CN115232208A - anti-BLyS antibodies, pharmaceutical compositions thereof, and uses thereof - Google Patents

anti-BLyS antibodies, pharmaceutical compositions thereof, and uses thereof Download PDF

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CN115232208A
CN115232208A CN202210443212.1A CN202210443212A CN115232208A CN 115232208 A CN115232208 A CN 115232208A CN 202210443212 A CN202210443212 A CN 202210443212A CN 115232208 A CN115232208 A CN 115232208A
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seq
amino acid
acid sequence
variable region
chain variable
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王莹
文珺
周岳华
向丹丹
开仁冬
李薇
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Shanghai Junshi Biosciences Co Ltd
Suzhou Junmeng Biosciences Co Ltd
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Shanghai Junshi Biosciences Co Ltd
Suzhou Junmeng Biosciences Co Ltd
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Abstract

The present invention provides anti-BLyS antibodies, pharmaceutical compositions thereof, and uses thereof. The anti-BLyS antibody or the antigen binding fragment thereof comprises a heavy chain variable region and a light chain variable region, the amino acid sequences of LCDR1, LCDR2 and LCDR3 are respectively shown as SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3, and the amino acid sequences of HCDR1, HCDR2 and HCDR3 are respectively shown as SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6. The anti-BLyS antibody or the antigen-binding fragment thereof of the present invention has excellent binding ability to the human BLyS protein and ability to competitively inhibit the binding of human BLyS to its receptor, human BR 3. The pharmaceutical composition of the present invention is a highly stable pharmaceutical composition comprising an anti-BLyS antibody and a buffer, and may further comprise at least one stabilizer and optionally a surfactant.

Description

anti-BLyS antibodies, pharmaceutical compositions thereof, and uses thereof
Technical Field
The invention belongs to the field of biomedicine, and relates to an anti-BLyS antibody, a pharmaceutical composition thereof and application thereof, wherein the anti-BLyS antibody has excellent binding capacity of human BLyS protein and capacity of competitively inhibiting the binding of human BLyS and a receptor human BR3 thereof.
Background
Systemic Lupus Erythematosus (SLE) is a chronically progressive, often remitting, and recurring autoimmune disease that affects multiple systems throughout the body, such as the skin, joints, heart, lungs, kidneys, blood, and brain. Systemic lupus erythematosus mainly affects african-caribbean, asian, and hispanic, while caucasians (caucasians) are affected to a lesser extent. Because the initial symptoms of SLE are more hidden, the actual number of patients may be far greater than currently estimated. Therefore, the clinical application has great objective requirements on the diagnosis and treatment of the systemic lupus erythematosus.
The cause of systemic lupus erythematosus is complex and undefined, is not caused by a single factor, and may be related to various factors such as heredity, environment, sex hormone, immunity and the like. Currently, systemic lupus erythematosus is recognized by the world's scientific community as autoimmune because self-reactive B cells are present in peripheral tissues for too long time at the cellular level to produce human autoantigens. Therefore, if the growth and proliferation of early B cells can be inhibited, lupus erythematosus can be treated.
B Lymphocyte Stimulator (BLyS), also known as toll-1 (TNF and Apol related leukocyte expressed ligand 1), BAFF (B cell activating factor cloning to the TNF family), THANK (TNF homologies at activating cytokines, NF-. Kappa.B and JNK), is a new cytokine first discovered and cloned by the United states of America and others in 1999. BLyS is used as a costimulatory factor of B lymphocyte, can specifically stimulate B cell proliferation and differentiation in the presence of anti-IgM and IL-4, and plays an extremely important role in humoral immunity; the overexpression of the polypeptide is closely related to autoimmune diseases in vivo.
In vitro experiments showed that BLyS induces massive proliferation and secretion of large amounts of IgM and IgA after preactivation of B cells with IgM, but this stimulatory effect is not significant for B cells during rest (3). Further studies showed that BLyS acts mainly on pre-B lymphocytes, immature B lymphocytes, activated lymphocytes, but not on plasma cells, lymphopluripotent stem cells. Like most cytokines, BLyS stimulates downstream signaling primarily through B cell surface receptors. Several groups have demonstrated that the receptors that bind to BLyS are: b cell activator receptor (BR 3, BLyS receptor 3 or BAFF-R), transmembrane activator-1and calcium modulator and cyclophilin ligand-intron (TACI), and B Cell Maturation Antigen (BCMA). This specificity determines BLyS to be a good target for B cell antibody mediated autoimmune diseases and lymphoma.
Therapeutic antibodies against BLyS have been shown to be effective in inhibiting B cell growth, igA and IgM secretion both in vitro and in vivo in order to treat systemic lupus erythematosus (Edwards BM et al, the rechargeable flexible nature of The human antibody reagent; isolation of one and two diagnostic reagents to a single protein, BLyS.J Mol biol.2003Nov 14 (1): 103-18 Baker KP et al, genetics and characterization of lymphoma-B, a human monoclonal antibodies of The biological activity of B lymphocyte specific tissue or of arthritis Rheum. Nov 2003; 11 3253-65): 48. The anti-BLyS antibody Benlysta developed by the American human genome company became the first new drug for treating lupus erythematosus in the past 60 years. The Benlysta only aims at B cells stimulated by BLyS, and compared with chemotherapeutic drugs, the Benlysta greatly reduces side effects in the treatment process, thereby providing a safe and effective treatment method for systemic lupus erythematosus patients. In recent years, targeted therapeutic research and clinical application of BLyS have been rapidly developed, and companies other than the american human genome company adopt fusion proteins modified based on BLyS or its receptor. The American Gene Take (Genentech) company developed BR3-FC drugs, the Zymogenetics company developed TACI-FC drugs, and the Anjin (AMGEN) company developed polypeptide-FC drugs. Compared with Benlysta, they have poor specificity, weak binding force, relatively poor curative effect and strong toxicity. All three drugs were stopped or terminated in the second phase of the clinical trial. Therefore, anti-BLyS antibody drugs are the only effective pharmaceutical approaches to this target.
Disclosure of Invention
The anti-BLyS antibody provided by the invention has excellent binding capacity of human BLyS protein and capacity of competitively inhibiting the binding capacity of human BLyS and a receptor human BR3 thereof. The pharmaceutical composition is a highly stable pharmaceutical composition comprising an anti-BLyS antibody, in particular, the present inventors have found that the humanized anti-BLyS antibody has an unexpected characteristic, i.e. high stability, in a combination of a histidine buffer system and L-arginine, L-arginine hydrochloride or methionine.
The present invention provides a humanized anti-BLyS antibody or antigen binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein:
the amino acid sequences of LCDR1, LCDR2 and LCDR3 of the humanized anti-BLyS antibody or the antigen binding fragment thereof are respectively shown as SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3;
the amino acid sequences of HCDR1, HCDR2 and HCDR3 of the humanized anti-BLyS antibody or the antigen binding fragment thereof are respectively shown as SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6;
the FR1 of the light chain variable region of the humanized anti-BLyS antibody or antigen binding fragment thereof is selected from the group consisting of the FR1 of any one of SEQ ID NOS: 7, 9 and 13, the FR2 is the FR2 of SEQ ID NO:7, the FR3 is selected from the FR3 of any one of SEQ ID NOS: 7, 9 and 11, and the FR4 is the FR4 of SEQ ID NO: 7;
FR1 of the heavy chain variable region of the humanized anti-BLyS antibody or an antigen-binding fragment thereof is selected from the group consisting of FR1 of any one of SEQ ID NOS: 8, 10, 12 and 15, FR2 is selected from the group consisting of FR2 of any one of SEQ ID NOS: 8, 10 and 15, FR3 is selected from the group consisting of FR3 of any one of SEQ ID NOS: 8, 12 and 14, FR4 is the FR4 of SEQ ID NO:8 or 10; and is
The humanized anti-BLyS antibody or antigen binding fragment thereof does not comprise: the light chain variable region is the amino acid sequence shown in SEQ ID NO. 7, while the heavy chain variable region is the humanized anti-BLyS antibody or antigen-binding fragment thereof of the amino acid sequence shown in SEQ ID NO. 8.
In some embodiments, in the humanized anti-BLyS antibody or antigen binding fragment thereof described above:
FR1 of the light chain variable region is selected from FR1 of any one of SEQ ID NO 7, 9 and 13, FR2 is FR2 of SEQ ID NO 7, FR3 is selected from FR3 of any one of SEQ ID NO 7, 9 and 11, FR4 is FR4 of SEQ ID NO 7; FR1-FR4 of the heavy chain variable region are FR1-FR4 of SEQ ID NO. 15, respectively; or
FR1 of the light chain variable region is selected from FR1 of SEQ ID NO. 7, 9 and 13, FR2 is FR2 of SEQ ID NO. 7, FR3 is selected from FR3 of SEQ ID NO. 7, 9 and 11, FR4 is FR4 of SEQ ID NO. 7; FR1-FR4 of the heavy chain variable region are respectively FR1-FR4 of SEQ ID NO. 10; or
FR1 of the light chain variable region is selected from FR1 of any one of SEQ ID NO 7, 9 and 13, FR2 is FR2 of SEQ ID NO 7, FR3 is selected from FR3 of any one of SEQ ID NO 7, 9 and 11, FR4 is FR4 of SEQ ID NO 7; FR1-FR4 of the heavy chain variable region are FR1-FR4 of SEQ ID NO. 12, respectively; or
FR1 of the light chain variable region is FR1 described by SEQ ID NO. 11 or 13, FR2 is FR2 described by SEQ ID NO. 11, FR3 is FR3 described by SEQ ID NO. 11 or 13, and FR4 is FR4 described by SEQ ID NO. 11; FR1-FR4 of the heavy chain variable region are respectively FR1-FR4 of SEQ ID NO. 14.
In some embodiments, 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 an amino acid sequence as set forth in SEQ ID NO: 7. SEQ ID NO: 9. SEQ ID NO:11 or SEQ ID NO:13, and the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 8. SEQ ID NO: 10. SEQ ID NO: 12. SEQ ID NO:14 or SEQ ID NO:15, or a pharmaceutically acceptable salt thereof.
In some embodiments, 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 and the heavy chain variable region are selected from the group consisting of:
(1) The light chain variable region comprises the amino acid sequence shown as SEQ ID NO:7, and the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 10. the amino acid sequence of SEQ ID NO: 12. SEQ ID NO:14 or SEQ ID NO: 15; or
(2) The light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:9, and the heavy chain variable region comprises the amino acid sequence set forth as SEQ ID NO: 8. SEQ ID NO: 10. the amino acid sequence of SEQ ID NO: 12. SEQ ID NO:14 or SEQ ID NO: 15; or
(3) The light chain variable region comprises the amino acid sequence shown as SEQ ID NO:11, and the heavy chain variable region comprises the amino acid sequence set forth as SEQ ID NO: 8. the amino acid sequence of SEQ ID NO: 10. the amino acid sequence of SEQ ID NO: 12. SEQ ID NO:14 or SEQ ID NO: 15; or
(4) The light chain variable region comprises the amino acid sequence shown as SEQ ID NO:13, and the heavy chain variable region comprises the amino acid sequence set forth as SEQ ID NO: 8. SEQ ID NO: 10. SEQ ID NO: 12. SEQ ID NO:14 or SEQ ID NO: 15;
preferably, the light chain variable region and the heavy chain variable region are selected from:
(5) The light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:9, and the heavy chain variable region comprises the amino acid sequence set forth as SEQ ID NO: 10; or
(6) The light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:11, and the heavy chain variable region comprises the amino acid sequence set forth as SEQ ID NO: 12; or
(7) The light chain variable region comprises the amino acid sequence shown as SEQ ID NO:13, and the heavy chain variable region comprises the amino acid sequence set forth as SEQ ID NO: 14; or
(8) The light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:7, and the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 15; or
(9) The light chain variable region comprises the amino acid sequence shown as SEQ ID NO:9, and the heavy chain variable region comprises the amino acid sequence set forth as SEQ ID NO: 15; or
(10) The light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:11, and the heavy chain variable region comprises the amino acid sequence set forth as SEQ ID NO: 15; or
(11) The light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:13, and the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:15, or a pharmaceutically acceptable salt thereof.
The humanized anti-BLyS antibody of the present invention further comprises a human light chain constant region and a human heavy chain constant region, and the light chain variable region and the heavy chain variable region are linked to the human light chain constant region and the heavy chain constant region, respectively. That is, the humanized anti-BLyS antibody comprises an intact light chain formed by the variable region of the light chain comprised in the anti-BLyS antibody linked to the constant region of the human light chain, and an intact heavy chain formed by the variable region of the heavy chain comprised in the anti-BLyS antibody linked to the constant region of the human heavy chain.
In some embodiments, the human light chain constant region is a human light chain kappa constant region.
In some embodiments, the human heavy chain constant region is a human heavy chain Fc fragment.
Wherein the human light chain kappa constant region and the human heavy chain Fc fragment are both derived from healthy human B lymphocytes. The light chain and the heavy chain of the complete humanized anti-BLyS antibody are obtained by connecting the variable region and the constant region through overlap extension PCR by using a genetic engineering technology.
In some embodiments, 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 an amino acid sequence as set forth in SEQ ID NO: 16. SEQ ID NO: 18. SEQ ID NO:20 or SEQ ID NO: 22. the amino acid sequence shown in SEQ ID NO: 17. SEQ ID NO: 19. the amino acid sequence of SEQ ID NO: 21. the amino acid sequence of SEQ ID NO:23 or SEQ ID NO: 24.
In some embodiments, the humanized anti-BLyS antibody or antigen binding fragment thereof comprises a light chain and a heavy chain, wherein:
(1) The light chain comprises the amino acid sequence set forth as SEQ ID NO:16, and the heavy chain comprises the amino acid sequence set forth as SEQ ID NO: 19. SEQ ID NO: 21. SEQ ID NO:23 or SEQ ID NO: 24; or
(2) The light chain comprises the amino acid sequence set forth as SEQ ID NO:18, and the heavy chain comprises the amino acid sequence set forth as SEQ ID NO: 17. the amino acid sequence of SEQ ID NO: 19. SEQ ID NO: 21. SEQ ID NO:23 or SEQ ID NO: 24; or
(3) The light chain comprises the amino acid sequence set forth as SEQ ID NO:20, and the heavy chain comprises the amino acid sequence set forth as SEQ ID NO: 17. SEQ ID NO: 19. SEQ ID NO: 21. SEQ ID NO:23 or SEQ ID NO: 24; or
(4) The light chain comprises the amino acid sequence as set forth in SEQ ID NO:22, and the heavy chain comprises the amino acid sequence set forth as SEQ ID NO: 17. SEQ ID NO: 19. the amino acid sequence of SEQ ID NO: 21. the amino acid sequence of SEQ ID NO:23 or SEQ ID NO: 24;
preferably:
(5) The light chain comprises the amino acid sequence as set forth in SEQ ID NO:18, and the heavy chain comprises the amino acid sequence set forth as SEQ ID NO: 19; or
(6) The light chain comprises the amino acid sequence as set forth in SEQ ID NO:20, and the heavy chain comprises the amino acid sequence set forth as SEQ ID NO: 21; or
(7) The light chain comprises the amino acid sequence as set forth in SEQ ID NO:22, and the heavy chain comprises the amino acid sequence set forth as SEQ ID NO: 23; or
(8) The light chain comprises the amino acid sequence as set forth in SEQ ID NO:16, and the heavy chain comprises the amino acid sequence set forth as SEQ ID NO: 24; or
(9) The light chain comprises the amino acid sequence as set forth in SEQ ID NO:18, and the heavy chain comprises the amino acid sequence set forth as SEQ ID NO: 24; or
(10) The light chain comprises the amino acid sequence set forth as SEQ ID NO:20, and the heavy chain comprises the amino acid sequence set forth as SEQ ID NO: 24; or
(11) The light chain comprises the amino acid sequence set forth as SEQ ID NO:22, and the heavy chain comprises the amino acid sequence set forth as SEQ ID NO: 24.
In some embodiments, the antigen binding fragment of the invention is selected from a Fab, fab '-SH, fv, scFv, F (ab') 2, sdAb, or diabody.
In a further aspect, the present invention provides a polynucleotide molecule selected from the group consisting of: a polynucleotide molecule or its complement encoding a humanized anti-BLyS antibody or antigen binding fragment thereof as described in any one of the schemes herein.
In a further aspect, the present invention provides an expression vector comprising a polynucleotide molecule as described herein, preferably said expression vector is a eukaryotic expression vector.
In a further aspect, the invention provides a host cell comprising a polynucleotide molecule or expression vector as described herein, preferably said host cell is a eukaryotic cell, more preferably a mammalian cell.
In yet another aspect, the 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 culturing a host cell as described herein under conditions suitable for expression of the antibody or antigen-binding fragment thereof, such that it expresses the antibody or antigen-binding fragment thereof, and recovering the expressed antibody or antigen-binding fragment thereof.
In yet another aspect, the invention provides a pharmaceutical composition comprising a humanized anti-BLyS antibody or antigen-binding fragment thereof as in any one of the embodiments herein, a polynucleotide described herein, an expression vector described herein, or a host cell described herein, and a pharmaceutically acceptable carrier or excipient.
In some embodiments, the present invention provides a pharmaceutical composition comprising: (1) a buffer solution; (2) A humanized anti-BLyS antibody or an antigen-binding fragment thereof having LCDR1, LCDR2 and LCDR3 with amino acid sequences shown as SEQ ID No. 1, SEQ ID No. 2 and SEQ ID No. 3, respectively, and HCDR1, HCDR2 and HCDR3 with amino acid sequences shown as SEQ ID No. 4, SEQ ID No. 5 and SEQ ID No. 6, respectively.
In some embodiments, the present invention provides a pharmaceutical composition comprising: (1) a buffer solution; (2) a humanized anti-BLyS antibody or antigen binding fragment thereof; the humanized anti-BLyS antibody or antigen-binding fragment thereof is a humanized anti-BLyS antibody or antigen-binding fragment thereof as described in any one of the embodiments herein.
In some embodiments, the concentration of the humanized anti-BLyS antibody or antigen-binding fragment thereof in the pharmaceutical composition described above is about 1 to about 300mg/mL, preferably about 10 to about 300mg/mL, more preferably about 20 to about 280mg/mL, more preferably about 30 to about 150mg/mL, more preferably about 80 to about 120mg/mL, more preferably about 180 to about 220mg/mL.
In some embodiments, the buffer is selected from one or more of an acetate buffer, a citrate buffer, a succinate buffer, a phosphate buffer, and a histidine buffer.
In some embodiments, the buffer is present at a concentration of about 1 mM to about 200mM, preferably about 1 mM to about 100mM, preferably about 5mM to about 50mM, preferably about 10mM to about 40mM, preferably about 20mM to about 40mM.
In some embodiments, the pH of the buffer is about 5.0 to about 6.5, preferably about 5.0 to about 6.0, and preferably about 5.5 to about 6.0.
In some embodiments, the pharmaceutical composition further comprises a stabilizer.
In some embodiments, the stabilizing agent is selected from one or more of arginine, arginine hydrochloride, methionine, proline, glycine, sodium chloride, mannitol, sorbitol, sucrose, maltose, xylitol, and trehalose.
In some embodiments, the stabilizing agent is selected from one or more of L-arginine, L-arginine hydrochloride, sucrose, methionine, and sodium chloride.
In some embodiments, the concentration of the stabilizer is about 10mM to 400mM, preferably about 50mM to 300mM, more preferably about 100mM to 300mM, and even more preferably about 100mM to 200mM.
In some embodiments, the above stabilizer is a combination of L-arginine hydrochloride at a concentration of about 10-30mM and sodium chloride at a concentration of about 50-200 mM; or the stabilizer is a combination of sodium chloride at a concentration of about 10-30mM L-arginine and about 50-200 mM; or the stabilizing agent is a combination of L-arginine at a concentration of about 10-30mM and sucrose at a concentration of about 80-220 mM; or said stabilizer is a combination of L-arginine hydrochloride at a concentration of about 10-30mM with sucrose at a concentration of about 80-220 mM; or the stabilizer is a combination of about 30-90mM methionine and about 50-200mM sodium chloride; or the stabilizer is about 10-120mM L-arginine hydrochloride; or the stabilizer is methionine at about 10-120 mM; preferably, the stabilizer is a combination of L-arginine hydrochloride at a concentration of about 10-30mM and sodium chloride at a concentration of about 80-120 mM; or the stabilizing agent is a combination of L-arginine at a concentration of about 10-30mM and sodium chloride at a concentration of about 80-120 mM; or the stabilizer is a combination of about 50-70mM methionine and about 80-120mM sodium chloride; or the stabilizer is a combination of L-arginine at a concentration of about 10-30mM and sucrose at a concentration of about 130-170 mM; or the stabilizer is a combination of L-arginine hydrochloride at a concentration of about 10-30mM with sucrose at a concentration of about 130-170 mM.
In some embodiments, the above pharmaceutical composition further comprises a surfactant, preferably, the surfactant is selected from one or more of polysorbate 80, polysorbate 20, and poloxamer 188.
In some embodiments, the surfactant concentration is from about 0.001% to about 0.1%, preferably from about 0.01% to about 0.1%, and preferably from about 0.01% to about 0.05%, calculated as w/v.
In some embodiments, the pharmaceutical composition further comprises water for injection.
In some embodiments, the pharmaceutical composition is a liquid formulation or a lyophilized formulation.
In some embodiments, the pharmaceutical composition is a liquid formulation.
In yet another aspect, the present invention provides an injection comprising a pharmaceutical composition as described in any one of the embodiments herein and a sodium chloride solution; preferably, the concentration of the sodium chloride solution is 0.85-0.9%; preferably, the concentration of the humanized anti-BLyS antibody in the injection is 3 to 100mg/mL, more preferably about 3 to 60mg/mL; preferably, the injection has a pH of 5.5 to 6.0.
In some embodiments, the pharmaceutical composition or injection is administered by subcutaneous injection.
In yet another aspect, the present invention provides a use of the humanized anti-BLyS antibody or antigen binding fragment thereof as described in any one of the embodiments herein, the polynucleotide molecule described herein, the expression vector described herein, the host cell described herein, the pharmaceutical composition described herein, or the injection described herein for the preparation of a medicament for preventing and/or treating a disease caused by B cell hyperproliferation.
In yet another aspect, the invention provides 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, an expression vector as described herein, a host cell as described herein, a pharmaceutical composition as described herein, or an injection as described herein for use in the prevention and/or treatment of a disease caused by an excessive B-cell proliferation.
In yet another aspect, the invention provides a method of preventing and/or treating a disease caused by B cell hyperproliferation, comprising administering to a subject in need thereof a humanized anti-BLyS antibody or antigen binding fragment thereof as described in any one of the regimens herein, 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.
Preferably, the disease caused by excessive B cell proliferation is systemic lupus erythematosus, rheumatoid arthritis, mandatory arthritis, or B cell lymphoma.
In yet another aspect, the 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 described herein, an expression vector described herein, a host cell described herein, a pharmaceutical composition described herein, or an injection described herein, and one or more additional therapeutic agents.
In yet another aspect, the 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 as described herein, an expression vector as described herein, a host cell as described herein, a pharmaceutical composition as described herein, or an injection as described herein, preferably further comprising a drug delivery device.
In yet another aspect, the present invention provides the use of a histidine buffer and one or more stabilizing agents selected from the group consisting of L-arginine, L-arginine hydrochloride, methionine, and sodium chloride, and optionally a surfactant (preferably polysorbate 80), to increase the stability of a pharmaceutical formulation of a humanized anti-BLyS antibody or antigen-binding fragment thereof, or to prepare a pharmaceutical formulation of a humanized anti-BLyS antibody or antigen-binding fragment thereof with increased stability. Preferably, the histidine buffer, stabilizer and surfactant and amounts thereof are as described in any embodiment herein; the increased stability is as described in any embodiment herein; the humanized anti-BLyS antibody or antigen binding fragment thereof is as described in any embodiment herein.
Detailed Description
Definitions and explanations
In order that the invention may be more readily understood, certain technical and scientific terms are specifically defined below. Unless otherwise defined herein, all other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It is to be understood that this invention is not limited to particular methods, reagents, compounds, compositions or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.
As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a polypeptide" includes a combination of two or more polypeptides and the like.
The term "pharmaceutical composition" or "formulation" means a mixture containing one or more of the antibodies described herein and other components, such as physiologically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to facilitate administration to an organism, facilitate absorption of the active ingredient and exert biological activity.
The term "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 invention are stable on storage and their stability is independent of lyophilization (or other state change methods, such as spray drying).
The term "aqueous liquid formulation" refers to a liquid formulation that uses water as a solvent. In some embodiments, the aqueous liquid formulation is one that does not require lyophilization, spray drying, and/or freezing to maintain stability (e.g., chemical and/or physical stability and/or biological activity).
The term "excipient" refers to an agent that may be added to a formulation to provide a desired characteristic (e.g., consistency, improved stability) and/or to adjust osmotic pressure. Examples of commonly used excipients include, but are not limited to, sugars, polyols, amino acids, surfactants, and polymers.
As used herein, "about" when referring to a measurable value (e.g., amount, duration, etc.) is intended to encompass variations of ± 20% or ± 10% from the particular value, including ± 5%, ± 1% and ± 0.1%, as such variations are suitable for carrying out the disclosed methods.
The term "buffer pH of about 5.0-6.5" refers to an agent that, through the action of its acid/base conjugate components, renders a solution containing the agent resistant to pH changes. The buffer used in the formulation 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.
Examples of "buffers" that control pH within this range herein include succinic acid, succinate (e.g., sodium succinate), gluconic acid, histidine hydrochloride, methionine, citric acid, citrate, phosphoric acid, phosphate, citrate/phosphate, imidazole, acetic acid, acetate, combinations thereof, and other organic acid buffers.
"histidine buffer" is a buffer comprising histidine ions. Examples of histidine buffers include salts of histidine and histidine, such as histidine hydrochloride, histidine acetate, histidine phosphate, and histidine sulfate, and the like, such as histidine buffer containing histidine and histidine hydrochloride; the histidine buffer of the present invention also includes histidine buffers comprising histidine and an acetate salt (e.g., sodium or potassium salt).
A "citrate buffer" is a buffer that includes citrate ions. Examples of citrate buffers include sodium citrate-citrate, potassium citrate-citrate, calcium citrate-citrate, magnesium citrate-citrate, and the like. Preferably the citrate buffer is a citric acid-sodium citrate buffer.
An "acetate buffer" is a buffer that includes acetate ions. Examples of acetate buffers include sodium acetate, potassium acetate, calcium acetate, magnesium acetate, and the like. Preferably, the acetate buffer is an acetic acid-sodium acetate buffer.
A "succinic acid buffer" is a buffer that includes succinate ions. Examples of the succinate buffer include sodium succinate-succinate, potassium succinate-succinate, calcium succinate-succinate, magnesium succinate-succinate, and the like. Preferably the succinate buffer is sodium succinate-succinate buffer.
A "phosphate buffer" is a buffer comprising phosphate ions. Examples of the phosphate buffer include disodium hydrogenphosphate-sodium dihydrogenphosphate, dipotassium hydrogenphosphate-potassium dihydrogenphosphate, and the like. Preferably the phosphate buffer is disodium hydrogen phosphate-sodium dihydrogen phosphate.
The term "stabilizer" means a pharmaceutically acceptable excipient that protects the active pharmaceutical ingredient and/or formulation from chemical and/or physical degradation during manufacture, storage and use. Stabilizers include, but are not limited to, sugars, amino acids, salts, polyols and their metabolites, such as sodium chloride, calcium chloride, magnesium chloride, mannitol, sorbitol, sucrose, trehalose, methionine, L-arginine or salts thereof (e.g., L-arginine hydrochloride), glycine, alanine (α -alanine, β -alanine), betaine, leucine, lysine, glutamic acid, aspartic acid, proline, 4-hydroxyproline, sarcosine, γ -aminobutyric acid (GABA), octopine (stromite), and N-oxide of Trimethylamine (TMAO), human serum albumin (hsa), bovine serum albumin (bsa), α -casein, globulin, α -lactalbumin, GABA, lysozyme, myoglobin, ovalbumin, and RNAaseA. Some stabilizers, such as sodium chloride, calcium chloride, magnesium chloride, mannitol, sorbitol, sucrose, etc., may also serve to control osmotic pressure. The stabilizer used in the present invention is selected from one or more of polyols, amino acids, salts, and sugars. Preferably the sugars are sucrose and trehalose, and preferably the polyol is mannitol. Preferably the amino acid is L-arginine or a salt thereof (e.g. L-arginine hydrochloride), methionine, glycine, proline. Preferably the stabilizer is sodium chloride, mannitol, sorbitol, sucrose, trehalose, methionine, L-arginine hydrochloride, glycine, proline, sodium chloride-L-arginine hydrochloride, sodium chloride-methionine, sodium chloride-sorbitol, sodium chloride-mannitol, sodium chloride-sucrose, sodium chloride-trehalose, L-arginine hydrochloride-mannitol, L-arginine hydrochloride-sucrose, more preferably L-arginine hydrochloride, L-arginine, methionine, sodium chloride-L-arginine hydrochloride, sodium chloride-methionine, more preferably sodium chloride-L-arginine, sodium chloride-L-arginine hydrochloride. In a particularly preferred embodiment, the stabilizer used in the present invention is selected from one or more of methionine, L-arginine hydrochloride and sodium chloride.
The term "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 antibodies or 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 (e.g., polysorbate 20 and polysorbate 80), polyethylene-polypropylene copolymers, polyethylene-polypropylene glycols, polyoxyethylene-stearates, polyoxyethylene alkyl ethers, such as polyoxyethylene monolauryl ether, alkylphenylpolyoxyethylene ether (Triton-X), polyoxyethylene-polyoxypropylene copolymers (poloxamers, pluronics), sodium Dodecyl Sulfate (SDS). In a particularly preferred embodiment, the surfactant used in the present invention is polysorbate 80.
The term "isotonic" means that the formulation has substantially the same osmotic pressure as human blood. Isotonic formulations generally have an osmotic pressure of about 250 to 350 mOsm. Isotonicity can be measured using an osmometer of the vapor pressure or subfreezing type.
The term "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. The pharmaceutical preparation may be stable even if the contained antibody fails to maintain 100% of its chemical structure or biological function after storage over a certain period of time. In certain instances, an antibody structure or function that maintains about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% after storage over a 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, vincent Lee, marcel Dekker, inc., new York, n.y., pubs. (1991)), and Jones, a., 1993, adv. 29-90 (both incorporated by reference).
After storage of the formulation at a temperature and for a period of time, its stability can be measured by determining the percentage of native antibody remaining therein (among other methods). The percentage of native antibody can be measured by size exclusion chromatography (e.g., size exclusion high performance liquid chromatography [ SEC-HPLC ]), among other methods, "native" referring to unaggregated and undegraded. In some embodiments, the stability of a protein is determined as a percentage of monomeric protein in a solution having a low percentage of degraded (e.g., fragmented) and/or aggregated protein. In some embodiments, the formulation is stable for storage at room temperature, about 25-30 ℃, or 40 ℃ 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 no more than 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 that migrates ("acidic form") in the fraction where this main fraction of antibody ("predominantly charged form") is acidic during ion exchange, where stability is inversely proportional to the percentage of acidic form antibody. The percentage of "acidified" antibody can be measured by, among other methods, ion exchange chromatography (e.g., cation exchange high performance liquid chromatography [ CEX-HPLC ]). In some embodiments, an acceptable degree of stability means that no more than about 49%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% of the antibody in acidic form is detectable in the formulation after storage of the formulation at a temperature and for a time. The certain time 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. When evaluating stability, a temperature that allows for storage of the pharmaceutical formulation can be any temperature in the range of about-80 ℃ to about 45 ℃, e.g., storage at about-80 ℃, about-30 ℃, about-20 ℃, about 0 ℃, about 2-8 ℃, about 5 ℃, about 25 ℃, or about 40 ℃.
An antibody "retains its physical stability" in a pharmaceutical composition if it shows substantially no signs of, for example, aggregation, precipitation and/or denaturation when visually inspected for color and/or clarity or measured by UV light scattering or by pore size exclusion chromatography. Aggregation is the process by which individual molecules or complexes associate, covalently or non-covalently, to form aggregates. Aggregation may proceed to the extent that a visible precipitate is formed.
Stability, e.g., physical stability, of a formulation can be assessed by methods well known in the art, including measuring the apparent extinction (absorbance or optical density) of a sample. Such extinction measurements correlate with the turbidity of the formulation. Turbidity of a formulation is, in part, an inherent property of proteins dissolved in solution and is typically measured by nephelometry and is measured in Nephelometric Turbidity Units (NTU).
The level of turbidity which varies with, for example, the concentration of one or more components in the solution (e.g., protein and/or salt concentration) is also referred to as the "opacification" or "opacified appearance" of the formulation. The turbidity level can be calculated with reference to a standard curve generated using suspensions of known turbidity. The reference standard for determining the turbidity level of a pharmaceutical composition may be based on the criteria of the European Pharmacopoeia (European Pharmacopoeia), fourth edition, "European commission on pharmaceutical Quality instruction" (directive for the Quality of the Medicine of the Council of Europe) (EDQM), strasbourg, france). According to the european pharmacopoeia standard, a clear solution is defined as a solution having a turbidity lower than or equal to that of a reference suspension according to the european pharmacopoeia standard having a turbidity of about 3. Nephelometric turbidity measurements can detect rayleigh scattering in the absence of association or non-ideal effects, which typically varies linearly with concentration. Other methods for assessing physical stability are well known in the art.
An antibody "retains its chemical stability" in a pharmaceutical composition if its chemical stability at a given point in time is such that the antibody is considered to still retain its biological activity as defined hereinafter. Chemical stability can be assessed, for example, by detecting or quantifying chemically altered forms of the antibody. Chemical changes may include size changes (e.g., clipping), which may be assessed using, for example, pore size exclusion chromatography, SDS-PAGE, and/or matrix-assisted laser desorption ionization/time of flight mass spectrometry (MALDI/TOF MS). Other types of chemical changes include charge changes (e.g., occurring as a result of deamidation or oxidation), which can be assessed by, for example, ion exchange chromatography.
An antibody in a pharmaceutical composition "retains its biological activity" if it is biologically active for its intended purpose. For example, a formulation of the invention may be considered stable if, after storage of the formulation for a certain period of time (e.g., 1 to 12 months) at a temperature, e.g., 5 ℃,25 ℃,45 ℃, etc., the formulation contains a humanized monoclonal antibody that binds to COVID-19 with an affinity that is at least 90%, 95% or more of the binding affinity of the antibody prior to said storage. Binding affinity can also be determined using, for example, ELISA or plasmon resonance techniques.
In the context of the present invention, a "therapeutically effective amount" or "effective amount" of an antibody, in a pharmacological sense, refers to an amount effective in the prevention or treatment or alleviation of the symptoms of the disorder that the antibody is effective to treat. In the present invention, a "therapeutically effective amount" or "therapeutically effective dose" of a drug is any amount of the drug that, when used alone or in combination with another therapeutic agent, protects a subject from the onset of a disease or promotes disease regression as evidenced by a reduction in the severity of disease symptoms, an increase in the frequency and duration of asymptomatic phases of the disease, or the prevention of injury or disability resulting from the affliction of the disease. The ability of a drug to promote disease regression can be evaluated using a variety of methods known to those skilled in the art, such as in human subjects during clinical trials, in animal model systems that predict human efficacy, or by assaying the activity of the agent in an in vitro assay. A therapeutically effective amount of a drug includes a "prophylactically effective amount," i.e., any amount of a drug that inhibits the development or recurrence of a disease when administered to a subject at risk of developing a disease or a subject with a relapse of a disease, either alone or as combined with other therapeutic drugs.
The term "subject" or "patient" is intended to include mammalian organisms. Examples of subjects/patients include human and non-human mammals, such as non-human primates, dogs, cows, horses, pigs, sheep, goats, cats, mice, rabbits, rats and transgenic non-human animals. In a particular embodiment of the invention, the subject is a human.
The terms "administering," "administering," and "treating" refer to introducing a composition comprising a therapeutic agent into a subject using any of a variety of methods or delivery systems known to those of skill in the art. Routes of administration of the humanized anti-BLyS antibody or antigen binding fragment thereof include intravenous, intramuscular, subcutaneous, peritoneal, spinal or other parenteral routes of administration, such as injection or infusion. "parenteral administration" refers to modes of administration other than enteral or topical administration, typically by injection, including, but not limited to, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraframe, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion, and in vivo electroporation.
Antibodies
The term "antibody" as used herein is to be understood as including whole antibody molecules and antigen-binding fragments thereof. The term "antigen-binding portion" or "antigen-binding fragment" of an antibody (or simply "antibody portion" or "antibody fragment"), as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind BLyS.
The term "full length antibody" or "whole antibody molecule" as used herein refers to an immunoglobulin molecule comprising four peptide chains, two heavy (H) chains (about 50-70kDa in full length) and two light (L) chains (about 25kDa in full length) linked to each other by disulfide bonds. Each heavy chain (abbreviated herein as HC) is composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (abbreviated herein as CH). The heavy chain constant region consists of 3 domains, CH1, CH2 and CH 3. Each light chain (abbreviated LC herein) consists of a light chain variable region (abbreviated VL herein) 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 Complementarity Determining Regions (CDRs) with high variability and regions called Framework Regions (FRs) that are spaced more conservatively. Each VH or VL region is formed by, in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 consist of 3 CDRs and 4 FRs arranged from amino terminus to carboxy terminus. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of the antibody can 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 of the classical complement system (Clq).
As used herein, the term "CDR" refers to complementarity determining regions within an antibody variable sequence. There are 3 CDRs in each variable region of the heavy and light chains, 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 for different systems.
The precise amino acid sequence boundaries of the variable region CDRs of the antibodies of the invention may be determined using any of a number of well-known protocols, including the Kabat protocol described by Kabat et al (1991), "Sequences of Proteins of Immunological Interest, 5 th edition Public Health Service, national Institutes of Health, bethesda, MD (" Kabat "numbering scheme) and the IMGT protocol described by Lefranc M. -P. et al (1999 nucleic Acids research,27, 209-212).
As used herein, an "antigen-binding fragment" includes a fragment of an antibody or derivative thereof, typically including at least one fragment of an antigen-binding region or variable region (e.g., one or more CDRs) of a parent antibody that retains at least some of the binding specificity of the parent antibody. Examples of antigen binding fragments include, but are not limited to, fab ', F (ab') 2, and Fv fragments; a diabody; a linear antibody; single chain antibody molecules, such as sc-Fv; nanobodies (nanobodies) and multispecific antibodies formed from antibody fragments. When the binding activity of an antibody is expressed on a molar concentration basis, the binding fragment or derivative thereof typically retains at least 10% of the antigen binding activity of the parent antibody. Preferably, 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. It is also contemplated that antigen-binding fragments of an antibody may include conservative or non-conservative amino acid substitutions (referred to as "conservative variants" or "functionally conservative variants" of the antibody) that do not significantly alter its biological activity.
When referring to ligand/receptor, antibody/antigen or other binding pairs, "specific" binding refers to determining the presence or absence of a protein in a heterogeneous population of proteins and/or other biological agents. For example, the binding reaction of a monoclonal antibody of the invention to a BLyS protein. Thus, under the conditions specified, a particular ligand/antigen binds to a particular receptor/antibody and does not bind in significant amounts to other proteins present in the sample.
The humanized monoclonal antibodies or antigen binding fragments thereof described herein include any of the anti-BLyS antibodies described in application No. CN201210160474.3, the entire disclosure of which is incorporated herein by reference. In some embodiments, the CDR sequences of the antibodies used in the methods and compositions of the invention comprise CDR sequences from antibody BLyS-IV described in CN 201210160474.3. In some embodiments, the non-limiting, exemplary antibody used in the examples herein is selected from the humanized anti-BLyS antibody BLyS-IV described in CN 201210160474.3.
In some embodiments, the non-limiting, exemplary humanized anti-BLyS antibody used in the examples herein has a light chain amino acid sequence as set forth in SEQ ID No. 18, and a heavy chain amino acid sequence as set forth in SEQ ID No. 19; the humanized anti-BLyS antibody was designated H482L472.
In some embodiments, the present invention provides a humanized anti-BLyS antibody or antigen binding fragment thereof, which is capable of binding to B lymphocyte stimulating factor and capable of inhibiting the binding of B lymphocyte stimulating factor to its receptor BR3-Fc, has excellent binding ability to human BLyS protein and competes for the inhibition of the binding of human BLyS to its receptor human BR 3. The humanized anti-BLyS antibody or the antigen binding fragment thereof provided by the invention can be obtained by constructing an expression vector by referring to the methods shown in the embodiment 7 and the embodiment 8 in the application number CN201210160474.3, performing transient expression by using 293F cells, performing a 100mL system and performing one-step affinity purification.
In some embodiments, the invention provides a humanized anti-BLyS antibody or antigen-binding fragment thereof, the amino acid sequences of LCDR1, LCDR2 and LCDR3 of which are set forth in SEQ ID No. 1, SEQ ID No. 2 and SEQ ID No. 3, respectively, and the amino acid sequences of HCDR1, HCDR2 and HCDR3 are set forth 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 FR1 of any one of SEQ ID NO 7, 9 and 13, FR2 is FR2 of SEQ ID NO 7, FR3 is selected from FR3 of any one of SEQ ID NO 7, 9 and 11, FR4 is FR4 of SEQ ID NO 7; FR1 of the heavy chain variable region is selected from FR1 of any one of SEQ ID NOs 8, 10, 12 and 15, FR2 is selected from FR2 of any one of SEQ ID NOs 8, 10 and 15, FR3 is selected from FR3 of any one of SEQ ID NOs 8, 12 and 14, and FR4 is FR4 of SEQ ID NOs 8 or 10.
In some embodiments, the humanized anti-BLyS antibody or antigen binding fragment thereof of the invention does not comprise: the light chain variable region is the amino acid sequence shown in SEQ ID NO. 7, while the heavy chain variable region is the humanized anti-BLyS antibody or antigen-binding fragment thereof of the amino acid sequence shown in SEQ ID NO. 8. In some embodiments, a humanized anti-BLyS antibody or antigen-binding fragment thereof described herein does not comprise: the light chain is the amino acid sequence shown in SEQ ID NO. 16, while the heavy chain is the humanized anti-BLyS antibody or antigen-binding fragment thereof of the amino acid sequence shown in SEQ ID NO. 17.
In some embodiments, in the humanized anti-BLyS antibody or antigen-binding fragment thereof of the invention, the amino acid sequences of LCDR1, LCDR2 and LCDR3 are set forth in SEQ ID No. 1, SEQ ID No. 2 and SEQ ID No. 3, respectively, and the amino acid sequences of HCDR1, HCDR2 and HCDR3 are set forth 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 FR1 of SEQ ID NO. 7, 9 and 13, FR2 is FR2 of SEQ ID NO. 7, FR3 is selected from FR3 of SEQ ID NO. 7, 9 and 11, FR4 is FR4 of SEQ ID NO. 7; FR1-FR4 of the heavy chain variable region are respectively FR1-FR4 of SEQ ID NO. 15.
In some embodiments, in the humanized anti-BLyS antibody or antigen-binding fragment thereof of the invention, the amino acid sequences of LCDR1, LCDR2 and LCDR3 are set forth in SEQ ID No. 1, SEQ ID No. 2 and SEQ ID No. 3, respectively, and the amino acid sequences of HCDR1, HCDR2 and HCDR3 are set forth 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 FR1 of SEQ ID NO. 7, 9 and 13, FR2 is FR2 of SEQ ID NO. 7, FR3 is selected from FR3 of SEQ ID NO. 7, 9 and 11, FR4 is FR4 of SEQ ID NO. 7; FR1-FR4 of the heavy chain variable region are FR1-FR4 of SEQ ID NO. 10, respectively.
In some embodiments, in the humanized anti-BLyS antibody or antigen-binding fragment thereof of the invention, the amino acid sequences of LCDR1, LCDR2 and LCDR3 are set forth in SEQ ID No. 1, SEQ ID No. 2 and SEQ ID No. 3, respectively, and the amino acid sequences of HCDR1, HCDR2 and HCDR3 are set forth 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 FR1 of SEQ ID NO. 7, 9 and 13, FR2 is FR2 of SEQ ID NO. 7, FR3 is selected from FR3 of SEQ ID NO. 7, 9 and 11, FR4 is FR4 of SEQ ID NO. 7; FR1-FR4 of the heavy chain variable region are FR1-FR4 of SEQ ID NO. 12, respectively.
In some embodiments, in the humanized anti-BLyS antibody or antigen-binding fragment thereof of the invention, the amino acid sequences of LCDR1, LCDR2 and LCDR3 are set forth in SEQ ID No. 1, SEQ ID No. 2 and SEQ ID No. 3, respectively, and the amino acid sequences of HCDR1, HCDR2 and HCDR3 are set forth in SEQ ID No. 4, SEQ ID No. 5 and SEQ ID No. 6, respectively; FR1 of the light chain variable region is FR1 described by SEQ ID NO. 11 or 13, FR2 is FR2 described by SEQ ID NO. 11, FR3 is FR3 described by SEQ ID NO. 11 or 13, and FR4 is FR4 described by SEQ ID NO. 11; FR1-FR4 of the heavy chain variable region are respectively FR1-FR4 of SEQ ID NO. 14.
In some embodiments, 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 an amino acid sequence as set forth in SEQ ID NO: 7. SEQ ID NO: 9. the amino acid sequence of SEQ ID NO:11 or SEQ ID NO:13, and the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 8. SEQ ID NO: 10. the amino acid sequence of SEQ ID NO: 12. the amino acid sequence of SEQ ID NO:14 or SEQ ID NO:15, or a pharmaceutically acceptable salt thereof.
In some embodiments, the humanized anti-BLyS antibody or antigen binding fragment thereof comprises a light chain variable region and a heavy chain variable region, wherein:
(1) The light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:7, and the heavy chain variable region comprises the amino acid sequence set forth as SEQ ID NO: 10. SEQ ID NO: 12. the amino acid sequence of SEQ ID NO:14 or SEQ ID NO: 15; or
(2) The light chain variable region comprises the amino acid sequence shown as SEQ ID NO:9, and the heavy chain variable region comprises the amino acid sequence set forth as SEQ ID NO: 8. SEQ ID NO: 10. SEQ ID NO: 12. the amino acid sequence of SEQ ID NO:14 or SEQ ID NO: 15; or
(3) The light chain variable region comprises the amino acid sequence shown as SEQ ID NO:11, and the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 8. SEQ ID NO: 10. the amino acid sequence of SEQ ID NO: 12. SEQ ID NO:14 or SEQ ID NO: 15; or
(4) The light chain variable region comprises the amino acid sequence shown as SEQ ID NO:13, and the heavy chain variable region comprises the amino acid sequence set forth as SEQ ID NO: 8. SEQ ID NO: 10. SEQ ID NO: 12. SEQ ID NO:14 or SEQ ID NO:15, or a pharmaceutically acceptable salt thereof.
Preferably, the light chain variable region and the heavy chain variable region are selected from one of the following groups:
(5) The light chain variable region comprises the amino acid sequence shown as SEQ ID NO:9, and the heavy chain variable region comprises the amino acid sequence set forth as SEQ ID NO: 10; or
(6) The light chain variable region comprises the amino acid sequence shown as SEQ ID NO:11, and the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 12; or
(7) The light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:13, and the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 14; or
(8) The light chain variable region comprises the amino acid sequence shown as SEQ ID NO:7, and the heavy chain variable region comprises the amino acid sequence set forth as SEQ ID NO: 15; or
(9) The light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:9, and the heavy chain variable region comprises the amino acid sequence set forth as SEQ ID NO: 15; or
(10) The light chain variable region comprises the amino acid sequence shown as SEQ ID NO:11, and the heavy chain variable region comprises the amino acid sequence set forth as SEQ ID NO: 15; or
(11) The light chain variable region comprises the amino acid sequence shown as SEQ ID NO:13, and the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:15, or a pharmaceutically acceptable salt thereof.
In some embodiments, the humanized anti-BLyS antibody or antigen binding fragment thereof comprises a light chain variable region and a heavy chain variable region, wherein:
(a) The heavy chain variable region comprises the amino acid sequence as shown in SEQ ID NO:8, and the light chain variable region comprises the amino acid sequence set forth as SEQ ID NO: 13;
(b) The heavy chain variable region comprises the amino acid sequence as shown in SEQ ID NO:10, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 7. 9, 11 or 13;
(c) The heavy chain variable region comprises the amino acid sequence as shown in SEQ ID NO:12, and the light chain variable region comprises the amino acid sequence set forth as SEQ ID NO: 7. 9, 11 or 13;
(d) The heavy chain variable region comprises the amino acid sequence set forth as 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
(e) The heavy chain variable region comprises the amino acid sequence set forth as SEQ ID NO:15, and 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 comprises a human light chain constant region and a human heavy chain constant region, and the light chain variable region and the heavy chain variable region are linked to the human light chain constant region and the heavy chain constant region, respectively. That is, the humanized anti-BLyS antibody comprises an intact light chain formed by the linkage of the light chain variable region of the anti-BLyS antibody to the human light chain constant region, and an intact heavy chain formed by the linkage of the heavy chain variable region of the anti-BLyS antibody to the human heavy chain constant region.
In some embodiments, the human light chain constant region is a human light chain kappa constant region.
In some embodiments, the human heavy chain constant region is a human heavy chain Fc fragment.
Wherein 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 the constant region are connected by overlap extension PCR through a genetic engineering technology to obtain the light chain and the heavy chain of the complete humanized anti-BLyS antibody.
In some embodiments, the humanized anti-BLyS antibody or antigen binding fragment thereof described above, comprising a light chain and a heavy chain, wherein the light chain comprises the amino acid sequence set forth in SEQ ID NO: 16. SEQ ID NO: 18. SEQ ID NO:20 or SEQ ID NO:22, and the heavy chain comprises the amino acid sequence shown as SEQ ID NO: 17. the amino acid sequence of SEQ ID NO: 19. SEQ ID NO: 21. SEQ ID NO:23 or SEQ ID NO: 24.
In some embodiments, the humanized anti-BLyS antibody or antigen binding fragment thereof comprises a light chain and a heavy chain, wherein:
(1) The light chain comprises the amino acid sequence as set forth in SEQ ID NO:16, and the heavy chain comprises the amino acid sequence set forth as SEQ ID NO: 19. SEQ ID NO: 21. SEQ ID NO:23 or SEQ ID NO: 24; or
(2) The light chain comprises the amino acid sequence set forth as SEQ ID NO:18, and the heavy chain comprises the amino acid sequence set forth as SEQ ID NO: 17. SEQ ID NO: 19. the amino acid sequence of SEQ ID NO: 21. the amino acid sequence of SEQ ID NO:23 or SEQ ID NO: 24; or
(3) The light chain comprises the amino acid sequence as set forth in SEQ ID NO:20, and the heavy chain comprises the amino acid sequence set forth as SEQ ID NO: 17. the amino acid sequence of SEQ ID NO: 19. the amino acid sequence of SEQ ID NO: 21. the amino acid sequence of SEQ ID NO:23 or SEQ ID NO: 24; or
(4) The light chain comprises the amino acid sequence as set forth in SEQ ID NO:22, and the heavy chain comprises the amino acid sequence set forth as SEQ ID NO: 17. the amino acid sequence of SEQ ID NO: 19. SEQ ID NO: 21. the amino acid sequence of SEQ ID NO:23 or SEQ ID NO: 24; or
Preferably, the light and heavy chains are selected from one of the following groups:
(5) The light chain comprises the amino acid sequence as set forth in SEQ ID NO:18, and the heavy chain comprises the amino acid sequence set forth as SEQ ID NO: 19; or
(6) The light chain comprises the amino acid sequence as set forth in SEQ ID NO:20, and the heavy chain comprises the amino acid sequence set forth as SEQ ID NO: 21; or
(7) The light chain comprises the amino acid sequence as set forth in SEQ ID NO:22, and the heavy chain comprises the amino acid sequence set forth as SEQ ID NO: 23; or
(8) The light chain comprises the amino acid sequence as set forth in SEQ ID NO:16, and the heavy chain comprises the amino acid sequence set forth as SEQ ID NO: 24; or
(9) The light chain comprises the amino acid sequence set forth as SEQ ID NO:18, and the heavy chain comprises the amino acid sequence set forth as SEQ ID NO: 24; or
(10) The light chain comprises the amino acid sequence as set forth in SEQ ID NO:20, and the heavy chain comprises the amino acid sequence set forth as SEQ ID NO: 24; or
(11) The light chain comprises the amino acid sequence as set forth in SEQ ID NO:22, and the heavy chain comprises the amino acid sequence set forth as SEQ ID NO: 24.
In some embodiments, the humanized anti-BLyS antibody or antigen binding fragment thereof comprises a light chain and a heavy chain, wherein:
(a) The heavy chain comprises the amino acid sequence as set forth in SEQ ID NO:17, and the light chain comprises the amino acid sequence set forth as SEQ ID NO: 22;
(b) The heavy chain comprises the amino acid sequence set forth as SEQ ID NO:19, and the light chain comprises the amino acid sequence set forth as SEQ ID NO: 16. 18, 20 or 22;
(c) The heavy chain comprises the amino acid sequence as set forth in SEQ ID NO:21, and the light chain comprises the amino acid sequence set forth as SEQ ID NO: 16. 18, 20 or 22;
(d) The heavy chain comprises the amino acid sequence as set forth in SEQ ID NO:23, and the light chain comprises the amino acid sequence set forth as SEQ ID NO:20 or 22; or
(e) The heavy chain comprises the amino acid sequence as set forth in SEQ ID NO:24, and the light chain comprises the amino acid sequence set forth as SEQ ID NO: 16. 18, 20 or 22.
Pharmaceutical preparation
The pharmaceutical composition of the invention is a high-stability pharmaceutical composition containing an anti-BLyS antibody. In particular, the present inventors have found that the anti-BLyS antibody has an unexpected characteristic, i.e., high stability, in a combination of a histidine buffer system and L-arginine, L-arginine hydrochloride, or methionine.
The present invention provides a pharmaceutical composition comprising a humanized anti-BLyS antibody or antigen-binding fragment thereof as described 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.
In some embodiments, the present invention provides a pharmaceutical composition comprising: (1) a buffer solution; (2) A humanized anti-BLyS antibody or an antigen-binding fragment thereof having LCDR1, LCDR2 and LCDR3 with amino acid sequences shown as SEQ ID No. 1, SEQ ID No. 2 and SEQ ID No. 3, respectively, and HCDR1, HCDR2 and HCDR3 with amino acid sequences shown as SEQ ID No. 4, SEQ ID No. 5 and SEQ ID No. 6, respectively.
Preferably, the humanized anti-BLyS antibody or antigen binding fragment thereof in the pharmaceutical composition of the invention is as described in any of the embodiments of the "antibodies" section of the present application; or 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 set forth in SEQ ID NO:7, and the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 8; or a 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 as set forth in SEQ ID NO:16, and the heavy chain comprises the amino acid sequence set forth as SEQ ID NO: 17.
For example, in some embodiments, the humanized anti-BLyS antibody or antigen binding fragment thereof described above, comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 7. SEQ ID NO: 9. SEQ ID NO:11 or SEQ ID NO:13, and the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 8. SEQ ID NO: 10. the amino acid sequence of SEQ ID NO: 12. SEQ ID NO:14 or SEQ ID NO:15, or a pharmaceutically acceptable salt thereof.
In some embodiments, the humanized anti-BLyS antibody or antigen binding fragment thereof described above, comprising a light chain variable region and a heavy chain variable region, wherein:
(1) The light chain variable region comprises the amino acid sequence shown as SEQ ID NO:7, and the heavy chain variable region comprises the amino acid sequence set forth as SEQ ID NO: 8. SEQ ID NO: 10. SEQ ID NO: 12. the amino acid sequence of SEQ ID NO:14 or SEQ ID NO: 15; or
(2) The light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:9, and the heavy chain variable region comprises the amino acid sequence set forth as SEQ ID NO: 8. SEQ ID NO: 10. the amino acid sequence of SEQ ID NO: 12. SEQ ID NO:14 or SEQ ID NO: 15; or
(3) The light chain variable region comprises the amino acid sequence shown as SEQ ID NO:11, and the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 8. SEQ ID NO: 10. SEQ ID NO: 12. SEQ ID NO:14 or SEQ ID NO: 15; or
(4) The light chain variable region comprises the amino acid sequence shown as SEQ ID NO:13, and the heavy chain variable region comprises the amino acid sequence set forth as SEQ ID NO: 8. the amino acid sequence of SEQ ID NO: 10. SEQ ID NO: 12. SEQ ID NO:14 or SEQ ID NO: 15; or
Preferably, the light chain variable region and the heavy chain variable region are selected from one of the following groups:
(5) The light chain variable region comprises the amino acid sequence shown as SEQ ID NO:9, and the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 10; or
(6) The light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:11, and the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 12; or
(7) The light chain variable region comprises the amino acid sequence shown as SEQ ID NO:13, and the heavy chain variable region comprises the amino acid sequence set forth as SEQ ID NO: 14; or
(8) The light chain variable region comprises the amino acid sequence shown as SEQ ID NO:7, and the heavy chain variable region comprises the amino acid sequence set forth as SEQ ID NO: 15; or
(9) The light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:9, and the heavy chain variable region comprises the amino acid sequence set forth as SEQ ID NO: 15; or
(10) The light chain variable region comprises the amino acid sequence shown as SEQ ID NO:11, and the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 15; or
(11) The light chain variable region comprises the amino acid sequence shown as SEQ ID NO:13, and the heavy chain variable region comprises the amino acid sequence set forth as SEQ ID NO: 15; or
(12) The light chain variable region comprises the amino acid sequence shown as SEQ ID NO:7, and the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 8.
The humanized anti-BLyS antibody of the present invention further comprises a human light chain constant region and a human heavy chain constant region, and the light chain variable region and the heavy chain variable region are linked to the human light chain constant region and the heavy chain constant region, respectively. That is, the humanized anti-BLyS antibody comprises an intact light chain formed by the linkage of the light chain variable region of the anti-BLyS antibody to the human light chain constant region, and an intact heavy chain formed by the linkage of the heavy chain variable region of the anti-BLyS antibody to the human heavy chain constant region.
In some embodiments, the human light chain constant region is a human light chain kappa constant region.
In some embodiments, the human heavy chain constant region is a human heavy chain Fc fragment.
Wherein the human light chain kappa constant region and the human heavy chain Fc fragment are both derived from healthy human B lymphocytes. The light chain and the heavy chain of the complete humanized anti-BLyS antibody are obtained by connecting the variable region and the constant region through overlap extension PCR by using a genetic engineering technology.
In some embodiments, the humanized anti-BLyS antibody or antigen binding fragment thereof described above, comprising a light chain and a heavy chain, wherein the light chain comprises an amino acid sequence as set forth in SEQ ID NO: 16. the amino acid sequence of SEQ ID NO: 18. SEQ ID NO:20 or SEQ ID NO:22, and the heavy chain comprises the amino acid sequence shown as SEQ ID NO: 17. SEQ ID NO: 19. SEQ ID NO: 21. SEQ ID NO:23 or SEQ ID NO: 24.
In some embodiments, the humanized anti-BLyS antibody or antigen binding fragment thereof described above, comprising a light chain and a heavy chain, wherein:
(1) The light chain comprises the amino acid sequence as set forth in SEQ ID NO:16, and the heavy chain comprises the amino acid sequence set forth as SEQ ID NO: 17. the amino acid sequence of SEQ ID NO: 19. SEQ ID NO: 21. SEQ ID NO:23 or SEQ ID NO: 24; or
(2) The light chain comprises the amino acid sequence set forth as SEQ ID NO:18, and the heavy chain comprises the amino acid sequence set forth as SEQ ID NO: 17. SEQ ID NO: 19. the amino acid sequence of SEQ ID NO: 21. SEQ ID NO:23 or SEQ ID NO: 24; or
(3) The light chain comprises the amino acid sequence set forth as SEQ ID NO:20, and the heavy chain comprises the amino acid sequence set forth as SEQ ID NO: 17. the amino acid sequence of SEQ ID NO: 19. the amino acid sequence of SEQ ID NO: 21. SEQ ID NO:23 or SEQ ID NO: 24; or
(4) The light chain comprises the amino acid sequence set forth as SEQ ID NO:22, and the heavy chain comprises the amino acid sequence set forth as SEQ ID NO: 17. SEQ ID NO: 19. the amino acid sequence of SEQ ID NO: 21. the amino acid sequence of SEQ ID NO:23 or SEQ ID NO: 24; or
Preferably, the light and heavy chains are selected from one of the following groups:
(5) The light chain comprises the amino acid sequence set forth as SEQ ID NO:18, and the heavy chain comprises the amino acid sequence set forth as SEQ ID NO: 19; or
(6) The light chain comprises the amino acid sequence as set forth in SEQ ID NO:20, and the heavy chain comprises the amino acid sequence set forth as SEQ ID NO: 21; or
(7) The light chain comprises the amino acid sequence set forth as SEQ ID NO:22, and the heavy chain comprises the amino acid sequence set forth as SEQ ID NO: 23; or
(8) The light chain comprises the amino acid sequence set forth as SEQ ID NO:16, and the heavy chain comprises the amino acid sequence set forth as SEQ ID NO: 24; or
(9) The light chain comprises the amino acid sequence as set forth in SEQ ID NO:18, and the heavy chain comprises the amino acid sequence set forth as SEQ ID NO: 24; or
(10) The light chain comprises the amino acid sequence as set forth in SEQ ID NO:20, and the heavy chain comprises the amino acid sequence set forth as SEQ ID NO: 24; or
(11) The light chain comprises the amino acid sequence as set forth in SEQ ID NO:22, and the heavy chain comprises the amino acid sequence set forth as SEQ ID NO: 24; or
(12) The light chain comprises the amino acid sequence as set forth in SEQ ID NO:16, and the heavy chain comprises the amino acid sequence set forth as SEQ ID NO:17, or a pharmaceutically acceptable salt thereof.
In some embodiments, the antigen binding fragment of the invention is selected from the group consisting of a Fab, fab '-SH, fv, scFv, F (ab') 2, sdAb, or diabody.
In some embodiments, the concentration of the humanized anti-BLyS antibody or antigen-binding fragment thereof in the pharmaceutical composition is about 1 to about 300mg/mL, preferably about 10 to about 300mg/mL, more preferably about 20 to about 280mg/mL, more preferably about 30 to about 150mg/mL, more preferably about 80 to about 120mg/mL, more preferably about 180 to about 220mg/mL; more preferably, the humanized monoclonal antibody or antigen-binding fragment thereof described above has a concentration of 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,90 mg/mL,100mg/mL,110mg/mL,120mg/mL,130mg/mL,140mg/mL,150mg/mL, 160mg/mL,170mg/mL,180mg/mL or 200mg/mL, preferably about 40mg/mL,60mg/mL,70 mg/mL,80mg/mL,90mg/mL,95mg/mL,100mg/mL,105mg/mL,110mg/mL,120 mg/mL,150mg/mL,160mg/mL,170mg/mL,180mg/mL,190mg/mL,195mg/mL,200 mg/mL,220mg/mL, or 200 mg/mL.
In some embodiments, the buffer is selected from one or more of an acetate buffer, a citrate buffer, a succinate buffer, a phosphate buffer, and a histidine buffer.
In some embodiments, the buffer has a concentration of about 1 mM to about 200mM, preferably about 1 mM to about 100mM, preferably about 5mM to about 50mM, preferably about 10mM to about 40mM, preferably about 20mM to about 40mM; non-limiting examples of the above buffer concentration are about 5mM,10mM,2 mM,25mM,30mM,35mM,40mM,45mM,50mM,60mM, 70mM,80mM,90mM,100mM,105mM,110mM,115mM,120mM,130mM,140mM, 150mM,160mM,170mM, or 180mM or a range in which any two of these values are formed as endpoints, and preferably about 10mM, 2 mM,25mM,30mM, or 35mM.
In some embodiments, the pH of the buffer is about 5.0 to about 6.5, preferably about 5.0 to about 6.0, preferably about 5.5 to about 6.0, and non-limiting examples of the pH of the 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.
In some embodiments, the buffer is a histidine buffer, preferably the histidine buffer is selected from a histidine-hydrochloride buffer or a histidine-acetate buffer, preferably a histidine-hydrochloride buffer.
In some embodiments, the histidine-hydrochloride buffer is prepared from histidine and histidine hydrochloride, preferably L-histidine and L-histidine monohydrochloride. In some embodiments, the histidine buffer is made up of 5-40mM L-histidine and 5-40mM L-histidine monohydrochloride. In some embodiments, the histidine buffer is made up of histidine and histidine hydrochloride in a molar ratio of 1 to 1. In some embodiments, the histidine buffer is made of histidine and histidine hydrochloride in a molar ratio of 1. In some embodiments, the histidine buffer is made from histidine and histidine hydrochloride in a molar ratio of 1. In some embodiments, the histidine buffer is: histidine buffer at pH5.8 prepared from about 10.0-11.0mM L-histidine and about 24.0-25.0 mM L-histidine monohydrochloride. In some embodiments, a 35mM histidine buffer at pH5.8 is prepared by: preparing 35mM L-histidine and L-histidine monohydrochloride solutions, respectively, and mixing the two solutions until the pH value is 5.8. In some embodiments, the histidine buffer is: histidine buffer at pH5.8 made from about 10.0mM histidine and about 25.0mM histidine hydrochloride.
In some embodiments, the buffer is an acetate buffer, preferably, the acetate buffer is an acetate-sodium acetate buffer or an acetate-potassium acetate buffer, preferably an acetate-sodium acetate buffer.
In some embodiments, the buffer is a citric acid buffer, preferably, the citric acid buffer is a citric acid-sodium citrate buffer.
In some embodiments, the buffer is a succinic acid buffer, and preferably, the succinic acid buffer is a succinic acid-sodium succinate buffer.
In some embodiments, the buffer is a phosphate buffer, and preferably, the phosphate buffer is a sodium dihydrogen phosphate-disodium hydrogen phosphate buffer or a potassium dihydrogen phosphate-dipotassium hydrogen phosphate buffer.
In some embodiments, the pharmaceutical composition further comprises a stabilizer.
In some embodiments, the stabilizing agent is selected from one or more of arginine, arginine hydrochloride, methionine, proline, glycine, sodium chloride, mannitol, sorbitol, sucrose, maltose, xylitol, and trehalose.
In some embodiments, the stabilizing agent is selected from one or more of L-arginine, L-arginine hydrochloride, sucrose, methionine, and sodium chloride.
In some embodiments, the concentration of the stabilizer is about 10mM to 400mM, preferably about 50mM to 300mM, more preferably about 100mM to 300mM, and even more preferably about 100mM to 200mM.
In some embodiments, the above stabilizer is a combination of L-arginine hydrochloride at a concentration of about 10-30mM and sodium chloride at a concentration of about 50-200 mM; or the stabilizer is a combination of sodium chloride at a concentration of about 10-30mM L-arginine and about 50-200 mM; or the stabilizer is a combination of L-arginine at a concentration of about 10-30mM and sucrose at a concentration of about 80-220 mM; or said stabilizer is a combination of L-arginine hydrochloride at a concentration of about 10-30mM with sucrose at a concentration of about 80-220 mM; or the stabilizer is a combination of about 30-90mM methionine and about 50-200mM sodium chloride; or the stabilizer is about 10-120mM L-arginine hydrochloride; or the stabilizer is methionine at about 10-120 mM; preferably, the stabilizer is a combination of L-arginine hydrochloride at a concentration of about 10-30mM and sodium chloride at a concentration of about 80-120 mM; or the stabilizing agent is a combination of L-arginine at a concentration of about 10-30mM and sodium chloride at a concentration of about 80-120 mM; or the stabilizer is a combination of about 50-70mM methionine and about 80-120mM sodium chloride; or the stabilizer is a combination of L-arginine at a concentration of about 10-30mM and sucrose at a concentration of about 130-170 mM; or the stabilizer is a combination of L-arginine hydrochloride at a concentration of about 10-30mM with sucrose at a concentration of about 130-170 mM.
In some embodiments, the stabilizing agent consists of sodium chloride at a concentration of about 50 to 200mM and L-arginine at a concentration of about 10 to 30 mM; preferably, the stabilizer consists of sodium chloride at a concentration of about 80-120mM and L-arginine at a concentration of about 10-30 mM; non-limiting examples of such stabilizers are composed of sodium chloride at a concentration of about 50mM,60mM,70mM,80mM, 90mM,95mM,100mM,105mM,110mM,115mM,120mM, or 130mM and L-arginine at a concentration of about 10mM,15mM,20mM,25mM, 30mM.
In some embodiments, the above stabilizer consists of sodium chloride at a concentration of about 50-200mM and L-arginine hydrochloride at a concentration of about 10-30 mM; preferably, said stabilizer consists of sodium chloride at a concentration of about 80-120mM and L-arginine hydrochloride at a concentration of about 10-30 mM; non-limiting examples of such stabilizers are composed of sodium chloride at a concentration of about 50mM,60mM,70mM,80mM, 90mM,95mM,100mM,105mM,110mM,115mM,120Mm or 130mM and L-arginine hydrochloride at a concentration of about 10mM,15mM,20mM,25mM, 30mM.
In some embodiments, the stabilizing agent consists of sodium chloride at a concentration of about 50 to 200mM and methionine at a concentration of about 30 to 90 mM; preferably, the stabilizer consists of sodium chloride at a concentration of about 80-120mM and methionine at a concentration of about 50-70 mM; non-limiting examples of such stabilizers are composed of sodium chloride at a concentration of about 50mM,60mM,70mM,80mM, 90mM,95mM,100mM,105mM,110mM,115mM,120Mm or 130mM and methionine at a concentration of about 30mM,40mM,50mM,55mM,60mM,65mM,70mM,75Mm or 80 mM.
In some embodiments, the stabilizer consists of L-arginine hydrochloride at a concentration of about 10 to 30mM with sucrose at a concentration of about 80 to 220 mM; preferably, the above stabilizer consists of sucrose at a concentration of about 130-170mM and L-arginine hydrochloride at a concentration of about 10-30 mM; non-limiting examples of such stabilizers are composed of sucrose at a concentration of about 80mM,90mM,100mM, 110mM,120Mm,125Mm,130Mm,135Mm,140Mm,145Mm,150Mm,155Mm, 160Mm or 170mM and L-arginine hydrochloride at a concentration of about 10mM,15mM,20mM,25mM, 30mM.
In some embodiments, the stabilizing agent is L-arginine. In some embodiments, the stabilizing agent is L-arginine at a concentration of about 10mM to about 100mM, preferably about 15mM to about 50mM, preferably about 20mM to about 30mM, non-limiting examples of such L-arginine concentrations are about 10mM,15mM,20mM,25mM,30mM,35mM,40mM,45mM,50mM,55mM,60mM,70mM,80mM,90mM,100mM, preferably 25mM or 30mM.
In some embodiments, the stabilizer is methionine. In some embodiments, the stabilizing agent is methionine at a concentration of about 20-200 mM, preferably about 20-180mM, preferably about 40-150mM, preferably about 45-100mM, preferably about 50-90mM, non-limiting examples of which are about 10mM,1 mM,20mM,25mM,30mM,35mM,40mM,45mM,50mM,55mM,60mM, 65mM,70mM,75mM,80mM,85mM,90mM,100mM, preferably 60mM or 65mM.
In some embodiments, the stabilizer is sodium chloride. In some embodiments, the stabilizing agent is sodium chloride at a concentration of about 30-200mM, preferably about 50-190mM, preferably about 60-180mM, preferably about 70-150 mM, preferably about 80-120mM, with non-limiting examples of such concentrations being about 60mM,70mM,80mM, 85mM,90mM,95mM,100mM,105mM,110mM,115mM,120mM,130 mM,140mM,150mM,160mM,170mM,180mM,190mM,200mM, preferably 100mM or 105mM.
In some embodiments, the stabilizing agent is L-arginine hydrochloride. In some embodiments, the stabilizing agent is L-arginine hydrochloride at a concentration of about 30mM to about 200mM, preferably at a concentration of about 50mM to about 190mM, preferably about 100mM to about 180mM, preferably about 120mM to about 170mM, preferably about 130mM to about 150mM, non-limiting examples of such concentrations of L-arginine hydrochloride are about 100mM,110mM,120mM,125mM,130mM,135mM, 140mM,145mM,150mM,155mM,160mM,170mM,180mM,190mM,200mM, preferably 135mM or 140mM.
In some embodiments, the above pharmaceutical composition further comprises a surfactant selected from one or more of polysorbate 80, polysorbate 20, and poloxamer 188.
In some embodiments, the surfactant is selected from polysorbate 80.
In some embodiments, the surfactant is selected from polysorbate 20.
In some embodiments, the surfactant concentration is from about 0.001% to about 0.1%, preferably from about 0.01% to about 0.05%; by way of non-limiting example, the concentration of the above surfactant is about 0.01%, 0.02%,0.03%,0.04%,0.05%.0.06%,0.07%,0.09% or 0.08%.
In some embodiments, the pharmaceutical composition further comprises water for injection.
In some embodiments, the pharmaceutical composition comprises: (a) About 20mg/mL to 280mg/mL of the humanized anti-BLyS antibody or antigen-binding fragment thereof described herein; (b) About 5-50mM histidine buffer or about 5-50mM acetic acid buffer, pH about 5.0-6.5; (c) About 10-30mM of L-arginine or about 10-30mM of L-arginine hydrochloride; (d) about 80 to 220mM sucrose; (e) and about 0.01% to 0.1% polysorbate 80.
In some embodiments, the pharmaceutical composition comprises: (a) About 20mg/mL to 280mg/mL of the humanized anti-BLyS antibody or antigen-binding fragment thereof according to any of the embodiments herein; (b) About 10-40mM histidine buffer or about 10-40mM acetic acid buffer, pH about 5.0-6.5; (c) About 10-30mM L-arginine or about 10-30mM L-arginine hydrochloride; (d) about 130 to 170mM sucrose; (e) and about 0.01% to 0.1% polysorbate 80.
In some embodiments, the pharmaceutical composition comprises: (a) About 30mg/mL to 150mg/mL of a humanized anti-BLyS antibody or antigen-binding fragment thereof according to any of the embodiments herein; (b) About 5-50mM histidine buffer or about 5-50mM acetic acid buffer, pH about 5.0-6.5; (c) About 10-30mM of L-arginine, about 10-30mM of L-arginine hydrochloride, or about 30-90mM of methionine; (d) about 50-200mM sodium chloride; (e) and about 0.01% to 0.1% polysorbate 80.
In some embodiments, the above pharmaceutical composition comprises: (a) About 30mg/mL to 150mg/mL of the humanized anti-BLyS antibody or antigen-binding fragment thereof according to any of the embodiments herein; (b) About 5-50mM histidine buffer, pH about 5.0-6.5; (c) About 10-30mM of L-arginine, about 10-30mM of L-arginine hydrochloride, or about 30-90mM of methionine; (d) about 50-200mM sodium chloride; (e) and about 0.01% to 0.1% polysorbate 80.
In some embodiments, the above pharmaceutical composition comprises: (a) About 30mg/mL to 150mg/mL of a humanized anti-BLyS antibody or antigen-binding fragment thereof according to any of the embodiments herein; (b) about 5 to about 50mM acetate buffer, pH about 5.0 to about 6.5; (c) About 10-30mM of L-arginine, about 10-30mM of L-arginine hydrochloride, or about 30-90mM of methionine; (d) about 50-200mM sodium chloride; (e) and about 0.01% to 0.1% polysorbate 80.
In some embodiments, the pharmaceutical composition comprises: (a) About 80mg/mL to 120mg/mL of a humanized anti-BLyS antibody or antigen-binding fragment thereof according to any of the embodiments herein; (b) About 20-40mM histidine buffer or about 20-40mM acetic acid buffer, pH about 5.5-6.0; (c) About 10-30mM of L-arginine, about 50-70mM of methionine, or about 10-30mM of L-arginine hydrochloride; (d) about 80 to 120mM sodium chloride; (e) and about 0.01% to 0.05% polysorbate 80.
In some embodiments, the pharmaceutical composition comprises: (a) About 80mg/mL to 120mg/mL of a humanized anti-BLyS antibody or antigen-binding fragment thereof according to any of the embodiments herein; (b) About 20-40mM histidine buffer or, pH about 5.5-6.0; (c) About 10-30mM of L-arginine, about 50-70mM of methionine, or about 10-30mM of L-arginine hydrochloride; (d) about 80-120mM sodium chloride; (e) and about 0.01% to 0.05% polysorbate 80.
In some embodiments, the pharmaceutical composition comprises: (a) About 80mg/mL to 120mg/mL of the humanized anti-BLyS antibody or antigen-binding fragment thereof according to any of the embodiments herein; (b) About 20-40mM acetate buffer, pH about 5.5-6.0; (c) About 10-30mM of L-arginine, about 50-70mM of methionine, or about 10-30mM of L-arginine hydrochloride; (d) about 80-120mM sodium chloride; (e) and about 0.01% to 0.05% polysorbate 80.
In some embodiments, the pharmaceutical composition comprises: (a) About 180mg/mL to 220mg/mL of the humanized anti-BLyS antibody or antigen-binding fragment thereof described herein; (b) About 20-40mM histidine buffer or about 20-40mM acetic acid buffer, pH about 5.5-6.0; (c) About 10-30mM of L-arginine or about 10-30mM of L-arginine hydrochloride; (d) about 130 to 170mM sucrose; (e) and about 0.01% to 0.05% polysorbate 80; or
In some embodiments, the pharmaceutical composition comprises: (a) About 200mg/mL of the humanized anti-BLyS antibody or antigen-binding fragment thereof according to any of the embodiments herein; (b) About 25mM histidine buffer, about 25mM acetate buffer, or about 35-36mM histidine buffer, pH about 5.5-6.0; (c) About 15mM L-arginine or about 15mM L-arginine hydrochloride; (d) 150mM sucrose; (e) and about 0.02% polysorbate 80.
In some embodiments, the pharmaceutical composition comprises: (a) About 100mg/mL of a humanized anti-BLyS antibody or antigen-binding fragment thereof according to any one of the embodiments herein; (b) about 25mM histidine buffer, at a pH of about 5.5 to about 6.0; (c) About 25mM L-arginine or about 25mM L-arginine hydrochloride; (d) about 100mM sodium chloride; (e) and about 0.02% polysorbate 80.
In some embodiments, the pharmaceutical composition comprises: (a) About 100mg/mL of the humanized anti-BLyS antibody or antigen-binding fragment thereof according to any of the embodiments herein; (b) about 25mM acetate buffer, at a pH of about 5.5 to about 6.0; (c) methionine at about 60 mM; (d) about 100mM sodium chloride; (e) and about 0.02% polysorbate 80.
In some embodiments, the pharmaceutical composition comprises: (a) About 100mg/mL of the humanized anti-BLyS antibody or antigen-binding fragment thereof according to any of the embodiments herein; (b) about 35-36mM histidine buffer, pH about 5.5-6.0; (c) About 15mM L-arginine hydrochloride or about 15mM L-arginine; (d) about 100mM sodium chloride; (e) and about 0.02% polysorbate 80.
In some embodiments, the pharmaceutical composition comprises a component as shown in any one of the following (1) to (20):
(1) (a) about 20mg/mL to 280mg/mL of a humanized anti-BLyS antibody or antigen-binding fragment thereof comprising a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:9, and the heavy chain variable region comprises the amino acid sequence set forth as SEQ ID NO: 8. the amino acid sequence of SEQ ID NO: 10. SEQ ID NO: 12. SEQ ID NO:14 or SEQ ID NO: 15; (b) About 5-50mM histidine buffer, pH about 5.0-6.5; (c) About 10-30mM L-arginine or about 10-30mM L-arginine hydrochloride; (d) about 80 to 220mM sucrose; (e) and about 0.01% to 0.1% polysorbate 80; or
(2) (a) about 30mg/mL to 150mg/mL of a humanized anti-BLyS antibody or antigen-binding fragment thereof comprising a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:9, and the heavy chain variable region comprises the amino acid sequence set forth as SEQ ID NO: 8. SEQ ID NO: 10. SEQ ID NO: 12. SEQ ID NO:14 or SEQ ID NO: 15; (b) about 5-50mM histidine buffer, pH about 5.0-6.5; (c) about 10-30mM of L-arginine; (d) about 50-200mM sodium chloride; (e) and about 0.01% to 0.1% polysorbate 80; or
(3) (a) about 30mg/mL to 150mg/mL of a humanized anti-BLyS antibody or antigen-binding fragment thereof comprising a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:9 and the heavy chain variable region comprises the amino acid sequence shown as SEQ ID NO: 8. the amino acid sequence of SEQ ID NO: 10. the amino acid sequence of SEQ ID NO: 12. SEQ ID NO:14 or SEQ ID NO: 15; (b) about 5-50mM histidine buffer, pH about 5.0-6.5; (c) about 10-30mM L-arginine hydrochloride; (d) about 50-200mM sodium chloride; (e) and about 0.01% to 0.1% polysorbate 80; or
(4) (a) about 30mg/mL to 150mg/mL of a humanized anti-BLyS antibody or antigen-binding fragment thereof comprising a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:9, and the heavy chain variable region comprises the amino acid sequence set forth as SEQ ID NO: 8. SEQ ID NO: 10. SEQ ID NO: 12. SEQ ID NO:14 or SEQ ID NO: 15; (b) about 5-50mM histidine buffer, at a pH of about 5.0-6.5; (c) methionine at a concentration of about 30 to about 90 mM; (d) about 50-200mM sodium chloride; (e) and about 0.01% to 0.1% polysorbate 80; or
(5) (a) about 30mg/mL to 150mg/mL of a humanized anti-BLyS antibody or antigen-binding fragment thereof comprising a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:9 and the heavy chain variable region comprises the amino acid sequence shown as SEQ ID NO: 8. SEQ ID NO: 10. SEQ ID NO: 12. SEQ ID NO:14 or SEQ ID NO: 15; (b) about 5 to about 50mM acetate buffer, at a pH of about 5.0 to about 6.5; (c) about 10-30mM L-arginine; (d) about 50-200mM sodium chloride; (e) and about 0.01% to 0.1% polysorbate 80; or
(6) (a) about 30mg/mL to 150mg/mL of a humanized anti-BLyS antibody or antigen-binding fragment thereof comprising a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:9 and the heavy chain variable region comprises the amino acid sequence shown as SEQ ID NO: 8. SEQ ID NO: 10. the amino acid sequence of SEQ ID NO: 12. the amino acid sequence of SEQ ID NO:14 or SEQ ID NO: 15; (b) about 5 to about 50mM acetate buffer, at a pH of about 5.0 to about 6.5; (c) about 10-30mM L-arginine hydrochloride; (d) about 50-200mM sodium chloride; (e) and about 0.01% to 0.1% polysorbate 80; or
(7) (a) about 30mg/mL to 150mg/mL of a humanized anti-BLyS antibody or antigen-binding fragment thereof comprising a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:9, and the heavy chain variable region comprises the amino acid sequence set forth as SEQ ID NO: 8. SEQ ID NO: 10. SEQ ID NO: 12. the amino acid sequence of SEQ ID NO:14 or SEQ ID NO: 15; (b) about 5 to 50mM acetate buffer, pH about 5.0 to 6.5; (c) methionine in an amount of about 30 to about 90 mM; (d) about 50-200mM sodium chloride; (e) and about 0.01% to 0.1% polysorbate 80; or
(8) (a) about 80mg/mL to 120mg/mL of a humanized anti-BLyS antibody or antigen-binding fragment thereof comprising a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:9, and the heavy chain variable region comprises the amino acid sequence set forth as SEQ ID NO: 10; (b) about 20 to 40mM histidine buffer, pH about 5.5 to 6.0; (c) about 10-30mM of L-arginine; (d) about 80 to 120mM sodium chloride; (e) and about 0.01% to 0.05% polysorbate 80; or
(9) (a) about 80mg/mL to 120mg/mL of a humanized anti-BLyS antibody or antigen-binding fragment thereof comprising a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:9 and the heavy chain variable region comprises the amino acid sequence shown as SEQ ID NO: 10; (b) about 20-40mM histidine buffer, at a pH of about 5.5-6.0; (c) about 10-30mM L-arginine hydrochloride; (d) about 80 to 120mM sodium chloride; (e) and about 0.01% to 0.05% polysorbate 80; or
(10) (a) about 80mg/mL to 120mg/mL of a humanized anti-BLyS antibody or antigen-binding fragment thereof comprising a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:9 and the heavy chain variable region comprises the amino acid sequence shown as SEQ ID NO: 10; (b) about 20-40mM acetate buffer, at a pH of about 5.5-6.0; (c) methionine at a concentration of about 50 to about 70 mM; (d) about 80 to 120mM sodium chloride; (e) and about 0.01% to 0.05% polysorbate 80; or
(11) (a) about 180mg/mL to 220mg/mL of a humanized anti-BLyS antibody or antigen-binding fragment thereof comprising a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:9 and the heavy chain variable region comprises the amino acid sequence shown as SEQ ID NO: 10; (b) about 20 to 40mM histidine buffer, pH about 5.5 to 6.0; (c) About 10-30mM L-arginine or about 10-30mM L-arginine hydrochloride; (d) sucrose at about 130-170 mM; (e) and about 0.01% to 0.05% polysorbate 80; or
(12) (a) about 200mg/mL of a humanized anti-BLyS antibody or antigen-binding fragment thereof comprising a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO:9, and the heavy chain variable region comprises the amino acid sequence set forth as SEQ ID NO: 10; (b) about 35-36mM histidine buffer, at a pH of about 5.5-6.0; (c) about 15mM L-arginine hydrochloride; (d) about 150mM sucrose; (e) and about 0.02% polysorbate 80; or
(13) (a) about 200mg/mL of a humanized anti-BLyS antibody or antigen-binding fragment thereof comprising a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO:9 and the heavy chain variable region comprises the amino acid sequence shown as SEQ ID NO: 10; (b) about 35-36mM histidine buffer, pH about 5.5-6.0; (c) about 15mM L-arginine; (d) about 150mM sucrose; (e) and about 0.02% polysorbate 80; or
(14) (a) about 200mg/mL of a humanized anti-BLyS antibody or antigen-binding fragment thereof comprising a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO:9, and the heavy chain variable region comprises the amino acid sequence set forth as SEQ ID NO: 10; (b) about 25mM histidine buffer, pH about 5.5-6.0; (c) about 15mM L-arginine hydrochloride; (d) sucrose at about 150 mM; (e) and about 0.02% polysorbate 80; or
(15) (a) about 100mg/mL of a humanized anti-BLyS antibody or antigen-binding fragment thereof comprising a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO:9 and the heavy chain variable region comprises the amino acid sequence shown as SEQ ID NO: 10; (b) about 25mM histidine buffer, at a pH of about 5.5 to about 6.0; (c) about 25mM L-arginine; (d) about 100mM sodium chloride; (e) and about 0.02% polysorbate 80; or
(16) (a) about 100mg/mL of a humanized anti-BLyS antibody or antigen-binding fragment thereof comprising a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO:9, and the heavy chain variable region comprises the amino acid sequence set forth as SEQ ID NO: 10; (b) about 25mM histidine buffer, pH about 5.5-6.0; (c) about 25mM L-arginine hydrochloride; (d) about 100mM sodium chloride; (e) and about 0.02% polysorbate 80; or
(17) (a) about 100mg/mL of a humanized anti-BLyS antibody or antigen-binding fragment thereof comprising a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO:9 and the heavy chain variable region comprises the amino acid sequence shown as SEQ ID NO: 10; (b) about 25mM acetate buffer, pH about 5.5-6.0; (c) methionine at about 60 mM; (d) about 100mM sodium chloride; (e) and about 0.02% polysorbate 80; or
(18) (a) about 100mg/mL of a humanized anti-BLyS antibody or antigen-binding fragment thereof comprising a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO:9, and the heavy chain variable region comprises the amino acid sequence set forth as SEQ ID NO: 10; (b) about 35-36mM histidine buffer, at a pH of about 5.5-6.0; (c) about 15mM L-arginine hydrochloride; (d) about 100mM sodium chloride; (e) and about 0.02% polysorbate 80; or
(19) (a) about 100mg/mL of a humanized anti-BLyS antibody or antigen-binding fragment thereof comprising a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO:9 and the heavy chain variable region comprises the amino acid sequence shown as SEQ ID NO: 10; (b) about 35-36mM histidine buffer, at a pH of about 5.5-6.0; (c) about 15mM L-arginine; (d) about 100mM sodium chloride; (e) and about 0.02% polysorbate 80; or
(20) (a) about 100mg/mL of a humanized anti-BLyS antibody or antigen-binding fragment thereof comprising a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO:9, and the heavy chain variable region comprises the amino acid sequence set forth as SEQ ID NO: 10; (b) about 35mM histidine buffer, pH about 5.8; (c) about 15mM L-arginine hydrochloride; (d) about 100mM sodium chloride; (e) and about 0.02% polysorbate 80;
preferably, in each of the above pharmaceutical compositions, the humanized anti-BLyS antibody has a light chain amino acid sequence as shown in SEQ ID NO. 18 and a heavy chain amino acid sequence as shown in SEQ ID NO. 19.
In some embodiments, the pharmaceutical composition is a liquid formulation or a lyophilized formulation.
In some embodiments, the pharmaceutical composition is a liquid formulation.
The liquid formulation or lyophilized formulation is stable at 2-8 ℃ for at least 3 months, at least 6 months, at least 12 months, at least 18 months or at least 24 months.
The aqueous solution or lyophilized formulation is stable at 40 ℃ for at least 7 days, at least 14 days or at least 28 days.
Medical use and method
The present invention provides the use of a humanized anti-BLyS antibody or antigen binding fragment thereof as described in any of the schemes herein, a polynucleotide as described herein, an expression vector as described herein, a host cell as described herein, a pharmaceutical composition as described herein, or an injectable formulation as described herein, in the manufacture of a medicament for the prevention and/or treatment of a disease caused by an excessive B-cell proliferation.
The present invention provides a humanized anti-BLyS antibody or antigen binding fragment thereof as described in any of the embodiments herein, a polynucleotide as described herein, an expression vector as described herein, a host cell as described herein, a pharmaceutical composition as described herein, or an injection as described herein for use in the prevention and/or treatment of a disease caused by an excessive B-cell proliferation.
The present invention provides a method of preventing and/or treating a disease caused by B cell hyperproliferation, comprising administering to a subject in need thereof a humanized anti-BLyS antibody or antigen binding fragment thereof as described in any one of the regimens herein, a polynucleotide described herein, an expression vector described herein, a host cell described herein, a pharmaceutical composition described herein, or an injection described herein.
In the present invention, diseases caused by excessive B cell proliferation include systemic lupus erythematosus, rheumatoid arthritis, mandatory arthritis, or B cell lymphoma.
The invention will be elucidated hereinafter by means of specific examples. It should be understood that these examples are illustrative only and are not intended to limit the scope of the present invention. The methods and materials used in the examples are, unless otherwise indicated, conventional in the art.
Detection method
The major pathways for protein degradation are the formation of aggregates, cleavage products and charged variants. The percentage of the native form (protein monomers) to the aggregated form was determined by size exclusion chromatography (SEC-HPLC) and the percentage of the acidic and basic forms of the antibody was determined by cation exchange chromatography (CEX-HPLC).
Stability was assessed by the following parameters: (1) visual appearance and visible foreign matter; (2) measuring the protein content by an ultraviolet spectrophotometry; (3) SEC-HPLC measurement of antibody monomer, dimer or fragment content; (4) CEX-HPLC measures antibody main charge, acidic charge or basic charge content; (5) detecting the molecular weight of the antibody by an NR-CE-SDS method; (6) detecting the molecular weight of the antibody by using an R-CE-SDS method; (7) detecting the antibody binding activity by ELISA method.
(1) Protein content
The protein concentration was measured using an ultraviolet spectrophotometer (Thermo, model: biomate 3S). The percentage extinction coefficient (E1%) was set at 1.47 (g/mL) -1cm-1. The cuvette was washed three times with ultrapure water using a BIO MATE 3S instrument, 150. Mu.L of ultrapure water was added to the cuvette, and the measurement was performed by click with blank correction with ultrapure water. 2 portions of solution were measured in parallel for each sample, and the measurement was repeated 3 times for each solution; each solution was assayed by rinsing the cuvette 2 times with 150. Mu.L of the solution and then taking 150. Mu.L of the solution. And calculating the concentration of the corresponding detection product according to the extinction coefficient and the OD value.
(2) SEC-HPLC purity
SEC purity was determined by HPLC (Ultimate 3000, DIONEX instrument) using a SEC column (TOSOH G4000sxl SEC 7.8mm ID x 30cm,5 μm). The mobile phase composition was 50mM PB/300mM NaCl pH 7.0. + -. 0.2. The relative percentages of the main peak, high polymer and low polymer were calculated using area normalization. Details are shown in table 1 below:
table 1: SEC-HPLC purity detection method
Figure RE-GDA0003822617550000321
Figure RE-GDA0003822617550000331
(3) CEX-HPLC purity
The results were quantitatively analyzed by peak area normalization to calculate the peak area percentages of K0, K1, and K2 (K0 being the peak with the highest peak area percentage, K1 being the first peak after K0, and the retention time difference between K2 and K1 being about 2.5 min). The sum of the peak area percentages of three peaks of K0, K1 and K2 is the content of lysine variant (namely the purity of the sample), the sum of all peak area percentages before the K0 peak is the content of acidic peak, and the sum of all peak area percentages after the K0 peak is removed from the K1 and K2 peaks is the content of alkaline peak. Details are shown in table 2 below:
table 2: CEX-HPLC purity detection method
Figure RE-GDA0003822617550000332
(4) Insoluble microparticles
The instrument was turned on and the illumination knob was adjusted to 1200lx.
Taking 20 samples to be tested, removing the labels of the containers, wiping the outer walls of the containers to ensure that the containers are clean and transparent, and standing for 12 hours at room temperature;
the test article is placed at the edge of the shading plate, the container is slightly rotated and turned over to suspend visible foreign matters possibly existing in the liquid medicine (no bubbles are generated in the liquid medicine), and the liquid medicine is observed in a black background and a white background at a position of about 25 cm, wherein the inspection time is 20 seconds.
Example 1: antibody preparation
Antibody sequence design:
starting with an antibody H48L47 (light chain as shown in SEQ ID NO:16 and heavy chain as shown in SEQ ID NO: 17), the Framework Regions (FRs) of the antibody were mutated to screen out the following amino acid sequences for the purposes of reducing the immunogenicity and improving the stability of the antibody: the FR region of light chain L47 (SEQ ID NO: 16) was mutated to produce three sequences L472 (SEQ ID NO: 18), L473 (SEQ ID NO: 20) and L474 (SEQ ID NO: 22), and the FR region of heavy chain H48 (SEQ ID NO: 17) was mutated to produce four sequences H482 (SEQ ID NO: 19), H483 (SEQ ID NO: 21), H484 (SEQ ID NO: 23) and H485 (SEQ ID NO: 24).
Pairwise pairing and combining the light chains and the heavy chains to construct an expression vector, wherein the combination of the light chains and the heavy chains for antibody expression is shown in the following table 3, the light chains and the heavy chains are subjected to transient expression by using 293F cells, a 100ml system is subjected to one-step affinity purification to obtain proteins, and twenty humanized antibodies are obtained and are shown in the following table 4.
Table 3: antibody expression light and heavy chain combination table
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
Table 4: variable region amino acid sequences and full-length sequences of antibodies
Figure RE-GDA0003822617550000341
Figure RE-GDA0003822617550000351
Example 2: binding ELISA method for determining Binding capacity of antibody and human BLyS protein
Human BLyS was coated on a hydrophobic ELISA plate, binding ELISA was used to determine the Binding capacity of the antibody to human BLyS protein, and EC50 was calculated by fitting a curve. The test results are shown in table 5 below.
Table 5: binding ability of antibodies to human BLyS protein
Figure RE-GDA0003822617550000352
Figure RE-GDA0003822617550000361
The results show that 20 antibodies of the invention bind to all recombinant human BLyS proteins, and that antibodies engineered with Framework Region (FR) sequences have an EC50 that is smaller or comparable to the EC50 of H48L 47.
Example 3: blocking ELISA method for determining ability of antibody to competitively inhibit binding of human BLyS and human BR3
Human BLyS is coated on a hydrophobic enzyme label plate, the ability of the antibody subjected to Framework Region (FR) sequence transformation to competitively inhibit the combination of the human BLyS and human BR3 is measured by a Blocking ELISA method, and an IC50 is calculated by fitting a curve. Binding ELISA results showed that the EC50 of the antibodies engineered with the Framework Region (FR) sequences was smaller than the EC50 of the non-engineered antibodies, so only the light and heavy chain combinations, H482L472, H483L473 and H484L474, were selected for Blocking ELISA assay and compared to the commercially available positive control antibody Benlysta. The test results are shown in table 6 below.
Positive control: benlysta, human Genome Sciences, see CN1279055C, CN106661111A.
Table 6: ability of antibodies to compete for inhibition of binding of human BLyS to human BR3
Name of antibody IC50(μg/mL)
H482L472 1.238
H483L473 0.8576
H484L474 1.275
Benlysta 4.714
From the test results, both the antibody modified by the Framework Region (FR) sequence and the positive drug Benlysta can inhibit the binding of BLyS and its receptor BR3, and the IC50 of the antibodies H482L472, H483L473 and H484L474 is significantly better than that of the control Benlysta.
Example 4: reporter gene method for determining downstream NF-kB signal stimulated by antibody competitive inhibition of human BLyS binding to TACI
Human soluble BLyS binds to TACI to stimulate a downstream NF-kB signal path, downstream luciferase is detected along with the released signal, the antibody blocks the combination of BLyS and TACI, the downstream NF-kB signal is inhibited, the change of the signal value can be detected by a luciferase detection kit, and the IC50 is calculated by fitting a curve. Combinations of light and heavy chains, all engineered, i.e., H482L472, H483L473, and H484L474, were also selected for cell biology assays and compared to the marketed positive control antibody Benlysta, with the results shown in table 7 below.
Positive control: benlysta, human Genome Sciences, see CN1279055C, CN106661111A.
Table 7: antibody competition inhibits human BLyS binding to TACI-stimulated downstream NF-kB signal
Name of antibody IC 50 (ng/mL)
H482L472 37.56
H483L473 29.27
H484L474 31.52
Benlysta 51.68
As can be seen from the test results, both the antibody engineered with the Framework Region (FR) sequence and the positive drug Benlysta inhibited the binding of human BLyS to TACI-stimulated downstream NF- κ B signal, and the IC50 of antibodies H482L472, H483L473 and H484L474 were significantly better than the control Benlysta.
Example 5: buffer solution System, pH screening experiment
In the liquid pharmaceutical composition, the buffer system and pH have a close influence on the stability of the antibody, and each antibody with unique physicochemical properties has the most suitable type and pH of the buffer. This example is directed to screening an optimal buffer system and pH for optimal stability of the antibodies disclosed herein for clinical use. The antibody in this example was H482L472.
1.1 buffer preparation
25mM sodium acetate (pH 5.0,5.5, 6.0), 25mM sodium phosphate (pH 6.0,6.5, 7.0), 35mM histidine (pH 5.5,6.0, 6.5), and 25mM succinic acid buffer (pH 5.5,6.0, 6.5) were prepared, and 50mL of each solution was prepared.
1.2 changing the liquid
A15 mL centrifugal ultrafiltration tube (Millipore, UCF 803096) was used for a centrifugal ultrafiltration exchange (5000 g. Times.15 min). Each group of samples contained 1mL of protein and 3 changes of buffer solution with 2 mL. After completion of the exchange, the concentration was measured with a spectrophotometer, and the final concentration of the preparation was adjusted to an antibody concentration of about 30mg/mL.
1.3 Sterilization filtration and lofting
The samples after the exchange were sterile filtered in 1.5mL cryopreserved tubes, about 1mL each, using a needle filter in a clean bench. Incubate at 37 ℃ for 7d, then detect SEC, CEX-HPLC.
The experimental protocol is shown in table 8 below.
Table 8: buffer system and pH screening protocol
Figure RE-GDA0003822617550000381
The results of the measurements are shown in Table 9 below.
Table 9: buffer system and pH screening test results
Figure RE-GDA0003822617550000382
From the experimental results, the SEC and CEX purity of the acetate buffer and the histidine buffer decreased less after standing at 37 ℃ for 6 days, and the purity decreased better than that of the phosphate and succinate buffers. Acetate and histidine buffers were therefore selected for the next round of screening.
Example 6: stabilizer screening experiments
According to the literature report, when the concentration of the protein in the monoclonal antibody preparation is higher, adverse effects such as protein agglutination, high polymer content increase and the like are caused. In the experiment, on the basis of a preparation buffer system screened in the first round, a plurality of different amino acids (L-arginine and methionine) are added, and the influence of the auxiliary materials on the stability of the preparation is inspected.
2.1 buffer preparation
The formulations were prepared according to the buffer formulations in tables 10 and 11, respectively. Wherein the content of the histidine salt buffer solution is 35mM; the acetate buffer content was 25mM, the antibody in this example was H482L472, the final antibody concentration in each formulation was 100mg/mL, and the sodium chloride concentration in each formulation was 100mM.
Table 10: experimental scheme for screening stabilizing agent in histidine buffer solution system
Figure RE-GDA0003822617550000391
Table 11: experimental scheme for screening stabilizer in acetic acid buffer solution system
Figure RE-GDA0003822617550000392
Figure RE-GDA0003822617550000401
2.2 changing the liquid
The concentrate was concentrated by ultrafiltration (5000 g. Times.25 min) using a 50mL centrifugal ultrafiltration tube (Millipore, UFC 903096). 3.5mL of each sample, ultrafiltering and concentrating to 1mL, and adding corresponding buffer solution 4mL for changing solution for 3 times. And after liquid replacement is finished, the volume is increased to 1mL for standby, and the final concentration of the antibody in the sample is about 100mg/mL.
2.3 sterile filtration
The samples after liquid change were aseptically filtered in a clean bench and then dispensed into 1mL pre-filled syringes, each of which was about 1mL.
2.4 lofting and detection
And (4) placing the sterilized and filtered sample in a constant-temperature incubator at 37 ℃ for incubation for 7d. Detecting SEC and CEX-HPLC.
The experimental results are shown in tables 12 and 13 below.
Table 12: experimental result of screening stabilizer in histidine buffer solution system
Figure RE-GDA0003822617550000402
Table 13: experimental result of screening stabilizer in acetic acid buffer solution system
Figure RE-GDA0003822617550000403
Figure RE-GDA0003822617550000411
According to the experimental results, the pH of the formulation has a significant effect on stability, whether histidine or acetic acid. The purity tends to increase and decrease with increasing pH, while the addition of L-arginine or methionine to the formulation is effective in reducing polymer production. According to the results, two groups of preparation prescriptions are screened out: 35mM histidine +15mM L-arginine +100mM sodium chloride, pH5.8;25mM acetic acid +60mM methionine +100mM sodium chloride, pH5.8.
Example 7: forced degradation experiment
Further forced degradation experiments were performed on the screened formulations to investigate the stability of the formulations at various screening pressures. The antibody in this example was H482L472 and the protocol is shown in table 14 below.
Table 14: forced degradation experimental scheme
Figure RE-GDA0003822617550000412
3.1 buffer preparation
500mL of each preparation buffer required for the experiment was prepared according to the formulation shown in Table 15 below, and filtered through a 0.2 μm filter.
Table 15: formulation buffer solution formula
Figure RE-GDA0003822617550000421
3.2 concentrate exchange solution
The concentrate was concentrated by ultrafiltration (5000 g. Times.25 min) using a 50mL centrifuge ultrafiltration tube (Millipore, UFC 903096). 4.5mL of the stock solution was added to each tube, and after concentration by ultrafiltration to about 1.5mL, the corresponding buffer solution was added 4mL and the solution was changed 3 times. After the liquid change is finished, the protein concentration is measured by mixing evenly, and the final concentration of the sample is adjusted to be about 100mg/mL.
3.3 preparation of mother liquor Tween-80
10mL of aqueous solution containing 2% of Tween-80 is prepared, and the mixture is shaken and mixed evenly. And adding a proper volume of prepared Tween-80 mother liquor into the sample after the liquid change, and adjusting the Tween-80 content in the sample to 0.02%.
3.4 Sterilization filtration and packaging
The prepared samples were filtered through a 0.2 μm sterile filter in a clean bench and dispensed into 1mL pre-filled syringes, each approximately 1mL.
3.5 lofting and detection
The samples were set out according to the protocol in Table 14 and tested.
The results of the experiments are shown in tables 16-20 below.
Table 16: results of shaking experiments
Figure RE-GDA0003822617550000422
Table 17: results of freeze thawing experiments
Figure RE-GDA0003822617550000423
Figure RE-GDA0003822617550000431
Table 18: experimental results of ultraviolet irradiation
Figure RE-GDA0003822617550000432
Table 19: SEC/CEX purity high-temperature accelerated experiment result
Figure RE-GDA0003822617550000433
Table 20: high temperature accelerated test results of appearance and visible foreign matter
Figure RE-GDA0003822617550000434
Figure RE-GDA0003822617550000441
According to experimental results, no obvious purity reduction is seen after shaking and freeze thawing of F1 and F2 prescriptions, but the SEC and CEX purities of two prescription preparations have a reduction trend after 8 hours of ultraviolet irradiation, which indicates that the prescription of the preparation is sensitive to ultraviolet light and needs to be stored in a dark place; the high-temperature accelerated test shows that the appearance of the sample is not obviously changed after the sample is placed at 4W and 8W under the high-temperature condition, but the acid peak and the polymer content are obviously increased, wherein the rising trend of the formula F1 is slightly lower than that of the formula F2. When the subsequent production is performed according to the formula F1, the pH value needs to be adjusted by hydrochloric acid because the L-arginine has slightly high alkalinity, then the L-arginine in the original formula F1 is replaced by L-arginine hydrochloride (CAS No.: 1119-34-2), the stability of the replaced formula F3 (the preparation formula: 100mg/mL antibody +35mM histidine buffer +15mM L-arginine hydrochloride +100mM sodium chloride +0.02% Tween-80, pH 5.8) is examined, and the SEC and CEX purities are detected after 4W of the preparation is placed at 4 ℃ and 37 ℃. The results of the measurements are shown in Table 21 below.
Table 21: stability detection result after replacing arginine hydrochloride
Figure RE-GDA0003822617550000451
From the experimental results, the trend of SEC and CEX decrease after arginine is replaced by arginine hydrochloride is basically consistent with the situation of arginine, and the pharmaceutical composition of the invention can keep better stability of the prescription preparation by adding the two stabilizing agents respectively.
Example 8: compatibility stability research experiment
4.1 preparation of physiological saline
500mL of 0.9% physiological saline was prepared with ultrapure water, and stirred with a magnetic stirrer until completely dissolved for use. 500mL of 0.9% physiological saline was prepared with ultrapure water, and stirred with a magnetic stirrer until completely dissolved for use.
4.2 sample dilution
Diluting a preparation sample (preparation formula: 100mg/mL antibody +35mM histidine buffer solution +15mM L-arginine hydrochloride +100mM sodium chloride +0.02% Tween-80, pH 5.8) to corresponding final concentration (60, 30, 10, 3 mg/mL) by using the physiological saline prepared in 5.4.1.1, wherein the volume of the diluted sample is about 25mL, and fully shaking the sample for standby; the antibody in this example was H482L472.
4.3 sample dispensing
And after the diluted sample is sterilized and filtered in a super-clean workbench, aseptically subpackaging the sample in 6mL penicillin bottles, subpackaging 5 bottles of each concentration sample, each bottle with the amount of 5mL, and capping by using a capping machine after plugging.
4.4 lofting and detection
Samples were taken after incubation at 25 ℃ and 30 ℃ for 0, 2, 4h, respectively, and the samples were stored at-80 ℃. After being frozen and stored for 5 days at the temperature of minus 80 ℃, the SEC purity, the bioactivity (blocking ELISA) and insoluble particles of the sample are detected.
The experimental results are shown in tables 22 and 23 below.
Table 22: detection results after incubation at 25 DEG C
Figure RE-GDA0003822617550000452
Figure RE-GDA0003822617550000461
Table 23: detection results after incubation at 30 DEG C
Figure RE-GDA0003822617550000462
The experimental results show that the detection results of samples of various concentration groups are not obviously reduced after incubation for 2h and 4h at 25 ℃ and 30 ℃, and the samples meet the requirements of product quality standards. Therefore, it was judged that the preparation was stable in properties when stored at room temperature for 4 hours after dilution with physiological saline.
Compatibility stability experiments show that the final prescription is diluted by 0.9% sodium chloride solution, and the sample property is kept stable within the range of 3-60 mg/ml of protein concentration and standing for 4 hours at room temperature.
In conclusion, by inspecting different buffer systems, different pH conditions, different antibody concentrations and different auxiliary material compositions, the optimal preparation formula is finally determined. Histidine buffer is adopted to adjust pH, arginine hydrochloride and sodium chloride are adopted to adjust osmotic pressure of the preparation, and polysorbate 20 is added to increase the solubility of the preparation.
Example 9: research on inhibition effect of UBP1213sc preparation on change of physiological index of human BLyS-induced Balb/c mice
The weight of the animal was randomly divided into 10 groups, and the total number of the animal was 7 groups, which were: g1
Figure RE-GDA0003822617550000473
The group, G2 Vehicle model control group, G3 KLH hIgG1 group, G4 UBP1213sc 5mg/kg group, G5 UBP1213sc 1.5mg/kg group, G6 UBP1213sc 0.5mg/kg group, and G7 UBP1213sc 0.15mg/kg group. Day 1and Day3 were administered 2 times in total. Day1 to Day4 were subcutaneously administered 0.3mg/kg human BLyS. During the experiment, the animal body weight was measured daily. At the end of the experiment, animals were euthanized, blood was sampled to detect IgA levels in animal serum, spleen was peeled off and weighed to calculate organ coefficients, FACS was used to detect B220 in spleen + /ThB + Cell ratio. Wherein, the UBP1213sc preparation formula: antibody H482L472+35mM histidine buffer +15mM L-arginine hydrochloride +100mM sodium chloride +0.02% Tween-80, pH5.8, 100mg/mL. The test results are shown in tables 24-26.
Table 24: effect of UBP1213sc on BLyS-stimulated spleen Wet weight and spleen organ coefficients in mice
(data are expressed as mean + -SEM, n = 10)
Figure RE-GDA0003822617550000471
Note: * :P<0.05； ** :P<0.01； *** :P<0.01vs
Figure RE-GDA0003822617550000474
# :P<0.05； ## :P<0.01； ### :P<0.01vs Vehicle。
table 25: b220 in spleen of BLyS-stimulated mice with UBP1213sc + /ThB + Influence of the ratio
(data are expressed as mean + -SEM, n = 10)
Figure RE-GDA0003822617550000472
Figure RE-GDA0003822617550000481
Note: * :P<0.05； ** :P<0.01； *** :P<0.01vs
Figure RE-GDA0003822617550000482
# :P<0.05； ## :P<0.01； ### :P<0.01vs Vehicle。
table 26: effect of UBP1213sc on IgA levels in serum of BLyS-stimulated mice
(data are expressed as mean + -SEM, n = 10)
Figure RE-GDA0003822617550000484
Note: * :P<0.05；**:P<0.01；***:P<0.001vs
Figure RE-GDA0003822617550000483
# :P<0.05； ## :P<0.01； ### :P<0.01 vs Vehicle。
the results of the experiments according to tables 24-26 show that
Figure RE-GDA0003822617550000485
Compared with the group, the results of the Vehicle group show that the spleen wet weight/spleen organ coefficient, the spleen B220+/ThB + cell ratio and the serum IgA level of Balb/c mice are obviously increased by successfully inducing 0.3mg/kg of human BLyS (subcutaneous injection). Compared with the Vehicle group, the composition can obviously inhibit the increase of each physiological index of the model mouse; 1.5mg/kg of UBP1213 can obviously inhibit the increase of various physiological indexes of a model mouse subcutaneously; UBP1213sc of 0.5,1.5 and 5mg/kg significantly inhibited the increase in various physiological indices in the model mice.
Subcutaneous injection of human BLyS induced significant increases in spleen wet weight/spleen organ coefficients, spleen B220+/ThB + cell ratios, and serum IgA levels in Balb/c mice. Different doses (0.15, 0.5,1.5,5 mg/kg) of the anti-BLyS monoclonal antibody UBP1213sc are injected subcutaneously, UBP1213sc with 1.5 and 5mg/kg can obviously inhibit the increase of the spleen wet weight/spleen organ coefficient of the mice, and UBP1213sc with 0.5,1.5 and 5mg/kg can obviously inhibit the increase of the B220+/ThB + cell ratio and the serum IgA level, and show good dose-effect relationship. And the weight average of each administration group animal body is not obviously changed.
Sequence listing
<110> Shanghai Junshi biomedical science and technology Co., ltd
SUZHOU JUNMENG BIOSCIENCES Co.,Ltd.
<120> anti-BLyS antibodies, pharmaceutical compositions thereof and uses thereof
<130> 213020Z1 1CNCN
<150> CN 202110441100.8
<151> 2021-04-23
<160> 24
<170> PatentIn version 3.5
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Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Ser Thr Leu Pro Trp
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 12
<211> 127
<212> PRT
<213> Artificial Sequence
<220>
<223> VH
<400> 12
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Ala
20 25 30
Trp Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Gly Glu Ile Arg Asn Lys Ala Asn Asn His Ala Thr Tyr Gln Ala Glu
50 55 60
Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Thr
65 70 75 80
Val Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr
85 90 95
Tyr Cys Thr Arg Ala Pro Phe Asp Leu Leu Val Arg Arg Gly Tyr Tyr
100 105 110
Ile Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 13
<211> 107
<212> PRT
<213> Artificial Sequence
<220>
<223> VL
<400> 13
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Thr Ser Gln Asp Ile Ser Ile Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Tyr Thr Ser Arg Leu Arg Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln Gly Ser Thr Leu Pro Trp
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 14
<211> 127
<212> PRT
<213> Artificial Sequence
<220>
<223> VH
<400> 14
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Ala
20 25 30
Trp Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Glu Ile Arg Asn Lys Ala Asn Asn His Ala Thr Tyr Gln Ala Glu
50 55 60
Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser
65 70 75 80
Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
85 90 95
Tyr Cys Thr Arg Ala Pro Phe Asp Leu Leu Val Arg Arg Gly Tyr Tyr
100 105 110
Ile Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 15
<211> 127
<212> PRT
<213> Artificial Sequence
<220>
<223> VH
<400> 15
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Ala
20 25 30
Trp Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Glu Ile Arg Asn Lys Ala Asn Asn His Ala Thr Tyr Gln Ala Glu
50 55 60
Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser
65 70 75 80
Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
85 90 95
Tyr Cys Thr Arg Ala Pro Phe Asp Leu Leu Val Arg Arg Gly Tyr Tyr
100 105 110
Ile Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 16
<211> 214
<212> PRT
<213> Artificial Sequence
<220>
<223> LC
<400> 16
Asp Ile Gln Met Thr Gln Thr Pro Ser Ser Leu Ser Ala Ser Leu Gly
1 5 10 15
Asp Arg Val Thr Ile Ser Cys Arg Thr Ser Gln Asp Ile Ser Ile Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Tyr Thr Ser Arg Leu Arg Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Ser Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln Gly Ser Thr Leu Pro Trp
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys
210
<210> 17
<211> 457
<212> PRT
<213> Artificial Sequence
<220>
<223> HC
<400> 17
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Met Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Ala
20 25 30
Trp Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Glu Ile Arg Asn Lys Ala Asn Asn His Ala Thr Tyr Gln Ala Glu
50 55 60
Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser
65 70 75 80
Val Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr
85 90 95
Tyr Cys Thr Arg Ala Pro Phe Asp Leu Leu Val Arg Arg Gly Tyr Tyr
100 105 110
Ile Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Ala
115 120 125
Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser
130 135 140
Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe
145 150 155 160
Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly
165 170 175
Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu
180 185 190
Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr
195 200 205
Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg
210 215 220
Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro
225 230 235 240
Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
245 250 255
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
260 265 270
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
275 280 285
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
290 295 300
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
305 310 315 320
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
325 330 335
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
340 345 350
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met
355 360 365
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
370 375 380
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
385 390 395 400
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
405 410 415
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
420 425 430
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
435 440 445
Lys Ser Leu Ser Leu Ser Pro Gly Lys
450 455
<210> 18
<211> 214
<212> PRT
<213> Artificial Sequence
<220>
<223> LC
<400> 18
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Thr Ser Gln Asp Ile Ser Ile Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Tyr Thr Ser Arg Leu Arg Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln Gly Ser Thr Leu Pro Trp
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys
210
<210> 19
<211> 457
<212> PRT
<213> Artificial Sequence
<220>
<223> HC
<400> 19
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Ala
20 25 30
Trp Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Gly Glu Ile Arg Asn Lys Ala Asn Asn His Ala Thr Tyr Gln Ala Glu
50 55 60
Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser
65 70 75 80
Val Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr
85 90 95
Tyr Cys Thr Arg Ala Pro Phe Asp Leu Leu Val Arg Arg Gly Tyr Tyr
100 105 110
Ile Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala
115 120 125
Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser
130 135 140
Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe
145 150 155 160
Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly
165 170 175
Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu
180 185 190
Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr
195 200 205
Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg
210 215 220
Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro
225 230 235 240
Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
245 250 255
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
260 265 270
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
275 280 285
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
290 295 300
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
305 310 315 320
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
325 330 335
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
340 345 350
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met
355 360 365
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
370 375 380
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
385 390 395 400
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
405 410 415
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
420 425 430
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
435 440 445
Lys Ser Leu Ser Leu Ser Pro Gly Lys
450 455
<210> 20
<211> 214
<212> PRT
<213> Artificial Sequence
<220>
<223> LC
<400> 20
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Thr Ser Gln Asp Ile Ser Ile Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Tyr Thr Ser Arg Leu Arg Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Ser Thr Leu Pro Trp
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys
210
<210> 21
<211> 457
<212> PRT
<213> Artificial Sequence
<220>
<223> HC
<400> 21
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Ala
20 25 30
Trp Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Gly Glu Ile Arg Asn Lys Ala Asn Asn His Ala Thr Tyr Gln Ala Glu
50 55 60
Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Thr
65 70 75 80
Val Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr
85 90 95
Tyr Cys Thr Arg Ala Pro Phe Asp Leu Leu Val Arg Arg Gly Tyr Tyr
100 105 110
Ile Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala
115 120 125
Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser
130 135 140
Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe
145 150 155 160
Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly
165 170 175
Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu
180 185 190
Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr
195 200 205
Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg
210 215 220
Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro
225 230 235 240
Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
245 250 255
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
260 265 270
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
275 280 285
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
290 295 300
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
305 310 315 320
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
325 330 335
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
340 345 350
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met
355 360 365
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
370 375 380
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
385 390 395 400
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
405 410 415
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
420 425 430
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
435 440 445
Lys Ser Leu Ser Leu Ser Pro Gly Lys
450 455
<210> 22
<211> 214
<212> PRT
<213> Artificial Sequence
<220>
<223> LC
<400> 22
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Thr Ser Gln Asp Ile Ser Ile Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Tyr Thr Ser Arg Leu Arg Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln Gly Ser Thr Leu Pro Trp
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys
210
<210> 23
<211> 457
<212> PRT
<213> Artificial Sequence
<220>
<223> HC
<400> 23
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Ala
20 25 30
Trp Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Glu Ile Arg Asn Lys Ala Asn Asn His Ala Thr Tyr Gln Ala Glu
50 55 60
Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser
65 70 75 80
Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
85 90 95
Tyr Cys Thr Arg Ala Pro Phe Asp Leu Leu Val Arg Arg Gly Tyr Tyr
100 105 110
Ile Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala
115 120 125
Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser
130 135 140
Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe
145 150 155 160
Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly
165 170 175
Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu
180 185 190
Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr
195 200 205
Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg
210 215 220
Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro
225 230 235 240
Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
245 250 255
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
260 265 270
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
275 280 285
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
290 295 300
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
305 310 315 320
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
325 330 335
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
340 345 350
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met
355 360 365
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
370 375 380
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
385 390 395 400
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
405 410 415
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
420 425 430
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
435 440 445
Lys Ser Leu Ser Leu Ser Pro Gly Lys
450 455
<210> 24
<211> 457
<212> PRT
<213> Artificial Sequence
<220>
<223> HC
<400> 24
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Ala
20 25 30
Trp Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Glu Ile Arg Asn Lys Ala Asn Asn His Ala Thr Tyr Gln Ala Glu
50 55 60
Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser
65 70 75 80
Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
85 90 95
Tyr Cys Thr Arg Ala Pro Phe Asp Leu Leu Val Arg Arg Gly Tyr Tyr
100 105 110
Ile Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala
115 120 125
Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser
130 135 140
Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe
145 150 155 160
Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly
165 170 175
Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu
180 185 190
Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr
195 200 205
Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg
210 215 220
Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro
225 230 235 240
Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
245 250 255
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
260 265 270
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
275 280 285
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
290 295 300
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
305 310 315 320
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
325 330 335
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
340 345 350
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met
355 360 365
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
370 375 380
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
385 390 395 400
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
405 410 415
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
420 425 430
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
435 440 445
Lys Ser Leu Ser Leu Ser Pro Gly Lys
450 455

Claims (19)

1. A humanized anti-BLyS antibody or antigen binding fragment thereof comprising a heavy chain variable region and a light chain variable region,
the amino acid sequences of LCDR1, LCDR2 and LCDR3 of the humanized anti-BLyS antibody or the antigen binding fragment thereof are respectively shown as SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3;
the amino acid sequences of HCDR1, HCDR2 and HCDR3 of the humanized anti-BLyS antibody or the antigen binding fragment thereof are respectively shown as SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6;
FR1 of the light chain variable region of the humanized anti-BLyS antibody or an antigen-binding fragment thereof is selected from the group consisting of FR1 of any one of SEQ ID NOS: 7, 9 and 13, FR2 is the FR2 of SEQ ID NO:7, FR3 is selected from the group consisting of FR3 of any one of SEQ ID NOS: 7, 9 and 11, FR4 is the FR4 of SEQ ID NO: 7;
FR1 of the heavy chain variable region of said humanized anti-BLyS antibody or antigen-binding fragment thereof is selected from the group consisting of FR1 of any one of SEQ ID NOs 8, 10, 12 and 15, FR2 is selected from the group consisting of FR2 of any one of SEQ ID NOs 8, 10 and 15, FR3 is selected from the group consisting of FR3 of any one of SEQ ID NOs 8, 12 and 14, FR4 is the FR4 of SEQ ID NOs 8 or 10; and is
The humanized anti-BLyS antibody or antigen binding fragment thereof does not comprise: the light chain variable region is the amino acid sequence shown in SEQ ID NO. 7, while the heavy chain variable region is the humanized anti-BLyS antibody or antigen-binding fragment thereof of the amino acid sequence shown in SEQ ID NO. 8.
2. The humanized anti-BLyS antibody or antigen-binding fragment thereof of claim 1,
in the humanized anti-BLyS antibody or antigen-binding fragment thereof, FR1 of the light chain variable region is selected from the FR1 of any one of SEQ ID NOs 7, 9 and 13, FR2 is the FR2 of SEQ ID NO 7, FR3 is selected from the FR3 of any one of SEQ ID NOs 7, 9 and 11, and FR4 is the FR4 of SEQ ID NO 7; FR1-FR4 of the heavy chain variable region are respectively FR1-FR4 of SEQ ID NO. 15; or
In the humanized anti-BLyS antibody or antigen-binding fragment thereof, FR1 of the light chain variable region is selected from the FR1 of any one of SEQ ID NOs 7, 9 and 13, FR2 is the FR2 of SEQ ID NO 7, FR3 is selected from the FR3 of any one of SEQ ID NOs 7, 9 and 11, and FR4 is the FR4 of SEQ ID NO 7; FR1-FR4 of the heavy chain variable region are respectively FR1-FR4 of SEQ ID NO. 10; or
In the humanized anti-BLyS antibody or antigen-binding fragment thereof, FR1 of the light chain variable region is selected from the FR1 of any one of SEQ ID NOs 7, 9 and 13, FR2 is the FR2 of SEQ ID NO 7, FR3 is selected from the FR3 of any one of SEQ ID NOs 7, 9 and 11, and FR4 is the FR4 of SEQ ID NO 7; FR1-FR4 of the heavy chain variable region are FR1-FR4 of SEQ ID NO. 12, respectively; or
In the humanized anti-BLyS antibody or the antigen binding fragment thereof, FR1 of the light chain variable region is FR1 of SEQ ID NO. 11 or 13, FR2 is FR2 of SEQ ID NO. 11, FR3 is FR3 of SEQ ID NO. 11 or 13, and FR4 is FR4 of SEQ ID NO. 11; FR1-FR4 of the heavy chain variable region are FR1-FR4 of SEQ ID NO. 14, respectively.
3. The humanized anti-BLyS antibody or antigen-binding fragment thereof of claim 1, wherein the light chain variable region of said humanized anti-BLyS antibody or antigen-binding fragment thereof comprises the amino acid sequence as set forth in SEQ ID NO: 7. SEQ ID NO: 9. SEQ ID NO:11 or SEQ ID NO:13, and the heavy chain variable region comprises the amino acid sequence set forth as SEQ ID NO: 8. SEQ ID NO: 10. SEQ ID NO: 12. SEQ ID NO:14 or SEQ ID NO:15, or a pharmaceutically acceptable salt thereof.
4. The humanized anti-BLyS antibody or antigen binding fragment thereof of claim 1, comprising a light chain variable region and a heavy chain variable region, wherein:
(1) The light chain variable region comprises the amino acid sequence shown as SEQ ID NO:7, and the heavy chain variable region comprises the amino acid sequence set forth as SEQ ID NO: 10. the amino acid sequence of SEQ ID NO: 12. the amino acid sequence of SEQ ID NO:14 or SEQ ID NO: 15; or
(2) The light chain variable region comprises the amino acid sequence shown as SEQ ID NO:9, and the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 8. the amino acid sequence of SEQ ID NO: 10. SEQ ID NO: 12. SEQ ID NO:14 or SEQ ID NO: 15; or
(3) The light chain variable region comprises the amino acid sequence shown as SEQ ID NO:11, and the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 8. SEQ ID NO: 10. SEQ ID NO: 12. the amino acid sequence of SEQ ID NO:14 or SEQ ID NO: 15; or
(4) The light chain variable region comprises the amino acid sequence shown as SEQ ID NO:13, and the heavy chain variable region comprises the amino acid sequence set forth as SEQ ID NO: 8. SEQ ID NO: 10. SEQ ID NO: 12. the amino acid sequence of SEQ ID NO:14 or SEQ ID NO: 15; or
(5) The light chain variable region comprises the amino acid sequence shown as SEQ ID NO:9, and the heavy chain variable region comprises the amino acid sequence set forth as SEQ ID NO: 10; or
(6) The light chain variable region comprises the amino acid sequence shown as SEQ ID NO:11, and the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 12; or
(7) The light chain variable region comprises the amino acid sequence shown as SEQ ID NO:13, and the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 14; or
(8) The light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:7, and the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 15; or
(9) The light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:9, and the heavy chain variable region comprises the amino acid sequence set forth as SEQ ID NO: 15; or
(10) The light chain variable region comprises the amino acid sequence shown as SEQ ID NO:11, and the heavy chain variable region comprises the amino acid sequence set forth as SEQ ID NO: 15; or
(11) The light chain variable region comprises the amino acid sequence shown as SEQ ID NO:13, and the heavy chain variable region comprises the amino acid sequence set forth as SEQ ID NO: 15; or
(12) The heavy chain variable region comprises the amino acid sequence as shown in SEQ ID NO:8, and the light chain variable region comprises the amino acid sequence set forth as SEQ ID NO: 13; or
(13) The heavy chain variable region comprises the amino acid sequence set forth as SEQ ID NO:10, and the light chain variable region comprises the amino acid sequence set forth as SEQ ID NO: 7. 9, 11 or 13; or
(14) The heavy chain variable region comprises the amino acid sequence as shown in SEQ ID NO:12, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 7. 9, 11 or 13; or
(15) The heavy chain variable region comprises the amino acid sequence as shown 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
(16) The heavy chain variable region comprises the amino acid sequence set forth as SEQ ID NO:15, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 7. 9, 11 or 13.
5. The humanized anti-BLyS antibody or antigen binding fragment thereof of claim 1, wherein said humanized anti-BLyS antibody further comprises a human light chain constant region and a heavy chain constant region, and said light chain variable region and heavy chain variable region are linked to a human light chain constant region and heavy chain constant region, respectively; preferably, the human light chain constant region is a human light chain kappa constant region; preferably, the human heavy chain constant region is a human heavy chain Fc fragment.
6. The humanized anti-BLyS antibody or antigen-binding fragment thereof of any of claims 1-5, comprising a light chain and a heavy chain, wherein the light chain comprises the amino acid sequence as set forth in SEQ ID NO: 16. SEQ ID NO: 18. SEQ ID NO:20 or SEQ ID NO:22, and the heavy chain comprises the amino acid sequence shown as SEQ ID NO: 17. SEQ ID NO: 19. SEQ ID NO: 21. SEQ ID NO:23 or SEQ ID NO: 24.
7. The humanized anti-BLyS antibody or antigen-binding fragment thereof of claim 6, comprising a light chain and a heavy chain, wherein:
(1) The light chain comprises the amino acid sequence set forth as SEQ ID NO:16, and the heavy chain comprises the amino acid sequence set forth as SEQ ID NO: 19. SEQ ID NO: 21. SEQ ID NO:23 or SEQ ID NO: 24; or
(2) The light chain comprises the amino acid sequence as set forth in SEQ ID NO:18, and the heavy chain comprises the amino acid sequence set forth as SEQ ID NO: 17. SEQ ID NO: 19. SEQ ID NO: 21. SEQ ID NO:23 or SEQ ID NO: 24; or
(3) The light chain comprises the amino acid sequence set forth as SEQ ID NO:20, and the heavy chain comprises the amino acid sequence set forth as SEQ ID NO: 17. SEQ ID NO: 19. SEQ ID NO: 21. SEQ ID NO:23 or SEQ ID NO: 24; or
(4) The light chain comprises the amino acid sequence set forth as SEQ ID NO:22, and the heavy chain comprises the amino acid sequence set forth as SEQ ID NO: 17. the amino acid sequence of SEQ ID NO: 19. the amino acid sequence of SEQ ID NO: 21. SEQ ID NO:23 or SEQ ID NO: 24; or
(5) The light chain comprises the amino acid sequence as set forth in SEQ ID NO:18, and the heavy chain comprises the amino acid sequence set forth as SEQ ID NO: 19; or
(6) The light chain comprises the amino acid sequence as set forth in SEQ ID NO:20, and the heavy chain comprises the amino acid sequence set forth as SEQ ID NO: 21; or
(7) The light chain comprises the amino acid sequence set forth as SEQ ID NO:22, and the heavy chain comprises the amino acid sequence set forth as SEQ ID NO: 23; or
(8) The light chain comprises the amino acid sequence as set forth in SEQ ID NO:16, and the heavy chain comprises the amino acid sequence set forth as SEQ ID NO: 24; or
(9) The light chain comprises the amino acid sequence as set forth in SEQ ID NO:18, and the heavy chain comprises the amino acid sequence set forth as SEQ ID NO: 24; or
(10) The light chain comprises the amino acid sequence set forth as SEQ ID NO:20, and the heavy chain comprises the amino acid sequence set forth as SEQ ID NO: 24; or
(11) The light chain comprises the amino acid sequence set forth as SEQ ID NO:22, and the heavy chain comprises the amino acid sequence set forth as SEQ ID NO: 24; or
(12) The heavy chain comprises the amino acid sequence as set forth in SEQ ID NO:17, and the light chain comprises the amino acid sequence set forth as SEQ ID NO: 22; or
(13) The heavy chain comprises the amino acid sequence set forth as SEQ ID NO:19, and the light chain comprises the amino acid sequence set forth as SEQ ID NO: 16. 18, 20 or 22; or
(14) The heavy chain comprises the amino acid sequence set forth as SEQ ID NO:21, and the light chain comprises the amino acid sequence set forth as SEQ ID NO: 16. 18, 20 or 22; or
(15) The heavy chain comprises the amino acid sequence as set forth in SEQ ID NO:23, and the light chain comprises the amino acid sequence set forth as SEQ ID NO:20 or 22; or
(16) The heavy chain comprises the amino acid sequence as set forth in SEQ ID NO:24, and the light chain comprises the amino acid sequence set forth as SEQ ID NO: 16. 18, 20 or 22.
8. A polynucleotide molecule selected from the group consisting of:
(1) A polynucleotide molecule encoding the humanized anti-BLyS antibody or antigen binding fragment thereof of any one of claims 1-7; and
(2) The complement of the polynucleotide molecule of (1).
9. An expression vector comprising the polynucleotide molecule of claim 8; preferably, the expression vector is a eukaryotic expression vector.
10. A host cell comprising the polynucleotide molecule of claim 8 or the expression vector of claim 9; preferably, the host cell is a eukaryotic cell; more preferably mammalian cells.
11. A pharmaceutical composition comprising: (1) a buffer solution; (2) A humanized anti-BLyS antibody or an antigen-binding fragment thereof having LCDR1, LCDR2 and LCDR3 with amino acid sequences shown as SEQ ID No. 1, SEQ ID No. 2 and SEQ ID No. 3, respectively, and HCDR1, HCDR2 and HCDR3 with amino acid sequences shown as SEQ ID No. 4, SEQ ID No. 5 and SEQ ID No. 6, respectively.
12. The pharmaceutical composition of claim 11, wherein the humanized anti-BLyS antibody or antigen binding fragment thereof is according to any one of claims 1-7.
13. The pharmaceutical composition of claim 11 or 12, wherein the pharmaceutical composition is characterized by one or more of the following:
the humanized anti-BLyS antibody or antigen-binding fragment thereof is present at a concentration of about 10 to about 300mg/mL, preferably about 20 to about 280mg/mL, preferably about 30 to about 150mg/mL, preferably about 80 to about 120mg/mL, preferably about 180 to about 220mg/mL;
the buffer solution is selected from one or more of acetic acid buffer solution, citric acid buffer solution, succinic acid buffer solution, phosphoric acid buffer solution and histidine buffer solution; preferably, the buffer has a concentration of about 5 to about 50mM, preferably about 10 to about 40mM, preferably about 20 to about 40mM; preferably, the pH of the buffer is about 5.0 to about 6.5, preferably about 5.0 to about 6.0, preferably about 5.5 to about 6.0;
the pharmaceutical composition further comprises a stabilizer selected from one or more of arginine, arginine hydrochloride, methionine, proline, glycine, sodium chloride, mannitol, sorbitol, sucrose, maltose, xylitol, and trehalose; preferably, the stabilizer is selected from one or more of L-arginine, L-arginine hydrochloride, sucrose, methionine and sodium chloride; preferably, the concentration of the stabilizer is about 50mM to 300mM, preferably about 100mM to 300mM, more preferably about 100mM to 200mM;
the pharmaceutical composition further comprises a surfactant; preferably, the surfactant is selected from one or more of polysorbate 80, polysorbate 20, poloxamer 188; preferably, the surfactant concentration is from about 0.001% to about 0.1%, preferably from about 0.01% to about 0.05%, calculated as w/v.
14. The pharmaceutical composition of claim 13, wherein the stabilizer is a combination of L-arginine hydrochloride at a concentration of about 10-30mM and sodium chloride at a concentration of about 50-200 mM; or the stabilizer is a combination of L-arginine at a concentration of about 10-30mM and sodium chloride at a concentration of about 50-200 mM; or the stabilizer is a combination of L-arginine at a concentration of about 10-30mM and sucrose at a concentration of about 80-220 mM; or said stabilizer is a combination of L-arginine hydrochloride at a concentration of about 10-30mM with sucrose at a concentration of about 80-220 mM; or the stabilizer is a combination of about 30-90mM methionine and about 50-200mM sodium chloride; or the stabilizer is about 10-120mM L-arginine hydrochloride; or the stabilizer is methionine at about 10-120 mM; preferably, the stabilizer is a combination of L-arginine hydrochloride at a concentration of about 10-30mM, and sodium chloride at a concentration of about 80-120 mM; or the stabilizer is a combination of L-arginine at a concentration of about 10-30mM and sodium chloride at a concentration of about 80-120 mM; or the stabilizer is a combination of about 50-70mM methionine and about 80-120mM sodium chloride; or the stabilizer is a combination of L-arginine at a concentration of about 10-30mM and sucrose at a concentration of about 130-170 mM; or the stabilizer is a combination of L-arginine hydrochloride at a concentration of about 10-30mM and sucrose at a concentration of about 130-170 mM.
15. The pharmaceutical composition of claim 11 or 12,
the pharmaceutical composition comprises: (a) About 20mg/mL to 280mg/mL of a humanized anti-BLyS antibody or antigen-binding fragment thereof; (b) About 5-50mM histidine buffer or about 5-50mM acetic acid buffer, pH about 5.0-6.5; (c) About 10-30mM of L-arginine or about 10-30mM of L-arginine hydrochloride; (d) about 80 to 220mM sucrose; (e) and about 0.01% to 0.1% polysorbate 80; or
The pharmaceutical composition comprises: (a) About 30mg/mL to 150mg/mL of a humanized anti-BLyS antibody or antigen-binding fragment thereof; (b) About 5-50mM histidine buffer or about 5-50mM acetic acid buffer, pH about 5.0-6.5; (c) About 10-30mM of L-arginine, about 10-30mM of L-arginine hydrochloride, or about 30-90mM of methionine; (d) about 50-200mM sodium chloride; (e) and about 0.01% to 0.1% polysorbate 80; or
The pharmaceutical composition comprises: (a) About 80mg/mL to 120mg/mL of a humanized anti-BLyS antibody or antigen-binding fragment thereof; (b) About 20-40mM histidine buffer or about 20-40mM acetic acid buffer, pH about 5.5-6.0; (c) About 10-30mM of L-arginine, about 50-70mM of methionine, or about 10-30mM of L-arginine hydrochloride; (d) about 80-120mM sodium chloride; (e) and about 0.01% to 0.05% polysorbate 80; or
The pharmaceutical composition comprises: (a) About 180mg/mL to 220mg/mL of a humanized anti-BLyS antibody or antigen-binding fragment thereof; (b) About 20-40mM histidine buffer or about 20-40mM acetic acid buffer, pH about 5.5-6.0; (c) About 10-30mM L-arginine or about 10-30mM L-arginine hydrochloride; (d) about 130 to 170mM sucrose; (e) and about 0.01% to 0.05% polysorbate 80; or
The pharmaceutical composition comprises: (a) About 200mg/mL of a humanized anti-BLyS antibody or antigen-binding fragment thereof; (b) About 25mM histidine buffer, about 25mM acetate buffer, or about 35-36mM histidine buffer, pH about 5.5-6.0; (c) About 15mM L-arginine or about 15mM L-arginine hydrochloride; (d) about 150mM sucrose; (e) and about 0.02% polysorbate 80; or
The pharmaceutical composition comprises: (a) About 100mg/mL of a humanized anti-BLyS antibody or antigen-binding fragment thereof; (b) about 25mM histidine buffer, pH about 5.5-6.0; (c) About 25mM L-arginine or about 25mM L-arginine hydrochloride; (d) about 100mM sodium chloride; (e) and about 0.02% polysorbate 80; or
The pharmaceutical composition comprises: (a) About 100mg/mL of a humanized anti-BLyS antibody or antigen-binding fragment thereof; (b) about 25mM acetate buffer, pH about 5.5-6.0; (c) methionine at about 60 mM; (d) about 100mM sodium chloride; (e) and about 0.02% polysorbate 80; or
The pharmaceutical composition comprises: (a) About 100mg/mL of a humanized anti-BLyS antibody or antigen-binding fragment thereof; (b) about 35-36mM histidine buffer, pH about 5.5-6.0; (c) About 15mM L-arginine hydrochloride or about 15mM L-arginine; (d) about 100mM sodium chloride; (e) and about 0.02% polysorbate 80.
16. The pharmaceutical composition according to claim 11, which comprises a component represented by any one of the following (1) to (20):
(1) (a) about 20mg/mL to 280mg/mL of a humanized anti-BLyS antibody or antigen-binding fragment thereof comprising a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:9, and the heavy chain variable region comprises the amino acid sequence set forth as SEQ ID NO: 8. SEQ ID NO: 10. SEQ ID NO: 12. SEQ ID NO:14 or SEQ ID NO: 15; (b) About 5-50mM histidine buffer, pH about 5.0-6.5; (c) About 10-30mM of L-arginine or about 10-30mM of L-arginine hydrochloride; (d) about 80 to 220mM sucrose; (e) and about 0.01% to 0.1% polysorbate 80; or
(2) (a) about 30mg/mL to 150mg/mL of a humanized anti-BLyS antibody or antigen-binding fragment thereof comprising a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:9, and the heavy chain variable region comprises the amino acid sequence set forth as SEQ ID NO: 8. the amino acid sequence of SEQ ID NO: 10. SEQ ID NO: 12. SEQ ID NO:14 or SEQ ID NO: 15; (b) about 5-50mM histidine buffer, at a pH of about 5.0-6.5; (c) about 10-30mM of L-arginine; (d) about 50-200mM sodium chloride; (e) and about 0.01% to 0.1% polysorbate 80; or
(3) (a) about 30mg/mL to 150mg/mL of a humanized anti-BLyS antibody or antigen-binding fragment thereof comprising a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:9, and the heavy chain variable region comprises the amino acid sequence set forth as SEQ ID NO: 8. SEQ ID NO: 10. the amino acid sequence of SEQ ID NO: 12. the amino acid sequence of SEQ ID NO:14 or SEQ ID NO: 15; (b) about 5-50mM histidine buffer, at a pH of about 5.0-6.5; (c) about 10-30mM L-arginine hydrochloride; (d) about 50-200mM sodium chloride; (e) and about 0.01% to 0.1% polysorbate 80; or
(4) (a) about 30mg/mL to 150mg/mL of a humanized anti-BLyS antibody or antigen-binding fragment thereof comprising a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:9, and the heavy chain variable region comprises the amino acid sequence set forth as SEQ ID NO: 8. the amino acid sequence of SEQ ID NO: 10. the amino acid sequence of SEQ ID NO: 12. SEQ ID NO:14 or SEQ ID NO: 15; (b) about 5-50mM histidine buffer, at a pH of about 5.0-6.5; (c) methionine at a concentration of about 30 to about 90 mM; (d) about 50-200mM sodium chloride; (e) and about 0.01% -0.1% polysorbate 80; or
(5) (a) about 30mg/mL to 150mg/mL of a humanized anti-BLyS antibody or antigen-binding fragment thereof comprising a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:9 and the heavy chain variable region comprises the amino acid sequence shown as SEQ ID NO: 8. the amino acid sequence of SEQ ID NO: 10. the amino acid sequence of SEQ ID NO: 12. the amino acid sequence of SEQ ID NO:14 or SEQ ID NO: 15; (b) about 5 to 50mM acetate buffer, pH about 5.0 to 6.5; (c) about 10-30mM of L-arginine; (d) about 50-200mM sodium chloride; (e) and about 0.01% to 0.1% polysorbate 80; or
(6) (a) about 30mg/mL to 150mg/mL of a humanized anti-BLyS antibody or antigen-binding fragment thereof comprising a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:9 and the heavy chain variable region comprises the amino acid sequence shown as SEQ ID NO: 8. SEQ ID NO: 10. SEQ ID NO: 12. the amino acid sequence of SEQ ID NO:14 or SEQ ID NO: 15; (b) about 5 to 50mM acetate buffer, pH about 5.0 to 6.5; (c) about 10-30mM L-arginine hydrochloride; (d) about 50-200mM sodium chloride; (e) and about 0.01% to 0.1% polysorbate 80; or
(7) (a) about 30mg/mL to 150mg/mL of a humanized anti-BLyS antibody or antigen-binding fragment thereof comprising a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:9 and the heavy chain variable region comprises the amino acid sequence shown as SEQ ID NO: 8. SEQ ID NO: 10. SEQ ID NO: 12. the amino acid sequence of SEQ ID NO:14 or SEQ ID NO: 15; (b) about 5 to 50mM acetate buffer, pH about 5.0 to 6.5; (c) methionine at a concentration of about 30 to about 90 mM; (d) about 50-200mM sodium chloride; (e) and about 0.01% -0.1% polysorbate 80; or
(8) (a) about 80mg/mL to 120mg/mL of a humanized anti-BLyS antibody or antigen-binding fragment thereof comprising a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:9, and the heavy chain variable region comprises the amino acid sequence set forth as SEQ ID NO: 10; (b) about 20 to 40mM histidine buffer, pH about 5.5 to 6.0; (c) about 10-30mM L-arginine; (d) about 80 to 120mM sodium chloride; (e) and about 0.01% to 0.05% polysorbate 80; or
(9) (a) about 80mg/mL to 120mg/mL of a humanized anti-BLyS antibody or antigen-binding fragment thereof comprising a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:9 and the heavy chain variable region comprises the amino acid sequence shown as SEQ ID NO: 10; (b) about 20 to 40mM histidine buffer, pH about 5.5 to 6.0; (c) about 10-30mM L-arginine hydrochloride; (d) about 80-120mM sodium chloride; (e) and about 0.01% to 0.05% polysorbate 80; or
(10) (a) about 80mg/mL to 120mg/mL of a humanized anti-BLyS antibody or antigen-binding fragment thereof comprising a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:9, and the heavy chain variable region comprises the amino acid sequence set forth as SEQ ID NO: 10; (b) about 20-40mM acetate buffer, at a pH of about 5.5-6.0; (c) methionine at a concentration of about 50 to about 70 mM; (d) about 80 to 120mM sodium chloride; (e) and about 0.01% to 0.05% polysorbate 80; or
(11) (a) about 180mg/mL to 220mg/mL of a humanized anti-BLyS antibody or antigen-binding fragment thereof comprising a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:9 and the heavy chain variable region comprises the amino acid sequence shown as SEQ ID NO: 10; (b) about 20 to 40mM histidine buffer, pH about 5.5 to 6.0; (c) About 10-30mM of L-arginine or about 10-30mM of L-arginine hydrochloride; (d) about 130 to 170mM sucrose; (e) and about 0.01% to 0.05% polysorbate 80; or
(12) (a) about 200mg/mL of a humanized anti-BLyS antibody or antigen-binding fragment thereof comprising a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO:9, and the heavy chain variable region comprises the amino acid sequence set forth as SEQ ID NO: 10; (b) about 35-36mM histidine buffer, at a pH of about 5.5-6.0; (c) about 15mM L-arginine hydrochloride; (d) sucrose at about 150 mM; (e) and about 0.02% polysorbate 80; or
(13) (a) about 200mg/mL of a humanized anti-BLyS antibody or antigen-binding fragment thereof comprising a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO:9, and the heavy chain variable region comprises the amino acid sequence set forth as SEQ ID NO: 10; (b) about 35-36mM histidine buffer, pH about 5.5-6.0; (c) about 15mM L-arginine; (d) about 150mM sucrose; (e) and about 0.02% polysorbate 80; or
(14) (a) about 200mg/mL of a humanized anti-BLyS antibody or antigen-binding fragment thereof comprising a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO:9 and the heavy chain variable region comprises the amino acid sequence shown as SEQ ID NO: 10; (b) about 25mM histidine buffer, pH about 5.5-6.0; (c) about 15mM L-arginine hydrochloride; (d) about 150mM sucrose; (e) and about 0.02% polysorbate 80; or
(15) (a) about 100mg/mL of a humanized anti-BLyS antibody or antigen-binding fragment thereof comprising a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO:9, and the heavy chain variable region comprises the amino acid sequence set forth as SEQ ID NO: 10; (b) about 25mM histidine buffer, at a pH of about 5.5 to about 6.0; (c) about 25mM L-arginine; (d) about 100mM sodium chloride; (e) and about 0.02% polysorbate 80; or
(16) (a) about 100mg/mL of a humanized anti-BLyS antibody or antigen-binding fragment thereof comprising a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO:9 and the heavy chain variable region comprises the amino acid sequence shown as SEQ ID NO: 10; (b) about 25mM histidine buffer, at a pH of about 5.5 to about 6.0; (c) about 25mM L-arginine hydrochloride; (d) about 100mM sodium chloride; (e) and about 0.02% polysorbate 80; or
(17) (a) about 100mg/mL of a humanized anti-BLyS antibody or antigen-binding fragment thereof comprising a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO:9 and the heavy chain variable region comprises the amino acid sequence shown as SEQ ID NO: 10; (b) about 25mM acetate buffer, pH about 5.5-6.0; (c) methionine at about 60 mM; (d) about 100mM sodium chloride; (e) and about 0.02% polysorbate 80; or
(18) (a) about 100mg/mL of a humanized anti-BLyS antibody or antigen-binding fragment thereof comprising a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO:9, and the heavy chain variable region comprises the amino acid sequence set forth as SEQ ID NO: 10; (b) about 35-36mM histidine buffer, pH about 5.5-6.0; (c) about 15mM L-arginine hydrochloride; (d) about 100mM sodium chloride; (e) and about 0.02% polysorbate 80; or
(19) (a) about 100mg/mL of a humanized anti-BLyS antibody or antigen-binding fragment thereof comprising a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO:9 and the heavy chain variable region comprises the amino acid sequence shown as SEQ ID NO: 10; (b) about 35-36mM histidine buffer, pH about 5.5-6.0; (c) about 15mM L-arginine; (d) about 100mM sodium chloride; (e) and about 0.02% polysorbate 80; or
(20) (a) about 100mg/mL of a humanized anti-BLyS antibody or antigen-binding fragment thereof comprising a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO:9, and the heavy chain variable region comprises the amino acid sequence set forth as SEQ ID NO: 10; (b) about 35mM histidine buffer, pH about 5.8; (c) about 15mM L-arginine hydrochloride; (d) about 100mM sodium chloride; (e) and about 0.02% polysorbate 80;
preferably, the light chain amino acid sequence of the humanized anti-BLyS antibody is shown as SEQ ID NO. 18, and the heavy chain amino acid sequence is shown as SEQ ID NO. 19.
17. An injection comprising the pharmaceutical composition of any one of claims 11-16 and a sodium chloride solution; preferably, the humanized anti-BLyS antibody is present in the injection at a concentration of 3 to 100mg/mL; preferably, the injection has a pH of 5.5 to 6.0.
18. A pharmaceutical composition according to any one of claims 11 to 16 or an injection according to claim 17, for administration by subcutaneous injection.
19. Use of a humanized anti-BLyS antibody or antigen-binding fragment thereof according to any of claims 1-7, or a polynucleotide molecule according to claim 8, or an expression vector according to claim 9, or a host cell according to claim 10, or a pharmaceutical composition according to any of claims 11-16, or an injection according to claim 17, for the manufacture of a medicament for the prevention and/or treatment of a disease caused by an excessive B-cell proliferation; preferably, the disease caused by excessive B cell proliferation is systemic lupus erythematosus, rheumatoid arthritis, mandatory arthritis or B cell lymphoma.
CN202210443212.1A 2021-04-23 2022-04-25 anti-BLyS antibodies, pharmaceutical compositions thereof, and uses thereof Pending CN115232208A (en)

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CN1273488C (en) * 2001-12-29 2006-09-06 梁瑞安 Gene reform immune globulin with low immunogenicity and its application
US7462697B2 (en) * 2004-11-08 2008-12-09 Epitomics, Inc. Methods for antibody engineering
CN103421113B (en) * 2012-05-22 2018-01-19 武汉华鑫康源生物医药有限公司 Anti- BLyS antibody
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