Anti-DKK1 antibody pharmaceutical composition and application thereof

Abstract

The present invention provides pharmaceutical compositions of anti-DKK1 antibodies and uses thereof. The pharmaceutical composition contains an anti-DKK1 antibody or an antigen binding fragment thereof and a buffer solution, the anti-DKK1 antibody or the antigen binding fragment thereof comprises HCDR1, HCDR2 and HCDR3 with amino acid sequences respectively shown as SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, and LCDR1, LCDR2 and LCDR3 with amino acid sequences respectively shown as SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, or HCDR1, HCDR2 and HCDR3 with amino acid sequences respectively shown as SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15, and LCDR1, LCDR2 and LCDR3 with amino acid sequences respectively shown as SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18. The invention also provides an injection containing the pharmaceutical composition and application of the pharmaceutical composition and the injection in preparation of drugs for treating diseases or symptoms by eliminating, inhibiting or reducing DKK1 activity.

Description

Technical Field

[0001] The present invention relates to the field of therapeutic pharmaceutical compositions, and in particular to anti-DKK1 antibody pharmaceutical compositions and uses thereof. Background Art

[0002] DKK1 is a secretory inhibitor of the Wnt / β-catenin signal transduction pathway. It belongs to the DKK (dickkopf) family, one of the protein families that inhibit Wnt signal transduction, and has the ability to inhibit Wnt-induced axis replication. The DKK family currently has 4 family members, namely DKK1, DKK2, DKK3 and DKK4.

[0003] The Wnt signaling pathway is involved in the control of embryonic development and tumorigenesis. Extracellular Wnt proteins are responsible for the growth and differentiation of multiple cell types during embryonic development and promote the development of multiple cancers. Genetic analysis showed that DKK1 inhibits Wnt signaling upstream, and DKK1 interacts antagonistically with LRP6, blocking Wnt-mediated signal activation.

[0004] When DKK1 protein is absent, Wnt, Frizzled, and LRP6 bind to form heterotrimers, activate downstream signaling pathways, maintain the stability of β-catenin, and aggregate after entering the nucleus, promoting the transcription and translation of osteoblast-related genes and tumor suppressor genes. When DKK1 protein is present, DKK1 competes with Wnt for binding to LRP6 molecules, and a series of downstream signal reactions cause β-catenin to phosphorylate and then be degraded. This blocks β-catenin from entering the nucleus, and then blocks the transcription and translation of osteoblast-related genes and tumor suppressor genes. When anti-DKK1 antibodies are present, antibodies compete for the binding of LRP6 and DKK1 molecules, which is conducive to the re-formation of heterotrimers by Wnt, Frizzled, and LRP6. The downstream signaling pathways are restored, so that the transcription and translation of osteoblast-related genes and tumor suppressor genes return to normal, which promotes the stability and proliferation of osteoblasts and inhibits the occurrence and proliferation of cancer cells. DKK1 can promote the formation of the tumor microenvironment by increasing the inhibitory activity of MDSC and Treg cells, downregulating the ligands that activate NK cells on the surface of tumor cells, promoting Th2 cell polarization, and reducing the production of IFNγ. In addition, DKK1 can also promote tumor growth and migration, promote the generation of CSC-like cells, and promote tumor angiogenesis, thereby directly promoting the occurrence and development of tumors (Yu et al., Signal Transduct Target Therapy, 2021, 6(1); Nikolai et al., Cancer Letters, 2020, 482). On March 22, 2021, Leap Therapeutics announced that its DKN-01 monoclonal antibody targeting the DKK1 target achieved positive results in the Phase 2 clinical treatment of advanced gynecological cancer. Clinical results show that DKN-01 is more effective in patients with high expression of DKK1, with an objective response rate of 14% and a disease control rate of 57%. To date, antibody drugs for this target have not yet been marketed. The two molecules with the fastest progress are in the clinical research stage, namely DKN-01 (CN110114087A) which is being studied for the treatment of multiple myeloma, gallbladder cancer, esophageal cancer, liver cancer, bladder cancer, and gynecological cancer, and BHQ-880 (WO2007084344) which is being studied for the treatment of multiple myeloma.

[0005] Anti-DKK1 antibodies must be prepared into antibody preparations before use in subjects, and must go through the process of storage and transportation. During storage and transportation, antibodies may undergo changes such as multimerization or modification, reduced activity, etc. These changes may cause immunotoxicity or reduced efficacy in subjects. Therefore, a stable preparation suitable for long-term storage and transportation of drugs is needed to ensure that the antibodies still have the biological activity required for treatment before they reach the body of the subject. However, there is currently no stable preparation on the market that is most suitable for the anti-DKK1 antibodies described in this article. Summary of the invention

[0006] The pharmaceutical composition provided by the present invention is a highly stable pharmaceutical composition containing an antibody that specifically binds to DKK1. In particular, the present invention develops a long-term stable anti-DKK1 antibody pharmaceutical composition by selecting an appropriate buffer system and pH, optimizing a stabilizer and a surfactant, and conducting studies on the antigen affinity, antigen binding activity, and cell activity of the pharmaceutical composition.

[0007] In one aspect, the present invention provides a pharmaceutical composition comprising:

[0008] (1) buffer; and

[0009] (2) anti-DKK1 antibodies or antigen-binding fragments thereof;

[0010] The anti-DKK1 antibody or antigen-binding fragment thereof comprises:

[0011] HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, respectively, and LCDR1, LCDR2 and LCDR3 with amino acid sequences as shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, respectively; or

[0012] The amino acid sequences of HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NO:11, SEQ ID NO:12 and SEQ ID NO:13, respectively, and the amino acid sequences of LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO:14, SEQ ID NO:15 and SEQ ID NO:16, respectively.

[0013] In some embodiments, in the pharmaceutical composition as described above, the concentration of the anti-DKK1 antibody or antigen-binding fragment thereof is about 2-200 mg / mL, preferably about 10-100 mg / mL, and more preferably about 20-60 mg / mL.

[0014] In some embodiments, the pH of the pharmaceutical composition as described above is about 5.0-7.0, preferably about 5.0-6.0, and more preferably about 5.4-5.6.

[0015] In some embodiments, the osmotic pressure of the pharmaceutical composition as described above is in the range of 250-350 mOsm / kg.

[0016] In some embodiments, in the pharmaceutical composition as described above, the buffer is selected from one or more of acetate buffer, histidine buffer, citrate buffer and phosphate buffer; preferably, the buffer is a histidine buffer, and the histidine buffer is selected from histidine-histidine hydrochloride buffer or histidine-histidine acetate buffer.

[0017] In some embodiments, in the pharmaceutical composition as described above, the concentration of the buffer is about 5-50 mM; more preferably, the concentration of the buffer is about 10-30 mM; more preferably, the concentration of the buffer is about 15-25 mM; preferably, the pH of the buffer is about 4.5-7.0, preferably about 5.0-6.5, more preferably about 5.0-6.0, more preferably about 5.4-5.6.

[0018] In some embodiments, the pharmaceutical composition as described above further comprises a stabilizer.

[0019] In some embodiments, the stabilizer is selected from one or more of arginine, arginine salt, sodium chloride, mannitol, sorbitol, sucrose, glycine and trehalose.

[0020] In some embodiments, the concentration of the stabilizer is about 50-300 mM, preferably about 120-280 mM, and more preferably about 130-250 mM.

[0021] In some embodiments, in the pharmaceutical composition as described above, the stabilizer is any one selected from the following (1) to (6):

[0022] (1) trehalose, at a concentration of about 120 to 280 mM, preferably, at a concentration of about 200 to 260 mM;

[0023] (2) sucrose, at a concentration of about 120 to 280 mM, preferably, at a concentration of about 200 to 260 mM;

[0024] (3) arginine or arginine salt, at a concentration of about 120 to 280 mM, preferably, at a concentration of about 120 to 160 mM; preferably, the arginine salt is arginine hydrochloride;

[0025] (4) a combination of sodium chloride and mannitol, wherein the concentration of sodium chloride is about 20-80 mM, and the concentration of mannitol is about 100-180 mM; preferably, the concentration of sodium chloride is about 30-70 mM, and the concentration of mannitol is about 120-160 mM;

[0026] (5) a combination of sodium chloride and sucrose, wherein the concentration of sodium chloride is about 20-80 mM and the concentration of sucrose is about 100-180 mM; preferably, the concentration of sodium chloride is about 30-70 mM and the concentration of sucrose is about 120-160 mM; or

[0027] (6) A combination of arginine hydrochloride and sucrose, wherein the concentration of arginine hydrochloride is about 20-80 mM, and the concentration of sucrose is about 100-180 mM; preferably, the concentration of arginine hydrochloride is about 30-70 mM, and the concentration of sucrose is about 120-160 mM.

[0028] In some embodiments, the pharmaceutical composition as described above further comprises a surfactant.

[0029] In some embodiments, the surfactant is selected from one or more of polysorbate 80, polysorbate 20 and poloxamer 188.

[0030] In some embodiments, the surfactant concentration is about 0.005% to 0.1% calculated on a w / v basis; more preferably, the surfactant concentration is about 0.01% to 0.08%; more preferably, the surfactant concentration is about 0.01% to 0.04%.

[0031] In some embodiments, the pharmaceutical composition as described above, wherein the anti-DKK1 antibody or antigen-binding fragment thereof comprises:

[0032] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:7, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:8; or

[0033] The heavy chain variable region with the amino acid sequence shown in SEQ ID NO: 11, and the light chain variable region with the amino acid sequence shown in SEQ ID NO: 8; or

[0034] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 19, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 20; or

[0035] The heavy chain variable region of the amino acid sequence shown in SEQ ID NO: 19, and the light chain variable region of the amino acid sequence shown in SEQ ID NO: 21; or

[0036] The heavy chain variable region has an amino acid sequence as shown in SEQ ID NO:22, and the light chain variable region has an amino acid sequence as shown in SEQ ID NO:20.

[0037] In some embodiments, the pharmaceutical composition as described above, wherein the anti-DKK1 antibody or antigen-binding fragment thereof comprises:

[0038] A heavy chain having an amino acid sequence as shown in SEQ ID NO:9, and a light chain having an amino acid sequence as shown in SEQ ID NO:10; or

[0039] A heavy chain having an amino acid sequence as shown in SEQ ID NO: 12, and a light chain having an amino acid sequence as shown in SEQ ID NO: 10; or

[0040] A heavy chain having an amino acid sequence as shown in SEQ ID NO: 23, and a light chain having an amino acid sequence as shown in SEQ ID NO: 24; or

[0041] A heavy chain having an amino acid sequence as shown in SEQ ID NO: 23, and a light chain having an amino acid sequence as shown in SEQ ID NO: 25; or

[0042] The heavy chain has an amino acid sequence as shown in SEQ ID NO:26, and the light chain has an amino acid sequence as shown in SEQ ID NO:25.

[0043] In some embodiments, the pharmaceutical composition as described above comprises the components shown in any one of the following (1) to (12):

[0044] (1) (a) about 10 to 100 mg / mL of the anti-DKK1 antibody or antigen-binding fragment thereof; (b) about 10 to 30 mM acetate buffer, pH 4.5 to 7.0; (c) about 120 to 280 mM sucrose; and (d) about 0.01% to 0.08% polysorbate 80; or

[0045] (2) (a) about 10 to 100 mg / mL of the anti-DKK1 antibody or antigen-binding fragment thereof; (b) about 10 to 30 mM histidine buffer, pH about 4.5 to 7.0; (c) 120 to 280 mM sucrose; and (d) about 0.01% to 0.08% polysorbate 80; or

[0046] (3) (a) about 10 to 100 mg / mL of the anti-DKK1 antibody or antigen-binding fragment thereof; (b) about 10 to 30 mM citric acid buffer, pH about 4.5 to 7.0; (c) 120 to 280 mM sucrose; and (d) about 0.01% to 0.08% polysorbate 80; or

[0047] (4) (a) about 10 to 100 mg / mL of the anti-DKK1 antibody or antigen-binding fragment thereof; (b) about 10 to 30 mM histidine buffer, pH about 4.5 to 7.0; (c) 120 to 280 mM trehalose; and (d) about 0.01% to 0.08% polysorbate 80; or

[0048] (5) (a) about 10 to 100 mg / mL of the anti-DKK1 antibody or antigen-binding fragment thereof; (b) about 10 to 30 mM acetate buffer, pH about 4.5 to 7.0; (c) 120 to 280 mM trehalose; and (d) about 0.01% to 0.08% polysorbate 80; or

[0049] (6) (a) about 20 to 60 mg / mL of the anti-DKK1 antibody or antigen-binding fragment thereof; (b) about 15 to 25 mM histidine buffer, pH about 5.0 to 6.0; (c) 130 to 250 mM sucrose; and (d) about 0.01% to 0.08% polysorbate 80; or

[0050] (7) (a) about 20 to 60 mg / mL of the anti-DKK1 antibody or antigen-binding fragment thereof; (b) about 15 to 25 mM histidine buffer, pH about 5.0 to 6.0; (c) 130 to 250 mM trehalose; and (d) about 0.01% to 0.08% polysorbate 80; or

[0051] (8) (a) about 20 to 60 mg / mL of the anti-DKK1 antibody or antigen-binding fragment thereof; (b) about 15 to 25 mM acetate buffer, pH about 5.0 to 6.0; (c) about 130 to 250 mM sucrose; and (d) about 0.01% to 0.08% polysorbate 80; or

[0052] (9) (a) about 20 to 60 mg / mL of the anti-DKK1 antibody or antigen-binding fragment thereof; (b) about 15 to 25 mM acetate buffer, pH about 5.0 to 6.0; (c) about 130 to 250 mM trehalose; and (d) about 0.01% to 0.08% polysorbate 80; or

[0053] (10) (a) about 40 to 60 mg / mL of the anti-DKK1 antibody or antigen-binding fragment thereof; (b) about 18 to 23 mM histidine buffer, pH about 5.4 to 5.6; (c) about 210 to 240 mM sucrose; and (d) about 0.01% to 0.04% polysorbate 80;

[0054] (11) (a) about 50 mg / mL of the anti-DKK1 antibody or antigen-binding fragment thereof; (b) about 20 mM histidine buffer, pH about 5.4-5.6; (c) about 230 mM sucrose; and (d) about 0.02% polysorbate 80; or

[0055] (12) (a) about 50 mg / mL of the anti-DKK1 antibody or antigen-binding fragment thereof; (b) about 20 mM histidine buffer, pH about 5.4-5.6; (c) about 230 mM sucrose; and (d) about 0.04% polysorbate 80; or

[0056] (13) (a) about 40 mg / mL of the anti-DKK1 antibody or antigen-binding fragment thereof; (b) about 20 mM histidine buffer, pH about 5.4-5.6; (c) about 230 mM sucrose; and (d) about 0.04% polysorbate 80; or

[0057] (14) (a) about 50 mg / mL of the anti-DKK1 antibody or antigen-binding fragment thereof; (b) about 20 mM histidine buffer, pH about 5.4-5.6; (c) about 230 mM sucrose; and (d) about 0.08% polysorbate 80; or

[0058] (15) (a) about 50 mg / mL of the anti-DKK1 antibody or antigen-binding fragment thereof; (b) about 20 mM histidine buffer, pH about 5.4-5.6; (c) about 230 mM trehalose; and (d) about 0.04% polysorbate 80; or

[0059] (16) (a) about 50 mg / mL of the anti-DKK1 antibody or antigen-binding fragment thereof; (b) about 20 mM acetate buffer, pH about 5.4-5.6; (c) about 230 mM sucrose; and (d) about 0.04% polysorbate 80; or

[0060] (17) (a) about 40 mg / mL of the anti-DKK1 antibody or antigen-binding fragment thereof; (b) about 20 mM acetate buffer, pH about 5.4-5.6; (c) about 230 mM sucrose; and (d) about 0.04% polysorbate 80; or

[0061] (18) (a) about 50 mg / mL of the anti-DKK1 antibody or its antigen-binding fragment; (b) about 20 mM acetate buffer, pH about 5.4-5.6; (c) about 230 mM trehalose; and (d) about 0.04% polysorbate 80.

[0062] Preferably, the anti-DKK1 antibody is as described in any embodiment herein; preferably, the histidine buffer is histidine-histidine hydrochloride buffer.

[0063] In some embodiments, the present invention provides a lyophilized formulation of the pharmaceutical composition described in any of the embodiments herein.

[0064] In another aspect, the present invention provides a reconstituted preparation, which is a preparation obtained by reconstitution of the lyophilized preparation described in any embodiment herein.

[0065] In another aspect, the present invention provides a liquid preparation comprising the pharmaceutical composition described in any embodiment herein, or a preparation obtained by reconstituting the lyophilized preparation described above with a sodium chloride solution or a glucose solution; preferably, the liquid preparation is an injection; preferably, the concentration of the sodium chloride solution is about 0.85-0.9% (w / v); preferably, the concentration of the glucose solution is about 5-25% (w / v); preferably, in the injection, the concentration of the anti-DKK1 antibody is about 0.1-50 mg / mL, more preferably about 0.2-20 mg / mL; preferably, the pH of the liquid preparation is about 4.5-7.0.

[0066] In some embodiments, in the liquid formulation as described above, the anti-DKK1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO:7, and a light chain variable region with an amino acid sequence as shown in SEQ ID NO:8.

[0067] In some embodiments, in the liquid formulation as described above, the anti-DKK1 antibody or antigen-binding fragment thereof comprises a heavy chain amino acid sequence as shown in SEQ ID NO:9, and a light chain amino acid sequence as shown in SEQ ID NO:10.

[0068] In some embodiments, the pH of the liquid formulation is about 5.0-6.0.

[0069] In some embodiments, in the pharmaceutical composition and liquid preparation as described above, the pharmaceutical composition or liquid preparation is administered by intravenous injection or subcutaneous injection.

[0070] In another aspect, provided herein is a use of the pharmaceutical composition, lyophilized preparation, reconstituted preparation or liquid preparation as described above in the preparation of a medicament for treating a disease or condition by eliminating, inhibiting or reducing DKK1 activity; preferably, the disease or condition is selected from cancer, infectious disease or inflammatory disease; more preferably, the disease is cancer.

[0071] In some embodiments, the liquid formulation or lyophilized formulation as described above is stable at 2-8° C. for at least 3 months, at least 6 months, at least 12 months, at least 18 months or at least 24 months.

[0072] In some embodiments, the liquid formulation or lyophilized formulation is stable at 40° C. for at least 7 days, at least 14 days, or at least 28 days.

[0073] In some embodiments, the liquid preparation is administered by intravenous injection or subcutaneous injection. BRIEF DESCRIPTION OF THE DRAWINGS

[0074] Figure 1 : Binding and dissociation curves of a pharmaceutical composition comprising an anti-DKK1 antibody and a human DKK1 antigen protein. In the figure, each curve corresponds to 16 nM, 8 nM, 4 nM, 2 nM, 1 nM and 0.5 nM from top to bottom.

[0075] Figure 2 : Binding curve of a pharmaceutical composition comprising an anti-DKK1 antibody and human DKK1 antigen protein.

[0076] Figure 3 : The luciferase reporter gene system was used to determine the activity of the pharmaceutical composition containing anti-DKK1 antibodies in activating the Wnt signaling pathway. DETAILED DESCRIPTION

[0077] Definition and Description

[0078] In order to make the present invention easier to understand, some technical and scientific terms are specifically defined below. Unless otherwise clearly defined in this article, all other technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art to which the present invention belongs. It should be understood that the present invention is not limited to specific methods, reagents, compounds, compositions or biological systems, and of course the above can be changed. It should also be understood that the terms used in this application are only for describing specific embodiments and are not intended to be limited. All references cited herein, including patents, patent applications, papers, textbooks and the like, and references cited therein, to the extent that they have not been cited, are incorporated herein by reference in their entirety. If one or more of the documents and similar materials incorporated therein are different or contradictory from the present application, including but not limited to defined terms, term usage, described technology and the like, then the present application shall prevail.

[0079] 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 combinations of two or more polypeptides, etc.

[0080] The term "pharmaceutical composition" or "preparation" refers to a mixture containing one or more antibodies described herein and other components, such as physiologically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration to an organism, facilitate the absorption of the active ingredient, and thus exert biological activity.

[0081] 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 formulation of the present invention is stable upon storage, and its stability is not dependent on lyophilization (or other state-changing methods, such as spray drying).

[0082] The term "aqueous liquid formulation" refers to a liquid formulation using water as a solvent. In some embodiments, the aqueous liquid formulation is a formulation that does not require lyophilization, spray drying and / or freezing to maintain stability (eg, chemical and / or physical stability and / or biological activity).

[0083] The term "excipient" refers to an agent that can be added to a formulation to provide desired properties (e.g., consistency, increased 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.

[0084] As used herein, "about" when referring to a measurable value (e.g., an amount, a duration, etc.) is intended to encompass variations of ±20% or ±10% relative to the particular value, including ±5%, ±1%, and ±0.1%, as such variations are suitable for performing the disclosed methods.

[0085] The term "buffer pH of about 5.0 to 7.0" refers to an agent that, through the action of its acid / base conjugate components, allows the solution containing the agent to resist 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 7.0, or 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.

[0086] Herein, examples of "buffers" that control the pH within this range include acetic acid, acetate salts (e.g., sodium acetate), succinic acid, succinate salts (e.g., sodium succinate), gluconic acid, histidine, histidine salts (e.g., histidine hydrochloride), methionine, citric acid (citric acid), citrate (citrate), phosphate, citrate / phosphate, imidazole, combinations thereof, and other organic acid buffers.

[0087] "Histidine buffer" is a buffer containing histidine ions. Examples of histidine buffers include histidine and histidine salts, such as histidine hydrochloride, histidine acetate, histidine phosphate and histidine sulfate, such as a histidine buffer containing histidine and histidine hydrochloride; the histidine buffer of the present invention also includes a histidine buffer containing histidine and acetate (such as sodium salt or potassium salt).

[0088] "Citrate buffer", also known as "citrate buffer", is a buffer comprising citrate ions. Examples of citrate buffers include citric acid-sodium citrate, citric acid-potassium citrate, citric acid-calcium citrate, citric acid-magnesium citrate, etc. The preferred citrate buffer is citric acid-sodium citrate buffer.

[0089] "Acetate buffer" is a buffer comprising acetate ions. Examples of acetate buffers include acetate-sodium acetate, acetate-potassium acetate, acetate-calcium acetate, acetate-magnesium acetate, etc. A preferred acetate buffer is acetate-sodium acetate buffer.

[0090] "Succinate buffer" is a buffer comprising succinate ions. Examples of succinate buffers include succinate-sodium succinate, succinate-potassium succinate, succinate-calcium succinate, succinate-magnesium succinate, and the like. A preferred succinate buffer is succinate-sodium succinate buffer.

[0091] 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 as defined below, such as sodium chloride, calcium chloride, magnesium chloride, mannitol, sorbitol, sucrose, trehalose, arginine or its salts (such as arginine hydrochloride), glycine, alanine (α-alanine, β-alanine), betaine, leucine, lysine, glutamic acid, aspartic acid, proline, 4-hydroxyproline, sarcosine, γ-aminobutyric acid (GABA), opines, alanine, octopine, strombine and trimethylamine N-oxide (TMAO), human serum albumin (hsa), bovine serum albumin (BSA), α-casein, globulin, α-lactalbumin, LDH, lysozyme, myoglobin, ovalbumin and RNAase A. Some stabilizers, such as sodium chloride, calcium chloride, magnesium chloride, mannitol, sorbitol, sucrose, etc., can also play a role in controlling osmotic pressure. The stabilizer specifically used in the present invention is selected from one or more of polyols, amino acids, salts, and sugars. Preferred salts are sodium chloride, preferred sugars are sucrose and trehalose, and preferred polyols are sorbitol and mannitol. Preferred amino acids are arginine, glycine, and proline. Amino acids can exist in their D- and / or L- forms, but are typically L-forms, and amino acids can exist in any suitable salt, such as hydrochlorides, such as arginine hydrochloride. Preferred stabilizers are sodium chloride, mannitol, sorbitol, sucrose, trehalose, arginine hydrochloride, glycine, proline, sodium chloride-sorbitol, sodium chloride-mannitol, sodium chloride-sucrose, sodium chloride-trehalose, arginine hydrochloride-mannitol, arginine hydrochloride-sucrose.

[0092] The term "surfactant" generally includes agents that protect proteins such as antibodies from stress induced by the air / solution interface, solution / surface induced stress to reduce the aggregation of antibodies or minimize the formation of particulate matter in the formulation. Exemplary surfactants include, but are not limited to, non-ionic surfactants such as polyoxyethylene sorbitan fatty acid esters (such as polysorbate 20 and polysorbate 80), polyethylene-polypropylene copolymers, polyethylene-polypropylene glycol, polyoxyethylene-stearate, polyoxyethylene alkyl ethers, such as polyoxyethylene monolauryl ether, alkylphenyl polyoxyethylene ether (Triton-X), polyoxyethylene-polyoxypropylene copolymers (poloxamers, Pluronic), sodium dodecyl sulfate (SDS). Herein, unless otherwise specified, the terms "polysorbate 20 concentration" and "polysorbate 80 concentration" all refer to mass volume concentration (w / v), such as "about 0.04% polysorbate 80" in "0.04%" means "100 mL of liquid contains 0.04g of polysorbate 80".

[0093] The term "viscosity" as used herein may be "kinematic viscosity" or "absolute viscosity". "Kinematic viscosity" is a measure of a fluid's resistance to flow under the influence of gravity. "Absolute viscosity", sometimes called dynamic viscosity or simple viscosity, is the product of kinematic viscosity and the density of the fluid (absolute viscosity = kinematic viscosity x density). The dimension of kinematic viscosity is L 2 / T, where L is the length and T is the time. Usually, kinematic viscosity is expressed in centistokes (cSt). The SI unit of kinematic viscosity is mm 2 / s, i.e. 1cSt. Absolute viscosity is expressed in centipoise (cP). The SI unit of absolute viscosity is milliPascal·second (mPa·s), where 1cP = 1mPa·s.

[0094] 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 / kg. Isotonicity can be measured using a vapor pressure or freezing point depression osmometer.

[0095] The term "stable" formulation is one in which the antibody substantially maintains its physical stability and / or chemical stability and / or biological activity during the manufacturing process and / or upon storage. A pharmaceutical formulation may be stable even if the antibody contained therein does not retain 100% of its chemical structure or biological function after a period of storage. In some cases, an antibody formulation that maintains about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% of its structure or function after a period of storage 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, ed., Marcel Dekker, Inc., New York, NY, Pubs. (1991), and Jones, A. (1993) Adv. Drug Delivery Rev. 10: 29-90 (both incorporated by reference).

[0096] The stability of the formulation can be measured by determining the percentage of natural antibodies remaining therein (and other methods) after the formulation has been stored at a certain temperature for a certain period of time. Among other methods, the percentage of natural antibodies can be measured by size exclusion chromatography (e.g., size exclusion high performance liquid chromatography [SEC-HPLC]), and "natural" means not aggregated and not degraded. In some embodiments, the stability of the protein is determined by the percentage of monomeric protein in a solution with a low percentage of degradation (e.g., fragmentation) and / or aggregated protein. In some embodiments, the formulation can be stably stored at room temperature, about 25-30°C or 40°C for at least 2 weeks, at least 28 days, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 months, at least 24 months, or longer, with no more than about 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% aggregated form of antibodies.

[0097] Stability can be measured by determining the percentage of antibody that migrates in the more acidic fraction of the main fraction of the antibody (the "major charged form") during ion exchange (among other methods), where stability is inversely proportional to the percentage of the antibody in the acidic form. The percentage of "acidified" antibody can be measured by ion exchange chromatography (e.g., cation exchange high performance liquid chromatography [CEX-HPLC]), among other methods. In some embodiments, an acceptable level of stability means that after a formulation is stored at a certain temperature for a certain period of time, 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 is detectable in the acidic form. The storage period before 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, the temperature at which the pharmaceutical preparation is allowed to be stored can be any temperature in the range of about -80°C to about 45°C, such as storage at about -80°C, about -30°C, about -20°C, about 0°C, about 2-8°C, about 5°C, about 25°C, or about 40°C.

[0098] An antibody "retains its physical stability" in the 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 when measured by UV light scattering or by 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.

[0099] The stability of the formulation, e.g., physical stability, can be assessed by methods known in the art, including measuring the apparent extinction (absorbance or optical density) of a sample. Such extinction measurements are related to the turbidity of the formulation. The turbidity of a formulation is, in part, an inherent property of the protein dissolved in solution, and is typically measured by turbidimetry and measured in nephelometric turbidity units (NTU).

[0100] The turbidity level that 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 "emulsification" or "emulsified appearance" of the formulation. The turbidity level can be calculated with reference to a standard curve generated using a suspension of known turbidity. The reference standard for determining the turbidity level of a pharmaceutical composition can be based on the European Pharmacopoeia standard (European Pharmacopoeia, 4th edition, "Directorate for the Quality of 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 less than or equal to the turbidity of a reference suspension having about 3 according to the European Pharmacopoeia standard. Turbidimetric turbidity measurements can detect Rayleigh scattering in the absence of association or non-ideal effects, which generally varies linearly with concentration. Other methods for evaluating physical stability are well known in the art.

[0101] An antibody "retains its chemical stability" in a pharmaceutical composition if its chemical stability at a given time point is such that the antibody is considered to still retain its biological activity as defined below. Chemical stability can be assessed, for example, by detecting or quantifying chemically altered forms of the antibody. Chemical alterations can include size changes (e.g., shortening), which can be assessed using, for example, size exclusion chromatography, SDS-PAGE, and / or matrix-assisted laser desorption ionization / time of flight mass spectrometry (MALDI / TOF MS). Other types of chemical alterations include charge changes (e.g., occurring as a result of deamidation or oxidation), which can be assessed by, for example, ion exchange chromatography.

[0102] An antibody "retains its biological activity" in a pharmaceutical composition if the antibody in the pharmaceutical composition is biologically active for its intended purpose. For example, a formulation of the invention is considered stable if, after storage at a temperature such as 5°C, 25°C, 45°C, etc. for a certain period of time (e.g., 1 to 12 months), the anti-DKK1 antibody contained in the formulation binds to DKK1 with an affinity that is at least 90%, 95% or more of the binding affinity of the antibody before the storage. Binding affinity can also be determined, for example, by ELISA or plasma resonance techniques.

[0103] In the context of the present invention, in a pharmacological sense, a "therapeutically effective amount" or "effective amount" of an antibody refers to an amount that is effective in preventing or treating or alleviating the symptoms of a disorder that the antibody can effectively treat. In the present invention, a "therapeutically effective amount" or "therapeutically effective dose" of a drug is any amount of the drug that protects a subject from the onset of the disease or promotes disease regression when used alone or in combination with another therapeutic agent, as evidenced by a reduction in the severity of disease symptoms, an increase in the frequency and duration of disease symptom-free periods, or the prevention of damage or disability caused by disease pain. 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 measuring 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 the drug that inhibits the development or recurrence of a disease when given alone or in combination with other therapeutic drugs to a subject at risk of the disease or a subject with a recurring disease.

[0104] The term "subject" or "patient" is intended to include mammalian organisms. Examples of subjects / patients include humans and non-human mammals, such as non-human primates, dogs, cows, horses, pigs, sheep, goats, cats, mice, rabbits, rats, and transgenic non-human animals. In a specific embodiment of the invention, the subject is a human.

[0105] 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 skilled in the art. Routes of administration of anti-DKK1 antibodies include intravenous, intramuscular, subcutaneous, peritoneal, spinal or other parenteral routes of administration, such as injection or infusion. "Parenteral administration" refers to administration other than enteral or topical administration, usually by injection, including but not limited to intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraframe, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, intradural and intrasternal injection and infusion and in vivo electroporation.

[0106] Anti-DKK1 Antibodies

[0107] The term "antibody" as used herein should be understood to include complete 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 to human DKK1 or its epitope. Therefore, it is used in the broadest sense, specifically including but not limited to monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (such as bispecific antibodies), humanized antibodies, fully human antibodies, chimeric antibodies and single domain antibodies.

[0108] The term "isolated antibody" refers to the purified state of the binding compound, and in this case means that the molecule is substantially free of other biomolecules, such as nucleic acids, proteins, lipids, sugars, or other substances such as cell debris and growth medium. The term "isolated" does not imply the complete absence of such substances or the absence of water, buffers, or salts unless they are present in amounts that significantly interfere with the experimental or therapeutic use of the binding compound described herein.

[0109] The term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic epitope. In contrast, conventional (polyclonal) antibody preparations typically include a large number of antibodies directed against (or specific for) different epitopes. The modifier "monoclonal" indicates the character of the antibody as being obtained from a population of substantially homogeneous antibodies, and is not to be construed as requiring production of the antibody by any particular method.

[0110] The term "murine antibody" or "hybridoma antibody" in the present disclosure refers to a monoclonal antibody against human DKK1 prepared according to the knowledge and skills in the art. During preparation, a test subject is injected with a DKK1 antigen, and then a hybridoma expressing an antibody with the desired sequence or functional properties is isolated.

[0111] The term "chimeric antibody" is an antibody having the variable domains of a first antibody and the constant domains of a second antibody, wherein the first antibody and the second antibody are from different species. Typically, the variable domains are obtained from antibodies such as rodents ("parent antibodies"), while the constant domain sequences are obtained from human antibodies, such that the resulting chimeric antibodies are less likely to induce adverse immune responses in human subjects than the parent rodent antibodies.

[0112] The term "humanized antibody" refers to an antibody form containing sequences from human and non-human (e.g., mouse, rat) antibodies. In general, a humanized antibody comprises substantially all of at least one, usually two variable domains, wherein all or substantially all of the hypervariable loops correspond to the hypervariable loops of non-human immunoglobulins, and all or substantially all of the framework (FR) regions are framework regions of human immunoglobulin sequences. A humanized antibody may optionally comprise at least a portion of a human immunoglobulin constant region (Fc).

[0113] The term "full-length antibody" or "complete antibody molecule" refers to an immunoglobulin molecule comprising four peptide chains, two heavy (H) chains (about 50-70 kDa at full length) and two light (L) chains (about 25 kDa at full length) interconnected by disulfide bonds. Each heavy chain consists 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 three domains, CH1, CH2, and CH3. Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region consists of one domain, CL. The VH and VL regions can be further subdivided into highly variable complementary determining regions (CDRs) and regions separated by more conservative framework regions (FRs). Each VH or VL region consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant regions of antibodies mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[0114] The term "CDR" refers to the complementarity determining region within the variable sequence of an antibody. There are three CDRs in each variable region of the heavy and light chains, which are named 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 according to different systems.

[0115] The precise amino acid sequence boundaries of the variable region CDRs of the antibodies of the invention can be determined using any of a number of well-known schemes, including Chothia (Chothia et al. (1989) Nature 342:877-883, Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997), Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 4th Edition, US Department of Health and Human Services, National Institutes of Health (1987), AbM (University of Bath), Contact (University College London), the international ImMunoGeneTics database (IMGT) (1999 Nucleic Acids ) based on the variability of antibody sequences. Research, 27, 209-212), and the North CDR definition based on affinity propagation clustering using a large number of crystal structures. The CDRs of the antibodies of the present invention can be defined by a person skilled in the art according to any scheme in the art (e.g., different assignment systems or combinations).

[0116] As used herein, "antigen-binding fragments" include fragments of antibodies or derivatives thereof, generally including at least one fragment of the antigen-binding region or variable region (e.g., one or more CDRs) of a parent antibody, which retains at least some of the binding specificity of the parent antibody. Examples of antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab') 2And Fv fragments; diabodies; linear antibodies; single-chain antibody molecules, such as sc-Fv; nanobodies and multispecific antibodies formed by antibody fragments. When the binding activity of the antibody is expressed on a molar concentration basis, the binding fragment or its derivative generally retains at least 10% of the antigen binding activity of the parent antibody. Preferably, the binding fragment or its derivative 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 expected that the antigen-binding fragment of the antibody may include conservative or non-conservative amino acid substitutions that do not significantly change its biological activity (referred to as "conservative variants" or "functional conservative variants" of the antibody).

[0117] The CDR sequence contained in the anti-DKK1 antibody or antigen-binding fragment thereof of the present invention is preferably from any CDR sequence or any CDR sequence combination (i.e., a combination of LCDR1-LCDR3 and HCDR1-HCDR3) described in application number PCT / CN2023 / 073335 (the entire contents disclosed in this article are incorporated herein by reference). In some embodiments, the antibody or antigen-binding fragment thereof used in the methods and compositions of the present invention is any anti-DKK1 antibody or antigen-binding fragment thereof described in PCT / CN2023 / 073335, preferably any anti-DKK1 humanized antibody or antigen-binding fragment thereof described in PCT / CN2023 / 073335.

[0118] In some embodiments, the anti-DKK1 antibody or antigen-binding fragment thereof used in the methods and compositions of the present invention comprises HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively, and LCDR1, LCDR2 and LCDR3 with amino acid sequences as shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, respectively. Preferably, the anti-DKK1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 7, and a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 8. Further preferably, the anti-DKK1 antibody or antigen-binding fragment thereof comprises a heavy chain amino acid sequence as shown in SEQ ID NO: 9, and a light chain amino acid sequence as shown in SEQ ID NO: 10.

[0119] In some embodiments, the anti-DKK1 antibody or antigen-binding fragment thereof used in the methods and compositions of the present invention comprises HCDR1, HCDR2 and HCDR3 having amino acid sequences as shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively, and LCDR1, LCDR2 and LCDR3 having amino acid sequences as shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, respectively. Preferably, the anti-DKK1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 11, and a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 8. Further preferably, the anti-DKK1 antibody or antigen-binding fragment thereof comprises a heavy chain amino acid sequence as shown in SEQ ID NO: 12, and a light chain amino acid sequence as shown in SEQ ID NO: 10.

[0120] In some embodiments, the anti-DKK1 antibody or antigen-binding fragment thereof used in the methods and compositions of the present invention comprises HCDR1, HCDR2 and HCDR3 having amino acid sequences as shown in SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15, respectively, and LCDR1, LCDR2 and LCDR3 having amino acid sequences as shown in SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18, respectively. Preferably, the anti-DKK1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 19, and a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 20. Further preferably, the anti-DKK1 antibody or antigen-binding fragment thereof comprises a heavy chain amino acid sequence as shown in SEQ ID NO: 23, and a light chain amino acid sequence as shown in SEQ ID NO: 24.

[0121] In some embodiments, the anti-DKK1 antibody or antigen-binding fragment thereof used in the methods and compositions of the present invention comprises HCDR1, HCDR2 and HCDR3 having amino acid sequences as shown in SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15, respectively, and LCDR1, LCDR2 and LCDR3 having amino acid sequences as shown in SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18, respectively. Preferably, the anti-DKK1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 19, and a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 21. Further preferably, the anti-DKK1 antibody or antigen-binding fragment thereof comprises a heavy chain amino acid sequence as shown in SEQ ID NO: 23, and a light chain amino acid sequence as shown in SEQ ID NO: 25.

[0122] In some embodiments, the anti-DKK1 antibody or antigen-binding fragment thereof used in the methods and compositions of the present invention comprises HCDR1, HCDR2 and HCDR3 having amino acid sequences as shown in SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15, respectively, and LCDR1, LCDR2 and LCDR3 having amino acid sequences as shown in SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18, respectively. Preferably, the anti-DKK1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 22, and a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 20. Further preferably, the anti-DKK1 antibody or antigen-binding fragment thereof comprises a heavy chain amino acid sequence as shown in SEQ ID NO: 26, and a light chain amino acid sequence as shown in SEQ ID NO: 25.

[0123] The amino acid sequences of SEQ ID NOs: 1-26 are shown below.

[0124] SEQ ID NO:1

[0125] EYTM

[0126] SEQ ID NO:2

[0127] VIDPSNGDTSYNQKFKG

[0128] SEQ ID NO:3

[0129] TTGPWFAS

[0130] SEQ ID NO:4<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0131] <h2 style=";text-align:left;direction:ltr"> RASQDISNYLN<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0132] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:5<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0133] <h2 style=";text-align:left;direction:ltr"> YKSRLHS<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0134] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:6<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0135] <h2 style=";text-align:left;direction:ltr"> QQVHTLPPT<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0136] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:7<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0137] <h2 style=";text-align:left;direction:ltr"> EVQLLQSAAEVKRPGESLRISCKTSGYTFT<h2 style=";text-align:left;direction:ltr"> EYTM <h2 style=";text-align:left;direction:ltr"> WVRQMPGKELEWMG<h2 style=";text-align:left;direction:ltr"> VIDPSNGDTSYNQKFKG <h2 style=";text-align:left;direction:ltr"> HVTISADKSSSSTAYLQWSSLKASDAAMYYCSS<h2 style=";text-align:left;direction:ltr"> TTGPWFAS <h2 style=";text-align:left;direction:ltr"> WGQGTLVTVSS<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0138] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:8<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0139] <h2 style=";text-align:left;direction:ltr"> EIVMTQSPPTLSLSPGERVTLSC<h2 style=";text-align:left;direction:ltr"> RASQDISNYLN <h2 style=";text-align:left;direction:ltr"> WYQQKPGQAPRLLIY<h2 style=";text-align:left;direction:ltr"> YKSRLHS <h2 style=";text-align:left;direction:ltr"> GIPARFSGSGSGTDFTLTISSLQPEDFAVYYC<h2 style=";text-align:left;direction:ltr"> QQVHTLPPT <h2 style=";text-align:left;direction:ltr"> FGQGTKLEIK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0140] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:9<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0141] <h2 style=";text-align:left;direction:ltr"> EVQLLQSAAEVKRPGESLRISCKTSGYTFT<h2 style=";text-align:left;direction:ltr"> EYTM <h2 style=";text-align:left;direction:ltr"> WVRQMPGKELEWMG<h2 style=";text-align:left;direction:ltr"> VIDPSNGDTSYNQKFKG <h2 style=";text-align:left;direction:ltr"> HVTISADKSSSSTAYLQWSSLKASDAAMYYCSS<h2 style=";text-align:left;direction:ltr"> TTGPWFASWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0142] SEQ ID NO:10

[0143] EIVMTQSPPTLSLSPGERVTLSC RASQDISNYLN WYQQKPGQAPRLLIY YKSRLHS GIPARFSGSGSGTDFTLTISSLQPEDFAVYYC QQVHTLPPT FGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0144] SEQ ID NO:11

[0145] QVQLVQSGAEVKKPGASVKVSCKASGYTFT EYTM WVRQAPGQRLEWMG VIDPSNGDTSYNQKFKG RVTITVDKSSSTAYMELSSLRSEDTAVYYCSS TTGPWFAS WGQGTLVTVSS

[0146] SEQ ID NO:12

[0147] QVQLVQSGAEVKKPGASVKVSCKASGYTFT EYTM WVRQAPGQRLEWMG VIDPSNGDTSYNQKFKG RVTITVDKSSSTAYMELSSLRSEDTAVYYCSS TTGPWFASWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0148] SEQ ID NO:13

[0149] SYNML

[0150] SEQ ID NO:14

[0151] VIFPGNGDTSYNPKFKG

[0152] SEQ ID NO:15

[0153] DGYPSMDY

[0154] SEQ ID NO:16

[0155] ITSTDIDDDMN

[0156] SEQ ID NO:17

[0157] EGNSLRP

[0158] SEQ ID NO:18

[0159] LQFNNMPLT

[0160] SEQ ID NO:19

[0161] QVQLVQSGAEVKKPGASVKVSCKASGYTFT SYNML WVRQAPGQRLEWMG VIFPGNGDTSYNPKFKG RVTLTADKSSSTAYMELSSLRSEDTAVYYCAR DGYPSMDY WGQGTLVTVSS

[0162] SEQ ID NO:20

[0163] DIQMTQSPSSVSASVGDRVTITC ITSTDIDDDMN WYQQKPGKAPKLLIS EGNSLRP GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC LQFNNMPLT FGQGTKLEIK

[0164] SEQ ID NO:21

[0165] DTQLTQSPSFLSASVGDRVTITC ITSTDIDDDMN WYQQKPGKAPKLLIS EGNSLRP GVPSRFSSSGSGTEFTLTISSLQPEDFATYYC LQFNNMPLT FGQGTKLEIK

[0166] SEQ ID NO:22

[0167] QVQLVQSGAEVKKPGASVKVSCKASGYTFT SYNML WVRQAPGQRLEWMG VIFPGNADTSYNPKFKG RVTLTADKSSSTAYMELSSLRSEDTAVYYCAR DGYPSMDY WGQGTLVTVSS

[0168] SEQ ID NO:23

[0169] QVQLVQSGAEVKKPGASVKVSCKASGYTFT SYNML WVRQAPGQRLEWMG VIFPGNGDTSYNPKFKG RVTLTADKSSSTAYMELSSLRSEDTAVYYCAR DGYPSMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0170] SEQ ID NO:24

[0171] DIQMTQSPSSVSASVGDRVTITC ITSTDIDDDMN WYQQKPGKAPKLLIS EGNSLRP GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC LQFNNMPLT FGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0172] SEQ ID NO:25

[0173] DTQLTQSPSFLSASVGDRVTITC ITSTDIDDDMN WYQQKPGKAPKLLIS EGNSLRP GVPSRFSSSGSGTEFTLTISSLQPEDFATYYC LQFNNMPLT FGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0174] SEQ ID NO:26

[0175] QVQLVQSGAEVKKPGASVKVSCKASGYTFT SYNML WVRQAPGQRLEWMG VIFPGNADTSYNPKFKG RVTLTADKSSSTAYMELSSLRSEDTAVYYCAR DGYPSMDY WGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWY VDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0176] In some embodiments, the anti-DKK1 antibody or antigen-binding fragment thereof used in the methods and compositions of the present invention is selected from a murine antibody or antigen-binding fragment thereof, a chimeric antibody or antigen-binding fragment thereof, a humanized antibody or antigen-binding fragment thereof, preferably a humanized antibody or antigen-binding fragment thereof.

[0177] In some embodiments, the anti-DKK1 antibody or antigen-binding fragment thereof used in the methods and compositions of the present invention is a humanized antibody or a chimeric antibody and may include a human constant region. In some embodiments, the constant region is selected from the group consisting of human IgG1, IgG2, IgG3 and IgG4 constant regions; preferably, the anti-DKK1 antibody or antigen-binding fragment thereof suitable for the methods and compositions of the present invention comprises a heavy chain constant region of human IgG1 or IgG4 isotype, more preferably a human IgG4 constant region. In some embodiments, one or more amino acid modifications may be introduced into the Fc region of the anti-DKK1 antibody provided herein to generate an Fc region variant, such as introducing an S228P mutation into the sequence of the IgG4 heavy chain constant region of the anti-DKK1 antibody or antigen-binding fragment thereof, which replaces the serine residue in the hinge region with a proline residue that is usually present at the corresponding position of an IgG1 isotype antibody.

[0178] Pharmaceutical preparations

[0179] The pharmaceutical composition of the present invention is a highly stable pharmaceutical composition containing an antibody that specifically binds to DKK1. The present invention finds that trehalose can significantly improve the stability of the pharmaceutical composition.

[0180] The pharmaceutical composition of the present invention comprises: (1) a buffer; and (2) an anti-DKK1 antibody or an antigen-binding fragment thereof.

[0181] The anti-DKK1 antibody or antigen-binding fragment thereof in the pharmaceutical composition of the present invention is as described in any embodiment of the "Anti-DKK1 Antibody" section of this application.

[0182] The concentration of the anti-DKK1 antibody or antigen-binding fragment thereof in the pharmaceutical composition of the present invention is about 2 to 200 mg / mL, preferably about 10 to 100 mg / mL, and more preferably about 20 to 60 mg / mL. In some embodiments, the concentration of the anti-DKK1 antibody or antigen-binding fragment thereof in the pharmaceutical composition of the present invention is about 40 to 60 mg / mL. Preferably, the concentration of the anti-DKK1 antibody or antigen-binding fragment thereof is about 15 mg / mL, 20 mg / mL, 25 mg / mL, 30 mg / mL, 35 mg / mL, 40 mg / mL, 45 mg / mL, 50 mg / mL, 55 mg / mL, 60 mg / mL, 65 mg / mL, 70 mg / mL, 75 mg / mL, 80 mg / mL, 85 mg / mL, 90 mg / mL or 95 mg / mL, more preferably 20 mg / mL, 25 mg / mL, 30 mg / mL, 35 mg / mL, 40 mg / mL, 45 mg / mL, 50 mg / mL or 55 mg / mL.

[0183] The buffer in the pharmaceutical composition of the present invention is selected from one or more of acetate buffer, citrate buffer, phosphate buffer and histidine buffer; preferably, the buffer is a histidine buffer. Preferably, the histidine buffer is selected from histidine-histidine hydrochloride buffer or histidine-histidine acetate buffer, preferably histidine-histidine hydrochloride buffer. Preferably, the concentration of the buffer is about 5 to 100 mM, preferably about 5 to 50 mM, more preferably about 10 to 30 mM; more preferably about 15 to 25 mM. Preferably, the pH of the buffer is about 4.5 to 7.0, preferably about 5.0 to 6.5, more preferably about 5.0 to 6.0, more preferably about 5.4 to 5.6. Herein, non-limiting examples of 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, 6.6, 6.7, 6.8, 6.9, 7.0, preferably about 5.4, 5.5 or 5.6.

[0184] In some embodiments, the histidine buffer is a histidine-histidine hydrochloride buffer. In some embodiments, the above-mentioned histidine-histidine hydrochloride buffer is made of histidine and histidine hydrochloride, preferably L-histidine and L-histidine monohydrochloride. In some embodiments, the histidine buffer is made of 1 to 30 mM L-histidine and 1 to 30 mM L-histidine monohydrochloride. In some embodiments, the histidine buffer is made of histidine and histidine hydrochloride in a molar ratio of 1:1 to 1:4. In some embodiments, the histidine buffer is made of histidine and histidine hydrochloride in a molar ratio of about 1:1. In some embodiments, the histidine buffer is made of histidine and histidine hydrochloride in a molar ratio of about 1:3. In some embodiments, the histidine preparation is: a histidine buffer made of about 4.5 mM L-histidine and about 15.5 mM L-histidine monohydrochloride. A histidine buffer having a pH of about 5.5 made from about 7.5 mM L-histidine and about 22.5 mM L-histidine monohydrochloride. In some embodiments, the histidine preparation is a histidine buffer having a pH of about 6.0 made from about 10 mM histidine and about 10 mM histidine hydrochloride. In some embodiments, the histidine preparation is a histidine buffer having a pH of about 6.5 made from about 14.7 mM L-histidine and about 5.3 mM L-histidine monohydrochloride.

[0185] In some embodiments, 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 acetate buffer is made of 1 to 30 mM acetic acid and 1 to 30 mM sodium acetate. In some embodiments, the acetate buffer is made of acetic acid and sodium acetate in a molar ratio of about 1:2.1. In some embodiments, the acetate buffer is made of acetic acid and sodium acetate in a molar ratio of about 1:5.7. In some embodiments, the acetate buffer is: an acetate buffer with a pH of about 5.0 made of about 6.5 mM acetic acid and about 13.5 mM sodium acetate. In some embodiments, the acetate buffer is: an acetate buffer with a pH of about 5.5 made of about 3 mM acetic acid and about 17 mM sodium acetate.

[0186] In some embodiments, the citric acid buffer is a citric acid-sodium citrate buffer. In some embodiments, the citric acid buffer is made of 1 to 30 mM citric acid and 1 to 30 mM sodium citrate. In some embodiments, the citric acid buffer is made of citric acid and sodium citrate in a molar ratio of about 1:1 to 1:4. In some embodiments, the citric acid buffer is a citric acid buffer with a pH of about 6.5 made from about 5.0 mM citric acid and about 15.0 mM sodium citrate. In some embodiments, the citric acid buffer is a citric acid buffer with a pH of about 6.0 made from about 10 mM citric acid and about 10 mM sodium citrate. In some embodiments, the citric acid buffer is a citric acid buffer with a pH of about 5.0 made from about 7.6 mM citric acid and about 12.4 mM sodium citrate. In some embodiments, the citric acid buffer is a citric acid buffer with a pH of about 5.5 made from about 5.3 mM citric acid and about 14.7 mM sodium citrate.

[0187] In some embodiments, the phosphate buffer is a sodium dihydrogen phosphate-sodium dihydrogen phosphate buffer. In some embodiments, the phosphate buffer is made of about 1 to 20 mM sodium dihydrogen phosphate and about 1 to 20 mM sodium dihydrogen phosphate. In some embodiments, the phosphate buffer is made of sodium dihydrogen phosphate and sodium dihydrogen phosphate in a molar ratio of about 1:1 to 1:4. In some embodiments, the phosphate buffer is a phosphate buffer with a pH of about 7.0 made of about 10 mM sodium dihydrogen phosphate and about 10 mM sodium dihydrogen phosphate. In some embodiments, the pharmaceutical composition as described above comprises a buffer, and the concentration of the buffer is about 5-100 mM, preferably about 10-50 mM, preferably about 10-30 mM; preferably about 15-25 mM; non-limiting examples of the above buffer concentration are about 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM or a range formed by any two values ​​within these ranges as endpoints, preferably about 15 mM, 20 mM or 25 mM.

[0188] Therefore, the pharmaceutical composition of the present invention may contain: a histidine buffer having a pH of about 5.0 to 6.5, the concentration of which in the pharmaceutical composition is about 10 to 30 mM; and about 10 to 100 mg / mL of the anti-DKK1 antibody or antigen-binding fragment thereof according to any of the above embodiments. Preferably, the amino acid sequence of the heavy chain variable region of the anti-DKK1 antibody or antigen-binding fragment thereof is as shown in SEQ ID NO: 7 or SEQ ID NO: 17, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 8 or SEQ ID NO: 18; more preferably, the heavy chain amino acid sequence of the anti-DKK1 antibody or antigen-binding fragment thereof is as shown in SEQ ID NO: 9 or SEQ ID NO: 19, and the light chain amino acid sequence is as shown in SEQ ID NO: 10 or SEQ ID NO: 20.

[0189] Therefore, the pharmaceutical composition of the present invention may contain: an acetate buffer having a pH of about 5.0 to 6.5, the concentration of which in the pharmaceutical composition is about 10 to 30 mM; and about 10 to 100 mg / mL of the anti-DKK1 antibody or antigen-binding fragment thereof according to any of the above embodiments. Preferably, the amino acid sequence of the heavy chain variable region of the anti-DKK1 antibody or antigen-binding fragment thereof is as shown in SEQ ID NO: 7 or SEQ ID NO: 17, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 8 or SEQ ID NO: 18; more preferably, the heavy chain amino acid sequence of the anti-DKK1 antibody or antigen-binding fragment thereof is as shown in SEQ ID NO: 9 or SEQ ID NO: 19, and the light chain amino acid sequence is as shown in SEQ ID NO: 10 or SEQ ID NO: 20.

[0190] In some embodiments, the pharmaceutical composition of the present invention further comprises a stabilizer, preferably, the stabilizer is selected from one or more of arginine, arginine salt, sodium chloride, mannitol, sorbitol, sucrose, glycine and trehalose. Preferably, the concentration of the stabilizer is about 10-400mM, preferably about 100-300mM, preferably about 120-280mM, preferably about 130-250mM or about 210-250mM. Non-limiting examples of the above-mentioned stabilizer concentration are about 100mM, 110mM, 120mM, 130mM, 140mM, 150mM, 160mM, 170mM, 180mM, 190mM, 200mM, 210mM, 220mM, 230mM, 240mM, 250mM or a range formed by any two values ​​in these ranges as endpoints, preferably about 220mM, 230mM, 240mM or 250mM.

[0191] In some embodiments, the stabilizer included in the pharmaceutical composition described herein is trehalose. In some embodiments, the stabilizer is trehalose at a concentration of about 100 to 300 mM, preferably about 120 to 280 mM, more preferably about 200 to 260 mM. Non-limiting examples of the above trehalose concentration are about 180 mM, 200 mM, 210 mM, 220 mM, 230 mM, 240 mM, 250 mM, 260 mM, 270 mM, 280 mM, preferably about 220 mM, 230 mM, 240 mM or 250 mM.

[0192] In some embodiments, the stabilizer included in the pharmaceutical composition described herein is sucrose. In some embodiments, the above stabilizer is sucrose at a concentration of about 100 to 300 mM, and the concentration of the above sucrose is preferably about 120 to 280 mM, more preferably 200 to 260 mM or 210 to 250 mM. Non-limiting examples of the above sucrose concentration are about 180 mM, 200 mM, 210 mM, 220 mM, 230 mM, 240 mM, 250 mM, 260 mM, 270 mM, 280 mM, preferably about 220 mM, 230 mM, 240 mM or 250 mM.

[0193] In some embodiments, the stabilizer contained in the pharmaceutical composition described herein is arginine or an arginine salt; preferably arginine hydrochloride. In some embodiments, the above-mentioned stabilizer is arginine or an arginine salt at a concentration of about 100 to 300 mM, preferably about 120 to 280 mM, and more preferably about 120 to 160 mM. Non-limiting examples of the above-mentioned arginine or arginine salt concentrations are about 120 mM, 125 mM, 130 mM, 135 mM, 140 mM, 145 mM, 150 mM, 155 mM, 160 mM, preferably about 135 mM, 140 mM, 145 mM, 150 mM or 155 mM.

[0194] In some embodiments, the stabilizer included in the pharmaceutical composition described herein is sodium chloride. In some embodiments, the above stabilizer is sodium chloride at a concentration of about 100-300mM, preferably about 100-200mM, more preferably about 120-180mM, and more preferably 130-150mM. Non-limiting examples of the above sodium chloride concentration are about 105mM, 110mM, 115mM, 120mM, 125mM, 130mM, 135mM, 140mM, 145mM, 150mM, 155mM, 160mM, 165mM, 170mM, 175mM, preferably about 135mM, 140mM, 145mM, 150mM or 155mM.

[0195] In some embodiments, the stabilizer included in the pharmaceutical composition described herein is mannitol. In some embodiments, the above-mentioned stabilizer is mannitol at a concentration of about 100-300mM, preferably about 200-300mM, preferably about 220-250mM. Non-limiting examples of the above-mentioned mannitol concentration are about 205mM, 210mM, 215mM, 220mM, 225mM, 230mM, 235mM, 240mM, 245mM, 250mM, 255mM, 260mM, 265mM, 270mM, 275mM, preferably about 235mM, 240mM, 245mM, 250mM or 255mM.

[0196] In some embodiments, the stabilizer included in the pharmaceutical composition described herein is sorbitol. In some embodiments, the above stabilizer is sorbitol at a concentration of about 100-300mM, preferably about 200-300mM, preferably about 220-250m. Non-limiting examples of the above mannitol concentration are about 205mM, 210mM, 215mM, 220mM, 225mM, 230mM, 235mM, 240mM, 245mM, 250mM, 255mM, 260mM, 265mM, 270mM, 275mM, preferably about 235mM, 240mM, 245mM, 250mM or 255mM.

[0197] In some embodiments, the stabilizer included in the pharmaceutical composition described herein is a combination of sodium chloride and mannitol. In some embodiments, the above stabilizer is a combination of about 20-200mM sodium chloride and about 20-200mM mannitol, preferably a combination of about 30-100mM sodium chloride and about 100-180mM mannitol, preferably a combination of about 20-80mM sodium chloride and about 100-180mM mannitol, and further preferably a combination of about 30-70mM sodium chloride and about 120-160mM mannitol. A non-limiting example of the above stabilizer is a combination of about 50mM sodium chloride and about 140mM, 145mM or 150mM mannitol.

[0198] In some embodiments, the stabilizer included in the pharmaceutical composition described herein is a combination of arginine hydrochloride and sucrose. In some embodiments, the above stabilizer is a combination of about 20-200mM arginine hydrochloride and about 20-200mM sucrose, preferably a combination of about 20-80mM arginine hydrochloride and about 100-180mM sucrose, preferably a combination of about 30-70mM arginine hydrochloride and about 100-160mM sucrose, more preferably a combination of about 30-70mM arginine hydrochloride and 120-160mM sucrose. A non-limiting example of the above stabilizer is a combination of about 50mM arginine hydrochloride and about 120mM, 125mM, 130mM or 135mM sucrose.

[0199] In some embodiments, the stabilizer included in the pharmaceutical composition described herein is a combination of arginine hydrochloride and glycine. In some embodiments, the above stabilizer is a combination of about 20-200mM arginine hydrochloride and about 20-200mM glycine, preferably a combination of about 20-80mM arginine hydrochloride and about 80-180mM glycine, more preferably about 20-80mM arginine hydrochloride and about 100-180mM glycine, more preferably about 30-70mM arginine hydrochloride and about 80-140mM glycine, and further preferably about 30-70mM arginine hydrochloride and about 100-120mM glycine. A non-limiting example of the above stabilizer is a combination of about 50mM arginine hydrochloride and about 100mM, 105mM, 110mM or 115mM glycine.

[0200] In some embodiments, the stabilizer included in the pharmaceutical composition described herein is a combination of sodium chloride and sucrose. In some embodiments, the above stabilizer is a combination of about 20-200mM sodium chloride and about 20-200mM sucrose, preferably a combination of about 20-100mM sodium chloride and about 100-180mM sucrose, preferably a combination of about 20-80mM sodium chloride and a sucrose concentration of about 100-180mM, more preferably a combination of about 30-70mM sodium chloride and about 120-160mM sucrose. A non-limiting example of the above stabilizer is a combination of about 50mM sodium chloride and about 120mM, 125mM, 130mM or 135mM sucrose.

[0201] In some embodiments, the stabilizer included in the pharmaceutical composition described herein is a combination of sodium chloride and trehalose. In some embodiments, the stabilizer is a combination of about 20-200mM sodium chloride and about 20-200mM trehalose, preferably a combination of about 20-80mM sodium chloride and about 100-180mM trehalose, preferably a combination of about 30-70mM sodium chloride and about 120-160mM trehalose. A non-limiting example of the stabilizer is a combination of about 50mM sodium chloride and about 120mM, 130mM or 140mM trehalose.

[0202] Therefore, the pharmaceutical composition of the present invention may contain: a histidine buffer having a pH of about 5.0 to 6.5, the concentration of which in the pharmaceutical composition is about 10 to 30 mM; about 10 to 100 mg / mL of the anti-DKK1 antibody or antigen-binding fragment thereof according to any of the above embodiments; and about 100 to 300 mM of a stabilizer, preferably, the stabilizer is selected from one or more of arginine, arginine salt, sodium chloride, mannitol, sorbitol, sucrose, glycine and trehalose. Preferably, the arginine salt is arginine hydrochloride. Preferably, the stabilizer is sucrose at a concentration of about 120-280 mM; or the stabilizer is trehalose at a concentration of about 120-280 mM; or the stabilizer is arginine hydrochloride at a concentration of about 120-280 mM; or the stabilizer is a combination of sodium chloride at a concentration of about 20-80 mM and mannitol at a concentration of about 100-180 mM; or the stabilizer is a combination of sodium chloride at a concentration of about 20-80 mM and sucrose at a concentration of about 100-180 mM; or the stabilizer is a combination of arginine hydrochloride at a concentration of about 20-80 mM and sucrose at a concentration of about 100-180 mM.

[0203] Therefore, the pharmaceutical composition of the present invention may contain: an acetate buffer having a pH of about 5.0 to 6.5, the concentration of which in the pharmaceutical composition is about 10 to 30 mM; about 10 to 100 mg / mL of the anti-DKK1 antibody or antigen-binding fragment thereof according to any of the above embodiments; and about 100 to 300 mM of a stabilizer, preferably, the stabilizer is selected from one or more of arginine, arginine salt, sodium chloride, mannitol, sorbitol, sucrose, glycine and trehalose. Preferably, the arginine salt is arginine hydrochloride. Preferably, the stabilizer is sucrose at a concentration of about 120-280 mM; or the stabilizer is trehalose at a concentration of about 120-280 mM; or the stabilizer is arginine hydrochloride at a concentration of about 120-280 mM; or the stabilizer is a combination of sodium chloride at a concentration of about 20-80 mM and mannitol at a concentration of about 100-180 mM; or the stabilizer is a combination of sodium chloride at a concentration of about 20-80 mM and sucrose at a concentration of about 100-180 mM; or the stabilizer is a combination of arginine hydrochloride at a concentration of about 20-80 mM and sucrose at a concentration of about 100-180 mM.

[0204] In some embodiments, the pharmaceutical composition further comprises a surfactant, preferably, the surfactant is selected from one or more of polysorbate 80, polysorbate 20 and poloxamer 188. Preferably, calculated by w / v, the concentration of the surfactant is about 0.001% to 0.1%, preferably about 0.005% to 0.1%, more preferably about 0.01% to 0.08%, more preferably about 0.01% to 0.04% or 0.02% to 0.06%. As a non-limiting example, the concentration of the surfactant is about 0.01%, 0.02%, 0.04% or 0.08%, preferably about 0.02%.

[0205] Therefore, the pharmaceutical composition of the present invention may contain: a histidine buffer having a pH of about 5.0 to 6.5, the concentration of which in the pharmaceutical composition is about 10 to 30 mM; about 10 to 100 mg / mL of the anti-DKK1 antibody or antigen-binding fragment thereof according to any of the above embodiments; about 100 to 300 mM of a stabilizer, preferably, the stabilizer is trehalose having a concentration of about 120 to 280 mM; or the stabilizer is sucrose having a concentration of about 120 to 280 mM; or the stabilizer is arginine hydrochloride having a concentration of about 120 to 280 mM; or the stabilizer is a combination of arginine hydrochloride having a concentration of about 20 to 80 mM and sucrose having a concentration of about 100 to 180 mM; or the stabilizer is a combination of sodium chloride having a concentration of about 20 to 80 mM and sucrose having a concentration of about 100 to 180 mM; and about 0.01% to 0.1% of polysorbate 80 on a w / v basis.

[0206] Therefore, the pharmaceutical composition of the present invention may contain: an acetate buffer having a pH of about 5.0 to 6.5, the concentration of which in the pharmaceutical composition is about 10 to 30 mM; about 10 to 100 mg / mL of the anti-DKK1 antibody or antigen-binding fragment thereof according to any of the above embodiments; about 100 to 300 mM of a stabilizer, preferably, the stabilizer is trehalose having a concentration of about 120 to 280 mM; or the stabilizer is sucrose having a concentration of about 120 to 280 mM; or the stabilizer is arginine hydrochloride having a concentration of about 120 to 280 mM; or the stabilizer is a combination of arginine hydrochloride having a concentration of about 20 to 80 mM and sucrose having a concentration of about 100 to 180 mM; or the stabilizer is a combination of sodium chloride having a concentration of about 20 to 80 mM and sucrose having a concentration of about 100 to 180 mM; and about 0.01% to 0.1% of polysorbate 80 on a w / v basis.

[0207] The osmotic pressure of the pharmaceutical composition of the present invention is in the range of 250 to 350 mOsm / kg, preferably in the range of 260 to 320 mOsm / kg, and more preferably in the range of 290 to 310 mOsm / kg.

[0208] The present invention provides a lyophilized preparation of the pharmaceutical composition of the present invention, and a reconstituted preparation obtained by reconstituted the lyophilized preparation using the components of the pharmaceutical composition of the present invention other than the antibody or its antigen-binding fragment. The reconstituted preparation can be further compounded with an injection excipient (such as a glucose solution or normal saline) conventionally used in the art to obtain a liquid preparation. In some embodiments, the liquid preparation is an injection.

[0209] Therefore, in some embodiments, the present invention provides a liquid preparation, which contains the pharmaceutical composition according to any embodiment, or contains a glucose solution or a sodium chloride solution and the reconstituted preparation according to any embodiment herein; preferably, the concentration of the sodium chloride solution is about 0.85-0.9% (w / v), and the concentration of the glucose solution is about 5-25% (w / v); preferably, in the liquid preparation, the concentration of the anti-DKK1 antibody is about 0.1-50 mg / mL, more preferably about 0.2-20 mg / mL; preferably, the pH of the liquid preparation is about 5.0-7.0.

[0210] Medical uses and methods

[0211] The present invention also provides use of the pharmaceutical composition or injection described in any embodiment herein in the preparation of a drug for treating a disease or condition by eliminating, inhibiting or reducing the activity of DKK1.

[0212] The present invention also provides the pharmaceutical composition or injection described in any one of the embodiments herein for use in a method for treating a disease or disorder by eliminating, inhibiting or reducing DKK1 activity.

[0213] The present invention also provides a method for treating a disease or disorder by eliminating, inhibiting or reducing DKK1 activity, which comprises administering the pharmaceutical composition or injection as described in any embodiment herein to a subject in need thereof.

[0214] In some embodiments, the above-mentioned disease or condition is selected from cancer, infectious disease or inflammatory disease. Preferably, the above-mentioned disease or condition is cancer.

[0215] As used herein, "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Included in this definition are benign and malignant cancers as well as dormant tumors or micrometastases. Examples of cancer include, but are not limited to, carcinomas, lymphomas, blastomas, sarcomas, and leukemias. More specific examples of such cancers include squamous cell carcinoma, lung cancer (including small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous cell carcinoma of the lung), peritoneal cancer, hepatocellular carcinoma, gastric cancer or stomach cancer (including gastrointestinal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer or renal cancer, nasopharyngeal cancer, esophageal squamous cell carcinoma, hepatoma, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, and various types of head and neck cancer, as well as B-cell lymphoma (including low-grade / follicular non-Hodgkin's lymphoma (NHL), small lymphocytic (SL) NHL, intermediate-grade / follicular NHL, intermediate-grade diffuse NHL, high-grade immunoblastic NHL, high-grade lymphoblastic NHL, high-grade small non-cleaved cell NHL, storage disease (bulky disease) NHL, mantle cell lymphoma, AIDS-related lymphoma, and Waldenstrom's macroglobulinemia), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, chronic myeloblastic leukemia, and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with phakomatoses, edema (such as that associated with brain tumors), and Meigs syndrome.

[0216] In some embodiments, the invention provides a pharmaceutical composition and one or more other therapies (e.g., treatment modalities and / or other therapeutic agents) administered in combination to a subject to treat a disease or condition. In some embodiments, the therapy includes surgical treatment and / or radiotherapy.

[0217] In some embodiments, the present invention provides a method of treating a disease or condition by administering to a subject a therapeutically effective amount of a pharmaceutical composition described in any embodiment of the present invention and one or more other therapeutic agents. In some embodiments, the other therapeutic agents include at least one of a chemotherapeutic agent, an immune checkpoint inhibitor, a tubulin inhibitor, and an anti-angiogenic agent. In some embodiments, the disease or condition is preferably cancer.

[0218] Example

[0219] The present invention will be described below in the form of specific examples. It should be understood that these examples are merely illustrative and are not intended to limit the scope of the present invention. The present invention has been described in detail herein, and specific embodiments thereof are also disclosed. For those skilled in the art, it will be obvious that various changes and improvements will be made to the specific embodiments of the present invention without departing from the spirit and scope of the present invention. Any modifications, equivalent substitutions, improvements, etc., should be included in the scope of protection of the present invention. The methods and materials used in the examples are conventional methods and materials in the art unless otherwise stated. A non-limiting anti-DKK1 antibody example used in the examples is JS015, which comprises a heavy chain with an amino acid sequence as shown in SEQ ID NO: 9, and a light chain with an amino acid sequence as shown in SEQ ID NO: 10.

[0220] In liquid pharmaceutical compositions, the buffer system and pH closely affect the stability of the antibody. Each antibody with unique physicochemical properties has the most suitable buffer type and pH. This example aims to preliminarily screen out the best buffer system and stabilizer so that the anti-DKK1 antibody disclosed in the present invention has the best stability for clinical application.

[0221] The present invention uses the following abbreviations:

[0222] hr means hours;

[0223] W stands for week;

[0224] M stands for month;

[0225] C represents the number of freeze-thaw cycles;

[0226] FT indicates freeze-thaw cycle;

[0227] RT means room temperature;

[0228] T0 represents the initial test of the prescription sample before the sample is placed;

[0229] Tagg indicates the temperature at which aggregation begins.

[0230] Example 1: Preliminary screening experiment of buffer system

[0231] 1.1 Experimental methods

[0232] First, the monoclonal antibody targeting DKK1 was replaced with a pH 6.0 histidine buffer system (FS1-7) using an ultrafiltration membrane, and then the protein was replaced with a different buffer system by dialysis. A 4× buffer containing a stabilizer was pre-prepared, and the protein was evenly distributed in the corresponding buffer by dilution. The final concentration was adjusted to about 40 mg / mL, and aseptically filled into 2R vials (2.0 ml / bottle) on a clean bench for stability sampling and testing. The preliminary screening plan is shown in Table 1 below.

[0233] The components of different drug compositions to be tested are shown in Table 1, wherein: acetate buffer refers to acetic acid-sodium acetate buffer; histidine buffer refers to L-histidine-L-histidine hydrochloride buffer; citric acid buffer refers to citric acid-sodium citrate buffer.

[0234] Table 1: Preliminary screening protocol for buffer systems

[0235]

[0236] Stability test conditions include:

[0237] (1) Placed in an environment at 5±3℃ for 2 weeks, 4 weeks or 3 months, hereinafter referred to as “long-term (5±3℃)”;

[0238] (2) Placed in an environment of 40±2℃ for 2W, 4W or 3M, hereinafter referred to as "high temperature (40±2℃)".

[0239] Test method: Take out 200μl of the sample and place it in a centrifuge tube and then centrifuge at 10000rpm for 5 minutes. Take 150μl of the supernatant and add it to a 96-well plate, centrifuge at 3000rpm for 5 minutes to remove the bubbles in the sample plate, and use a multi-hole plate direct reading dynamic light scattering instrument (DynaPro Plate Reader III) to perform Tagg value analysis.

[0240] Protein content test

[0241] Test method: Use NanoDrop one ultra-micro spectrophotometer to detect protein concentration, and set the percentage extinction coefficient (E1%) at 15.0 (g / 100ml) -1 cm -1 . Clean the detection wells with ultrapure water and use 3μl ultrapure water for blank correction. Each sample is measured twice in parallel. Before each measurement, the detection wells need to be cleaned with ultrapure water; read the protein concentration of each measurement (Note: The first round of prescription screening, i.e. FS1, uses this method for detection).

[0242] Appearance test

[0243] Test method: Visual inspection is used to inspect the appearance. Ensure that the light intensity of the clarity tester is maintained between 1000lux and 1500lux. Keep the sample at eye level and gently shake or invert it to avoid bubbles. Perform visual inspection in front of a black background and a white background. Record the results in terms of color, opalescence and visible foreign matter.

[0244] SEC-HPLC (Size Exclusion High Performance Liquid Chromatography) Purity Test

[0245] Test method: SEC purity is measured by installing a SEC column ( SEC 7.8mm×30cm,3.5μm) HPLC (Waters instrument) was used for detection. The mobile phase composition was 50mM phosphate, 200mM sodium chloride, pH 6.8±0.2. The peak area percentages of monomers, polymers and fragments were calculated by area normalization method. Detailed test parameters are shown in Table 2.

[0246] Table 2: SEC-HPLC purity test parameters

[0247]

[0248] icIEF purity test

[0249] Test method:

[0250] (1) Prepare according to the ratio shown in Table 3 (for one sample, this volume can be injected multiple times). If there are multiple samples, multiply the volume of each reagent by the corresponding multiple. Add the reagent to the centrifuge tube according to the calculated volume and vortex for at least 15 seconds to ensure complete mixing. Store at 2-8℃ and discard after use on the same day.

[0251] Table 3: icIEF Master Mix Preparation

[0252] Reagent name Required volume per sample (μl) 1% methylcellulose 35 Pharmalyte 3-10 4 pI Marker 5.85 0.5 pI Marker 9.46 0.5 500 mM arginine 2 <![CDATA[H 2 The]]> 38

[0253] (2) Dilute the sample (reference substance / test substance) to 1 mg / mL with ultrapure water.

[0254] (3) Take 20 μl of system suitability reference solution (1 mg / mL) and mix with 80 μl of premixed solution, centrifuge at 10,000 rpm for 5 min, take 75 μl of supernatant and add it to a 96-well plate, taking care to avoid bubbles, and then centrifuge the 96-well plate at 1,000 g for 5 min to remove bubbles.

[0255] (4) Detection was performed using a capillary electrophoresis instrument (Maurice).

[0256] rCE-SDS purity test

[0257] Test method: Dilute the sample / reference product to 4 mg / mL with 40mM phosphate buffer (pH6.5) for use; add 70μl 1% SDS, 40mM phosphate buffer, 5μl 2-mercaptoethanol, and 25μl diluted sample / reference product in a 1.5ml EP tube and mix well according to the final volume of 100μl; ultrapure water is used as a blank control, and the dilution ratio is consistent with the reference product. (Use 1% SDS, 40mM phosphate buffer to dilute the test product by at least 2 times). Centrifuge at 3000rpm at room temperature for 30sec, then incubate at 70℃ for 15min. After incubation, cool to room temperature and centrifuge at 12000rpm at room temperature for 5min. Take out 75μl of sample solution from the sample tube and add it to a 96-well plate to avoid bubbles, centrifuge at 1000g at room temperature for 10min, and detect using a capillary electrophoresis instrument (Maurice).

[0258] nrCE-SDS purity test

[0259] Test method: nrCE-SDS purity was tested using a capillary electrophoresis instrument (Maurice Instrument). Dilute the sample / reference product with 40mM phosphate buffer (pH6.5) to 4mg / mL for use; add 70μl 1% SDS, 40mM phosphate buffer, 5μl 0.25M NEM, and 25μl diluted sample / reference product to a 1.5ml EP tube and mix well according to the final volume of 100μl; use ultrapure water as a blank control, and keep the dilution ratio the same as the reference product. (Use 1% SDS, 40mM phosphate buffer to dilute the test product by at least 2 times). Centrifuge at 3000rpm at room temperature for 30s, then incubate at 70℃ for 5min. After incubation, cool to room temperature and centrifuge at 12000rpm at room temperature for 5min. 75 μl of sample solution was taken out from the sample tubes and placed in a 96-well plate to avoid bubbles. The plates were centrifuged at 1000 g for 10 min at room temperature and detected using a capillary electrophoresis instrument (Maurice).

[0260] Binding activity test

[0261] Test method: This method uses an indirect method, using recombinant human DKK1 protein (His tag) as an antigen coated on a 96-well plate. JS015 antibody can bind to recombinant human DKK1 protein (His tag), and mouse anti-human IgG4 Fc-HRP can specifically bind to JS015 antibody bound to the solid phase antigen (recombinant human DKK1 protein (His tag)). Under the action of hydrogen peroxide, it can catalyze TMB to develop blue. The depth of blue is positively correlated with the amount of mouse anti-human IgG4 Fc-HRP binding, and it turns yellow after termination with 2M hydrochloric acid. The absorbance (OD value) is measured at a wavelength of 450nm / 620nm using an enzyme marker, and the ability of JS015 antibody to bind to recombinant human DKK1 protein (His tag) is examined by the effective binding activity (EC50) of the standard curve. See Table 4 below for details.

[0262] Table 4: JS015 binding to DKK1 ELISA experimental method

[0263]

[0264] Cell viability test

[0265] Test method: DKK1 (Dickkopf-related protein 1) is a secretory protein and a member of the Dickkopf family. It antagonizes typical Wnt signals by inhibiting the interaction between LRP5 / 6 and Wnt and forming a ternary complex with the transmembrane protein KREMEN to promote the internalization of LRP5 / 6. DKK1 is highly expressed in a variety of tumors. It can upregulate the expression of VEGFR2 by activating the Akt signaling pathway, thereby promoting angiogenesis and providing nutrients for tumor survival and proliferation. On the other hand, it enhances the immunosuppressive ability of myeloid-derived suppressor cells (MDSC) by regulating the Wnt signaling pathway.

[0266] In this study, target cells 293TOP overexpressing LRP5 / 6 and luciferase reporter gene system and recombinant Wnt-3a (R&D systems, cat#5036-WN) and human DKK1 (Sino Biological, cat#10170-H08H) proteins were used to detect the biological activity of JS015 cells. Target cells 293TOP cells were seeded into 96-well flat-bottom white plates (corning, cat#3917) at 50,000 cells per well and cultured overnight. Then the supernatant was discarded and the test antibodies were added with gradient dilutions and pre-incubated at 37°C for 0.5h; then the Wnt-3a and human DKK1 proteins were added with diluted working concentrations of 0.2μg / ml and 0.1μg / ml, respectively, and incubated at 37°C for 6-8h. Finally, the luciferase substrate One-Lite (Vazyme, cat#DD1203-03-AB) was added and chemiluminescence was detected.

[0267] Insoluble particle test

[0268] Test method: HIAC insoluble particle counter was used as the detection instrument, and the insoluble particles in each pharmaceutical composition (sample) were statistically determined by the light obscuration method. The operation mode used was Run counter, and the setting parameters were shown in Table 5 below.

[0269] Table 5: Insoluble Particle Test Parameters

[0270] parameter Settings Number of runs 4 Dilution Factor 1 First run flush volume (mL) 0 Whether to discard the first run result yes

[0271] First, the sample was Millipore Pellicon3 0.11m 2 The membrane coating solution was replaced with a pH 6.0 His buffer system (FS1-7), and then the protein was replaced into a different buffer system by dialysis. A 4-fold concentration buffer containing a stabilizer was pre-prepared, and the protein was evenly distributed into the corresponding buffer by dilution. The final concentration was adjusted to about 40 mg / ml, and the protein was aseptically filled into 2R vials (2.0 ml / bottle) on a clean bench for stability sampling and testing.

[0272] 1.2 Experimental Results

[0273] 1.2.1 Thermal stability study

[0274] The overall aggregation start temperature (Tagg) of the histidine system (FS1-6, FS1-7, FS1-8) is higher than that of other prescriptions, and the aggregation start temperature of the FS1-3 prescription of the citric acid system is relatively low as a whole.

[0275] Table 6: First round of prescription screening - thermal stability study

[0276]

[0277]

[0278] 1.2.2 Protein content and appearance results

[0279] After 4 weeks of storage under high temperature and long-term conditions, the protein content of all samples did not change significantly, as shown in Table 7.

[0280] Table 7: First round of prescription screening - protein content test

[0281]

[0282] 1.2.3 Appearance Results

[0283] According to the appearance results in Table 8, the samples of the citric acid buffer system (FS1-3, FS1-4, FS1-5) showed strong opalescence under T0, high temperature and long-term conditions; after being placed under high temperature and long-term conditions for 4 weeks, FS1-7 of the histidine system produced a small amount of particles, and the appearance of the other prescription samples did not change significantly.

[0284] Table 8: First round of prescription screening - appearance test

[0285]

[0286] 1.2.4 SEC-HPLC purity results

[0287] According to the SEC-HPLC purity results in Table 9, after being placed under high temperature conditions for 4 weeks, the proportions of fragments and polymers of the samples used increased slightly, and the proportions of monomer purity decreased; among them, the degree of decrease in the purity of FS1-3 monomers in the citric acid system was relatively large. There was no significant change in the monomer purity of the samples of each prescription after being placed under long-term conditions for 4 weeks.

[0288] Table 9: First round of prescription screening - SEC-HPLC test

[0289]

[0290] 1.2.5 icIEF purity results

[0291] According to the icIEF purity results in Table 10, after being placed under high temperature conditions for 4 weeks, the main peak purity of all samples decreased significantly; the main peak purity of FS1-1, FS1-2 of the acetic acid system and FS1-6 of the histidine system decreased relatively less, and performed better; the main peak purity of FS1-3 of the citric acid system decreased most significantly, followed by FS1-4 of the citric acid system and FS1-8 of the histidine system. After being placed under long-term conditions for 4 weeks, the icIEF purity of each prescription sample did not change significantly.

[0292] Table 10: First round of prescription screening - icIEF test

[0293]

[0294]

[0295] 1.2.6 R-CE-SDS purity results

[0296] According to the rCE-SDS results in Table 11, the purity of all formulation samples decreased slightly after being placed under high temperature conditions for 4 weeks. The FS1-3 formulation of the citric acid system performed relatively poorly, with a significantly higher degree of decrease than other formulations. All formulation samples did not decrease significantly after being placed under long-term conditions for 4 weeks.

[0297] Table 11: First round of prescription screening—R-CE-SDS test

[0298]

[0299] 1.2.7 NR-CE-SDS purity results

[0300] According to the nrCE-SDS results in Table 12, the purity of all formulations decreased slightly after being placed under high temperature conditions for 4 weeks. The FS1-3 formulation of the citric acid system performed relatively poorly, with a significantly higher degree of decrease than other formulations. All samples showed no significant changes after being placed under long-term conditions for 4 weeks.

[0301] Table 12: First round of prescription screening—NR-CE-SDS test

[0302]

[0303] 1.2.8 Binding activity results

[0304] According to the test results of binding activity and cell biological activity in Table 13, the activities of all samples placed under high temperature and long-term conditions for 4 weeks had no significant changes and were all within a reasonable range.

[0305] Table 13: First round of formulation screening - combined activity test

[0306]

[0307] 1.2.9 Cell activity results

[0308] According to the test results of cell biological activity in Table 14, the activity of all samples placed under high temperature and long-term conditions for 4 weeks had no significant change and was within a reasonable range.

[0309] Table 14: First round of prescription screening - cell biology testing

[0310]

[0311] 1.2.10 Insoluble particle test

[0312] According to the sub-visible particle detection results in Table 15, the number of sub-visible particles in all prescriptions did not change significantly after being placed under high temperature and long-term conditions, and were all within a reasonable range.

[0313] Table 15: First round of prescription screening - sub-visible particle testing

[0314]

[0315] 1.3 Conclusion of the first round of prescription screening

[0316] Judging from the detection results of aggregation temperature data (Tagg), the initial aggregation temperature of the histidine buffer system (FS1-6, FS1-7, FS1-8) is higher than that of other formulations; judging from the appearance data, the samples of the citric acid buffer system (FS1-3, FS1-4, FS1-5) showed obvious opalescence, while the FS1-7 sample of the histidine system produced particles after being placed under high temperature and long-term conditions for 4 weeks; in the tests of SEC-HPLC, icIEF, r-CE-SDS and nr-CE-SDS, the proteins in the acetic acid system (FS1-1, FS1-2) and the FS1-6 sample of the histidine system were relatively stable compared with other formulations; the overall performance of the FS1-3 sample in the citric acid system was relatively poor.

[0317] In summary, acetate buffer and histidine buffer were selected, and a relatively low pH of 5.5 was adopted as the buffer system for the next step of prescription screening.

[0318] Example 2: Excipient screening experiment

[0319] In order to further explore the effects of different excipients on antibody stability, we selected sucrose and trehalose as stabilizers, and performed comparative tests using surfactants of different concentrations.

[0320] 2.1 Experimental procedures

[0321] Samples were prepared using Millipore Pellicon3 0.11m 2 The membrane was replaced with a 20 mM histidine buffer system by UF / DF and concentrated to about 50 mg / ml. The protein was then evenly distributed into the corresponding buffer (as shown in Table 16) and the final concentration was adjusted to about 40 mg / ml. The protein was aseptically filled into 2R vials of 1.0 ml / bottle and 2.0 ml / bottle on a clean bench for stability sampling and testing.

[0322] Table 16: Second round of prescription screening - excipient screening experimental plan

[0323]

[0324] 2.2 Experimental Results

[0325] 2.2.1 Appearance test

[0326] Test results: According to the appearance results in Table 17, there is no obvious change in appearance after being placed under high temperature and long-term conditions for 4 weeks.

[0327] Table 17: Second round of prescription screening - appearance test

[0328]

[0329] 2.2.2 SEC-HPLC purity results

[0330] According to the SEC-HPLC purity results in Table 18, after being placed under high temperature conditions for 4 weeks, the proportion of fragments and polymers of all samples increased slightly, and the proportion of monomer purity decreased. Among them, the degree of decrease of the prescriptions FS2-3 and FS2-4 of the histidine system was more significant; FS2-1 of the histidine system and FS2-6, FS2-7 and FS2-8 of the acetate system performed relatively well. After being placed under long-term conditions for 4 weeks, the monomer purity of all samples did not change significantly.

[0331] Table 18: Second round of prescription screening - SEC-HPLC test

[0332]

[0333] 2.2.3 icIEF purity results

[0334] According to the icIEF purity results in Table 19, after being placed under high temperature conditions for 4 weeks, the main peak purity of all samples decreased significantly; among them, the decrease in the histidine system prescriptions FS2-2 and FS2-4 was more significant; the histidine system FS2-5 and the acetic acid system FS2-6 and FS2-8 performed relatively well. After being placed under long-term conditions for 4 weeks, the icIEF purity of all samples did not change significantly.

[0335] Table 19: Second round of prescription screening - icIEF test

[0336]

[0337]

[0338] 2.2.4 R-CE-SDS purity results

[0339] According to the rCE-SDS results in Table 20, the purity of all formulations decreased slightly after being placed under high temperature conditions for 4 weeks, and there was no significant difference between the samples. All samples were placed under long-term conditions for 4 weeks, and there was no significant change in purity.

[0340] Table 20: Second round of prescription screening—R-CE-SDS test

[0341]

[0342] 2.2.5 NR-CE-SDS purity results

[0343] According to the nrCE-SDS results in Table 21, the purity of all formulations decreased slightly after being placed under high temperature conditions for 4 weeks, and the degree of decrease in the histidine system formulation FS2-4 was relatively high. All samples were placed under long-term conditions for 4 weeks, and the purity did not change significantly.

[0344] Table 21: Second round of prescription screening—NR-CE-SDS test

[0345]

[0346]

[0347] 2.2.6 Binding activity results

[0348] According to the binding activity test results in Table 22, after being placed under high temperature and long-term conditions for 4 weeks, the activity of all samples did not change significantly and was within a reasonable range.

[0349] Table 22: Second round of formulation screening - combined activity test

[0350]

[0351] 2.2.7 Cell activity results

[0352] According to the test results of cell biological activity in Table 23, the activity of all samples placed under high temperature and long-term conditions for 4 weeks had no significant change and was within a reasonable range.

[0353] Table 23: Second round of prescription screening - cell activity test

[0354]

[0355] 2.2.8 Insoluble particles results

[0356] According to the sub-visible particle test results in Table 24, the number of sub-visible particles in all prescriptions did not change significantly after being placed under high temperature and long-term conditions, and were all within a reasonable range.

[0357] Table 24: Second round of prescription screening - insoluble particulate test

[0358]

[0359] 2.3 Conclusion of the second round of prescription screening

[0360] From the appearance data of the samples, there was no significant difference among all the samples. The results of SEC-HPLC, icIEF, r-CE-SDS and nr-CE-SDS showed that the stability of the histidine system FS2-1, FS2-5 and the acetic acid system FS2-6, FS2-7 and FS2-8 was relatively good. There was no significant difference in the performance of all the samples in the binding activity, biological activity and sub-visible particle tests.

[0361] In summary, in the histidine system formulations (FS2-1 to FS2-5), the results of SEC-HPLC, icIEF, r-CE-SDS and nr-CE-SDS suggested that too high Tween-80 content may be detrimental to the stability of human DKK1 protein in the system; when the antibody concentration was 50 mg / mL and the surfactant was 0.04% TW80, the performance of 230 mM trehalose in the histidine system was better than that of 230 mM sucrose, and reducing the concentration of the surfactant to 0.02% could improve the stability of the sucrose system. In the acetic acid system formulations (FS2-6 to FS2-8), when the other conditions were the same, there was no significant difference between 230 mM trehalose and 230 mM sucrose. The effect of 40 mg / mL and 50 mg / mL antibody concentrations on stability was only slightly different in icIEF purity. There were no significant differences in biological activity, rCE-SDS and subvisible particles among all formulations.

[0362] Example 3: Prescription Confirmation

[0363] FS2-1 was further tested for performance confirmation using the optimized formulation according to Example 2. The non-limiting formulation used in this implementation method comprises 50 mg / mL anti-DKK1 antibody, 20 mM histidine buffer, 230 mM sucrose, 0.02% TW-80, and a pH of 5.5.

[0364] Table 25 below shows the prescription regimen.

[0365] Table 25: Experimental plan for formula performance confirmation

[0366]

[0367] The test results of the samples in the long-term test at 5°C are shown in Tables 26-36 below.

[0368] Table 26: Antibody concentration (unit: mg / mL)

[0369]

[0370] Table 27: Osmotic pressure (unit: mOsmol / kg)

[0371]

[0372] *NA means no data were detected under this condition.

[0373] Table 28: pH

[0374]

[0375] Table 29: Appearance

[0376]

[0377]

[0378] Table 30: SEC-HPLC (unit: %)

[0379]

[0380] Table 31: CEX-HPLC (unit: %)

[0381]

[0382] Table 32: R-CE-SDS (unit: %)

[0383]

[0384] Table 33: NR-CE-SDS (unit: %)

[0385]

[0386] Table 34: Binding activity

[0387]

[0388] Table 35: Cell viability

[0389]

[0390] Table 36: Insoluble particles (unit: particles / mL)

[0391]

[0392] According to the experimental results, when placed at 3M, 6M, and 30M for a long time (5±2℃), the antibody concentration and pH did not change significantly, the appearance change was within an acceptable range, the SEC-HPLC monomer and CEX-HPLC main peak content only slightly decreased, and R-CE-SDS, NR-CE-SDS, binding activity, cell activity, and insoluble particles did not change significantly. When placed at 30M for a long time (5±2℃), the antibody concentration did not change significantly.

[0393] The above results show that the formulation composition is 50 mg / mL anti-DKK1 antibody, 20 mM histidine buffer, 230 mM sucrose, 0.02% TW-80, and the pH is about 5.5, and the formulation has excellent stability performance.

[0394] Example 4: Antigen affinity determination of pharmaceutical compositions

[0395] 4.1 Experimental methods

[0396] This experiment used the T200 molecular interaction analyzer Biacore from GE Healthcare Life Sciences. Using surface plasmon resonance technology, 40 μg / mL sheep anti-human Fc fragment antibody (Jackson ImmunoResearch) was coupled to the surface of the CM5 chip, and then the JS015 antibody drug composition (1 μg / mL) was captured. Then, the gradient dilution of human DKK1 antigen protein was injected to detect the binding signal. The gradient dilution concentrations of human DKK1 antigen protein were 16nM, 8nM, 4nM, 2nM, 1nM and 0.5nM, of which 16nM was repeated. The affinity K was obtained by fitting using the kinetic model analysis of the Biacore analysis software Biacore T200 Evaluation Software 3.0. D value.

[0397] 4.2 Experimental Results

[0398] The experimental results are shown in Table 37 and Figure 1 As shown in FIG. 1 , it is shown that the pharmaceutical composition containing the anti-DKK1 antibody has a strong antigen affinity with the human DKK1 antigen protein, and its binding constant K D The value is 2.92E-10M.

[0399] Table 37: Summary of affinity between the pharmaceutical composition and human DKK1 antigen protein

[0400] Antigen name <![CDATA[k a (1 / Ms)]]> <![CDATA[k d (1 / s)]]> <![CDATA[K D (M)]]> Human DKK1 1.33E+06 3.89E-04 2.92E-10

[0401] Example 5: Antigen Binding Activity of Pharmaceutical Compositions

[0402] 5.1 Experimental Methods

[0403] This experiment uses a Thermo Scientific or Molecular Devices microplate reader. The plate is coated with 2.0 μg / mL human DKK1 (His tag), blocked with 2% BSA, and then gradiently diluted JS015 antibody and negative control antibody (starting at 3.0 μg / mL, 4-fold gradient dilution for a total of 12 concentrations) are added. Mouse Anti-Human IgG4 Fc-HRP is diluted 5000 times as the detection antibody for detection, and then 0.1 mg / mL TMB is used for color development. Finally, the reaction is terminated with 2M HCl and the plate is read at 450nm / 620nm. The four-parameter logarithmic regression (4PL) model is used to fit the EC 50 .

[0404] 5.2 Experimental Results

[0405] In this experiment, the binding of the pharmaceutical composition containing the anti-DKK1 antibody JS015 and the negative control antibody (hIgG4 isotype) to human DKK1 (His tag) was measured. The experimental results are shown in Tables 38 and Figure 2 As shown, the pharmaceutical composition specifically binds to human DKK1 (His tag), EC 50 The value was 2.6 ng / mL, and the negative control had no binding.

[0406] Table 38: Summary of ELISA binding between drug composition and recombinant human DKK1 protein

[0407]

[0408] Analysis of the binding curve shape characteristics shows that the binding curve of the drug composition to human DKK1 (His tag) is "S" shaped, with specific binding. 50 The value was 2.6 ng / mL. There was almost no binding signal between Anti-KLH hIgG4 and human DKK1 (His tag), so it did not bind to human DKK1 (His tag).

[0409] Example 6: Cellular activity of the pharmaceutical composition

[0410] 6.1 Experimental methods

[0411] The cellular biological activity of the drug composition was detected using target cells 293TOP cells overexpressing LRP5 / 6 and a luciferase reporter gene system, and recombinant mouse Wnt-1 / sFRP-1 (R&D systems, cat#9765-WN) and human DKK1 (Sino Biological, cat#10170-H08H) proteins. The target cell 293TOP cells were inoculated into a 96-well flat-bottom white plate (corning, cat#3917) at 50,000 cells per well and cultured overnight, then the supernatant was discarded and a serially diluted pharmaceutical composition containing the anti-DKK1 antibody JS015 (lot number: 01520211101) was added and pre-incubated at 37°C for 0.5h; after the pre-incubation, the diluted working concentrations of 0.2μg / ml and 0.2μg / ml of mouse Wnt-1 / sFRP-1 and human DKK1 proteins were added to the cells and antibodies, and incubated at 37°C for 6-8h. Finally, the luciferase substrate One-Lite (Vazyme, cat#DD1203-03-AB) was added and chemiluminescence was detected.

[0412] 6.2 Experimental Results

[0413] The TCF-Luc luciferase reporter gene system is one of the important ways to detect Wnt pathway transduction signals, which can reflect Wnt pathway dephosphorylation, response gene activation, and β-catenin nuclear translocation. This experiment used the luciferase reporter gene method to study the ability of the drug composition to bind to DKK1 and activate the transcriptional expression of TCF downstream of the Wnt pathway. The experimental data showed that the drug composition can specifically bind to DKK1 and effectively promote the activation of the transcription factor TCF downstream of the Wnt pathway. Its EC 50 The values ​​were 368.0ng / ml ( Figure 3 ).

Claims

1. A pharmaceutical composition comprising: (1) buffer; and (2) anti-DKK1 antibodies or antigen-binding fragments thereof; The anti-DKK1 antibody or antigen-binding fragment thereof comprises: HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, respectively, and LCDR1, LCDR2 and LCDR3 with amino acid sequences as shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, respectively; or The amino acid sequences of HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15, respectively, and the amino acid sequences of LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18, respectively. 2 . The pharmaceutical composition according to claim 1 , wherein the concentration of the anti-DKK1 antibody or antigen-binding fragment thereof is about 2 to 200 mg / mL, preferably about 10 to 100 mg / mL, and more preferably about 20 to 60 mg / mL.

3. The pharmaceutical composition according to claim 1, wherein the pH of the pharmaceutical composition is about 4.5 to 7.0, preferably about 5.0 to 6.5, more preferably about 5.0 to 6.0, and more preferably about 5.4 to 5.

6.

4. The pharmaceutical composition of claim 1, wherein the osmotic pressure of the pharmaceutical composition is in the range of 250 to 350 mOsm / kg.

5. The pharmaceutical composition according to claim 1, wherein: The buffer is selected from one or more of acetate buffer, histidine buffer, citrate buffer and phosphate buffer; preferably, the buffer is a histidine buffer, and the histidine buffer is selected from histidine-histidine hydrochloride buffer or histidine-histidine acetate buffer; and / or The concentration of the buffer is about 5-50 mM; more preferably, the concentration of the buffer is about 10-30 mM; more preferably, the concentration of the buffer is about 15-25 mM; preferably, the pH of the buffer is about 4.5-7.0, preferably about 5.0-6.5, more preferably about 5.0-6.0, more preferably about 5.4-5.

6.

6. The pharmaceutical composition according to any one of claims 1 to 5, wherein the pharmaceutical composition further comprises a stabilizer; Preferably, the stabilizer is selected from one or more of arginine, arginine salt, sodium chloride, mannitol, sorbitol, sucrose, glycine and trehalose; Preferably, the concentration of the stabilizer is about 50-300 mM, preferably about 120-280 mM, and more preferably about 130-250 mM.

7. The pharmaceutical composition according to claim 6, wherein the stabilizer is: (1) trehalose at a concentration of about 120 to 280 mM, preferably about 200 to 260 mM; or (2) Sucrose, at a concentration of about 120 to 280 mM, preferably, at a concentration of about 200 to 260 mM.

8. The pharmaceutical composition of claim 6, wherein the pharmaceutical composition further comprises a surfactant; Preferably, the surfactant is selected from one or more of polysorbate 80, polysorbate 20 and poloxamer 188; Preferably, the surfactant concentration is about 0.005% to 0.1% calculated on a w / v basis; more preferably, the surfactant concentration is about 0.01% to 0.08%; more preferably, the surfactant concentration is about 0.01% to 0.04%.

9. The pharmaceutical composition according to any one of claims 1 to 8, wherein The anti-DKK1 antibody or antigen-binding fragment thereof comprises: The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:7, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:8; or The heavy chain variable region with the amino acid sequence shown in SEQ ID NO: 11, and the light chain variable region with the amino acid sequence shown in SEQ ID NO: 8; or The heavy chain variable region of the amino acid sequence shown in SEQ ID NO: 19, and the light chain variable region of the amino acid sequence shown in SEQ ID NO: 20; or The heavy chain variable region of the amino acid sequence shown in SEQ ID NO: 19, and the light chain variable region of the amino acid sequence shown in SEQ ID NO: 21; or The heavy chain variable region has an amino acid sequence as shown in SEQ ID NO:22, and the light chain variable region has an amino acid sequence as shown in SEQ ID NO:

20.

10. The pharmaceutical composition according to claim 9, wherein The anti-DKK1 antibody or antigen-binding fragment thereof comprises A heavy chain having an amino acid sequence as shown in SEQ ID NO:9, and a light chain having an amino acid sequence as shown in SEQ ID NO:10; or A heavy chain having an amino acid sequence as shown in SEQ ID NO: 12, and a light chain having an amino acid sequence as shown in SEQ ID NO: 10; or A heavy chain having an amino acid sequence as shown in SEQ ID NO: 23, and a light chain having an amino acid sequence as shown in SEQ ID NO: 24; or A heavy chain having an amino acid sequence as shown in SEQ ID NO: 23, and a light chain having an amino acid sequence as shown in SEQ ID NO: 25; or The heavy chain has an amino acid sequence as shown in SEQ ID NO:26, and the light chain has an amino acid sequence as shown in SEQ ID NO:

25.

11. The pharmaceutical composition according to any one of claims 1 to 10, comprising the components as shown in any one of the following (1) to (12): (1) (a) about 10 to 100 mg / mL of the anti-DKK1 antibody or antigen-binding fragment thereof; (b) about 10 to 30 mM acetate buffer, pH 4.5 to 7.0; (c) about 120 to 280 mM sucrose; and (d) about 0.01% to 0.08% polysorbate 80; or (2) (a) about 10 to 100 mg / mL of the anti-DKK1 antibody or antigen-binding fragment thereof; (b) about 10 to 30 mM histidine buffer, pH about 4.5 to 7.0; (c) 120 to 280 mM sucrose; and (d) about 0.01% to 0.08% polysorbate 80; or (3) (a) about 10 to 100 mg / mL of the anti-DKK1 antibody or antigen-binding fragment thereof; (b) about 10 to 30 mM citric acid buffer, pH about 4.5 to 7.0; (c) 120 to 280 mM sucrose; and (d) about 0.01% to 0.08% polysorbate 80; or (4) (a) about 10 to 100 mg / mL of the anti-DKK1 antibody or antigen-binding fragment thereof; (b) about 10 to 30 mM histidine buffer, pH about 4.5 to 7.0; (c) 120 to 280 mM trehalose; and (d) about 0.01% to 0.08% polysorbate 80; or (5) (a) about 10 to 100 mg / mL of the anti-DKK1 antibody or antigen-binding fragment thereof; (b) about 10 to 30 mM acetate buffer, pH about 4.5 to 7.0; (c) 120 to 280 mM trehalose; and (d) about 0.01% to 0.08% polysorbate 80; or (6) (a) about 20 to 60 mg / mL of the anti-DKK1 antibody or antigen-binding fragment thereof; (b) about 15 to 25 mM histidine buffer, pH about 5.0 to 6.0; (c) 130 to 250 mM sucrose; and (d) about 0.01% to 0.08% polysorbate 80; or (7) (a) about 20 to 60 mg / mL of the anti-DKK1 antibody or antigen-binding fragment thereof; (b) about 15 to 25 mM histidine buffer, pH about 5.0 to 6.0; (c) 130 to 250 mM trehalose; and (d) about 0.01% to 0.08% polysorbate 80; or (8) (a) about 20 to 60 mg / mL of the anti-DKK1 antibody or antigen-binding fragment thereof; (b) about 15 to 25 mM acetate buffer, pH about 5.0 to 6.0; (c) about 130 to 250 mM sucrose; and (d) about 0.01% to 0.08% polysorbate 80; or (9) (a) about 20 to 60 mg / mL of the anti-DKK1 antibody or antigen-binding fragment thereof; (b) about 15 to 25 mM acetate buffer, pH about 5.0 to 6.0; (c) about 130 to 250 mM trehalose; and (d) about 0.01% to 0.08% polysorbate 80; or (10) (a) about 40 to 60 mg / mL of the anti-DKK1 antibody or antigen-binding fragment thereof; (b) about 18 to 23 mM histidine buffer, pH about 5.4 to 5.6; (c) about 210 to 240 mM sucrose; and (d) about 0.01% to 0.04% polysorbate 80; (11) (a) about 50 mg / mL of the anti-DKK1 antibody or antigen-binding fragment thereof; (b) about 20 mM histidine buffer, pH about 5.4-5.6; (c) about 230 mM sucrose; and (d) about 0.02% polysorbate 80; or (12) (a) about 50 mg / mL of the anti-DKK1 antibody or antigen-binding fragment thereof; (b) about 20 mM histidine buffer, pH about 5.4-5.6; (c) about 230 mM sucrose; and (d) about 0.04% polysorbate 80; or (13) (a) about 40 mg / mL of the anti-DKK1 antibody or antigen-binding fragment thereof; (b) about 20 mM histidine buffer, pH about 5.4-5.6; (c) about 230 mM sucrose; and (d) about 0.04% polysorbate 80; or (14) (a) about 50 mg / mL of the anti-DKK1 antibody or antigen-binding fragment thereof; (b) about 20 mM histidine buffer, pH about 5.4-5.6; (c) about 230 mM sucrose; and (d) about 0.08% polysorbate 80; or (15) (a) about 50 mg / mL of the anti-DKK1 antibody or antigen-binding fragment thereof; (b) about 20 mM histidine buffer, pH about 5.4-5.6; (c) about 230 mM trehalose; and (d) about 0.04% polysorbate 80; or (16) (a) about 50 mg / mL of the anti-DKK1 antibody or antigen-binding fragment thereof; (b) about 20 mM acetate buffer, pH about 5.4-5.6; (c) about 230 mM sucrose; and (d) about 0.04% polysorbate 80; or (17) (a) about 40 mg / mL of the anti-DKK1 antibody or antigen-binding fragment thereof; (b) about 20 mM acetate buffer, pH about 5.4-5.6; (c) about 230 mM sucrose; and (d) about 0.04% polysorbate 80; or (18) (a) about 50 mg / mL of the anti-DKK1 antibody or its antigen-binding fragment; (b) about 20 mM acetate buffer, pH about 5.4-5.6; (c) about 230 mM trehalose; and (d) about 0.04% polysorbate 80.

12. A lyophilized preparation of the pharmaceutical composition according to any one of claims 1 to 11.

13. A reconstituted preparation obtained by reconstitution of the lyophilized preparation according to claim 12.

14. A liquid preparation, characterized in that The liquid preparation contains the pharmaceutical composition according to any one of claims 1 to 11, or contains a glucose solution or a sodium chloride solution and the reconstituted preparation according to claim 13; preferably, the concentration of the sodium chloride solution is about 0.85-0.9% (w / v), and the concentration of the glucose solution is about 5-25% (w / v); preferably, in the liquid preparation, the concentration of the anti-DKK1 antibody is about 0.1-50 mg / mL, more preferably about 0.2-20 mg / mL; preferably, the pH of the liquid preparation is about 5.0-7.0; preferably, the liquid preparation is an injection.

15. Use of the pharmaceutical composition of any one of claims 1 to 11, the lyophilized preparation of claim 12, the reconstituted preparation of claim 13 or the liquid preparation of claim 14 in the preparation of a medicament for treating a disease or condition by eliminating, inhibiting or reducing the activity of DKK1; preferably, the disease or condition is selected from cancer, infectious disease or inflammatory disease; more preferably, the disease is cancer.

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