Stable pharmaceutical formulations comprising anti-GREMLIN1 antibodies

Abstract

The invention discloses a pharmaceutical preparation which comprises an anti-GREM1 antibody, a buffer solution, a stabilizer, a surfactant and, optionally, an antioxidant. The pharmaceutical formulation can still maintain the stability of the anti-GREM1 antibody after long-term storage, high temperature (e.g., 40 DEG C), room temperature, oscillation and / or multiple freeze thaws. The invention also discloses application of the pharmaceutical preparation in prevention and / or treatment of GREM1 related diseases, especially cancers (e.g., GREM1 related cancers), and a method for preparing the pharmaceutical preparation.

Description

Technical Field

[0001] The present application relates to a pharmaceutical preparation, in particular to a stable pharmaceutical preparation containing an anti-gremlin1 (GREM1) antibody. The present application also relates to a preparation method of the pharmaceutical preparation and use thereof. Background Art

[0002] Gremlin1 (GREM1) is a highly conserved secreted protein with a cysteine-rich region and a cysteine ​​knot (Wordinger et al., Exp Eye Res. 2008 Aug;87(2):78-79.). It is a member of the differential screening-selected gene aberration in neuroblastoma (DAN) family that acts as an antagonist of bone morphogenetic protein (BMP) (Wordinger et al., Exp Eye Res. 2008 Aug;87(2):78-79.). GREM1 can physically bind to BMP-2, BMP-4 or BMP-7 to form heterodimers and prevent BMP ligands from interacting with their corresponding BMP receptors, and then sequentially inhibit the activation of the BMP signaling pathway.

[0003] GREM1 is closely associated with fibrotic lesions in the kidney, lung, liver, and retina, as well as several tumor types, including pancreatic cancer, colon cancer, lung cancer, glioma, gastric cancer, and prostate cancer (Sneddon et al., Proceedings of the National Academy of Sciences of the United States of America (PNAS) October 2006; 103(40):14842-14847). For example, aberrant GREM1 upregulation confers tumorigenicity to colon cells outside the stem cell niche. Tumor stem cells have also been found to highly express and secrete GREM1 to maintain their stemness in gliomas (Yan, K. et al., Genes Dev 28, 1085-1100 (2014)). Therefore, GREM1 has been used as a therapeutic target in the treatment of GREM1-related diseases, for example, using anti-GREM1 antibodies.

[0004] Since antibody molecules have a complex multi-level protein structure and are prone to physically stick together, which may cause undesirable immune responses or may clog syringes or pumps during administration and be unsafe for patients, one of the problems that liquid formulations of antibodies have long faced is the stability problem caused by aggregation.

[0005] Therefore, there is a need for pharmaceutical formulations of anti-GREM1 antibodies with stability and consistent quality. SUMMARY OF THE INVENTION

[0007] The present application provides a stable pharmaceutical formulation comprising an anti-GREM1 antibody that remains homogeneous and stable after long-term storage, high temperature (eg, 40° C.), room temperature, shaking, and / or multiple freeze-thaw cycles.

[0008] In one aspect, the present application provides a pharmaceutical formulation comprising an anti-GREM1 antibody and a buffer having a pH value of 4.5-6.5.

[0009] In one aspect, the present application provides a pharmaceutical preparation comprising an anti-GREM1 antibody and a buffer, wherein the buffer is an acetate buffer or a histidine buffer, and the pH value is 5.5-6.5. In some embodiments, the concentration of the buffer in the pharmaceutical preparation is 5-50 mM, or 10-30 mM.

[0010] In some embodiments, the pharmaceutical formulation further comprises a stabilizer.

[0011] In some embodiments, the concentration of the stabilizer in the pharmaceutical formulation is 0.5-20% (w / v), or 0.5-10% (w / v).

[0012] In some embodiments, the stabilizer is selected from the group consisting of: sucrose, trehalose, sorbitol, NaCl, and arginine hydrochloride.

[0013] In some embodiments, the stabilizer is sucrose, and the concentration of sucrose in the pharmaceutical formulation is 1-10% (w / v). In other embodiments, the stabilizer is arginine hydrochloride, and the concentration of arginine hydrochloride in the pharmaceutical formulation is 1-5% (w / v). In other embodiments, the stabilizer is trehalose, and the concentration of trehalose in the pharmaceutical formulation is 5-10% (w / v). In other embodiments, the stabilizer is sorbitol, and the concentration of sorbitol in the pharmaceutical formulation is 2-8% (w / v). In other embodiments, the stabilizer is NaCl, and the concentration of NaCl in the pharmaceutical formulation is 0.5-1.5% (w / v).

[0014] In some embodiments, the pharmaceutical formulation further comprises a surfactant.

[0015] In some embodiments, the concentration of the surfactant in the pharmaceutical preparation is 0.005% (w / v) to 0.4% (w / v), or 0.01% (w / v) to 0.2% (w / v).

[0016] In some embodiments, the surfactant is selected from the group consisting of: polysorbate 80 and polysorbate 20.

[0017] In some embodiments, the surfactant is polysorbate 80, and the concentration of polysorbate 80 and / or polysorbate 20 in the pharmaceutical formulation is 0.025% (w / v)-0.1% (w / v).

[0018] In some embodiments, the pharmaceutical formulation further comprises an antioxidant.

[0019] In some embodiments, the concentration of the antioxidant in the pharmaceutical formulation is 0.01-0.2% (w / v), or 0.02-0.06% (w / v).

[0020] In some embodiments, the antioxidant is selected from the group consisting of: methionine, cysteine, glutathione, sodium thiosulfate, and ascorbic acid.

[0021] In some embodiments, the concentration of the anti-GREM1 antibody in the pharmaceutical preparation is 1 to 200 mg / ml.

[0022] In some embodiments, the concentration of the anti-GREM1 antibody in the pharmaceutical preparation is 20-40 mg / ml.

[0023] In some embodiments, the anti-GREM1 antibody comprises a heavy chain CDR1 (HCDR1) as shown in SEQ ID NO:1, a HCDR2 as shown in SEQ ID NO:2, and a HCDR3 as shown in SEQ ID NO:3, and / or a light chain CDR1 (LCDR1) as shown in SEQ ID NO:4, a LCDR2 as shown in SEQ ID NO:5, and a LCDR3 as shown in SEQ ID NO:6.

[0024] In some embodiments, the anti-GREM1 antibody comprises CDR1, CDR2, and CDR3 of the heavy chain variable region as shown in SEQ ID NO:7, and / or CDR1, CDR2, and CDR3 of the light chain variable region as shown in SEQ ID NO:8.

[0025] In some embodiments, the anti-GREM1 antibody comprises a heavy chain framework region 1 (HFR1) as shown in QX1QLVQSGSELKKPGASVKVSCKASGX2TFT (SEQ ID NO: 24), an HFR2 as shown in WMX3QAPGQGLX4WMG (SEQ ID NO: 25), an HFR3 as shown in RFX5FSLDTSVSTAYLQISSLKAEDTAVYYCAR (SEQ ID NO: 26), and an HFR4 as shown in WGQGTMVTVSS (SEQ ID NO: 17), and / or a light chain framework region 1 (LFR1) as shown in DVVMTQSPLSLPVTLGQPASISC (SEQ ID NO: 27), an LFR2 as shown in WLQQRPGQSPRRLIX6 (SEQ ID NO: 32), an LFR3 as shown in GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC (SEQ ID NO: 29), and an HFR4 as shown in FGQGTKLEIK (SEQ ID NO: 30). NO:30) shown in LFR4, wherein X1 is V or I; X2 is Y or S; X3 is R or K; X4 is E or T; X5 is V or A; X6 is Y or S.

[0026] In some embodiments, the anti-GREM1 antibody comprises a heavy chain framework region 1 (HFR1) as shown in SEQ ID NO: 14, 18 or 21, HFR2 as shown in SEQ ID NO: 15, 19 or 22, HFR3 as shown in SEQ ID NO: 16, 20 or 23, and HFR4 as shown in SEQ ID NO: 17, and / or a light chain framework region 1 (LFR1) as shown in SEQ ID NO: 27, LFR2 as shown in SEQ ID NO: 28 or 31, LFR3 as shown in SEQ ID NO: 29, and LFR4 as shown in SEQ ID NO: 30.

[0027] In some embodiments, the anti-GREM1 antibody comprises FR1, FR2, FR3 and FR4 of the heavy chain variable region as shown in SEQ ID NO:7, 11 or 12, and / or FR1, FR2, FR3 and FR4 of the light chain variable region as shown in SEQ ID NO:8 or 13.

[0028] In some embodiments, the anti-GREM1 antibody comprises a heavy chain variable region as shown in SEQ ID NO:7, 11 or 12, and / or a light chain variable region as shown in SEQ ID NO:8 or 13.

[0029] In some embodiments, the anti-GREM1 antibody comprises a heavy chain constant region as shown in SEQ ID NO:33, and / or a light chain constant region as shown in SEQ ID NO:34.

[0030] In some embodiments, the anti-GREM1 antibody comprises a heavy chain as shown in SEQ ID NO:9, and / or a light chain as shown in SEQ ID NO:10.

[0031] In some embodiments, the pharmaceutical preparation comprises an anti-GREM1 antibody, a buffer, a stabilizer, and a surfactant, wherein the buffer is a histidine buffer, the stabilizer is arginine hydrochloride or sucrose, the surfactant is polysorbate 80 or polysorbate 20, and the pH value is about 4.5-6.5.

[0032] In some embodiments, the concentration of the anti-GREM1 antibody in the pharmaceutical preparation is 20-40 mg / ml, the concentration of the histidine buffer in the pharmaceutical preparation is 10-30 mM, the concentration of the arginine hydrochloride or sucrose in the pharmaceutical preparation is 1-10% (w / v), and / or the concentration of the polysorbate 80 or polysorbate 20 in the pharmaceutical preparation is 0.025% (w / v) to 0.1% (w / v).

[0033] In some embodiments, the concentration of the anti-GREM1 antibody in the pharmaceutical formulation is about 33 mg / ml, the concentration of the histidine buffer in the pharmaceutical formulation is about 20 mM, the stabilizer is sucrose, and its concentration in the pharmaceutical formulation is about 8-9% (w / v), the surfactant is polysorbate 80, and its concentration in the pharmaceutical formulation is about 0.05% (w / v), and the pH value is about 5.5-6.5.

[0034] In some embodiments, the pharmaceutical formulation further comprises an antioxidant, wherein the antioxidant is methionine, and the concentration of methionine in the pharmaceutical formulation is about 0.04% (w / v).

[0035] In some embodiments, the concentration of the anti-GREM1 antibody in the pharmaceutical formulation is about 33 mg / ml, the concentration of the histidine buffer in the pharmaceutical formulation is about 20 mM, the stabilizer is sucrose, and its concentration in the pharmaceutical formulation is about 8.2% (w / v), the surfactant is polysorbate 80, and its concentration in the pharmaceutical formulation is about 0.05% (w / v), the concentration of methionine in the pharmaceutical formulation is about 0.04% (w / v), and the pH value is about 6.0.

[0036] In another aspect, the present application provides use of the pharmaceutical formulation described herein in the preparation of a medicament for preventing and / or treating a GREM1-related disease.

[0037] In some embodiments, the GREM1-associated disease is selected from the group consisting of cancer, fibrotic disease, angiogenesis, glaucoma or retinal disease, kidney disease, pulmonary hypertension or osteoarthritis (OA), or the GREM1-associated disease is associated with an increased level of GREM1 selected from the group consisting of scleroderma, idiopathic pulmonary fibrosis, diabetic nephropathy, IgAN, lupus nephritis, Alport syndrome, glioma, head and neck cancer, prostate cancer, lung cancer, gastric cancer, pancreatic cancer, esophageal cancer, bladder cancer, breast cancer and colorectal cancer.

[0038] In some embodiments, the drug further comprises a second therapeutic agent, which is selected from chemotherapy drugs (such as cisplatin), radiotherapy, immunotherapy drugs (such as immune checkpoint regulators, such as PD-1 / PD-L1 axis inhibitors, TGF-β inhibitors), anti-angiogenic drugs (such as antagonists of VEGFR-1, VEGFR-2 and VEGFR-3), targeted therapy drugs, cell therapy drugs, gene therapy drugs, hormone therapy drugs, cytokines, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 The stability data of the protein measured by SEC in the pH screening experiment using three different buffer systems of acetic acid, histidine and citric acid are shown.

[0040] Figure 2 The stability data of the protein measured by CEX in pH screening experiments using three different buffer systems: acetic acid, histidine, and citric acid.

[0041] Figure 3 The protein stability data measured by SEC in the excipient screening experiment in which NaCl, arginine hydrochloride, sucrose, sorbitol, trehalose and mannitol were selected as stabilizers were shown.

[0042] Figure 4 The data show the protein stability data measured by CEX in a histidine-histidine hydrochloride buffer system under different pH conditions, in which arginine hydrochloride and sucrose are selected as stabilizers and methionine is used as an auxiliary material screening experiment for an antioxidant.

[0043] Figure 5 The data show the protein stability data measured by CEX in a histidine-histidine hydrochloride buffer system under different pH conditions, in which arginine hydrochloride and sucrose are selected as stabilizers and methionine is used as an auxiliary material screening experiment for an antioxidant.

[0044] Figure 6 The data show the protein stability data measured by SEC in a histidine-histidine hydrochloride buffer system under different pH conditions, in which arginine hydrochloride and sucrose are selected as stabilizers and methionine is used as an auxiliary material screening experiment for an antioxidant.

[0045] Figure 7 The data show the protein stability data measured by NR CE-SDS in a histidine-histidine hydrochloride buffer system under different pH conditions, in which arginine hydrochloride and sucrose are selected as stabilizers and methionine is used as an auxiliary material screening experiment for an antioxidant.

[0046] Figure 8 The protein stability data of the target preparation measured by SEC under long-term 5°C experimental conditions are shown.

[0047] Fig. 9 The protein stability data of the target preparation measured by NR CE-SDS under long-term 5°C experimental conditions are shown.

[0048] Fig.10 The protein stability data of the target preparation measured by CEX under long-term 5°C experimental conditions are shown.

[0049] Fig.11 The protein stability data of the target preparation measured by SEC under accelerated experimental conditions at 25°C are shown.

[0050] Fig.12 The protein stability data of the target preparation measured by NR CE-SDS under accelerated experimental conditions at 25°C are shown.

[0051] Fig.13 The protein stability data of the target formulation measured by CEX under accelerated experimental conditions at 25°C are shown.

[0052] Fig.14 The protein stability data of the target preparation measured by CEX under the experimental condition of 40°C are shown. DETAILED DESCRIPTION

[0053] The following description of the application is only intended to illustrate each embodiment of the application. The specific embodiments described should not be construed as limiting the scope of the application. Without departing from the spirit and essence of the application, those skilled in the art may perform various equivalent substitutions, changes or variations, and should be understood that these equivalent embodiments are also included herein. All documents cited herein, including publications, patents and patent applications, are incorporated herein by reference in their entirety. When the method mentioned in the present disclosure includes the situation of two or more limiting steps, the defined steps may be performed in any order or simultaneously (unless the context excludes this possibility), and the method may include one or more other steps, and the step may be performed before any defined step, between two defined steps or after all defined steps (unless the context excludes this possibility).

[0054] References to "about" (a certain value or a certain numerical range) herein include embodiments for the value or the numerical range itself, for example, "about X" includes "X". In general, the term "about" refers to the numerical value of a certain variable and all numerical values ​​within the experimental error range of the variable (for example, within the 95% confidence interval of the mean value) or all numerical values ​​within 10% of the variable, whichever is greater. For example, "about X" includes "110% × X", "109% × X", "108% × X", "107% × X", "106% × X", "105% × X", "104% × X", "103% × X", "102% × X", "101% × X", "99% × X", "98% × X", "97% × X", "96% × X", "95% × X", "94% × X", "93% × X", "92% × X", "91% × X" or "90% × X".

[0055] The term "at least" followed by a number is used herein to indicate the beginning of a range that begins with that number (which can be a range with an upper limit or without an upper limit, depending on the variables defined). For example, "at least 1" means 1 or greater than 1. The term "at most" followed by a number is used herein to indicate the end of a range that ends with that number (which can be a range with 1 or 0 as a lower limit, or a range with no lower limit, depending on the variables defined). For example, "at most 4" means 4 or less than 4, and "at most 40%" means 40% or less than 40%. In the present disclosure, when a range is set to "(first number) to (second number)" or "(first number) to (second number)", this means that the lower limit of the range is the first number and the upper limit is the second number. For example, It refers to a range with a lower limit of 5 mg / mL and an upper limit of 50 mg / mL. The term "less than" or "greater than" a certain value includes the value in the present invention.

[0056] As used herein, the term "benchmark antibody" refers to any existing anti-GREM1 antibody (such as 6245P), which is generated according to the sequence of H4H6245P disclosed in WO2014159010, the disclosure of which is incorporated herein by reference in its entirety.

[0057] Antibody

[0058] As used herein, the term "antibody" includes any immunoglobulin, monoclonal antibody, polyclonal antibody, multivalent antibody, bivalent antibody, monovalent antibody, multispecific antibody or bispecific antibody that binds to a specific antigen. A complete antibody comprises two heavy chains and two light chains. Each heavy chain consists of a heavy chain variable region (V H ) and the first, second and third heavy chain constant regions (C H1 , C H2 , C H3 ), and each light chain consists of a light chain variable region (V L ) and the light chain constant region (C L ) is composed of heavy and light chains. H and V L There are also three complementary determining regions (CDRs) in each region, and the three CDRs are separated by a continuous portion called a framework region (FR), which is more highly conserved than the CDR and forms a scaffold to support the hypervariable loop. The six CDRs of a heavy chain and a light chain together constitute the antigen binding site of the antibody, which determines the specificity of the antibody. The antibodies described herein also include fragments or derivatives with antigen binding function of the complete antibody, and the fragments or derivatives have the same antigen binding specificity as the complete antibody, but the affinity of the fragments or derivatives to their specific antigens may be the same or different from that of the complete antibody.

[0059] In some embodiments, the antibodies described herein include antigen-binding fragments. Antigen-binding fragments refer to one or more antibody fragments that retain the ability to specifically bind to an antigen. Examples of antigen-binding fragments include, but are not limited to (i) Fab fragments, which refer to fragments consisting of V L 、V H , C L and C H1 (ii) a Fab' fragment, which refers to a Fab fragment containing a portion of the hinge region; (iii) a F(ab')2 fragment, which refers to a bivalent fragment containing two Fab fragments connected by a disulfide bond in the hinge region; (iv) a fragment consisting of V H and C H1 The Fd fragment consists of a single-arm V L and V H(vi) dAb fragments (Ward et al., Nature 341: 544-546 (1989); PCT Publication WO 90 / 05144), which contain a single variable domain; (vii) isolated CDRs; (viii) single-chain Fv fragments, which refer to V L and V H The domains are directly linked or connected through a peptide chain to form a monovalent fragment (Huston JS et al., Proc Natl Acad Sci USA, 85: 5879 (1988)).

[0060] In some embodiments, the antibodies described herein include chimeric antibodies, a portion of which the heavy and / or light chains are identical or homologous to the corresponding sequences of antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remaining chains are identical or homologous to the corresponding sequences of antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, as long as they exhibit the desired functional activity.

[0061] In some embodiments, the antibodies described herein include humanized antibodies. Humanized forms of non-human (e.g., mouse) antibodies are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (e.g., Fv, Fab, Fab', F(ab')2, or other antigen-binding sequences of antibodies) that contain minimal sequences obtained from non-human immunoglobulins. In some examples, humanized antibodies can be CDR-grafted antibodies, in which the amino acid sequences of human CDRs are introduced into non-human V H and V L The amino acid sequence of a humanized antibody is replaced by an amino acid sequence of a corresponding non-human CDR. In other examples, most of the amino acid sequence of a humanized antibody can be derived from a human immunoglobulin (receptor antibody), wherein the amino acid residues of the CDR of the receptor are replaced by amino acid residues of the CDR of a non-human (e.g., mouse, rat, rabbit) antibody having the desired specificity, affinity, and ability. Usually, a humanized antibody substantially comprises at least one, generally comprising two variable domains, wherein all or substantially all of the CDR regions correspond to the sequence of a non-human immunoglobulin, and all or substantially all of the framework (FR) regions are sequences of human immunoglobulins. In some examples, the framework region residues of the variable region of a human immunoglobulin are replaced by corresponding non-human residues. Moreover, a humanized antibody may include residues that are not present in both the original antibody and the introduced CDR or framework region sequences.

[0062] The anti-GREM1 antibody described herein refers to an antibody that can specifically bind to gremlin1 (GREM1) protein, such as humanized 14E3 (hzd 14E3), such as Hu14E3_HaLa, Hu14E3_HaLb, Hu14E3_HbLa, Hu14E3_HbLb, Hu14E3_HcLa and Hu14E3_HcLb.

[0063] "Hu14E3_HaLa" described herein refers to humanized 14E3, which comprises a heavy chain variable region having amino acids as shown in SEQ ID NO: 7 and a light chain variable region having amino acids as shown in SEQ ID NO: 8. In some embodiments, "Hu14E3_HaLa" described herein further comprises a heavy chain constant region having amino acids as shown in SEQ ID NO: 33 and a light chain constant region having amino acids as shown in SEQ ID NO: 34. In some embodiments, "Hu14E3_HaLa" described herein comprises a heavy chain having amino acids as shown in SEQ ID NO: 9 and a light chain having amino acids as shown in SEQ ID NO: 10.

[0064] "Hu14E3_HaLb" described herein refers to humanized 14E3, which comprises a heavy chain variable region having amino acids as shown in SEQ ID NO: 7 and a light chain variable region having amino acids as shown in SEQ ID NO: 13. In some embodiments, "Hu14E3_HaLb" described herein further comprises a heavy chain constant region having amino acids as shown in SEQ ID NO: 33 and a light chain constant region having amino acids as shown in SEQ ID NO: 34.

[0065] "Hu14E3_HbLa" described herein refers to humanized 14E3, which comprises a heavy chain variable region having amino acids as shown in SEQ ID NO: 11 and a light chain variable region having amino acids as shown in SEQ ID NO: 8. In some embodiments, "Hu14E3_HbLa" described herein further comprises a heavy chain constant region having amino acids as shown in SEQ ID NO: 33 and a light chain constant region having amino acids as shown in SEQ ID NO: 34.

[0066] "Hu14E3_HbLb" described herein refers to humanized 14E3, which comprises a heavy chain variable region having amino acids as shown in SEQ ID NO: 11 and a light chain variable region having amino acids as shown in SEQ ID NO: 13. In some embodiments, "Hu14E3_HbLb" described herein further comprises a heavy chain constant region having amino acids as shown in SEQ ID NO: 33 and a light chain constant region having amino acids as shown in SEQ ID NO: 34.

[0067] "Hu14E3_HcLa" described herein refers to humanized 14E3, which comprises a heavy chain variable region having amino acids as shown in SEQ ID NO: 12 and a light chain variable region having amino acids as shown in SEQ ID NO: 8. In some embodiments, "Hu14E3_HcLa" described herein further comprises a heavy chain constant region having amino acids as shown in SEQ ID NO: 33 and a light chain constant region having amino acids as shown in SEQ ID NO: 34.

[0068] "Hu14E3_HcLb" described herein refers to humanized 14E3, which comprises a heavy chain variable region having amino acids as shown in SEQ ID NO: 12 and a light chain variable region having amino acids as shown in SEQ ID NO: 13. In some embodiments, "Hu14E3_HcLb" described herein further comprises a heavy chain constant region having amino acids as shown in SEQ ID NO: 33 and a light chain constant region having amino acids as shown in SEQ ID NO: 34.

[0069] The term "gremlin1" or "GREM1" refers to variant 1 of gremlin, and encompasses gremlin1 in different species, such as humans, mice, monkeys, etc. GREM1 is evolutionarily conserved and the human gremlin1 gene (hGREM1) has been mapped to chromosome 15q13-q15 (Topol LZ et al., (1997) Mol. Cell Biol., 17:4801-4810; Topol LZ et al., Cytogenet Cell Genet., 89:79-84). The amino acid sequence of hGREM1 can be accessed through the GenBank database with accession number NP-037504 or the Uniprot database via accession number O60565. The term "human gremlin1" and the term "hGREM1" are used interchangeably in the present disclosure.

[0070] In some embodiments, the anti-GREM1 antibodies described herein include a heavy chain variable region V H , the heavy chain variable region V H It has a CDR1 as shown in SEQ ID NO:1, a CDR2 as shown in SEQ ID NO:2, and / or a CDR3 as shown in SEQ ID NO:3.

[0071] In some embodiments, the anti-GREM1 antibodies described herein include a light chain variable region V L , the light chain variable region V LIt has a CDR1 as shown in SEQ ID NO:4, a CDR2 as shown in SEQ ID NO:5, and / or a CDR3 as shown in SEQ ID NO:6.

[0072] In some embodiments, the anti-GREM1 antibodies described herein include a heavy chain variable region V H and light chain variable region V L , the heavy chain variable region V H comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO: 1, a CDR2 having an amino acid sequence as shown in SEQ ID NO: 2, and / or a CDR3 having an amino acid sequence as shown in SEQ ID NO: 3, wherein the light chain variable region V L comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO:4, a CDR2 having an amino acid sequence as shown in SEQ ID NO:5, and / or a CDR3 having an amino acid sequence as shown in SEQ ID NO:6. In some embodiments, the anti-GREM1 antibody comprises a heavy chain CDR1 (HCDR1) as shown in SEQ ID NO:1, a HCDR2 as shown in SEQ ID NO:2, and a HCDR3 as shown in SEQ ID NO:3, and a light chain CDR1 (LCDR1) as shown in SEQ ID NO:4, a LCDR2 as shown in SEQ ID NO:5, and a LCDR3 as shown in SEQ ID NO:6. In some embodiments, the anti-GREM1 antibody comprises CDR1, CDR2, and CDR3 of the heavy chain variable region as shown in SEQ ID NO:7, and CDR1, CDR2, and CDR3 of the light chain variable region as shown in SEQ ID NO:8.

[0073] In some embodiments, the anti-GREM1 antibodies described herein comprise a heavy chain framework region 1 (HFR1) as set forth in QX1QLVQSGSELKKPGASVKVSCKASGX2TFT (SEQ ID NO: 24), an HFR2 as set forth in WMX3QAPGQGLX4WMG (SEQ ID NO: 25), an HFR3 as set forth in RFX5FSLDTSVSTAYLQISSLKAEDTAVYYCAR (SEQ ID NO: 26), and an HFR4 as set forth in WGQGTMVTVSS (SEQ ID NO: 17), and a light chain framework region 1 (LFR1) as set forth in DVVMTQSPLSLPVTLGQPASISC (SEQ ID NO: 27), an LFR2 as set forth in WLQQRPGQSPRRLIX6 (SEQ ID NO: 32), an LFR3 as set forth in GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC (SEQ ID NO: 29), and an HFR4 as set forth in FGQGTKLEIK (SEQ ID NO: 30). NO:30) shown in LFR4, wherein X1 is V or I; X2 is Y or S; X3 is R or K; X4 is E or T; X5 is V or A; X6 is Y or S.

[0074] In some embodiments, the anti-GREM1 antibodies described herein comprise a heavy chain framework region 1 (HFR1) as set forth in SEQ ID NO: 14, 18 or 21, an HFR2 as set forth in SEQ ID NO: 15, 19 or 22, an HFR3 as set forth in SEQ ID NO: 16, 20 or 23, and an HFR4 as set forth in SEQ ID NO: 17, and a light chain framework region 1 (LFR1) as set forth in SEQ ID NO: 27, an LFR2 as set forth in SEQ ID NO: 28 or 31, an LFR3 as set forth in SEQ ID NO: 29, and an LFR4 as set forth in SEQ ID NO: 30. In some embodiments, the anti-GREM1 antibodies comprise FR1, FR2, and FR3 of the heavy chain variable region as set forth in SEQ ID NO: 7, 11 or 12, and FR1, FR2, and FR3 of the light chain variable region as set forth in SEQ ID NO: 8 or 13.

[0075] In some embodiments, the anti-GREM1 antibodies described herein include a heavy chain variable region V having an amino acid sequence as shown in SEQ ID NO: 7, 11 or 12. H In some embodiments, the anti-GREM1 antibodies described herein include a light chain variable region V having an amino acid sequence as shown in SEQ ID NO: 8 or 13. LIn some embodiments, the anti-GREM1 antibodies described herein include a heavy chain variable region V having an amino acid sequence as shown in SEQ ID NO: 7, 11 or 12. H and a light chain variable region V having an amino acid sequence as shown in SEQ ID NO: 8 or 13 L In some embodiments, the anti-GREM1 antibodies described herein comprise a heavy chain variable region as shown in SEQ ID NO:7, 11 or 12 and a light chain variable region as shown in SEQ ID NO:8 or 13.

[0076] In some embodiments, the anti-GREM1 antibodies described herein further include an immunoglobulin constant region. In some embodiments, the immunoglobulin constant region includes a heavy chain and / or a light chain constant region. The heavy chain constant region includes C H1 , C H1 -C H2 or C H1 -C H3 region, the light chain constant region comprising C L In some embodiments, the anti-GREM1 antibodies described herein comprise a heavy chain constant region as shown in SEQ ID NO:33 and a light chain constant region as shown in SEQ ID NO:34.

[0077] In some embodiments, the anti-GREM1 antibody described herein 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. In some embodiments, the anti-GREM1 antibody comprises a heavy chain as shown in SEQ ID NO: 9 and a light chain as shown in SEQ ID NO: 10.

[0078] Exemplary amino acid sequences in some embodiments are listed in Table A below:

[0079] Table A Exemplary amino acid sequences

[0080]

[0081]

[0082]

[0083] Table B Exemplary hzd 14E3 (hu14E3) framework region (FR) amino acid sequences

[0084]

[0085]

[0086] Wherein X1 is V or I; X2 is Y or S; X3 is R or K; X4 is E or T; X5 is V or A; X6 is Y or S.

[0087] The present application relates to a pharmaceutical preparation comprising an anti-GREM1 antibody (e.g., an anti-GREM1 antibody described herein). In some embodiments, the concentration of the anti-GREM1 antibody in the pharmaceutical preparation of the present application may be 1-200 mg / ml, 1-190 mg / ml, 10-190 mg / ml, 20-180 mg / ml, 20-170 mg / ml, 20-160 mg / ml, 20-150 mg / ml, 20-140 mg / ml, 20-130 mg / ml, 20-120 mg / ml, 20-110 mg / ml, 20-100 mg / ml, 20-90 mg / ml, 20-80 mg / ml, 20-70 mg / ml, 20-60 mg / ml, 20-50 mg / ml, 20-40 mg / ml, 20-30 mg / ml. In some embodiments, the concentration of the anti-GREM1 antibody is any concentration value within the above range. For example, as desired, the concentration of the anti-GREM1 antibody in the pharmaceutical preparation may be at least 5 mg / ml, at least 10 mg / ml, at least 20 mg / ml, at least 30 mg / ml, at least 40 mg / ml, at least 50 mg / ml, at least 60 mg / ml, at least 70 mg / ml, at least 80 mg / ml, at least 90 mg / ml, at least 100 mg / ml, at least 110 mg / ml, at least 120 mg / ml, at least 130 mg / ml, at least 140 mg / ml, at least 150 mg / ml, at least 160 mg / ml, at least 170 mg / ml, at least 180 mg / ml, at least 190 mg / ml and / or up to 200 mg / ml. In some embodiments, the concentration of the anti-GREM1 antibody in the pharmaceutical preparation of the present application is 21 mg / ml. In some embodiments, the concentration of the anti-GREM1 antibody in the pharmaceutical preparation of the present application is 33 mg / ml.

[0088] Buffer

[0089] The term "buffer" generally refers to a buffer solution that tolerates changes in pH through the action of its acid-base conjugate components. "Buffer" as used herein refers to a compound solution that is known to be safely used in pharmaceutical preparations and has the ability to maintain or control the pH of the preparation within a desired range. Acceptable buffers that can control the pH in the range of mildly acidic pH to mildly alkaline pH (e.g., pH 4.5 to 8.0) include, but are not limited to, one or a combination of any of succinate buffer, citrate buffer, phosphate buffer, acetate buffer, arginine buffer, 2-amino-2-hydroxymethyl-1,3-propanediol (TRIS) buffer, histidine buffer, etc.

[0090] The buffer in the formulation solvent can be prepared using any appropriate method known in the art. In some embodiments, specific acidic and alkaline ion pairs can be used to prepare the buffer described in the present application. In one example, the excipients of the acidic and alkaline ion pairs can be accurately weighed, and pure water of about 60% of the target buffer volume can be added. After mixing evenly, the pH of the solution is measured. If the pH value deviates from the target value, the corresponding ion pair can be used to adjust the pH. The solution is then diluted with pure water to the target weight or target volume. Finally, the conductivity, osmotic pressure and pH of the solution are measured for verification.

[0091] The stable pharmaceutical formulation of the present application may include a buffer solution that allows the pharmaceutical formulation to have a pH value of 4.5 to 8.0, such as a buffer solution that allows the pharmaceutical formulation to have a pH value of 4.5 to 6.0, 4.5 to 6.5, 5.7 to 6.3, 6.0 to 7.0, or 7.0 to 8.0. In some embodiments, a suitable buffer solution allows the pharmaceutical formulation of the present application to have a pH value of 4.5 to 6.5. In particular, the pH value of the pharmaceutical formulation of the present application can be any pH value within the pH ranges listed above, for example 4.5, 4.6, 4.7, 4.8, 4.9, 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, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9 or 8.0.

[0092] Examples of buffers that can control the pH value of the pharmaceutical preparation within the desired range include acetate buffer, histidine buffer, citrate buffer and other organic acid or inorganic acid buffers. These buffers can be used alone or in combination of two or more. Preferably, the pharmaceutical preparation of the present application comprises acetate buffer and histidine buffer. More preferably, the pharmaceutical preparation of the present application comprises histidine buffer.

[0093] "Acetate buffer" is a buffer containing acetate ions. The acetate buffer may contain one or more of acetic acid (e.g., glacial acetic acid), potassium acetate, sodium acetate (e.g., sodium acetate trihydrate), etc. In some embodiments, the acetate buffer is an acetic acid-sodium acetate buffer, specifically a glacial acetic acid-sodium acetate trihydrate buffer. In some embodiments, the pH value of the acetate buffer may be any pH value in the range of 4.5 to 6.5, such as 4.5, 4.6, 4.7, 4.8, 4.9, 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, or 6.5.

[0094] "Histidine buffer" is a buffer containing histidine ions. The histidine buffer may include one or more of histidine, histidine hydrochloride (e.g., histidine hydrochloride monohydrate), histidine acetate, histidine phosphate, histidine sulfate, etc. In some embodiments, the histidine buffer may be a histidine-histidine hydrochloride buffer. In some embodiments, the pH value of the histidine buffer may be any pH value in the range of 4.5 to 6.5, such as 4.5, 4.6, 4.7, 4.8, 4.9, 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, or 6.5.

[0095] "Citrate buffer" is a buffer containing citrate ions. The citric acid buffer may contain one or more of citric acid, monosodium citrate, disodium citrate, trisodium citrate, monopotassium citrate, dipotassium citrate, tripotassium citrate, sodium chloride, potassium chloride, etc. In some embodiments, the citric acid buffer is a citric acid-trisodium citrate buffer. In some embodiments, the pH value of the citric acid buffer may be any pH value in the range of 4.5 to 6.5, such as 4.5, 4.6, 4.7, 4.8, 4.9, 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 or 6.5.

[0096] In some embodiments, the buffer used in the pharmaceutical preparation of the present application is an acetate buffer or a histidine buffer, and the pH value of the pharmaceutical preparation of the present application is 4.5 to 6.5. Without wishing to be bound by theory, it is believed that acetate buffer and histidine buffer are superior to citric acid buffer. For example, when the buffer of the pharmaceutical preparation of the present application is an acetate buffer or a histidine buffer, the anti-GREM1 antibody in the pharmaceutical preparation is more stable (for example, more stable at high temperatures), the force between the antibody molecules is repulsive rather than attractive, and there is a smaller risk of intermolecular aggregation.

[0097] The concentration of buffer as described herein refers to the concentration of buffer ions in the buffer. In some embodiments, the concentration of a suitable buffer for the pharmaceutical preparation of the present application may be 5-50mM, 10-50mM, 10-40mM, 10-30mM, 10-20mM. In some embodiments, the concentration of the buffer is any concentration value within the above range. For example, as required, the concentration of the buffer may be 5mM, at least 10mM, at least 15mM, at least 20mM, at least 25mM, at least 30mM, at least 35mM, at least 40mM, at least 45mM and / or at most 50mM, depending on the stability required for the specific buffer and pharmaceutical preparation.

[0098] In some embodiments, the buffer used in the pharmaceutical preparation of the present application is an acetate buffer, such as an acetic acid-sodium acetate buffer, and its concentration may be 5 to 50 mM. In some embodiments, the concentration of the acetate buffer may be 5 to 50 mM, 5 to 45 mM, 5 to 40 mM, 5 to 35 mM, 5 to 30 mM, 5 to 25 mM, 5 to 20 mM, 5 to 15 mM, 5 to 10 mM.

[0099] In some embodiments, the buffer used in the pharmaceutical preparation of the present application is a histidine buffer, such as a histidine-histidine hydrochloride buffer, and its concentration may be 5 to 50 mM. In some embodiments, the concentration of the histidine buffer may be 5 to 50 mM, 5 to 45 mM, 5 to 40 mM, 5 to 35 mM, 5 to 30 mM, 5 to 25 mM, 5 to 20 mM, 5 to 15 mM, 5 to 10 mM.

[0100] In some embodiments, the buffer used in the pharmaceutical preparation of the present application is an acetate buffer or a histidine buffer, and the concentration is 5 to 50 mM. In some embodiments, the buffer used in the pharmaceutical preparation of the present application is an acetate buffer or a histidine buffer, and the concentration is 10 to 30 mM. In some embodiments, the buffer used in the pharmaceutical preparation of the present application is an acetate buffer or a histidine buffer, and the concentration is about 20 mM. In some embodiments, the buffer used in the pharmaceutical preparation of the present application is a histidine buffer, and the concentration is about 20 mM.

[0101] Stabilizer

[0102] As used herein, the term "stabilizer" refers to an agent that, when combined with a protein of interest, prevents or reduces the chemical and / or physical instability of the protein. Examples of stabilizers include sugars, alcohols, acids, salts, polymers, and the like. Examples of sugars include glucose, sucrose, trehalose, lactose, dextran, and the like. Examples of alcohols include sorbitol, and the like. Examples of acids include citric acid, phosphoric acid, tartaric acid, amino acids, ethylenediaminetetraacetic acid, arginine hydrochloride, and the like. Examples of salts include sodium sulfate, sodium glutamate, sodium chloride (NaCl), potassium chloride, ammonium acetate, and the like. Examples of polymers include polyethylene glycol, povidone, and the like.

[0103] In some embodiments, the stabilizer for the pharmaceutical preparation of the present application is selected from sugars. In some embodiments, the stabilizer for the pharmaceutical preparation of the present application is selected from alcohols. In some embodiments, the stabilizer for the pharmaceutical preparation of the present application is not mannitol. In some embodiments, the stabilizer for the pharmaceutical preparation of the present application is selected from salts. In some embodiments, the stabilizer for the pharmaceutical preparation of the present application is selected from amino acids. In some embodiments, the stabilizer for the pharmaceutical preparation of the present application is selected from sucrose, trehalose, sorbitol, NaCl, arginine hydrochloride or a combination thereof.

[0104] In some embodiments, the concentration of the stabilizer used in the pharmaceutical preparation of the present application in the pharmaceutical preparation may be 0.5-20% (w / v), 0.5-10% (w / v), 0.5-5% (w / v), 0.5-2.5% (w / v), 0.5-1.5% (w / v), 1-15% (w / v), 1-10% (w / v), 1-5% (w / v), 5-10% (w / v), 2-8% (w / v) or 2-5% (w / v), depending on the specific stabilizer and the stability required for the pharmaceutical preparation. In some embodiments, the concentration of the stabilizer is any concentration value within the above range.

[0105] In some embodiments, the stabilizer used in the pharmaceutical preparation of the present application is sucrose, and its concentration in the pharmaceutical preparation may be 0.5-20% (w / v). In some embodiments, the concentration of sucrose in the pharmaceutical preparation may be 0.5-10% (w / v), 1-10% (w / v), 10-20% (w / v) or 5-15% (w / v). In some embodiments, the concentration of sucrose in the pharmaceutical preparation is 1-10% (w / v). In some embodiments, the concentration of sucrose in the pharmaceutical preparation is about 5% (w / v). In some embodiments, the concentration of sucrose in the pharmaceutical preparation is about 8.2% (w / v). In some embodiments, the concentration of sucrose in the pharmaceutical preparation is about 9% (w / v).

[0106] In some embodiments, the stabilizer for the pharmaceutical preparation of the present application is arginine hydrochloride, and its concentration in the pharmaceutical preparation may be 0.5-20% (w / v). In some embodiments, the concentration of arginine hydrochloride in the pharmaceutical preparation may be 0.5-10% (w / v), 1-10% (w / v), 10-20% (w / v) or 5-15% (w / v). In some embodiments, the concentration of arginine hydrochloride in the pharmaceutical preparation is 1-5% (w / v). In some embodiments, the concentration of arginine hydrochloride in the pharmaceutical preparation is about 3% (w / v).

[0107] In some embodiments, the stabilizer used in the pharmaceutical preparation of the present application is trehalose, and its concentration in the pharmaceutical preparation may be 0.5-20% (w / v). In some embodiments, the concentration of trehalose in the pharmaceutical preparation may be 0.5-10% (w / v), 1-10% (w / v), 10-20% (w / v) or 5-15% (w / v). In some embodiments, the concentration of trehalose in the pharmaceutical preparation is 5-10% (w / v). In some embodiments, the concentration of trehalose in the pharmaceutical preparation is about 9% (w / v).

[0108] In some embodiments, the stabilizer for the pharmaceutical preparation of the present application is sorbitol, and its concentration in the pharmaceutical preparation may be 0.5-20% (w / v). In some embodiments, the concentration of sorbitol in the pharmaceutical preparation may be 0.5-10% (w / v), 1-10% (w / v), 10-20% (w / v) or 5-15% (w / v). In some embodiments, the concentration of sorbitol in the pharmaceutical preparation is 2-8% (w / v). In some embodiments, the concentration of sorbitol in the pharmaceutical preparation is about 5% (w / v).

[0109] In some embodiments, the stabilizer for the pharmaceutical preparation of the present application is NaCl, and its concentration in the pharmaceutical preparation may be 0.5-20% (w / v). In some embodiments, the concentration of NaCl in the pharmaceutical preparation may be 0.5-10% (w / v), 1-10% (w / v), 10-20% (w / v) or 5-15% (w / v). In some embodiments, the concentration of NaCl in the pharmaceutical preparation is 0.5-1.5% (w / v). In some embodiments, the concentration of NaCl in the pharmaceutical preparation is about 0.9% (w / v).

[0110] Surfactants

[0111] As used herein, the term "surfactant" refers to organic substances having both hydrophilic and hydrophobic structures, i.e., they contain groups with opposite solubility tendencies, e.g., oil-soluble hydrocarbon chains and water-soluble ionic groups. Surfactants can be divided into anionic, cationic and nonionic surfactants depending on the charge of the surface active part.

[0112] Examples of surfactants include polysorbates (e.g., polysorbate 20 or 80); Poloxamers (e.g., poloxamer 188); Triton; and polyethylene glycol, polypropylene glycol, and copolymers of ethylene glycol and propylene glycol (e.g., Pluronics, PF68, etc.). In some embodiments, the surfactant of the pharmaceutical formulation of the present invention is selected from polysorbate 20 (polysorbate 20, PS20, or Tween 20), polysorbate 80 (polysorbate 80, PS 80, or Tween 80), or a combination thereof.

[0113] In some embodiments, the concentration of the surfactant in the pharmaceutical preparation of the present application may be 0.005% (w / v) to 0.4% (w / v), 0.01% (w / v) to 0.3% (w / v), 0.01% (w / v) to 0.2% (w / v), 0.025% (w / v) to 0.1% (w / v), 0.05% (w / v) to 0.1% (w / v) or 0.01% (w / v) to 0.1% (w / v), depending on the specific surfactant and the stability required for the pharmaceutical preparation. In some embodiments, the concentration of the surfactant is any concentration value within the above range.

[0114] In some embodiments, the surfactant of the pharmaceutical preparation of the present application is polysorbate 20 and / or polysorbate 80, and its concentration in the pharmaceutical preparation may be 0.005% (w / v) to 0.4% (w / v). In some embodiments, the concentration of polysorbate 20 and / or polysorbate 80 in the pharmaceutical preparation may be 0.01% (w / v) to 0.2% (w / v). In some embodiments, the concentration of polysorbate 20 and / or polysorbate 80 in the pharmaceutical preparation may be 0.025% (w / v) to 0.1% (w / v). In some embodiments, the concentration of polysorbate 20 and / or polysorbate 80 in the pharmaceutical preparation may be about 0.05% (w / v). In some embodiments, the surfactant of the pharmaceutical preparation of the present application is polysorbate 80. In some embodiments, the concentration of polysorbate 80 in the pharmaceutical preparation is about 0.05% (w / v).

[0115] Antioxidants

[0116] As used herein, the term "antioxidant" refers to a substance that can delay the oxidation of oxygen to a pharmaceutical preparation. It affects the various stages of the self-oxidation process from different angles, plays the role of a reducing agent, a retarder, a synergist and / or a chelating agent, and provides electrons or effective hydrogen atoms for free radicals to accept, interrupts the chain reaction of self-oxidation, thereby reducing the oxidation reaction of the protein. Examples of antioxidants include, but are not limited to, methionine, cysteine, glutathione, sodium thiosulfate, ascorbic acid. In some embodiments, the antioxidant of the pharmaceutical preparation of the present application can be methionine.

[0117] In some embodiments, the concentration of the antioxidant in the pharmaceutical preparation of the present application may be 0.01-0.2% (w / v), 0.01-0.1% (w / v) or 0.02-0.06% (w / v), depending on the specific antioxidant and the stability required for the pharmaceutical preparation. In some embodiments, the concentration of the antioxidant is any concentration value within the above range.

[0118] In some embodiments, the antioxidant of the pharmaceutical preparation of the present application is methionine and its concentration in the pharmaceutical preparation is 0.01-0.2% (w / v). In some embodiments, the antioxidant of the pharmaceutical preparation of the present application is methionine and its concentration in the pharmaceutical preparation is 0.02-0.06% (w / v). In some embodiments, the antioxidant of the pharmaceutical preparation of the present application is methionine and its concentration in the pharmaceutical preparation is about 0.04% (w / v).

[0119] Other Materials

[0120] The pharmaceutical preparation of the present application may also optionally contain other materials, such as but not limited to isotonicity agents, diluents, and the like.

[0121] The term "isotonic agent" refers to a compound or composition that can impart a suitable osmotic tension to a drug to avoid a net flow of water across a cell membrane in contact with the drug. In some embodiments, the formulation of the present application has an osmotic pressure substantially the same as human blood. Suitable isotonic agents include, but are not limited to, glycerol, amino acids or proteins (such as glycine or albumin), salts (such as sodium chloride), and sugars (such as glucose, sucrose, and lactose).

[0122] The term "diluent" is a pharmaceutically acceptable agent that can be used to dilute the pharmaceutical preparation of the present application. Typical diluents include water, physiological saline, antibacterial agents for injection, pH buffer, sterile saline solution, Ringer's solution or glucose solution.

[0123] preparation

[0124] In one aspect, the present application provides a stable pharmaceutical formulation comprising an anti-GREM1 antibody (e.g., a specific anti-GREM1 antibody provided herein), a buffer, a stabilizer, and a surfactant. The pharmaceutical formulation has a pH of 4.5 to 8.0, and in some embodiments, a pH of 4.5 to 6.5, to achieve sufficient stability.

[0125] In some embodiments, the pharmaceutical preparation of the present application comprises:

[0126] i) an anti-GREM1 antibody (e.g., a specific anti-GREM1 antibody provided herein) at a concentration of 1 to 200 mg / ml, preferably 20 to 40 mg / ml, more preferably about 21 mg / ml or 33 mg / ml; and / or

[0127] ii) a buffer, which is preferably an acetate buffer or a histidine buffer, more preferably a histidine buffer, and the concentration of the buffer in the pharmaceutical preparation is 5 to 100 mmol / L, preferably 5 to 50 mmol / L or 10 to 30 mmol / L, more preferably about 20 mmol / L; and / or

[0128] iii) a stabilizer, which is preferably sucrose, trehalose, sorbitol, NaCl or arginine hydrochloride,

[0129] a) is preferably sucrose, and the concentration of the stabilizer in the pharmaceutical preparation is 0.5-20% (w / v), preferably 1-10% (w / v), more preferably about 5% (w / v), about 8.2% (w / v) or about 9% (w / v), or

[0130] b) preferably arginine hydrochloride, and the concentration of the stabilizer in the pharmaceutical preparation is 0.5-20% (w / v), preferably 1-5% (w / v), more preferably about 3% (w / v), or

[0131] c) preferably trehalose, and the concentration of the stabilizer in the pharmaceutical preparation is 0.5-20% (w / v), preferably 5-10% (w / v), more preferably about 9% (w / v), or

[0132] d) is preferably sorbitol, and the concentration of the stabilizer in the pharmaceutical preparation is 0.5 to 20% (w / v), preferably 2 to 8% (w / v), more preferably about 5% (w / v), or

[0133] e) preferably NaCl, and the concentration of the stabilizer in the pharmaceutical preparation is 0.5-20% (w / v), preferably 0.5-1.5% (w / v), more preferably about 0.9% (w / v); and / or

[0134] iv) a surfactant, which is preferably polysorbate 80 or polysorbate 20, more preferably polysorbate 80, and the concentration of the surfactant in the pharmaceutical preparation is 0.005% (w / v) to 0.4% (w / v), preferably 0.01% (w / v) to 0.2% (w / v), more preferably 0.025% (w / v) to 0.1% (w / v), more preferably about 0.05% (w / v), and / or

[0135] v) an antioxidant, preferably methionine, cysteine, glutathione, sodium thiosulfate and ascorbic acid, more preferably methionine, and the concentration of the antioxidant in the pharmaceutical preparation is 0.01% (w / v) to 0.2% (w / v), preferably 0.02% (w / v) to 0.06% (w / v), more preferably about 0.04% (w / v), and

[0136] The pharmaceutical preparation has a pH value of about 4.5 to 6.5, preferably about 5.5 to 6.5.

[0137] In some embodiments, the pharmaceutical preparation of the present application comprises an anti-GREM1 antibody (e.g., a specific anti-GREM1 antibody provided herein) at a concentration of about 1 to 200 mg / ml, and an acetate buffer or a histidine buffer at a concentration of about 5 to 50 mmol / L, wherein the pH of the pharmaceutical preparation is 4.5 to 6.5. In some embodiments, the above-mentioned pharmaceutical preparation comprises an anti-GREM1 antibody at a concentration of about 20 to 40 mg / ml. In some embodiments, the above-mentioned pharmaceutical preparation comprises an acetate buffer or a histidine buffer at a concentration of about 10 to 30 mg / ml.

[0138] In some embodiments, the pharmaceutical preparation of the present application comprises an anti-GREM1 antibody (e.g., a specific anti-GREM1 antibody provided herein) at a concentration of about 1 to 200 mg / ml, an acetate buffer or histidine buffer at a concentration of about 5 to 50 mmol / L, and sucrose, trehalose and sorbitol, NaCl or arginine hydrochloride at a concentration of about 0.5 to 20% (w / v), wherein the pH of the pharmaceutical preparation is 4.5 to 6.5. In some embodiments, the above-mentioned pharmaceutical preparation comprises an anti-GREM1 antibody at a concentration of about 20 to 40 mg / ml. In some embodiments, the above-mentioned pharmaceutical preparation comprises an acetate buffer or histidine buffer at a concentration of about 10 to 30 mg / ml. In some embodiments, the above-mentioned pharmaceutical preparation comprises sucrose at a concentration of about 1 to 10% (w / v). In some embodiments, the above-mentioned pharmaceutical preparation comprises arginine hydrochloride at a concentration of about 1 to 5% (w / v). In some embodiments, the above-mentioned pharmaceutical preparation comprises trehalose at a concentration of about 5 to 10% (w / v). In some embodiments, the pharmaceutical preparation comprises sorbitol at a concentration of about 2-8% (w / v). In some embodiments, the pharmaceutical preparation comprises NaCl at a concentration of about 0.5-1.5% (w / v).

[0139] In some embodiments, the pharmaceutical preparation of the present application comprises an anti-GREM1 antibody (e.g., a specific anti-GREM1 antibody provided herein) at a concentration of about 1 to 200 mg / ml, an acetate buffer or histidine buffer at a concentration of about 5 to 50 mmol / L, sucrose, trehalose, sorbitol, NaCl or arginine hydrochloride at a concentration of about 0.5 to 20% (w / v), and polysorbate 80 or polysorbate 20 at a concentration of about 0.005% (w / v) to 0.4% (w / v), wherein the pH of the pharmaceutical preparation is 4.5 to 6.5. In some embodiments, the above-mentioned pharmaceutical preparation comprises an anti-GREM1 antibody at a concentration of about 20 to 40 mg / ml. In some embodiments, the above-mentioned pharmaceutical preparation comprises an acetate buffer or histidine buffer at a concentration of about 10 to 30 mg / ml. In some embodiments, the pharmaceutical preparation comprises sucrose at a concentration of about 1-10% (w / v), arginine hydrochloride at a concentration of about 1-5% (w / v), trehalose at a concentration of about 5-10% (w / v), sorbitol at a concentration of about 2-8% (w / v), or NaCl at a concentration of about 0.5-1.5% (w / v). In some embodiments, the pharmaceutical preparation comprises polysorbate 80 or polysorbate 20 at a concentration of about 0.025% (w / v) to 0.1% (w / v).

[0140] In some embodiments, the pharmaceutical preparation of the present application comprises an anti-GREM1 antibody (e.g., a specific anti-GREM1 antibody provided herein) at a concentration of about 1 to 200 mg / ml, an acetate buffer or histidine buffer at a concentration of about 5 to 50 mmol / L, sucrose, trehalose, sorbitol, NaCl or arginine hydrochloride at a concentration of about 0.5 to 20% (w / v), polysorbate 80 or polysorbate 20 at a concentration of about 0.005% (w / v) to 0.4% (w / v), and methionine at a concentration of about 0.01% (w / v) to 0.2% (w / v), wherein the pH of the pharmaceutical preparation is 4.5 to 6.5. In some embodiments, the above-mentioned pharmaceutical preparation comprises an anti-GREM1 antibody at a concentration of about 20 to 40 mg / ml. In some embodiments, the above-mentioned pharmaceutical preparation comprises an acetate buffer or histidine buffer at a concentration of about 10 to 30 mg / ml. In some embodiments, the pharmaceutical preparation comprises sucrose at a concentration of about 1-10% (w / v), arginine hydrochloride at a concentration of about 1-5% (w / v), trehalose at a concentration of about 5-10% (w / v), sorbitol at a concentration of about 2-8% (w / v), or NaCl at a concentration of about 0.5-1.5% (w / v). In some embodiments, the pharmaceutical preparation comprises polysorbate 80 or polysorbate 20 at a concentration of about 0.025% (w / v) to 0.1% (w / v). In some embodiments, the pharmaceutical preparation comprises methionine at a concentration of about 0.02% (w / v) to 0.06% (w / v).

[0141] In some embodiments, the pharmaceutical preparation of the present application comprises an anti-GREM1 antibody at a concentration of about 20-40 mg / ml, a histidine buffer at a concentration of about 10-30 mmol / L, arginine hydrochloride at a concentration of about 1-10% (w / v), and polysorbate 80 or polysorbate 20 at a concentration of about 0.025% (w / v) to 0.1% (w / v), wherein the pH of the pharmaceutical preparation is about 4.5-6.5 or about 5.5-6.5.

[0142] In some embodiments, the pharmaceutical preparation of the present application comprises an anti-GREM1 antibody at a concentration of about 20-40 mg / ml, a histidine buffer at a concentration of about 10-30 mmol / L, arginine hydrochloride at a concentration of about 1-10% (w / v), polysorbate 80 or polysorbate 20 at a concentration of about 0.025% (w / v) to 0.1% (w / v), and methionine at a concentration of about 0.01% (w / v) to 0.2% (w / v), wherein the pH of the pharmaceutical preparation is about 4.5-6.5 or about 5.5-6.5.

[0143] In some embodiments, the pharmaceutical formulation of the present application comprises an anti-GREM1 antibody at a concentration of about 20-40 mg / ml, a histidine buffer at a concentration of about 10-30 mmol / L, sucrose at a concentration of about 1-10% (w / v), and polysorbate 80 or polysorbate 20 at a concentration of about 0.025% (w / v) to 0.1% (w / v), wherein the pH of the pharmaceutical formulation is about 4.5-6.5 or about 5.5-6.5.

[0144] In some embodiments, the pharmaceutical formulation of the present application comprises an anti-GREM1 antibody at a concentration of about 20-40 mg / ml, a histidine buffer at a concentration of about 10-30 mmol / L, sucrose at a concentration of about 1-10% (w / v), polysorbate 80 or polysorbate 20 at a concentration of about 0.025% (w / v) to 0.1% (w / v), and methionine at a concentration of about 0.01% (w / v) to 0.2% (w / v), wherein the pH of the pharmaceutical formulation is about 4.5-6.5 or about 5.5-6.5.

[0145] In some embodiments, the pharmaceutical formulation of the present application comprises an anti-GREM1 antibody at a concentration of about 33 mg / ml, a histidine buffer at a concentration of about 20 mmol / L, sucrose at a concentration of about 8-9% (w / v), and polysorbate 80 at a concentration of about 0.05%, wherein the pH of the pharmaceutical formulation is about 5.5-6.5.

[0146] In some embodiments, the pharmaceutical formulation of the present application comprises an anti-GREM1 antibody at a concentration of about 33 mg / ml, a histidine buffer at a concentration of about 20 mmol / L, sucrose at a concentration of about 8.2% (w / v), polysorbate 80 at a concentration of about 0.05% (w / v), and methionine at a concentration of about 0.04% (w / v), wherein the pH of the pharmaceutical formulation is about 6.0.

[0147] The chemical degradation of antibody molecules, or the aggregation to form polymers, or the deglycosylation, glycosylation, oxidation or other structural modifications of antibody molecules that may reduce at least one functional activity of the monomeric protein, may cause instability of the antibody preparation. For pharmaceutical preparations containing anti-GREM1 antibodies, during the storage of the pharmaceutical preparations, the anti-GREM1 antibodies may undergo chemical degradation, resulting in a decrease in the concentration of the antibody; the anti-GREM1 antibodies may also aggregate to form polymers that are sometimes insoluble in the form of multimeric molecules containing multiple antibody molecules, resulting in a decrease in the content of monomers containing single antibody molecules. Therefore, an increase in the content of polymer antibodies will lead to a decrease in the purity of the monomeric antibodies. In addition, due to the formation of insoluble polymers, the turbidity of the pharmaceutical preparation may increase.

[0148] In some embodiments, the pharmaceutical preparations comprising anti-GREM1 antibodies of the present application can maintain stability after long-term storage, high temperature, room temperature, oscillation and / or multiple freeze-thaw cycles, wherein the physical and / or chemical stability and / or functional activity of the anti-GREM1 antibodies remain relatively constant over time. In some embodiments, antibody protein concentration, protein purity, protein activity, pH value of the preparation, osmotic pressure of the preparation, appearance of the preparation, insoluble particles in the preparation, etc. can be used as markers of the stability of the pharmaceutical preparation. There are a variety of analytical techniques for determining protein stability in the art, which are reviewed in Peptide and Protein Drug Delivery, 247-301, Vincent Lee, ed., Marcel Dekker Inc., New York, New York Press (1991) and Jones, A. Adv. Drug Delivery Rev. 10: 29-90 (1993).

[0149] In some embodiments, the stability of the pharmaceutical formulation can be measured under selected conditions and time by methods known in the art. Exemplary methods include, but are not limited to, dynamic light scattering (DLS), size exclusion chromatography (SEC), cation exchange chromatography (CEX), non-reducing capillary electrophoresis (NR CE-SDS), pH measurement, protein concentration (Protein Conc) measurement, visual inspection (Visual Inspection) and microfluidic imaging (MFI).

[0150] As shown in the examples of the present application, the pharmaceutical preparations of the present application comprising anti-GREM1 antibodies show high stability in visual inspection, for example, high stability under long-term storage, high stability at high temperature (e.g., 40° C.) or at room temperature, high stability after shaking, and high stability after freezing and thawing.

[0151] In some embodiments, a stable pharmaceutical preparation refers to a pharmaceutical preparation in which the protein particle size does not change significantly during storage in a DLS test, and the KD is positive, that is, the force between the monoclonal antibody molecules is a repulsive force, and the monoclonal antibody molecules do not aggregate. As shown in the examples of the present application, the pharmaceutical preparation comprising an anti-GREM1 antibody of the present application shows high stability in a DLS test, for example, high stability under long-term storage, high stability at high temperature (e.g., 40° C.) or at room temperature, high stability after shaking, and high stability after freezing and thawing.

[0152] In some embodiments, a stable pharmaceutical preparation refers to a pharmaceutical preparation that has only a small amount of protein degradation during storage, and a slow increase rate of polymers and oligomers in the SEC test. As shown in the examples of the present application, the pharmaceutical preparation comprising an anti-GREM1 antibody of the present application shows high stability in the SEC test, for example, high stability under long-term storage, high stability at high temperature (e.g., 40° C.) or at room temperature, high stability after shaking, and high stability after freezing and thawing.

[0153] In some embodiments, a stable pharmaceutical preparation refers to a pharmaceutical preparation in which the charge heterogeneity of the pharmaceutical preparation does not change significantly during storage, and the degree of separation of the acidic peak and the basic peak does not change significantly in the CEX test. As shown in the examples of the present application, the pharmaceutical preparation comprising the anti-GREM1 antibody of the present application shows high stability in the CEX test, for example, high stability under long-term storage, high stability at high temperature (e.g., 40° C.), and high stability after freeze-thaw.

[0154] In some embodiments, a stable pharmaceutical preparation refers to a pharmaceutical preparation that has only a small amount of protein degradation during storage, and a slow increase rate of polymers and oligomers in the NR CE-SDS test. As shown in the examples of the present application, the pharmaceutical preparation comprising an anti-GREM1 antibody of the present application shows high stability in the NR CE-SDS test, for example, high stability under long-term storage, high stability at high temperature (e.g., 40° C.) or at room temperature, high stability after shaking, and high stability after freezing and thawing.

[0155] In some embodiments, a stable pharmaceutical preparation means that when the pharmaceutical preparation is visually inspected, no significant change in appearance of the pharmaceutical preparation is observed during storage, and the pharmaceutical preparation still appears as a colorless clear liquid.

[0156] In some embodiments, a stable pharmaceutical formulation means that when the pharmaceutical formulation is tested in an MFI test, there is no obvious increase in subvisible particles in different particle size ranges during storage of the pharmaceutical formulation.

[0157] In some embodiments, a stable pharmaceutical formulation means that when the pharmaceutical formulation is tested for protein concentration, the change in protein concentration in the pharmaceutical formulation does not exceed + / -20%, does not exceed + / -19%, does not exceed + / -18%, does not exceed + / -17%, does not exceed + / -16%, does not exceed + / -15%, does not exceed + / -14%, does not exceed + / -13%, does not exceed + / -12%, does not exceed + / -11%, does not exceed + / -10%, does not exceed + / -9%, does not exceed + / -8%, does not exceed + / -7%, does not exceed + / -6%, does not exceed + / -5%, does not exceed + / -4%, does not exceed + / -3%, does not exceed + / -2%, does not exceed + / -1%, or does not exceed + / -0.5%, wherein the protein concentration can be determined by UV-visible spectrophotometry according to the General Rule 0401 of the Chinese Pharmacopoeia 2010 edition, Part III.

[0158] Preparation

[0159] The pharmaceutical preparation provided in the present application can be prepared by the following method, which comprises:

[0160] 1) providing a formulation medium and an anti-GREM1 antibody stock solution, wherein the formulation medium comprises a buffer;

[0161] 2) using the formulation solvent to replace the anti-GREM1 antibody stock solution, thereby obtaining a pharmaceutical preparation as described herein. In some embodiments, the replacement of the solution is a buffer exchange, such as buffer exchange by dialysis. In some embodiments, the replacement of the solution is a filtration replacement. In some embodiments, the filtration is sterile filtration, and / or other filtration methods known to those skilled in the art.

[0162] 3) After the liquid exchange is completed, the required amount of formulation excipients (such as stabilizers, surfactants, and optionally antioxidants) are added to the anti-GREM1 antibody stock solution, and diluted with a buffer to the target concentration.

[0163] In some embodiments, the buffer in the formulation vehicle is a histidine buffer at a concentration of about 20 mM.

[0164] In some embodiments, the stabilizer is sucrose at a concentration of about 8.2% (w / v), the surfactant is polysorbate 80 at a concentration of about 0.05% (w / v), and the antioxidant is methionine at a concentration of about 0.04% (w / v).

[0165] In some embodiments, the fluid replacement is buffer replacement. In some embodiments, the anti-GREM1 antibody stock solution is dialyzed using the formulation medium. For example, a certain volume of sample can be placed in a dialysis bag (e.g., Snake The dialysis bag is sealed and placed in a target buffer with a volume of ≧100 times the volume, and stirred continuously to promote substitution. The dialysis is performed for an appropriate number of times (e.g., 3 times), for an appropriate time (e.g., 4 h, 4 h, and overnight), and is completed at an appropriate stirring speed (e.g., 300 rpm).

[0166] In some embodiments, the liquid exchange is a filtration liquid exchange. In some embodiments, the filtration is a sterile filtration (e.g., through a filter membrane with a pore size of 0.22 microns, the bacteria are retained on the membrane, and the anti-GREM1 antibody solution will pass through the filter membrane, thereby sterilizing), and / or other filtration methods known to those skilled in the art.

[0167] Drug combinations

[0168] On the other hand, the present application provides a drug combination comprising the drug preparation described herein and a second therapeutic agent, wherein the second therapeutic agent is selected from chemotherapeutic drugs (such as cisplatin), radiotherapy, immunotherapy drugs (such as immune checkpoint regulators, such as PD-1 / PD-L1 axis inhibitors, TGF-β inhibitors), anti-angiogenic drugs (such as antagonists of VEGFR-1, VEGFR-2 and VEGFR-3), targeted therapy drugs, cell therapy drugs, gene therapy drugs, hormone therapy drugs, cytokines, etc.

[0169] application

[0170] In another aspect, the present application also provides a method for treating a disease in a subject, comprising administering a therapeutically effective amount of the pharmaceutical preparation or pharmaceutical combination of the present application to the subject, wherein the subject has or may have a disease that requires treatment with an antibody against GREM1.

[0171] As used herein, the term "treat" refers to reducing or ameliorating the severity and / or duration of a disorder or one or more symptoms thereof, inhibiting or preventing the progression of a disorder, causing regression of a disorder, inhibiting or preventing the recurrence, development, onset or progression of one or more symptoms associated with a disorder. Subjects in need of treatment include those already suffering from the disease.

[0172] The term "therapeutically effective amount" refers to the minimum concentration required to achieve a measurable improvement or prevention of a particular condition.

[0173] The pharmaceutical preparation or pharmaceutical combination of the present application can be used to treat GREM1-related diseases, including chronic and acute diseases. GREM1-related diseases include cancer, etc. In some embodiments, the GREM1-related disease is a cancer expressing GREM1. Examples of cancers expressing GREM1 include, but are not limited to, cancer, fibrotic diseases, angiogenesis, glaucoma or retinal diseases, kidney disease, pulmonary hypertension or osteoarthritis (OA), or the GREM1-related disease or condition is associated with an increased level of GREM1, which is selected from the group consisting of: scleroderma, idiopathic pulmonary fibrosis, diabetic nephropathy, IgAN, lupus nephritis, Alport syndrome, glioma, head and neck cancer, prostate cancer, lung cancer, gastric cancer, pancreatic cancer, esophageal cancer, bladder cancer, breast cancer and colorectal cancer

[0174] The pharmaceutical preparation or pharmaceutical combination of the present application can be administered to a subject by any suitable route. For example, the pharmaceutical preparation can be administered to a subject by intravenous injection.

[0175] In yet another aspect, the present application provides use of a pharmaceutical preparation or a pharmaceutical combination in the preparation of a drug for preventing and / or treating a GREM1-related disease.

[0176] In some embodiments, the drug further includes a second therapeutic agent, which is selected from chemotherapy drugs (such as cisplatin), radiotherapy, immunotherapy drugs (such as immune checkpoint regulators, such as PD-1 / PD-L1 axis inhibitors, TGF-β inhibitors), anti-angiogenic drugs (such as antagonists of VEGFR-1, VEGFR-2 and VEGFR-3), targeted therapy drugs, cell therapy drugs, gene therapy drugs, hormone therapy drugs, cytokines, etc.

[0177] Example

[0178] Overview of experimental methods:

[0179] In the following examples, anti-GREM1 antibodies were subjected to formulation screening tests. The anti-GREM1 antibody used in the following examples is Hu14E3_HaLa. Since the amino acid sequences of other humanized anti-GREM1 antibodies (e.g., Hu14E3_HaLb, Hu14E3_HbLa, Hu14E3_HbLb, Hu14E3_HcLa, and Hu14E3_HcLb) are substantially identical to those of Hu14E3_HaLa, it can be expected that the following formulation screening experiments are also applicable to these humanized anti-GREM1 antibodies. In other words, the above-mentioned humanized anti-GREM1 antibodies (e.g., Hu14E3_HaLa, Hu14E3_HaLb, Hu14E3_HbLa, Hu14E3_HbLb, Hu14E3_HcLa and Hu14E3_HcLb) can also achieve the expected high stability (e.g., high stability under long-term storage, high stability at high temperature (e.g., 40° C.) or room temperature, high stability after shaking, high stability after freezing and thawing) in the formulation provided in the present application. The anti-GREM1 antibodies described in the present application are prepared by the following method: 1×10 6 HEK293E cells at 10 cells / ml were transfected with equal amounts of heavy chain vector and light chain vector DNA at a final concentration of 0.5 μg / ml and 1.0 μg / ml of PEI (polyethyleneimine-straight chain, Polyscience). The DNA to PEI ratio was 1:2. The period of DNA and PEI complex formation with optimal MEM should be 15 minutes at room temperature. The transfected cells were cultured in flasks at 5% CO2, 37°C and 125rpm shaking speed. 1% peptone medium was added 22 to 26 hours after transfection. The conditioned medium was collected on the 6th day and the supernatant was centrifuged at 3,000rpm for 30 minutes. The clarified conditioned medium was then loaded onto an nProteinA column (GE Healthcare), washed with PBS plus 0.1% triton-X100, and finally eluted with a solution containing 0.1M glycine at pH 3.5 to bind IgG. The eluted antibody protein was dialyzed into PBS and stored at -80 ° C. To remove endotoxins, the purified protein was further processed through a Hitrap DEAE Sepharose FF column and the resulting antibody was analyzed using size exclusion chromatography (Superdex 200 5 / 150GL, GE Healthcare) to determine the purity level. For a detailed description, please refer to PCT / CN2022 / 072297, the disclosure of which is incorporated herein by reference in its entirety.

[0180] The anti-GREM1 antibody formulations tested in the following examples of this application were prepared as follows:

[0181] 1. Prepare the target formulation solution:

[0182] a) Using dialysis to replace the buffer containing the anti-GREM1 antibody (i.e., stock solution, also referred to as DS in the following examples) into the target formulation buffer. Specifically, a certain volume of DS sample is placed in Snake Into the dialysis bag, sealed, placed

[0183] ≧100 times the volume of the target buffer, stirring continuously to facilitate replacement. Dialysis was performed three times, with durations of 4 h, 4 h and overnight, respectively, and stirring speed of 300 rpm.

[0184] b) After the dialysis is completed, the required amount of stabilizer and surfactant mother solution is added to DS.

[0185] Then, the DS is diluted to the target concentration using the target buffer system to obtain the target preparation solution.

[0186] The following examples of the present application analyze the anti-GREM1 antibody formulations tested using the following analytical methods:

[0187] 2. Dynamic light scattering (DLS): The protein particle size and distribution were measured by dynamic light scattering (DLS) with the following parameters: acquisition time 5 s, 20 acquisitions per measurement, and measurement temperature 25 °C.

[0188] 3. Size Exclusion Chromatography (SEC): Protein aggregation was determined by SEC using Waters' ultra-high performance liquid chromatography system (UPLC) and a Tosoh TSKgelG3000SWXL SEC column (7.8×300mm, 5μm). The mobile phase was 50mM sodium phosphate buffer, 300mM NaCl, pH 6.8±0.1. The flow rate was 1.0mL / min. The sample was diluted to 10mg / mL, the detection volume was 10μL, and the detection wavelength was 280nm.

[0189] 4. Cation exchange chromatography (CEX): The charge heterogeneity of the protein was determined by CEX using a Thermo Propac Elite WCX-10 4 mm×150 mm 5 um column in an Agilent 1260 Infinity system. The sample was diluted to 2.00 mg / mL with a mixed solution of mobile phases A and B.

[0190] 5. Non-reducing capillary electrophoresis (NR CE-SDS): Protein fragmentation was determined by CE-SDS (NR) method. The standard or test sample was diluted to 4 mg / mL with phosphate-citrate buffer, and then 25 μL was vortexed with 75 μL SDS sample buffer and 5 μL NEM (100 mM N-ethylmaleimide) for denaturation. The denatured sample was centrifuged, incubated at 70 ± 2 ° C for 10 ± 2 min, cooled at room temperature, and then centrifuged again. Separation was performed on PA800 plus using SDS separation gel kit and uncoated fused silica capillaries.

[0191] 6. Visual Inspection: Check the appearance of the sample against a black background using a YB-2 clarity tester. Report transparency and color.

[0192] 7. Microfluidic Imaging (MFI): The number of subvisible particles of specified sizes (2-10 μm, 10-25 μm and >25 μm) was determined by Microfluidic Imaging 5200. Briefly, 500 μL of each sample was injected into the MFI instrument according to the instrument manual. The average number of particles per milliliter was reported.

[0193] 8. Activity detection: The binding activity of anti-GREM1 antibody to Gremlin antigen epitope was detected by ELISA binding method. Human Gremlin / GREM1 Protein (Acro) was used as coating antigen, detection antibody was Goat anti-Human IgG-Fc HRP conjugated (Bethyl laboratories), and the substrate of enzyme reaction was TMB (3,3', 5,5'-tetramethylbenzidine). The antigen was coated and adsorbed on the ELISA solid phase carrier adsorption plate, and the test sample was added to bind to it after washing and blocking. After incubation, washing, detection antibody was added, incubation and washing were performed to remove unbound detection antibody, and then substrate was added for color development. Finally, the reaction termination solution was added, and the absorbance value was read on the microplate reader. The detection wavelength was 450nm / 650nm.

[0194] Example 1: pH screening experiment

[0195] The anti-GREM1 antibody protein was subjected to a pH screening test for the formulation. The stability of the anti-GREM1 antibody in acetic acid, histidine and citric acid buffer systems was investigated in the range of pH 4.5 to pH 6.5. The specific formulation design is shown in Table 1. All formulations were investigated for stability (detected by SEC, NR CE-SDS, CEX and / or MFI) at the initial time T0, 40°C for 3 days, 5 days, 7 days (40°C (3D, 5D, 7D)) and 3 cycles and 5 cycles of freeze-thaw (FT (3C, 5C)).

[0196] Table 1. pH screening experiment design

[0197]

[0198] The experimental results are as follows:

[0199] Freeze-thaw (5C): Under the freeze-thaw condition of 5 cycles (5C), there was no significant change in the protein appearance, concentration, SEC, DLS and NR CE-SDS results of each formulation. In the MFI experiment, after 5 cycles of freeze-thaw stress, the sub-visible particles in different particle size ranges of formulations F6, F7 and F8 showed a significant increase trend, and were significantly worse than other formulations. Overall, there was no significant difference between formulations F1 to F5 (data shown in the table below). Therefore, it is shown that the citric acid-sodium citrate buffer system is not conducive to the low-temperature storage of proteins.

[0200]

[0201]

[0202] 40℃ (3D, 5D, 7D): At 40℃, there was no significant change in protein appearance, concentration, and DLS results for each formulation. Figure 1 As shown in Figure 2, SEC data showed that the polymer content and increase rate of formulations F6, F7, and F8 were slightly faster than those of other formulations. Figure 2 As shown in the figure, the CEX main peaks of formulations F1 and F2 degraded fastest. The main peak purity and polymer content of other formulations remained basically unchanged and there was basically no difference between the formulations.

[0203] In summary, through freeze-thaw and 40°C stability tests, the citric acid-sodium citrate buffer system is not conducive to low-temperature storage of proteins. Under 40°C conditions, the increase rate of high molecular weight polymer HMW and small molecular fragment LMW of the citric acid-sodium citrate buffer system F8 is slightly faster than that of other prescriptions, and the CEX main peaks of F1 and F2 in the acetic acid-sodium acetate buffer system are degraded the fastest. Therefore, the preferred buffer system is histidine-histidine hydrochloride buffer.

[0204] Example 2: Screening of excipients

[0205] The anti-GREM1 antibody protein was subjected to an excipient screening test for the preparation. The protective effects of inorganic salts (sodium chloride), amino acids (arginine hydrochloride), alcohols (sorbitol and mannitol) and sugars (sucrose and trehalose) on the protein were studied. The specific formulation design is shown in Table 2. The samples were tested after incubation, and all formulations were investigated for stability (detected by SEC, NR CE-SDS, CEX and / or MFI) at the initial time T0, 3 days, 7 days, 14 days (40°C (3D, 7D, 14D)) at 40°C, and 1 cycle, 3 cycles, and 5 cycles (FT (1C, 3C, 5C)) of freeze-thaw.

[0206] Table 2

[0207]

[0208]

[0209] The experimental results are as follows:

[0210] 1. 5 freeze-thaw cycles: There was no significant change in the concentrations of each formulation, CEX and NR CE-SDS results. The DLS results showed (Table 3) that the protein of formulation F15 (mannose) showed a polydisperse system (PDI%>25%) under T0 and freeze-thaw stress, indicating that the protein had the risk of aggregation in this formulation. During the 5 cycles of freeze-thaw stress on SEC, formulation F15 showed a significant increase in polymers ( Figure 3 ), no significant differences were observed in the other preparations and the main peak did not change.

[0211] Table 3

[0212]

[0213] 2. Experimental results at 40℃:

[0214] During the 14-day stress at 40°C, the experimental results show that after incubation at 40°C for 14 days, the HWM of the high molecular weight polymer of each formulation did not change significantly, the LMW of the small molecule fragments increased slightly, the CEX main peak decreased significantly, and there was no significant difference between the formulations ( Fig.14 During the 4-week incubation at 40°C, formulations F10 and F13 were selected to investigate the oxidation tendency. LC-MS results showed that these two formulations had obvious oxidation tendency at sites HC_M101, HC_M109, HC_M249, and HC_M425 (Table 4).

[0215] Table 4: Excipient screening study oxidation results_40℃

[0216]

[0217] Note: NT means not tested

[0218] In summary, through freeze-thaw and 40°C stability tests, mannitol is not conducive to protein stability, and there is no obvious difference in the other stabilizers. Considering that sucrose is widely used in biological preparations, and arginine hydrochloride can protect proteins through hydrogen bonding, sucrose and arginine hydrochloride are preferred for subsequent experiments. In addition, LC-MS results show that the protein has a clear tendency to oxidize at some sites, and the antioxidant methionine is added for subsequent formulation prescription screening.

[0219] Example 3. Surfactant screening

[0220] The anti-GREM1 antibody protein was subjected to surfactant screening test. The protective effect of 0.025% to 0.1% (w / v) polysorbate 80 and polysorbate 20 was studied. The formulation design is detailed in Table 5. All formulations were investigated for stability (detected by SEC, NR CE-SDS, CEX) after storage at the initial time T0, 3 days, 7 days, 14 days at 40°C (40°C (3D, 7D, 14D)) and shaking at 200 rpm at 25°C for 1 day, 3 days, and 5 days (shaking (200 rpm, 25°C: 1D, 3D, 5D)).

[0221] Table 5

[0222]

[0223]

[0224] 1. Analysis of shaking sample results

[0225] From the experimental results, it can be seen that after shaking for 5 days, the high molecular weight polymer HWM, small molecule fragments LMW and charge isomers of each formulation did not change significantly, and there was no obvious difference between the formulations.

[0226] 2. Analysis of the results of 40℃ samples

[0227] From the experimental results, we can see that after incubation at 40°C for 14 days, the high molecular weight polymer HWM of each formulation did not change significantly, the small molecule fragment LMW increased slightly, the CEX main peak decreased significantly, and there was no obvious difference between the formulations.

[0228] In summary, through shaking and 40°C stability tests, polysorbate 80 and polysorbate 20 had good protective effects on proteins in the concentration range of 0.025% (w / v) to 0.1% (w / v) and there was no obvious difference, both met the experimental requirements, and 0.05% (w / v) polysorbate 80 was selected for subsequent research.

[0229] Example 4. pH screening, stabilizer and antioxidant experiments

[0230] This experiment further investigated the histidine-histidine hydrochloride buffer system and two stabilizers, arginine hydrochloride and sucrose, under different pH conditions, and investigated the antioxidant effect of methionine. The formulation design is detailed in Table 6. The stability of all formulations was investigated at the initial time T0, storage at 40°C for 2 weeks and 4 weeks (40°C (2W, 4W)), storage at 25°C for 2 weeks, 4 weeks, and 6 weeks (25°C (2W, 4W, 6W)), storage at 5°C for 2 weeks, 4 weeks, 6 weeks, and 3 months (5°C (2W, 4W, 6W, 3M)), freeze-thaw for 1 cycle, 3 cycles, and 5 cycles (FT (1C, 3C, 5C)), and shaking at 1000 rpm at 25°C for 8 hours (shaking (1000 rpm, 25°C: 8h)), stirring at 400 rpm at 25°C for 1 hour (stirring (400 rpm, 25°C: 1h)) (by SEC, NR CE-SDS, CEX, visual inspection (visible foreign matter) and / or MFI detection, activity detection).

[0231] Table 6

[0232]

[0233] 1. Freeze-thaw, shaking and stirring results

[0234] From the experimental results, it can be seen that under the conditions of freeze-thaw, shaking and stirring, the high molecular weight polymer HWM, small molecule fragments LMW and charge isomers of each formulation did not change significantly, and there was no obvious difference between the formulations.

[0235] 2. Lighting Results

[0236] From the experimental results, it can be seen that the high molecular weight polymer HMW and small molecular fragment LMW of each prescription have no obvious changes, the CEX main peak decreases slightly, and there is no obvious difference between the prescriptions.

[0237] 3. 40℃ Results

[0238] From the experimental results, it can be seen that at 40℃, the HMW of the high molecular weight polymer of each formulation increased slightly, the LMW of the small molecular fragments increased significantly, and the main peak of CEX decreased ( Figure 4 ), there was no significant difference between the formulations. In addition, there was no significant difference in the activity results between the formulations (the activity test used T0 sample as the reference. As shown in Table 7, after incubation at 40°C for 4 weeks, the combined activity results of all preparations were within the acceptance standard range (between 50%-150% relative to the reference). As shown in Table 8, by comparing the LC-MS data of formulations F24 and F26, it can be found that methionine has a significant antioxidant effect.

[0239] Table 7

[0240] prescription 40℃_4 weeks_(%) F22 73％ F23 75％ F24 72％ F25 81％ F26 84％ F27 79％

[0241] Table 8

[0242]

[0243] Table 8

[0244]

[0245] Note: NT means not tested

[0246] 4. 25℃ Results

[0247] From the experimental results, it can be seen that at 25℃, the high molecular weight polymer HMW and small molecular fragment LMW of each formulation did not change significantly, the CEX main peak decreased to a certain extent, and there was no significant difference between the formulations ( Figure 5-7 ).

[0248] 5. 5℃ Results

[0249] From the experimental results, it can be seen that at 5°C, the high molecular weight polymer HMW, small molecular fragment LMW and charge isomers of each prescription did not change significantly, and there was no significant difference between the prescriptions. However, at 3 months (3M), prescriptions F22, F24, F26 and F27, that is, all prescriptions containing arginine hydrochloride, were observed to have visible foreign matter (Table 9), while prescriptions F23 and F25 containing sucrose did not have visible foreign matter.

[0250] Table 9

[0251]

[0252] Note: UPS---Uncountable Particles; NVP---No Visible Foreign Matter

[0253] Taking into account the results of each condition, after being placed at 5°C for 3 months, visible foreign matter was observed in all formulations containing arginine hydrochloride, so the preferred stabilizer is sucrose. In order to make the osmotic pressure of the preparation as close as possible to the plasma osmotic pressure of the human body, the concentration of sucrose was adjusted from 9% (w / v) to 8.2% (w / v). There was no significant difference in the histidine-histidine hydrochloride buffer system (formulations 22, 24 and 27) at different pH (pH5.7, pH6.0 and pH6.5), so the middle pH, pH6.0, was selected as the preferred pH. Methionine has a significant antioxidant effect. Therefore, the preferred formulation is 20mM histidine / histidine hydrochloride, 8.2% (w / v) sucrose, 0.04% (w / v) methionine, 0.05% (w / v) polysorbate 80, pH6.0 for formulation confirmation research.

[0254] Example 5: Prescription Confirmation Experiment

[0255] This experiment further investigated the stability of the preferred formulation in Example 4, i.e., 20 mM histidine / histidine hydrochloride, 8.2% (w / v) sucrose, 0.04% (w / v) methionine, 0.05% (w / v) polysorbate 80, pH 6.0, under various conditions. The formulation design is detailed in Table 10. The stability of all formulations was investigated at the initial time T0, storage at 40°C for 2 weeks and 4 weeks (40°C (2W, 4W)), storage at 25°C for 2 weeks, 4 weeks, and 6 weeks (25°C (2W, 4W, 6W)), storage at 5°C for 2 weeks, 4 weeks, 6 weeks, 3 months, and 6 months (5°C (2W, 4W, 6W, 3M, 6M)), freeze-thaw for 1 cycle, 3 cycles, and 5 cycles (FT (1C, 3C, 5C)), and shaking at 200 rpm at 25°C for 3 days and 7 days (shaking (200 rpm, 25°C: 3D, 7D)), stirring at 400 rpm at 25°C for 2 hours and 4 hours (400 rpm, 25°C: 2H, 4H), and after illumination for 5 days and 10 days (detected by SEC, NR CE-SDS, CEX, visual inspection (visible foreign matter), and / or MFI).

[0256] Table 10

[0257]

[0258]

[0259] The experimental results show that the final preparation has a long-term temperature of 5°C ( Figure 8-10 ) and accelerated 25℃( Figure 11-13 ), freeze-thaw, shaking and stirring experimental conditions, and no significant changes occurred in the test indicators (Tables 11-14). Under light conditions, although the main peak of CEX decreased significantly, the binding activity results of the preparation were within the acceptance standard range (between 50% and 150% relative to the reference product), so there was no significant change in activity, meeting the stability requirements. Under 40°C conditions (Table 15), although the LMW increased significantly and the main peak of CEX decreased significantly, the 4-week binding activity results of the preparation were within the acceptance standard range (between 50% and 150% relative to the reference product), so there was no significant change in activity. . Therefore, the selected buffer system and prescription components can provide good stability for the GREM1 protein.

[0260] Table 11: Freeze-thaw results

[0261]

[0262] Table 12: Stirring results

[0263]

[0264]

[0265] Table 13: Shaking results

[0266]

[0267] Table 14: Lighting and Light Control

[0268]

[0269] Note: NT means not tested

[0270] Table 15: 40°C

[0271]

[0272] Note: NT means not tested

[0273] Example 6: Activity characterization of the anti-GREM1 antibody used in this application

[0274] Binding affinity analysis of anti-GREM1 antibodies to human GREM1 by ELISA

[0275] Transparent polystyrene culture plates (BEAVER) were coated overnight at 4°C with 100 microliters / well of high pH coating buffer containing 0.5 μg / ml hGREM1 (ACRO) and mouse gremlin (R&D). The culture plates were then washed once using PBS+0.1% Tween 20 (Sigma) on an automatic culture plate washer. 100 μl of blocking solution consisting of PBS+1% BSA+1% standard goat serum+0.5% Tween 20 (Sigma) was added to each well and incubated for 2 hours at room temperature. Then 100 μl of antibodies (starting at 2 μg / ml and serially diluted) in an antibody dilution buffer containing PBS+1% BSA+1% standard goat serum+0.01% Tween 20 was added to each well of the culture plate and incubated for 1 hour at room temperature. Then, the culture plate was washed three times with 200 μl of PBS+0.1% Tween20, followed by the addition of 100 μl / well of 1:10000 goat anti-mouse IgG-HRP (Abcam), and incubated at room temperature for 1 hour. It was then washed 3 times with PBS+0.1% Tween20. Finally, 100 μl / well of TMB (Pierce) was added to each well, and after several minutes, 50 μl of stop solution was added to each well. The culture plate was read at 450 nM on a Multiscan FC microplate reader (Thermo Scientific). As shown in Tables 16 and 17, the anti-GREM1 antibodies used in the present application and other humanized anti-GREM1 antibodies (e.g., Hu14E3_HaLb, Hu14E3_HbLa, Hu14E3_HbLb, Hu14E3_HcLa and Hu14E3_HcLb) have better binding affinity to human GREM1 than the benchmark antibody 6245P.

[0276] Measuring affinity of humanized antibodies by Fortebio

[0277] Human GREM1 protein was diluted with kinetic buffer to obtain a concentration of 2 μg / ml. 0 nM was used as a reference control. The antibodies to be tested were diluted with ForteBio kinetic buffer (PBS pH 7.4, 0.1% BSA + 0.002% Tween-20) to concentrations of 100 nM, 50 nM, and 25 nM. Human GREM1-his was immobilized on the NTA biosensor. The baseline was detected for 60 seconds, and then the anti-GREM1 antibody association was detected for 120 seconds to obtain K on The K factor was then obtained by dissociation in kinetic buffer for 90 seconds. offFactor data. As shown in Table 16, the anti-GREM1 antibody used in the present application has a KD value of less than 1 nM, which is much lower than the KD value of the benchmark antibody, that is, compared with the benchmark antibody 6245P, the anti-GREM1 antibody used in the present application has a better binding affinity to human GREM1.

[0278] The ability of anti-GREM1 antibodies to block GREM1 binding to human BMP2 / 4 / 7 was tested by ELISA

[0279] The culture plate was coated with recombinant human BMP2 / 4 (0.5 μg / ml) overnight and then serial dilutions of the antibody were incubated with 1 μg / ml of modified human GREM1-his for 1 h at room temperature, after which the complex was added to the coated culture plate and incubated for another hour at room temperature. The culture plate was then washed and anti-his HRP (GenScript) was added. The culture plate was then developed with TMB solution and stopped by adding stop solution. The culture plate was read at 450 nm on a plate reader. The results are shown in Table 16, indicating that the anti-GREM1 antibodies provided herein can better inhibit the binding of GREM1 to BMP2 or BMP4 than the benchmark antibody 6245P, that is, compared to the benchmark antibody 6245P, the anti-GREM1 antibodies used in this application have better blocking activity for the binding of human GREM1 to BMP2 or BMP4.

[0280] Table 16

[0281]

[0282]

[0283] Table 17: EC50 of binding to hGremlin-his

[0284]

[0285] Efficacy of the combination of anti-GREM1 antibody and cisplatin in esophageal cancer PDX model

[0286] Human gremlin IHC, specifically positive esophageal tumor tissue (E7) was obtained from NOD / SCID mice passaged at Beijing Cancer Hospital and an established PDX bank. Upon testing, the E7 esophageal PDX model was found to be positive in GREM1 expression but not PD-L1 expression.

[0287] Each mouse was inoculated subcutaneously with a small tumor tissue piece of approximately 3 mm in diameter, which was cut from the whole tumor exfoliation of the tumor-bearing mouse. 18 days after inoculation, tumors of approximately 70 mm in size were selected.3 The animals were randomly divided into 4 groups, each consisting of 8 mice. Then, the mice were treated with isotype control + PBS, 20 mg / kg dose of the anti-GREM1 antibody of the present application, 3 mg / kg dose of cisplatin, and the combination of the anti-GREM1 antibody and cisplatin of the present application. Isotype control and anti-GREM1 antibody of the present application were administered by intraperitoneal injection and PBS twice a week for 4 weeks, while cisplatin was administered by intravenous injection once a week for 4 weeks. Animals were killed by CO2 inhalation at the end of the study. Tumor size was measured in two dimensions using a caliper (INSIZE) twice or three times a week, and the following formula was used to express mm 3 Indicates volume: V = 0.5a × b 2 , where a and b are the long and short diameters of the tumor, respectively. Results were analyzed using Prism GraphPad and expressed as mean ± SEM. Comparisons between two groups were performed by T-test, and differences were considered significant if p was * < 0.05 and ** < 0.01.

[0288] Table 18 shows that compared with the isotype control, when the anti-GREM1 antibody of the present application was used alone in this experiment, tumor growth inhibition was significantly enhanced. Compared with the anti-GREM1 antibody of the present application alone (63.97% TGI vs. 42.92% TGI) or compared with cisplatin alone (63.97% TGI vs. 59.79% TGI), the combination of the anti-GREM1 antibody of the present application and cisplatin further inhibited tumor growth, thereby indicating the synergistic effect of the combination treatment of the anti-GREM1 antibody of the present application and cisplatin on esophageal cancer.

[0289] So far, the first-line therapy for esophageal cancer generally includes esophagectomy, chemotherapy, targeted therapy, immunotherapy (e.g., targeting PD-1 or PD-L1), and / or a combination thereof. Second-line therapy and subsequent therapy for esophageal cancer may involve targeted therapy, such as ramucirumab targeting vascular endothelial growth factor (VEGF) receptors or trastuzumab for overexpression of HER2 for metastatic adenocarcinoma (NCCN Clinical Practice Guidelines in Oncology, Esophageal and Esophagogastric Junction Cancers, National Comprehensive Cancer Network. V1.2020). The data described above show that the anti-GREM1 antibodies provided herein can effectively treat tumors that do not express PD-L1, such as esophageal cancers that do not overexpress PD-L1, and can further achieve a synergistic effect when combined with chemotherapy (e.g., cisplatin). This shows that the anti-GREM1 antibodies provided herein can serve as a new option for first-line therapy or second-line therapy for esophageal cancer.

[0290] Table 18: Tumor growth inhibition in the esophageal cancer PDX model by the combination of anti-GREM1 antibodies and cisplatin used in the present application on day 26

[0291]

Claims

1. A pharmaceutical preparation comprising an anti-GREM1 antibody and a buffer, wherein the buffer is an acetate buffer or a histidine buffer and has a pH value of 4.5 to 6.

5. 2 . The pharmaceutical preparation according to claim 1 , wherein the concentration of the buffer in the pharmaceutical preparation is 5 to 50 mM, or 10 to 30 mM.

3. The pharmaceutical preparation according to claim 1 or 2, further comprising a stabilizer.

4. The pharmaceutical preparation according to claim 3, wherein the concentration of the stabilizer in the pharmaceutical preparation is 0.5-20% (w / v), or 0.5-10% (w / v).

5. The pharmaceutical preparation according to claim 3 or 4, wherein the stabilizer is selected from the group consisting of sucrose, trehalose, sorbitol, NaCl and arginine hydrochloride.

6. The pharmaceutical preparation according to claim 5, wherein: a) the stabilizer is sucrose, and the concentration of the sucrose in the pharmaceutical preparation is 1-10% (w / v); and / or b) the stabilizer is arginine hydrochloride, and the concentration of arginine hydrochloride in the pharmaceutical preparation is 1-5% (w / v); and / or c) the stabilizer is trehalose, and the concentration of the trehalose in the pharmaceutical preparation is 5-10% (w / v); and / or d) the stabilizer is sorbitol, and the concentration of sorbitol in the pharmaceutical preparation is 2-8% (w / v); and / or e) The stabilizer is NaCl, and the concentration of NaCl in the pharmaceutical preparation is 0.5-1.5% (w / v).

7. A pharmaceutical formulation according to any preceding claim, further comprising a surfactant.

8. The pharmaceutical preparation according to claim 7, wherein the concentration of the surfactant in the pharmaceutical preparation is 0.005% (w / v)-0.4% (w / v), or 0.01% (w / v)-0.2% (w / v).

9. The pharmaceutical preparation according to claim 7 or 8, wherein the surfactant is selected from the group consisting of polysorbate 80 and polysorbate 20.

10. The pharmaceutical preparation according to claim 9, wherein: a) the surfactant is polysorbate 80, and the concentration of polysorbate 80 and / or polysorbate 20 in the pharmaceutical preparation is 0.025% (w / v)-0.1% (w / v).

11. A pharmaceutical formulation according to any preceding claim, further comprising an antioxidant.

12. The pharmaceutical preparation according to claim 11, wherein the concentration of the antioxidant in the pharmaceutical preparation is 0.01-0.2% (w / v), or 0.02-0.06% (w / v).

13. The pharmaceutical formulation according to claim 11 or 12, wherein the antioxidant is selected from the group consisting of methionine, cysteine, glutathione, sodium thiosulfate and ascorbic acid.

14. The pharmaceutical formulation according to any one of the preceding claims, wherein the concentration of the anti-GREM1 antibody in the pharmaceutical formulation is 1 to 200 mg / ml. 15 . The pharmaceutical preparation according to claim 14 , wherein the concentration of the anti-GREM1 antibody in the pharmaceutical preparation is 20 to 40 mg / ml.

16. The pharmaceutical formulation according to any of the preceding claims, wherein the anti-GREM1 antibody comprises a heavy chain CDR1 (HCDR1) as shown in SEQ ID NO: 1, a HCDR2 as shown in SEQ ID NO: 2, and a HCDR3 as shown in SEQ ID NO: 3, and / or a light chain CDR1 (LCDR1) as shown in SEQ ID NO: 4, a LCDR2 as shown in SEQ ID NO: 5, and a LCDR3 as shown in SEQ ID NO:

6.

17. The pharmaceutical formulation according to any of the preceding claims, wherein the anti-GREM1 antibody comprises CDR1, CDR2 and CDR3 of the heavy chain variable region as shown in SEQ ID NO:7, and / or CDR1, CDR2 and CDR3 of the light chain variable region as shown in SEQ ID NO:

8.

18. The pharmaceutical formulation according to any of the preceding claims, wherein the anti-GREM1 antibody comprises a heavy chain framework region 1 (HFR1) as shown in QX1QLVQSGSELKKPGASVKVSCKASGX2TFT (SEQ ID NO: 24), an HFR2 as shown in WMX3QAPGQGLX4WMG (SEQ ID NO: 25), an HFR3 as shown in RFX5FSLDTSVSTAYLQISSLKAEDTAVYYCAR (SEQ ID NO: 26), and an HFR4 as shown in WGQGTMVTVSS (SEQ ID NO: 17), and / or a light chain framework region 1 (LFR1) as shown in DVVMTQSPLSLPVTLGQPASISC (SEQ ID NO: 27), an LFR2 as shown in WLQQRPGQSPRRLIX6 (SEQ ID NO: 32), an HFR3 as shown in GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC (SEQ ID NO: 33). NO:29) and LFR4 as shown in FGQGTKLEIK (SEQ ID NO:30), wherein, X1 is V or I; X2 is Y or S; X3 is R or K; X4 is E or T; X5 is V or A; X6 is Y or S.

19. The pharmaceutical formulation of claim 18, wherein the anti-GREM1 antibody comprises a heavy chain framework region 1 (HFR1) as shown in SEQ ID NO: 14, 18 or 21, an HFR2 as shown in SEQ ID NO: 15, 19 or 22, an HFR3 as shown in SEQ ID NO: 16, 20 or 23, and an HFR4 as shown in SEQ ID NO: 17, and / or a light chain framework region 1 (LFR1) as shown in SEQ ID NO: 27, an LFR2 as shown in SEQ ID NO: 28 or 31, an LFR3 as shown in SEQ ID NO: 29, and an LFR4 as shown in SEQ ID NO:

30.

20. The pharmaceutical formulation according to any preceding claim, wherein the anti-GREM1 antibody comprises FR1, FR2, FR3 and FR4 of the heavy chain variable region as shown in SEQ ID NO: 7, 11 or 12, and / or FR1, FR2, FR3 and FR4 of the light chain variable region as shown in SEQ ID NO: 8 or 13.

21. The pharmaceutical formulation according to any one of the preceding claims, wherein the anti-GREM1 antibody comprises a heavy chain variable region as shown in SEQ ID NO: 7, 11 or 12, and / or a light chain variable region as shown in SEQ ID NO: 8 or 13.

22. The pharmaceutical formulation according to any one of the preceding claims, wherein the anti-GREM1 antibody comprises a heavy chain constant region as shown in SEQ ID NO: 33 and / or a light chain constant region as shown in SEQ ID NO:

34.

23. The pharmaceutical formulation according to any of the preceding claims, wherein the anti-GREM1 antibody comprises the heavy chain shown in SEQ ID NO:9 and / or the light chain shown in SEQ ID NO:

10.

24. The pharmaceutical formulation according to any one of the preceding claims, comprising an anti-GREM1 antibody, a buffer, a stabilizer and a surfactant, wherein the buffer is a histidine buffer, the stabilizer is arginine hydrochloride or sucrose, the surfactant is polysorbate 80 or polysorbate 20, and the pH value is about 4.5-6.

5.

25. The pharmaceutical preparation according to claim 24, wherein the concentration of the anti-GREM1 antibody in the pharmaceutical preparation is 20-40 mg / ml, the concentration of the histidine buffer in the pharmaceutical preparation is 10-30 mM, the concentration of the arginine hydrochloride or sucrose in the pharmaceutical preparation is 1-10% (w / v), and / or the concentration of the polysorbate 80 or polysorbate 20 in the pharmaceutical preparation is 0.025% (w / v)-0.1% (w / v).

26. The pharmaceutical formulation according to claim 25, wherein the concentration of the anti-GREM1 antibody in the pharmaceutical formulation is about 33 mg / ml, the concentration of the histidine buffer in the pharmaceutical formulation is about 20 mM, the stabilizer is sucrose, and its concentration in the pharmaceutical formulation is about 8-9% (w / v), the surfactant is polysorbate 80, and its concentration in the pharmaceutical formulation is about 0.05% (w / v), and the pH value is about 5.5-6.

5.

27. The pharmaceutical formulation according to any one of the preceding claims, further comprising an antioxidant, said antioxidant being methionine.

28. The pharmaceutical formulation according to any one of the preceding claims, wherein the concentration of methionine in the pharmaceutical formulation is about 0.04% (w / v).

29. The pharmaceutical formulation according to claim 27, wherein the concentration of the anti-GREM1 antibody in the pharmaceutical formulation is about 33 mg / ml, the concentration of the histidine buffer in the pharmaceutical formulation is about 20 mM, the stabilizer is sucrose, and its concentration in the pharmaceutical formulation is about 8.2% (w / v), the surfactant is polysorbate 80, and its concentration in the pharmaceutical formulation is about 0.05% (w / v), the concentration of methionine in the pharmaceutical formulation is about 0.04% (w / v), and the pH value is about 6.

0.

30. Use of the pharmaceutical preparation according to any one of claims 1 to 29 in the preparation of a medicament for preventing and / or treating a GREM1-related disease.

31. The use according to claim 30, wherein the GREM1-related disease is selected from the group consisting of cancer, fibrotic disease, angiogenesis, glaucoma or retinal disease, kidney disease, pulmonary hypertension or osteoarthritis (OA), or the GREM1-related disease is associated with an increased level of GREM1 selected from the group consisting of scleroderma, idiopathic pulmonary fibrosis, diabetic nephropathy, IgAN, lupus nephritis, Alport syndrome, glioma, head and neck cancer, prostate cancer, lung cancer, gastric cancer, pancreatic cancer, esophageal cancer, bladder cancer, breast cancer and colorectal cancer.

32. The use according to claim 30 or 31, wherein the drug further comprises a second therapeutic agent, which is selected from chemotherapeutic drugs (such as cisplatin), radiotherapy, immunotherapy drugs (such as immune checkpoint regulators, such as PD-1 / PD-L1 axis inhibitors, TGF-β inhibitors), anti-angiogenic drugs (such as antagonists of VEGFR-1, VEGFR-2 and VEGFR-3), targeted therapy drugs, cell therapy drugs, gene therapy drugs, hormone therapy drugs, cytokines, etc.

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