Stable pharmaceutical formulations containing anti-gremlin 1 antibodies

Abstract

Pharmaceutical formulations are provided. The pharmaceutical formulations include an anti-GREM1 antibody, a buffer, a stabilizer, a surfactant, and optionally an antioxidant. The pharmaceutical formulations provided herein can maintain the stability of the anti-GREM1 antibody after long-term storage, storage at high temperatures (e.g., 40°C), storage at room temperature, vibration, and / or multiple freeze-thaw cycles. Use of the pharmaceutical formulations in the prevention and / or treatment of GREM1-associated diseases, particularly cancer (e.g., GREM1-associated cancer), as well as methods for preparing the pharmaceutical formulations, are provided.

Description

[Technical Field]

[0001] The present disclosure relates to pharmaceutical formulations, and in particular to stable pharmaceutical formulations containing anti-Gremlin 1 (GREM1) antibodies. The present disclosure also relates to methods for preparing the pharmaceutical formulations and uses thereof. [Background technology]

[0002] Gremlin 1 (GREM1) is a highly conserved secreted protein with a cysteine-rich region and cysteine junctions (Wordinger et al., Experimental Eye Research (Exp Eye Res.), August 2008;87(2):78-79). As a member of the differential screening-selected gene aberrative in neuroblastoma (DAN) family, GREM1 is used as a bone morphogenetic protein (BMP) antagonist (Wordinger et al., Exp Eye Res., August 2008;87(2):78-79). GREM1 physically binds to BMP-2, BMP-4, or BMP-7 to form heterodimers, preventing BMP ligands from interacting with their corresponding BMP receptors and subsequently inhibiting 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 various 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 (PNAS), October 2006; 103(40):14842-14847). For example, abnormal upregulation of GREM1 confers tumorigenic potential to colon cells outside the stem cell niche. It has also been found that GREM1 is highly expressed and secreted by tumor stem cells to maintain stem cell properties in glioma (Yan, K. et al., Genes Development (Genes Dev), 28, 1085-1100 (2014)). Therefore, GREM1 has been used as a therapeutic target in the treatment of GREM1-related diseases, such as the use of anti-GREM1 antibodies.

[0004] Because antibody molecules have a complex multilevel protein structure and tend to be physically associated, this can cause unwanted immune responses or be dangerous to patients due to clogging of syringes or pumps during administration. Thus, one of the long-standing problems with liquid antibody formulations is stability issues due to aggregation.

[0005] Therefore, there is a need for pharmaceutical formulations of anti-GREM1 antibodies that have stability and consistent quality. Summary of the Invention

[0006] The present disclosure provides stable pharmaceutical formulations comprising anti-GREM1 antibodies that maintain homogeneity and stability after long-term storage, storage at elevated temperatures (e.g., 40°C), storage at room temperature, shaking, and / or multiple freeze-thaw cycles.

[0007] In one aspect, the present disclosure provides a pharmaceutical formulation comprising an anti-GREM1 antibody and a buffer, the pharmaceutical formulation having a pH value of 4.5 to 6.5.

[0008] In one aspect, the disclosure provides a pharmaceutical formulation 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 5.5 to 6.5. In some embodiments, the concentration of the buffer in the pharmaceutical formulation is 5 mM to 50 mM or 10 mM to 30 mM.

[0009] In some embodiments, the pharmaceutical formulation further comprises a stabilizer.

[0010] In some embodiments, the concentration of the stabilizer in the pharmaceutical formulation is between 0.5% (w / v) and 20% (w / v), or between 0.5% (w / v) and 10% (w / v).

[0011] In some embodiments, the stabilizer is selected from the group consisting of sucrose, trehalose, sorbitol, NaCl, and arginine hydrochloride.

[0012] In some embodiments, the stabilizer is sucrose, and the concentration of sucrose in the pharmaceutical formulation is 1% (w / v) to 10% (w / v). In other embodiments, the stabilizer is arginine hydrochloride, and the concentration of arginine hydrochloride in the pharmaceutical formulation is 1% (w / v) to 5% (w / v). In other embodiments, the stabilizer is trehalose, and the concentration of trehalose in the pharmaceutical formulation is 5% (w / v) to 10% (w / v). In other embodiments, the stabilizer is sorbitol, and the concentration of sorbitol in the pharmaceutical formulation is 2% (w / v) to 8% (w / v). In other embodiments, the stabilizer is NaCl, and the concentration of NaCl in the pharmaceutical formulation is 0.5% (w / v) to 1.5% (w / v).

[0013] In some embodiments, the pharmaceutical formulation further comprises a surfactant.

[0014] In some embodiments, the concentration of surfactant in the pharmaceutical formulation is between 0.005% (w / v) and 0.4% (w / v), or between 0.01% (w / v) and 0.2% (w / v).

[0015] In some embodiments, the surfactant is selected from the group consisting of polysorbate 80 and polysorbate 20.

[0016] In some embodiments, the surfactant is polysorbate 80, and the concentration of polysorbate 80 and / or polysorbate 20 in the pharmaceutical formulation is between 0.025% (w / v) and 0.1% (w / v).

[0017] In some embodiments, the pharmaceutical formulation further comprises an antioxidant.

[0018] In some embodiments, the concentration of the antioxidant in the pharmaceutical formulation is between 0.01% (w / v) and 0.2% (w / v), or between 0.02% (w / v) and 0.06% (w / v).

[0019] In some embodiments, the antioxidant is selected from the group consisting of methionine, cysteine, glutathione, sodium thiosulfate, and ascorbic acid.

[0020] In some embodiments, the concentration of the anti-GREM1 antibody in the pharmaceutical formulation is between 1 mg / ml and 200 mg / ml.

[0021] In some embodiments, the concentration of the anti-GREM1 antibody in the pharmaceutical formulation is 20 mg / ml to 40 mg / ml.

[0022] In some embodiments, the anti-GREM1 antibody comprises a heavy chain CDR1 (HCDR1) set forth in SEQ ID NO: 1, an HCDR2 set forth in SEQ ID NO: 2, and an HCDR3 set forth in SEQ ID NO: 3, and / or a light chain CDR1 (LCDR1) set forth in SEQ ID NO: 4, an LCDR2 set forth in SEQ ID NO: 5, and an LCDR3 set forth in SEQ ID NO: 6.

[0023] In some embodiments, the anti-GREM1 antibody comprises CDR1, CDR2 and CDR3 of the heavy chain variable region set forth in SEQ ID NO:7, and / or CDR1, CDR2 and CDR3 of the light chain variable region set forth in SEQ ID NO:8.

[0024] In some embodiments, the anti-GREM1 antibody comprises a heavy chain framework region 1 (HFR1) set forth in QX1QLVQSGSELKKPGASVKVSCKASGX2TFT (SEQ ID NO: 24), an HFR2 set forth in WMX3QAPGQGLX4WMG (SEQ ID NO: 25), an HFR3 set forth in RFX5FSLDTSVSTAYLQISSLKAEDTAVYYCAR (SEQ ID NO: 26), and an HFR4 set forth in WGQGTMVTVSS (SEQ ID NO: 17), and / or DVVMTQSPLSLPVTLGQPASI X4 is E or T; X5 is V or A; and X6 is Y or S.

[0025] In some embodiments, the anti-GREM1 antibody comprises heavy chain framework region 1 (HFR1) set forth in SEQ ID NO: 14, 18 or 21, HFR2 set forth in SEQ ID NO: 15, 19 or 22, HFR3 set forth in SEQ ID NO: 16, 20 or 23, and HFR4 set forth in SEQ ID NO: 17, and / or light chain framework region 1 (LFR1) set forth in SEQ ID NO: 27, LFR2 set forth in SEQ ID NO: 28 or 31, LFR3 set forth in SEQ ID NO: 29, and LFR4 set forth in SEQ ID NO: 30.

[0026] In some embodiments, the anti-GREM1 antibody comprises FR1, FR2, FR3 and FR4 of the heavy chain variable region set forth in SEQ ID NO: 7, 11 or 12, and / or FR1, FR2, FR3 and FR4 of the light chain variable region set forth in SEQ ID NO: 8 or 13.

[0027] In some embodiments, the anti-GREM1 antibody comprises a heavy chain variable region set forth in SEQ ID NO:7, 11, or 12, and / or a light chain variable region set forth in SEQ ID NO:8 or 13.

[0028] In some embodiments, the anti-GREM1 antibody comprises a heavy chain constant region set forth in SEQ ID NO:33 and / or a light chain constant region set forth in SEQ ID NO:34.

[0029] In some embodiments, the anti-GREM1 antibody comprises a heavy chain set forth in SEQ ID NO:9 and / or a light chain set forth in SEQ ID NO:10.

[0030] In some embodiments, the pharmaceutical formulation 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 is about 4.5 to 6.5.

[0031] In some embodiments, the concentration of the anti-GREM1 antibody in the pharmaceutical formulation is 20 mg / ml to 40 mg / ml, the concentration of the histidine buffer in the pharmaceutical formulation is 10 mM to 30 mM, the concentration of arginine hydrochloride or sucrose in the pharmaceutical formulation is 1% (w / v) to 10% (w / v), and / or the concentration of polysorbate 80 or polysorbate 20 in the pharmaceutical formulation is 0.025% (w / v) to 0.1% (w / v).

[0032] In some embodiments, the concentration of the anti-GREM1 antibody in the pharmaceutical formulation is 33 mg / ml, the concentration of the histidine buffer in the pharmaceutical formulation is about 20 mM, the stabilizer is sucrose, the concentration of sucrose in the pharmaceutical formulation is 8% (w / v) to 9% (w / v), the surfactant is polysorbate 80, the concentration of polysorbate 80 in the pharmaceutical formulation is about 0.05% (w / v), and the pH value is about 5.5 to 6.5.

[0033] In some embodiments, the pharmaceutical formulation further comprises an antioxidant, wherein the antioxidant is methionine. In some embodiments, the concentration of methionine in the pharmaceutical formulation is about 0.04% (w / v).

[0034] 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, the concentration of sucrose in the pharmaceutical formulation is about 8.2% (w / v), the surfactant is polysorbate 80, the concentration of polysorbate 80 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.

[0035] In another aspect, the disclosure provides the use of a pharmaceutical formulation described herein in the manufacture of a medicament for preventing and / or treating a GREM1-associated disease.

[0036] 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 increased levels of GREM1 and 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.

[0037] In some embodiments, the medicament further comprises a second therapeutic agent, wherein the second therapeutic agent is selected from the group consisting of a chemotherapeutic agent (e.g., cisplatin), a radiotherapeutic agent, an immunotherapeutic agent (e.g., an immune checkpoint modulator, e.g., a PD-1 / PD-L1 axis inhibitor and a TGF-β inhibitor), an anti-angiogenic agent (e.g., an antagonist of VEGFR-1, VEGFR-2, and VEGFR-3), a targeted therapy agent, a cell therapy agent, a gene therapy agent, a hormone therapy agent, a cytokine, and the like. [Brief explanation of the drawings]

[0038] [Figure 1] FIG. 1 shows protein stability data measured by SEC in pH screening experiments using acetate, histidine, and citrate buffer systems, respectively. [Figure 2] FIG. 2 shows protein stability data measured by CEX in pH screening experiments using acetate, histidine, and citrate buffer systems, respectively. [Figure 3] FIG. 3 shows protein stability data measured by SEC in excipient screening experiments using NaCl, arginine hydrochloride, sucrose, sorbitol, trehalose, and mannitol as stabilizers, respectively. [Figure 4] Figure 4 shows protein stability data measured by CEX in an excipient screening experiment using arginine hydrochloride and sucrose as stabilizers, respectively, and methionine as an antioxidant in a histidine-histidine hydrochloride buffer system under different pH conditions. [Figure 5] FIG. 5 shows protein stability data measured by CEX in an excipient screening experiment using arginine hydrochloride and sucrose as stabilizers, respectively, and methionine as an antioxidant in a histidine-histidine hydrochloride buffer system under different pH conditions. [Figure 6] FIG. 6 shows protein stability data measured by SEC in an excipient screening experiment using arginine hydrochloride and sucrose as stabilizers, respectively, and methionine as an antioxidant in a histidine-histidine hydrochloride buffer system under different pH conditions. [Figure 7] Figure 7 shows protein stability data measured by NR CE-SDS in an excipient screening experiment using arginine hydrochloride and sucrose as stabilizers, respectively, and methionine as an antioxidant in a histidine-histidine hydrochloride buffer system under different pH conditions. [Figure 8]FIG. 8 shows protein stability data of the targeted formulations measured by SEC under long-term experimental conditions at 5° C. [Figure 9] Figure 9 shows protein stability data of targeted formulations measured by NR CE-SDS under long-term experimental conditions at 5°C. [Figure 10] FIG. 10 shows protein stability data of targeted formulations measured by CEX under long-term experimental conditions at 5° C. [Figure 11] FIG. 11 shows protein stability data of the targeted formulations measured by SEC under accelerated experimental conditions at 25° C. [Figure 12] FIG. 12 shows protein stability data of targeted formulations measured by NR CE-SDS under accelerated experimental conditions at 25° C. [Figure 13] FIG. 13 shows protein stability data of targeted formulations measured by CEX under accelerated experimental conditions at 25° C. [Figure 14] FIG. 14 shows the protein stability data of the targeted formulations measured by CEX under experimental conditions at 40° C. DETAILED DESCRIPTION OF THE INVENTION

[0039] The following description of the present disclosure is intended solely to describe various embodiments of the present disclosure. The specific examples described should not be construed as limiting the scope of the present disclosure. It should be understood that various equivalent substitutions, modifications, or variations may be made by those skilled in the art without departing from the spirit and essence of the present disclosure, and all such equivalent embodiments are also included herein. All documents cited herein, including publications, patents, and patent applications, are incorporated herein by reference in their entirety. Where a method referred to in this disclosure includes two or more eligible steps, the specified steps may be performed in any order or simultaneously (unless the context precludes this possibility). Furthermore, the method may include one or more of the other steps, which may be performed before any specified step, between two specified steps, or after all specified steps (unless the context precludes this possibility).

[0040] References herein to "about" (a value or range of values) include examples of that value or range of values. For example, "about X" includes "X". In general, the term "about" refers to the value of a variable, any value within the experimental error range of that variable (e.g., within a 95% confidence interval of the mean), or any value within 10% of that variable, based on a larger value. For example, "about X" includes "110% x X", "109% x X", "108% x X", "107% x X", "106% x X", "105% x X", "104% x X", "103% x X", "102% x X", "101% x X", "99% x X", "98% x X", "97% x X", "96% x X", "95% x X", "94% x X", "93% x X", "92% x X", "91% x X", or "90% x X".

[0041] The term "at least" followed by a number is used herein to indicate the beginning of a range beginning with that number (which may be a range with or without an upper limit, depending on the variable specified). For example, "at least 1" refers to a value of 1 or greater than 1. The term "at most" followed by a number is used herein to indicate the end of a range ending with that number (which may be a range with a lower limit of 1 or 0, or a range without a lower limit, depending on the variable specified). For example, "at most 4" refers to a value of 4 or less than 4, and "at most 40%" refers to a value of 40% or less than 40%. In the present disclosure, when a range is stated as "from (a first number) to (a second number)" or "from (a first number) to (a second number)," this indicates that the lower limit of the range is the first number and the upper limit is the second number. For example, 5 to 50 mg / mL refers to a range having a lower limit of 5 mg / mL and an upper limit of 50 mg / mL. As used herein, the terms "less than" or "greater than" a value are inclusive of the value in question.

[0042] As used herein, the term "reference antibody" refers to any existing anti-GREM1 antibody (e.g., 6245P) produced based on the sequence of H4H6245P disclosed in WO2014159010, the entire disclosure of which is incorporated herein by reference.

[0043] antibody As used herein, the term "antibody" includes any immunoglobulin, monoclonal, polyclonal, multivalent, bivalent, monovalent, multispecific, or bispecific antibody that binds to a specific antigen. A complete antibody comprises two heavy chains and two light chains. Each heavy chain comprises a heavy chain variable region (V H ), heavy chain first constant region (C H1 ), heavy chain second constant region (C H2 ) and the third constant region of the heavy chain (C H3 Each light chain consists of a light chain variable region (V L ) and the light chain constant region (C L ) V of the heavy chain H Region and V of the light chainL Each region has three complementary determinant regions (CDRs) interposed between flanking stretches known as framework regions (FRs). The framework regions are more highly conserved than the CDRs and form a scaffold to support the hypervariable loops. The six CDRs of one heavy chain and one light chain together constitute the antigen-binding site of an antibody and determine the specificity of the antibody. The antibodies described herein also include fragments or derivatives that retain the antigen-binding function of the intact antibody. The fragment or derivative has the same antigen-binding specificity as the intact antibody, but the binding affinity of the fragment or derivative for a specific antigen may be the same as or different from the binding affinity of the intact antibody.

[0044] In some embodiments, the antibodies described herein comprise antigen-binding fragments. An antigen-binding fragment refers to one or more types of antibody fragments that retain binding specificity for an antigen. Examples of antigen-binding fragments include: (i) V L , V H , C L and C H1 (ii) a Fab' fragment, which is a monovalent fragment consisting of a Fab domain; (iii) a F(ab')2 fragment, which is a bivalent fragment containing two Fab fragments connected by disulfide bonds in the hinge region; and (iv) a V H and C H1 (v) a single arm V of an antibody; L and V H (vi) dAb fragments containing a single variable domain (Ward et al., Nature 341:544-546 (1989); PCT Publication WO 90 / 05144); (vii) isolated CDRs; and (viii) V fragments directly or via peptide chains. L and V H Examples include, but are not limited to, single-chain Fv fragments (Huston JS et al., Proc Natl Acad Sci USA, 85:5879 (1988)), which are monovalent fragments formed by the connection between domains.

[0045] In some embodiments, the antibodies described herein include chimeric antibodies having portions of the heavy and / or light chains that are identical or homologous to corresponding sequences in antibodies from a particular species or belonging to a particular antibody class or subclass, and the remaining portions of the chains that are identical or homologous to corresponding sequences in antibodies from another class or belonging to another antibody class or subclass, and fragments thereof, provided that they possess the desired functional activity.

[0046] In some embodiments, the antibodies described herein include humanized antibodies. Humanized forms of non-human (e.g., murine) antibodies can be 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 sequence derived from non-human immunoglobulin. In some examples, humanized antibodies replace the amino acid sequences of human CDRs with those of non-human V. H and V L A humanized antibody may be a CDR-grafted antibody, in which amino acid residues of a CDR of a human immunoglobulin have been introduced into the human immunoglobulin (i.e., acceptor antibody) to replace the amino acid sequence of the corresponding non-human CDR. In other examples, the majority of the amino acid sequence of a humanized antibody can be derived from a human immunoglobulin (i.e., acceptor antibody), with the amino acid residues of the acceptor antibody's CDR replaced by amino acid residues of the CDR of a non-human (e.g., mouse, rat, or rabbit) antibody having the desired specificity, affinity, and capacity. Typically, a humanized antibody contains at least one, and generally two, variable domains, with all or substantially all of the CDR sequences derived from a non-human immunoglobulin and all or substantially all of the framework region (FR) sequences derived from a human immunoglobulin. In some examples, residues in the framework regions of the human immunoglobulin variable regions are replaced by corresponding non-human residues. Furthermore, a humanized antibody can contain residues that are found neither in the original antibody nor in the imported CDR or framework region sequences.

[0047] The anti-GREM1 antibodies described herein refer to antibodies capable of specifically binding to Gremlin 1 (GREM1) protein, e.g., humanized 14E3 (hzd 14E3), e.g., Hu14E3_HaLa, Hu14E3_HaLb, Hu14E3_HbLa, Hu14E3_HbLb, Hu14E3_HcLa and Hu14E3_HcLb.

[0048] "Hu14E3_HaLa" as described herein refers to humanized 14E3 comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 7 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 8. In some embodiments, "Hu14E3_HaLa" as described herein further comprises a heavy chain constant region having the amino acid sequence set forth in SEQ ID NO: 33 and a light chain constant region having the amino acid sequence set forth in SEQ ID NO: 34. In some embodiments, "Hu14E3_HaLa" as described herein comprises a heavy chain having the amino acid sequence set forth in SEQ ID NO: 9 and a light chain having the amino acid sequence set forth in SEQ ID NO: 10.

[0049] "Hu14E3_HaLb" as described herein refers to humanized 14E3 comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 7 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 13. In some embodiments, "Hu14E3_HaLb" as described herein further comprises a heavy chain constant region having the amino acid sequence set forth in SEQ ID NO: 33 and a light chain constant region having the amino acid sequence set forth in SEQ ID NO: 34.

[0050] "Hu14E3_HbLa" as described herein refers to humanized 14E3 comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 11 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 8. In some embodiments, "Hu14E3_HbLa" as described herein further comprises a heavy chain constant region having the amino acid sequence set forth in SEQ ID NO: 33 and a light chain constant region having the amino acid sequence set forth in SEQ ID NO: 34.

[0051] "Hu14E3_HbLb" as described herein refers to humanized 14E3 comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 11 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 13. In some embodiments, "Hu14E3_HbLb" as described herein further comprises a heavy chain constant region having the amino acid sequence set forth in SEQ ID NO: 33 and a light chain constant region having the amino acid sequence set forth in SEQ ID NO: 34.

[0052] "Hu14E3_HcLa" as described herein refers to humanized 14E3 comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 12 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 8. In some embodiments, "Hu14E3_HcLa" as described herein further comprises a heavy chain constant region having the amino acid sequence set forth in SEQ ID NO: 33 and a light chain constant region having the amino acid sequence set forth in SEQ ID NO: 34.

[0053] "Hu14E3_HcLb" as described herein refers to humanized 14E3 comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 12 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 13. In some embodiments, "Hu14E3_HcLb" as described herein further comprises a heavy chain constant region having the amino acid sequence set forth in SEQ ID NO: 33 and a light chain constant region having the amino acid sequence set forth in SEQ ID NO: 34.

[0054] The term "Gremlin 1" or "GREM1" refers to Gremlin variant 1 and encompasses Gremlin 1 in different species, such as humans, mice, monkeys, and others. GREM1 is evolutionarily conserved, and the human Gremlin 1 gene (hGREM1) has been mapped to chromosome 15q13-q15 (Topol LZ, et al., (1997) Molecular Cell Biology (Mol. Cell Biol.), 17:4801-4810; Topol LZ, et al., Cytogenetic Cell Genetics (Cytogenet Cell Genet.), 89:79-84). The amino acid sequence of hGREM1 can be accessed in the GenBank database under accession number NP-037504 or in the Uniprot database under accession number O60565. The terms "human Gremlin 1" and "hGREM1" can be used interchangeably in the present disclosure.

[0055] In some embodiments, the anti-GREM1 antibodies described herein comprise a heavy chain variable region (V H ), and a heavy chain variable region (V H ) comprises the CDR1 set forth in SEQ ID NO: 1, the CDR2 set forth in SEQ ID NO: 2 and / or the CDR3 set forth in SEQ ID NO: 3.

[0056] In some embodiments, the anti-GREM1 antibodies described herein comprise a light chain variable region (V L ), and a light chain variable region (V L ) comprises a CDR1 set forth in SEQ ID NO:4, a CDR2 set forth in SEQ ID NO:5 and / or a CDR3 set forth in SEQ ID NO:6.

[0057] In some embodiments, the anti-GREM1 antibodies described herein comprise a heavy chain variable region (V H ) and the light chain variable region (V L ), and a heavy chain variable region (V H) comprises a CDR1 having the amino acid sequence shown in SEQ ID NO: 1, a CDR2 having the amino acid sequence shown in SEQ ID NO: 2, and / or a CDR3 having the amino acid sequence shown in SEQ ID NO: 3, and a light chain variable region (V L ) comprises a CDR1 having the amino acid sequence set forth in SEQ ID NO:4, a CDR2 having the amino acid sequence set forth in SEQ ID NO:5, and / or a CDR3 having the amino acid sequence set forth in SEQ ID NO:6. In some embodiments, the anti-GREM1 antibody comprises a heavy chain CDR1 (HCDR1) set forth in SEQ ID NO:1, an HCDR2 set forth in SEQ ID NO:2, and an HCDR3 set forth in SEQ ID NO:3, and a light chain CDR1 (LCDR1) set forth in SEQ ID NO:4, an LCDR2 set forth in SEQ ID NO:5, and an LCDR3 set forth in SEQ ID NO:6. In some embodiments, the anti-GREM1 antibody comprises CDR1, CDR2, and CDR3 of a heavy chain variable region set forth in SEQ ID NO:7, and CDR1, CDR2, and CDR3 of a light chain variable region set forth in SEQ ID NO:8.

[0058] In some embodiments, the anti-GREM1 antibodies described herein have a heavy chain framework region 1 (HFR1) set forth in QX1QLVQSGSELKKPGASVKVSCKASGX2TFT (SEQ ID NO: 24), a HFR2 set forth in WMX3QAPGQGLX4WMG (SEQ ID NO: 25), a HFR3 set forth in RFX5FSLDTSVSTAYLQISSLKAEDTAVYYCAR (SEQ ID NO: 26), and a HFR4 set forth in WGQGTMVTVSS (SEQ ID NO: 17), and a HFR5 set forth in DVVMTQSPLSLPVTLGQ X4 is E or T; X5 is V or A; and X6 is Y or S.

[0059] In some embodiments, the anti-GREM1 antibodies described herein comprise a heavy chain framework region 1 (HFR1) set forth in SEQ ID NO: 14, 18, or 21, an HFR2 set forth in SEQ ID NO: 15, 19, or 22, an HFR3 set forth in SEQ ID NO: 16, 20, or 23, and an HFR4 set forth in SEQ ID NO: 17, and a light chain framework region 1 (LFR1) set forth in SEQ ID NO: 27, an LFR2 set forth in SEQ ID NO: 28 or 31, an LFR3 set forth in SEQ ID NO: 29, and an LFR4 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 set forth in SEQ ID NO: 7, 11, or 12, and FR1, FR2, and FR3 of the light chain variable region set forth in SEQ ID NO: 8 or 13.

[0060] In some embodiments, the anti-GREM1 antibodies described herein comprise a heavy chain variable region (V) having the amino acid sequence set forth in SEQ ID NO: 7, 11, or 12. H In some embodiments, the anti-GREM1 antibodies described herein comprise a light chain variable region (V) having the amino acid sequence set forth in SEQ ID NO: 8 or 13. L In some embodiments, the anti-GREM1 antibodies described herein comprise a heavy chain variable region (V) having the amino acid sequence set forth in SEQ ID NO: 7, 11, or 12. H ), and a light chain variable region (V) having the amino acid sequence set forth in SEQ ID NO: 8 or 13 L In some embodiments, the anti-GREM1 antibodies described herein comprise a heavy chain variable region set forth in SEQ ID NO: 7, 11, or 12, and a light chain variable region set forth in SEQ ID NO: 8 or 13.

[0061] In some embodiments, the anti-GREM1 antibodies described herein further comprise an immunoglobulin constant region. In some embodiments, the immunoglobulin constant region comprises a heavy chain constant region and / or a light chain constant region. The heavy chain constant region comprises a C H1 , C H1 ~C H2 or C H1 ~C H3 The light chain constant region comprises the CL In some embodiments, the anti-GREM1 antibodies described herein comprise a heavy chain constant region set forth in SEQ ID NO:33 and a light chain constant region set forth in SEQ ID NO:34.

[0062] In some embodiments, the anti-GREM1 antibodies described herein comprise a heavy chain having the amino acid sequence set forth in SEQ ID NO: 9 and a light chain having the amino acid sequence set forth in SEQ ID NO: 10. In some embodiments, the anti-GREM1 antibodies comprise a heavy chain set forth in SEQ ID NO: 9 and a light chain set forth in SEQ ID NO: 10.

[0063] Exemplary amino acid sequences for use in some embodiments are listed in Table A below.

[0064] [Table A-1] [Table A-2]

[0065] [Table B]

[0066] The present disclosure relates to pharmaceutical formulations containing 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 formulation of the present disclosure is 1 mg / ml to 200 mg / ml, 1 mg / ml to 190 mg / ml, 10 mg / ml to 190 mg / ml, 20 mg / ml to 180 mg / ml, 20 mg / ml to 170 mg / ml, 20 mg / ml to 160 mg / ml, 20 mg / ml to 150 mg / ml, 20 mg / ml to 140 mg / ml, 20 mg / ml to 130 mg / ml, or 20 mg / ml to 240 mg / ml. The concentration of the anti-GREM1 antibody may be 0 mg / ml, 20 mg / ml to 120 mg / ml, 20 mg / ml to 110 mg / ml, 20 mg / ml to 100 mg / ml, 20 mg / ml to 90 mg / ml, 20 mg / ml to 80 mg / ml, 20 mg / ml to 70 mg / ml, 20 mg / ml to 60 mg / ml, 20 mg / ml to 50 mg / ml, 20 mg / ml to 40 mg / ml, or 20 mg / ml to 30 mg / ml. In some embodiments, the concentration of the anti-GREM1 antibody is any concentration value within the above-mentioned ranges. For example, depending on the requirements, the concentration of the anti-GREM1 antibody in the pharmaceutical formulation can 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 at most 200 mg / ml. In some embodiments, the concentration of the anti-GREM1 antibody in the pharmaceutical formulation is 21 mg / ml. In some embodiments, the concentration of the anti-GREM1 antibody in the pharmaceutical formulation is 33 mg / ml.

[0067] buffer The term "buffer" generally refers to a buffered solution that resists changes in pH through the action of its acid-base conjugate components. As used herein, "buffer" refers to a complex solution known to be safe when used in pharmaceutical formulations to maintain or control the pH of the formulation within a desired range. Acceptable buffers capable of controlling the pH within a range from slightly acidic to slightly alkaline pH values (e.g., pH values between 4.5 and 8.0) include, but are not limited to, succinate buffer, citrate buffer, phosphate buffer, acetate buffer, arginine buffer, 2-amino-2-hydroxymethyl-1,3-propanediol (TRIS) buffer, histidine buffer, and others, either alone or in any combination.

[0068] The buffer in the formulation solvent can be prepared using any suitable method known in the art. In some embodiments, the buffer of the present disclosure can be prepared using a specific acid-base ion pair. In one example, the excipient of the acid-base ion pair can be accurately weighed and added to pure water that is approximately 60% of the volume of the target buffer, mixed uniformly, and then the pH value of the resulting solution can be determined. If the pH value deviates from the target value, the pH value can be adjusted using an appropriate ion pair. The solution is then diluted with pure water to reach the target weight or volume. Finally, the conductivity, osmolality, and pH value of the solution are measured for verification.

[0069] The stable pharmaceutical formulations of the present disclosure can include a buffer such that the pharmaceutical formulation has a pH value of 4.5 to 8.0, such as 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 is used such that the pharmaceutical formulation has a pH value of 4.5 to 6.5. In particular, the pH value of the pharmaceutical formulations of the present disclosure can be any pH value within the pH ranges listed above, 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, 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.

[0070] Examples of buffers that can control the pH value of pharmaceutical formulations within a desired range include acetate buffers, histidine buffers, citrate buffers, and other organic acid buffers or inorganic acid buffers. Any one of these buffers can be used alone, or two or more of these buffers can be combined for use. Preferably, the pharmaceutical formulation of the present disclosure includes acetate buffers and histidine buffers. More preferably, the pharmaceutical formulation of the present disclosure includes histidine buffers.

[0071] "Acetate buffer" refers to a buffer containing acetate radical ions. Acetate buffers can include one or more of acetic acid (e.g., glacial acetic acid), potassium acetate, sodium acetate (e.g., sodium acetate trihydrate), and others. In some embodiments, the acetate buffer is an acetic acid-sodium acetate buffer, such as glacial acetic acid-sodium acetate trihydrate buffer. In some embodiments, the pH value of the acetate buffer can be any pH value within 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.

[0072] "Histidine buffer" refers to a buffer containing histidine radical ions. Histidine buffers can include one or more of histidine, histidine hydrochloride (e.g., histidine hydrochloride monohydrate), histidine acetate, histidine phosphate, histidine sulfate, and the like. In some embodiments, the histidine buffer can be a histidine-histidine hydrochloride buffer. In some embodiments, the pH value of the histidine buffer can be any pH value within 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.

[0073] "Citrate buffer" refers to a buffer containing citrate radical ions. Citrate buffers can include one or more of citric acid, monosodium citrate, disodium citrate, trisodium citrate, monopotassium citrate, dipotassium citrate, tripotassium citrate, sodium chloride, potassium chloride, and the like. In some embodiments, the citrate buffer is a citric acid-trisodium citrate buffer. In some embodiments, the pH value of the citrate buffer can be any pH value within 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.

[0074] In some embodiments, the buffer used in the pharmaceutical formulation of the present disclosure is an acetate buffer or a histidine buffer, and the pH value of the pharmaceutical formulation of the present disclosure is 4.5 to 6.5. Without wishing to be bound by theory, acetate buffers and histidine buffers are thought to be superior to citrate buffers. For example, when the buffer of the pharmaceutical formulation of the present disclosure is an acetate buffer or a histidine buffer, the anti-GREM1 antibody in the pharmaceutical formulation is more stable (e.g., more stable at high temperatures), the force between the antibody molecules is repulsive rather than attractive, and the risk of molecular aggregation is lower.

[0075] The buffer concentration described herein refers to the concentration of buffer ions in the buffer. In some embodiments, the concentration of a suitable buffer used in the pharmaceutical formulation of the present disclosure may be 5 mM to 50 mM, 10 mM to 50 mM, 10 mM to 40 mM, 10 mM to 30 mM, or 10 mM to 20 mM. In some embodiments, the buffer concentration is any concentration value within the above range. For example, depending on the specific buffer and the stability required for the pharmaceutical formulation, the buffer concentration may be 5 mM, at least 10 mM, at least 15 mM, at least 20 mM, at least 25 mM, at least 30 mM, at least 35 mM, at least 40 mM, at least 45 mM, and / or at most 50 mM.

[0076] In some embodiments, the buffer used in the pharmaceutical formulation of the present disclosure is an acidic buffer, such as an acetic acid-sodium acetate buffer, and may have a concentration of 5 mM to 50 mM. In some embodiments, the concentration of the acidic buffer may be 5 mM to 50 mM, 5 mM to 45 mM, 5 mM to 40 mM, 5 mM to 35 mM, 5 mM to 30 mM, 5 mM to 25 mM, 5 mM to 20 mM, 5 mM to 15 mM, or 5 mM to 10 mM.

[0077] In some embodiments, the buffer used in the pharmaceutical formulations of the present disclosure is a histidine buffer, such as histidine-histidine hydrochloride buffer, and the concentration can be 5 mM to 50 mM. In some embodiments, the concentration of the histidine buffer can be 5 mM to 50 mM, 5 mM to 45 mM, 5 mM to 40 mM, 5 mM to 35 mM, 5 mM to 30 mM, 5 mM to 25 mM, 5 mM to 20 mM, 5 mM to 15 mM, and 5 mM to 10 mM.

[0078] In some embodiments, the buffer used in the pharmaceutical formulation of the present disclosure is an acidic acid buffer or a histidine buffer, and has a concentration of 5 mM to 50 mM. In some embodiments, the buffer used in the pharmaceutical formulation of the present disclosure is an acidic acid buffer or a histidine buffer, and has a concentration of 10 mM to 30 mM. In some embodiments, the buffer used in the pharmaceutical formulation of the present disclosure is an acidic acid buffer or a histidine buffer, and has a concentration of about 20 mM. In some embodiments, the buffer used in the pharmaceutical formulation of the present disclosure is a histidine buffer, and has a concentration of about 20 mM.

[0079] stabilizers As used herein, the term "stabilizer" refers to an agent capable of preventing or reducing the chemical and / or physical instability of a protein of interest when bound to the protein. Examples of stabilizers include sugars, alcohols, acids, salts, polymers, and the like. Examples of sugars include glucose, sucrose, trehalose, lactose, glucans, 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.

[0080] In some embodiments, the stabilizer used in the pharmaceutical formulation of the present disclosure is selected from sugars.In some embodiments, the stabilizer used in the pharmaceutical formulation of the present disclosure is selected from alcohols.In some embodiments, the stabilizer used in the pharmaceutical formulation of the present disclosure is not mannitol.In some embodiments, the stabilizer used in the pharmaceutical formulation of the present disclosure is selected from salts.In some embodiments, the stabilizer used in the pharmaceutical formulation of the present disclosure is selected from amino acids.In some embodiments, the stabilizer used in the pharmaceutical formulation of the present disclosure is selected from sucrose, trehalose, sorbitol, NaCl, arginine hydrochloride, or a combination thereof.

[0081] In some embodiments, the concentration of stabilizer used in the pharmaceutical formulations of the present disclosure can be 0.5% (w / v) to 20% (w / v), 0.5% (w / v) to 10% (w / v), 0.5% (w / v) to 5% (w / v), 0.5% (w / v) to 2.5% (w / v), 0.5% (w / v) to 1.5% (w / v), 1% (w / v) to 15% (w / v), 1% (w / v) to 10% (w / v), 1% (w / v) to 5% (w / v), 5% (w / v) to 10% (w / v), 2% (w / v) to 8% (w / v), or 2% (w / v) to 5% (w / v), depending on the specific stabilizer and the stability desired for the pharmaceutical formulation. In some embodiments, the stabilizer concentration is any concentration value within the above ranges.

[0082] In some embodiments, the stabilizer used in the pharmaceutical formulation of the present disclosure is sucrose, and the concentration of sucrose in the pharmaceutical formulation may be 0.5% (w / v) to 20% (w / v). In some embodiments, the concentration of sucrose in the pharmaceutical formulation may be 0.5% (w / v) to 10% (w / v), 1% (w / v) to 10% (w / v), 10% (w / v) to 20% (w / v), or 5% (w / v) to 15% (w / v). In some embodiments, the concentration of sucrose in the pharmaceutical formulation is 1% (w / v) to 10% (w / v). In some embodiments, the concentration of sucrose in the pharmaceutical formulation is about 5% (w / v). In some embodiments, the concentration of sucrose in the pharmaceutical formulation is about 8.2% (w / v). In some embodiments, the concentration of sucrose in the pharmaceutical formulation is about 9% (w / v).

[0083] In some embodiments, the stabilizer used in the pharmaceutical formulation of the present disclosure is arginine hydrochloride, and the concentration of arginine hydrochloride in the pharmaceutical formulation may be 0.5% (w / v) to 20% (w / v). In some embodiments, the concentration of arginine hydrochloride in the pharmaceutical formulation may be 0.5% (w / v) to 10% (w / v), 1 to 10% (w / v), 10 to 20% (w / v), or 5 to 15% (w / v). In some embodiments, the concentration of arginine hydrochloride in the pharmaceutical formulation is 1% (w / v) to 5% (w / v). In some embodiments, the concentration of arginine hydrochloride in the pharmaceutical formulation is about 3% (w / v).

[0084] In some embodiments, the stabilizer used in the pharmaceutical formulation of the present disclosure is trehalose, and the concentration of trehalose in the pharmaceutical formulation may be 0.5% (w / v) to 20% (w / v). In some embodiments, the concentration of trehalose in the pharmaceutical formulation may be 0.5% (w / v) to 10% (w / v), 1% (w / v) to 10% (w / v), 10% (w / v) to 20% (w / v), or 5% (w / v) to 15% (w / v). In some embodiments, the concentration of trehalose in the pharmaceutical formulation is 5% (w / v) to 10% (w / v). In some embodiments, the concentration of trehalose in the pharmaceutical formulation is about 9% (w / v).

[0085] In some embodiments, the stabilizer used in the pharmaceutical formulation of the present disclosure is sorbitol, and the concentration of sorbitol in the pharmaceutical formulation may be 0.5% (w / v) to 20% (w / v). In some embodiments, the concentration of sorbitol in the pharmaceutical formulation may be 0.5% (w / v) to 10% (w / v), 1% (w / v) to 10% (w / v), 10% (w / v) to 20% (w / v), or 5% (w / v) to 15% (w / v). In some embodiments, the concentration of sorbitol in the pharmaceutical formulation is 2% (w / v) to 8% (w / v). In some embodiments, the concentration of sorbitol in the pharmaceutical formulation is about 5% (w / v).

[0086] In some embodiments, the stabilizer used in the pharmaceutical formulation of the present disclosure is NaCl, and the concentration of NaCl in the pharmaceutical formulation may be 0.5% (w / v) to 20% (w / v). In some embodiments, the concentration of NaCl in the pharmaceutical formulation may be 0.5% (w / v) to 10% (w / v), 1% (w / v) to 10% (w / v), 10% (w / v) to 20% (w / v), or 5% (w / v) to 15% (w / v). In some embodiments, the concentration of NaCl in the pharmaceutical formulation is 0.5% (w / v) to 1.5% (w / v). In some embodiments, the concentration of NaCl in the pharmaceutical formulation is about 0.9% (w / v).

[0087] surfactant As used herein, the term "surfactant" refers to an organic substance having an amphiphilic structure, possessing both hydrophilic and hydrophobic properties; in other words, the organic substance contains groups with opposite solubility tendencies, such as oil-soluble hydrocarbon chains and water-soluble ionic groups. Depending on the charge of the surfactant moiety, surfactants can include anionic surfactants, cationic surfactants, and nonionic surfactants.

[0088] Exemplary surfactants include polysorbates (e.g., polysorbate 20 or 80), poloxamers (e.g., poloxamer 188), Triton, polyethylene glycol, polypropylene glycol, and copolymers of ethylene glycol and propylene glycol (e.g., Pluronic, PF68, etc.). In some embodiments, the surfactant used in the pharmaceutical formulations of the present disclosure is selected from polysorbate 20 (also known as PS 20 or Tween 20), polysorbate 80 (also known as PS 80 or Tween 80), or a combination thereof.

[0089] In some embodiments, the concentration of surfactant used in the pharmaceutical formulations of the present disclosure can 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 particular surfactant and the stability desired for the pharmaceutical formulation. In some embodiments, the surfactant concentration is any concentration value within the above ranges.

[0090] In some embodiments, the surfactant(s) used in the pharmaceutical formulations of the present disclosure are polysorbate 20 and / or polysorbate 80, and the concentration of polysorbate 20 and / or polysorbate 80 in the pharmaceutical formulation 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 formulation 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 formulation 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 formulation may be about 0.05% (w / v).

[0091] In some embodiments, the surfactant used in the pharmaceutical formulation of the present disclosure is polysorbate 80. In some embodiments, the concentration of polysorbate 80 in the pharmaceutical formulation is about 0.05% (w / v).

[0092] antioxidants As used herein, the term "antioxidant" refers to a substance capable of retarding the oxidation of pharmaceutical formulations by oxygen. Antioxidants are used to affect various stages of the autoxidation process from different aspects, such as achieving the effects of reducing agents, blockers, synergists, and / or chelators, and providing electrons or available hydrogen atoms for free radicals, so that the autoxidation chain reaction is interrupted, thereby reducing protein oxidation. Exemplary antioxidants include, but are not limited to, methionine, cysteine, glutathione, sodium thiosulfate, and ascorbic acid. In some embodiments, the antioxidant used in the pharmaceutical formulations of the present disclosure may be methionine.

[0093] In some embodiments, the concentration of antioxidant used in the pharmaceutical formulations of the present disclosure can be 0.01% (w / v) to 0.2% (w / v), 0.01% (w / v) to 0.1% (w / v), or 0.02% (w / v) to 0.06% (w / v), depending on the particular antioxidant and the stability desired for the pharmaceutical formulation. In some embodiments, the concentration of the antioxidant is any concentration value within the above ranges.

[0094] In some embodiments, the antioxidant used in the pharmaceutical formulation of the present disclosure is methionine, and the concentration of methionine in the pharmaceutical formulation is 0.01% (w / v) to 0.2% (w / v). In some embodiments, the antioxidant used in the pharmaceutical formulation of the present disclosure is methionine, and the concentration of methionine in the pharmaceutical formulation is 0.02% (w / v) to 0.06% (w / v). In some embodiments, the antioxidant used in the pharmaceutical formulation of the present disclosure is methionine, and the concentration of methionine in the pharmaceutical formulation is about 0.04% (w / v).

[0095] Other materials The pharmaceutical formulations of the present disclosure may further contain other excipients, as needed, including, but not limited to, isotonicity agents, diluents, and the like.

[0096] The term "isotonicity agent" refers to a compound or composition that provides a drug with an appropriate osmotic tension to prevent the net flow of water through the cell membrane in contact with the drug. In some embodiments, the pharmaceutical formulation of the present disclosure has the same osmotic pressure as human blood. Suitable isotonicity agents include, but are not limited to, glycerol, amino acids or proteins (e.g., glycine or albumin), salts (e.g., sodium chloride), and sugars (e.g., glucose, sucrose, and lactose).

[0097] The term "diluent" refers to a pharmaceutically acceptable reagent that can be used to dilute the pharmaceutical formulation of the present disclosure. Typical diluents include water, saline, bacteriostatic agents for injection, pH buffers, sterile salt solutions, Ringer's solution, or glucose solutions.

[0098] formulation In one aspect, the present disclosure provides a stable pharmaceutical formulation comprising an anti-GREM1 antibody (e.g., a specific anti-GREM1 antibody provided by the present disclosure), a buffer, a stabilizer, and a surfactant. The pharmaceutical formulation has a pH value of 4.5 to 8.0. In some embodiments, the pH value is 4.5 to 6.5 to achieve suitable stability.

[0099] In some embodiments, the pharmaceutical formulation of the present disclosure comprises: (i) an anti-GREM1 antibody (e.g., a specific anti-GREM1 antibody provided by the present disclosure), wherein the concentration of the anti-GREM1 antibody is between 1 mg / ml and 200 mg / ml, preferably between 20 mg / ml and 40 mg / ml, and more preferably about 21 mg / ml or 33 mg / ml; and / or (ii) a buffer, preferably an acetate buffer or a histidine buffer, more preferably a histidine buffer, and the concentration of the buffer in the pharmaceutical formulation is 5 mmol / L to 100 mmol / L, preferably 5 mmol / L to 50 mmol / L or 10 mmol / L to 30 mmol / L, more preferably about 20 mmol / L; and / or (iii) a stabilizer, preferably sucrose, trehalose, sorbitol, NaCl, or arginine hydrochloride; (a) the stabilizer is preferably sucrose, and the concentration of the stabilizer in the pharmaceutical formulation is 0.5% (w / v) to 20% (w / v), preferably 1% (w / v) to 10% (w / v), more preferably about 5% (w / v), about 8.2% (w / v), or about 9% (w / v); or (b) the stabilizer is preferably arginine hydrochloride, and the concentration of the stabilizer in the pharmaceutical formulation is 0.5% (w / v) to 20% (w / v), preferably 1% (w / v) to 5% (w / v), more preferably about 3% (w / v); or (c) the stabilizer is preferably trehalose, and the concentration of the stabilizer in the pharmaceutical formulation is 0.5% (w / v) to 20% (w / v), preferably 5% (w / v) to 10% (w / v), more preferably about 9% (w / v); or (d) the stabilizer is preferably sorbitol, and the concentration of the stabilizer in the pharmaceutical formulation is 0.5% (w / v) to 20% (w / v), preferably 2% (w / v) to 8% (w / v), more preferably about 5% (w / v); or (e) a stabilizer, preferably NaCl, having a concentration of 0.5% (w / v) to 20% (w / v), preferably 0.5% (w / v) to 1.5% (w / v), more preferably about 0.9% (w / v); and / or (iv) a surfactant, wherein the surfactant is preferably polysorbate 80 or polysorbate 20, more preferably polysorbate 80, and the concentration of the surfactant in the pharmaceutical formulation 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 (v) an antioxidant, wherein the antioxidant is preferably methionine, cysteine, glutathione, sodium thiosulfate, and ascorbic acid, more preferably methionine, and the concentration of the antioxidant in the pharmaceutical formulation 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); The pharmaceutical preparation has a pH value of about 4.5 to 6.5, preferably about 5.5 to 6.5.

[0100] In some embodiments, a pharmaceutical formulation of the present disclosure comprises an anti-GREM1 antibody (e.g., a specific anti-GREM1 antibody provided by the present disclosure) having a concentration of about 1 mg / ml to 200 mg / ml, and an acetate buffer or histidine buffer having a concentration of about 5 mmol / L to 50 mmol / L, and the pH of the pharmaceutical formulation is 4.5 to 6.5. In some embodiments, the pharmaceutical formulation comprises an anti-GREM1 antibody having a concentration of about 20 mg / ml to 40 mg / ml. In some embodiments, the pharmaceutical formulation comprises an acetate buffer or histidine buffer having a concentration of about 10 mg / ml to 30 mg / ml.

[0101] In some embodiments, the pharmaceutical formulation of the present disclosure comprises an anti-GREM1 antibody (e.g., a specific anti-GREM1 antibody provided by the present disclosure) having a concentration of about 1 mg / ml to 200 mg / ml, an acetate buffer or histidine buffer having a concentration of about 5 mmol / L to 50 mmol / L, and sucrose, trehalose, sorbitol, NaCl, or arginine hydrochloride having a concentration of about 0.5% (w / v) to 20% (w / v), and the pH of the pharmaceutical formulation is 4.5 to 6.5. In some embodiments, the pharmaceutical formulation comprises an anti-GREM1 antibody having a concentration of about 20 mg / ml to 40 mg / ml. In some embodiments, the pharmaceutical formulation comprises an acetate buffer or histidine buffer having a concentration of about 10 mg / ml to 30 mg / ml. In some embodiments, the pharmaceutical formulation comprises sucrose having a concentration of about 1% (w / v) to 10% (w / v). In some embodiments, the pharmaceutical formulation comprises arginine hydrochloride at a concentration of about 1% (w / v) to 5% (w / v). In some embodiments, the pharmaceutical formulation comprises trehalose at a concentration of about 5% (w / v) to 10% (w / v). In some embodiments, the pharmaceutical formulation comprises sorbitol at a concentration of about 2% (w / v) to 8% (w / v). In some embodiments, the pharmaceutical formulation comprises NaCl at a concentration of about 0.5% (w / v) to 1.5% (w / v).

[0102] In some embodiments, the pharmaceutical formulation of the present disclosure comprises an anti-GREM1 antibody (e.g., a specific anti-GREM1 antibody provided by the present disclosure) having a concentration of about 1 mg / ml to 200 mg / ml, an acetate buffer or histidine buffer having a concentration of about 5 mg / ml to 50 mmol / L, sucrose, trehalose, sorbitol, NaCl, or arginine hydrochloride having a concentration of about 0.5% (w / v) to 20% (w / v), and polysorbate 80 or polysorbate 20 having a concentration of about 0.005% (w / v) to 0.4% (w / v), and the pH of the pharmaceutical formulation is 4.5 to 6.5. In some embodiments, the pharmaceutical formulation comprises an anti-GREM1 antibody having a concentration of about 20 mg / ml to 40 mg / ml. In some embodiments, the pharmaceutical formulation comprises an acetate buffer or histidine buffer having a concentration of about 10 mg / ml to 30 mg / ml. In some embodiments, the pharmaceutical formulation comprises sucrose at a concentration of about 1% to 10% (w / v), arginine hydrochloride at a concentration of about 1% to 5% (w / v), trehalose at a concentration of about 5% to 10% (w / v), sorbitol at a concentration of about 2% to 8% (w / v), or NaCl at a concentration of about 0.5% to 1.5% (w / v). In some embodiments, the pharmaceutical formulation comprises polysorbate 80 or polysorbate 20 at a concentration of about 0.025% to 0.1% (w / v).

[0103] In some embodiments, the pharmaceutical formulation of the present disclosure comprises an anti-GREM1 antibody (e.g., a specific anti-GREM1 antibody provided herein) having a concentration of about 1 mg / ml to 200 mg / ml, an acetate buffer or histidine buffer having a concentration of about 5 mmol / L to 50 mmol / L, sucrose, trehalose, sorbitol, NaCl, or arginine hydrochloride having a concentration of about 0.5% (w / v) to 20% (w / v), polysorbate 80 or polysorbate 20 having a concentration of about 0.005% (w / v) to 0.4% (w / v), and methionine having a concentration of about 0.01% (w / v) to 0.2% (w / v), and the pH of the pharmaceutical formulation is 4.5 to 6.5. In some embodiments, the pharmaceutical formulation comprises an anti-GREM1 antibody having a concentration of about 20 mg / ml to 40 mg / ml. In some embodiments, the pharmaceutical formulation comprises an acetate buffer or histidine buffer at a concentration of about 10 mg / ml to 30 mg / ml. In some embodiments, the pharmaceutical formulation comprises sucrose at a concentration of about 1% (w / v) to 10% (w / v), arginine hydrochloride at a concentration of about 1% (w / v) to 5% (w / v), trehalose at a concentration of about 5% (w / v) to 10% (w / v), sorbitol at a concentration of about 2% (w / v) to 8% (w / v), or NaCl at a concentration of about 0.5% (w / v) to 1.5% (w / v). In some embodiments, the pharmaceutical formulation 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 formulation comprises methionine having a concentration of about 0.02% (w / v) to 0.06% (w / v).

[0104] In some embodiments, the pharmaceutical formulation of the present disclosure comprises an anti-GREM1 antibody having a concentration of about 20 mg / ml to 40 mg / ml, a histidine buffer having a concentration of about 10 mmol / L to 30 mmol / L, arginine hydrochloride having a concentration of about 1% (w / v) to 10% (w / v), and polysorbate 80 or polysorbate 20 having a concentration of about 0.025% (w / v) to 0.1% (w / v), and the pH value of the pharmaceutical formulation is about 4.5 to 6.5 or about 5.5 to 6.5.

[0105] In some embodiments, the pharmaceutical formulation of the present disclosure comprises an anti-GREM1 antibody having a concentration of about 20 mg / ml to 40 mg / ml, a histidine buffer having a concentration of about 10 mg / ml to 30 mmol / L, arginine hydrochloride having a concentration of about 1% (w / v) to 10% (w / v), polysorbate 80 or polysorbate 20 having a concentration of about 0.025% (w / v) to 0.1% (w / v), and methionine having a concentration of about 0.01% (w / v) to 0.2% (w / v), and the pH value of the pharmaceutical formulation is about 4.5 to 6.5 or about 5.5 to 6.5.

[0106] In some embodiments, the pharmaceutical formulation of the present disclosure comprises an anti-GREM1 antibody having a concentration of about 20 mg / ml to 40 mg / ml, a histidine buffer having a concentration of about 10 mmol / L to 30 mmol / L, sucrose having a concentration of about 1% (w / v) to 10% (w / v), and polysorbate 80 or polysorbate 20 having a concentration of about 0.025% (w / v) to 0.1% (w / v), and the pH value of the pharmaceutical formulation is about 4.5 to 6.5 or about 5.5 to 6.5.

[0107] In some embodiments, the pharmaceutical formulation of the present disclosure comprises an anti-GREM1 antibody having a concentration of about 20 mg / ml to 40 mg / ml, a histidine buffer having a concentration of about 10 mmol / L to 30 mmol / L, sucrose having a concentration of about 1% (w / v) to 10% (w / v), polysorbate 80 or polysorbate 20 having a concentration of about 0.025% (w / v) to 0.1% (w / v), and methionine having a concentration of about 0.01% (w / v) to 0.2% (w / v), and the pH value of the pharmaceutical formulation is about 4.5 to 6.5 or about 5.5 to 6.5.

[0108] In some embodiments, the pharmaceutical formulation of the present disclosure comprises an anti-GREM1 antibody having a concentration of about 33 mg / ml, a histidine buffer having a concentration of about 20 mmol / L, sucrose having a concentration of about 8% (w / v) to 9% (w / v), and polysorbate 80 having a concentration of about 0.05% (w / v), and the pH value of the pharmaceutical formulation is about 5.5 to 6.5.

[0109] In some embodiments, the pharmaceutical formulation of the present disclosure comprises an anti-GREM1 antibody having a concentration of about 33 mg / ml, a histidine buffer having a concentration of about 20 mmol / L, sucrose having a concentration of about 8.2% (w / v), polysorbate 80 having a concentration of about 0.05% (w / v), and methionine having a concentration of about 0.04% (w / v), and the pH value of the pharmaceutical formulation is about 6.0.

[0110] Polymer formation due to chemical degradation or aggregation of antibody molecules, or deglycosylation, glycosylation modification, oxidation, or other structural modifications of antibody molecules, which can reduce at least one functional activity of the monomeric protein, can lead to instability of antibody formulations. For pharmaceutical formulations containing anti-GREM1 antibodies, the anti-GREM1 antibodies may be chemically degraded during storage of the pharmaceutical formulation, resulting in a decrease in antibody concentration. Anti-GREM1 antibodies may aggregate to form polymers that may be insoluble in the form of polymer 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 polymeric antibodies will result in a decrease in the purity of the monomeric antibody. Furthermore, the formation of insoluble polymers may increase the turbidity of the pharmaceutical formulation.

[0111] In some embodiments, pharmaceutical formulations comprising the anti-GREM1 antibodies of the present disclosure can remain stable after long-term storage, treatment (e.g., storage) at elevated temperatures, room temperature, vibration, and / or multiple freeze-thaw cycles, where the physical and / or chemical stability and / or functional activity of the anti-GREM1 antibody remain relatively constant over time. In some embodiments, antibody protein concentration, protein purity, protein activity, formulation pH, formulation osmolality, formulation appearance, insoluble particles in the formulation, and the like can be used as indicators of pharmaceutical formulation stability. Various analytical techniques for determining protein stability are available in the art and are described in Peptide and Protein Drug Delivery, 247-301, edited by Vincent Lee, Marcel Dekker Inc., New York, New York Press (1991) and Jones, A. Adv. Drug Delivery Rev. 10:29-90 (1993).

[0112] In some embodiments, the stability of a pharmaceutical formulation can be determined over a selected period of time under selected conditions 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 value determination, protein concentration determination, visual inspection, and microfluidic imaging (MFI).

[0113] As shown in the examples of the present disclosure, pharmaceutical formulations comprising the anti-GREM1 antibodies provided herein have high stability by visual inspection, for example, high stability under long-term storage, high stability at high temperatures (e.g., 40°C) or room temperature, high stability after shaking, and high stability after freezing and thawing.

[0114] In some embodiments, a stable pharmaceutical formulation refers to one in which, in a DLS test for the pharmaceutical formulation, the protein particle size does not change significantly during storage; the KD value is positive, in other words, the force of interaction between single antibody molecules is repulsive, and aggregation of single antibody molecules does not occur. As shown in the examples of the present disclosure, pharmaceutical formulations containing anti-GREM1 antibodies provided herein have high stability in DLS tests, for example, high stability under long-term storage, high stability at high temperatures (e.g., 40°C) or room temperature, high stability after shaking, and high stability after freezing and thawing.

[0115] In some embodiments, a stable pharmaceutical formulation refers to a pharmaceutical formulation in which, in an SEC test for the pharmaceutical formulation, only a small amount of protein is degraded during storage of the pharmaceutical formulation, and the content of high polymers or oligomers increases slowly. As shown in the examples of the present disclosure, the pharmaceutical formulations comprising the anti-GREM1 antibodies provided herein have high stability as shown in SEC tests, for example, high stability under long-term storage, high stability at high temperatures (e.g., 40°C) or room temperature, high stability after shaking, and high stability after freezing and thawing.

[0116] In some embodiments, a stable pharmaceutical formulation refers to a pharmaceutical formulation in which, in a CEX test of the pharmaceutical formulation, the charge heterogeneity of the pharmaceutical formulation does not change significantly during storage, and the resolution of the acidic and alkaline peaks does not change significantly. As shown in the examples of the present disclosure, pharmaceutical formulations comprising the anti-GREM1 antibodies provided herein have high stability as shown in CEX tests, for example, high stability under long-term storage, high stability at high temperatures (e.g., 40°C), and high stability after freeze-thawing.

[0117] In some embodiments, a stable pharmaceutical formulation refers to one in which, in an NR CE-SDS test of the pharmaceutical formulation, only a small amount of protein is degraded during storage of the pharmaceutical formulation, and the content of high polymers or oligomers increases slowly. As shown in the examples of the present disclosure, pharmaceutical formulations containing anti-GREM1 antibodies provided herein have high stability as shown in an NR CE-SDS test, for example, high stability under long-term storage, high stability at high temperatures (e.g., 40°C) or room temperature, high stability after shaking, and high stability after freeze-thawing.

[0118] In some embodiments, a stable pharmaceutical formulation refers to one in which, upon visual inspection of the pharmaceutical formulation, no obvious change in appearance of the pharmaceutical formulation is observed during storage, and the pharmaceutical formulation remains a clear, colorless liquid.

[0119] In some embodiments, a stable pharmaceutical formulation refers to one in which, in an MFI test of the pharmaceutical formulation, the content of sub-visible particles of different particle size ranges does not tend to increase significantly during storage of the pharmaceutical formulation.

[0120] In some embodiments, a stable pharmaceutical formulation refers to a protein concentration test of the pharmaceutical formulation, wherein the protein concentration in the pharmaceutical formulation varies by no more than + / -20%, no more than + / -19%, no more than + / -18%, no more than + / -17%, no more than + / -16%, no more than + / -15%, no more than + / -14%, no more than + / -13%, no more than + / -12%, no more than + / -11%, no more than + / -10%, no more than + / -9%, no more than + / -8%, no more than + / -7%, no more than + / -6%, no more than + / -5%, no more than + / -4%, no more than + / -3%, no more than + / -2%, no more than + / -1%, or no more than + / -0.5%, and wherein the protein concentration can be determined by ultraviolet-visible spectrophotometry in accordance with General Rule 0401 of Chinese Pharmacopoeia (2010 edition), Volume III.

[0121] Preparation of formulations The present disclosure provides a method for preparing a pharmaceutical formulation, comprising: (1) Prepare a formulation solvent containing a buffer and an anti-GREM1 antibody stock solution; (2) Subjecting the anti-GREM1 antibody stock solution to solvent exchange with a formulation solvent to obtain a pharmaceutical formulation described herein. In some embodiments, solvent exchange refers to buffer exchange, such as buffer exchange by dialysis methods. In some embodiments, solvent exchange refers to solvent exchange by filtration. In some embodiments, filtration refers to sterile filtration and / or other filtration methods familiar to those skilled in the art. (3) Add the required amount of formulation excipients (e.g., stabilizers, surfactants, and, if necessary, antioxidants) to the solvent-exchanged anti-GREM1 antibody stock solution, followed by dilution with buffer to the target concentration. The present invention provides a method comprising:

[0122] In some embodiments, the buffer in the formulation solvent is a histidine buffer having a concentration of about 20 mM.

[0123] In some embodiments, the stabilizer is sucrose having a concentration of about 8.2% (w / v), the surfactant is polysorbate 80 having a concentration of about 0.05% (w / v), and the antioxidant is methionine having a concentration of about 0.04% (w / v).

[0124] In some embodiments, solvent exchange refers to buffer replacement. In some embodiments, an anti-GREM1 antibody stock solution is subjected to solvent exchange by dialysis with a formulation solvent. For example, a certain volume of sample can be placed in a dialysis bag (e.g., a Snake Skin® dialysis bag), the dialysis bag is sealed, and placed in a target buffer with a volume 100 times or more, and continuously stirred to promote the replacement. Dialysis is performed an appropriate number of times (e.g., three times) for an appropriate period (e.g., 4 hours, 4 hours, and overnight), under stirring at an appropriate speed (e.g., 300 rpm).

[0125] In some embodiments, solvent exchange refers to solvent exchange by filtration. In some embodiments, filtration refers to sterile filtration (e.g., through a filter membrane with a pore size of 0.22 microns, bacteria are retained on the membrane, and the anti-GREM1 antibody solution passes through the filter membrane, thereby achieving an antibacterial effect) and / or other filtration methods well known to those skilled in the art.

[0126] Pharmaceutical Composition In another aspect, the present disclosure provides a pharmaceutical composition comprising a pharmaceutical formulation described herein and a second therapeutic agent, wherein the second therapeutic agent is selected from the group consisting of a chemotherapeutic agent (e.g., cisplatin), a radiotherapeutic agent, an immunotherapeutic agent (e.g., an immune checkpoint modulator, e.g., a PD-1 / PD-L1 axis inhibitor, a TGF-β inhibitor), an anti-angiogenic agent (e.g., an antagonist of VEGFR-1, VEGFR-2, and VEGFR-3), a targeted therapy agent, a cell therapy agent, a gene therapy agent, a hormone therapy agent, a cytokine, etc.

[0127] Applicable In another aspect, the present disclosure further provides a method for treating a disease in a subject in need thereof, the method comprising administering a therapeutically effective amount of a pharmaceutical formulation or pharmaceutical composition provided herein to the subject, wherein the subject has or is suspected of having a disease requiring treatment with an antibody against GREM1.

[0128] As used herein, the term "treatment" refers to reducing or alleviating the severity and / or duration of a disease state or one or more of its symptoms, inhibiting or preventing the progression of a disease state, reducing or terminating the symptoms associated with a condition, and inhibiting or preventing the recurrence, onset, onset, or progression of one or more symptoms associated with a disease state. A subject in need thereof includes a subject who has had the disease.

[0129] The term "therapeutically effective amount" refers to the minimum measurable concentration required to treat (eg, ameliorate or prevent) a particular disease state.

[0130] The pharmaceutical preparations or compositions of the present disclosure can be used to treat GREM1-related diseases, such as chronic and acute diseases. GREM1-related diseases include cancer and others. In some embodiments, the GREM1-related disease refers to a GREM1-expressing cancer. Examples of GREM1-expressing cancers include, but are not limited to, cancer, fibrotic diseases, angiogenesis, glaucoma or retinal diseases, kidney diseases, pulmonary hypertension or osteoarthritis (OA), or GREM1-related diseases or disease states associated with increased GREM1 content, and are 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.

[0131] The pharmaceutical formulations or compositions of the present disclosure can be administered to a subject by any suitable route. For example, the pharmaceutical formulations can be administered to a subject intravenously.

[0132] In another aspect, the disclosure provides the use of the pharmaceutical formulation or composition in the manufacture of a medicament for preventing and / or treating a GREM1-associated disease.

[0133] In some embodiments, the medicament further comprises a second therapeutic agent, wherein the second therapeutic agent is selected from the group consisting of a chemotherapeutic agent (e.g., cisplatin), a radiotherapeutic agent, an immunotherapeutic agent (e.g., an immune checkpoint modulator, e.g., a PD-1 / PD-L1 axis inhibitor and a TGF-β inhibitor), an anti-angiogenic agent (e.g., an antagonist of VEGFR-1, VEGFR-2, and VEGFR-3), a targeted therapy agent, a cell therapy agent, a gene therapy agent, a hormone therapy agent, a cytokine, and the like. [Example]

[0134] Experimental method summary: In the following embodiment, formulation screening experiments were carried out on formulations of anti-GREM1 antibodies. The anti-GREM1 antibody used in the following examples is Hu14E3_HaLa. Other humanized anti-GREM1 antibodies (e.g., Hu14E3_HaLb, Hu14E3_HbLa, Hu14E3_HbLb, Hu14E3_HcLa, and Hu14E3_HcLb) have substantially the same amino acid sequence as Hu14E3_HaLa, so it can be expected that the following formulation screening experiments will also be applicable to these humanized anti-GREM1 antibodies. In other words, the above 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 temperatures (e.g., 40°C) or room temperature, high stability after shaking, and high stability after freeze-thawing) in the formulations provided herein. The anti-GREM1 antibodies of the present disclosure are prepared by the following method: 1 x 10 6HEK293E cells at a concentration of 100 cells / ml were cultured in Freestyle 293 expression culture medium containing 10% Pluronic F-68 and transfected with equal amounts of heavy and light chain vector DNA at a final concentration of 0.5 μg / ml and PEI (polyethyleneimine-linear, Polyscience) at a concentration of 1.0 μg / ml. The DNA to PEI ratio was 1:2. DNA and PEI complexes were formed in optimized MEM at room temperature for 15 minutes. Transfected cells were cultured in flasks at 37°C with 5% CO2 and a shaking speed of 125 rpm. 22–26 hours after transfection, 1% peptone culture medium was added. On day 6, the conditioned culture medium was collected, and the conditioned culture medium supernatant was centrifuged at 3,000 rpm for 30 minutes. The clarified conditioned culture medium was then loaded onto an nProtein A column (GE Healthcare), the column was washed with PBS containing 0.1% triton-X100, and finally, the bound IgG was eluted with a solution containing 0.1 M glycine with a pH value of 3.5. The eluted antibody protein was dialyzed into PBS and stored at -80°C. To remove endotoxin, the purified protein was further processed through a Hitrap DEAE Sepharose FF column, and the resulting antibody was analyzed by size exclusion chromatography (Superdex 200 5 / 150 GL, GE Healthcare) to determine the purity level. A detailed description can be found in PCT / CN2022 / 072297, the entire disclosure of which is incorporated herein by reference.

[0135] The formulations of anti-GREM1 antibodies tested in the following examples of this disclosure were prepared by the following method: 1. Preparation of target formulation solution: a) A buffer containing an anti-GREM1 antibody (i.e., stock solution, also abbreviated as DS in the following examples) was replaced with a target formulation buffer by dialysis. Specifically, a certain volume of DS sample was placed in a Snake Skin® dialysis bag, the dialysis bag was sealed, and then placed in a target buffer with a volume 100 times or more. Continuous stirring was performed to promote the replacement. Dialysis was performed three times, for 4 hours, 4 hours, and overnight, respectively, with a stirring speed of 300 rpm. b) After dialysis was completed, the required amount of stabilizer and surfactant stock solution was added to the DS, followed by dilution with the target buffer system to the target concentration to obtain the target formulation solution.

[0136] The anti-GREM1 antibody formulations tested in the following examples of this disclosure were analyzed by the following analytical methods: 2. Dynamic Light Scattering (DLS): Protein particle size and distribution were determined by DLS, the parameters of the method were as follows: collection for 5 seconds per measurement, 20 times in total, and measurements were performed at a temperature of 25°C. 3. Size Exclusion Chromatography (SEC): Protein aggregation was determined by SEC using a Waters Ultra Performance Liquid Chromatography System (UPLC) and a Tosoh TSKgel G3000SWXL SEC column (7.8 x 300 mm, 5 μm) as the chromatography column. The mobile phase contained 50 mM sodium phosphate buffer and 300 mM NaCl, with a pH value of 6.8 ± 0.1. The flow rate was 1.0 mL / min. The sample was diluted to 10 mg / mL and detected in a volume of 10 μL at a wavelength of 280 nm. 4. Cation exchange chromatography (CEX): Protein charge heterogeneity was determined by CEX using a Thermo Propac Elite WCX-10 4 mm × 150 mm 5 μm column on an Agilent 1260 Infinity system. The sample was diluted to 2.00 mg / mL with a mixture of mobile phases A and B. 5. Non-reducing capillary electrophoresis (NR CE-SDS): Protein fragments were determined by the CE-SDS (NR) method. Standard or test samples were diluted to 4 mg / mL with phosphate-citrate buffer, and then 25 μL of the sample was vortex-mixed with 75 μL of SDS sample buffer and 5 μL of NEM (100 mM N-ethylmaleimide), followed by denaturation. The denatured sample was centrifuged, followed by incubation at 70 ± 2°C for 10 ± 2 minutes, cooling to room temperature, and centrifugation again. Separation was performed on a PA800 plus using an SDS separation gel kit and uncoated fused silica capillaries. 6. Visual inspection: The appearance of the sample was inspected with a YB-2 clarity detector under a black background. Clarity and color were reported. 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. 8. Efficacy detection: The binding efficacy of the anti-GREM1 antibody to the Gremlin antigen epitope was detected by ELISA binding method. Detection was performed using human Gremlin / GREM1 protein (Acro) as the coating antigen, goat anti-human IgG-Fc HRP conjugate (Bethyl Laboratories) as the detection antibody, and 3,3',5,5'-tetramethylbenzidine (TMB) as the enzyme reaction substrate. The coating antigen was adsorbed onto an ELISA solid support adsorption plate, washed, and sealed. The test sample was added to bind to the coating antigen, followed by incubation and washing. The detection antibody was added, followed by incubation and washing to remove unbound detection antibody. Next, a substrate was added for color development. Finally, a reaction stop solution was added, and the absorbance value was read in a microplate reader at detection wavelengths of 450 nm / 650 nm.

[0137] [Example 1] pH screening experiment A pH screening test was performed on formulations of anti-GREM1 antibody protein. The stability of anti-GREM1 antibody in acetate, histidine, or citrate buffer systems was investigated within the pH range of 4.5 to 6.5. The specific formulation design is shown in Table 1. The stability (tested by SEC, NR CE-SDS, CEX, and / or MFI) of all formulations was investigated at the initial time point (T0), after 3, 5, and 7 days of storage at 40°C (40°C (3D, 5D, and 7D)), and after 3 and 5 cycles of freeze-thaw (FT (3C, 5C)).

[0138] [Table 1]

[0139] The experimental results are shown below.

[0140] Freeze-thaw (5C): Under 5 cycles of freeze-thaw (5C), all formulations showed no significant changes in protein appearance, protein concentration, SEC, DLS, and NR CE-SDS results. In the MFI experiment, the content of subvisible particles of different particle size ranges in formulations F6, F7, and F8 significantly increased after 5 cycles of freeze-thaw stress; the stability of formulations F6, F7, and F8 was significantly worse than that of the other formulations. Overall, there was no significant difference between formulations F1 to F5 (data shown in the table below). Therefore, the citric acid-sodium citrate buffer system was not conducive to protein storage at low temperatures.

[0141] [Table C]

[0142] 40°C (3D, 5D, 7D): Under the condition of 40°C, all formulations showed no significant changes in protein appearance, protein concentration, and DLS results. As shown in Figure 1, the SEC data indicate that the content and increasing rate of high molecular weight (HMW) polymer in formulations F6, F7, and F8 were slightly higher than those of the other formulations. As shown in Figure 2, the CEX main peak of formulations F1 and F2 decomposed at the highest rate. The purity of the main peak and the content of high polymer in the other formulations did not change substantially, and the formulations were not significantly different from each other.

[0143] In conclusion, stability studies based on freeze-thaw and 40°C temperature showed that the citric acid-sodium citrate buffer system was not conducive to protein storage at low temperatures. At 40°C, the rate of increase of high molecular weight (HMW) polymers and low molecular weight (LMW) fragments of formulation F8 in the citric acid-sodium citrate buffer system was slightly higher than that of the other formulations, and the CEX main peak of formulations F1 and F2 in the acetic acid-sodium acetate buffer system degraded at the highest rate. Therefore, the histidine-histidine hydrochloride buffer solution was the preferred buffer system.

[0144] [Example 2] Excipient screening Excipient screening experiments were performed on formulations of anti-GREM1 antibody protein. The protective effects of inorganic salts (e.g., sodium chloride), amino acids (e.g., arginine hydrochloride), alcohols (e.g., sorbitol and mannitol), and sugars (e.g., sucrose and trehalose) on the protein were investigated. The specific formulation design is shown in Table 2. Samples were examined after incubation. The stability (examined by SEC, NR CE-SDS, CEX, and / or MFI) of all formulations was investigated at the initial time point TO, after 3, 7, and 14 days of storage at 40°C (40°C (3D, 7D, and 14D)), and after 1, 3, and 5 cycles of freeze-thaw (FT (1C, 3C, 5C)).

[0145] [Table 2]

[0146] The experimental results are shown below.

[0147] 1. Five cycles of freeze-thaw: All formulations showed no significant changes in concentration, CEX results, and NR CE-SDS results. DLS results (as shown in Table 3) showed that the protein in formulation F15 (mannitol) appeared as polydisperse (PDI%>25%) after treatment under freeze-thaw stress at TO, indicating that the protein in the formulation was at risk of aggregation. During treatment under freeze-thaw stress for five cycles in SEC, formulation F15 had a significantly increased content of HMW polymer (Figure 3), while the other formulations showed no significant difference and no change in the main peak.

[0148] [Table 3]

[0149] 2. Experimental results at 40℃: During the 14-day period under 40°C stress, the experimental results showed that all formulations had no significant changes in high molecular weight (HMW) polymers; the content of low molecular weight (LMW) fragments increased slightly; and the CEX main peak decreased significantly; no significant differences were observed between the formulations (Figure 14). During the 4-week period under 40°C incubation, formulations F10 and F13 were selected for oxidation tendency investigation. LC-MS results showed that the two formulations had significant oxidation tendency at the sites HC_M101, HC_M109, HC_M249, and HC_M425 (Table 4).

[0150] [Table 4]

[0151] In conclusion, freeze-thaw and 40°C stability studies showed that mannitol did not aid protein stability, and there was no significant difference between the other stabilizers. Considering that sucrose is widely used in biological formulations and arginine hydrochloride can protect proteins by binding through hydrogen bonds, sucrose and arginine hydrochloride were therefore selected for subsequent experiments. In addition, LC-MS results showed that the protein had a significant tendency to oxidize at some sites, so an antioxidant (i.e., methionine) was added for subsequent formulation screening.

[0152] [Example 3] Surfactant screening Surfactant screening experiments were performed on anti-GREM1 antibody protein. The protective effects of polysorbate 80 and polysorbate 20 at concentrations of 0.025 to 0.1% (w / v) were investigated. The specific formulation design is shown in Table 5. The stability of all formulations (tested by SEC, NR CE-SDS, and CEX) was investigated at the initial time point TO, after storage at 40°C for 3, 7, and 14 days (40°C (3D, 7D, and 14D)), and after shaking at 25°C at a speed of 200 rpm for 1, 3, and 5 days (Shaking (200 rpm, 25°C: 1D, 3D, 5D)).

[0153] [Table 5]

[0154] 1. Analysis of the results of the vibrated sample: The experimental results showed that after 5 days of shaking, all formulations had no significant changes in high molecular weight (HMW) polymer, low molecular weight (LMW) fragments and charge isomers, and there were significant differences between the formulations.

[0155] 2. Analysis of the results for samples at 40°C: The experimental results showed that after 14 days of incubation at 40°C, all formulations showed no significant changes in high molecular weight (HMW) polymers; the content of low molecular weight (LMW) fragments increased slightly; the content of the CEX main peak decreased significantly; and there was no significant difference between the formulations.

[0156] In conclusion, the vibration and 40°C stability studies showed that polysorbate 80 and polysorbate 20 with concentrations of 0.025% (w / v) to 0.1% (w / v) had excellent protective effects on proteins (with no significant difference between them), and both of them met the experimental requirements. 0.05% (w / v) polysorbate 80 was selected for further studies.

[0157] Example 4: pH screening and stabilizer and antioxidant experiments This experiment was carried out to further investigate the antioxidant effects of the histidine-histidine hydrochloride buffer system, two stabilizers (arginine hydrochloride and sucrose), and methionine under different pH conditions. The specific design of the formulation is shown in Table 6. The stability of all formulations (tested by SEC, NR CE-SDS, CEX, visual inspection (visible particles) and / or MFI and potency detection) was investigated at an initial time point TO, after 2 and 4 weeks of storage at 40°C (40°C (2W, 4W)), after 2, 4 and 6 weeks of storage at 25°C (25°C (2W, 4W, 6W)), after 2, 4, 6 and 3 months of storage at 5°C (5°C (2W, 4W, 6W, 3M)), after 1, 3 and 5 cycles of freeze-thaw (FT (1C, 3C, 5C)), after 8 hours of shaking at 25°C at a speed of 1,000 rpm (Shaking (1,000 rpm, 25°C: 8h)), and after 1 hour of stirring at 25°C at a speed of 400 rpm (Stirring (400 rpm, 25°C: 1h)).

[0158] [Table 6]

[0159] 1. Freeze-thaw, shaking and stirring results: Experimental results showed that under the conditions of freezing and thawing, shaking, and stirring, all formulations showed no significant changes in high molecular weight (HMW) polymers, low molecular weight (LMW) fragments, and charge isomers, and no significant differences were observed between the formulations.

[0160] 2. Lighting results: The experimental results showed that all formulations showed no significant changes in high molecular weight (HMW) polymers and low molecular weight (LMW) fragments; the content of the CEX main peak decreased slightly; and there was no significant difference between the formulations.

[0161] 3. 40℃ result: The experimental results showed that at 40°C, all formulations had a slightly increased content of high molecular weight (HMW) polymers, a significantly increased content of low molecular weight (LMW) fragments, and a decreased content of the CEX main peak (Figure 4); no significant differences were observed between the formulations. In addition, the potency detection results showed no significant differences between the formulations (the TO sample was used as the reference sample in potency detection). As shown in Table 7, after 4 weeks of incubation at a high temperature of 40°C, the binding potency results of all formulations were within the acceptable standard range (50-150% of the potency of the reference sample). As shown in Table 8, after comparing the liquid chromatography-mass spectrometry (LC-MS) data of formulations F24 and F26, it was observed that methionine had a significant antioxidant effect.

[0162] [Table 7]

[0163] [Table 8]

[0164] 4. 25℃ results: The experimental results showed that at 25°C, all formulations showed no significant changes in high molecular weight (HMW) polymers and low molecular weight (LMW) fragments; the content of the CEX main peak decreased to some extent; and there was no significant difference between the formulations (Figures 5 to 7).

[0165] 5. 5℃ results: The experimental results showed that at 5°C, all formulations showed no significant changes in high molecular weight (HMW) polymer, low molecular weight (LMW) fragment, and charge isomers; and no significant differences were observed between formulations. However, at 3 months (3M), formulations F22, F24, F26, and F27 (i.e., all formulations containing arginine hydrochloride) had visible particles (Table 9), while formulations F23 and F25 containing sucrose had no visible particles.

[0166] [Table 9]

[0167] Considering the results under all conditions comprehensively, all formulas containing arginine hydrochloride had visible particles after 3 months at 5°C. Therefore, sucrose is the preferred stabilizer. To make the osmolality of the formulations as similar as possible to that of human plasma, the sucrose concentration was adjusted from 9% (w / v) to 8.2% (w / v). There was no obvious difference between histidine-histidine hydrochloride buffer systems (including formulas 22, 24, and 27) with different pH values (pH 5.7, pH 6.0, and pH 6.5). Therefore, the intermediate pH, i.e., pH 6.0, was selected as the preferred pH value. Methionine has a clear antioxidant effect. Therefore, the preferred formula for formula validation studies contains 20 mM histidine / histidine hydrochloride, 8.2% (w / v) sucrose, 0.04% (w / v) methionine and 0.05% (w / v) polysorbate 80, with a pH of 6.0.

[0168] [Example 5] Formulation verification experiment This experiment was carried out to further investigate the stability of the preferred formulation selected in Example 4 under various conditions. The preferred formulation contains 20 mM histidine / histidine hydrochloride, 8.2% (w / v) sucrose, 0.04% (w / v) methionine and 0.05% (w / v) polysorbate 80, with a pH value of 6.0. The specific design of the formulation is shown in Table 10. The stability (SEC, NR) of all formulations was The CE-SDS, CEX, visual inspection (visible particles) and / or MFI) were investigated at an initial time point TO, after 2 and 4 weeks of storage at 40°C (40°C (2W, 4W)), after 2, 4 and 6 weeks of storage at 25°C (25°C (2W, 4W, 6W)), after 2, 4, 6 weeks, 3 and 6 months of storage at 5°C (5°C (2W, 4W, 6W, 3M, 6M)), after 1, 3 and 5 cycles of freeze-thaw (FT (1C, 3C, 5C)), after 3 and 7 days of shaking at 25°C at a speed of 200 rpm (Shaking (200 rpm, 25°C: 3D, 7D)), after 2 and 4 hours of agitation at 25°C at a speed of 400 rpm (400 rpm, 25°C: 2H, 4H), and after 5 and 10 days of illumination.

[0169] [Table 10]

[0170] The experimental results showed that the final formulation had excellent stability under the experimental conditions of long-term storage at 5°C (Figures 8-10), accelerated storage at 25°C (Figures 11-13), freeze-thaw, shaking, and agitation; various test indices did not change significantly (Tables 11-14). Under light conditions, the CEX major peak significantly decreased, but the binding potency results of the formulation were within the acceptable standard range (50%-150% of the potency of the reference sample). Therefore, the potency did not change significantly, and the stability requirements were met. At 40°C (Table 15), the LMW significantly increased and the CEX major peak significantly decreased, but the binding potency results of the formulation after 4 weeks were within the acceptable standard range (50%-150% of the potency of the reference sample). Therefore, the potency did not change significantly. Therefore, the selected buffer system and formulation components can provide excellent stability for GREM1 protein.

[0171] [Table 11]

[0172] [Table 12]

[0173] [Table 13]

[0174] [Table 14]

[0175] [Table 15]

[0176] Example 6: Efficacy characteristics of anti-GREM1 antibodies used in the present disclosure Analysis of binding affinity between anti-GREM1 antibodies and human GREM1 by ELISA Clear polystyrene culture plates (BEAVER) were coated overnight at 4°C with 100 μl / 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 with PBS and 0.1% Tween 20 (Sigma) in an automated culture plate washer. 100 μl of blocking solution consisting of PBS, 1% BSA, 1% normal goat serum, and 0.5% Tween 20 (Sigma) was added to each well for 2 hours of incubation at room temperature. Next, 100 μl of serially diluted antibody (starting at 2 μg / ml) in antibody dilution buffer containing PBS, 1% BSA, 1% normal goat serum, and 0.01% Tween 20 was added to each well of the culture plate for 1 hour of incubation at room temperature. The culture plate was then washed three times with 200 μl of a solution containing PBS and 0.1% Tween 20, and 100 μl / well of goat anti-mouse IgG-HRP (Abcam) at a ratio of 1:10,000 was added for 1 hour of incubation at room temperature. The culture plate was then washed three times with a solution containing PBS and 0.1% Tween 20. Finally, 100 μl / well of TMB (Pierce) was added to each well, and after a few minutes, 50 μl of stop solution was added to each well. The culture plate was read at 450 nM in a Multiscan FC microplate reader (Thermo Scientific). As shown in Tables 16 and 17, compared to the reference antibody 6245P, the anti-GREM1 antibodies used in the present disclosure and other humanized anti-GREM1 antibodies (e.g., Hu14E3_HaLb, Hu14E3_HbLa, Hu14E3_HbLb, Hu14E3_HcLa and Hu14E3_HcLb) have superior binding affinity to human GREM1.

[0177] Determining the affinity of humanized antibodies with Fortebio Human GREM1 protein was diluted in kinetic buffer to a concentration of 2 μg / ml. A concentration of 0 nM was used as a reference control. The antibodies to be tested were diluted in ForteBio kinetic buffer (containing PBS with a pH value of 7.4, 0.1% BSA, and 0.002% Tween-20) to concentrations of 100 nM, 50 nM, and 25 nM. Human GREM1-his was immobilized on an NTA biosensor. Detection was performed at baseline for 60 seconds, and the association of anti-GREM1 antibodies was detected for 120 seconds to obtain the K. on The fractional data were then obtained. Dissociation was then performed in kinetic buffer for 90 seconds to obtain K off Factor data was obtained. As shown in Table 16, the anti-GREM1 antibody used in the present disclosure had a KD value of less than 1 nM, which was significantly lower than the KD value of the reference antibody. In other words, compared with the reference antibody 6245P, the anti-GREM1 antibody used in the present disclosure had a better binding affinity with human GREM1.

[0178] Detection of the ability of anti-GREM1 antibodies to block the binding of GREM1 to human BMP2 / 4 / 7 by ELISA Culture plates were coated overnight with recombinant human BMP2 / 4 (0.5 μg / ml), serially diluted with buffer and incubated with 1 μg / ml modified human GREM1-his at room temperature for 1 hour, and the resulting complex was then added to the coated culture plate and incubated at room temperature for another 1 hour. The culture plate was then washed, and anti-his HRP (GenScript) was added. The culture plate was then developed with TMB solution, and the development was stopped by adding stop solution. The culture plate was read at 450 nm in a microplate reader. The results shown in Table 16 indicated that the anti-GREM1 antibody provided herein was better able to inhibit the binding of GREM1 to BMP2 or BMP4 than the reference antibody 6245P. In other words, the anti-GREM1 antibody used in the present disclosure had a better ability to block the binding of human GREM1 to BMP2 or BMP4 than the reference antibody 6245P.

[0179] [Table 16]

[0180] [Table 17]

[0181] Efficacy of the combination of anti-GREM1 antibodies of the present disclosure and cisplatin in an esophageal cancer PDX model Human Gremlin IHC-specific positive esophageal tumor tissue (E7) was obtained from Beijing Cancer Hospital passage in NOD / SCID mice and an established PDX bank. After testing, the esophageal cancer PDX model E7 was found to be positive for GREM1 expression but not for PD-L1 expression.

[0182] Each mouse was subcutaneously inoculated with a small tumor tissue block, approximately 3 mm in diameter, which had been sheared from a tumor-bearing mouse by integrated tumor dissection. 18 days after inoculation, approximately 70 mm3 Animals with tumor sizes of 1000-12000 were selected and randomly divided into four groups of 8 mice each. The mice were then treated with an isotype control plus PBS, an anti-GREM1 antibody of the present disclosure at a dose of 20 mg / kg, cisplatin at a dose of 3 mg / kg, and a combination of an anti-GREM1 antibody of the present disclosure and cisplatin. The isotype control and PBS, as well as the anti-GREM1 antibody of the present disclosure, were administered by ip injection twice a week for 4 weeks, while cisplatin was administered by iv injection once a week for 4 weeks. Animals were sacrificed at the end of the study by CO2 inhalation. Tumor size was measured twice or three times a week (triple time) in two dimensions using a caliper (INSIZE) and calculated using the formula: V = 0.5a * b 2 Volume was expressed in mm^3 using the formula: (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 made by T-test, and differences were considered significant when p<0.05 and **<0.01.

[0183] Table 18 shows significantly enhanced tumor growth inhibition when the anti-GREM1 antibody of the present disclosure was used alone in this experiment compared to the isotype control. The combination of the anti-GREM1 antibody of the present disclosure and cisplatin further inhibited tumor growth compared to either the anti-GREM1 antibody of the present disclosure alone (63.97% TGI vs. 42.92% TGI) or cisplatin alone (63.97% TGI vs. 59.79% TGI). This suggests a synergistic effect of combined treatment with the anti-GREM1 antibody of the present disclosure and cisplatin in esophageal cancer.

[0184] To date, first-line treatments for esophageal cancer generally include esophagectomy, chemotherapy, targeted therapy, immunotherapy (e.g., targeting PD-1 or PD-L1), and / or a combination thereof. Second-line and subsequent treatments for esophageal cancer may involve targeted therapy, such as ramucirumab for targeting vascular endothelial growth factor (VEGF) receptors or trastuzumab for metastatic adenocarcinomas that overexpress HER2 (NCCN Clinical Practice Guidelines in Oncology. Esophageal and Esophagogastric Junction Cancers. National Comprehensive Cancer Network. V1.2020). Applicants' data described above indicate 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 synergistic effects when combined with chemotherapy, such as cisplatin. This suggested that the anti-GREM1 antibodies provided herein could serve as a new option for either first-line or second-line treatment for esophageal cancer.

[0185] [Table 18]

Claims

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

3. 3. The pharmaceutical formulation of claim 1 or 2, further comprising a stabilizer.

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

5. 5. The pharmaceutical formulation of claim 3, wherein the stabilizer is selected from the group consisting of sucrose, trehalose, sorbitol, NaCl, and arginine hydrochloride.

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

7. 10. The pharmaceutical formulation according to any one of the preceding claims, further comprising a surfactant.

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

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

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

11. 10. The pharmaceutical formulation according to any one of the preceding claims, further comprising an antioxidant.

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

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

14. 10. The pharmaceutical formulation of any one of the preceding claims, wherein the concentration of the anti-GREM1 antibody in the pharmaceutical formulation is from 1 mg / ml to 200 mg / ml.

15. The pharmaceutical formulation of claim 14, wherein the concentration of the anti-GREM1 antibody in the pharmaceutical formulation is 20 mg / ml to 40 mg / ml.

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

6.

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

8.

18. The anti-GREM1 antibody is １ QLVQSGSELKKPGASVKVSCKASGX ２ Heavy chain framework region 1 (HFR1) shown in TFT (SEQ ID NO: 24), WMX ３ QAPGQGLX ４ HFR2, RFX shown in WMG (SEQ ID NO: 25) ５ HFR3 represented by FSLDTSVSTAYLQISSLKAEDTAVYYCAR (SEQ ID NO: 26) and HFR4 represented by WGQGTMVTVSS (SEQ ID NO: 17), and / or light chain framework region 1 (LFR1) represented by DVVMTQSPLSLPVTLGQPASISC (SEQ ID NO: 27), WLQQRPGQSPRRLIX ６ (SEQ ID NO: 32), LFR3 represented by GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC (SEQ ID NO: 29), and LFR4 represented by FGQGTKLEIK (SEQ ID NO: 30), １ is V or I, and X ２ is Y or S, and X ３ is R or K, and X ４ is E or T, and X ５ is V or A, and X ６ 10. The pharmaceutical formulation of any one of the preceding claims, wherein is Y or S.

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

30.

20. 10. The pharmaceutical formulation of claim 9, wherein the anti-GREM1 antibody comprises FR1, FR2, FR3 and FR4 of the heavy chain variable region set forth in SEQ ID NO: 7, 11 or 12, and / or FR1, FR2, FR3 and FR4 of the light chain variable region set forth in SEQ ID NO: 8 or 13.

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

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

34.

23. 10. The pharmaceutical formulation of any one of the preceding claims, wherein the anti-GREM1 antibody comprises a heavy chain set forth in SEQ ID NO:9 and / or a light chain set forth in SEQ ID NO:

10.

24. 10. The pharmaceutical formulation of 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 to 6.

5.

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

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

5.

27. 10. The pharmaceutical formulation of any one of the preceding claims, further comprising an antioxidant, wherein said antioxidant is methionine.

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

29. 28. The pharmaceutical formulation of 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, the concentration of the sucrose in the pharmaceutical formulation is about 8.2% (w / v), the surfactant is polysorbate 80, the concentration of the polysorbate 80 in the pharmaceutical formulation is about 0.05% (w / v), the concentration of the methionine in the pharmaceutical formulation is about 0.04% (w / v), and the pH value is about 6.

0.

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

31. The use of claim 30, wherein 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 increased levels of GREM1 and 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.

32. The use of claim 30 or 31, wherein the medicament further comprises a second therapeutic agent, and the second therapeutic agent is selected from the group consisting of chemotherapeutic agents (e.g., cisplatin), radiotherapeutic agents, immunotherapeutic agents (e.g., immune checkpoint modulators, e.g., PD-1 / PD-L1 axis inhibitors and TGF-β inhibitors), anti-angiogenic agents (e.g., VEGFR-1, VEGFR-2 and VEGFR-3 antagonists), targeted therapeutic agents, cell therapeutic agents, gene therapeutic agents, hormone therapeutic agents, cytokines, and the like.

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