Combination of Gremlin-1 antagonist and inhibitor of Ras-Raf-MEK-ERK signaling

Abstract

The present invention relates to anti-GREM1 antagonists for use in methods of treating or preventing cancer in combination with inhibitors of Ras-Raf-MEK-ERK signaling.

Description

[Technical field]

[0001] The present invention relates to combination therapy for treating or preventing cancer. In particular, the present invention relates to anti-GREM1 antagonists for use in methods for treating or preventing cancer in combination with inhibitors of Ras-Raf-MEK-ERK signaling, and related compositions and kits. The present invention also relates to a method for predicting whether a patient is likely to respond to combination therapy based on stromal GREM1 overexpression and signaling of the Ras-Raf-MEK-ERK signaling pathway or mutations in RAS or RAF genes. [Background technology]

[0002] Mitogen-activated protein kinase (MAPK) signaling pathways (e.g., Ras-Raf-MEK-ERK signaling pathway) play important roles in cell survival, proliferation and differentiation. Dysregulated signaling in these pathways is associated with various diseases, including cancer, due to uncontrolled cell proliferation. In addition, upregulated signaling in the MAPK pathway has been reported as a resistance mechanism in response to a wide range of anticancer drug therapies, including cytotoxic agents, immunomodulators and EGFR inhibitors (Martinelli, E. et al., 2017; Kozar, I. et al., 2019; Kobayashi, Y. et al., 2020). Inhibitors of the MAPK signaling pathway, particularly inhibitors of the Ras-Raf-MEK-ERK signaling pathway, such as MEK inhibitors and ERK inhibitors, have been reported to treat cancer. For example, MEK inhibitors have been approved for use in the treatment of BRAF-mutated melanoma and KRAS / BRAF-mutated colorectal cancer. However, acquired resistance is common in patients who are treated with inhibitors of MAPK signaling pathways, such as Ras-Raf-MEK-ERK signaling pathways.Therefore, there is a need to develop new therapies for treating and preventing cancer.In addition, there is a need for combination therapy that can improve the effectiveness of existing MAPK pathway inhibitors.

[0003] One cancer that poses a significant challenge is pancreatic cancer. It is a fatal disease whose incidence has increased in recent years. Environmental risk factors associated with pancreatic cancer include smoking, pancreatitis, alcohol, obesity, infections, and diet, meaning that pancreatic cancer is becoming increasingly common in developed countries. Surgical resection (pancreaticoduodenectomy) combined with chemotherapy presents the only long-term treatment for pancreatic cancer. However, limited screening coupled with a lack of symptoms means that patients are typically diagnosed with late-stage, advanced disease with limited chances for surgical resection. As a result, pancreatic cancer has the poorest survival rate of all common cancers, with a 5-year survival rate of less than 7% and a 10-year survival rate as low as 5%. Over the past 40 years, there has been limited improvement in survival rates, reinforcing the urgent need for new therapies for pancreatic cancer. In unresectable tumors, chemotherapy regimens are the standard treatment for pancreatic cancer. However, chemotherapy is associated with side effects and cancer recurrence is common. Improved treatment of pancreatic cancer is needed due to late diagnosis, poor prognosis, and limited treatment strategies. Summary of the Invention

[0004] The inventors have G12D / + ;LSL-Trp53 R172H / +In a Pdx1-Cre (KPC) mouse model, we demonstrated that exposure to anti-GREM1 antagonists (e.g., Ab7326) upregulated genes involved in Ras-Raf-MEK-ERK signaling, such as NRas, Myc, and Mapk1. Furthermore, we surprisingly showed that GREM1 antagonists can be advantageously administered in combination with inhibitors of Ras-Raf-MEK-ERK signaling, such as MEK inhibitors, for the treatment or prevention of pancreatic cancer. Based on these results, we envision that combination therapy comprising GREM1 antagonists and inhibitors of Ras-Raf-MEK-ERK signaling, such as MEK inhibitors and ERK inhibitors, is generally useful in the treatment and prevention of cancers in which Ras-Raf-MEK-ERK signaling is induced / upregulated after treatment with anti-GREM1 antagonists. Furthermore, based on these results, the inventors envision that combination therapy comprising a GREM1 antagonist and an inhibitor of Ras-Raf-MEK-ERK signaling, such as a MEK inhibitor and an ERK inhibitor, will be generally useful in the treatment and prevention of cancers associated with dysregulation of the Ras-Raf-MEK-ERK pathway or cancers containing mutations in the Ras or Raf genes. The in vivo results provided herein demonstrate the inhibition of LSL-Kras signaling by administration of a GREM1 antagonist in combination with a MEK inhibitor. G12D / + ;LSL-Trp53 R172H / + show significantly increased survival, as well as tumor growth delay and tumor shrinkage in the Pdx1-Cre (KPC) mouse model. Our findings provide an improved approach to the prevention and treatment of cancer, particularly pancreatic cancer.

[0005] Thus, in a first aspect of the present invention there is provided an anti-GREM1 antagonist for use in a method of treating or preventing cancer, the method further comprising administering an inhibitor of Ras-Raf-MEK-ERK signalling.

[0006] In a further aspect of the invention there is provided an inhibitor of Ras-Raf-MEK-ERK signalling for use in a method of treating or preventing cancer, the method further comprising administering an anti-GREM1 antagonist.

[0007] Another aspect of the present invention provides a method for treating cancer, comprising administering to a subject in need thereof a therapeutically effective amount of an anti-GREM1 antagonist in combination with a therapeutically effective amount of an inhibitor of Ras-Raf-MEK-ERK signaling.

[0008] In yet another aspect of the invention, there is provided a composition or kit comprising an anti-GREM1 antagonist and an inhibitor of Ras-Raf-MEK-ERK signaling.

[0009] In a further aspect of the present invention, there is provided a method for determining whether a patient suffering from or suspected of suffering from cancer, or at risk of developing cancer, is likely to respond to combined treatment with a GREM1 antagonist and an inhibitor of Ras-Raf-MEK-ERK signaling, the method comprising measuring Ras-Raf-MEK-ERK signaling in the patient in response to treatment with an anti-GREM1 antagonist, thereby predicting whether the patient is likely to respond to the combined treatment.

[0010] In a further aspect of the present invention, there is provided a method for determining whether a patient suffering from or suspected of suffering from cancer, or at risk of developing cancer, is likely to respond to combined treatment with a GREM1 antagonist and an inhibitor of Ras-Raf-MEK-ERK signaling, the method comprising measuring stromal expression of GREM1, epithelial expression of GREM1 and / or Ras-Raf-MEK-ERK signaling in the patient, thereby predicting whether the patient is likely to respond to the combined treatment.

[0011] In yet another aspect of the present invention, there is provided a method for determining whether a patient suffering from or suspected of suffering from cancer, or at risk of developing cancer, is likely to respond to combined treatment with a GREM1 antagonist and an inhibitor of Ras-Raf-MEK-ERK signaling, the method comprising measuring stromal expression of GREM1 and / or epithelial expression of GREM1 in the patient, determining whether the patient has a mutation in a RAS gene or a RAF gene, thereby predicting whether the patient is likely to respond to the combined treatment. [Brief description of the drawings]

[0012] [Figure 1] Kaplan-Meier analysis showing survival of PDAC patients with Gremlin-1 expression above or below the indicated median values. Patients with tumors expressing high levels of Gremlin-1 have a significantly worse prognosis compared to patients with low expression. Generated from KMplotter. [Diagram 2] Schematic diagram showing (a) breeding strategy and (b) experimental design. [Figure 3A] Gene expression in the KPC mouse model for 46 genes profiled by NanoString technology: A. Unadjusted p-values ​​depicted in a Volcano plot. [Figure 3B] B. Linear model of Nras expression. [Figure 3C] C. Linear model of Myc expression. [Figure 3D] D. Linear model of Mapk1 expression. [Figure 4] FIG. 13 depicts pathway enrichment showing the effect of Ab7326 mIgG1 exposure on the KRAS pathway. [Diagram 5] FIG. 1 is a graph showing tumor burden as measured by high-resolution ultrasound in individual mice during treatment (as indicated) in a pharmacodynamic study. [Figure 6]FIG. 13 is a graph showing tumor burden as measured by high resolution ultrasound in individual mice during treatment (as indicated) in survival study cohorts. [Figure 7] FIG. 13 shows a Kaplan-Meier analysis showing survival of Pdx1-Cre; LSL-KrasG12D / +; LSL-Trp53R172H / + (KPC) mice from the initiation of treatment with the indicated Ab7326 mIgG1 (n=9), selumetinib (n=8), or Ab7326 mIgG1 in combination with selumetinib (n=9). [Figure 8] Graph of IHC using antibodies against markers of cancer-associated fibroblasts (aSMA and podoplanin) and scoring of picrosirius red staining for collagen I and III (by HALO software analysis) in mice treated as indicated. [Figure 9a] FIG. 13 shows a Kaplan-Meier analysis showing survival of Pdx1-Cre; LSL-KrasG12D / +; LSL-Trp53R172H / + (KPC) mice from the initiation of treatment with vehicle control (n=10), Ab7326 mIgG1 (n=10), UCB-554 (n=10), or Ab7326 mIgG1 in combination with UCB-554 (n=11), as indicated. [Figure 9b] FIG. 1 shows boxes and whiskers depicting tumor volumes 7 days after initiation of treatment as measured by high-resolution ultrasound in individual KPC mice treated with vehicle control (n=10), Ab7326 mIgG1 (n=8), UCB-554 (n=10), or Ab7326 mIgG1 in combination with UCB-554 (n=11). Each box represents the interquartile range and whiskers represent the minimum and maximum observed values. Solid bars represent median values ​​and dashed bars represent mean volumes. [Figure 9c] FIG. 13 is a graph showing tumor burden over time as measured by high resolution ultrasound in individual KPC mice upon treatment with vehicle control (n=10) or Ab7326 mIgG1 in combination with UCB-554 (n=11). [Figure 10a]Figure 5 shows a Kaplan-Meier analysis showing survival of Pdx1-Cre;LSL-KrasG12D / +;LSL-Trp53R172H / + (KPC) mice from the initiation of treatment with vehicle control (n=10), Ab7326 mIgG1 (n=19), selumetinib (n=17), or Ab7326 mIgG1 in combination with selumetinib (n=19) as indicated. Data is a combined dataset including that shown in Figure 7. [Figure 10b] FIG. 1 shows boxes and whiskers depicting tumor volumes 7 days after initiation of treatment as measured by high-resolution ultrasound in individual KPC mice treated with vehicle control (n=10), Ab7326 mIgG1 (n=8), selumetinib (n=9), or Ab7326 mIgG1 in combination with selumetinib (n=10). Each box represents the interquartile range and whiskers represent the minimum and maximum observed values. Solid bars represent median values ​​and dashed bars represent mean volumes. [Figure 10c] FIG. 13 is a graph showing tumor burden over time as measured by high resolution ultrasound in individual KPC mice upon treatment with vehicle control (n=10) or Ab7326 mIgG1 in combination with selumetinib (n=10). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] Brief explanation of the table Table 1-Kras G12D+ Table showing grem1 upregulated in KPC tumors and all pancreatic mouse models combined compared to mouse pancreatic ductal cells. Table 2 - Table showing doses and given treatment schedules, number of mice receiving each treatment, and median survival in each treatment cohort. Table 3 - Table showing doses and given treatment schedule, number of mice per arm, and sampling time points. Table 4 - Table showing doses and given treatment schedules, number of mice in each treatment, and median survival in each treatment cohort. * Indicate the historical data used. Table 5 - Table comparing survival statistics between treatment cohorts (log-rank analysis). Mice treated with the combination of Ab7326 mIgG1 and selumetinib show a significant increase in survival compared to treatment with selumetinib alone. Table 6 - Table showing survival data and censoring of individual mice during treatment. Table 7 - Table showing individual mouse samples analyzed. Table 8 - Table showing doses and given treatment schedules, number of mice in each treatment, and median survival in each treatment cohort. Table 9 - Summary of survival statistics for KPC mice treated with a combination of Ab7326 and UCB-554. Table 10 - Summary survival statistics of KPC mice treated with a combination of Ab7326 and selumetinib.

[0014] Detailed Description of the Invention It is to be understood that different applications of the disclosed products and methods can be tailored to the particular needs of the art, and that the terminology used herein is for the purpose of describing particular embodiments of the invention only, and is not intended to be limiting.

[0015] Further, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to an "inhibitor" includes two or more such inhibitors or reference to an "oligonucleotide" includes two or more such oligonucleotides, and so forth.

[0016] All publications, patents, and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.

[0017] Combination therapy with anti-GREM1 antagonist and inhibitor of Ras-Raf-MEK-ERK signaling The present invention provides an anti-GREM1 antagonist in combination with an inhibitor of Ras-Raf-MEK-ERK signaling, such as an MEK or ERK inhibitor, for use in a method of treating or preventing cancer. The cancer is typically a cancer with overexpression of GREM1 and / or a cancer with dysregulation of the Ras-Raf-MEK-ERK signaling pathway. The cancer may additionally or alternatively be a cancer that exhibits dysregulation of the Ras-Raf-MEK-ERK pathway after exposure / treatment to an anti-GREM1 antagonist. In particular, the cancer may be a cancer in which Ras-Raf-MEK-ERK pathway signaling is induced / upregulated after treatment with an anti-GREM1 antagonist as described herein. Such a cancer may not exhibit dysregulation of the Ras-Raf-MEK-ERK pathway in the absence of treatment with an anti-GREM1 antagonist. In other words, the cancer may exhibit normal Ras-Raf-MEK-ERK pathway signaling prior to treatment with an anti-GREM1 antagonist described herein, as compared to a reference sample or value. The cancer may additionally or alternatively be characterized as including a mutation in a RAS gene or a RAF gene.

[0018] The present invention further provides an inhibitor of Ras-Raf-MEK-ERK signaling for use in a method for treating or preventing cancer, the method comprising separate, sequential or simultaneous administration of an anti-GREM1 antagonist.

[0019] Inhibitors of Ras-Raf-MEK-ERK signaling An inhibitor of Ras-Raf-MEK-ERK signaling can be any inhibitor that targets the Ras-Raf-MEK-ERK mitogen-activated protein kinase (MAPK) signaling cascade (sometimes referred to herein as the classical MAPK pathway, Raf / MEK / ERK, Raf-MEK-ERK, Ras-regulated Raf-MEK-ERK pathway, MAPK / ERK(MEK), Ras / Raf / MAPK(MEK) / ERK, Ras-Raf-MAPK or Raf-MEK-ERK pathway), which is involved in cell survival, proliferation and differentiation. The MAPK cascade consists of multiple intracellular signaling pathways. Typically, the binding of growth factors to their cognate receptors (e.g., EGF / EGFR) at the cell surface leads to the recruitment of the small GTPase Ras, which activates the serine serone kinase RAF, leading to the phosphorylation and activation of the mitogen-activated protein kinase kinase (MEK) enzymes MEK1 and MEK2. MEK1 and MEK2 (also known as Map2k1 and Map2k2, respectively) are dual specificity kinases that phosphorylate Erk1 and Erk2 (MAPK), leading to their translocation to the nucleus and activation of multiple transcription factors. Thus, MEK inhibitors typically block MEK1 / MEK2 phosphorylation of Erk1 / Erk2 and downstream signaling pathways. ERK inhibitors can also prevent phosphorylation of Erk1 / Erk2 and / or inhibit the catalytic activity of Erk1 / Erk2.

[0020] The present inventors identified that KRAS signaling is upregulated in the KPC mouse model after exposure to anti-GREM1 antagonist (Figure 4). Furthermore, genes involved in the Ras-Raf-MEK-ERK pathway, such as Mapk1, Nras and Myc, are upregulated by exposure to anti-GREM1 antibody Ab7326 in the KPC mouse model (Figure 3). The present inventors further showed that the combination of anti-GREM1 antibody and MEK inhibitor has significant anti-tumor activity in this model. Therefore, combination therapy including anti-GREM1 antagonist combined with inhibitor of Ras-Raf-MEK-ERK signaling can be used to treat cancers associated with GREM1 overexpression, dysregulation of the Ras-Raf-MEK-ERK pathway, and / or mutations in Ras and Raf genes.

[0021] Furthermore, a combination therapy comprising an anti-GREM1 antagonist as defined herein and an inhibitor of Ras-Raf-MEK-ERK signaling, such as a MEK inhibitor or an ERK inhibitor, may be used to treat or prevent cancers in which the Ras-Raf-MEK-ERK pathway is dysregulated after earlier exposure to / treatment with the anti-GREM1 antagonist. In particular, the cancer may be a cancer in which the Ras-Raf-MEK-ERK pathway signaling is induced / regulated after treatment with the anti-GREM1 antagonist as described herein. Such a cancer may not exhibit dysregulation of the Ras-Raf-MEK-ERK pathway in the absence of treatment with the anti-GREM1 antagonist. In other words, the cancer may exhibit normal Ras-Raf-MEK-ERK pathway signaling compared to a reference sample or reference value before treatment with the anti-GREM1 antagonist as described herein.

[0022] Dysregulated means that the Ras-Raf-MEK-ERK pathway is upregulated or downregulated compared to a reference sample or reference level. The Ras-Raf-MEK-ERK pathway may be upregulated or downregulated at the mRNA or protein level. For example, the cancer may be a cancer that shows upregulation of genes / proteins of the Ras-Raf-MEK-ERK pathway after exposure to the anti-GREM1 antagonist of the present invention. Alternatively or additionally, the cancer may be a cancer that shows increased phosphorylation of Map2k1, Map2k2, Erk1 and / or Erk2 in response to treatment with the anti-GREM1 antagonist of the present invention. The cancer may be a cancer or tumor previously described for treatment with a GREM1 antagonist. Such cancers include, but are not limited to, pancreatic cancer, multiple myeloma, colon cancer, breast cancer and prostate cancer. In a preferred embodiment, the cancer is pancreatic cancer.

[0023] As used herein, inhibitors of Ras-Raf-MEK-ERK signaling include pharma- ceutically acceptable salts and solvates thereof.

[0024] Any protein in the Ras-Raf-MEK-ERK pathway (classical MAPK cascade) can be a target of an inhibitor for use in the present invention. Thus, the inhibitor targets at least one component of the Ras-Raf-MEK-ERK pathway to inhibit Ras-Raf-MEK-ERK signaling. Inhibition of Ras-Raf-MEK-ERK signaling typically involves a reduction in the activity of at least one component of the pathway. Thus, the inhibitor can be a direct or indirect inhibitor of any node (typically any protein) in the Ras-Raf-MEK-ERK pathway (classical MAPK cascade). For example, the inhibitor of the Ras-Raf-MEK-ERK pathway can be a direct or indirect RAS inhibitor, such as an inhibitor of KRAS (e.g., ARS-1620 or MRTX849). Alternatively, the inhibitor of Ras-Raf-MEK-ERK signaling can be a RAF inhibitor, such as sorafenib, vemurafenib, dabrafenib, or encorafenib. In another embodiment, the inhibitor of Ras-Raf-MEK-ERK signaling can be a MEK inhibitor. Exemplary MEK inhibitors are detailed below. In yet a further embodiment, the inhibitor of Ras-Raf-MEK-ERK signaling can be an ERK inhibitor, such as ulixertinib (4-[5-chloro-2-[(1-methylethyl)amino]-4-pyridinyl]-N-[(1S)-1-(3-chlorophenyl)-2-hydroxyethyl]-1H-pyrrole-2-carboxamide, monohydrochloride; (S)-4-(5-chloro-2-(isopropylamino)pyridin-4-yl) -N-(1-(3-chlorophenyl)-2-hydroxyethyl)-1H-pyrrole-2-carboxamide; BVD-523; VRT-752271), temtelqib (LY3214996; 4H-thieno[2,3-c]pyrrol-4-one, 5,6-dihydro-6,6-dimethyl-2-[2-[(1-methyl-1H-pyrazol-5-yl)amino]-4-pyrimidinyl]-5-[2-(4-morpholinyl)ethyl];6,6-dimethyl-2-[2-[(2-methylpyrazol-3-yl)amino]pyrimidin-4-yl]-5-(2-morpholin-4-ylethyl)thieno[2,3-c]pyrrol-4-one), JSI-1187, lavoxertinib (1-[(1S)-1-(4-chloro-3-fluorophenyl)-2-hydroxyethyl]-4-[2-[(2-methylpyrazol-3-yl)amino]pyrimidin-4 -yl]pyridin-2-one;(S)-1-(1-(4-chloro-3-fluorophenyl)-2-hydroxyethyl)-4-(2-((1-methyl-1H-pyrazol-5-yl)amino)pyrimidin-4-yl)pyridin-2(1H)-one;MK-8353 ((3S)-3-methylsulfanyl-1-[2-[4-[4-(1-methyl-1,2,4-triazol-3-yl)phenyl]-3,6 -dihydro-2H-pyridin-1-yl]-2-oxoethyl]-N-[3-(6-propan-2-yloxypyridin-3-yl)-1H-indazol-5-yl]pyrrolidine-3-carboxamide;(S)-N-(3-(6-isopropoxypyridin-3-yl)-1H-indazol-5-yl)-1-(2-(4-(4-(1-methyl-1H-1,2,4-triazol-3-yl)phenyl) )-3,6-dihydropyridin-1(2H)-yl)-2-oxoethyl)-3-(methylthio)pyrrolidine-3-carboxamide), or ASN007 ((S)-N-(2-amino-1-(3-chloro-5-fluorophenyl)ethyl)-1-(5-methyl-2-((tetrahydro-2H-pyran-4-yl)amino)pyrimidin-4-yl)-1H-imidazole-4-carboxamide). In a preferred embodiment, the ERK inhibitor is ulixertinib. In a particularly preferred embodiment of the present invention, the ERK inhibitor is ulixertinib and the cancer to be treated is pancreatic cancer, preferably pancreatic ductal adenocarcinoma (PDAC). Other inhibitors of the Ras-Raf-MEK-ERK signaling pathway are well known in the art.;

[0025] In a preferred embodiment of the present invention, the inhibitor of Ras-Raf-MEK-ERK signaling is a MEK inhibitor. The MEK inhibitor for use in combination with the anti-GREM1 antagonist can be an allosteric MEK inhibitor and / or a MEK1 inhibitor and / or a MEK2 inhibitor. The term "allosteric MEK inhibitor" refers to an inhibitor that binds to an allosteric site outside the ATP-binding pocket of MEK. Allosteric MEK inhibitors (otherwise known as type III MEK inhibitors) are highly selective and typically function by reducing the movement of the activation loop of MEK1 / 2. Other MEK inhibitors include type I and type II MEK inhibitors, which compete for ATP binding and either occupy the catalytic site of MEK (type I) or bind to an adjacent site within the ATP-binding pocket (type II). All three types of MEK inhibitors are contemplated for use in the methods of the present invention. That is, type I, type II, or type III MEK inhibitors can be used in the methods of the present invention.

[0026] Exemplary allosteric MEK inhibitors include selumetinib (AZD6244; ARRY-142886; 6-(4-bromo-2-chloroanilino)-7-fluoro-N-(2-hydroxyethoxy)-3-methylbenzimidazole-5-carboxamide) or trametinib (GSK-1120212; N-(3-{3-cyclopropyl-5-[(2-fluoro-4-iodophenyl)amino]-6,8-dimethyl-2,4,7-trioxo-3,4 Other exemplary MEK inhibitors for use in the present invention include selumetinib (AZD6244), trametinib (GSK-1120212), cobimetinib (GDC-0973; (S)-[3,4-difluoro-2-(2-fluoro-4-iodophenylamino)phenyl][3-hydroxy-3-(piperidin-2-yl)azetidine]. -1-yl]methanone), binimetinib (MEK162; 5-((4-bromo-2-fluorophenyl)amino)-4-fluoro-N-(2-hydroxyethoxy)-1-methyl-1H-benzo[d]imidazole-6-carboxamide), CI-1040 (PD184352), mirdametinib (PD0325901; N-[(2R)-2,3-dihydroxypropoxy]-3,4-difluoro-2-[(2-fluoro-4-iodophenyl)amino]beta amide), TAK733 ((R)-3-(2,3-dihydroxypropyl)-6-fluoro-5-(2-fluoro-4-iodophenylamino)-8-methylpyrido[2,3-d]pyrimidine-4,7(3H,8H)-dione; 3-[(2R)-2,3-dihydroxypropyl]-6-fluoro-5-(2-fluoro-4-iodoanilino)-8-methylpyrido[2,3-d]pyrimidine-4,7-dione), refametinib (RDEA119 / Bay 86-9766;N-[3,4-difluoro-2-(2-fluoro-4-iodoanilino)-6-methoxyphenyl]-1-[(2S)-2,3-dihydroxypropyl]cyclopropane-1-sulfonamide), RO-5126766 (3-[[2-[(methylaminosulfonyl)amino]-3-fluoropyridin-4-yl]methyl]-4-methyl-7-[(pyrimidin-2-yl)oxy]-2H -1-benzopyran-2-one; N-[3-fluoro-4-[[4-methyl-2-oxo-7-(2-pyrimidinyloxy)-2H-1-benzopyran-3-yl]methyl]-2-pyridinyl]-N'-methylsulfamide), RO5068760, pimasertib (AS703026; N-[(2S)-2,3-dihydroxypropyl]-3-(2-fluoro-4-iodoanilino)pi lysine-4-carboxamide), AZD8330 (2-(2-fluoro-4-iodoanilino)-N-(2-hydroxyethoxy)-1,5-dimethyl-6-oxopyridine-3-carboxamide; 2-((2-fluoro-4-iodophenyl)amino)-N-(2-hydroxyethoxy)-1,5-dimethyl-6-oxo-1,6-dihydropyridine-3-carboxamide), GDC- 0623 (5-(2-fluoro-4-iodoanilino)-N-(2-hydroxyethoxy)imidazo[1,5-a]pyridine-6-carboxamide), RO-4987655 (3,4-difluoro-2-(2-fluoro-4-iodoanilino)-N-(2-hydroxyethoxy)-5-[(3-oxooxazinan-2-yl)methyl]benzamide), WX-554 (UCB-554);[3-(aminomethyl)azetidin-1-yl]-{2-[(2-fluoro-4-iodophenyl)amino]thieno[2,3-b]pyridin-3-yl}-methanone), HL-085, ARRY-300, ClnQ-03, G-573, PD184161 (5-bromo-2-[(2-chloro-4-iodophenyl)amino]-N-(cyclopropylmethoxy)-3,4-difluorobenzamide), PD318088 (5-bromo-N-(2,3-dihydroxy propoxy)-3,4-difluoro-2-[(2-fluoro-4-iodophenyl)amino]benzamide), PD98059 (2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one), U0126 (1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene; (2Z,3Z)-2,3-bis[amino[(2-aminophenyl)thio]methylene]butanedinitrile) or SL327.

[0027] In a preferred embodiment of the invention, the MEK inhibitor is selumetinib (AZD6244) or trametinib (GSK-1120212). In a particularly preferred embodiment of the invention, the MEK inhibitor is selumetinib (AZD6244) or trametinib (GSK-1120212) and the cancer to be treated is pancreatic cancer, preferably pancreatic ductal adenocarcinoma (PDAC).

[0028] In a preferred embodiment of the invention, the MEK inhibitor belongs to the class of thieno[2,3-b]pyridine derivatives substituted at the 2-position with a substituted anilino moiety. Exemplary MEK inhibitors belonging to this class include: - N-(2-fluoro-4-iodophenyl)-3-(morpholin-4-ylcarbonyl)thieno[2,3-b]pyridin-2-amine; - N-(2-fluoro-4-iodophenyl)-3-[(4-methylpiperazin-1-yl)carbonyl]thieno[2,3-b]pyridin-2-amine; - [2-[((2-fluoro-4-iodophenyl)amino]thieno[2,3-b]pyridin-3-yl](pyrrolidin-1-yl)methanone; - (1-{2-[(2-fluoro-4-iodophenyl)amino]thieno[2,3-b]pyridine-3-carbonyl}piperidin-4-yl)-carbamic acid tert-butyl ester; - 2-[((2-fluoro-4-iodophenyl)amino]thieno[2,3-b]pyridine-3-carboxylic acid (4-aminopiperidin-1-yl)amide dihydrochloride; - (1-{2-[(2-fluoro-4-iodophenyl)amino]thieno[2,3-b]pyridine-3-carbonyl}azetidin-3-ylmethyl)carbamic acid tert-butyl ester; - [3-(aminomethyl)azetidin-1-yl]-{2-[(2-fluoro-4-iodophenyl)amino]thieno[2,3-b]pyridin-3-yl}-methanone; - (1-{2-[(2-fluoro-4-iodophenyl)amino]thieno[2,3-b]pyridine-3-carbonyl}-(3R)-pyrrolidin-3-yl)carbamic acid tert-butyl ester; - (1-{2-[(2-fluoro-4-iodophenyl)amino]thieno[2,3-b]pyridine-3-carbonyl}-(3S)-pyrrolidin-3-yl)carbamic acid tert-butyl ester; - (1-{2-[(2-fluoro-4-iodophenyl)amino]thieno[2,3-b]pyridine-3-carbonyl}azetidin-3-yl)-carbamic acid tert-butyl ester; - [(3R)-3-aminopyrrolidin-1-yl]-{2-[(2-fluoro-4-iodophenyl)amino]thieno[2,3-b]pyridin-3-yl}-methanone; - [(3S)-3-aminopyrrolidin-1-yl]-{2-[(2-fluoro-4-iodophenyl)amino]thieno[2,3-b]pyridin-3-yl}-methanone; - (3-aminoazetidin-1-yl)-{2-[(2-fluoro-4-iodophenyl)amino]thieno[2,3-b]pyridin-3-yl]-methanone; - {2-[((2-fluoro-4-iodophenyl)amino]thieno[2,3-b]pyridin-3-yl}-(3-hydroxyazetidin-1-yl)-methanone; - {2-[((2-fluoro-4-iodophenyl)amino]thieno[2,3-b]pyridin-3-yl}-[(3R)-3-hydroxypyrrolidin-1-yl]-methanone; - {2-[((2-fluoro-4-iodophenyl)amino]thieno[2,3-b]pyridin-3-yl}-[(3S)-3-hydroxypyrrolidin-1-yl]-methanone; - {2-[((2-fluoro-4-iodophenyl)amino]thieno[2,3-b]pyridin-3-yl}-[2-(hydroxymethyl)-piperidin-1-yl]-methanone; - {2-[((2-fluoro-4-iodophenyl)amino]thieno[2,3-b]pyridin-3-yl}-[(3S)-3-(hydroxymethyl)-morpholin-4-yl]-methanone; - 4-{2-[(2-fluoro-4-iodophenyl)amino]thieno[2,3-b]pyridine-3-carbonyl}-(3R)-3-(hydroxymethyl)piperazine-1-carboxylic acid tert-butyl ester; - 2-[4({2-[(2-fluoro-4-iodophenyl)amino]thieno[2,3-b]pyridin-3-yl}carbonyl)piperazin-1-yl]ethanol; - 3-((1,4-diazepan-1-ylcarbonyl)-N-(2-fluoro-4-iodophenyl)thieno[2,3-b]pyridin-2-amine; - N-(2-fluoro-4-iodophenyl)-3-(piperazin-1-ylcarbonyl)thieno[2,3-b]pyridin-2-amine; - ethyl [4-({2-[(2-fluoro-4-iodophenyl)amino]thieno[2,3-b]pyridin-3-yl}carbonyl)-piperazin-1-yl]acetate; - [4-({2-[(2-fluoro-4-iodophenyl)amino]thieno[2,3-b]pyridin-3-yl}carbonyl)piperazin-1-yl]acetic acid; {2-[((2-fluoro-4-iodophenyl)amino]thieno[2,3-b]pyridin-3-yl}-[(2R)-2-(methoxymethyl)-pyrrolidin-1-yl]-methone; and - {2-[((2-fluoro-4-iodophenyl)amino]thieno[2,3-b]pyridin-3-yl}-[(2R)-2-(hydroxymethyl)-piperazin-1-yl]-methanone Examples include:

[0029] Methods for producing these MEK inhibitors are disclosed in International Publication No. WO2007 / 088345, which is incorporated herein by reference in its entirety.

[0030] A preferred MEK inhibitor for use in the present invention belonging to the class of thieno[2,3-b]pyridine derivatives substituted at the 2-position with a substituted anilino moiety has the formula (II): [ka] [In the formula, R 12 represents a halogen; R 3 CONR b R c represents; R b and R c taken together with the nitrogen atom to which they are both attached represent azetidin-1-yl, pyrrolidin-1-yl, piperidin-1-yl, morpholin-4-yl, thiomorpholin-4-yl, piperazin-1-yl, homopiperidin-1-yl, homo-morpholin-4-yl or homopiperazin-1-yl, any of which groups may optionally be represented by C 1-6 Alkyl, C 1-6 Alkoxy, hydroxy, hydroxy(C 1-6 ) alkyl, amino-(C 1-6 ) alkyl, (amino) (hydroxy) (C 1-6 ) Alkyl, halogen, oxo, C 2-6 Alkylcarbonyl, Carboxy, C 2-6Alkoxycarbonyl, di(C 1-6 ) Alkylhydrazinylcarbonyl, amino, C 1-6 Alkylamino, di(C 1-6 ) Alkylamino, C 2-6 Alkylcarbonylamino, Aminocarbonylamino, Aminocarbonyl, C 1-6 Alkylaminocarbonyl, di(C 1-6 ) Alkylaminocarbonyl, aminosulfonyl, C 1-6 Alkylsulfonyl, C 1-6 Alkylaminocarbonyl (C 1-6 ) Alkyl, C 1-6 Alkoxy(C 1-6 ) alkyl, carboxy (C 1-6 ) Alkyl, C 2-6 Alkoxycarbonyl (C 1-6 ) Alkyl, C 2-6 Alkoxycarbonylamino and C 2-6 Alkoxycarbonylamino-(C 1-6 ) alkyl, and pharma- ceutically acceptable salts, solvates, and N-oxides thereof.

[0031] In one embodiment, R 12 is bromo. In another embodiment, R 12 is iodine.

[0032] Methods for preparing compounds of formula (II) are disclosed in International Publication No. WO2007 / 088345, which is incorporated herein by reference in its entirety.

[0033] In a preferred embodiment of the invention, the MEK inhibitor is a compound of formula (II) or a pharma- ceutically acceptable salt, solvate or N-oxide thereof. In a particularly preferred embodiment of the invention, the MEK inhibitor is a compound of formula (II) or a pharma- ceutically acceptable salt, solvate or N-oxide thereof, and the cancer to be treated is pancreatic cancer, preferably pancreatic ductal adenocarcinoma (PDAC).

[0034] In a preferred embodiment of the invention, the MEK inhibitor is WX-554 (UCB-554). In a particularly preferred embodiment of the invention, the MEK inhibitor is WX-554 (UCB-554) and the cancer to be treated is pancreatic cancer, preferably pancreatic ductal adenocarcinoma (PDAC).

[0035] The structure of WX-554 (UCB-554); [3-(aminomethyl)azetidin-1-yl]-{2-[(2-fluoro-4-iodophenyl)amino]thieno[2,3-b]pyridin-3-yl}-methanone) is shown here. [ka]

[0036] Methods for producing WX-554 are disclosed in International Publication No. WO2007 / 088345, which is incorporated herein by reference in its entirety.

[0037] The above-mentioned MEK inhibitors, which belong to the class of thieno[2,3-b]pyridine derivatives substituted at the 2-position with a substituted anilino moiety, exhibited excellent binding affinity (IC 50 ) of 50 μM or less, generally 20 μM or less, usually 5 μM or less, typically 1 μM or less, suitably 500 nM or less, ideally 100 nM or less, preferably 20 nM or less. (Those skilled in the art will recognize that 50 (It is understood that the lower the numerical value, the more active the compound.) These MEK inhibitors for use in the present invention may have at least a 10-fold selective affinity for the human MEK1 and / or MEK2 enzymes compared to other human kinases, typically at least a 20-fold selective affinity, preferably at least a 50-fold selective affinity, and ideally at least a 100-fold selective affinity.

[0038] Various methods can be used to determine the activity of MEK inhibitors, such as an in vitro MEK assay. An exemplary in vitro MEK assay is described in International Publication No. WO2007 / 088345, which is incorporated herein by reference in its entirety.

[0039] It is also contemplated that combination therapy comprising a GREM1 antagonist as defined herein and an inhibitor of Ras-Raf-MEK-ERK signaling, such as a MEK inhibitor or an ERK inhibitor, can be used to treat or prevent cancers that are typically targeted with inhibitors of Ras-Raf-MEK-ERK signaling. The cancer can be a cancer or tumor previously described for treatment with inhibitors of Ras-Raf-MEK-ERK signaling. Such cancers include, but are not limited to, pancreatic cancer, multiple myeloma, colon cancer, lung cancer, and melanoma.

[0040] The cancer may be a cancer that is responsive to treatment with an inhibitor of Ras-Raf-MEK-ERK signaling and / or a cancer that is intended to be treated with an inhibitor of Ras-Raf-MEK-ERK signaling. Alternatively, the cancer may be a cancer that is poorly responsive, non-responsive, or refractory to treatment with an inhibitor of Ras-Raf-MEK-ERK signaling. The cancer may be one that has been previously described as not suitable for treatment with an inhibitor of Ras-Raf-MEK-ERK signaling. In some cases, the cancer may be initially responsive to treatment with an inhibitor of Ras-Raf-MEK-ERK signaling, but develop resistance to the inhibitor of Ras-Raf-MEK-ERK signaling. Responsiveness may be measured by any means. For example, responsiveness to treatment may be evaluated by measuring tumor size before and after treatment using X-ray, CT, or MRI scan, or by measuring tumor markers. Blood tests to determine organ function may also be used to evaluate responsiveness to treatment. One exemplary blood marker for monitoring the responsiveness of pancreatic cancer is CA19-9. Those skilled in the art know how to measure the responsiveness of a cancer to a treatment.

[0041] cancer In the context of a combination therapy comprising an anti-GREM1 antagonist and an inhibitor of Ras-Raf-MEK-ERK signaling, such as an MEK or ERK inhibitor, the cancer may be any cancer or tumor. In particular, in the context of a combination therapy comprising an anti-GREM1 antagonist and an inhibitor of Ras-Raf-MEK-ERK signaling, the cancer may be a cancer or tumor with stroma, typically a detumorized stroma. The cancer may be any cancer or tumor that is GREM1-inducible. The cancer may be any cancer in which overexpression of stromal and / or epithelial GREM1 is observed. The cancer or tumor may have stromal GREM1 overexpression and no epithelial GREM1 overexpression. The cancer or tumor may have epithelial GREM1 overexpression and no stromal GREM1 overexpression. In a preferred embodiment, the cancer or tumor has overexpression of GREM1 in detumorized stroma. The cancer or tumor may be any cancer or tumor suitable for targeting with a GREM1 antagonist, a MEK inhibitor and / or an ERK inhibitor, including any cancer or tumor known in the art to be suitable for targeting with such an agent(s), and any cancer or tumor known in the art to be suitable for treatment with such an agent(s).

[0042] The cancer or tumor may be a solid tumor. A solid tumor may have a decellularized stroma.

[0043] Anti-GREM1 antagonists have previously been shown to be effective in treating various cancers in International Publication No. WO2019 / 243801, filed June 18, 2019, the disclosure of which is incorporated by reference in its entirety herein.

[0044] Particularly preferred cancers that can be treated include colorectal cancer, multiple myeloma, pancreatic cancer, bladder cancer, breast cancer, lung cancer, gastric cancer, duodenal cancer, esophageal cancer, head and neck cancer, prostate cancer, glioma, endometrial cancer, ovarian cancer, liver cancer, splenic cancer, bone cancer, melanoma and osteosarcoma. The cancer that can be treated can be intestinal cancer, colon cancer or rectal cancer. The cancer to be treated can be disseminated cancer, for example, metastatic cancer. Disseminated cancer should be understood as cancer that has spread from its original site of origin in the body. For example, disseminated cancer can originate from the pancreas, bone marrow, colon, prostate or breast tissue of a patient and metastasize to the patient's liver or lung, etc.

[0045] Combination therapy including a GREM1 antagonist and an inhibitor of Ras-Raf-MEK-ERK signaling can also be used to prevent cancer dissemination.

[0046] Combination therapy including a GREM1 antagonist and an inhibitor of Ras-Raf-MEK-ERK signaling can be used to prevent cancer-associated polyposis.

[0047] Grading systems are used in cancer biology and medicine to classify cancer cells with respect to their lack of cellular differentiation. This reflects how different the morphology of cancer cells is from healthy cells found in the tissue in which they originate. Grading systems can be used as an indication of how fast a particular cancer will grow. Typically used cancer grades are grades (G)X and 1-4. GX indicates that the cancer grade cannot be assessed. G1 (low grade) cancer cells have a similar morphology to normal, healthy cells (i.e., well differentiated) and are expected to grow slowly and not likely to spread. G2 (intermediate grade) cancer cells are moderately differentiated; i.e., they look more abnormal and are expected to grow slightly faster than G1 cells. G3 (high grade) cancer cells have a very different morphology compared to normal cells (i.e., they are less differentiated) and are expected to grow faster than G1 and G2 cells. G4 (high-grade) cancer cells are undifferentiated (also called anaplastic) and are predicted to have the highest proliferation potential.

[0048] Cancer grade is different from cancer staging, which gives an indication of how the cancer will spread. A common cancer staging system has five stages: Stage 0: cancer cells are in situ (located in normal tissue); Stage I: cancer is limited to one part of the body; Stage II: cancer is locally advanced; Stage III: cancer is more locally advanced (whether it is designated as stage II or stage III may depend on the particular type of cancer); Stage IV: cancer has often metastasized, or spread to other organs or the whole body.

[0049] The skilled artisan knows how to determine the grade and / or stage of cancer. In one embodiment, the present invention relates to the use of an anti-GREM1 antagonist in combination with an inhibitor of Ras-Raf-MEK-ERK signaling for the treatment and / or prevention of established cancer. In one embodiment, the cancer is an established cancer. The established cancer may be a high-grade cancer, such as a G3 or G4 cancer. The established cancer may be a stage II or higher cancer. The established cancer may be a stage III or IV cancer. In one embodiment, the established cancer is a metastatic stage IV cancer. In one embodiment, the established cancer is an established pancreatic cancer.

[0050] In addition to the specifically exemplified uses in the treatment and prevention of pancreatic cancer, the inventors envisage that the therapeutic effects of GREM1 antagonists in combination with inhibitors of Ras-Raf-MEK-ERK signalling as exemplified in the Examples are also applicable to the treatment of other cancers having corresponding characteristics as described herein.

[0051] In particular, it is envisaged that the combination comprising a GREM1 antagonist is useful for the prevention or treatment of cancers in which the Ras-Raf-MEK-ERK pathway is dysregulated in response to treatment / exposure with an anti-GREM1 antagonist as described herein. In particular, the cancer may be a cancer in which the Ras-Raf-MEK-ERK signaling is induced / upregulated after treatment with an anti-GREM1 antagonist as described herein. Such a cancer may not show dysregulation of the Ras-Raf-MEK-ERK pathway in the absence of treatment with an anti-GREM1 antagonist. In other words, the cancer may show normal Ras-Raf-MEK-ERK signaling compared to a reference sample or reference value before treatment with an anti-GREM1 antagonist as described herein.

[0052] It is also envisaged that combinations comprising GREM1 antagonists are useful for the prevention or treatment of cancers in which there is stromal and / or epithelial overexpression of GREM1 and this overexpression contributes to the proliferation of malignant cells, including pancreatic cancer, bladder cancer, lung cancer, gastric cancer, duodenal cancer, esophageal cancer, head and neck cancer, glioma, endometrial cancer, liver cancer, splenic cancer, bone resident cancer and osteosarcoma.

[0053] The antagonist of the present invention is used in combination with an inhibitor of Ras-Raf-MEK-ERK signal transduction (e.g., MEK inhibitor or ERK inhibitor) to treat or prevent cancer. Preventing cancer also includes preventing a subject from being diagnosed with cancer or delaying the onset of cancer. Preventing cancer can also include preventing the relapse or recurrence of cancer in a subject previously diagnosed with cancer. Preventing cancer additionally includes increasing the survival of a subject who has not been diagnosed with cancer or who has previously been diagnosed with cancer.

[0054] Treating cancer can improve one or more symptoms of cancer, induce or prolong remission of cancer, or delay recurrence or recurrence of cancer. Treating cancer can cure, alleviate, or partially halt cancer. It may result in a decrease in the severity of disease symptoms, or an increase in the frequency or duration of symptom-free periods. Treating cancer can also include preventing the spread of cancer (e.g., established cancer) from a site of origin in the patient's body to one or more secondary sites in the patient's body. Thus, treating cancer can include preventing dissemination or metastasis of an existing cancer. In a preferred embodiment, the antagonist of the present invention is used to treat or prevent pancreatic cancer in combination with an inhibitor of Ras-Raf-MEK-ERK signaling (e.g., a MEK inhibitor or an ERK inhibitor). Treating pancreatic cancer can result in a reduction in primary tumor size, for example, as assayed by CT or endoscopic ultrasound. Such a reduction may facilitate Whipple procedure (pancreaticoduodenectomy), which removes tumors from the head of the pancreas. The present inventors have demonstrated that treatment with anti-GREM1 antagonists in combination with MEK inhibitors can increase survival in KPC mouse models.Therefore, treatment of pancreatic cancer with anti-GREM1 antagonists and MEK inhibitors can also improve patient survival.It is expected that treatment of cancer, especially pancreatic cancer, with ERK inhibitors will also produce similar beneficial results.

[0055] Pancreatic cancer The present invention is preferably directed to the treatment or prevention of pancreatic cancer. As described in more detail in the Examples, the inventors confirmed the overexpression of GREM1 in human pancreatic ductal adenocarcinoma samples, and demonstrated that high expression of GREM1 mRNA is significantly associated with poor prognosis (Figure 1). Furthermore, the inventors demonstrated that in KPC mouse models, after exposure with anti-GREM1 antagonist, KRAS signaling is upregulated (Figure 4), and in KPC mouse models, upon exposure with anti-GREM1 antibody Ab7326, genes involved in the Ras-Raf-MEK-ERK signaling pathway, such as Mapk1, Nras and Myc, are upregulated (Figure 3). The inventors also showed that combination therapy comprising anti-GREM1 antagonist and MEK inhibitor significantly increases survival, delays tumor growth, and promotes tumor shrinkage in KPC mouse models. KPC mice develop tumors that are histologically and pathologically similar to human pancreatic tumors and mimic human pancreatic cancer.

[0056] Thus, in one embodiment, pancreatic cancer is pancreatic cancer characterized by having overexpression of GREM1. In another embodiment, pancreatic cancer can be characterized as having exocrine tumors or neuroendocrine tumors. Pancreatic neuroendocrine cancer (also known as pancreatic islet cell tumors) arise in the endocrine glands of the pancreas. A particularly preferred form of pancreatic cancer is exocrine pancreatic cancer, such as pancreatic ductal adenocarcinoma (PDAC). PDAC has been reported to account for 90% of all pancreatic carcinomas (Feldmann et al. J Hepatobiliary Pancreat Surg. 2007; 14(3): 224-32). Pancreatic cancer can be any cancer that arises in the lining of the pancreatic duct.

[0057] The Ras-Raf-MEK-ERK signaling pathway has been reported to play an important role in pancreatic ductal adenocarcinoma (Collisson EA. et al. (2012) A central role for RAF→MEK→ERK signaling in the genesis of pancreatic ductal adenocarcinoma. Cancer Discov. 2012 2(8):685-93). For example, activating mutations in the KRAS oncogene are known to drive PDAC formation by promoting increased cell proliferation, motility and survival, but targeting KRAS signaling remains challenging and clinical trials with MEK inhibitors have rarely yielded statistically significant results in patients (Brauswetter et al. (2017) Molecular subtype specific efficacy of MEK inhibitors in pancreatic cancers. PLoS ONE 12(9): e0185687). Thus, in one embodiment, the pancreatic cancer to be treated is a pancreatic cancer with dysregulation and / or mutations of the Ras-Raf-MEK-ERK pathway in the Ras (e.g., KRAS, NRAS, and / or HRAS) or Raf genes (e.g., ARAF, BRAF, and / or CRAF) as described herein. For example, the pancreatic cancer may be a pancreatic cancer with dysregulation and / or mutations of the Ras-Raf-MEK-ERK pathway in the Ras (e.g., KRAS, NRAS, and / or HRAS) or Raf genes (e.g., ARAF, BRAF, and / or CRAF) as described herein. G12D or KRAS G12R The pancreatic cancer may be a pancreatic cancer tumor having a BRAF mutation / substitution. V600E It may be a pancreatic cancer tumor with a mutation / substitution.

[0058] In another embodiment, the pancreatic cancer is a pancreatic cancer in which Ras-Raf-MEK-ERK pathway is dysregulated in response to treatment / exposure with anti-GREM1 antagonists as described herein.In particular, the pancreatic cancer can be a pancreatic cancer in which Ras-Raf-MEK-ERK signaling is induced / upregulated after treatment with anti-GREM1 antagonists.Such pancreatic cancer may not show dysregulation of Ras-Raf-MEK-ERK pathway in the absence of treatment with anti-GREM1 antagonists.In other words, the pancreatic cancer may show normal Ras-Raf-MEK-ERK pathway signaling compared to a reference sample or reference value before treatment with anti-GREM1 antagonists as described herein.

[0059] Other exocrine pancreatic cancers include squamous cell carcinoma forming in the pancreatic duct; adenosquamous carcinoma; signet ring cell carcinoma; and colloid carcinoma, which typically arises from an intraductal papillary mucinous neoplasm.

[0060] A preferred type of pancreatic cancer to be treated may be resistant to one or more known anti-cancer agents (e.g., chemotherapy). The pancreatic cancer may be disseminated pancreatic cancer. The pancreatic cancer may be metastatic pancreatic cancer. Metastatic cancer should be understood as cancer that has spread from its original site of origin in the living body. Thus, metastatic pancreatic cancer refers to cancer that starts in the pancreas and spreads to other organs, such as the lung, liver, bone, and brain. Typically, metastatic pancreatic cancer refers to cancer that starts in the pancreas and spreads to the lung and / or liver. The pancreatic cancer may also be recurrent pancreatic cancer. In other words, pancreatic cancer to be treated by the method of the present invention includes pancreatic cancer that has recurred after months or even years of previous treatment, such as chemotherapy, radiotherapy, or radical surgery. A preferred type of pancreatic cancer to be treated may be resistant to one or more known anti-cancer agents (e.g., chemotherapy), as further described below.

[0061] In some aspects, the pancreatic cancer may be a pancreatic cancer that is responsive to treatment with an inhibitor of Ras-Raf-MEK-ERK signaling, e.g., a pancreatic cancer that is responsive to treatment with a MEK or ERK inhibitor as described herein. Alternatively, the pancreatic cancer may be a pancreatic cancer that is poorly responsive, non-responsive, or refractory to treatment with an inhibitor of Ras-Raf-MEK-ERK signaling, e.g., a pancreatic cancer that is poorly responsive, non-responsive, or refractory to treatment with a MEK or ERK inhibitor as described herein. The pancreatic cancer may be one that was previously considered unsuitable for treatment with an inhibitor of Ras-Raf-MEK-ERK signaling, e.g., a pancreatic cancer that was previously considered unsuitable for treatment with a MEK or ERK inhibitor as described herein. In some cases, the pancreatic cancer may initially be responsive to treatment with an inhibitor of Ras-Raf-MEK-ERK signaling, but develop resistance to the inhibitor of Ras-Raf-MEK-ERK signaling. For example, pancreatic cancer may be initially responsive to treatment with a MEK or ERK inhibitor, but may acquire resistance to the MEK or ERK inhibitors described herein.

[0062] lung cancer In a further aspect, the present invention relates to the treatment or prevention of lung cancer. The lung cancer is most preferably non-small cell lung cancer (NSCLC). The NSCLC can be advanced NSCLC, such as stage III or stage IV NSCLC. The NSCLC can be squamous cell carcinoma, adenocarcinoma or large cell carcinoma. The cancer can be small cell lung cancer. The lung cancer can be unresectable. The lung cancer can be non-angiogenic and / or can exhibit vascular coupling. The lung cancer can include alveolar and / or stromal tumor growth. The lung cancer can be primary lung cancer or any secondary cancer that has spread to the lung, such as breast cancer or pancreatic cancer. The lung cancer can be disseminated lung cancer. The lung cancer can be metastatic lung cancer. The lung cancer can be lung cancer characterized by having overexpression of GREM1. The lung cancer can also be recurrent lung cancer. In other words, lung cancers to be treated by the methods of the present invention include lung cancers that have recurred months or even years after previous treatments such as chemotherapy, radiotherapy or curative surgery. Preferred types of lung cancers to be treated may be resistant to one or more known anti-cancer agents (e.g., chemotherapeutic agents), as described further below.

[0063] In some aspects, the lung cancer may be a lung cancer that is responsive to treatment with an inhibitor of Ras-Raf-MEK-ERK signaling, e.g., a lung cancer that is responsive to treatment with a MEK or ERK inhibitor as described herein. Alternatively, the lung cancer may be a lung cancer that is poorly responsive, non-responsive, or refractory to treatment with an inhibitor of Ras-Raf-MEK-ERK signaling, e.g., a lung cancer that is poorly responsive, non-responsive, or refractory to treatment with a MEK or ERK inhibitor as described herein. The lung cancer may be a lung cancer that was previously considered unsuitable for treatment with an inhibitor of Ras-Raf-MEK-ERK signaling, e.g., a lung cancer that was previously considered unsuitable for treatment with a MEK or ERK inhibitor as described herein. In some cases, the lung cancer may initially be responsive to treatment with an inhibitor of Ras-Raf-MEK-ERK signaling, but may become resistant to the inhibitor of Ras-Raf-MEK-ERK signaling. For example, lung cancers may be initially responsive to treatment with MEK or ERK inhibitors, but may acquire resistance to the MEK or ERK inhibitors described herein.

[0064] In a further aspect, the lung cancer to be treated is a lung cancer having dysregulation of Ras-Raf-MEK-ERK pathway and / or mutation in Ras (e.g., KRAS, NRAS and / or HRAS) or Raf gene (e.g., ARAF, BRAF and / or CRAF) as described herein.For example, the lung cancer to be treated can be a non-small cell lung cancer having dysregulation of MAPK / ERK pathway and / or mutation in Ras (e.g., KRAS, NRAS and / or HRAS) or Raf gene (e.g., ARAF, BRAF and / or CRAF) as described herein.

[0065] In another embodiment, the lung cancer is a lung cancer in which Ras-Raf-MEK-ERK signaling pathway is dysregulated in response to treatment / exposure with the anti-GREM1 antagonist described herein. In particular, the lung cancer can be a lung cancer in which Ras-Raf-MEK-ERK signaling is induced / upregulated after treatment with the anti-GREM1 antagonist. Such lung cancer may not show dysregulation of Ras-Raf-MEK-ERK pathway in the absence of treatment with the anti-GREM1 antagonist. In other words, the lung cancer may show normal Ras-Raf-MEK-ERK pathway signaling compared to a reference sample or reference value before treatment with the anti-GREM1 antagonist described herein.

[0066] melanoma In a further aspect, the present invention relates to the treatment or prevention of melanoma. The melanoma can be cutaneous / skin melanoma, such as superficial spreading melanoma, nodular melanoma, lentigo maligna melanoma, or lentigo acuminata melanoma. The cutaneous melanoma can be amelanotic melanoma, nevus melanoma, patchy melanoma, or detumorous melanoma. In some aspects, the melanoma can be eye / ocular melanoma, such as conjunctival melanoma, choroidal melanoma, or iris melanoma. In other aspects, the melanoma can be mucosal melanoma.

[0067] The melanoma may be metastatic melanoma. The melanoma may be characterized by having overexpression of GREM1. The melanoma may also be recurrent melanoma. In other words, the melanoma to be treated by the method of the present invention includes melanoma that recurs after several months or even years after previous treatment such as chemotherapy, radiotherapy or radical surgery. The preferred type of melanoma to be treated may be resistant to one or more known anti-cancer agents (e.g., chemotherapeutic agents), as described further below.

[0068] In some aspects, the melanoma may be a melanoma that is responsive to treatment with an inhibitor of Ras-Raf-MEK-ERK signaling, e.g., a melanoma that is responsive to treatment with a MEK or ERK inhibitor as described herein. Alternatively, the melanoma may be a melanoma that is poorly responsive, non-responsive, or refractory to treatment with an inhibitor of Ras-Raf-MEK-ERK signaling, e.g., a melanoma that is poorly responsive, non-responsive, or refractory to treatment with a Ras-Raf-MEK-ERK inhibitor as described herein. The melanoma may be a melanoma that was previously considered unsuitable for treatment with an inhibitor of Ras-Raf-MEK-ERK signaling, e.g., a melanoma that was previously considered unsuitable for treatment with a MEK or ERK inhibitor as described herein. In some cases, the melanoma may initially be responsive to treatment with an inhibitor of Ras-Raf-MEK-ERK signaling, but may acquire resistance to the inhibitor of Ras-Raf-MEK-ERK signaling. For example, melanomas may be initially responsive to treatment with MEK or ERK inhibitors, but may acquire resistance to the MEK or ERK inhibitors described herein.

[0069] In a further aspect, the melanoma to be treated is a melanoma with dysregulation of the MAPK / ERK pathway and / or mutations in the Ras (e.g., KRAS, NRAS and / or HRAS) or Raf genes (e.g., ARAF, BRAF and / or CRAF), as described herein.

[0070] In another embodiment, the melanoma is a melanoma in which MAPK / ERK pathway is dysregulated in response to treatment / exposure with an anti-GREM1 antagonist as described herein. In particular, the melanoma may be a melanoma in which MAPK / ERK signaling is induced / upregulated after treatment with an anti-GREM1 antagonist. Such melanoma may not show MAPK / ERK pathway dysregulation in the absence of treatment with an anti-GREM1 antagonist. In other words, the melanoma may show normal MAPK / ERK pathway signaling compared to a reference sample or reference value before treatment with an anti-GREM1 antagonist as described herein.

[0071] Colorectal cancer The present invention relates in one aspect to the prevention or treatment of colorectal cancer. By way of background, the intestinal mucosa is a complex ecosystem in which the epithelium is in an interdependent relationship with its microenvironment, particularly the underlying stroma. Mesenchymal-epithelial crosstalk is deeply involved in controlling homeostasis and is dynamically altered during intestinal regeneration and cancer. Cell signaling networks, which are effector pathways of intercompartmental crosstalk and control epithelial cell fate decisions, can be coupled and disrupted by the tumor microenvironment in colorectal cancer.

[0072] Current chemotherapy management of colorectal cancer has not changed substantially in the past 20 years and is primarily based on combinations of cytotoxic agents directed against the proliferating tumour epithelium (e.g. FOLFOX and FOLFIRI regimens http: / / www.cancerresearchuk.org / about-cancer / cancer-in-general / treatment / cancer-drugs / drugs), with the potential for resistance to these epithelium-targeting agents to develop. Identifying novel treatments for use in colorectal cancer has never been more important.

[0073] Thus, the cancer or tumor to be treated is a colon cancer or a colon tumor. A particularly preferred form of colon cancer to be treated is a colon cancer characterized by overexpression of GREM1 in stromal cells, i.e., stromal GREM1 overexpression. The stromal cells may be cancer-associated fibroblasts. A colon cancer with stromal GREM1 overexpression may not show epithelial GREM1 overexpression. A colon cancer with stromal GREM1 overexpression may include stromal Foxl1 overexpression. A particularly suitable form of colon cancer to be treated is a colon cancer of mesenchymal subtype, also described as CMS4 (Guinney et al, Nat Med 2015). Any other subtype of colon cancer may also be treated, including any of CMS1, CMS2 and CMS3, as described in Guinney et al. supra. The colon cancer described herein may be a proximal colon cancer (or proximal colon tumor). The proximal colon is the region of the large intestine upstream from the splenic flexure, and refers to the cecum, ascending colon, and transverse colon. Cancers or tumors in this region are also called right-sided cancers or tumors. The present invention may relate to the treatment of right-sided colon cancer or right-sided colon tumors.

[0074] The colon cancer may be a distal colon cancer (or a distal colon tumor). Distal colon is the region of the colon downstream from the splenic flexure, meaning the descending colon, the sigmoid colon and the rectum. Cancers or tumors in this region are also called left-sided cancers or tumors. The present invention may relate to the treatment of left-sided colon cancers or tumors. The cancer with stromal overexpression of GREM1 may preferably be a sporadic cancer. Sporadic cancers may be caused by somatic mutations. Sporadic cancers may be caused by carcinogens. Sporadic cancers are not due to inherited genetic mutations. Sporadic cancers may cause stromal overexpression of GREM1. The growth of sporadic cancers may depend on stromal overexpression of GREM1 in the cancer.

[0075] At least three single nucleotide polymorphisms (SNPs) near GREM1 are independently associated with risk of colorectal cancer (CRC) in Northern European Caucasians and possibly other ethnicities (Tomlinson et al, PLos Genet, 2011). There is a direct link with GREM1 expression, and other SNPs are likely to have similar effects. In addition, two common SNPs near BMP2, two near BMP4, and one near BMP7 affect expression of BMP ligands and thus CRC risk. Thus, cancers may contain one or more of the above SNPs.

[0076] Further types of cancer or tumors amenable to treatment according to the invention are those that show overexpression of GREM1 in epithelial cells. Overexpression of GREM1 in epithelial cells may cause cancer. Cancer growth may depend on overexpression of GREM1 in the epithelium. Thus, the cancer may be of epithelial origin. The cancer may be colon cancer or duodenal cancer. The cancer may be GREM1-induced. By GREM1-induced, it is meant that the cancer is caused by a mutagenic event that enhances the activity or expression of GREM1. Such cancers may result from inherited genetic mutations. Thus, the cancer may be familial (see below).

[0077] Preferred types of colon cancer to be treated may be resistant to one or more known anti-cancer agents (eg, chemotherapeutic agents), as described further below.

[0078] The colorectal cancer can be disseminated colorectal cancer. The colorectal cancer can be metastatic colorectal cancer. The colorectal cancer can be lung metastatic colorectal cancer. The colorectal cancer can be liver metastatic colorectal cancer. The colorectal cancer can be bone metastatic colorectal cancer.

[0079] Colon cancer can be characterized by stromal overexpression of Foxl1. Colon cancer can be characterized by stromal overexpression of one or more Wnt ligands. For example, colon cancer can be characterized by stromal overexpression of Wnt5A and / or Wnt2B. When colon cancer has stromal overexpression of Foxl1 and / or Wnt ligand, for example, Wnt5A or Wnt2B, the colon cancer is particularly suitable for prevention or treatment with GREM1 antagonist.

[0080] In some aspects, the colon cancer may be a colon cancer that is responsive to treatment with an inhibitor of Ras-Raf-MEK-ERK signaling, e.g., a colon cancer that is responsive to treatment with a MEK or ERK inhibitor as described herein. Alternatively, the colon cancer may be a colon cancer that is poorly responsive, non-responsive, or refractory to treatment with an inhibitor of Ras-Raf-MEK-ERK signaling, e.g., a colon cancer that is poorly responsive, non-responsive, or refractory to treatment with a MEK inhibitor or ERK inhibitor as described herein. The colon cancer may be one that was previously considered unsuitable for treatment with an inhibitor of Ras-Raf-MEK-ERK signaling, e.g., a colon cancer that was previously considered unsuitable for treatment with a MEK inhibitor or ERK inhibitor as described herein. In some cases, the colon cancer may initially respond to treatment with an inhibitor of Ras-Raf-MEK-ERK signaling, but may acquire resistance to the inhibitor of Ras-Raf-MEK-ERK signaling. For example, colon cancer may initially be responsive to treatment with a MEK inhibitor or ERK inhibitor as described herein, but may acquire resistance to the MEK inhibitor or ERK inhibitor.

[0081] In a further aspect, the colon cancer to be treated is a colon cancer having dysregulation of the Ras-Raf-MEK-ERK pathway and / or a mutation in a Ras gene (e.g., KRAS, NRAS and / or HRAS) or a Raf gene (e.g., ARAF, BRAF and / or CRAF) as described herein. For example, the colon cancer may be a KRAS / BRAF mutated colon cancer. KRAS mutated colon cancer may be characterized as having any of the following mutations in KRAS: G12D, G12V, G12C, G12A, G13D, Q61H, Q61L, Q61R, A146T and / or A146V. BRAF mutated colon cancer may be characterized as having any of the following mutations in BRAF: G12D, G12V, G12C, G12A, G13D, Q61H, Q61L, Q61R, A146T and / or A146V. V600E It may be a mutation.

[0082] In another embodiment, the colon cancer is a colon cancer in which the Ras-Raf-MEK-ERK pathway is dysregulated in response to treatment / exposure with the anti-GREM1 antagonist described herein. In particular, the colon cancer may be a colon cancer in which Ras-Raf-MEK-ERK signaling is induced / upregulated after treatment with the anti-GREM1 antagonist. Such a colon cancer may not show dysregulation of the Ras-Raf-MEK-ERK pathway in the absence of treatment with the anti-GREM1 antagonist. In other words, the colon cancer may show normal Ras-Raf-MEK-ERK pathway signaling compared to a reference sample or reference value before treatment with the anti-GREM1 antagonist described herein.

[0083] Familial cancer Familial cancers include those resulting from a mutation or mutations in the gene encoding GREM1 or any other mutation affecting expression of the GREM1 gene. Autosomal dominant mixed polyposis syndrome (HMPS) is caused by a 40 kb duplication upstream of GREM1, resulting in a shift in pathological compartment expression from a mesenchymal-restricted gradient to ectopic GREM1 gene expression throughout the epithelium.

[0084] A subject to be treated with an anti-GREM1 antagonist may have previously been determined to be at risk for developing a familial cancer. For example, the subject may have been determined to be at risk based on family history and / or because the subject has a mutation in a gene known to cause or increase the risk of developing a familial cancer.

[0085] The familial cancer may be Lynch syndrome, also called hereditary nonpolyposis colorectal cancer (HNPCC). The familial cancer may be familial adenomatous polyposis (FAP).

[0086] Patients or subjects suffering from familial adenomatous polyposis (FAP) may be particularly suitable for treatment with a combination therapy comprising an anti-GREM1 antagonist. The familial cancer to be treated or prevented with a combination therapy comprising an anti-GREM1 antagonist (e.g., an anti-GREM1 antibody) and an inhibitor of Ras-Raf-MEK-ERK signaling may be FAP. Subjects previously suffering from FAP may be administered prophylactically with an anti-GREM1 antagonist in combination with an inhibitor of Ras-Raf-MEK-ERK signaling, for example, to prevent recurrence. Subjects not previously suffering from FAP, but previously determined to be at risk for developing FAP, may be administered prophylactically with an anti-GREM1 antagonist in combination with an inhibitor of Ras-Raf-MEK-ERK signaling. A subject is determined to be at risk for developing FAP because the subject is found to have a fulminant mutation in the Apc gene.

[0087] In some aspects, the familial cancer may be a familial cancer that is responsive to treatment with an inhibitor of Ras-Raf-MEK-ERK signaling, e.g., a familial cancer that is responsive to treatment with a MEK or ERK inhibitor as described herein. The familial cancer may alternatively be a familial cancer that is poorly responsive, non-responsive, or refractory to treatment with an inhibitor of Ras-Raf-MEK-ERK signaling, e.g., a familial cancer that is poorly responsive, non-responsive, or refractory to treatment with a MEK or ERK inhibitor as described herein. The familial cancer may have previously been considered unsuitable for treatment with an inhibitor of Ras-Raf-MEK-ERK signaling, e.g., may have been considered unsuitable for treatment with a MEK or ERK inhibitor as described herein. In some cases, the familial cancer may initially respond to treatment with an inhibitor of Ras-Raf-MEK-ERK signaling, but may acquire resistance to the inhibitor of Ras-Raf-MEK-ERK signaling. For example, a familial cancer may initially be responsive to treatment with a MEK or ERK inhibitor as described herein, but may acquire resistance to the MEK or ERK inhibitor.

[0088] In a further aspect, the familial cancer to be treated is a familial cancer with dysregulation of the Ras-Raf-MEK-ERK pathway and / or a mutation in a Ras gene (e.g., KRAS, NRAS and / or HRAS) or a Raf gene (e.g., ARAF, BRAF and / or CRAF), as described herein.

[0089] In another embodiment, the familial cancer is a familial cancer in which the Ras-Raf-MEK-ERK pathway is dysregulated in response to treatment / exposure with the anti-GREM1 antagonist described herein. In particular, the familial cancer may be a familial cancer in which Ras-Raf-MEK-ERK signaling is induced / upregulated after treatment with the anti-GREM1 antagonist. Such familial cancer may not show dysregulation of the Ras-Raf-MEK-ERK pathway in the absence of treatment with the anti-GREM1 antagonist. In other words, the familial cancer may show normal Ras-Raf-MEK-ERK pathway signaling compared to a reference sample or reference value before treatment with the anti-GREM1 antagonist described herein.

[0090] Multiple myeloma In another aspect, the present invention relates to the treatment or prevention of multiple myeloma. Multiple myeloma (MM) is a hematological malignancy characterized by clonal proliferation of plasma cells (PC) within the bone marrow (BM). It is well known that BM supports MM tumor growth, and bidirectional signaling between tumor cells and BM is essential for the continued proliferation, proliferation, and survival of MM PC. Cellular and non-cellular BM components differentially affect the proliferation and proliferation of MMPC. Although recent studies have identified components of BM that play a role in disease progression and developed treatments that target these, standard treatments in MM still rely primarily on targeting the tumor cells themselves. Although such therapies are effective in prolonging patient survival, the large role played by BM in MM cell proliferation, proliferation, survival, and drug resistance necessitates more effective therapies that target key aspects of the disease. Indeed, MM is largely incurable, and disease relapse is a major problem faced in effectively treating the disease.

[0091] Therefore, the present invention is also directed to the treatment or prevention of multiple myeloma. Multiple myeloma typically includes the presence of multiple plasma cell masses in the bone marrow. Thus, multiple myeloma is typically associated with abnormal proliferation of plasma cells in the bone marrow. Particularly preferred forms of multiple myeloma to be treated are characterized by having overexpression of GREM1 in the bone marrow. Thus, multiple myeloma may include overexpression of stromal GREM1. Stromal GREM1 overexpression may be present in the dense matter compartment of bone. Stromal GREM1 overexpression may reflect an increase in the number of stromal cells or an increase in the expression level of GREM1 in existing GREM1-expressing stromal cells. Bone marrow may include osteochondral (OCR) stem cells. Stromal cells that overexpress GREM1 may include OCR stem cells. Preferred types of multiple myeloma to be treated may be resistant to one or more known anti-cancer agents (e.g., chemotherapeutic agents), as further described below.

[0092] In some embodiments, the multiple myeloma may be a multiple myeloma that is responsive to treatment with an inhibitor of Ras-Raf-MEK-ERK signaling, e.g., a multiple myeloma that is responsive to treatment with a MEK or ERK inhibitor as described herein. Alternatively, the multiple myeloma may be a multiple myeloma that is poorly responsive, non-responsive, or refractory to treatment with an inhibitor of Ras-Raf-MEK-ERK signaling, e.g., a multiple myeloma that is poorly responsive, non-responsive, or refractory to treatment with a MEK or ERK inhibitor as described herein. The multiple myeloma may be a multiple myeloma that was previously thought to be unsuitable for treatment with an inhibitor of Ras-Raf-MEK-ERK signaling, e.g., a multiple myeloma that was previously thought to be unsuitable for treatment with a MEK or ERK inhibitor. In some cases, multiple myeloma may initially respond to treatment with an inhibitor of Ras-Raf-MEK-ERK signaling, but may acquire resistance to the inhibitor of Ras-Raf-MEK-ERK signaling. For example, multiple myeloma may initially respond to treatment with a MEK or ERK inhibitor described herein, but may acquire resistance to the MEK or ERK inhibitor.

[0093] In a further aspect, the multiple myeloma to be treated is a multiple myeloma having dysregulation of the Ras-Raf-MEK-ERK pathway and / or a mutation in a Ras gene (e.g., KRAS, NRAS and / or HRAS) or a Raf gene (e.g., ARAF, BRAF and / or CRAF), as described herein.

[0094] In another embodiment, the multiple myeloma is a multiple myeloma in which the Ras-Raf-MEK-ERK pathway is dysregulated in response to treatment / exposure with an anti-GREM1 antagonist as described herein. In particular, the multiple myeloma may be a multiple myeloma in which Ras-Raf-MEK-ERK signaling is induced / upregulated after treatment with an anti-GREM1 antagonist. Such multiple myeloma may not show dysregulation of the Ras-Raf-MEK-ERK pathway in the absence of treatment with an anti-GREM1 antagonist. In other words, the multiple myeloma may show normal Ras-Raf-MEK-ERK pathway signaling compared to a reference sample or reference value before treatment with an anti-GREM1 antagonist as described herein.

[0095] Breast cancer In another aspect, the present invention relates to the treatment or prevention of breast cancer. The breast cancer may be invasive breast cancer, e.g., invasive lobular breast cancer. The breast cancer may be triple-negative breast cancer. The breast cancer may be inflammatory breast cancer. The breast cancer may be breast angiosarcoma. The breast cancer may be in situ ductal carcinoma or in situ lobular carcinoma.

[0096] The present invention provides for the treatment and prevention of breast cancer by administering an anti-GREM1 antagonist in combination with an inhibitor of Ras-Raf-MEK-ERK signaling. The breast cancer may comprise stromal GREM1 overexpression. The stromal breast cells overexpressing GREM1 may comprise stromal fibroblasts, also described herein as cancer-associated fibroblasts. The breast cancer may also be recurrent breast cancer. In other words, the breast cancer to be treated by the method of the present invention includes breast cancer that has recurred after months or even years following previous treatments such as chemotherapy, radiotherapy or curative surgery. The preferred types of breast cancer to be treated may be resistant to one or more known anti-cancer agents (e.g., chemotherapeutic agents), as further described below. The breast cancer may be disseminated breast cancer. The breast cancer may be metastatic breast cancer. The breast cancer may be lung metastatic breast cancer. The breast cancer may be liver metastatic breast cancer. The breast cancer may be bone metastatic breast cancer.

[0097] In some aspects, the breast cancer may be a breast cancer that is responsive to treatment with an inhibitor of Ras-Raf-MEK-ERK signaling, e.g., a breast cancer that is responsive to treatment with a MEK or ERK inhibitor as described herein. Alternatively, the breast cancer may be a breast cancer that is poorly responsive, non-responsive, or refractory to treatment with an inhibitor of Ras-Raf-MEK-ERK signaling, e.g., a breast cancer that is poorly responsive, non-responsive, or refractory to treatment with a MEK or ERK inhibitor as described herein. The breast cancer may be a breast cancer that was previously considered unsuitable for treatment with an inhibitor of Ras-Raf-MEK-ERK signaling, e.g., a breast cancer that was previously considered unsuitable for treatment with a MEK or ERK inhibitor as described herein. In some cases, the breast cancer may initially be responsive to treatment with an inhibitor of Ras-Raf-MEK-ERK signaling, but may acquire resistance to the inhibitor of Ras-Raf-MEK-ERK signaling. For example, breast cancers may be initially responsive to treatment with MEK or ERK inhibitors as described herein, but may acquire resistance to the MEK or ERK inhibitors.

[0098] In another embodiment, the breast cancer is a breast cancer in which the Ras-Raf-MEK-ERK pathway is dysregulated in response to treatment / exposure with an anti-GREM1 antagonist as described herein. In particular, the breast cancer may be a breast cancer in which Ras-Raf-MEK-ERK signaling is induced / upregulated after treatment with an anti-GREM1 antagonist. Such breast cancer may not show dysregulation of the Ras-Raf-MEK-ERK pathway in the absence of treatment with an anti-GREM1 antagonist. In other words, the breast cancer may show normal Ras-Raf-MEK-ERK pathway signaling compared to a reference sample or reference value before treatment with an anti-GREM1 antagonist as described herein.

[0099] Prostate cancer In a further aspect, the present invention relates to the treatment or prevention of prostate cancer. The prostate cancer may be prostate adenocarcinoma. The prostate cancer may be transitional cell carcinoma or urothelial carcinoma that has spread to the prostate.

[0100] Prostate cancer can be characterized by having overexpression of GREM1.Prostate cancer can also be recurrent prostate cancer.In other words, the prostate cancer to be treated by the method of the present invention includes prostate cancer that recurs after several months or even years after previous treatment such as chemotherapy, radiotherapy or radical surgery.The preferred type of prostate cancer to be treated can be resistant to one or more known anti-cancer agents (e.g., chemotherapeutic agents), as described further below.

[0101] The present invention further provides for the treatment and prevention of prostate cancer by administering an anti-GREM1 antagonist in combination with an inhibitor of Ras-Raf-MEK-ERK signaling. The prostate cancer can be disseminated prostate cancer. The prostate cancer can be metastatic prostate cancer. The prostate cancer can be lung metastatic prostate cancer. The prostate cancer can be liver metastatic prostate cancer. The prostate cancer can be bone metastatic prostate cancer.

[0102] In some aspects, the prostate cancer may be a prostate cancer that is responsive to treatment with an inhibitor of Ras-Raf-MEK-ERK signaling, e.g., a prostate cancer that is responsive to treatment with a MEK or ERK inhibitor as described herein. Alternatively, the prostate cancer may be a prostate cancer that is poorly responsive, non-responsive, or refractory to treatment with an inhibitor of Ras-Raf-MEK-ERK signaling, e.g., a prostate cancer that is poorly responsive, non-responsive, or refractory to treatment with a MEK or ERK inhibitor as described herein. The prostate cancer may be a prostate cancer that was previously considered unsuitable for treatment with an inhibitor of Ras-Raf-MEK-ERK signaling, e.g., a prostate cancer that was previously considered unsuitable for treatment with a MEK or ERK inhibitor as described herein. In some cases, the prostate cancer may initially be responsive to treatment with an inhibitor of Ras-Raf-MEK-ERK signaling, but may acquire resistance to the inhibitor of Ras-Raf-MEK-ERK signaling. For example, prostate cancer may initially be responsive to treatment with the MEK or ERK inhibitors described herein, but may acquire resistance to the MEK or ERK inhibitors.

[0103] In another embodiment, the prostate cancer is a prostate cancer in which Ras-Raf-MEK-ERK pathway is dysregulated in response to treatment / exposure with anti-GREM1 antagonists as described herein.In particular, the prostate cancer can be a prostate cancer in which Ras-Raf-MEK-ERK signaling is induced / upregulated after treatment with anti-GREM1 antagonists.Such prostate cancer may not show dysregulation of Ras-Raf-MEK-ERK pathway in the absence of treatment with anti-GREM1 antagonists.In other words, the prostate cancer may show normal Ras-Raf-MEK-ERK pathway signaling compared to a reference sample or reference value before treatment with anti-GREM1 antagonists as described herein.

[0104] Bladder cancer In a further aspect, the present invention relates to the treatment or prevention of bladder cancer. The bladder cancer may be transitional cell (urothelial) bladder cancer. The bladder cancer may arise from the epithelial lining of the bladder. The bladder cancer may be non-muscle invasive bladder cancer. The bladder cancer may be squamous cell bladder cancer. The bladder cancer may be adenocarcinoma. The bladder cancer may be a high-grade T1 tumor that has grown from the lining of the bladder into the lamina propria of the bladder. The bladder cancer may be superficial or invasive bladder cancer. The bladder cancer may be recurrent bladder cancer. The term recurrent bladder cancer as used herein refers to bladder cancer that has recurred after treatment, such as surgical treatment.

[0105] The present invention further provides for the treatment and prevention of bladder cancer by administering an anti-GREM1 antagonist in combination with an inhibitor of Ras-Raf-MEK-ERK signaling. The bladder cancer may be disseminated bladder cancer. The bladder cancer may be metastatic bladder cancer. The bladder cancer may be lung metastatic bladder cancer. The bladder cancer may be liver metastatic bladder cancer. The bladder cancer may be bone metastatic bladder cancer. The bladder cancer may be bladder cancer characterized by having overexpression of GREM1. It may also be recurrent bladder cancer. In other words, the bladder cancer to be treated by the method of the present invention includes bladder cancer that has recurred after months or even years following previous treatments such as chemotherapy, radiotherapy or radical surgery. The preferred type of bladder cancer to be treated may be resistant to one or more known anticancer agents (e.g., chemotherapeutic agents), as further described below.

[0106] In some aspects, the bladder cancer may be a bladder cancer that is responsive to treatment with an inhibitor of Ras-Raf-MEK-ERK signaling, e.g., a bladder cancer that is responsive to treatment with a MEK or ERK inhibitor as described herein. Alternatively, the bladder cancer may be a bladder cancer that is poorly responsive, non-responsive, or refractory to treatment with an inhibitor of Ras-Raf-MEK-ERK signaling, e.g., a bladder cancer that is poorly responsive, non-responsive, or refractory to treatment with a MEK or ERK inhibitor. The bladder cancer may be one that was previously considered unsuitable for treatment with an inhibitor of Ras-Raf-MEK-ERK signaling, e.g., a bladder cancer that was previously considered unsuitable for treatment with a MEK or ERK inhibitor as described herein. In some cases, the bladder cancer may be initially responsive to treatment with an inhibitor of Ras-Raf-MEK-ERK signaling, but may acquire resistance to the inhibitor of Ras-Raf-MEK-ERK signaling. For example, bladder cancer may be initially responsive to treatment with the MEK or ERK inhibitors described herein, but may acquire resistance to the MEK or ERK inhibitors.

[0107] In another embodiment, the bladder cancer is a bladder cancer in which the Ras-Raf-MEK-ERK pathway is dysregulated in response to treatment / exposure with an anti-GREM1 antagonist as described herein. In particular, the bladder cancer can be a bladder cancer in which Ras-Raf-MEK-ERK signaling is induced / upregulated after treatment with an anti-GREM1 antagonist. Such a bladder cancer may not show dysregulation of the Ras-Raf-MEK-ERK pathway in the absence of treatment with an anti-GREM1 antagonist. In other words, the bladder cancer can show normal Ras-Raf-MEK-ERK pathway signaling compared to a reference sample or reference value before treatment with an anti-GREM1 antagonist as described herein.

[0108] Ovarian cancer In a further aspect, the present invention relates to the treatment or prevention of ovarian cancer. The ovarian cancer may be epithelial ovarian cancer, germ cell ovarian cancer or sex cord stromal ovarian cancer. The ovarian cancer may be primary peritoneal cancer. The ovarian cancer may be fallopian tube cancer. The ovarian cancer may be characterized by borderline ovarian tumors. The ovarian cancer may be characterized by germ cell ovarian tumors. The ovarian cancer may be clear cell ovarian cancer. The ovarian cancer may be serous ovarian cancer. The ovarian cancer may be mucinous ovarian cancer. The ovarian cancer may be endometrial cancer.

[0109] The ovarian cancer may be characterized by having overexpression of GREM1.The ovarian cancer may also be recurrent ovarian cancer.In other words, the ovarian cancer to be treated by the method of the present invention includes ovarian cancer that recurs after several months or even years after previous treatment such as chemotherapy, radiotherapy or radical surgery.The preferred type of ovarian cancer to be treated may be resistant to one or more known anti-cancer agents (e.g., chemotherapeutic agents), as described further below.

[0110] The present invention further provides for the treatment and prevention of ovarian cancer by administering an anti-GREM1 antagonist in combination with an inhibitor of Ras-Raf-MEK-ERK signaling. The ovarian cancer can be disseminated ovarian cancer. The ovarian cancer can be metastatic ovarian cancer. The ovarian cancer can be lung metastatic ovarian cancer. The ovarian cancer can be liver metastatic ovarian cancer. The ovarian cancer can be bone metastatic ovarian cancer.

[0111] In some aspects, the ovarian cancer may be an ovarian cancer that is responsive to treatment with an inhibitor of Ras-Raf-MEK-ERK signaling, e.g., an ovarian cancer that is responsive to treatment with a MEK or ERK inhibitor as described herein. Alternatively, the ovarian cancer may be an ovarian cancer that is poorly responsive, non-responsive, or refractory to treatment with an inhibitor of Ras-Raf-MEK-ERK signaling, e.g., an ovarian cancer that is poorly responsive, non-responsive, or refractory to treatment with a MEK or ERK inhibitor as described herein. The ovarian cancer may be one that was previously considered unsuitable for treatment with an inhibitor of Ras-Raf-MEK-ERK signaling, e.g., an ovarian cancer that was previously considered unsuitable for treatment with a MEK or ERK inhibitor as described herein. In some cases, the ovarian cancer may be initially responsive to treatment with an inhibitor of Ras-Raf-MEK-ERK signaling, but may acquire resistance to the inhibitor of Ras-Raf-MEK-ERK signaling. For example, ovarian cancer may be initially responsive to treatment with the MEK or ERK inhibitors described herein, but may acquire resistance to the MEK or ERK inhibitors.

[0112] In another embodiment, the ovarian cancer is an ovarian cancer in which the Ras-Raf-MEK-ERK pathway is dysregulated in response to treatment / exposure with the anti-GREM1 antagonist described herein. In particular, the ovarian cancer may be an ovarian cancer in which Ras-Raf-MEK-ERK signaling is induced / upregulated after treatment with the anti-GREM1 antagonist. Such ovarian cancer may not show dysregulation of the Ras-Raf-MEK-ERK pathway in the absence of treatment with the anti-GREM1 antagonist. In other words, the ovarian cancer may show normal Ras-Raf-MEK-ERK pathway signaling compared to a reference sample or reference value before treatment with the anti-GREM1 antagonist described herein.

[0113] Stroma and epithelium The cancers described herein for prevention or treatment using GREM1 antagonists in combination with inhibitors of MAPK / ERK signaling may involve stromal and / or epithelial overexpression of GREM1.

[0114] As used herein, the term "stromal cell(s)" or "stroma" refers to the structural and / or connective parts of a tissue or organ.

[0115] The interstitial tissue is composed of an extracellular matrix that contains primarily connective tissue cells. The extracellular matrix is ​​composed primarily of the stroma, a porous hydrated gel made up of proteoglycan aggregates, and connective tissue fibers. Three types of fibers are commonly found within the interstitium: type I collagen fibers, elastic fibers, and reticular fibers (type III collagen). Fibroblasts and pericytes are the most common types of interstitial cells.

[0116] In the case of cancer or tumors (e.g., arising from the epithelium of a tissue or organ), the stroma of the tissue or organ may aid in the growth or progression of the cancer. Stroma associated with cancer or tumors may be decellularized stroma caused by the proliferation of fibrous or connective tissue surrounding the cancer or tumor.

[0117] Overexpression of GREM1 can be observed in any part of stroma / any stromal cell. Stromal cells can be fibroblasts or fibroblast-like supporting cells. Stromal cells can be fibroblasts or fibroblast-like supporting cells isolated from the detumorized stroma of any of the above cancers or tumors, for example, from pancreas, colon or rectum in colorectal cancer, or bone marrow in multiple myeloma. Stromal cells can be cancer-associated fibroblasts.

[0118] The term "epithelium" as used herein refers to cells originating from the outer or inner lining of a tissue or organ. In the context of the large intestine, the intestinal epithelium is the layer of cells that forms the luminal surface or lining of both the small and large intestines of the digestive tract. It is composed of a simple columnar epithelium. The "upper barrier" is an intestinal epithelial monolayer of columnar cells consisting of four types of intestinal epithelial cells: absorptive enterocytes, goblet cells, Paneth cells and enteroendocrine cells. The properties of the upper barrier are similar in the small and large intestines. The main difference is established by the presence of ridges or projections (circular folds, villi, microvilli) in the duodenum, jejunum and ileum that allow an increase in the absorption area. This is not observed in the colon, which instead shows a flat surface. Among the projections of the mucosa called villi are inflexions called crypts of Lieberkuhn, which are distinct glandular ductal invasions. The cells in which overexpression of epithelial GREM1 is observed can be any epithelial cell, such as any intestinal epithelial cell.

[0119] Without being bound by theory, the inventors hypothesize that overexpression of GREM1 in epithelium and / or stroma may promote stem cell / progenitor cell phenotype (increase stem cell / progenitor cell number), promote epithelial stem cell behavior, and drive cancer progression and / or resistance to chemotherapeutic agents. Thus, a combination therapy comprising a GREM1 antagonist and an inhibitor of Ras-Raf-MEK-ERK signaling used according to the present invention can prevent the induction of abnormal cancer stem cell / progenitor cell phenotype, reduce epithelial stem cell behavior, and / or reduce the number of stem cells / progenitor cells in the epithelium of the subject's tissue or organ in which cancer is to be prevented or treated. The ability of GREM1 antagonists to affect stem cell behavior can be clinically assayed by evaluation of known epithelial stem cell and cancer stem cell markers.

[0120] Overexpression of GREM1 in stroma and / or epithelium can be determined by any method. Overexpression of GREM1 is typically determined by comparison with the level of the marker in normal cells of the same tissue type, i.e., basal expression level. Expression is typically normalized to the expression levels of other genes, preferably one or more housekeeping genes. GREM1 can also be classified as showing overexpression or underexpression in a threshold percentage of a cancer patient population. Overexpression in each patient in the population may be higher than 2 from the geometric mean. At least 10% of the patients in the population, more preferably at least 15% or more, may show such overexpression.

[0121] GREM1 stromal overexpression refers to stromal GREM1 levels that are higher than the levels in matched normal tissue, for example, stromal GREM1 levels may be at least two-fold higher than the levels in matched normal tissue.

[0122] When GREM1 is overexpressed, its amount can be increased to any amount compared to basal state.For example, GREM1-induced cancers such as HMPS can contain epithelial GREM1 upregulation of several thousand times, while no expression of GREM1 is observed in normal epithelium.Sporadic cancers containing stromal GREM overexpression can contain any level of stromal overexpression that exceeds the physiological GREM1 expression level in the normal stroma of an organ.Those skilled in the art can evaluate the presence of overexpression in stroma or epithelium by comparing it with the level of GREM1 in normal cells of the same type.

[0123] The amount determined may be the amount of mRNA. Thus, the cancer may include overexpression of GREM1 mRNA. The cancer may include an increased amount of GREM1 mRNA compared to normal cells of the same tissue type. The mRNA may be increased by any amount. The amount of mRNA may be measured using quantitative reverse transcription polymerase chain reaction (qRT-PCR), e.g., real-time qRT-PCR, QuantiGene assay (Affymetrix / Thermo Fisher), by Northern blotting, or by microarray, RNA sequencing. The mRNA expression is preferably determined by comparing the gene expression of the sample to the distribution of expression levels of a particular gene across a reference sample composed of tumors that are diploid for that gene. The z-score may be derived using the RNAseq by expectation maximisation (RSEM) algorithm (cBioportal for Cancer Genomics, www.cbioportal.org; Gao et al, 2013 and Serami eta al 2012). A z-score 2SD above or below the mean of the reference set is preferably considered as overexpression or underexpression, respectively.

[0124] The amount determined may be the amount of protein. The cancer may include overexpression of GREM1 protein compared to normal cells of the same tissue type. The protein may be increased by any amount. The amount of protein may be measured using immunohistochemistry, Western blotting, mass spectrometry or fluorescence activated cell sorting (FACS), including the use of the anti-GREM1 antibody of the present invention. The threshold for determining expression may differ between the techniques used and may be verified against the immunohistochemistry score.

[0125] Thus, the use of a GREM1 antagonist in combination with an inhibitor of Ras-Raf-MEK-ERK signaling to treat or prevent cancer in a patient as described herein may include (a) measuring the amount of GREM1 in the cancer, and (b) if the cancer comprises overexpression of GREM1, administering to the patient a GREM1 antagonist in combination with an inhibitor of Ras-Raf-MEK-ERK signaling, thereby treating or preventing the cancer. The amount of GREM1 may be the amount of mRNA or protein, and the overexpression may be any of the overexpressions discussed above.

[0126] The above measurements can be performed on any suitable sample from a patient. Measurements can be performed on a cancer or tumor biopsy obtained from a patient. Stroma and / or epithelium (stromal and / or epithelial cells) can be isolated from the biopsy. The biopsy tissue can be formalin-fixed, paraffin-embedded (FFPE) tissue or fresh tissue. The tissue can be pancreatic tissue, bladder tissue, lung tissue, endometrial tissue, breast tissue, stomach tissue, duodenal tissue, esophageal tissue, bone marrow or colon tissue. Any of the methods discussed above can be performed on a cancer biopsy. Such methods can also be performed on cancer cells circulating in the patient's blood. RNA methods can be performed on exosomes in urine or blood.

[0127] Cancers associated with dysregulation of the Ras-Raf-MEK-ERK signaling pathway As mentioned above, the present inventors have demonstrated that KRAS signaling is upregulated in KPC mouse model after exposure to the anti-GREM1 antagonist (Ab7326) according to the present invention. Furthermore, genes involved in the Ras-Raf-MEK-ERK pathway, such as Mapk1, Nras and Myc, are upregulated in KPC mouse model after exposure to Ab7326. Therefore, combination therapy targeting both Gremlin-1 and Ras-Raf-MEK-ERK pathway is advantageous.

[0128] The present invention encompasses the use of anti-GREM1 antagonists in combination with inhibitors of Ras-Raf-MEK-ERK signaling to treat or prevent cancers associated with dysregulation of the Ras-Raf-MEK-ERK pathway and / or containing mutations in the Ras or Raf genes. The Ras genes can be, for example, KRAS, NRAS and / or HRAS. The Raf genes can be, for example, ARAF, BRAF and / or CRAF. Cancers associated with dysregulation of the MAPK / ERK pathway and / or containing mutations in the Ras or Raf genes include, for example, colon cancer, multiple myeloma, pancreatic cancer, bladder cancer, breast cancer, lung cancer, gastric cancer, ovarian cancer, duodenal cancer, esophageal cancer, head and neck cancer, prostate cancer, glioma, endometrial cancer, liver cancer, splenic cancer, bone resident cancer, osteosarcoma, etc.

[0129] The present invention also encompasses combination therapy comprising an anti-GREM1 antagonist as defined herein and an inhibitor of Ras-Raf-MEK-ERK signaling for the treatment or prevention of cancers in which the Ras-Raf-MEK-ERK pathway is dysregulated after prior exposure / treatment to an anti-GREM1 antagonist. In particular, the cancer may be a cancer in which the Ras-Raf-MEK-ERK pathway signaling is induced / upregulated after treatment with an anti-GREM1 antagonist as described herein. Such a cancer may not exhibit dysregulation of the Ras-Raf-MEK-ERK pathway in the absence of treatment with an anti-GREM1 antagonist. In other words, the cancer may exhibit normal Ras-Raf-MEK-ERK pathway signaling compared to a reference sample or reference value before treatment with an anti-GREM1 antagonist as described herein.

[0130] In a preferred embodiment, the present invention is directed to the treatment or prevention of pancreatic cancer. For example, the present invention encompasses a combination therapy comprising an anti-GREM1 antagonist as defined herein and an inhibitor of Ras-Raf-MEK-ERK signaling for the treatment or prevention of pancreatic cancer in which the Ras-Raf-MEK-ERK pathway is dysregulated after prior exposure / treatment to the anti-GREM1 antagonist. Pancreatic cancer may alternatively or additionally be characterized by constitutive activation of the classical MAPK pathway. In many cases, pancreatic cancer is associated with gain-of-function mutations in KRAS, which encodes RAS. Exemplary gain-of-function mutations include G12D or G12R substitutions and are found in pancreatic ductal adenocarcinoma. In other cases, pancreatic cancer is associated with gain-of-function mutations in BRAF, which encodes B-RAF. Exemplary gain-of-function mutations are found at the V600E position, resulting in constitutive kinase activity of RAF.

[0131] As mentioned above, the present invention may also be directed to the treatment or prevention of KRAS / BRAF mutant colon cancer. In another embodiment, the present invention is directed to BRAF mutant melanoma, e.g., melanoma carrying a V600E mutation in BRAF.

[0132] GREM1 The term GREM1 or Gremlin-1 as used herein in the context of a protein typically refers to human GREM1, a protein having the amino acid sequence shown in UniProt entry O60565 (SEQ ID NO: 1). The terms GREM1 and Gremlin-1 may also refer to the Gremlin-1 polypeptide, (a) comprising or consisting of the amino acid sequence of SEQ ID NO:1, with or without the N-terminal signal peptide, i.e. comprising or consisting of the mature peptide sequence shown in SEQ ID NO:21; or (b) a derivative having one or more amino acid substitutions, modifications, deletions, or insertions relative to the amino acid sequence of SEQ ID NO:1, with or without the N-terminal signal peptide (shown in SEQ ID NO:21), which retains the activity of Gremlin-1, such as the amino acid sequence of SEQ ID NO:20; (c) variants thereof, such variants typically retain at least about 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94% or 95% identity (or even about 96%, 97%, 98% or 99% identity) to SEQ ID NO: 1 (or SEQ ID NO: 20 or 21). In other words, such variants may retain about 60% to about 99% identity to SEQ ID NO: 1, preferably about 80% to about 99% identity to SEQ ID NO: 1, more preferably about 90% to about 99% identity to SEQ ID NO: 1, and most preferably about 95% to about 99% identity to SEQ ID NO: 1. Variants are further described below.

[0133] As discussed further below, residue numbers are typically cited based on the sequence of SEQ ID NO: 1. However, the residue numbering can be easily extrapolated by one of skill in the art to derivative or variant sequences, as discussed above. When residue numbers are cited, the present invention also encompasses those residues on the variant or derivative sequences.

[0134] The GREM1 or Gremlin-1 nucleic acid sequence may comprise or consist of the sequence of SEQ ID NO: 36 or SEQ ID NO: 37 or variants thereof. Variant nucleic acid sequences are further described below. The GREM1 or Gremlin-1 nucleic acid sequence may comprise or consist of any GREM1 transcript variant. Examples of GREM1 transcript variants are transcript 1 (NCBI: NM_013372.6; ENSEMBL: ENST00000560677.5); transcript 2: NCBI: NM_001191323.1; ENSEMBL: ENST00000560830.1); transcript 3: NCBI: NM_001191322.1; ENSEMBL: ENST00000622074.1. The sequences available under the above accession numbers as of June 18, 2018 are incorporated herein by reference.

[0135] Antagonist An anti-GREM1 antagonist is any molecule that reduces the function or activity of GREM1. An anti-GREM1 antagonist can reduce the function or activity of GREM1 by any amount. An anti-GREM1 antagonist can reduce the function or activity of GREM1 by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%, or can prevent any function or activity of GREM1. The extent to which an anti-GREM1 antagonist reduces the function or activity of GREM1 can be determined by measuring the function or activity of GREM1 in cells in the presence and absence of the anti-GREM1 antagonist. The cells can be normal cells or cancer cells. The cells can be cancer cells as described above. They can be pancreatic cancer cells. The pancreatic cancer cells can be in the KPC mouse model described in the examples. Thus, in vivo assays for the activity of GREM1 antagonists in combination therapy in pancreatic cancer can be performed in mouse models. More generally, GREM1 antagonists shown to reduce GREM1 function or activity by any means can then be assayed in vitro or in vivo for their ability to prevent or reduce the proliferation of cancer cells, such as pancreatic cancer cells, or to prevent, reduce or eliminate cancer or tumors.

[0136] Antagonists may reduce GREM1 function by any means. They may increase or decrease the activity or amount of any molecule that directly or indirectly affects GREM1 function. They may reduce the amount of GREM1 at the mRNA or protein level. They may increase the degradation of GREM1. They may reduce GREM1 function by inhibitory modifications. They may reduce transcription of molecules that enhance GREM1 function. They may destroy DNA encoding GREM1 or molecules that enhance GREM1 function using agents such as zinc finger nucleases.

[0137] The antagonist can be an agent that interacts with Gremlin-1. An agent that interacts with Gremlin-1 is typically an agent that binds to Gremlin-1. An agent that interacts with Gremlin-1 can modulate Gremlin-1. An inhibitory regulator can affect any of the functions of Gremlin-1, but typically reduces the binding of Gremlin-1 to BMPs (BMP 2 / 4 / 7). The antagonist can be a BMP-7 mimetic molecule. Gremlin-1 is a negative regulator of BMPs, so reduced binding increases signaling through BMPs. Activation of the regulator can increase the binding of Gremlin-1 to BMPs.

[0138] BMP binding and signaling can be detected by any method known in the art.

[0139] Antagonists can act by binding to the active site of GREM1 or allosterically by binding to a different site. Antagonists can act by binding to a modulator or ligand of GREM1, thereby decreasing activation of GREM1. Antagonists can be reversible or irreversible.

[0140] The GREM1 antagonist can be a small molecule inhibitor, a peptide, a protein, an antibody, a polynucleotide, an oligonucleotide, an antisense RNA, a small interfering RNA (siRNA) or a small hairpin RNA (shRNA).

[0141] The GREM1 antagonist may be an oligonucleotide that specifically hybridizes to the mRNA encoding GREM1 or to the mRNA encoding a molecule that enhances the activity of GREM1. The GREM1 antagonist may be a polynucleotide encoding any molecule that reduces GREM1 function. For example, the GREM1 antagonist may be a polynucleotide encoding an anti-GREM1 antibody described herein.

[0142] The antagonist of GREM1 can be an antibody that specifically binds to any target molecule (typically a protein) so as to directly or indirectly reduce the function of GREM1. The antagonist can be an antibody that specifically binds to GREM1. In this embodiment, the antibody can reduce GREM1 function by allosteric inactivation or by blocking the interaction between its target and a ligand required for activity.

[0143] The interaction of the antagonist agent with the protein residue can be determined by any suitable method known in the art, such as the distance between the residue and the agent (typically less than 6 Å, or less than 4 Å) as determined by X-ray crystallography. Regions of Gremlin-1 that may be targeted by a therapeutic agent may include amino acids Asp92-Leu99, Arg116-His130, Ser137-Ser142, Cys176-Cys178, which are within 6 Å of the amino acids that are mutated on the surface of Gremlin-1.

[0144] Antibody antagonists The term "antibody" as referred to herein includes whole antibodies, any antigen-binding fragment (i.e., "antigen-binding portion"), or single chains thereof. An antibody refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains linked by disulfide bonds, or an antigen-binding portion thereof. Each heavy chain contains a heavy chain variable region (referred to herein as HCVR or V H Each light chain comprises a light chain variable region (abbreviated herein as LCVR or V L The heavy and light chain variable regions contain a binding domain that interacts with an antigen. H and V L The regions can be further subdivided into regions of hypervariability called complementarity determining regions (CDRs) and regions of high conserved activity called framework regions (FRs).

[0145] The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (Clq) of the classical complement system.

[0146] The antibody used according to the present invention may be a monoclonal or polyclonal antibody, typically a monoclonal antibody. The antibody used according to the present invention may be a chimeric antibody, a CDR-grafted antibody, a nanobody, a human antibody or a humanized antibody, or any antigen-binding portion thereof. For the production of monoclonal and polyclonal antibodies, the experimental animal is usually a non-human mammal, such as a goat, rabbit, rat or mouse, although the antibody can also be bred in other species.

[0147] Polyclonal antibodies can be produced in a routine manner, such as by immunizing a suitable animal with the antigen of interest, followed by blood removal from the animal and purification of the IgG fraction.

[0148] Antibodies against Gremlin-1 can be obtained by administering the polypeptide to an animal, e.g., a non-human animal, using well-known and routine protocols, if immunization of the animal is required, see, e.g., Handbook of Experimental Immunology, DM Weir (ed.), Vol 4, Blackwell Scientific Publishers, Oxford, England, 1986. Many warm-blooded animals can be immunized, such as rabbits, mice, rats, sheep, cattle, camels or pigs. Generally, however, mice, rabbits, pigs and rats are most suitable.

[0149] Monoclonal antibodies can be prepared by any method known in the art, such as the hybridoma technique (Kohler & Milstein, 1975, Nature, 256:495-497), the trioma technique, the human B cell hybridoma technique (Kozbor et al., 1983, Immunology Today, 4:72), and the EBV hybridoma technique (Cole et al., Monoclonal Antibodies and Cancer Therapy, pp77-96, Alan R Liss, Inc., 1985).

[0150] Antibodies to be used in accordance with the present invention can also be produced using a single lymphocyte antibody technique, for example, by cloning and expressing immunoglobulin variable region cDNA generated from a single lymphocyte selected to produce a particular antibody, by methods described by Babcook, J. et al., 1996, Proc. Natl. Acad. Sci. USA 93(15): 7843-7848l; International Publication No. WO92 / 02551; International Publication No. WO2004 / 051268 and International Publication No. WO2004 / 106377.

[0151] Antibodies can also be generated using various phage display methods known in the art, see, for example, Brinkman et al. (in J. Immunol. Methods, 1995, 182: 41-50), Ames et al. (J. Immunol. Methods, 1995, 184: 177-186), Kettleborough et al. (Eur. J. Immunol. 1994, 24: 952-958), Persic et al. (Gene, 1997 187 9-18), Burton et al. (Advances in Immunology, 1994, 57:191-280), as well as International Publication Nos. WO 90 / 02809; International Publication No. WO 91 / 10737; International Publication No. WO 92 / 01047; International Publication No. WO 92 / 18619; International Publication No. WO 93 / 11236; International Publication No. WO 95 / 15982; International Publication No. WO 95 / 20401; and U.S. Patent Nos. 5,698,426 and 5,223,409. ;5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969,108.

[0152] A fully human antibody is one in which the variable and constant regions (if present) of both the heavy and light chains are all of human origin or are substantially identical to sequences of human origin, although not necessarily derived from the same antibody. Examples of fully human antibodies include, for example, antibodies produced by the phage display methods described above, and antibodies produced in mice in which the mouse immunoglobulin variable region genes, and optionally the constant region genes, have been replaced with their human counterparts, such as those generally described in EP 0546073, U.S. Patent No. 5,545,806, U.S. Patent No. 5,569,825, U.S. Patent No. 5,625,126, U.S. Patent No. 5,633,425, U.S. Patent No. 5,661,016, U.S. Patent No. 5,770,429, EP 0438474, and EP 0463151.

[0153] Alternatively, antibodies for use in accordance with the present invention may be produced by a method comprising immunizing a non-human mammal with a Gremlin-1 immunogen; obtaining an antibody preparation from said mammal; and inducing therefrom a monoclonal antibody which recognizes Gremlin-1.

[0154] The antibody molecule used according to the present invention may comprise a complete antibody molecule having full-length heavy and light chains, or a fragment or antigen-binding portion thereof. The term "antigen-binding portion" of an antibody refers to one or more fragments of an antibody that retain the ability to selectively bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Antibodies and fragments and antigen-binding portions thereof may be, but are not limited to, Fab, modified Fab, Fab', modified Fab', F(ab')2, Fv, single domain antibodies (e.g., VH or VL or VHH), scFv, bivalent, trivalent or tetravalent antibodies, Bis-scFv, diabodies, triabodies, tetrabodies, and epitope-binding fragments of any of the above (see, e.g., Holliger and Hudson, 2005, Nature Biotech. 23(9):1126-1136; Adair and Lawson, 2005, Drug Design Reviews - Online 2(3), 209-217). Methods for generating and producing these antibody fragments are well known in the art (see, for example, Verma et al., 1998, Journal of Immunological Methods, 216, 165-181). Other antibody fragments for use in the present invention include Fab and Fab' fragments described in International Patent Application Publication Nos. WO2005 / 003169, WO2005 / 003170 and WO2005 / 003171, and Fab-dAb fragments described in International Patent Application Publication No. WO2009 / 040562. Multivalent antibodies may contain multiple specificities or may be monospecific (see, for example, International Patent Application Publication Nos. WO92 / 22853 and WO05 / 113605). These antibody fragments may be obtained using conventional techniques known to those of skill in the art and may be screened for utility in the same manner as intact antibodies.

[0155] The constant region domain of the antibody molecule, if present, can be selected taking into account the proposed function of the antibody molecule, in particular the effector function that may be required. For example, the constant region domain can be a human IgA, IgD, IgE, IgG or IgM domain. In particular, when the antibody molecule is intended for therapeutic use and antibody effector function is required, human IgG constant region domains, in particular IgG1 and IgG3 isotypes, can be used. Alternatively, when the antibody molecule is intended for therapeutic purposes and antibody effector function is not required, IgG2 and IgG4 isotypes can be used.

[0156] Antibodies used in accordance with the present invention may be prepared, expressed, created or isolated by recombinant means, such as (a) antibodies isolated from animals (e.g., mice) that are transgenic or transchromosomal for the immunoglobulin genes of interest or hybridomas prepared therefrom; (b) antibodies isolated from host cells, e.g., transfectomas, that have been transformed to express the antibody of interest; (c) antibodies isolated from recombinant, combinatorial antibody libraries; and (d) antibodies prepared, expressed, created or isolated by other means involving splicing of immunoglobulin gene sequences into other DNA sequences.

[0157] The antibody used according to the present invention may be a human antibody or a humanized antibody. The term "human antibody" as used herein is intended to include antibodies having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region is also derived from a human germline immunoglobulin sequence. The human antibodies described herein may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by in vitro random or site-specific mutagenesis or in vivo somatic mutation). However, the term "human antibody" as used herein is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

[0158] Such human antibodies can be human monoclonal antibodies, which can be produced by hybridomas comprising B cells obtained from a transgenic non-human animal, e.g., a transgenic mouse, whose genome comprises human heavy chain and light chain transgenes fused to an immortalized cell.

[0159] Human antibodies can be prepared by in vitro immunization of human lymphocytes, followed by transformation of the lymphocytes with Epstein-Barr virus.

[0160] The term "human antibody derivatives" refers to any modified form of human antibodies, such as conjugates of antibodies with other agents or antibodies.

[0161] The term "humanized antibody" is intended to refer to CDR-grafted antibody molecules in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences within which further modifications of the framework regions may be made.

[0162] As used herein, the term "CDR-grafted antibody molecule" refers to an antibody molecule in which the heavy and / or light chains comprise one or more CDRs (optionally including one or more modified CDRs) from a donor antibody (e.g., a mouse or rat monoclonal antibody) grafted onto the heavy and / or light chain variable region framework of an acceptor antibody (e.g., a human antibody). For a review, see Vaughan et al, Nature Biotechnology, 16, 535-539, 1998. In one embodiment, instead of transferring the entire CDR, only one or more of the specificity-determining residues from any one of the CDRs described herein above are transferred to the human antibody framework (see, e.g., Kashmiri et al., 2005, Methods, 36, 25-34). In one embodiment, only the specificity-determining residues from one or more of the CDRs described herein above are transferred to the human antibody framework. In another embodiment, only the specificity-determining residues from each CDR described herein above are introduced into the human antibody framework.

[0163] Where CDRs or specificity determining residues are grafted, any suitable acceptor variable region framework sequence may be used, including murine, primate and human framework regions, taking into account the class / type of the donor antibody from which the CDRs are derived. Suitably, a CDR-grafted antibody according to the invention has a variable domain comprising human acceptor framework regions as well as one or more of the above CDRs or specificity determining residues. Thus, in one embodiment, a neutralising CDR-grafted antibody is provided, the variable domain of which comprises human acceptor framework regions and a non-human donor CDR.

[0164] Examples of human frameworks that can be used in the present invention are KOL, NEWM, REI, EU, TUR, TEI, LAY and POM (Kabat et al., supra). For example, KOL and NEWM can be used for the heavy chain, REI can be used for the light chain, and EU, LAY and POM can be used for both the heavy and light chains. Alternatively, human germline sequences can be used. These are available, for example, at http: / / www.vbase2.org / (see Retter et al, Nucl. Acids Res. (2005) 33 (supplement 1), D671-D674).

[0165] In the CDR-grafted antibodies described herein, the acceptor heavy and light chains are not necessarily from the same antibody and may, if desired, comprise composite chains having framework regions from different chains.

[0166] Also, in the CDR-grafted antibodies described herein, the framework regions need not have the exact same sequence as those of the acceptor antibody. For example, unusual residues can be changed to residues that occur more frequently for that acceptor chain class or type. Alternatively, selected residues in the acceptor framework regions can be changed to correspond to residues found at the same positions in the donor antibody (see Reichmann et al., 1998, Nature, 332, 323-324). Such changes should be the minimum necessary to restore the affinity of the donor antibody. Protocols for selecting acceptor framework region residues that may require change are described in International Publication No. WO 91 / 09967.

[0167] Those skilled in the art will also appreciate that antibodies may undergo various post-translational modifications. The type and extent of these modifications often depend on the host cell line used to express the antibody, as well as the culture conditions. Such modifications may include changes in glycosylation, methionine oxidation, diketopiperazine formation, aspartic acid isomerization, and asparagine deamidation. A frequent modification is the loss of basic residues (e.g., lysine or arginine) at the carboxy terminus by the action of carboxypeptidases (described in Harris, RJ. Journal of Chromatography 705:129-134, 1995).

[0168] In one embodiment, the antibody heavy chain comprises a CH1 domain and the antibody light chain comprises a CL domain, either kappa or lambda.

[0169] Biological molecules such as antibodies or fragments thereof contain acidic and / or basic functional groups, thereby conferring a net positive or negative charge to the molecule. The amount of overall "observed" charge depends on the absolute amino acid sequence of the entity, the local environment of the charged groups in the 3D structure, and the environmental conditions of the molecule. The isoelectric point (pI) is the pH at which a particular molecule or surface has no net charge. In one embodiment, the antibody or fragment according to the present disclosure has an isoelectric point (pI) of at least 7. In one embodiment, the antibody or fragment has an isoelectric point of at least 8, such as 8.5, 8.6, 8.7, 8.8 or 9. In one embodiment, the antibody has a pI of 8. ** Programs such as ExPASY http: / / www.expasy.ch / tools / pi_tool.html (see Walker, The Proteomics Protocols Handbook, Humana Press (2005), 571-607) can be used to predict the isoelectric point of an antibody or fragment.

[0170] To characterize the preferred Gremlin-1 epitope, we crystallized human Gremlin-1 alone and in complex with an antibody (Fab fragment) called Ab 7326. Crystallization of Gremlin-1 allowed us to determine the putative residues of the BMP binding site. Furthermore, crystallization with Ab 7326, an allosteric inhibitor antibody, allowed us to determine the residues of the antibody epitope. Antibodies that bind to this epitope are particularly promising as therapeutic agents in the treatment of diseases associated with Gremlin-1.

[0171] Preferred Abs as described herein The 7326 antibody was confirmed to bind to the following residues of Gremlin-1: Ile110(131), Lys126(147), Lys127(148), Phe128(149), Thr129(150), Thr130(151), Arg148(169), Lys153(174), and Gln154(175), with Lys126(147), Lys127(148), Phe128(149), Thr129(150), Thr130(151), Arg148(169), Lys153(174), and Gln154(175) present on one Gremlin-1 monomer and Ile110(131) present on a second Gremlin-1 monomer. Numbering not in parentheses is based on the structure file and (consistent with mouse Gremlin-2 numbering based on structural alignment). Numbers in parentheses represent residues based on UniProt entry O60565 of SEQ ID NO: 1. These epitope residues were identified using 4 Å NCONT analysis from the Gremlin-1-Ab 7326 Fab complex.

[0172] Thus, the antibodies described herein may bind to an epitope comprising at least one residue selected from Ile131, Lys147, Lys148, Phe149, Thr150, Thr151, Arg169, Lys174 and Gln175 (residue numbering based on SEQ ID NO: 1). The antibodies described herein may bind to an epitope comprising 2, 3, 4, 5, 6, 7, 8 or all 9 residues (preferably at least 5 residues) of these residues.

[0173] The antibodies described herein may also recognize an epitope present on the Gremlin-1 monomer where Ile131 is distinct from other residues.

[0174] Although these residues are provided for a particular sequence of human Gremlin-1, one of skill in the art can readily extrapolate the positions of these residues to other corresponding Gremlin sequences (e.g., mouse) using routine techniques. Thus, antibodies that bind epitopes that include corresponding residues in these other Gremlin sequences are also provided by the invention.

[0175] To screen for antibodies that bind to a specific epitope, routine cross-blocking assays such as those described in Antibodies, Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harb., NY) can be performed. Other methods include alanine scanning mutants, peptide blots (Reineke (2004) Methods Mol Biol 248:443-63), or peptide truncation analysis. In addition, methods such as epitope excision, epitope extraction, and chemical modification of antigens can be employed (Tomer (2000) Protein Science 9: 487-496). Such methods are well known in the art.

[0176] The epitope of an antibody can also be determined by X-ray crystallography. Thus, the antibodies of the present disclosure can be evaluated by X-ray crystallography of the antibody bound to Gremlin-1. The epitope can be identified in this way, particularly by determining the residues on Gremlin-1 that are within 4 Å of the antibody paratope residues.

[0177] Thus, the antibodies described herein can bind to an epitope on Gremlin-1 that includes at least one residue selected from Trp93, Phe117, Tyr119, Phe125, Tyr126, and Phe138, where residue numbering is according to SEQ ID NO: 1. Further described herein are antibodies that bind to an epitope that includes all of Trp93, Phe117, Tyr119, Phe125, Tyr126, and Phe138. Further described are antibodies that bind to an epitope that includes the following residues: Ile131, Lys147, Lys148, Phe149, Thr150, Thr151, Arg169, Lys174, and Gln175. Preferably, Lys147, Lys148, Phe149, Thr150, Thr151, Arg169, Lys174 and Gln175 are located on one monomer of Gremlin-1 and Ile131 is located on the other monomer of Gremlin-1 (the Gremlin-1 dimer binds to the BMP dimer).

[0178] An antibody is capable of binding to a Gremlin-1 residue if the paratope of the antibody is within 4 Å of said Gremlin-1 residue as determined by x-ray crystallography.

[0179] Antibodies that bind to the epitopes disclosed herein may comprise at least one, at least two, or all three of the heavy chain CDR sequences of SEQ ID NOs: 4-6 (HCDR1 / HCDR2 / HCDR3, respectively), which are the HCDR1 / HCDR2 / HCDR3 sequences of the Ab 7326 antibody of the Examples, as determined using the Kabat method.

[0180] The Kabat and Chothia methods (as well as other techniques) for determining CDR sequences are well known in the art. CDR sequences can be determined using any suitable method, and in the present invention, Kabat is typically employed, although other techniques can be used as well. In this example, SEQ ID NO:3 shows the Ab 7326 HCDR1 sequence determined using the combined definition of Chothia & Kabat.

[0181] Antibodies for use according to the invention may comprise at least one, at least two, or all three light chain CDR sequences of SEQ ID NOs: 7-9 (LCDR1 / LCDR2 / LCDR3, respectively), which are the LCDR1 / LCDR2 / LCDR3 sequences of Ab 7326 using the Kabat method.

[0182] The antibody preferably comprises at least the HCDR3 sequence of SEQ ID NO:6.

[0183] Typically, the antibody comprises at least one heavy chain CDR sequence selected from SEQ ID NOs: 4 to 6 and at least one light chain CDR sequence selected from SEQ ID NOs: 7 to 9. The antibody may comprise at least two heavy chain CDR sequences selected from SEQ ID NOs: 4 to 6 and at least two light chain CDR sequences selected from SEQ ID NOs: 7 to 9. The antibody typically comprises all three heavy chain CDR sequences (HCDR1 / HCDR2 / HCDR3, respectively) of SEQ ID NOs: 4 to 6 and all three light chain CDR sequences (LCDR1 / LCDR2 / LCDR3, respectively) of SEQ ID NOs: 7 to 9. The antibody may be a chimeric antibody, a human antibody or a humanized antibody.

[0184] The antibody may comprise a heavy chain variable region (HCVR) sequence of SEQ ID NO: 10 or 12 (HCVR of Ab 7326 variants 1 and 2). The antibody may comprise a light chain variable region (LCVR) sequence of SEQ ID NO: 11 or 13 (LCVR of Ab 7326 variants 1 and 2). The antibody preferably comprises a heavy chain variable region sequence of SEQ ID NO: 10 or 12 and a light chain variable region sequence of SEQ ID NO: 11 or 13 (particularly the HCVR / LVCR pair of SEQ ID NO: 10 / 11 or 12 / 13).

[0185] The antibody is SEQ ID NO: 14 Mouse full length IgG1 heavy chain variant 1, or SEQ ID NO: 28 Mouse full length IgG1 heavy chain variant 2, or SEQ ID NO: 30 Human full length IgG1 heavy chain variant 1, or SEQ ID NO: 16 human full length IgG1 heavy chain variant 2, or SEQ ID NO: 22 Human full length IgG4P heavy chain variant 1, or SEQ ID NO: 34 human full length IgG4P heavy chain variant 2, or SEQ ID NO: 18 Fab heavy chain variant 1, or SEQ ID NO: 32 Fab heavy chain variant 2 It may comprise the heavy chain (H chain) sequence of

[0186] The antibody is SEQ ID NO: 15 mouse full length IgG1 light chain variant 1, or SEQ ID NO: 29 Mouse full length IgG1 light chain variant 2, or SEQ ID NO: 31 human full length IgG1 light chain variant 1, or SEQ ID NO: 17 human full length IgG1 light chain variant 2, or SEQ ID NO: 23 human full length IgG4P light chain variant 1, or SEQ ID NO: 35 human full length IgG4P light chain variant 2, or SEQ ID NO: 19 Fab light chain variant 1, or SEQ ID NO: 33 Fab light chain variant 2 It may comprise a light chain (L chain) sequence of

[0187] In one example, the antibody is SEQ ID NO: 14 / 15 Mouse full length IgG1 variant 1, or SEQ ID NO: 28 / 29 Mouse full length IgG1 variant 2, or SEQ ID NO: 30 / 31 human full length IgG1 variant 1, or SEQ ID NO: 16 / 17 human full length IgG1 variant 2, or SEQ ID NO: 22 / 23 human full length IgG4P variant 1, or SEQ ID NO: 34 / 35 human full-length IgG4P variant 2, or SEQ ID NO: 18 / 19 Fab light chain variant 1, or SEQ ID NO: 32 / 33 Fab light chain variant 2 The heavy chain / light chain sequence pair is

[0188] Variant forms of the corresponding sequences may be exchanged. For example, an antibody may SEQ ID NO: 14 / 29 Mouse full length IgG1 heavy chain variant 1 / light chain variant 2, or SEQ ID NO: 28 / 15 mouse full length IgG1 heavy chain variant 2 / light chain variant 1, or SEQ ID NO: 30 / 17 human full length IgG1 heavy chain variant 1 / light chain variant 2, or SEQ ID NO: 16 / 31 human full length IgG1 heavy chain variant 2 / light chain variant 1, or SEQ ID NO: 22 / 35 human full length IgG4P heavy chain variant 1 / light chain variant 2, or SEQ ID NO: 34 / 23 human full length IgG4P heavy chain variant 2 / light chain variant 1, or SEQ ID NO: 18 / 33 Fab heavy chain variant 1 / light chain variant 2, or SEQ ID NO: 32 / 19 Fab heavy chain variant 2 / light chain variant 1 The heavy chain / light chain sequence pair may include:

[0189] The antibody can be a chimeric antibody, a human antibody, or a humanized antibody.

[0190] The antibody may alternatively be or comprise a variant of one of the specific sequences described above. The following discussion of antibody variants is also applicable to the selection of GREM1 polypeptide variants described above.

[0191] For example, the variants can be substitution, deletion or addition variants of any of the above amino acid sequences.

[0192] A variant antibody may include 1, 2, 3, 4, 5, up to 10, up to 20 or more (typically up to 50) amino acid substitutions and / or deletions from the specific sequences discussed above. "Deletion" variants may include deletion of individual amino acids, deletion of small groups of amino acids such as 2, 3, 4 or 5 amino acids, or deletion of larger regions of amino acids such as deletion of specific amino acid domains or other properties. "Substitution" variants typically involve the replacement of one or more amino acids with the same number of amino acids, and making conservative amino acid substitutions. For example, an amino acid may be replaced with an alternative amino acid having similar properties, such as another basic amino acid, another acidic amino acid, another neutral amino acid, another charged amino acid, another hydrophilic amino acid, another hydrophobic amino acid, another polar amino acid, another aromatic amino acid, or another aliphatic amino acid. Some properties of the 20 main amino acids that can be used to select appropriate replacements are as follows: [Table A] "Derivatives" or "variants" generally include those in which an amino acid that appears in the sequence in place of a naturally occurring amino acid is a structural analog thereof. The amino acids used in the sequence may be derivatized or modified, for example labeled, so long as this does not significantly adversely affect the function of the antibody.

[0193] The above derivatives and variants can be prepared by modification during synthesis or after production of the antibody, or when the antibody is in recombinant form, using known techniques of site-directed mutagenesis, random mutagenesis, or enzymatic cleavage and / or ligation of nucleic acids.

[0194] A variant antibody may have an amino acid sequence that has more than about 60%, or more than about 70%, such as 75% or 80%, typically more than about 85%, such as more than about 90% or 95% amino acid identity with the amino acid sequences disclosed herein (particularly the HCVR / LCVR sequences, and the H-chain and L-chain sequences). Furthermore, the antibody may be a variant that has more than about 60%, or more than about 70%, such as 75% or 80%, typically more than about 85%, such as more than about 90% or 95% amino acid identity with the HCVR / LCVR sequences and the H-chain and L-chain sequences disclosed herein, while retaining the exact CDRs disclosed for these sequences. A variant may retain at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the HCVR / LCVR sequences and the H-chain and L-chain sequences disclosed herein (in some cases, while retaining the exact CDRs).

[0195] Variants typically retain about 60% to about 99% identity, about 80% to about 99% identity, about 90% to about 99% identity, or about 95% to about 99% identity. This level of amino acid identity can be found over the entire length of the sequence of the relevant SEQ ID NO, or over a portion of the sequence, for example, over about 20, 30, 50, 75, 100, 150, 200 or more amino acids, depending on the size of the full-length polypeptide.

[0196] In the context of amino acid sequences, "sequence identity" refers to sequences having the specified values ​​when assessed using ClustalW (Thompson et al., 1994, supra) with the following parameters: pairwise alignment parameters - method: exact, matrix: PAM, gap opening penalty: 10.00, gap extension penalty: 0.10; multiple alignment parameters - matrix: PAM, gap opening penalty: 10.00, delay % identity: 30, end gap penalty: on, gap separation distance: 0, negative matrix: none, gap extension penalty: 0.20, residue specific gap penalty: on, hydrophilic gap penalty: on, hydrophilic residues: GPSNDQEKR. Sequence identity at a particular residue is intended to include identical residues that have only been derivatized.

[0197] Thus, antibodies with specific sequences and variants that maintain the function or activity of these chains are provided.

[0198] The antibody may compete for binding to Gremlin-1 or bind to the same epitope as those defined above in terms of H / L chain, HCVR / LCVR or CDR sequences. In particular, the antibody may compete for binding to Gremlin-1 or bind to the same epitope as an antibody comprising the HCDR1 / HCDR2 / HCDR3 / LCDR1 / LCDR2 / LCDR3 sequence combination of SEQ ID NO: 4 / 5 / 6 / 7 / 8 / 9. The antibody may compete for binding to Gremlin-1 or bind to the same epitope as an antibody comprising the HCVR and LCVR sequence pairs of SEQ ID NO: 10 / 11 or 12 / 13, or the full length chains of SEQ ID NO: 14 / 15 or 16 / 17.

[0199] An "epitope" is a region of an antigen to which an antibody binds. Epitopes may be defined as structural or functional. Functional epitopes are generally a subset of structural epitopes and have residues that directly contribute to the affinity of the interaction. Epitopes may also be conformational, i.e., composed of non-linear amino acids. In certain embodiments, epitopes may include determinants that are chemically active surface groups of molecules such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and in certain embodiments may have specific three-dimensional structural characteristics, and / or specific charge characteristics.

[0200] Whether an antibody binds to the same epitope as a reference antibody or competes for binding can be easily determined using routine methods known in the art. For example, to determine whether a test antibody binds to the same epitope as a reference antibody of the present invention, the reference antibody is bound to a protein or peptide under saturating conditions. The ability of the test antibody to bind to the protein or peptide is then evaluated. If the test antibody can bind to the protein or peptide after saturation binding with the reference antibody, it can be concluded that the test antibody binds to a different epitope than the reference antibody. On the other hand, if the test antibody cannot bind to the protein or peptide after saturation binding with the reference antibody, the test antibody may bind to the same epitope as the epitope bound by the reference antibody of the present invention.

[0201] To determine whether an antibody competes with a reference antibody for binding, the above-mentioned binding method is carried out in two directions. In the first direction, the reference antibody is bound to the protein / peptide under saturating conditions, and then the binding of the test antibody to the protein / peptide molecule is evaluated. In the second direction, the test antibody is bound to the protein / peptide under saturating conditions, and then the binding of the reference antibody to the protein / peptide is evaluated. If in both directions only the first (saturating) antibody can bind to the protein / peptide, it is concluded that the test antibody and the reference antibody compete for binding to the protein / peptide. As will be understood by those skilled in the art, an antibody that competes with a reference antibody for binding does not necessarily bind to the same epitope as the reference antibody, but can sterically block the binding of the reference antibody by binding to an overlapping or adjacent epitope.

[0202] Two antibodies bind to the same or overlapping epitopes if each competitively inhibits (blocks) the binding of the other antibody to the antigen. That is, a 1-fold, 5-fold, 10-fold, 20-fold, or 100-fold excess of one antibody inhibits the binding of the other by at least 50%, 75%, 90%, or 99%, as measured by competitive binding assays (see, e.g., Junghans et al., Cancer Res, 1990:50:1495-1502). Alternatively, two antibodies have the same epitope if essentially all amino acid mutations in the antigen that reduce or eliminate binding of one antibody reduce or eliminate binding of the other antibody. Two antibodies have overlapping epitopes if some amino acid mutations that reduce or eliminate binding of one antibody reduce or eliminate binding of the other antibody.

[0203] Additional routine experiments (e.g., peptide mutations and binding analysis) can then be performed to confirm whether the observed lack of binding of the test antibody is indeed due to binding to the same epitope as the reference antibody, or whether the observed lack of binding is due to steric blocking (or another phenomenon). These types of experiments can be performed using ELISA, RIA, surface plasmon resonance, flow cytometry, or any other quantitative or qualitative antibody binding assay available in the art.

[0204] Antibodies can be tested for binding to Gremlin-1, for example, by standard ELISA or Western blotting. ELISA assays can also be used to screen for hybridomas that show positive reactivity with the target protein. The binding selectivity of the antibody can also be determined by monitoring the binding of the antibody to cells expressing the target protein, for example, by flow cytometry. Thus, screening methods can include identifying antibodies capable of binding to Gremlin-1 by ELISA or Western blot, or by flow cytometry.

[0205] The antibody selectively (or specifically) recognizes Gremlin-1. An antibody or other compound "selectively binds" or "selectively recognizes" a protein if it binds preferentially or with high affinity to that protein and does not substantially bind or binds with low affinity to other proteins. The selectivity of an antibody can be further studied by determining whether the antibody binds to or discriminates between other related proteins as discussed above. Antibodies used in accordance with the present invention typically recognize human Gremlin-1.

[0206] The antibodies may also be cross-reactive to related proteins, or to human Gremlin-1 and Gremlin-1 from other species.

[0207] Specific (or selective) means that the antibody binds to the protein of interest and does not exhibit significant cross-reactivity with any other molecule. Cross-reactivity can be assessed by any suitable method described herein. The cross-reactivity of an antibody can be considered significant if it binds to another molecule at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 100% as strongly as the antibody binds to the protein of interest. An antibody that is specific (or selective) can bind to another molecule less than about 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25% or 20% as strongly as it binds to the protein of interest. An antibody may bind to other molecules with less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 2%, or less than about 1% of the strength with which it binds to the protein of interest.

[0208] Anti-Gremlin antibodies have been previously reported, for example, International Publication No. WO2014 / 159010A1 (Regeneron) describes an anti-Gremlin antibody that inhibits Gremlin-1 activity, with a binding affinity of K D The binding affinity K ranges from 625 pM to 270 nM at 25 °C. Ciuclan et al. (2013) D is 5.6×10 -10 We describe an anti-Gremlin-1 monoclonal antibody, M.

[0209] The anti-gremlin-1 antibody described herein (as well as in International Publication Nos. WO2018 / 115017, filed December 19, 2017, and WO2019 / 243801, filed June 18, 2019, both of which are incorporated herein by reference in their entireties) is an allosteric inhibitor of gremlin-1 activity and binds to a novel epitope as described above, distal to the BMP binding site. The antibody binds to gremlin-1 with very high affinity, with a Kd value of less than 100 pM. Thus, the antibody is a significant improvement over currently available antibodies and is expected to be particularly useful in treating gremlin-1 mediated diseases.

[0210] Thus, antibodies suitable for use in the present invention are likely to have high affinity binding to (human) Gremlin-1. The antibodies should have a dissociation constant (K D In one example, the antibody can have a dissociation constant (K D In one example, the antibody has a dissociation constant (K D ). Various methods can be used to determine the binding affinity of an antibody to a target antigen, as is well known to those skilled in the art, such as surface plasmon resonance assays, saturation assays, or immunoassays such as ELISA or RIA. An exemplary method for determining binding affinity is by surface plasmon resonance on a BIAcore™ 2000 instrument (Biacore AB, Freiburg, Germany) using a CM5 sensor chip, as described in Krinner et al., (2007) Mol. Immunol. February; 44 (5):916-25 (Epub 2006 May 11)).

[0211] Antibodies used in accordance with the present invention are typically inhibitory antibodies. Gremlin-1 negatively regulates BMP-2, 4 and 7, so inhibition of Gremlin-1 results in increased signaling through BMPs.

[0212] Particular functional assays that can be used to screen whether an antibody can inhibit Gremlin 1 include SMAD phosphorylation assays and Hek Id1 reporter gene assays. Typically, inhibitory antibodies restore SMAD phosphorylation and / or restore BMP signaling in a Hek Id1 reporter gene assay. SMAD phosphorylation can be restored to at least 80%, 90% or 100% compared to a BMP control. In the Hek Id1 reporter gene assay, inhibitory antibodies demonstrate an IC 50 may be less than 10 nM, preferably less than 5 nM.

[0213] Once a suitable antibody is identified and selected, the amino acid sequence of the antibody can be identified by methods known in the art. The gene encoding the antibody can be cloned using degenerate primers. The antibody can be produced recombinantly by routine methods.

[0214] The present disclosure also provides isolated DNA sequences encoding the heavy and / or light chain variable region(s) (or full length H and L chains) of the antibody molecules newly described herein.

[0215] Variant polynucleotides can contain 1, 2, 3, 4, 5, up to 10, up to 20, up to 30, up to 40, up to 50, up to 75 or more nucleic acid substitutions and / or deletions from any of the nucleic acid sequences shown in the Sequence Listing (including GREM1 and anti-GREM1 antibody nucleic acid sequences). Typically, variants have 1-20, 1-50, 1-75 or 1-100 substitutions and / or deletions.

[0216] A suitable variant may be at least about 70%, typically at least about 80 or 90%, more preferably at least about 95%, 97% or 99% homologous to any one of the polynucleotides of the nucleic acid sequences disclosed herein. A variant may retain at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity. A variant typically retains about 60% to about 99% identity, about 80% to about 99% identity, about 90% to about 99% identity, or about 95% to about 99% identity. Homology and identity at these levels generally exist at least with respect to the coding region of the polynucleotide. Methods for measuring homology are well known in the art, and the skilled artisan will understand that in the present context, homology is calculated based on nucleic acid identity. Such homology may exist over a region of at least about 15, at least about 30, for example, at least about 40, 60, 100, 200 or more contiguous nucleotides (depending on the length). Such homology may exist over the entire length of the unmodified polynucleotide sequence.

[0217] Methods for measuring polynucleotide homology or identity are known in the art. For example, the UWGCG package provides the BESTFIT program, which can be used to calculate homology (e.g., used on its default settings) (Devereux et al (1984) Nucleic Acids Research 12, p387-395).

[0218] The PILEUP and BLAST algorithms can also be used (typically on their default settings) to calculate homology or align sequences, e.g., as described in Altschul SF (1993) J Mol Evol 36:290-300; Altschul, S, F et al (1990) J Mol Biol 215:403-10.

[0219] Software for performing BLAST analysis is publicly available from the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ). The algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that, when aligned with words of the same length in the database sequences, match or meet some positive threshold score T. T is called the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds to initiate searches to find HSPs containing the word. The word hits are extended in both directions along each sequence for as long as the cumulative alignment score increases. The extension of the word hits in each direction is stopped when the accumulation of one or more negatively scoring residue alignments causes the cumulative alignment score to fall below zero, or when the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLAST program uses as defaults a wordlength (W) of 11, the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1992) Proc. Natl. Acad. Sci. USA 89:10915-10919) of 50, alignment (B) of 10, expectation (E) of 10, M=5, N=4, and a comparison of both strands.

[0220] The BLAST algorithm performs a statistical analysis of the similarity between two sequences; see, for example, Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5787. One measure of similarity provided by the BLAST algorithm is the minimum sum probability (P(N)), which provides an indication of the probability that a match between two nucleotide or amino acid sequences would occur by chance. For example, a sequence is considered to be similar to another sequence if the minimum sum probability in a comparison of a first sequence to a second sequence is less than about 1, typically less than about 0.1, preferably less than about 0.01, and most preferably less than about 0.001. For example, the minimum sum probability can be in the range of about 1 to about 0.001, and often about 0.01 to about 0.001.

[0221] A homologue may differ from a sequence in the related polynucleotide by less than about 3, 5, 10, 15, 20 or more mutations, each of which may be a substitution, deletion or insertion. For example, a homologue may differ by 3 to 50 mutations, often by 3 to 20 mutations. These mutations may be measured over a region of at least 30, e.g., at least about 40, 60 or 100 or more contiguous nucleotides of the homologue.

[0222] In one embodiment, variant sequences may differ from the specific sequences shown in the sequence listing due to redundancy in the genetic code. There are four main nucleic acid residues in the DNA code (A, T, C, and G) that are used to "spell" three-letter codons that represent amino acids encoded in an organism's genes for proteins. The linear sequence of codons along a DNA molecule is translated into the linear sequence of amino acids in the protein(s) encoded by those genes. The code is highly degenerate, with 61 codons encoding the 20 natural amino acids and three codons representing "stop" signals. Thus, most amino acids are encoded by more than one codon, and in fact some amino acids are encoded by four or more different codons. Thus, a variant polynucleotide of the invention may encode the same polypeptide sequence as another polynucleotide of the invention, but may have a different nucleic acid sequence due to the use of different codons to encode the same amino acids.

[0223] The DNA sequence may include, for example, synthetic DNA produced by chemical processes, cDNA, genomic DNA, or a combination thereof.

[0224] DNA sequences encoding the antibody molecules described herein can be obtained by methods well known to those skilled in the art. For example, DNA sequences encoding part or all of the heavy and light chains of the antibodies can be synthesized, if desired, from determined DNA sequences or based on the corresponding amino acid sequences.

[0225] General methods by which vectors can be constructed, transfection methods and culture methods are well known to those skilled in the art, see in this regard "Current Protocols in Molecular Biology", 1999, FM Ausubel (ed), Wiley Interscience, New York and the Maniatis Manual produced by Cold Spring Harbor Publishing.

[0226] Nucleic Acid Antagonists A polynucleotide, such as a nucleic acid, is a polymer that includes two or more nucleotides. Nucleotides can be naturally occurring or artificial. Nucleotides typically contain a nucleobase, a sugar, and at least one linking group, such as a phosphate group, a 2'O-methyl group, a 2'methoxy-ethyl group, a phosphoramidate group, a methylphosphonate group, or a phosphorothioate group. Nucleobases are typically heterocyclic. Nucleobases include, but are not limited to, purines and pyrimidines, more specifically adenine (A), guanine (G), thymine (T), uracil (U), and cytosine (C). Sugars are typically pentose sugars. Nucleotide sugars include, but are not limited to, ribose and deoxyribose. Nucleotides are typically ribonucleotides or deoxyribonucleotides. Nucleotides typically contain monophosphates, diphosphates, or triphosphates. The phosphate can be attached to the 5' or 3' side of the nucleotide.

[0227] Nucleotides include, but are not limited to, adenosine monophosphate (AMP), adenosine diphosphate (ADP), adenosine triphosphate (ATP), guanosine monophosphate (GMP), guanosine diphosphate (GDP), guanosine triphosphate (GTP), thymidine monophosphate (TMP), thymidine diphosphate (TDP), thymidine triphosphate (TTP), uridine monophosphate (UMP), uridine diphosphate (UDP), uridine triphosphate (UTP), cytidine monophosphate (CMP), cytidine triphosphate (CTMP), cytidine monophosphate (CTMP), cytidine diphosphate (CTMP), cytidine triphosphate (CTMP), cytidine monophosphate (CTMP), cytidine triphosphate (CTMP), cytidine monophosphate (CTMP), cytidine monophosphate (CTMP), cytidine diphosphate (CTMP), cytidine triphosphate (CTMP), cytidine mono ... ), cytidine diphosphate (CDP), cytidine triphosphate (CTP), 5-methylcytidine monophosphate, 5-methylcytidine diphosphate, 5-methylcytidine triphosphate, 5-hydroxymethylcytidine monophosphate, 5-hydroxymethylcytidine diphosphate, 5-hydroxymethylcytidine triphosphate, cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP), deoxyadenosine monophosphate (dAMP), deoxyadenosine diphosphate (dADP) ), deoxyadenosine triphosphate (dATP), deoxyguanosine monophosphate (dGMP), deoxyguanosine diphosphate (dGDP), deoxyguanosine triphosphate (dGTP), deoxythymidine monophosphate (dTMP), deoxythymidine diphosphate (dTDP), deoxythymidine triphosphate (dTTP), deoxyuridine monophosphate (dUMP), deoxyuridine diphosphate (dUDP), deoxyuridine triphosphate (dUTP), deoxycytidine The nucleotides include 5-methyl-2'-deoxycytidine monophosphate (dCMP), deoxycytidine diphosphate (dCDP) and deoxycytidine triphosphate (dCTP), 5-methyl-2'-deoxycytidine monophosphate, 5-methyl-2'-deoxycytidine diphosphate, 5-methyl-2'-deoxycytidine triphosphate, 5-hydroxymethyl-2'-deoxycytidine monophosphate, 5-hydroxymethyl-2'-deoxycytidine diphosphate and 5-hydroxymethyl-2'-deoxycytidine triphosphate. The nucleotides are preferably selected from AMP, TMP, GMP, UMP, dAMP, dTMP, dGMP or dCMP.

[0228] Nucleotides may contain additional modifications. In particular, suitable modified nucleotides include, but are not limited to, 2'aminopyrimidines (e.g., 2'-aminocytidine and 2'-aminouridine), 2'-hydroxyl purines (e.g., 2'-fluoropyrimidines (e.g., 2'-fluorocytidine and 2'fluorouridine), hydroxylpyrimidines (e.g., 5'-α-P-boranouridine), 2'-O-methyl nucleotides (e.g., 2'-O-methyladenosine, 2'-O-methylguanosine, 2'-O-methylcytidine and 2'-O-methyluridine), 4'-thiopyrimidines (e.g., 4'-thiouridine and 4'-thiocytidine), and nucleotides having nucleobase modifications (e.g., 5-pentynyl-2'-deoxyuridine, 5-(3-aminopropyl)-uridine and 1,6-diaminohexyl-N-5-carbamoylmethyluridine).

[0229] The nucleotides in a polynucleotide can be linked together in any way. They can be linked by phosphate, 2'O-methyl, 2'methoxy-ethyl, phosphoramidate, methylphosphonate or phosphorothioate linkages. Nucleotides are typically linked by their sugar and phosphate groups as in nucleic acids. Nucleotides can be connected through their nucleobases as in pyrimidine dimers.

[0230] The GREM1 antagonist may be a polynucleotide encoding an anti-GREM1 antibody described herein.

[0231] The polynucleotide may be a nucleic acid, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). The polynucleotide may be any synthetic nucleic acid known in the art, such as peptide nucleic acid (PNA), glycerol nucleic acid (GNA), threose nucleic acid (TNA), locked nucleic acid (LNA), morpholino nucleic acid, or other synthetic polymers with nucleotide side chains. The polynucleotide may be single-stranded or double-stranded.

[0232] The polynucleotide sequence can be cloned into any suitable expression vector. In the expression vector, the polynucleotide sequence encoding the construct is typically operably linked to a control sequence capable of providing the expression of the coding sequence by the host cell. Such an expression vector can be used to express the construct.

[0233] In one embodiment, the anti-GREM1 antagonist is a polynucleotide encoding the anti-GREM1 antibody described herein. The polynucleotide can be provided for use in gene therapy. The polynucleotide can be provided in any suitable vector that can provide the expression of the anti-GREM1 antibody in vivo.

[0234] The polynucleotide encoding the anti-GREM1 antibody may be a DNA sequence. The DNA sequence may be provided in any suitable vector, e.g., an expression vector, for administration to a subject in need thereof. For example, the DNA sequence may be administered to a subject in an expression vector capable of providing expression of the anti-GREM1 antibody in vivo. The expression vector may be a viral expression vector, e.g., an adeno-associated virus (AAV) vector. In one embodiment, the anti-GREM1 antagonist is a DNA sequence encoding the anti-GREM1 antibody described herein. In one embodiment, the anti-GREM1 antagonist is a DNA sequence for use in gene therapy, the DNA sequence encoding the anti-GREM1 antibody described herein. In one embodiment, the anti-GREM1 antagonist is an AAV comprising a DNA sequence encoding the anti-GREM1 antibody described herein. In one embodiment, the anti-GREM1 antagonist is an AAV for use in gene therapy, the AAV comprising a DNA sequence encoding the anti-GREM1 antibody described herein.

[0235] The polynucleotide encoding the anti-GREM1 antibody may be an RNA sequence. The RNA sequence may be administered to a subject in need thereof in any suitable vector. The RNA sequence may be a messenger RNA (mRNA) sequence. The mRNA sequence may be administered to a subject in need thereof in a stabilized form. For example, the mRNA sequence may be provided in a lipid nanoparticle (LNP) composition. The LNP composition may comprise any suitable LNP capable of encapsulating the mRNA sequence to provide increased stability of said mRNA sequence. Thus, in one embodiment, the anti-GREM1 antagonist is a stabilized mRNA sequence encoding the anti-GREM1 antibody described herein. In one embodiment, the anti-GREM1 antagonist is a stabilized mRNA sequence for use in gene therapy, the mRNA sequence encoding the anti-GREM1 antibody described herein. In one embodiment, the anti-GREM1 antagonist is a LNP composition comprising an mRNA encoding the anti-GREM1 antibody described herein. In one embodiment, the anti-GREM1 antagonist is a LNP composition for use in gene therapy, the LNP composition comprising an mRNA encoding the anti-GREM1 antibody described herein.

[0236] The term "operably linked" refers to a juxtaposition where the described components are in a relationship permitting them to function in their intended manner. A control sequence "operably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences. Multiple copies of the same or different polynucleotides can be introduced into the vector.

[0237] The expression vector can then be introduced into a suitable host cell. Thus, a construct can be produced by inserting a polynucleotide sequence encoding the construct into an expression vector, introducing the vector into a compatible bacterial host cell, and growing the host cell under conditions that result in expression of the polynucleotide sequence.

[0238] Nucleic acid-based GREM1 antagonists can reduce the expression of GREM1. Antisense and RNA interference (RNAi) techniques for knocking down protein expression are well known in the art, and standard methods can be employed to knock down the expression of a molecule of interest. Antisense and siRNA techniques both interfere with mRNA. Antisense oligonucleotides interfere with mRNA by binding (hybridizing) to a portion of the mRNA. Thus, antisense oligonucleotides are designed to be complementary to the mRNA (although, as described below, the oligonucleotides do not need to be 100% complementary). In other words, the antisense oligonucleotide can be part of a cDNA. Again, the oligonucleotide sequence does not need to be 100% identical to the cDNA sequence. This is also discussed below. RNAi involves the use of double-stranded RNA, such as small interfering RNA (siRNA) or small hairpin RNA (shRNA), which can bind to mRNA and inhibit protein expression.

[0239] Thus, an antagonist can be an oligonucleotide that specifically hybridizes to an mRNA encoding GREM1, e.g., the coding sequence of SEQ ID NO: 36 or SEQ ID NO: 37, or a variant thereof. An oligonucleotide "specifically hybridizes" to a target sequence if it hybridizes preferentially or with high affinity to the target sequence, but does not substantially hybridize, does not hybridize, or hybridizes only with low affinity to other sequences. More preferably, the oligonucleotide has a T-type specificity relative to other nucleic acids. m T at least 5° C., at least 10° C., at least 20° C., at least 30° C., or at least 40° C. higher than mThe hybridization condition is a stringent condition as described in the art. The conditions that allow hybridization are well known in the art (e.g., Sambrook et al., 2001, Molecular Cloning: a laboratory manual, 3rd edition, Cold Spring Harbour Laboratory Press; and Current Protocols in Molecular Biology, Chapter 2, Ausubel et al., Eds., Greene Publishing and Wiley-Interscience, New York (1995)). The hybridization condition can be a stringent condition as described in the art.

[0240] Oligonucleotides are short nucleotide polymers, typically having 50 or less nucleotides, such as 40 or less, 30 or less, 22 or less, 21 or less, 20 or less, 10 or less, or 5 or less. The oligonucleotides used may be 20-25 nucleotides in length, more preferably 21 or 22 nucleotides in length. The nucleotides may be naturally occurring or artificial. The nucleotides may be any of those described above.

[0241] The GREM1 antagonist can be an antibody that binds to GREM1, typically an antibody that specifically binds to GREM1. An antibody "specifically binds" to a protein if it binds preferentially or with high affinity to the protein, but does not substantially bind, does not bind, or only binds with low affinity to other proteins. For example, an antibody "specifically binds" to a target molecule if it binds preferentially or with high affinity to the target molecule, but does not substantially bind, does not bind, or only binds with low affinity to other human proteins.

[0242] An antibody binds preferentially or with high affinity if the Kd is 1×10 M or less, more preferably 5×10 M or less, even more preferably 1×10 M or less, and even more preferably 5×10 M or less. An antibody binds with low affinity if the Kd is 1×10 M or more, more preferably 1×10 M or more, more preferably 1×10 M or more, more preferably 1×10 M or more, and even more preferably 1×10 M or more.

[0243] The antibody may be, for example, a monoclonal antibody, a polyclonal antibody, a single chain antibody, a chimeric antibody, a bispecific antibody, a CDR-grafted antibody, or a humanized antibody. The antibody may be an intact immunoglobulin molecule or a fragment, such as a Fab, F(ab')2 or Fv fragment.

[0244] patient Any patient can be treated according to the present invention. The patient is typically a human. However, the patient may also be another mammal, for example a commercially farmed animal such as a horse, cow, sheep, fish, chicken or pig, a laboratory animal such as a mouse or rat, or a pet such as a guinea pig, hamster, rabbit, cat or dog.

[0245] Pharmaceutical Compositions, Dosages and Dosing Regimens The GREM1 antagonist for use in the method of the present invention may be provided in a pharmaceutical composition. The inhibitor of Ras-Raf-MEK-ERK signaling for use in the method of the present invention (e.g., MEK inhibitor or ERK inhibitor described herein) may also be provided as part of the same pharmaceutical composition or in a separate pharmaceutical composition. For example, the GREM1 antagonist for use in the method of the present invention may be provided in a pharmaceutical composition together with the MEK inhibitor or ERK inhibitor described herein, or other inhibitor of Ras-Raf-MEK-ERK signaling. The pharmaceutical composition is usually sterile and typically includes a pharmaceutically acceptable carrier and / or adjuvant. These compositions may include, in addition to the therapeutically active ingredient(s), pharmaceutically acceptable excipients, carriers, diluents, buffers, stabilizers, or other materials well known to those skilled in the art. Such materials should be non-toxic and not interfere with the efficacy of the active ingredient. The pharmaceutical carrier or diluent may be, for example, an isotonic solution.

[0246] As used herein, "pharmaceutically acceptable carrier" includes any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc. The precise nature of the carrier or other material may depend on the route of administration, e.g., oral, intravenous, cutaneous or subcutaneous, nasal, intramuscular, and intraperitoneal routes.

[0247] The carrier may be suitable for parenteral administration, e.g., intravenous, intramuscular, intradermal, intraocular, intraperitoneal, subcutaneous, spinal or other parenteral routes of administration, e.g., by injection or infusion. Alternatively, the carrier may be suitable for parenteral administration, such as topical, epidermal, or mucosal routes of administration. The carrier may be suitable for oral administration. Depending on the route of administration, the modulator may be coated in a material to protect the compound from the action of acids and other natural conditions which may inactivate the compound. For example, solid oral dosage forms may contain, together with the active substance, diluents such as lactose, dextrose, saccharose, cellulose, corn starch or potato starch; lubricants such as silica, talc, stearic acid, magnesium or calcium stearate, and / or polyethylene glycol; binders such as starch, gum arabic, gelatin, methylcellulose, carboxymethylcellulose or polyvinylpyrrolidone; disaggregating agents such as starch, alginic acid, alginates or sodium starch glycolate; effervescent mixtures; dyes; sweeteners; wetting agents such as lecithin, polysorbates, lauryl sulfate; and non-toxic and pharmacologically inactive substances generally used in pharmaceutical preparations. Such pharmaceutical preparations can be produced by known methods, such as mixing, granulating, tableting, sugar-coating or film-coating processes.

[0248] Other oral formulations include commonly employed excipients, such as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain 10%-95%, preferably 25%-70%, of the active ingredient. If the pharmaceutical composition is lyophilized, the lyophilized material can be reconstituted prior to administration, e.g., as a suspension. Reconstitution is preferably carried out in a buffer solution.

[0249] Capsules, tablets and pills for oral administration to an individual may be provided with an enteric coating comprising, for example, Eudragit "S", Eudragit "L", cellulose acetate, cellulose acetate phthalate, or hydroxypropyl methylcellulose.

[0250] Liquid dispersions for oral administration may be syrups, emulsions or suspensions. The syrups may contain as carriers, for example, saccharose or saccharose with glycerine and / or mannitol and / or sorbitol.

[0251] Suspensions and emulsions may contain as a carrier, for example, natural gum, agar, sodium alginate, pectin, methylcellulose, carboxymethylcellulose, or polyvinyl alcohol. Suspensions or solutions for intramuscular injections may contain, together with the active substance, a pharma- ceutically acceptable carrier, for example, sterile water, olive oil, ethyl oleate, glycols, for example, propylene glycol, and, if desired, an appropriate amount of lidocaine hydrochloride.

[0252] The solutions for intravenous administration or infusion may contain as carrier, for example, sterile water or preferably they may be in the form of sterile, aqueous, isotonic saline solutions.

[0253] For suppositories, traditional binders and carriers may include, for example, polyalkylene glycols or triglycerides; such suppositories may be formed from mixtures containing the active ingredient in the range of 0.5% to 10%, preferably 1% to 2%.

[0254] The polynucleotide or oligonucleotide inhibitors may be naked nucleotide sequences or may be combined with cationic lipids, polymers or targeting systems. They may be delivered by any available technique. For example, polynucleotides or oligonucleotides may be introduced by needle injection, preferably intradermally, subcutaneously or intramuscularly. Alternatively, polynucleotides or oligonucleotides may be delivered directly across the skin using a delivery device such as particle-mediated gene delivery. Polynucleotides or oligonucleotides may be administered topically to the skin or to mucosal surfaces, for example, by nasal, oral or rectal administration.

[0255] The uptake of polynucleotide or oligonucleotide constructs can be enhanced by several known transfection techniques, including those that involve the use of transfection agents. Examples of these agents include cationic agents, such as calcium phosphate, DEAE-dextran, and lipofectants, such as lipofectam and transfectam. The dosage of the polynucleotide or oligonucleotide to be administered can be modified.

[0256] The pharmaceutical composition of the present invention may contain one or more pharma-ceutically acceptable salts. "Pharmaceutically acceptable salts" refers to salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects. Examples of such salts include acid addition salts and base addition salts.

[0257] Pharmaceutically acceptable carriers include aqueous carriers or diluents. Examples of suitable aqueous carriers that can be used in the pharmaceutical compositions of the present invention include water, buffered water and saline. Examples of other carriers include ethanol, polyol (e.g., glycerol, propylene glycol, polyethylene glycol, etc.) and their appropriate mixtures, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. In many cases, it is desirable to include isotonicity agents, such as sugars, polyhydric alcohols such as mannitol, sorbitol, or sodium chloride in the composition.

[0258] Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration.

[0259] The pharmaceutical compositions of the invention may contain additional active ingredients.

[0260] Also included within the scope of this disclosure are kits that include the combination therapies described herein and instructions for use. The kits may further contain one or more additional reagents, such as additional therapeutic or prophylactic agents as discussed herein.

[0261] The antagonists described herein or formulations or compositions thereof can be administered for prophylactic and / or therapeutic treatments.

[0262] In therapeutic applications, compounds are administered to subjects already suffering from the above disorders or conditions in an amount sufficient to cure, alleviate or partially halt the condition or one or more of its symptoms. Such therapeutic treatment may result in a decrease in the severity of disease symptoms or an increase in the frequency or duration of symptom-free periods. An amount sufficient to achieve this is defined as a "therapeutically effective amount".

[0263] In prophylactic applications, the formulations are administered to a subject at risk of the above disorders or conditions in an amount sufficient to prevent or reduce the subsequent effects of the condition or one or more of its symptoms. An amount sufficient to accomplish this is defined as a "prophylactically effective amount." Effective amounts for each purpose depend on the severity of the disease or injury, as well as the weight and general condition of the subject.

[0264] The subject of administration can be a human or a non-human animal. The term "non-human animal" includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc. Administration to humans is typical.

[0265] The antagonists, inhibitors of Ras-Raf-MEK-ERK signaling (e.g., MEK or ERK inhibitors), or pharmaceutical compositions of the present invention can be administered via one or more routes of administration using one or more of a variety of methods known in the art. As will be appreciated by those of skill in the art, the route and / or mode of administration will vary depending on the desired outcome. Examples of routes of administration of the compounds or pharmaceutical compositions of the present invention include intravenous, intramuscular, intradermal, intraocular, intraperitoneal, subcutaneous, spinal or other parenteral routes of administration, such as administration by injection or infusion. As used herein, the phrase "parenteral administration" refers to modes of administration other than enteral and topical administration, which are typically administration by injection. Alternatively, the antibodies / modulators or pharmaceutical compositions of the present invention can be administered via parenteral routes, such as topical, epidermal or mucosal routes of administration. The antibodies / modulators or pharmaceutical compositions of the present invention may be for oral administration.

[0266] The appropriate dosage of the antibody / modulator or pharmaceutical composition of the present invention can be determined by a skilled medical practitioner. The actual dosage level of the active ingredient in the pharmaceutical composition of the present invention can be varied to obtain an amount of the active ingredient effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without causing toxicity to the patient. The dosage level selected will depend on various pharmacokinetic factors, such as the activity of the particular composition of the present invention employed, the route of administration, the time of administration, the excretion rate of the particular compound employed, the duration of treatment, other drugs, compounds and / or materials used in combination with the particular composition employed, the age, sex, weight, condition, general health and previous medical history of the patient being treated, and similar factors well known in the medical field.

[0267] Suitable doses may range, for example, from about 0.01 μg / kg to about 1000 mg / kg, typically from about 0.1 μg / kg to about 100 mg / kg of the patient's body weight to be treated depending on the above conditions. For example, suitable dosages may be from about 1 μg / kg to about 10 mg / kg of body weight per day, or from about 10 μg / kg to about 5 mg / kg of body weight per day.

[0268] Dosage regimes can be adjusted to provide the optimum desired response (e.g., therapeutic response). For example, a single dose can be administered, several divided doses can be administered over time, or the dose can be proportionally reduced or increased according to the exigencies of the therapeutic situation. The dose can be provided as multiple doses, for example, at regular intervals, such as 2, 3 or 4 divided doses administered hourly. The multiple doses can be administered by the same or different routes, to the same or different locations. Alternatively, administration can be by sustained release formulation, which requires less frequent administration. Dosage and frequency can vary depending on the half-life of the antagonist in the patient and the desired duration of treatment.

[0269] Typically, polynucleotide or oligonucleotide inhibitors are administered in the range of 1 pg to 1 mg, preferably 1 pg to 10 μg of nucleic acid for particle-mediated delivery, and 10 μg to 1 mg for other routes.

[0270] Dosage unit form, as used herein, refers to physically discrete units suited as unitary dosages for the subjects to be treated, each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.

[0271] As noted above, the modulators / antibodies or pharmaceutical compositions of the invention may be co-administered with one or more other therapeutic agents.

[0272] Examples of the techniques and protocols mentioned above can be found in Remington's Pharmaceutical Sciences, 20th Edition, 2000, pub. Lippincott, Williams & Wilkins.

[0273] The co-administration of two or more agents can be accomplished in many different ways. Both can be administered together in a single composition, or in separate compositions as part of a combination therapy. For example, one can be administered before or separately, after or consecutively, or concurrently or simultaneously with the other. For example, an anti-GREM1 antagonist can be administered before or separately, after or consecutively, or concurrently or simultaneously with an inhibitor of Ras-Raf-MERK-ERK signaling. For example, an anti-GREM1 antagonist can be administered before or separately, after or consecutively, or concurrently or simultaneously with a MEK inhibitor as described herein. In another aspect of the invention, an anti-GREM1 antagonist can be administered before or separately, after or consecutively, or concurrently or simultaneously with an ERK inhibitor as described herein.

[0274] Compositions and kits Further provided is a composition or kit comprising an anti-GREM1 antagonist and an inhibitor of Ras-Raf-MEK-ERK signaling. The anti-GREM1 antagonist can be any anti-GREM1 antagonist described herein. The inhibitor of Ras-Raf-MEK-ERK signaling can be any inhibitor of Ras-Raf-MEK-ERK signaling described herein. For example, the present invention provides a composition or kit comprising an anti-GREM1 antagonist and a MEK inhibitor. In another aspect, the present invention provides a composition or kit comprising an anti-GREM1 antagonist and an ERK inhibitor. The composition or kit can be suitable for treating pancreatic cancer. In a preferred embodiment, the MEK inhibitor is selumetinib (AZD6244), trametinib (GSK-1120212) or WX-554 (UCB-554), particularly as part of a composition or kit for treating pancreatic cancer. A preferred combination comprises an anti-GREM1 antagonist and selumetinib (AZD6244), trametinib (GSK-1120212) or WX-554 (UCB-554). In another preferred embodiment, the ERK inhibitor is ulixertinib.

[0275] Bispecific antibodies combining an anti-GREM1 specificity with one of the other above-mentioned specificities may be provided in a composition or kit as described herein. The anti-GREM1 antagonist in any of the above compositions and kits may preferably be an anti-GREM1 antibody.

[0276] Additional therapeutic combinations The combination therapy of the present invention described above can be used / administered in combination with an additional therapeutic composition for treatment, e.g., as an adjunct therapy. The other therapeutic composition or treatment can be, e.g., one or more of those discussed herein, and can be administered simultaneously or sequentially with the composition of the present invention.

[0277] GREM1 antagonists are particularly useful in combination treatments, as they are used to sensitize cancers or tumors to additional anti-cancer drugs, such as radiation therapy or surgery, and in the absence of the GREM1 antagonist, the cancer may be resistant to other anti-cancer drugs or cancer treatments.

[0278] Thus, the anti-GREM1 antagonist in combination with an inhibitor of Ras-Raf-MEK-ERK signaling can further be used in combination with any other cancer treatment or any other therapeutic agent for cancer. The other cancer treatment can be selected from known treatments for related cancers, such as any known treatment for pancreatic cancer. The other cancer treatment can be radiation treatment. Suitable radiation therapy is described, for example, in Van Cutsem (and others) Annals of Oncology, 2014. Vol 25, Issue 3. Radiation therapy can be performed before cancer surgery or after cancer surgery. Radiation therapy can be adjuvant radiation therapy. Radiation therapy can be performed in combination with an inhibitor of Ras-Raf-MEK-ERK signaling described herein. For example, a combination therapy comprising a GREM1 antagonist and selumetinib (AZD6244) can be used in combination with radiation therapy. In addition, a combination therapy comprising a GREM1 antagonist and trametinib (GSK-1120212) can be used in combination with radiation therapy. Alternatively, the combination therapy comprising GREM1 antagonist and WX-554 (UCB-554) can be used in combination with radiation therapy. The combination therapy comprising GREM1 antagonist and any MEK inhibitor described herein can be used in combination with radiation therapy. Alternatively, the combination therapy comprising GREM1 antagonist and any ERK inhibitor described herein can be used in combination with radiation therapy. For example, the combination therapy comprising GREM1 antagonist and ulixertinib can be used in combination with radiation therapy.

[0279] The cancer may be resistant to radiation therapy unless administered with a combination therapy that includes a GREM1 antagonist as described herein.

[0280] As part of the above aspects, the present invention provides an anti-GREM1 antagonist in combination with an inhibitor of Ras-Raf-MEK-ERK signaling for use in a method for treating and / or preventing cancer, such as pancreatic cancer, according to the present invention, the method further comprising separate, sequential or simultaneous administration of an additional anti-cancer agent. In a preferred embodiment, the present invention provides an anti-GREM1 antagonist in combination with one or more MEK inhibitors (e.g., selumetinib (AZD6244), trametinib (GSK-1120212) and / or WX-554 (UCB-554)) for use in a method for treating and / or preventing cancer, such as pancreatic cancer, according to the present invention, the method further comprising separate, sequential or simultaneous administration of an additional anti-cancer agent.

[0281] As part of the above aspects, the present invention provides an anti-GREM1 antagonist in combination with one or more inhibitors of Ras-Raf-MEK-ERK signaling for use in a method of treating and / or preventing cancer, such as pancreatic cancer. For example, the present invention can provide an anti-GREM1 antagonist in combination with the MEK inhibitor selumetinib (AZD6244) for use in a method of treating and / or preventing cancer, such as pancreatic cancer, according to the present invention, the method further comprising the inclusion of an additional MEK inhibitor, such as trametinib (GSK-1120212), cobimetinib (GDC-0973), binimetinib (MEK162), CI-1040 (PD184352), mirdametinib (PD0325901), TAK733, rifametinib (RDEA119 / Bay), or a combination thereof. 86-9766), RO-5126766, RO5068760, pimasertib (AS703026), AZD8330, GDC-0623, RO-4987655, WX-554 (UCB-554), HL-085, ARRY-300, ClnQ-03, G-573, PD184161, PD318088, PD98059, U0126 or SL327.

[0282] In an alternative embodiment, the present invention may provide an anti-GREM1 antagonist in combination with the MEK inhibitor trametinib (GSK-1120212) for use in a method of treating and / or preventing cancer, such as pancreatic cancer, according to the present invention, the method further comprising the addition of an additional MEK inhibitor, for example selumetinib (AZD6244), cobimetinib (GDC-0973), binimetinib (MEK162), CI-1040 (PD184352), mirdametinib (PD0325901), TAK733, rifametinib (RDEA119 / Bay), or a combination thereof. 86-9766), RO-5126766, RO5068760, pimasertib (AS703026), AZD8330, GDC-0623, RO-4987655, WX-554 (UCB-554), HL-085, ARRY-300, ClnQ-03, G-573, PD184161, PD318088, PD98059, U0126 or SL327.

[0283] In still further alternative embodiments, the present invention may provide an anti-GREM1 antagonist in combination with the MEK inhibitor WX-554 (UCB-554) for use in the method of treatment and / or prevention of cancer, such as pancreatic cancer, according to the present invention, the method further comprising the addition of an additional MEK inhibitor, such as trametinib (GSK-1120212), selumetinib (AZD6244), cobimetinib (GDC-0973), binimetinib (MEK162), CI-1040 (PD18). 4352), mirdametinib (PD0325901), TAK733, refametinib (RDEA119 / Bay86-9766), RO-5126766, RO5068760, pimasertib (AS703026), AZD8330, GDC-0623, RO-4987655, HL-085, ARRY-300, ClnQ-03, G-573, PD184161, PD318088, PD98059, U0126 or SL327.

[0284] In a still further alternative embodiment, the present invention may provide an anti-GREM1 antagonist in combination with the ERK inhibitor ulixertinib for use in a method for the treatment and / or prevention of cancer, such as pancreatic cancer, according to the present invention, the method further comprising the separate, sequential or simultaneous administration of an additional ERK inhibitor, such as ulixertinib, temtelqib, JSI-1187, ravoxartinib, ASN007 or MK-8353.

[0285] The invention may further provide an anti-GREM1 antagonist in combination with a MEK inhibitor, as well as an anti-GREM1 antagonist in combination with an ERK inhibitor, as described herein.

[0286] Detection and Diagnosis Based on the correlation between stromal GREM1 and cancer, coupled with the finding that genes involved in the Ras-Raf-MEK-ERK pathway are upregulated following treatment with GREM1 antagonists, the present invention also provides additional means for predicting the responsiveness of cancer to treatment, which may include measuring Ras-Raf-MEK-ERK pathway signaling, stromal expression and / or epithelial expression of GREM1 in samples obtained from patients.

[0287] The present invention provides a method for determining whether a patient suffering from or suspected of suffering from cancer, or at risk of developing cancer, is likely to respond to combined treatment with a GREM1 antagonist and an inhibitor of Ras-Raf-MEK-ERK signaling, the method comprising measuring stromal and / or epithelial expression of GREM1 and Ras-Raf-MEK-ERK signaling in the patient, thereby predicting whether the patient is likely to respond to combined treatment.

[0288] The present invention further provides a method for determining whether a patient suffering from or suspected to suffer from cancer or at risk of developing cancer is likely to respond to a combination treatment with an anti-GREM1 antagonist and an inhibitor of Ras-Raf-MEK-ERK signaling, comprising measuring Ras-Raf-MEK-ERK pathway signaling in the patient after treatment / exposure with an anti-GREM1 antagonist, thereby predicting whether the patient is likely to respond to the combination treatment. For example, upregulation / induction of Ras-Raf-MEK-ERK signaling in response to treatment with an anti-GREM1 antagonist may indicate that the patient is a good candidate for combination therapy according to the present invention. The present invention further provides a method for determining whether a patient suffering from or suspected to suffer from pancreatic cancer or at risk of developing pancreatic cancer is likely to respond to a combination treatment with an anti-GREM1 antagonist and an inhibitor of Ras-Raf-MEK-ERK signaling, comprising measuring Ras-Raf-MEK-ERK pathway signaling in the patient after treatment / exposure with an anti-GREM1 antagonist, thereby predicting whether the patient is likely to respond to the combination treatment. For example, upregulation of Ras-Raf-MEK-ERK signaling in response to treatment with an anti-GREM1 antagonist may indicate that the patient is a good candidate for combination therapy according to the present invention. The present invention further provides a method for determining whether a patient suffering from or suspected of suffering from pancreatic cancer, or at risk of developing pancreatic cancer, is likely to respond to a combination treatment with a GREM1 antagonist and an inhibitor of Ras-Raf-MEK-ERK signaling, the method comprising measuring stromal and / or epithelial expression of GREM1 in the patient and determining whether the patient has a mutation in a RAS gene or a RAF gene, thereby predicting whether the patient is likely to respond to the combination treatment.

[0289] A predicted responsiveness of an individual to a given therapy means that the individual is expected to benefit or to benefit to a sufficient degree from receiving the therapy. A predicted non-responsiveness of an individual to a therapy means that the individual is not expected to benefit or to benefit to a sufficient degree from receiving the therapy. The method of predicting response can be performed before administration of a combination treatment of a GREM1 antagonist and an inhibitor of Ras-Raf-MEK-ERK signaling. The prediction can then be taken into account when selecting or recommending an appropriate treatment for the individual. Alternatively, the method is performed after a therapeutic treatment and used to monitor and predict the individual's response to the treatment. Typically, the method is for predicting whether an individual will have a primary response to the therapy, i.e., whether the individual will respond when first receiving the treatment. In some examples, the method is for predicting secondary non-responsiveness, i.e., whether an individual who initially responded to the treatment will later become unresponsive or less responsive to the treatment.

[0290] In some cases, overexpression of GREM1 compared to a reference sample or reference level indicates that the individual will respond to the combination treatment therapy described herein. Dysregulation of Ras-Raf-MEK-ERK signaling, for example, upregulation of Nras, Mapk1, and / or Myc, may also indicate that the individual will respond to the combination treatment therapy described herein, compared to a reference sample or reference level. Ras-Raf-MEK-ERK signaling can be measured by examining phosphorylation levels, for example, using phosphorylation-specific antibodies, such as phospho-specific antibodies against MEK and ERK. For example, increased phosphorylation of Map2k1 / Map2k2 / Erk1 / Erk2 in response to treatment with the anti-GREM1 antagonist of the present invention, compared to a reference sample or reference level, indicates that the individual will respond to the combination treatment therapy described herein. A combination therapy comprising a GREM1 antagonist in combination with an inhibitor of Ras-Raf-MEK-ERK signaling, such as a MEK inhibitor or an ERK inhibitor, can then be selected or recommended and further administered to the individual.

[0291] In other cases, reduced or normal levels of GREM1 compared to a reference sample or reference level indicate that the individual will not respond to treatment with a GREM1 antagonist. Normal or reduced Ras-Raf-MEK-ERK signaling compared to a reference sample or reference level may indicate that the individual will not respond to treatment with an inhibitor of Ras-Raf-MEK-ERK signaling. Then, a combination therapy comprising a GREM1 antagonist and an inhibitor of Ras-Raf-MEK-ERK signaling is not administered to the individual. Furthermore, a therapeutic treatment other than the combination therapy described herein may be selected or recommended for the treatment of the individual and then further administered to the individual.

[0292] In yet other cases, mutations in RAS genes (e.g., KRAS, NRAS, and / or HRAS) or RAF genes (e.g., ARAF, BRAF, and / or CRAF) indicate that the individual will respond to treatment with a combination treatment described herein. A combination therapy comprising a GREM1 antagonist in combination with an inhibitor of Ras-Raf-MEK-ERK signaling may then be selected or recommended and further administered to the individual.

[0293] In all aspects of the present invention, individuals with cancer (e.g., pancreatic cancer) or individuals suspected of having a disease or condition and / or individuals at risk of developing a disease or condition can be selected or identified for treatment. For example, an individual may not have been formally diagnosed, but may be suspected of having a disease or condition because one or more symptoms are present. If an individual has one or more risk factors associated with cancer and / or one or more predisposing factors that increase susceptibility to cancer, the individual may be considered at risk of developing cancer. Risk factors associated with pancreatic cancer may include inherited genetic mutations, such as familial pancreatitis caused by a mutation in the PRSS1 gene, a mutation or mutations in the gene encoding GREM1, or any other mutation that affects the expression of the GREM1 gene.

[0294] The following examples illustrate the invention. EXAMPLES

[0295] Materials and Methods Anti-Gremlin-1 antibody - Ab7326 mIgG1 - APP.4405.IgG.mFc Vehicle - Phosphate buffered saline pH 7.4 (provided by the Beatson Institute) Selumetinib (AZD6244) - Provided by Beatson Institute (purchased from SYNthesis Pharm) in 0.5% HPMC + 0.1% Tween-80. UCB-554 MEK inhibitor (UCB1366554-000, batch ID B10476792, provided by UCB) was provided in 10% DMSO + 90% 1% (w / v) methylcellulose. Dosage was 10 mg / kg, 125 μl, po, q2d. The vehicle for Example 5 was 125 μl of 10% DMSO+90% of 1% (w / v) methylcellulose, po, q2d.

[0296] Genetically modified mice LSL-Kras G12D / + ;Trp53 R172H / + ;Pdx1-Cre(KPC) mice have been described previously (Hingorani et al., 2005). Mice were generated by crossing mice carrying Pdx1-Cre with conditional LSL-KrasG12D or LSL-Trp53R172H alleles (strains 01XJ6 and 01XL9, Mouse Models of Human Cancer Consortium [MMHCC], NCI-Frederick, Frederick, MD, USA). Mice on mixed backgrounds were bred in-house at the CRUK Beatson Institute and maintained in normal cages with environmental enrichment and free access to standard food and water. Genotyping was performed at Transnetyx (Cordoba, TN, USA). Mice of both sexes were included in the study. All animal experiments were performed under UK Home Office permission and approved by the University of Glasgow Animal Welfare Ethics Committee.

[0297] Treatment of Examples 3 and 4 Mice were monitored three times a week until pancreatic cancer was diagnosed by abdominal palpation and confirmed by ultrasound imaging. Mice were randomized into treatment groups and received 30 mg / kg Ab7326 mIgG1 sc twice weekly; or 30 mg / kg Ab7326 mIgG1 sc twice weekly and 25 mg / kg selumetinib po, bid. Historical data was available for mice treated with 25 mg / kg selumetinib po, bid. There were ≥12 mice per group. Mice were monitored daily and sacrificed on day 7 after treatment initiation (4 mice / group) or when ethical endpoints were reached (≥8 mice / group, symptoms included abdominal distension, cachexia, intermittent hunched back or reduced mobility, porosity, mild diarrhea, anemia). Statistical evaluation of survival from treatment initiation in these mice was performed by Kaplan-Meier and log-rank analysis.

[0298] Ultrasound Imaging A VisualSonics Vevo 3100 preclinical imaging platform (FUJIFILM VisualSonics, Toronto, Canada) was used for high-resolution ultrasound imaging to confirm tumor diagnosis and weekly monitoring of tumor progression. Anesthesia was induced and maintained with a mixture of isoflurane and medical air. Tumor volumes were calculated weekly for each mouse and plotted longitudinally.

[0299] sampling Mice were culled using a schedule 1 method according to institutional guidelines. Postmortem tumor burden was assessed by gross pathology and histology. Organs were removed and fixed in 10% buffered formalin. At sacrifice, blood samples were taken by cardiac puncture, if possible, and serum processed. The majority of the tumors were fixed in 10% buffered formalin for FFPE processing, and any remaining tissues were harvested in RNAlater® (Sigma-Aldrich) for RNA preparation and / or snap frozen. Fixed tissues were paraffin embedded and 5 μm sections were placed on poly-L-lysine slides for IHC analysis.

[0300] Histology and immunohistochemistry H&E and picrosirius red staining were performed on formalin-fixed, paraffin-embedded tissues as previously described. Immunohistochemistry was performed using standard protocols. Briefly, formalin-fixed, paraffin-embedded sections were deparaffinized and rehydrated through xylene and a graded alcohol series. Endogenous peroxidase activity was inactivated by treatment with hydrogen peroxide, followed by antigen retrieval with citrate buffer. Sections were blocked with serum and incubated with primary antibodies. Sections were incubated with secondary antibodies for 30 min, and staining was visualized with 3,3'-diaminobenzidine tetrahydrochloride. Primary antibodies used were anti-alpha-SMA (1A4, Sigma-Aldrich) 1:20,000) and anti-podoplanin (RTD4E10, AbCam 1:250).

[0301] Processing Example 5 Mice were monitored three times weekly until pancreatic cancer was diagnosed by abdominal palpation and confirmed by ultrasound imaging. Mice were randomized (n=9-11 / group) into treatment groups and received 125μl vehicle, po, q2d; 30mg / kg Ab7326 mIgG1 sc, twice weekly; 25mg / kg selumetinib po, bid; 30mg / kg Ab7326 mIgG1 sc, twice weekly and 25mg / kg selumetinib po, bid; UCB-554 10mg / kg in 125μl, po, q2d; or 30mg / kg Ab7326 mIgG1 sc twice weekly and UCB-554 10mg / kg in 125μl, po, q2d. Mice were monitored daily and sacrificed when ethical endpoints were reached.

[0302] Statistics for Example 5 Statistical evaluation of the survival of these mice from the start of treatment was performed using Cox proportional hazards models to calculate hazard ratios. Day 7 tumor volume data were analyzed using a linear model appropriate for the randomized design, with day 0 treated tumor volume used as a covariate to refine the estimates.

[0303] (Example 1) Confirmation of Grem1 mRNA expression in pancreatic cancer To investigate whether Gremlin-1 plays a role in pancreatic cancer, Grem1 mRNA expression, as determined by RNAseq analysis, was determined in a cohort of human pancreatic ductal adenocarcinoma (PDAC) patients. This confirmed that Grem1 mRNA is expressed in human PDAC and high expression was significantly associated with poor prognosis (Figure 1). The results were consistent with a published study (Yu et al., 2018) that reported an association between Grem1 expression and poor prognosis in pancreatic cancer.

[0304] To determine whether grem1 mRNA is expressed in the KPC genetically engineered mouse pancreatic cancer model, gene expression was examined in KPC tumors (and other autologous pancreatic cancer models) and compared with control KrasG12D-expressing normal pancreatic ductal epithelium. The results show that grem1 mRNA expression was elevated in KPC tumors (and other mouse pancreatic tumors) compared with controls (Table 1). [Table 1]

[0305] (Example 2) Changes in gene expression in KPC mouse model by treatment with Ab7326 mIgG1 KPC mice with confirmed pancreatic tumors were established as described in Materials and Methods. Mice were enrolled into cohorts for treatment with anti-Gremlin antibody Ab7326 mIgG1 or vehicle control, as shown in Table 2.

[0306] Mice were sacrificed at ethical endpoints and primary tumor tissue was harvested into RNAlater® (Sigma-Aldrich) for RNA extraction. RNA samples were interrogated using Nanostring nCounter gene expression technology with a panel of approximately 750 probes. [Table 2]

[0307] Analysis of gene expression changes in a KPC mouse model exposed to Ab7326 mIgG1 showed upregulation of the MAPK / ERK (MEK) pathway and its canonical members.

[0308] Key members of the MAPK / ERK (MEK) pathway, Mapk1, Nras, and Myc, were upregulated (Figure 3). Due to insufficient power (three samples per group, i.e., Ab7326 mIgG1-exposed and control), these genes lost statistical significance after multiple hypothesis correction (FDR<5%). However, box plots showing the difference in gene expression distribution for these three genes clearly showed upregulated expression when comparing the Ab7326 mIgG1 group with the control group (Figure 3).

[0309] This finding was supported by pathway enrichment analysis (Figure 4). Downregulation of KRAS signaling was enriched in downregulated gene sets (p=0.03), whereas upregulation of KRAS signaling was associated with upregulated differentially expressed gene sets (p=0.01). Although these enriched pathways were not statistically significant after multiple hypothesis correction, the consistent pattern in the effects of upregulation and downregulation was striking.

[0310] Furthermore, when KPC mice were exposed to Ab7326 mIgG1, proliferation signals were elevated (G2M, E2F and MYC targets), consistent with downstream effects of MAPK / ERK (MEK) pathway activation. In Figure 4, -log10(p) scores are presented and only pathways with p<0.05 are retained. No multiple hypothesis correction was performed. However, the MYC_TARGETS, E2F_TARGETS and G2M_CHECKPOINT pathways remained significant after this correction (FDR<5%).

[0311] This finding supported the potential of combining Ab7326 mIgG1 with MEK inhibitors in the treatment of pancreatic cancer. Moreover, exposure to Ab7326 mIgG1 may render pancreatic cancer more sensitive to this targeted treatment.

[0312] (Example 3) Treatment with the combination of Ab7326 mIgG1 and selumetinib results in slower tumor growth compared to treatment with Ab7326 mIgG1 as a single agent LSL-Kras G12D / + ;LSL-Trp53 R172H / + KPC mice have been previously described (Hingorani et al., 2005). These mice develop tumors that are histologically and pathologically similar to human pancreatic tumors and are highly aggressive, frequently metastatic, and highly resistant to chemotherapy, again mimicking human pancreatic cancer. A cohort of KPC mice was established and the mice were monitored until they developed pancreatic cancer detectable by palpation. The breeding strategy and experimental design required to generate these mice are shown in Figure 2.

[0313] Mice were monitored at least weekly by palpation until pancreatic tumors were detected, at which point high-resolution ultrasound imaging was used to confirm the presence of pancreatic cancer and mice were enrolled into cohorts for treatment with anti-Gremlin 1 antibody Ab7326 mIgG1 or the combination of Ab7326 mIgG1 + selumetinib (detailed in Tables 3 and 4). [Table 3] [Table 4]

[0314] Four mice from each cohort underwent follow-up ultrasound examination one week later and were then sacrificed to generate samples for pharmacodynamic evaluation (Table 3). Analysis of gross pathology revealed no differences in tumor burden or phenotype between the different experimental conditions. Analysis of 3D tumor images by ultrasound revealed that tumor growth was delayed in mice treated with the combination of Ab7326 mIgG1 + selumetinib (Figure 5).

[0315] (Example 4) Treatment with the combination of Ab7326 mIgG1 and selumetinib results in delayed tumor growth and tumor shrinkage with prolonged responses compared to treatment with Ab7326 mIgG1 alone, and results in a significant increase in median survival compared to treatment with selumetinib and Ab7326 mIgG1 as single agents. In the surviving cohort (Table 4), high-resolution ultrasound imaging was performed weekly throughout the treatment period to monitor the tumor burden of each individual mouse during treatment. Mice continued in the study and were closely monitored until they reached the ethical endpoint, at which point they were sacrificed according to institutional guidelines. Clinical characteristics exhibited included abdominal distension, poor body condition indicative of cachexia, decreased exercise capacity, and occasionally jaundice.

[0316] Analysis of 3D tumor images by ultrasound revealed that in some mice treated with the combination of Ab7326 mIgG1 and selumetinib, there was a delay in tumor growth, tumor shrinkage, along with an extended response (Figure 6). Furthermore, we observed that mice treated with the combination of Ab7326 mIgG1 and selumetinib showed a significant increase in median survival (33 days) compared to mice treated with selumetinib alone (17 days, log-rank, p=0.050), and an increase in median survival compared to mice treated with Ab7326 alone (19 days, see Figure 7, Tables 5 and 6 for individual mouse data). Serum samples from both the pharmacodynamic evaluation and survival study were sent to UCB for analysis (Table 7). [Table 5] [Table 6] [Table 7]

[0317] After sacrificing the experimental mice, gross pathological examination did not show any differences in tumor burden and phenotype between the different experimental conditions. Immunohistochemical (IHC) analysis was performed on formalin-fixed, paraffin-embedded tumor tissue to examine the tumor microenvironment and evaluate any changes in the number of alpha-SMA-positive and podoplanin-positive tumor-associated fibroblasts, or the quality or quantity of collagen I and III (as measured by picrosirius red staining). Staining was scored using HALO digital software; however, none of the tested regimens had a significant effect on any of these parameters (Figure 8).

[0318] result In summary, the results presented here show that exposure to Ab7326 upregulates MAPK / ERK pathway genes in the KPC mouse model. Furthermore, Ab7326 mIgG1 can be safely given to KPC mice in combination with selumetinib. When mice were treated with the combination of Ab7326 mIgG1 and selumetinib, a significant improvement in survival was observed compared to treatment with selumetinib alone (see individual mouse data in Figure 7, Table 5, and Table 6). Furthermore, tumor shrinkage was observed in several mice treated with the combination of Ab7326 mIgG1 and selumetinib, and these mice had prolonged responses. This suggests that the combination of Ab7326 mIgG1 and selumetinib represents a very promising treatment for pancreatic cancer.

[0319] Example 5: Treatment of KPC mice with a combination of Ab7326 mIgG1 and either selumetinib or the UCB-554 MEK inhibitor results in delayed tumor growth and extended survival compared to treatment with single agents. To determine whether the increased survival seen with the combination of Ab7326 and S-selumetinib in Example 3 was applicable to other MEK inhibitors, and to further increase the robustness of the study by increasing the number of KPC mice in each cohort, a study was performed comparing the combination of Ab7326 with either the MEK inhibitor selumetinib or the MEK inhibitor UCB-554. KPC mice with confirmed pancreatic tumors were established as described in Materials and Methods, enrolled in treatment cohorts as shown in Table 8, and monitored for tumor growth by ultrasound and ethical survival endpoints. [Table 8]

[0320] Treatment with the MEK inhibitor UCB-554 resulted in a modest increase in survival of KPC mice compared to vehicle-only controls or Ab7326 treatment (Figure 9a and Table 8 median survival). However, combined treatment with Ab7326 and UCB-554 resulted in further improved survival of KPC mice compared to UCB-554 alone (Figure 9a and Table 8 median survival). Cox proportional hazards models were fitted to the survival data and hazard ratios (HRs) were estimated for comparison of the different treatment groups (Table 9). The hazard ratios for Ab7326+UCB-554 versus vehicle controls (HR=3.57, p=0.005) or Ab7326 alone (HR=4.38, p=0.001) indicated a statistically significant improvement in survival with the combination. The hazard ratio for Ab7326+UCB-554 versus UCB-554 alone was also greater than 1 (HR=1.75), suggesting improved survival with the combination over MEK inhibition alone, but did not reach statistical significance (p=0.211). [Table 9] Pancreatic tumor volumes in control or treated KPC mice were measured over time using 3D tumor imaging by ultrasound. Comparison of tumor volumes at 7 days after treatment initiation showed that tumor volumes were reduced in the UCB-554 treatment group compared to vehicle or Ab7326 treatment alone, consistent with survival data (Figure 9b). Furthermore, the combination of Ab7326+UCB-554 had a statistically significant reduction in tumor volume at 7 days compared to UCB-554 alone (p=0.040).

[0321] Measurement of tumor volume over time up to the ethical endpoint for mice in this cohort showed that combined treatment with Ab7326 and UCB-554 resulted in tumor shrinkage or reduced tumor growth in a subset of mice compared to vehicle controls (Figure 9c).

[0322] Survival data of KPC mice collected in the selumetinib MEK inhibitor study shown in Example 3 (Table 4) was combined with data from the cohort in Example 5 (Table 8) to increase the robustness of the analysis by including a larger number of animals. As shown in Figure 10a and Table 10, the combination of Ab7326 and selumetinib significantly increased survival of KPC mice compared to vehicle alone (HR = 5.16, p < 0.001) or Ab7326 treatment (HR = 3.82, p < 0.001). Furthermore, the combination of Ab7326 and selumetinib significantly increased survival compared to treatment with selumetinib alone, with a hazard ratio of 2.92 (p = 0.006), indicating a significant beneficial effect of the combination of Gremlin-1 inhibition and MEK pathway inhibition in this pancreatic cancer model. [Table 10]

[0323] Comparison of tumor volumes 7 days after treatment initiation showed that, consistent with the survival data, mean tumor volumes were reduced in the selumetinib and selumetinib + Ab7326 treatment groups compared to vehicle or Ab7326 treatment alone (Figure 10b).

[0324] Measurement of tumor volume over time up to the ethical endpoint for mice in this cohort showed that combined treatment with Ab7326 and selumetinib resulted in tumor shrinkage or reduced tumor growth in a subset of mice compared to vehicle controls (Figure 10c).

[0325] In conclusion, these data show that the anti-gremlin-1 antibody Ab7326, administered in combination with MEK inhibitors such as selumetinib or UCB-554, reduces tumor growth and increases survival in the KPC mouse model of pancreatic cancer compared with treatment with the individual agents, suggesting that simultaneous targeting of the gremlin-1 and MEK pharmacological pathways may provide additive or synergistic effects in the treatment of pancreatic cancer.

[0326] Aspects 1. An anti-GREM1 antagonist for use in a method for treating or preventing cancer, the method further comprising administering an inhibitor of Ras-Raf-MEK-ERK signaling. 2. An anti-GREM1 antagonist for use according to embodiment 1, wherein the cancer is a solid cancer. 3. An anti-GREM1 antagonist for use according to embodiment 1 or embodiment 2, wherein the cancer has stromal GREM1 overexpression. 4. The anti-GREM1 antagonist for use according to any one of the preceding aspects, wherein the cancer is a cancer associated with dysregulation of the Ras-Raf-MEK-ERK pathway. 5. An anti-GREM1 antagonist for use according to any one of the preceding aspects, wherein the cancer exhibits dysregulation of the Ras-Raf-MEK-ERK pathway following exposure to the anti-GREM1 antagonist. 6. An anti-GREM1 antagonist for use according to any one of the preceding aspects, wherein the cancer contains a mutation in the Ras gene, optionally KRAS, NRAS and / or HRAS. 7. An anti-GREM1 antagonist for use according to any one of the preceding aspects, wherein the cancer contains a mutation in the Raf genes; optionally ARAF, BRAF and / or CRAF. 8. The anti-GREM1 antagonist for use according to any one of the preceding aspects, wherein the cancer is a cancer that is poorly responsive, non-responsive or refractory to treatment with an inhibitor of Ras-Raf-MEK-ERK signaling. 9. The anti-GREM1 antagonist for use according to any one of the preceding aspects, wherein the cancer is selected from colorectal cancer, multiple myeloma, pancreatic cancer, bladder cancer, breast cancer, lung cancer, gastric cancer, duodenal cancer, esophageal cancer, head and neck cancer, prostate cancer, glioma, endometrial cancer, liver cancer, splenic cancer, bone resident cancer, melanoma and osteosarcoma. 10. The anti-GREM1 antagonist for use according to aspect 9, wherein the cancer is pancreatic cancer. 11. The anti-GREM1 antagonist for use according to aspect 10, wherein the pancreatic cancer is exocrine pancreatic cancer. 12. An anti-GREM1 antagonist for use according to embodiment 10 or embodiment 11, wherein the pancreatic cancer is pancreatic ductal adenocarcinoma (PDAC). 13. An anti-GREM1 antagonist for use according to aspect 9, wherein the cancer is multiple myeloma or colorectal cancer, optionally KRAS / BRAF mutated colorectal cancer. 14. An anti-GREM1 antagonist for use according to embodiment 9, wherein the cancer is lung cancer; optionally the cancer is non-small cell lung cancer (NSCLC). 15. An anti-GREM1 antagonist for use according to embodiment 9, wherein the cancer is melanoma; optionally BRAF mutated melanoma. 16. The anti-GREM1 antagonist for use according to any one of the preceding aspects, wherein the cancer has epithelial GREM1 overexpression. 17. An anti-GREM1 antagonist for use according to aspect 16, wherein the cancer is a GREM1-induced cancer. 18. The anti-GREM1 antagonist for use according to any one of the preceding aspects, wherein the cancer is a disseminated cancer. 19. The anti-GREM1 antagonist for use according to any one of the preceding aspects, wherein the cancer is an established cancer. 20. The anti-GREM1 antagonist for use according to any one of the preceding aspects, wherein the antagonist is a peptide, a protein, an antibody, a polynucleotide, an oligonucleotide, an antisense RNA, a small interfering RNA (siRNA), a small molecule inhibitor, or a small hairpin RNA (shRNA). 21. An anti-GREM1 antagonist for use according to aspect 20, wherein the antagonist is an antibody that binds to an epitope on Gremlin-1 comprising at least one residue selected from Ile131, Lys147, Lys148, Phe149, Thr150, Thr151, Arg169, Lys174 and Gln175, wherein the residue numbering is according to SEQ ID NO: 1. 22. An anti-GREM1 antagonist for use according to aspect 21, wherein the antibody binds to an epitope comprising all of Ile131, Lys147, Lys148, Phe149, Thr150, Thr151, Arg169, Lys174 and Gln175. 23. An anti-GREM1 antagonist for use according to aspect 21 or 22, wherein Lys147, Lys148, Phe149, Thr150, Thr151, Arg169, Lys174 and Gln175 are located on the same Gremlin-1 monomer, and Ile131 is located on a second Gremlin-1 monomer. 24. An anti-GREM1 antagonist for use according to aspect 20, wherein the antagonist is an anti-Gremlin-1 antibody comprising a heavy chain complementarity determining region (HCDR) sequence contained within the heavy chain variable region (HCVR) of SEQ ID NO: 10 or 12, and / or a light chain complementarity determining region (LCDR) sequence contained within the light chain variable region (LCVR) of SEQ ID NO: 11 or 13. 25. An anti-GREM1 antagonist for use according to aspect 20, wherein the antagonist is an anti-Gremlin-1 antibody comprising at least one HCDR sequence selected from SEQ ID NOs: 3, 4, 5 and 6, and / or at least one LCDR sequence selected from SEQ ID NOs: 7, 8 and 9. 26. An anti-GREM1 antagonist for use according to aspect 25, wherein the anti-Gremlin-1 antibody comprises the HCDR3 sequence of SEQ ID NO:6. 27. An anti-GREM1 antagonist for use according to aspect 25 or 26, wherein the anti-Gremlin-1 antibody comprises a combination of HCDR1 / HCDR2 / HCDR3 sequences selected from SEQ ID NO: 4 / 5 / 6 or SEQ ID NO: 3 / 5 / 6, and / or a combination of LCDR1 / LCDR2 / LCDR3 sequences selected from SEQ ID NO: 7 / 8 / 9. 28. An anti-GREM1 antagonist for use according to any one of aspects 25 to 27, wherein the anti-Gremlin-1 antibody comprises a combination of HCDR1 / HCDR2 / HCDR3 / LCDR1 / LCDR2 / LCDR3 sequences of SEQ ID NO: 4 / 5 / 6 / 7 / 8 / 9 or SEQ ID NO: 3 / 5 / 6 / 7 / 8 / 9. 29. An anti-GREM1 antagonist for use according to any one of aspects 25 to 28, wherein the anti-Gremlin-1 antibody comprises a heavy chain variable region (HCVR) sequence of SEQ ID NO: 10 or 12, and / or a light chain variable region (LCVR) sequence of SEQ ID NO: 11 or 13, or a sequence at least 95% identical thereto. 30. An anti-GREM1 antagonist for use according to aspect 29, wherein the anti-Gremlin-1 antibody comprises the HCVR and LCVR sequence pairs of SEQ ID NOs: 10 / 11 or 12 / 13, or sequences which are at least 95% identical thereto. 31. An anti-GREM1 antagonist for use according to aspect 30, wherein the anti-Gremlin-1 antibody comprises an HCDR1 / HCDR2 / HCDR3 / LCDR1 / LCDR2 / LCDR3 sequence consisting of SEQ ID NO: 4 / 5 / 6 / 7 / 8 / 9 or SEQ ID NO: 3 / 5 / 6 / 7 / 8 / 9, and the remainder of the HCVR and LCVR are at least 95% identical to SEQ ID NO: 10, 11, 12 and / or 13, respectively. 32. An anti-GREM1 antagonist for use according to aspects 29 to 31, wherein the anti-Gremlin-1 antibody comprises a heavy chain of SEQ ID NO: 14, 16, 18, 22, 28, 30, 32 or 34, and / or a light chain of SEQ ID NO: 15, 17, 19, 23, 29, 31, 33 or 35, or a sequence at least 95% identical thereto. 33. An anti-GREM1 antagonist for use according to aspect 32, wherein the anti-Gremlin-1 antibody comprises a heavy and light chain pair of SEQ ID NOs: 14 / 15, 16 / 17, 18 / 19, 22 / 23, 28 / 29 or 30 / 31, 32 / 33, 34 / 35, or a sequence which is at least 95% identical. 34. An anti-GREM1 antagonist for use according to aspect 33, wherein the HCDR1 / HCDR2 / HCDR3 / LCDR1 / LCDR2 / LCDR3 sequences of the antibody consist of SEQ ID NO: 4 / 5 / 6 / 7 / 8 / 9 or SEQ ID NO: 3 / 5 / 6 / 7 / 8 / 9, and the remainder of the heavy and light chains are at least 95% identical to SEQ ID NO: 14, 15, 16 and / or 17, respectively. 35. An anti-GREM1 antagonist for use according to aspect 20, wherein the antagonist is an antibody which competes for binding to Gremlin-1 with an antibody as defined in any one of aspects 22 to 32. 36. An anti-GREM1 antagonist for use according to aspect 20, wherein the antagonist is an antibody that binds to the same epitope on Gremlin-1 as an antibody defined in any one of aspects 22 to 32. 37. An anti-GREM1 antagonist for use according to any one of aspects 20 to 36, wherein the antagonist antibody is a chimeric antibody, a human antibody or a humanized antibody. 38. An anti-GREM1 antagonist for use according to any one of aspects 20 to 37, wherein the antagonist antibody is a Fab, a modified Fab, a Fab', a modified Fab', F(ab')2, an Fv, a single domain antibody or an scFv. 39. An anti-GREM1 antagonist for use according to aspect 20, wherein the antagonist is a polynucleotide encoding an antibody as defined in any one of aspects 21 to 38, or an expression vector comprising said polynucleotide. 40. An anti-GREM1 antagonist for use according to any one of aspects 20 to 38, wherein the antagonist antibody is comprised in a pharmaceutical composition further comprising a pharma- ceutically acceptable adjuvant and / or carrier. 41. An anti-GREM1 antagonist for use according to any one of the preceding aspects, wherein the inhibitor of Ras-Raf-MEK-ERK signaling downregulates Mapk1, Nras, Kras and / or Myc signaling. 42. An anti-GREM1 antagonist for use according to any one of the preceding embodiments, wherein the inhibitor of Ras-Raf-MEK-ERK signaling is a MEK inhibitor, optionally an allosteric MEK inhibitor. 43. An anti-GREM1 antagonist for use according to aspect 42, wherein the MEK inhibitor is a MEK1 and / or MEK2 inhibitor. 44. MEK inhibitors include selumetinib (AZD6244), trametinib (GSK-1120212), cobimetinib (GDC-0973), binimetinib (MEK162), CI-1040 (PD184352), mirdametinib (PD0325901), TAK733, refametinib (RDEA119 / Bay86-9766), RO-5126766, and RO506876. 0, pimasertib (AS703026), AZD8330, GDC-0623, RO-4987655, WX-554 (UCB-554), HL-085, ARRY-300, ClnQ-03, G-573, PD184161, PD318088, PD98059, U0126 or SL327. 45. MEK inhibitors, (a) N-(2-fluoro-4-iodophenyl)-3-(morpholin-4-ylcarbonyl)thieno[2,3-b]pyridin-2-amine; (b) N-(2-fluoro-4-iodophenyl)-3-[(4-methylpiperazin-1-yl)carbonyl]thieno[2,3-b]pyridin-2-amine; (c) [2-[((2-fluoro-4-iodophenyl)amino]thieno[2,3-b]pyridin-3-yl](pyrrolidin-1-yl)methanone; (d) (1-{2-[(2-fluoro-4-iodophenyl)amino]thieno[2,3-b]pyridine-3-carbonyl}piperidin-4-yl)-carbamic acid tert-butyl ester; (e) 2-[((2-fluoro-4-iodophenyl)amino]thieno[2,3-b]pyridine-3-carboxylic acid (4-aminopiperidin-1-yl)amide dihydrochloride; (f) (1-{2-[(2-fluoro-4-iodophenyl)amino]thieno[2,3-b]pyridine-3-carbonyl}azetidin-3-ylmethyl)carbamic acid tert-butyl ester; (g) [3-((aminomethyl)azetidin-1-yl]-{2-[(2-fluoro-4-iodophenyl)amino]thieno[2,3-b]pyridin-3-yl}-methanone; (h) (1-{2-[(2-fluoro-4-iodophenyl)amino]thieno[2,3-b]pyridine-3-carbonyl}-(3R)-pyrrolidin-3-yl)carbamic acid tert-butyl ester; (i) (1-{2-[(2-fluoro-4-iodophenyl)amino]thieno[2,3-b]pyridine-3-carbonyl}-(3S)-pyrrolidin-3-yl)carbamic acid tert-butyl ester; (j) (1-{2-[(2-fluoro-4-iodophenyl)amino]thieno[2,3-b]pyridine-3-carbonyl}azetidin-3-yl)-carbamic acid tert-butyl ester; (k) [(3R)-3-aminopyrrolidin-1-yl]-{2-[(2-fluoro-4-iodophenyl)amino]thieno[2,3-b]pyridin-3-yl}-methanone; (l) [(3S)-3-aminopyrrolidin-1-yl]-{2-[(2-fluoro-4-iodophenyl)amino]thieno[2,3-b]pyridin-3-yl}-methanone; (m) (3-aminoazetidin-1-yl)-{2-[(2-fluoro-4-iodophenyl)amino]thieno[2,3-b]pyridin-3-yl]-methanone; (n) {2-[((2-fluoro-4-iodophenyl)amino]thieno[2,3-b]pyridin-3-yl}-(3-hydroxyazetidin-1-yl)-methanone; (o) {2-[((2-fluoro-4-iodophenyl)amino]thieno[2,3-b]pyridin-3-yl}-[(3R)-3-hydroxypyrrolidin-1-yl]-methanone; (p) {2-[((2-fluoro-4-iodophenyl)amino]thieno[2,3-b]pyridin-3-yl}-[(3S)-3-hydroxypyrrolidin-1-yl]-methanone; (q) {2-[((2-fluoro-4-iodophenyl)amino]thieno[2,3-b]pyridin-3-yl}-[2-(hydroxymethyl)-piperidin-1-yl]-methanone; (r) {2-[((2-fluoro-4-iodophenyl)amino]thieno[2,3-b]pyridin-3-yl}-[(3S)-3-(hydroxymethyl)-morpholin-4-yl]-methanone; (s) 4-{2-[(2-fluoro-4-iodophenyl)amino]thieno[2,3-b]pyridine-3-carbonyl}-(3R)-3-(hydroxymethyl)piperazine-1-carboxylic acid tert-butyl ester; (t) 2-[4({2-[(2-fluoro-4-iodophenyl)amino]thieno[2,3-b]pyridin-3-yl}carbonyl)piperazin-1-yl]ethanol; (u) 3-((1,4-diazepan-1-ylcarbonyl)-N-(2-fluoro-4-iodophenyl)thieno[2,3-b]pyridin-2-amine; (v) N-(2-fluoro-4-iodophenyl)-3-(piperazin-1-ylcarbonyl)thieno[2,3-b]pyridin-2-amine; (w) ethyl [4-({2-[(2-fluoro-4-iodophenyl)amino]thieno[2,3-b]pyridin-3-yl}carbonyl)-piperazin-1-yl]acetate; (x) [4-({2-[(2-fluoro-4-iodophenyl)amino]thieno[2,3-b]pyridin-3-yl}carbonyl)piperazin-1-yl]acetic acid; (y) {2-[((2-fluoro-4-iodophenyl)amino]thieno[2,3-b]pyridin-3-yl}-[(2R)-2-(methoxymethyl)-pyrrolidin-1-yl]-methone; and (z) {2-[((2-fluoro-4-iodophenyl)amino]thieno[2,3-b]pyridin-3-yl}-[(2R)-2-(hydroxymethyl)-piperazin-1-yl]-methanone 43. The anti-GREM1 antagonist for use according to embodiment 42, selected from the group consisting of: 46. ​​The MEK inhibitor is represented by the formula (II): [ka] [In the formula, R 12 represents a halogen; R 3 CONR b R c represents; R b and R c taken together with the nitrogen atom to which they are both attached represent azetidin-1-yl, pyrrolidin-1-yl, piperidin-1-yl, morpholin-4-yl, thiomorpholin-4-yl, piperazin-1-yl, homopiperidin-1-yl, homo-morpholin-4-yl or homopiperazin-1-yl, any of which groups may optionally be represented by C 1-6 Alkyl, C 1-6 Alkoxy, hydroxy, hydroxy(C 1-6 ) alkyl, amino-(C 1-6 ) alkyl, (amino) (hydroxy) (C 1-6 ) Alkyl, halogen, oxo, C 2-6Alkylcarbonyl, Carboxy, C 2-6 Alkoxycarbonyl, di(C 1-6 ) Alkylhydrazinylcarbonyl, amino, C 1-6 Alkylamino, di(C 1-6 ) Alkylamino, C 2-6 Alkylcarbonylamino, Aminocarbonylamino, Aminocarbonyl, C 1-6 Alkylaminocarbonyl, di(C 1-6 ) Alkylaminocarbonyl, aminosulfonyl, C 1-6 Alkylsulfonyl, C 1-6 Alkylaminocarbonyl (C 1-6 ) Alkyl, C 1-6 Alkoxy(C 1-6 ) alkyl, carboxy (C 1-6 ) Alkyl, C 2-6 Alkoxycarbonyl (C 1-6 ) Alkyl, C 2-6 Alkoxycarbonylamino and C 2-6 and optionally substituted by one or more substituents selected from alkoxycarbonylamino-(C1-6)alkyl. and pharma- ceutically acceptable salts, solvates and N-oxides thereof. 47.R 12 An anti-GREM1 antagonist for use as claimed in claim 46, wherein represents bromo or iodo. 48. An anti-GREM1 antagonist for use according to embodiment 44, wherein the MEK inhibitor is selumetinib (AZD6244). 49. An anti-GREM1 antagonist for use according to embodiment 44, wherein the MEK inhibitor is trametinib (GSK-1120212). 50. An anti-GREM1 antagonist for use according to embodiment 44, wherein the MEK inhibitor is WX-554 (UCB-554). 51. An anti-GREM1 antagonist for use according to aspect 44, wherein the MEK inhibitor is selumetinib (AZD6244) and the cancer is pancreatic cancer, optionally PDAC. 52. An anti-GREM1 antagonist for use according to aspect 44, wherein the MEK inhibitor is trametinib (GSK-1120212) and the cancer is pancreatic cancer, optionally PDAC. 53. An anti-GREM1 antagonist for use according to aspect 44, wherein the MEK inhibitor is WX-554 (UCB-554) and the cancer is pancreatic cancer, optionally PDAC. 54. An anti-GREM1 antagonist for use according to any one of aspects 1 to 41, wherein the inhibitor of Ras-Raf-MEK-ERK signaling is an ERK inhibitor. 55. An anti-GREM1 antagonist for use according to aspect 54, wherein the ERK inhibitor is ulixertinib, temtelkib, JSI-1187, ravoxertinib, ASN007 or MK-8353. 56. An anti-GREM1 antagonist for use according to aspect 54 or 55, wherein the ERK inhibitor is ulixertinib. 57. An anti-GREM1 antagonist for use according to embodiment 56, wherein the ERK inhibitor is ulixertinib and the cancer is pancreatic cancer, optionally PDAC. 58. The anti-GREM1 antagonist for use according to any one of the preceding aspects, wherein the method further comprises administering an additional anti-cancer agent. 59. An inhibitor of Ras-Raf-MEK-ERK signaling for use in a method for treating or preventing cancer, the method further comprising administering an anti-GREM1 antagonist. 60. An inhibitor of Ras-Raf-MEK-ERK for use according to aspect 59, wherein said cancer, said antagonist and / or said method are as defined in any one of aspects 1 to 58. 61. An inhibitor of Ras-Raf-MEK-ERK signaling for use according to embodiment 59 or 60, wherein the inhibitor of Ras-Raf-MEK-ERK signaling is a MEK inhibitor. 62. An inhibitor of Ras-Raf-MEK-ERK signaling for use according to embodiment 61, wherein said MEK inhibitor is selumetinib (AZD6244). 63. An inhibitor of Ras-Raf-MEK-ERK signaling for use according to embodiment 61, wherein said MEK inhibitor is trametinib (GSK-1120212). 64. The inhibitor of Ras-Raf-MEK-ERK signaling for use according to aspect 61, wherein said MEK inhibitor is WX-554 (UCB-554). 65. An inhibitor of Ras-Raf-MEK-ERK signaling for use according to embodiment 59 or 60, wherein the inhibitor of Ras-Raf-MEK-ERK signaling is an ERK inhibitor. 66. An inhibitor of Ras-Raf-MEK-ERK signaling for use according to embodiment 65, wherein said ERK inhibitor is ulixertinib. 67. A method for treating cancer, comprising administering to a subject in need thereof a therapeutically effective amount of an anti-GREM1 antagonist in combination with a therapeutically effective amount of an inhibitor of Ras-Raf-MEK-ERK signaling. 68. The method according to aspect 67, wherein said cancer, said antagonist and / or said method are as defined in any one of aspects 1 to 66. 69. A composition or kit comprising an anti-GREM1 antagonist and an inhibitor of Ras-Raf-MEK-ERK signaling. 70. The composition or kit according to aspect 69, wherein the anti-GREM1 antagonist is as defined in any one of aspects 20 to 39. 71. The composition or kit according to embodiment 69 or 70, wherein the inhibitor of Ras-Raf-MEK-ERK signaling is as defined in any one of embodiments 41 to 57. 72. A method for determining whether a patient suffering from or suspected of suffering from cancer, or at risk of developing cancer, is likely to respond to combined treatment with a GREM1 antagonist and an inhibitor of Ras-Raf-MEK-ERK signaling, comprising measuring stromal expression of GREM1, epithelial expression of GREM1, and / or Ras-Raf-MEK-ERK pathway signaling in the patient, thereby predicting whether the patient is likely to respond to the combined treatment. 73. A method for determining whether a patient suffering from or suspected of suffering from cancer, or at risk of developing cancer, is likely to respond to a combination treatment with a GREM1 antagonist and an inhibitor of Ras-Raf-MEK-ERK signaling, comprising measuring stromal expression of GREM1 and / or epithelial expression of GREM1 in the patient, determining whether the patient has a mutation in the RAS gene or RAF gene, thereby predicting whether the patient is likely to respond to the combination treatment. 74. A method for determining whether a patient suffering from or suspected of suffering from cancer, or at risk of developing cancer, is likely to respond to combined treatment with a GREM1 antagonist and an inhibitor of Ras-Raf-MEK-ERK signaling, comprising measuring the patient's Ras-Raf-MEK-ERK pathway signaling in response to treatment with an anti-GREM1 antagonist, thereby predicting whether the patient is likely to respond to the combined treatment.

[0327] [Sequence table] SEQ ID NO:1 (human gremlin-1; Uniprot ID: O60565) MSRTAYTVGALLLLLGTLLPAAEGKKKGSQGAIPPPDKAQHNDSEQTQSPQQPGSRNRRGGQGRGTAMPGEEVLESSQEALHVTERKYLKRDWCKTQPLKQTIHEEGCNSRTIINRFCYGQCNSFYIPRHIRKEEGSFQSCSFCKPKKFTTMMVTLNCPELQPPTKKKRVTRVKQCRCISIDLD SEQ ID NO:2 (N-terminally tagged human truncated Gremlin-1 used for crystal structure analysis) MGSSHHHHHHSSGENLYFQGSAMPGEEVLESSQEALHVTERKYLKRDWCKTQPLKQTIHEEGCNSRTIINRFCYGQCNSFYIPRHIRKEEGSFQSCSFCKPKKFTTMMVTLNCPELQPPTKKKRVTRVKQCRCISIDLD SEQ ID NO:3 (Ab7326 HCDR1 Kabat & Chothia composite) GYTFTDYYMH SEQ ID NO:4 (Ab7326 HCDR1 Kabat) DYYMH SEQ ID NO:5 (Ab7326 HCDR2 Kabat) LVDPEDGETIYAEKFQG SEQ ID NO:6 (Ab7326 HCDR3 Kabat) DARGSGSYYPNHFDY SEQ ID NO:7 (Ab7326 LCDR1 Kabat) KSSQSVLYSSNNKNYLA SEQ ID NO:8 (Ab7326 LCDR2 Kabat) WASTRES SEQ ID NO:9 (Ab7326 LCDR3 Kabat) QQYYDTPT SEQ ID NO: 10 (Ab7326 heavy chain modified region variant 1) QVQLVESGAEVKKPGATVKISCKVSGYTFTDYYMHWVQQAPGKGLEWMGLVDPEDGETIYAEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCATDARGSGSYYPNHFDYWGQGTLVTVSS SEQ ID NO: 11 (Ab7326 light chain modified region variant 1) DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTINSLQAEDVAVYFCQQYYDTPTFGQGTRLEIK SEQ ID NO: 12 (Ab7326 heavy chain modified region variant 2) QVQLVQSGAEVKKPGATVKISCKVSGYTFTDYYMHWVQQAPGKGLEWMGLVDPEDGETIYAEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCATDARGSGSYYPNHFDYWGQGTLVTVSS SEQ ID NO: 13 (Ab7326 light chain modified region variant 2) DIVMTQTPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTINSLQAEDVAVYFCQQYYDTPTFGQGTRLEIK SEQ ID NO: 14 (Mouse full length IgG1 heavy chain variant 1) QVQLVESGAEVKKPGATVKISCKVSGYTFTDYYMHWVQQAPGKGLEWMGLVDPEDGETIYAEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCATDARGSGSYYPNHFD YWGQGTLVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPR DCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKT ISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK SEQ ID NO: 15 (Mouse full length IgG1 light chain variant 1) DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTINSLQAEDVAVYFCQQYYDTPTFGQGTRL EIKRTDAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC SEQ ID NO: 16 (human full length IgG1 heavy chain variant 2) QVQLVQSGAEVKKPGATVKISCKVSGYTFTDYYMHWVQQAPGKGLEWMGLVDPEDGETIYAEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCATDARGSGSYYPNHFDY WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 17 (human full length IgG1 light chain variant 2) DIVMTQTPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTINSLQAEDVAVYFCQQYYDTPTFGQGTRL EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 18 (Fab heavy chain variant 1) QVQLVESGAEVKKPGATVKISCKVSGYTFTDYYMHWVQQAPGKGLEWMGLVDPEDGETIYAEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCATDARGSGSYYPNHFDY WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC SEQ ID NO: 19 (Fab light chain variant 1) DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTINSLQAEDVAVYFCQQYYDTPTFGQGTRL EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO:20 (N-terminally tagged human truncated Gremlin-1 used for crystal structure analysis) AMPGEEVLESSQEALHVTERKYLKRDWCKTQPLKQTIHEEGCNSRTIINRFCYGQCNSFYIPRHIRKEEGSFQSCSFCKPKKFTTMMVTLNCPELQPPTKKKRVTRVKQCRCISIDLD SEQ ID NO:21 (mature Gremlin-1 sequence of SEQ ID NO:1 lacking the signal peptide of amino acids 1 to 21) KKKGSQGAIPPPDKAQHNDSEQTQSPQQPGSRNRRGGQGRGTAMPGEEVLESSQEALHVTERKYLKRDWCKTQPLKQTIHEEGCNSRTIINRFCYGQCNSFYIPRHIRKEEGSFQSCSFCKPKKFTTMMVTLNCPELQPPTKKKRVTRVKQCRCISIDLD SEQ ID NO: 22 (Human IgG4P heavy chain variant 1) QVQLVESGAEVKKPGATVKISCKVSGYTFTDYYMHWVQQAPGKGLEWMGLVDPEDGETIYAEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCATDARGSGSYYPNHFD YWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESK YGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEK TISKAKGQPREPQVYTLPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK SEQ ID NO: 23 (human IgG4P light chain variant 1) DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTINSLQAEDVAVYFCQQYYDTPTFGQGTRL EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 24 (Human IgG1 heavy chain DNA variant 1) SEQ ID NO: 25 (Human IgG1 kappa chain DNA variant 1) gacattgtgatgacccagtcccccgattcgcttgcggtgtccctgggagaacgggccaccattaactgcaagagctcacagtccgtcctgtattcatcgaacaacaagaattacctcgcatggtatcagcagaagcctggacagcctcccaagctgctcatctactgggctagcacccgcgaatccggggtgccggatagattctccggatcgggttcgggcactgacttcactctgactatcaactcactgcaagccgaggatgtcgcggtgtacttctgtcagcagtactacgacaccccgacctttggacaaggcaccagactggagattaagcgtacggtggccgctccctccgtgttcatcttcccaccctccgacgagcagctgaagtccggcaccgcctccgtcgtgtgcctgctgaacaacttctacccccgcgaggccaaggtgcagtggaaggtggacaacgccctgcagtccggcaactcccaggaatccgtcaccgagcaggactccaaggacagcacctactccctgtcctccaccctgaccctgtccaaggccgactacgagaagcacaaggtgtacgcctgcgaagtgacccaccagggcctgtccagccccgtgaccaagtccttcaaccggggcgagtgc SEQ ID NO: 26 (Human IgG4 heavy chain DNA variant 1) SEQ ID NO: 27 (Human IgG4 P kappa light chain DNA variant 1) gacattgtgatgacccagtcccccgattcgcttgcggtgtccctgggagaacgggccaccattaactgcaagagctcacagtccgtcctgtattcatcgaacaacaagaattacctcgcatggtatcagcagaagcctggacagcctcccaagctgctcatctactgggctagcacccgcgaatccggggtgccggatagattctccggatcgggttcgggcactgacttcactctgactatcaactcactgcaagccgaggatgtcgcggtgtacttctgtcagcagtactacgacaccccgacctttggacaaggcaccagactggagattaagcgtacggtggccgctccctccgtgttcatcttcccaccctccgacgagcagctgaagtccggcaccgcctccgtcgtgtgcctgctgaacaacttctacccccgcgaggccaaggtgcagtggaaggtggacaacgccctgcagtccggcaactcccaggaatccgtcaccgagcaggactccaaggacagcacctactccctgtcctccaccctgaccctgtccaaggccgactacgagaagcacaaggtgtacgcctgcgaagtgacccaccagggcctgtccagccccgtgaccaagtccttcaaccggggcgagtgc SEQ ID NO: 28 (Mouse full-length IgG1 heavy chain variant 2) QVQLVQSGAEVKKPGATVKISCKVSGYTFTDYYMHWVQQAPGKGLEWMGLVDPEDGETIYAEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCATDARGSGSYYPNHFD YWGQGTLVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPR DCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKT ISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK SEQ ID NO: 29 (Mouse full length IgG1 light chain variant 2) DIVMTQTPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTINSLQAEDVAVYFCQQYYDTPTFGQGTRL EIKRTDAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC SEQ ID NO: 30 (human full length IgG1 heavy chain variant 1) QVQLVESGAEVKKPGATVKISCKVSGYTFTDYYMHWVQQAPGKGLEWMGLVDPEDGETIYAEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCATDARGSGSYYPNHFDY WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 31 (human full length IgG1 light chain variant 1) DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTINSLQAEDVAVYFCQQYYDTPTFGQGTRL EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO:32 (Fab heavy chain variant 2) QVQLVQSGAEVKKPGATVKISCKVSGYTFTDYYMHWVQQAPGKGLEWMGLVDPEDGETIYAEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCATDARGSGSYYPNHFDY WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC SEQ ID NO: 33 (Fab light chain variant 2) DIVMTQTPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTINSLQAEDVAVYFCQQYYDTPTFGQGTRL EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 34 (Human IgG4P heavy chain variant 2) QVQLVQSGAEVKKPGATVKISCKVSGYTFTDYYMHWVQQAPGKGLEWMGLVDPEDGETIYAEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCATDARGSGSYYPNHFD YWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESK YGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEK TISKAKGQPREPQVYTLPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK SEQ ID NO: 35 (human IgG4P light chain variant 2) DIVMTQTPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTINSLQAEDVAVYFCQQYYDTPTFGQGTRL EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO:36 (human gremlin-1; full length) SEQ ID NO:37 (human gremlin-1; coding sequence) Atgagccgcacagcctacacggtgggagccctgcttctcctcttggggaccctgctgccggctgctgaagggaaaaagaaagggtcccaaggtgccatccccccgccagacaaggcccagcacaatgactcagagcag actcagtcgccccagcagcctggctccaggaaccgggggcggggccaagggcggggcactgccatgcccggggaggaggtgctggagtccagccaagaggccctgcatgtgacggagcgcaaatacctgaagcgagact ggtgcaaaacccagccgcttaagcagaccatccacgaggaaggctgcaacagtcgcaccatcatcaaccgcttctgttacggccagtgcaactctttctacatccccaggcacatccggaaggaggaaggttcctttca gtcctgctccttctgcaagcccaagaaattcactaccatgatggtcacactcaactgccctgaactacagccacctaccaagaagaagagagtcacacgtgtgaagcagtgtcgttgcatatccatcgatttggattaa

[0328] References Hingorani, SR, L. Wang, AS Multani, C. Combs, TB Deramaudt, RH Hruban, et al. Trp53R172H and KrasG12D cooperate to promote chromosomal instability and widely metastatic pancreatic ductal adenocarcinoma in mice Cancer Cell 2005;7:469-83. Kobayashi, Y., SO Lim and H. Yamaguchi. Oncogenic signaling pathways associated with immune evasion and resistance to immune checkpoint inhibitors in cancer Semin Cancer Biol;2020;65:51-64. Kozar, I., C. Margue, S. Rothengatter, et al. Many ways to resistance: How melanoma cells evade targeted therapies Biochim Biophys Acta Rev Cancer;2019;1871(2):313-322. Martinelli, E., F. Morgillo, T. Troiani, et al. Cancer resistance to therapies against the EGFR-RAS-RAF pathway: The role of MEK Cancer Treat Rev;2017;53:61-69. Yu, Y., L. Cheng, B. Yan, et al. Overexpression of Gremlin 1 by sonic hedgehog signaling promotes pancreatic cancer progression Int J Oncol;2018;53(6):2445-2457).

Claims

1. A drug for treating or preventing cancer, comprising an anti-gremlin 1 antibody, (a) The anti-gremlin 1 antibody comprises the combination of the HCDR1 / HCDR2 / HCDR3 / LCDR1 / LCDR2 / LCDR3 sequences of SEQ ID NO: 4 / 5 / 6 / 7 / 8 / 9 or SEQ ID NO: 3 / 5 / 6 / 7 / 8 / 9; and (b) The drug described above, which is used in combination with a MEK inhibitor.

2. (a) The cancer is a solid tumor; (b) The cancer has stromal GREM1 overexpression; (c) The cancer is characterized by a dysregulation of the Ras-Raf-MEK-ERK pathway; (d) After exposure to an anti-GREM1 antagonist, cancer showed dysregulation of the Ras-Raf-MEK-ERK pathway; (e) The cancer contains mutations in the Ras gene, and possibly in KRAS, NRAS and / or HRAS; (f) The cancer contains mutations in the Raf gene; possibly in ARAF, BRAF and / or CRAF; (g) The cancer is unresponsive, unresponsive, or refractory to treatment with Ras-Raf-MEK-ERK signaling inhibitors; and / or (h) The agent according to claim 1, wherein the cancer is selected from colorectal cancer, multiple myeloma, pancreatic cancer, bladder cancer, breast cancer, lung cancer, stomach cancer, duodenal cancer, esophageal cancer, head and neck cancer, prostate cancer, glioma, endometrial cancer, liver cancer, splenic cancer, bone cancer, melanoma, and osteosarcoma.

3. (a) The cancer is pancreatic cancer; in some cases, (i) the pancreatic cancer is exocrine pancreatic cancer; and / or (ii) The pancreatic cancer is pancreatic ductal adenocarcinoma (PDAC); (b) The cancer is multiple myeloma or colorectal cancer; and possibly KRAS / BRAF mutant colorectal cancer; (c) the cancer is lung cancer; in some cases the cancer is non-small cell lung cancer (NSCLC); or (d) The agent according to claim 2(h), wherein the cancer is melanoma, and in some cases is BRAF-mutated melanoma.

4. (a) The cancer has epithelial GREM1 overexpression; and in some cases the cancer is GREM1-induced; (b) the cancer is disseminated; and / or (c) The drug according to claim 1, wherein the cancer is an established cancer.

5. The anti-gremlin-1 antibody comprises the heavy chain variable region (HCVR) sequence of SEQ ID NO: 10 or 12, and / or the light chain variable region (LCVR) sequence of SEQ ID NO: 11 or 13, or a sequence that is at least 95% identical thereto. The drug according to claim 1.

6. (a) The anti-gremlin-1 antibody comprises the HCVR and LCVR sequence pair of SEQ ID NOs. 10 / 11 or 12 / 13, or a sequence that is at least 95% identical thereto; optionally, the anti-gremlin-1 antibody comprises the HCDR1 / HCDR2 / HCDR3 / LCDR1 / LCDR2 / LCDR3 sequence of SEQ ID NOs. 4 / 5 / 6 / 7 / 8 / 9 or SEQ ID NOs. 3 / 5 / 6 / 7 / 8 / 9: the remainder of the HCVR and LCVR comprises at least 95% identity with SEQ ID NOs. 10, 11, 12 and / or 13, respectively; and / or (b) The anti-gremlin-1 antibody comprises the heavy chain of SEQ ID NO: 14, 16, 18, 22, 28, 30, 32, or 34, and / or the light chain of SEQ ID NO: 15, 17, 19, 23, 29, 31, 33, or 35, or a sequence that is at least 95% identical thereto. The drug according to claim 1.

7. The anti-gremlin-1 antibody contains a heavy-chain and light-chain pair of sequence numbers 14 / 15, 16 / 17, 18 / 19, 22 / 23, 28 / 29 or 30 / 31, 32 / 33, 34 / 35, or a sequence that is at least 95% identical thereto; optionally, the HCDR1 / HCDR2 / HCDR3 / LCDR1 / LCDR2 / LCDR3 sequence of the antibody consists of sequence numbers 4 / 5 / 6 / 7 / 8 / 9 or 3 / 5 / 6 / 7 / 8 / 9, and the remainder of the heavy-chain and light-chain each contains at least 95% identity with sequence numbers 14, 15, 16 and / or 17. The drug according to claim 6(b)

8. (a) The antibody is a chimeric antibody, a human antibody, or a humanized antibody; and / or (b) Antibodies are Fab, modified Fab, Fab', modified Fab', F(ab') ２ , Fv, or scFv, The drug according to claim 1.

9. The agent according to claim 1, wherein the antibody is contained in a pharmaceutical composition further comprising a pharmaceutically acceptable adjuvant and / or carrier.

10. (a) The MEK inhibitor is a MEK1 and / or MEK2 inhibitor; and / or (b) MEK inhibitors include WX-554 (UCB-554), selumetinib (AZD6244), cobimetinib (GDC-0973), trametinib (GSK-1120212), binimetinib (MEK162), CI-1040 (PD184352), mildametinib (PD0325901), TAK733, refametinib (RDEA119 / Bay86-9766), RO-5126766, RO5068760, Pimasertib (AS703026), AZD8330, GDC-0623, RO-4987655, HL-085, ARRY-300, ClnQ-03, G-573, PD184161, PD318088, PD98059, U0126 or SL327; in some cases (i) The MEK inhibitor is selumetinib (AZD6244); (ii) The MEK inhibitor is trametinib (GSK-1120212); (iii) The MEK inhibitor is selumetinib (AZD6244), and the cancer is pancreatic cancer; possibly PDAC; or (iv) The MEK inhibitor is trametinib (GSK-1120212), and the cancer is pancreatic cancer; in some cases, it is PDAC. The drug according to claim 1.

11. The agent according to claim 1, wherein the MEK inhibitor is cobimetinib (GDC-0973).

12. The agent according to claim 1, wherein the MEK inhibitor is cobimetinib (GDC-0973), and the cancer is pancreatic cancer, and optionally PDAC.

13. The agent according to claim 1, wherein the MEK inhibitor is WX-554 (UCB-554).

14. The agent according to claim 1, wherein the MEK inhibitor is WX-554 (UCB-554), and the cancer is pancreatic cancer, and optionally PDAC.

15. (A) MEK inhibitor, (a) N-(2-fluoro-4-iodophenyl)-3-(morpholine-4-ylcarbonyl)thieno[2,3-b]pyridine-2-amine; (b) N-(2-fluoro-4-iodophenyl)-3-[(4-methylpiperazine-1-yl)carbonyl]thieno[2,3-b]pyridine-2-amine; (c) [2-[((2-fluoro-4-iodophenyl)amino]thieno[2,3-b]pyridine-3-yl](pyrrolidine-1-yl)methanone; (d) (1-{2-[(2-fluoro-4-iodophenyl)amino]thieno[2,3-b]pyridine-3-carbonyl}piperidine-4-yl)-carbamate tert-butyl ester; (e) 2-[((2-fluoro-4-iodophenyl)amino]thieno[2,3-b]pyridine-3-carboxylic acid (4-aminopiperidine-1-yl)amide dihydrochloride; (f) (1-{2-[(2-fluoro-4-iodophenyl)amino]thieno[2,3-b]pyridine-3-carbonyl}azetidine-3-ylmethyl)carbamate tert-butyl ester; (g) [3-((aminomethyl)azetidine-1-yl]-{2-[(2-fluoro-4-iodophenyl)amino]thieno[2,3-b]pyridine-3-yl}methanone; (h) (1-{2-[(2-fluoro-4-iodophenyl)amino]thieno[2,3-b]pyridine-3-carbonyl}-(3R)-pyrrolidine-3-yl)carbamate tert-butyl ester; (i) (1-{2-[(2-fluoro-4-iodophenyl)amino]thieno[2,3-b]pyridine-3-carbonyl}-(3S)-pyrrolidine-3-yl)carbamate tert-butyl ester; (j) (1-{2-[(2-fluoro-4-iodophenyl)amino]thieno[2,3-b]pyridine-3-carbonyl}azetidine-3-yl)-carbamate tert-butyl ester; (k) [(3R)-3-aminopyrrolidine-1-yl]-{2-[(2-fluoro-4-iodophenyl)amino]thieno[2,3-b]pyridine-3-yl}methanone; (l) [(3S)-3-aminopyrrolidine-1-yl]-{2-[(2-fluoro4-iodophenyl)amino]thieno[2,3-b]pyridine-3-yl}methanone; (m) (3-aminoazetidine-1-yl)-{2-[(2-fluoro-4-iodophenyl)amino]thieno[2,3-b]pyridine-3-yl]-methanone; (n) {2-[((2-fluoro-4-iodophenyl)amino]thieno[2,3-b]pyridine-3-yl}-(3-hydroxyazetidine-1-yl)-methanone; (o) {2-[((2-fluoro-4-iodophenyl)amino]thieno[2,3-b]pyridine-3-yl}-[(3R)-3-hydroxypyrrolidine-1-yl]-methanone; (p) {2-[(2-fluoro-4-iodophenyl)amino]thieno[2,3-b]pyridine-3-yl}-[(3S)-3-hydroxypyrrolidine-1-yl]-methanone; (q) {2-[(2-fluoro-4-iodophenyl)amino]thieno[2,3-b]pyridine-3-yl}-[2-(hydroxymethyl)-piperidine-1-yl]-methanone; (r) {2-[((2-fluoro-4-iodophenyl)amino]thieno[2,3-b]pyridine-3-yl}-[(3S)-3-(hydroxymethyl)-morpholine-4-yl]-methanone; (s) 4-{2-[(2-fluoro-4-iodophenyl)amino]thieno[2,3-b]pyridine-3-carbonyl}-(3R)-3-(hydroxymethyl)piperazine-1-carboxylate tert-butyl ester; (t) 2-[4({2-[(2-fluoro-4-iodophenyl)amino]thieno[2,3-b]pyridine-3-yl}carbonyl)piperazine-1-yl]ethanol; (u) 3-((1,4-diazepan-1-ylcarbonyl)-N-(2-fluoro-4-iodophenyl)thieno[2,3-b]pyridine-2-amine; (v) N-(2-fluoro-4-iodophenyl)-3-(piperazine-1-ylcarbonyl)thieno[2,3-b]pyridine-2-amine; (w) Ethyl [4-({2-[(2-fluoro-4-iodophenyl)amino]thieno[2,3-b]pyridine-3-yl}carbonyl)-piperazine-1-yl]acetate; (x) [4-({2-[(2-fluoro-4-iodophenyl)amino]thieno[2,3-b]pyridine-3-yl}carbonyl)piperazine-1-yl]acetic acid; (y) {2-[((2-fluoro-4-iodophenyl)amino]thieno[2,3-b]pyridine-3-yl}-[(2R)-2-(methoxymethyl)-pyrrolidine-1-yl]-methone; and (z) {2-[((2-fluoro-4-iodophenyl)amino]thieno[2,3-b]pyridine-3-yl}-[(2R)-2-(hydroxymethyl)-piperazine-1-yl]-methanone Selected from the group consisting of, (B) MEK inhibitors, formula (II): 【Chemistry 1】 [In the formula, R 12 represents halogen; R3 represents CONR b R c; Rb and Rc, when together with the nitrogen atom to which they are bonded, represent azetidine-1-yl, pyrrolidine-1-yl, piperidine-1-yl, morpholine-4-yl, thiomorpholine-4-yl, piperazine-1-yl, homopiperidine-1-yl, homomorpholine-4-yl, or homopiperazine-1-yl, and either of these groups may represent C1-6 alkyl, C1-6 alkoxy, hydroxy, hydroxy(C1-6)alkyl, amino-(C1-6)alkyl, (amino)(hydroxy)(C1-6)alkyl, halogen, oxo, C2-6 alkylcarbonyl, carboxy, C2-6 alkoxycarbonyl, di(C1-6)alkylhydrazinylcarbonyl, amino, C1-6 alkylamino, di(C1-6)alkylamino, C2-6 They may be substituted with one or more substituents selected from alkylcarbonylamino, aminocarbonylamino, aminocarbonyl, C1-6 alkylaminocarbonyl, di(C1-6)alkylaminocarbonyl, aminosulfonyl, C1-6 alkylaminocarbonyl(C1-6)alkyl, C1-6 alkoxy(C1-6)alkyl, carboxy(C1-6)alkyl, C2-6 alkoxycarbonyl(C1-6)alkyl, C2-6 alkoxycarbonylamino and C2-6 alkoxycarbonylamino-(C1-6)alkyl; Depending on the context, R 12 may represent bromo or iodine. The agent according to claim 1, which is represented by and a pharmaceutically acceptable salt, solvate, and N-oxide thereof.

16. The agent according to claim 1, used in combination with an additional anticancer agent.

17. A drug for treating or preventing cancer, comprising a polynucleotide or an expression vector having the polynucleotide, (a) The polynucleotide encodes an antibody as defined in any one of claims 1 and 5 to 8, (b) The agent is used in combination with a MEK inhibitor, or optionally a MEK inhibitor as defined in any one of claims 10 to 15.

18. A drug for treating or preventing cancer, comprising a MEK inhibitor, which is used in combination with an anti-gremlin 1 antibody as defined in claim 1, a polynucleotide encoding the antibody, or an expression vector having the polynucleotide.

19. The cancer, the anti-gremlin 1 antibody, and / or the treatment or prevention are as defined in any one of claims 2 to 16, and optionally, (a) The MEK inhibitor, and optionally the cancer, is as defined in claims 10 to 15, or (b) The MEK inhibitor is (i) Is it WX-554 (UCB-554)? (ii) Is it selumetinib (AZD6244)? (iii) Cobimetinib (GDC-0973), or The agent according to claim 18, wherein (iv) trametinib (GSK-1120212).

20. A composition or kit comprising an anti-gremlin 1 antibody and a MEK inhibitor, wherein the anti-gremlin 1 antibody is as defined in any one of claims 1 and 5 to 8, and optionally the MEK inhibitor is as defined in claim 10.

21. (a) A method for determining whether a patient who has cancer, is suspected of having cancer, or is at risk of developing cancer is likely to respond to combination therapy with an anti-gremlin 1 antibody and a MEK inhibitor, comprising measuring the interstitial expression of GREM1, the epithelial expression of GREM1, and / or Ras-Raf-MEK-ERK pathway signaling in a sample obtained from the patient, thereby predicting whether the patient is likely to respond to the combination therapy; or (b) A method for determining whether a patient who has cancer, is suspected of having cancer, or is at risk of developing cancer is likely to respond to combination therapy with an anti-gremlin 1 antibody and a MEK inhibitor, comprising measuring the interstitial expression and / or epithelial expression of GREM1 in a sample obtained from the patient, determining whether the patient has a mutation in the RAS gene or the RAF gene, and thereby predicting whether the patient is likely to respond to the combination therapy; or (c) A method for determining whether a patient who has cancer, is suspected of having cancer, or is at risk of developing cancer is likely to respond to combination therapy with an anti-gremlin 1 antibody and a MEK inhibitor, comprising measuring Ras-Raf-MEK-ERK signaling pathway signaling in a sample obtained from the patient in response to treatment with an anti-GREM1 antagonist, thereby predicting whether the patient is likely to respond to the combination therapy. The method wherein the anti-gremlin 1 antibody is as defined in any one of claims 1 and 5 to 8, and optionally the MEK inhibitor is as defined in claim 9 or 10.

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