施例8：配伍稳定性研究实验
4.1生理盐水配制
用超纯水配制0.9％生理盐水500mL，用磁力搅拌器搅拌至完全溶解后备用。用超纯水配制0.9％生理盐水500mL，用磁力搅拌器搅拌至完全溶解后备用。
4.2样品稀释
用5.4.1.1中所配制的生理盐水将制剂样品(制剂处方：100mg/mL抗体+35mM组氨酸缓冲液+15mM的L-精氨酸盐酸盐+100mM氯化钠+0.02％吐温-80，pH5.8)稀释至对应的终浓度(60、30、10、3mg/mL)，稀释后的样品体积约为25mL，将样品充分摇匀后备用；本实施例中的抗体为H482L472。
4.3样品分装
稀释后的样品在超净工作台中除菌过滤后，无菌分装于6mL西林瓶中，每个浓度样品分装5瓶，每瓶装量5mL，加塞后用轧盖机压盖。
4.4放样和检测
将各组样品分别于25℃和30℃孵育0、2、4h后取样，样品保存于-80℃。于-80℃冻存5天后送检，检测样品SEC纯度、生物活性(blocking ELISA)和不溶性微粒。
实验结果如下表22和表23所示。
表22：25℃孵育后检测结果
Figure PCTCN2022088507-appb-000022
Figure PCTCN2022088507-appb-000023
表23：30℃孵育后检测结果
Figure PCTCN2022088507-appb-000024
从实验结果看出，各浓度组别的样品在25℃和30℃孵育2h和4h后各项检测结果无明显的下降，均符合产品质量标准的要求。因此，可以判断制剂用生理盐水稀释后在室温保存4h内性质稳定。
配伍稳定性实验显示，最终处方经0.9％氯化钠溶液稀释，在蛋白浓度3～60mg/ml的范围内，室温放置4小时内样品性质保持稳定。
综上所述，通过对不同缓冲体系、不同pH条件、不同抗体浓度和不同辅料组成进行考察，最终确定了最优制剂配方。采用组氨酸缓冲液来调节pH，盐酸精氨酸和氯化钠调节制剂渗透压，添加聚山梨酯20来增加制剂溶解性。
实施例9：UBP1213sc制剂对人BLyS诱导的Balb/c小鼠生理指标改变的抑制作用研究
根据体重随机分组，每组10只，共7组，分别为：G1
Figure PCTCN2022088507-appb-000025
组、G2 Vehicle模型对照组、G3 KLH hIgG1组、G4 UBP1213sc 5mg/kg组、G5 UBP1213sc 1.5mg/kg组、G6 UBP1213sc 0.5mg/kg组和G7 UBP1213sc 0.15mg/kg组。Day1和Day3给药，共给药2次。Day1至Day4皮下给予0.3mg/kg人BLyS。实验期间，每天检测动物体重。实验结束时，动物安乐死，采血检测动物血清中IgA水平，剥取脾脏称重计算脏器系数、FACS检测脾脏中B220 +/ThB +细胞比例。其中，UBP1213sc制剂处方：100mg/mL抗体H482L472+35mM组氨酸缓冲液+15mM的L-精氨酸盐酸盐+100mM氯化钠+0.02％吐温-80，pH5.8。测试结果见表24-26。
表24：UBP1213sc对BLyS刺激的小鼠脾脏湿重和脾脏脏器系数的影响(数据采用mean±SEM表示，n＝10)
Figure PCTCN2022088507-appb-000026
注： *:P<0.05； **:P<0.01； ***:P<0.01vs
Figure PCTCN2022088507-appb-000027
#:P<0.05； ##:P<0.01； ###:P<0.01vs Vehicle。
表25：UBP1213sc对BLyS刺激的小鼠脾脏中B220 +/ThB +比例的影响(数据采用mean±SEM表示，n＝10)
Figure PCTCN2022088507-appb-000028
Figure PCTCN2022088507-appb-000029
注： *:P<0.05； **:P<0.01； ***:P<0.01vs
Figure PCTCN2022088507-appb-000030
#:P<0.05； ##:P<0.01； ###:P<0.01vs Vehicle。
表26：UBP1213sc对BLyS刺激的小鼠血清中IgA水平的影响(数据采用mean±SEM表示，n＝10)
Figure PCTCN2022088507-appb-000031
注： *:P<0.05；**:P<0.01；***:P<0.001vs
Figure PCTCN2022088507-appb-000032
#:P<0.05； ##:P<0.01； ###:P<0.01vs Vehicle。
根据表24-26的实验结果显示，与
Figure PCTCN2022088507-appb-000033
组相比，Vehicle组结果显示0.3mg/kg的人BLyS(皮下注射)成功的诱导了Balb/c小鼠脾脏湿重/脾脏脏器系数、脾脏B220+/ThB+细胞比例、血清IgA水平显著增加。相对于Vehicle组能显著抑制模型小鼠各生理指标的增加；1.5mg/kg的UBP1213皮下能显著抑制模型小鼠各生理指标的增加；0.5、1.5和5mg/kg的UBP1213sc能显著抑制模型小鼠各生理指标的增加。
皮下注射人BLyS可以诱导Balb/c小鼠脾脏湿重/脾脏脏器系数、脾脏B220+/ThB+细胞比例以及血清IgA水平显著增加。皮下注射给予不同剂量(0.15，0.5，1.5，5mg/kg)的抗BLyS单克隆抗体UBP1213sc，1.5和5mg/kg的UBP1213sc能显著抑制小鼠脾脏湿重/脾脏脏器系数的增加，0.5、1.5和5mg/kg的UBP1213sc均能显著抑制B220+/ThB+细胞比例以及血清IgA水平的增加，并呈现出良好的剂量效应关系。且各给药组动物体重均无显著改变。

Claims (19)

  1. 一种人源化抗BLyS抗体或其抗原结合片段，其包含重链可变区和轻链可变区，其特征在于，
    所述人源化抗BLyS抗体或其抗原结合片段的LCDR1、LCDR2和LCDR3的氨基酸序列分别如SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3所示；
    所述人源化抗BLyS抗体或其抗原结合片段的HCDR1、HCDR2和HCDR3的氨基酸序列分别如SEQ ID NO:4、SEQ ID NO:5和SEQ ID NO:6所示；
    所述人源化抗BLyS抗体或其抗原结合片段的轻链可变区的FR1选自SEQ ID NO:7、9和13中任一所述的FR1，FR2为SEQ ID NO:7所述的FR2，FR3选自SEQ ID NO:7、9和11中任一所述的FR3，FR4为SEQ ID NO:7所述的FR4；
    所述人源化抗BLyS抗体或其抗原结合片段的重链可变区的FR1选自SEQ ID NO:8、10、12和15中任一所述的FR1，FR2选自SEQ ID NO:8、10和15中任一所述的FR2，FR3选自SEQ ID NO:8、12和14中任一所述的FR3，FR4为SEQ ID NO:8或10所述的FR4；且
    所述人源化抗BLyS抗体或其抗原结合片段不包括：轻链可变区为SEQ ID NO:7所示的氨基酸序列，同时重链可变区为SEQ ID NO:8所示的氨基酸序列的人源化抗BLyS抗体或其抗原结合片段。
  2. 如权利要求1所述的人源化抗BLyS抗体或其抗原结合片段，其特征在于，
    所述人源化抗BLyS抗体或其抗原结合片段中，轻链可变区的FR1选自SEQ ID NO:7、9和13中任一所述的FR1，FR2为SEQ ID NO:7所述的FR2，FR3选自SEQ ID NO:7、9和11中任一所述的FR3，FR4为SEQ ID NO:7所述的FR4；重链可变区的FR1-FR4分别为SEQ ID NO:15的FR1-FR4；或
    所述人源化抗BLyS抗体或其抗原结合片段中，轻链可变区的FR1选自SEQ ID NO:7、9和13中任一所述的FR1，FR2为SEQ ID NO:7所述的FR2，FR3选自SEQ ID NO:7、9和11中任一所述的FR3，FR4为SEQ ID NO:7所述的FR4；重链可变区的FR1-FR4分别为SEQ ID NO:10的FR1-FR4；或
    所述人源化抗BLyS抗体或其抗原结合片段中，轻链可变区的FR1选自SEQ ID NO:7、9和13中任一所述的FR1，FR2为SEQ ID NO:7所述的FR2，FR3选自SEQ ID NO:7、9和11中任一所述的FR3，FR4为SEQ ID NO:7所述的FR4；重链可变区的FR1-FR4分别为SEQ ID NO:12的FR1-FR4；或
    所述人源化抗BLyS抗体或其抗原结合片段中，轻链可变区的FR1为SEQ ID NO:11 或13所述的FR1，FR2为SEQ ID NO:11所述的FR2，FR3为SEQ ID NO:11或13所述的FR3，FR4为SEQ ID NO:11所述的FR4；重链可变区的FR1-FR4分别为SEQ ID NO:14的FR1-FR4。
  3. 如权利要求1所述的人源化抗BLyS抗体或其抗原结合片段，其特征在于，所述的人源化抗BLyS抗体或其抗原结合片段的轻链可变区包含如SEQ ID NO：7、SEQ ID NO：9、SEQ ID NO：11或SEQ ID NO：13所示的氨基酸序列，所述重链可变区包含如SEQ ID NO：8、SEQ ID NO：10、SEQ ID NO：12、SEQ ID NO：14或SEQ ID NO：15所示的氨基酸序列。
  4. 如权利要求1所述的人源化抗BLyS抗体或其抗原结合片段，其包含轻链可变区和重链可变区，其中：
    (1)所述轻链可变区包含如SEQ ID NO：7所示的氨基酸序列，和所述重链可变区包含如SEQ ID NO：10、SEQ ID NO：12、SEQ ID NO：14或SEQ ID NO：15所示的氨基酸序列；或
    (2)所述轻链可变区包含如SEQ ID NO：9所示的氨基酸序列，和所述重链可变区包含如SEQ ID NO：8、SEQ ID NO：10、SEQ ID NO：12、SEQ ID NO：14或SEQ ID NO：15所示的氨基酸序列；或
    (3)所述轻链可变区包含如SEQ ID NO：11所示的氨基酸序列，和所述重链可变区包含如SEQ ID NO：8、SEQ ID NO：10、SEQ ID NO：12、SEQ ID NO：14或SEQ ID NO：15所示的氨基酸序列；或
    (4)所述轻链可变区包含如SEQ ID NO：13所示的氨基酸序列，和所述重链可变区包含如SEQ ID NO：8、SEQ ID NO：10、SEQ ID NO：12、SEQ ID NO：14或SEQ ID NO：15所示的氨基酸序列；或
    (5)所述轻链可变区包含如SEQ ID NO：9所示的氨基酸序列，和所述重链可变区包含如SEQ ID NO：10所示的氨基酸序列；或
    (6)所述轻链可变区包含如SEQ ID NO：11所示的氨基酸序列，和所述重链可变区包含如SEQ ID NO：12所示的氨基酸序列；或
    (7)所述轻链可变区包含如SEQ ID NO：13所示的氨基酸序列，和所述重链可变区包含如SEQ ID NO：14所示的氨基酸序列；或
    (8)所述轻链可变区包含如SEQ ID NO：7所示的氨基酸序列，和所述重链可变区包含如SEQ ID NO：15所示的氨基酸序列；或
    (9)所述轻链可变区包含如SEQ ID NO：9所示的氨基酸序列，和所述重链可变区包含如SEQ ID NO：15所示的氨基酸序列；或
    (10)所述轻链可变区包含如SEQ ID NO：11所示的氨基酸序列，和所述重链可变区包含如SEQ ID NO：15所示的氨基酸序列；或
    (11)所述轻链可变区包含如SEQ ID NO：13所示的氨基酸序列，和所述重链可变区包含如SEQ ID NO：15所示的氨基酸序列；或
    (12)所述重链可变区包含如SEQ ID NO：8所示的氨基酸序列，和所述轻链可变区包含如SEQ ID NO：13所示的氨基酸序列；或
    (13)所述重链可变区包含如SEQ ID NO：10所示的氨基酸序列，和所述轻链可变区包含如SEQ ID NO：7、9、11或13所示的氨基酸序列；或
    (14)所述重链可变区包含如SEQ ID NO：12所示的氨基酸序列，和所述轻链可变区包含如SEQ ID NO：7、9、11或13所示的氨基酸序列；或
    (15)所述重链可变区包含如SEQ ID NO：14所示的氨基酸序列，和所述轻链可变区包含如SEQ ID NO：11或13所示的氨基酸序列；或
    (16)所述重链可变区包含如SEQ ID NO：15所示的氨基酸序列，和所述轻链可变区包含如SEQ ID NO：7、9、11或13所示的氨基酸序列。
  5. 如权利要求1所述的人源化抗BLyS抗体或其抗原结合片段，其中，所述人源化抗BLyS抗体还含有人类轻链恒定区和重链恒定区，且所述轻链可变区及重链可变区分别连接至人类轻链恒定区和重链恒定区；优选地，所述人类轻链恒定区为人类轻链κ恒定区；优选地，所述人类重链恒定区为人类重链Fc片段。
  6. 如权利要求1-5中任一项所述的人源化抗BLyS抗体或其抗原结合片段，其包含轻链和重链，其中，所述轻链包含如SEQ ID NO：16、SEQ ID NO：18、SEQ ID NO：20或SEQ ID NO：22所示的氨基酸序列，所述重链包含如SEQ ID NO：17、SEQ ID NO：19、SEQ ID NO：21、SEQ ID NO：23或SEQ ID NO：24所示的氨基酸序列。
  7. 如权利要求6所述的人源化抗BLyS抗体或其抗原结合片段，其包含轻链和重链，其中：
    (1)所述轻链包含如SEQ ID NO：16所示的氨基酸序列，和所述重链包含如SEQ ID NO：19、SEQ ID NO：21、SEQ ID NO：23或SEQ ID NO：24所示的氨基酸序列；或
    (2)所述轻链包含如SEQ ID NO：18所示的氨基酸序列，和所述重链包含如SEQ ID NO：17、SEQ ID NO：19、SEQ ID NO：21、SEQ ID NO：23或SEQ ID NO：24所示的氨基酸序列；或
    (3)所述轻链包含如SEQ ID NO：20所示的氨基酸序列，和所述重链包含如SEQ ID NO：17、SEQ ID NO：19、SEQ ID NO：21、SEQ ID NO：23或SEQ ID NO：24所示的氨基酸序列；或
    (4)所述轻链包含如SEQ ID NO：22所示的氨基酸序列，和所述重链包含如SEQ ID NO：17、SEQ ID NO：19、SEQ ID NO：21、SEQ ID NO：23或SEQ ID NO：24所示的氨基酸序列；或
    (5)所述轻链包含如SEQ ID NO：18所示的氨基酸序列，和所述重链包含如SEQ ID NO：19所示的氨基酸序列；或
    (6)所述轻链包含如SEQ ID NO：20所示的氨基酸序列，和所述重链包含如SEQ ID NO：21所示的氨基酸序列；或
    (7)所述轻链包含如SEQ ID NO：22所示的氨基酸序列，和所述重链包含如SEQ ID NO：23所示的氨基酸序列；或
    (8)所述轻链包含如SEQ ID NO：16所示的氨基酸序列，和所述重链包含如SEQ ID NO：24所示的氨基酸序列；或
    (9)所述轻链包含如SEQ ID NO：18所示的氨基酸序列，和所述重链包含如SEQ ID NO：24所示的氨基酸序列；或
    (10)所述轻链包含如SEQ ID NO：20所示的氨基酸序列，和所述重链包含如SEQ ID NO：24所示的氨基酸序列；或
    (11)所述轻链包含如SEQ ID NO：22所示的氨基酸序列，和所述重链包含如SEQ ID NO：24所示的氨基酸序列；或
    (12)所述重链包含如SEQ ID NO：17所示的氨基酸序列，和所述轻链包含如SEQ ID NO：22所示的氨基酸序列；或
    (13)所述重链包含如SEQ ID NO：19所示的氨基酸序列，和所述轻链包含如SEQ ID NO：16、18、20或22所示的氨基酸序列；或
    (14)所述重链包含如SEQ ID NO：21所示的氨基酸序列，和所述轻链包含如SEQ ID NO：16、18、20或22所示的氨基酸序列；或
    (15)所述重链包含如SEQ ID NO：23所示的氨基酸序列，和所述轻链包含如SEQ ID NO：20或22所示的氨基酸序列；或
    (16)所述重链包含如SEQ ID NO：24所示的氨基酸序列，和所述轻链包含如SEQ ID NO：16、18、20或22所示的氨基酸序列。
  8. 一种多核苷酸分子，选自：
    (1)编码如权利要求1-7中任一项所述的人源化抗BLyS抗体或其抗原结合片段的多核苷酸分子；和
    (2)如(1)中所述多核苷酸分子的互补序列。
  9. 表达载体，其包含如权利要求8所述的多核苷酸分子；优选地，所述表达载体为 真核表达载体。
  10. 宿主细胞，其包含如权利要求8所述的多核苷酸分子或如权利要求9所述的表达载体；优选地，所述宿主细胞是真核细胞；更优选是哺乳动物细胞。
  11. 药物组合物，包含：(1)缓冲液；(2)人源化抗BLyS抗体或其抗原结合片段，所述人源化抗BLyS抗体或其抗原结合片段具有氨基酸序列分别如SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3所示的LCDR1、LCDR2和LCDR3，和氨基酸序列分别如SEQ ID NO:4、SEQ ID NO:5和SEQ ID NO:6所示的HCDR1、HCDR2和HCDR3。
  12. 如权利要求11所述的药物组合物，其中，所述人源化抗BLyS抗体或其抗原结合片段如权利要求1-7中任一项所述。
  13. 如权利要求11或12所述的药物组合物，其中，所述药物组合物具有以下一项或多项特征：
    所述人源化抗BLyS抗体或其抗原结合片段的浓度约为10-300mg/mL，优选约为20-280mg/mL，优选约为30-150mg/mL，优选约为80-120mg/mL，优选约为180-220mg/mL；
    所述缓冲液选自醋酸缓冲液、柠檬酸缓冲液、琥珀酸缓冲液、磷酸缓冲液和组氨酸缓冲液中的一种或多种；优选地，所述缓冲液的浓度约为5-50mM，优选约为10-40mM，优选约为20-40mM；优选地，所述缓冲液的pH约为5.0-6.5，优选约为5.0-6.0，优选约为5.5-6.0；
    所述药物组合物还包括稳定剂，所述稳定剂选自精氨酸、精氨酸盐酸盐、甲硫氨酸、脯氨酸、甘氨酸、氯化钠、甘露醇、山梨醇、蔗糖、麦芽糖、木糖醇和海藻糖中的一种或多种；优选地，所述稳定剂选自L-精氨酸、L-精氨酸盐酸盐、蔗糖、甲硫氨酸和氯化钠中的一种或多种；优选地，所述稳定剂的浓度为约50mM～300mM，优选约100mM～300mM，更优选约100mM～200mM；
    所述药物组合物还包括表面活性剂；优选地，所述表面活性剂选自聚山梨醇酯80、聚山梨醇酯20、泊洛沙姆188中的一种或多种；优选地，以w/v计算，所述表面活性剂浓度约为0.001％-0.1％，优选约为0.01％-0.1％，优选约为0.01％-0.05％。
  14. 如权利要求13所述的药物组合物，其中，所述稳定剂为浓度约10-30mM的L-精氨酸盐酸盐与约50-200mM的氯化钠的组合；或所述稳定剂为浓度约10-30mM的L-精氨酸与约50-200mM的氯化钠的组合；或所述稳定剂为浓度约10-30mM的L-精氨酸与约80-220mM的蔗糖的组合；或所述稳定剂为浓度约10-30mM的L-精氨酸盐酸盐与约80-220mM的蔗糖的组合；或所述稳定剂为约30-90mM的甲硫氨酸与约50-200mM的氯化钠的组合；或所述稳定剂为约10-120mM的L-精氨酸盐酸盐；或所述稳定剂为约10-120mM的甲硫氨酸；优选地，所述稳定剂为浓度约10-30mM的L-精氨酸盐酸盐与约 80-120mM的氯化钠的组合；或所述稳定剂为浓度约10-30mM的L-精氨酸与约80-120mM的氯化钠的组合；或所述稳定剂为约50-70mM的甲硫氨酸与约80-120mM的氯化钠的组合；或所述稳定剂为浓度约10-30mM的L-精氨酸与约130-170mM的蔗糖的组合；或所述稳定剂为浓度约10-30mM的L-精氨酸盐酸盐与约130-170mM的蔗糖的组合。
  15. 如权利要求11或12所述的药物组合物，其特征在于，
    所述药物组合物包含：(a)约20mg/mL-280mg/mL的人源化抗BLyS抗体或其抗原结合片段；(b)约5-50mM的组氨酸缓冲液或约5-50mM的醋酸缓冲液，pH约为5.0-6.5；(c)约10-30mM的L-精氨酸或约10-30mM的L-精氨酸盐酸盐；(d)约80-220mM的蔗糖；(e)以及约0.01％-0.1％的聚山梨醇酯80；或
    所述药物组合物含有：(a)约30mg/mL-150mg/mL的人源化抗BLyS抗体或其抗原结合片段；(b)约5-50mM的组氨酸缓冲液或约5-50mM的醋酸缓冲液，pH约为5.0-6.5；(c)约10-30mM的L-精氨酸、约10-30mM的L-精氨酸盐酸盐或约30-90mM的甲硫氨酸；(d)约50-200mM的氯化钠；(e)以及约0.01％-0.1％的聚山梨醇酯80；或
    所述药物组合物含有：(a)约80mg/mL-120mg/mL的人源化抗BLyS抗体或其抗原结合片段；(b)约20-40mM的组氨酸缓冲液或约20-40mM的醋酸缓冲液，pH约为5.5-6.0；(c)约10-30mM的L-精氨酸、约50-70mM的甲硫氨酸或约10-30mM的L-精氨酸盐酸盐；(d)约80-120mM的氯化钠；(e)以及约0.01％-0.05％的聚山梨醇酯80；或
    所述药物组合物含有：(a)约180mg/mL-220mg/mL的人源化抗BLyS抗体或其抗原结合片段；(b)约20-40mM的组氨酸缓冲液或约20-40mM的醋酸缓冲液，pH约为5.5-6.0；(c)约10-30mM的L-精氨酸或约10-30mM的L-精氨酸盐酸盐；(d)约130-170mM的蔗糖；(e)以及约0.01％-0.05％的聚山梨醇酯80；或
    所述药物组合物含有：(a)约200mg/mL的人源化抗BLyS抗体或其抗原结合片段；(b)约25mM组氨酸缓冲液、约25mM醋酸缓冲液或约35-36mM组氨酸缓冲液，pH约为5.5-6.0；(c)约15mM的L-精氨酸或约15mM的L-精氨酸盐酸盐；(d)约150mM的蔗糖；(e)以及约0.02％的聚山梨醇酯80；或
    所述药物组合物含有：(a)约100mg/mL的人源化抗BLyS抗体或其抗原结合片段；(b)约25mM组氨酸缓冲液，pH约为5.5-6.0；(c)约25mM的L-精氨酸或约25mM的L-精氨酸盐酸盐；(d)约100mM的氯化钠；(e)以及约0.02％的聚山梨醇酯80；或
    所述药物组合物含有：(a)约100mg/mL的人源化抗BLyS抗体或其抗原结合片段；(b)约25mM醋酸缓冲液，pH约为5.5-6.0；(c)约60mM的甲硫氨酸；(d)约100mM的氯化钠；(e)以及约0.02％的聚山梨醇酯80；或
    所述药物组合物含有：(a)约100mg/mL的人源化抗BLyS抗体或其抗原结合片段； (b)约35-36mM组氨酸缓冲液，pH约为5.5-6.0；(c)约15mM的L-精氨酸盐酸盐或约15mM的L-精氨酸；(d)约100mM的氯化钠；(e)以及约0.02％的聚山梨醇酯80。
  16. 如权利要求11所述的药物组合物，其包含如下(1)-(20)任一项所示的组分：
    (1)(a)约20mg/mL-280mg/mL的人源化抗BLyS抗体或其抗原结合片段，所述人源化抗BLyS抗体或其抗原结合片段包含轻链可变区和重链可变区，所述轻链可变区包含如SEQ ID NO：9所示的氨基酸序列，所述重链可变区包含如SEQ ID NO：8、SEQ ID NO：10、SEQ ID NO：12、SEQ ID NO：14或SEQ ID NO：15所示的氨基酸序列；(b)约5-50mM的组氨酸缓冲液，pH约为5.0-6.5；(c)约10-30mM的L-精氨酸或约10-30mM的L-精氨酸盐酸盐；(d)约80-220mM的蔗糖；(e)以及约0.01％-0.1％的聚山梨醇酯80；或
    (2)(a)约30mg/mL-150mg/mL的人源化抗BLyS抗体或其抗原结合片段，所述人源化抗BLyS抗体或其抗原结合片段包含轻链可变区和重链可变区，所述轻链可变区包含如SEQ ID NO：9所示的氨基酸序列，所述重链可变区包含如SEQ ID NO：8、SEQ ID NO：10、SEQ ID NO：12、SEQ ID NO：14或SEQ ID NO：15所示的氨基酸序列；(b)约5-50mM组氨酸缓冲液，pH约为5.0-6.5；(c)约10-30mM的L-精氨酸；(d)约50-200mM的氯化钠；(e)以及约0.01％-0.1％的聚山梨醇酯80；或
    (3)(a)约30mg/mL-150mg/mL的人源化抗BLyS抗体或其抗原结合片段，所述人源化抗BLyS抗体或其抗原结合片段包含轻链可变区和重链可变区，所述轻链可变区包含如SEQ ID NO：9所示的氨基酸序列，所述重链可变区包含如SEQ ID NO：8、SEQ ID NO：10、SEQ ID NO：12、SEQ ID NO：14或SEQ ID NO：15所示的氨基酸序列；(b)约5-50mM组氨酸缓冲液，pH约为5.0-6.5；(c)约10-30mM的L-精氨酸盐酸盐；(d)约50-200mM的氯化钠；(e)以及约0.01％-0.1％的聚山梨醇酯80；或
    (4)(a)约30mg/mL-150mg/mL的人源化抗BLyS抗体或其抗原结合片段，所述人源化抗BLyS抗体或其抗原结合片段包含轻链可变区和重链可变区，所述轻链可变区包含如SEQ ID NO：9所示的氨基酸序列，所述重链可变区包含如SEQ ID NO：8、SEQ ID NO：10、SEQ ID NO：12、SEQ ID NO：14或SEQ ID NO：15所示的氨基酸序列；(b)约5-50mM组氨酸缓冲液，pH约为5.0-6.5；(c)约30-90mM的甲硫氨酸；(d)约50-200mM的氯化钠；(e)以及约0.01％-0.1％的聚山梨醇酯80；或
    (5)(a)约30mg/mL-150mg/mL的人源化抗BLyS抗体或其抗原结合片段，所述人源化抗BLyS抗体或其抗原结合片段包含轻链可变区和重链可变区，所述轻链可变区包含如SEQ ID NO：9所示的氨基酸序列，所述重链可变区包含如SEQ ID NO：8、SEQ ID NO：10、SEQ ID NO：12、SEQ ID NO：14或SEQ ID NO：15所示的氨基酸序列；(b)约 5-50mM醋酸缓冲液，pH约为5.0-6.5；(c)约10-30mM的L-精氨酸；(d)约50-200mM的氯化钠；(e)以及约0.01％-0.1％的聚山梨醇酯80；或
    (6)(a)约30mg/mL-150mg/mL的人源化抗BLyS抗体或其抗原结合片段，所述人源化抗BLyS抗体或其抗原结合片段包含轻链可变区和重链可变区，所述轻链可变区包含如SEQ ID NO：9所示的氨基酸序列，所述重链可变区包含如SEQ ID NO：8、SEQ ID NO：10、SEQ ID NO：12、SEQ ID NO：14或SEQ ID NO：15所示的氨基酸序列；(b)约5-50mM醋酸缓冲液，pH约为5.0-6.5；(c)约10-30mM的L-精氨酸盐酸盐；(d)约50-200mM的氯化钠；(e)以及约0.01％-0.1％的聚山梨醇酯80；或
    (7)(a)约30mg/mL-150mg/mL的人源化抗BLyS抗体或其抗原结合片段，所述人源化抗BLyS抗体或其抗原结合片段包含轻链可变区和重链可变区，所述轻链可变区包含如SEQ ID NO：9所示的氨基酸序列，所述重链可变区包含如SEQ ID NO：8、SEQ ID NO：10、SEQ ID NO：12、SEQ ID NO：14或SEQ ID NO：15所示的氨基酸序列；(b)约5-50mM醋酸缓冲液，pH约为5.0-6.5；(c)约30-90mM的甲硫氨酸；(d)约50-200mM的氯化钠；(e)以及约0.01％-0.1％的聚山梨醇酯80；或
    (8)(a)约80mg/mL-120mg/mL的人源化抗BLyS抗体或其抗原结合片段，所述人源化抗BLyS抗体或其抗原结合片段包含轻链可变区和重链可变区，所述轻链可变区包含如SEQ ID NO：9所示的氨基酸序列，所述重链可变区包含如SEQ ID NO：10所示的氨基酸序列；(b)约20-40mM组氨酸缓冲液，pH约为5.5-6.0；(c)约10-30mM的L-精氨酸；(d)约80-120mM的氯化钠；(e)以及约0.01％-0.05％的聚山梨醇酯80；或
    (9)(a)约80mg/mL-120mg/mL的人源化抗BLyS抗体或其抗原结合片段，所述人源化抗BLyS抗体或其抗原结合片段包含轻链可变区和重链可变区，所述轻链可变区包含如SEQ ID NO：9所示的氨基酸序列，所述重链可变区包含如SEQ ID NO：10所示的氨基酸序列；(b)约20-40mM组氨酸缓冲液，pH约为5.5-6.0；(c)约10-30mM的L-精氨酸盐酸盐；(d)约80-120mM的氯化钠；(e)以及约0.01％-0.05％的聚山梨醇酯80；或
    (10)(a)约80mg/mL-120mg/mL的人源化抗BLyS抗体或其抗原结合片段，所述人源化抗BLyS抗体或其抗原结合片段包含轻链可变区和重链可变区，所述轻链可变区包含如SEQ ID NO：9所示的氨基酸序列，所述重链可变区包含如SEQ ID NO：10所示的氨基酸序列；(b)约20-40mM醋酸缓冲液，pH约为5.5-6.0；(c)约50-70mM的甲硫氨酸；(d)约80-120mM的氯化钠；(e)以及约0.01％-0.05％的聚山梨醇酯80；或
    (11)(a)约180mg/mL-220mg/mL的人源化抗BLyS抗体或其抗原结合片段，所述人源化抗BLyS抗体或其抗原结合片段包含轻链可变区和重链可变区，所述轻链可变区包 含如SEQ ID NO：9所示的氨基酸序列，所述重链可变区包含如SEQ ID NO：10所示的氨基酸序列；(b)约20-40mM组氨酸缓冲液，pH约为5.5-6.0；(c)约10-30mM的L-精氨酸或约10-30mM的L-精氨酸盐酸盐；(d)约130-170mM的蔗糖；(e)以及约0.01％-0.05％的聚山梨醇酯80；或
    (12)(a)约200mg/mL的人源化抗BLyS抗体或其抗原结合片段，所述人源化抗BLyS抗体或其抗原结合片段包含轻链可变区和重链可变区，所述轻链可变区包含如SEQ ID NO：9所示的氨基酸序列，所述重链可变区包含如SEQ ID NO：10所示的氨基酸序列；(b)约35-36mM组氨酸缓冲液，pH约为5.5-6.0；(c)约15mM的L-精氨酸盐酸盐；(d)约150mM的蔗糖；(e)以及约0.02％的聚山梨醇酯80；或
    (13)(a)约200mg/mL的人源化抗BLyS抗体或其抗原结合片段，所述人源化抗BLyS抗体或其抗原结合片段包含轻链可变区和重链可变区，所述轻链可变区包含如SEQ ID NO：9所示的氨基酸序列，所述重链可变区包含如SEQ ID NO：10所示的氨基酸序列；(b)约35-36mM组氨酸缓冲液，pH约为5.5-6.0；(c)约15mM的L-精氨酸；(d)约150mM的蔗糖；(e)以及约0.02％的聚山梨醇酯80；或
    (14)(a)约200mg/mL的人源化抗BLyS抗体或其抗原结合片段，所述人源化抗BLyS抗体或其抗原结合片段包含轻链可变区和重链可变区，所述轻链可变区包含如SEQ ID NO：9所示的氨基酸序列，所述重链可变区包含如SEQ ID NO：10所示的氨基酸序列；(b)约25mM组氨酸缓冲液，pH约为5.5-6.0；(c)约15mM的L-精氨酸盐酸盐；(d)约150mM的蔗糖；(e)以及约0.02％的聚山梨醇酯80；或
    (15)(a)约100mg/mL的人源化抗BLyS抗体或其抗原结合片段，所述人源化抗BLyS抗体或其抗原结合片段包含轻链可变区和重链可变区，所述轻链可变区包含如SEQ ID NO：9所示的氨基酸序列，所述重链可变区包含如SEQ ID NO：10所示的氨基酸序列；(b)约25mM组氨酸缓冲液，pH约为5.5-6.0；(c)约25mM的L-精氨酸；(d)约100mM的氯化钠；(e)以及约0.02％的聚山梨醇酯80；或
    (16)(a)约100mg/mL的人源化抗BLyS抗体或其抗原结合片段，所述人源化抗BLyS抗体或其抗原结合片段包含轻链可变区和重链可变区，所述轻链可变区包含如SEQ ID NO：9所示的氨基酸序列，所述重链可变区包含如SEQ ID NO：10所示的氨基酸序列；(b)约25mM组氨酸缓冲液，pH约为5.5-6.0；(c)约25mM的L-精氨酸盐酸盐；(d)约100mM的氯化钠；(e)以及约0.02％的聚山梨醇酯80；或
    (17)(a)约100mg/mL的人源化抗BLyS抗体或其抗原结合片段，所述人源化抗BLyS抗体或其抗原结合片段包含轻链可变区和重链可变区，所述轻链可变区包含如SEQ ID NO：9所示的氨基酸序列，所述重链可变区包含如SEQ ID NO：10所示的氨基酸序列； (b)约25mM醋酸缓冲液，pH约为5.5-6.0；(c)约60mM的甲硫氨酸；(d)约100mM的氯化钠；(e)以及约0.02％的聚山梨醇酯80；或
    (18)(a)约100mg/mL的人源化抗BLyS抗体或其抗原结合片段，所述人源化抗BLyS抗体或其抗原结合片段包含轻链可变区和重链可变区，所述轻链可变区包含如SEQ ID NO：9所示的氨基酸序列，所述重链可变区包含如SEQ ID NO：10所示的氨基酸序列；(b)约35-36mM组氨酸缓冲液，pH约为5.5-6.0；(c)约15mM的L-精氨酸盐酸盐；(d)约100mM的氯化钠；(e)以及约0.02％的聚山梨醇酯80；或
    (19)(a)约100mg/mL的人源化抗BLyS抗体或其抗原结合片段，所述人源化抗BLyS抗体或其抗原结合片段包含轻链可变区和重链可变区，所述轻链可变区包含如SEQ ID NO：9所示的氨基酸序列，所述重链可变区包含如SEQ ID NO：10所示的氨基酸序列；(b)约35-36mM组氨酸缓冲液，pH约为5.5-6.0；(c)约15mM的L-精氨酸；(d)约100mM的氯化钠；(e)以及约0.02％的聚山梨醇酯80；或
    (20)(a)约100mg/mL人源化抗BLyS抗体或其抗原结合片段，所述人源化抗BLyS抗体或其抗原结合片段包含轻链可变区和重链可变区，所述轻链可变区包含如SEQ ID NO：9所示的氨基酸序列，所述重链可变区包含如SEQ ID NO：10所示的氨基酸序列；(b)约35mM组氨酸缓冲液，pH约为5.8；(c)约15mM的L-精氨酸盐酸盐；(d)约100mM的氯化钠；(e)以及约0.02％的聚山梨醇酯80；
    优选地，所述人源化抗BLyS抗体的轻链氨基酸序列如SEQ ID NO:18所示，重链氨基酸序列如SEQ ID NO:19所示。
  17. 一种注射剂，其含有权利要求11-16中任一项所述的药物组合物与氯化钠溶液；优选地，所述注射剂中，所述人源化抗BLyS抗体的浓度为3～100mg/mL；优选地，所述注射剂的pH为5.5～6.0。
  18. 如权利要求11-16中任一项所述的药物组合物或权利要求17所述的注射剂，其经皮下注射施用。
  19. 如权利要求1-7中任一项所述的人源化抗BLyS抗体或其抗原结合片段、或如权利要求8所述的多核苷酸分子、或如权利要求9所述的表达载体、或如权利要求10所述的宿主细胞、或如权利要求11-16中任一项所述的药物组合物、或如权利要求17所述的注射剂在制备预防和/或治疗B细胞增殖过量引起的疾病的药物中的应用；优选地，所述B细胞增殖过量引起的疾病为系统性红斑狼疮、类风湿性关节炎、强制性关节炎或B细胞淋巴癌。
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