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

Abstract

The present invention relates to an anti-GREM1 antagonist in combination with a Ras-Raf-MEK-ERK signaling inhibitor for use in a method of treating or preventing cancer.

Description

Field of the Invention

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

[0003] Mitogen-activated protein kinase (MAPK) signal transduction pathways (such as Ras-Raf-MEK-ERK signal transduction pathways) play a key role in cell survival, proliferation and differentiation. Due to uncontrolled cell proliferation, dysregulated signal transduction in these pathways is associated with various diseases including cancer. In addition, it has been reported that upregulated signal transduction in the MAPK pathway is a resistance mechanism in response to a variety of anticancer 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 signal transduction pathway, particularly the Ras-Raf-MEK-ERK signal transduction pathway, such as MEK and ERK inhibitors, have been described for the treatment of cancer. For example, MEK inhibitors have been approved for the treatment of BRAF-mutated melanoma and KRAS / BRAF-mutated colorectal cancer. However, acquired resistance is common in patients treated with inhibitors of the MAPK signaling pathway, such as the Ras-Raf-MEK-ERK signaling pathway. Therefore, there is a need to develop new therapies for the treatment and prevention of cancer. There is also a need for combination therapies that can improve the efficacy of existing MAPK pathway inhibitors.

[0004] One cancer that presents a huge challenge is pancreatic cancer. Pancreatic cancer is a deadly disease with an increasing incidence in recent years. Environmental risk factors associated with pancreatic cancer include smoking, pancreatitis, alcohol consumption, obesity, infection and diet, which means that pancreatic cancer is becoming increasingly common in developed countries. Surgical resection (pancreaticoduodenectomy) combined with chemotherapy is the only long-term potential treatment for pancreatic cancer. However, limited screening coupled with the lack of symptoms means that patients are often diagnosed with advanced disease at a later stage, limiting the opportunity for surgical resection. As a result, pancreatic cancer has one of the lowest survival rates of all common cancers, with a 5-year survival rate of less than 7% and a survival rate of only 5% for 10 years or more. Over the past 40 years, there has been limited improvement in survival rates, which has highlighted the urgent need for new therapeutic treatments for pancreatic cancer. In unresectable tumors, the standard of care for pancreatic cancer is a chemotherapy regimen. However, chemotherapy is associated with side effects, and cancer recurrence is common. Due to the late diagnosis of pancreatic cancer, poor prognosis and limited treatment strategies, improved therapies are needed. SUMMARY OF THE INVENTION

[0006] The inventors have demonstrated that genes involved in Ras-Raf-MEK-ERK signaling, such as NRas, Myc, and Mapk1, are upregulated in LSL-Kras upon exposure to anti-GREM1 antagonists, such as Ab7326. G12D / + , LSL-Trp53 R172H / + , Pdx1-Cre (KPC) mouse model. In addition, the inventors have unexpectedly shown that GREM1 antagonists can be advantageously administered in combination with Ras-Raf-MEK-ERK signaling inhibitors (such as MEK inhibitors) for the treatment or prevention of pancreatic cancer. Based on these results, the inventors envision that combination therapies comprising GREM1 antagonists and Ras-Raf-MEK-ERK signaling inhibitors (such as MEK inhibitors and ERK inhibitors) will have general utility in the treatment and prevention of cancers in which Ras-Raf-MEK-ERK signaling is induced / upregulated after treatment with anti-GREM1 antagonists. In addition, based on these results, the inventors envision that combination therapies comprising GREM1 antagonists and Ras-Raf-MEK-ERK signaling inhibitors (such as MEK and ERK inhibitors) will have general utility in the treatment and prevention of cancers associated with dysregulation of the Ras-Raf-MEK-ERK pathway or cancers containing Ras or Raf gene mutations. The in vivo results presented here demonstrate that GREM1 antagonists, administered in combination with MEK inhibitors, inhibit the expression of GREM1 in LSL-Kras G12D / + , LSL-Trp53 R172H / +The survival rate in the Pdx1-Cre(KPC) mouse model was significantly improved, and tumor growth was slowed and tumors were reduced. The inventors' discovery provides an improved solution for the prevention and treatment of cancer, especially pancreatic cancer.

[0007] Thus, in a first aspect of the present invention, there is provided an anti-GREMl antagonist for use in a method of treating or preventing cancer, wherein the method further comprises administering a Ras-Raf-MEK-ERK signalling inhibitor.

[0008] In another aspect of the present invention, there is provided a Ras-Raf-MEK-ERK signaling inhibitor for use in a method for treating or preventing cancer, wherein the method further comprises administering an anti-GREM1 antagonist.

[0009] In another aspect of the invention, a method of treating cancer is provided, 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 a Ras-Raf-MEK-ERK signaling inhibitor.

[0010] In yet another aspect of the present invention, a composition or kit comprising an anti-GREM1 antagonist and a Ras-Raf-MEK-ERK signaling inhibitor is provided.

[0011] In another aspect of the invention, a method is provided for determining whether a patient having or suspected of having cancer or at risk of developing cancer is likely to respond to a combination treatment with a GREM1 antagonist and a Ras-Raf-MEK-ERK signaling inhibitor, the method comprising measuring Ras-Raf-MEK-ERK signaling in the patient in response to anti-GREM1 antagonist treatment, and thereby predicting whether the patient is likely to respond to treatment with the combination.

[0012] In another aspect of the present invention, a method is provided for determining whether a patient having or suspected of having cancer or at risk of developing cancer is likely to respond to a combination treatment with a GREM1 antagonist and a Ras-Raf-MEK-ERK signaling inhibitor, the method comprising measuring the mesenchymal expression of GREM1, the epithelial expression of GREM1 and / or Ras-Raf-MEK-ERK signaling in the patient, and thereby predicting whether the patient is likely to respond to treatment with the combination.

[0013] In yet another aspect of the present invention, a method is provided for determining whether a patient having or suspected of having cancer or at risk of developing cancer is likely to respond to a combination treatment with a GREM1 antagonist and a Ras-Raf-MEK-ERK signaling inhibitor, the method comprising measuring the mesenchymal expression of GREM1 and / or the epithelial expression of GREM1 in the patient, and determining whether the patient has a RAS gene or RAF gene mutation, and thereby predicting whether the patient is likely to respond to treatment with the combination.

[0014] BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 - Kaplan-Meier analysis showing the survival of PDAC patients with gremlin-1 expression above or below the median shown. Patients with tumors expressing high levels of gremlin 1 had a significantly worse prognosis compared to patients with low expression. Generated from KMplotter.

[0016] Figure 2 - Schematic diagram showing (a) the breeding strategy and (b) the experimental design.

[0017] Figure 3 - Gene expression of 46 genes in the KPC mouse model analyzed by NanoString technology. A. Unadjusted p-values ​​depicted in a volcano plot. B. Linear model for Nras expression. C. Linear model for Myc expression. D. Linear model for Mapk1 expression.

[0018] Figure 4 - Pathway enrichment showing the effect of Ab7326 mIgG1 exposure on the KRAS pathway.

[0019] Figure 5 - The figure shows the tumor burden measured by high-resolution ultrasound in individual mice treated as indicated in the pharmacodynamic studies.

[0020] Figure 6 - This figure shows tumor burden measured by high-resolution ultrasound in individual mice receiving treatment (as indicated) in the survival study cohort.

[0021] Figure 7 - Kaplan-Meier analysis showing Pdx1-Cre, LSL-Kras from the start of treatment with Ab7326 mIgG1 (n=9), selumetinib (n=8), or Ab7326 mIgG1 in combination with selumetinib (n=9) as indicated G12D / + , LSL-Trp53 R172H / +(KPC) Survival rate of mice.

[0022] Figure 8 - Graphs showing IHC scoring (analyzed by HALO software) using antibodies against markers of cancer-associated fibroblasts (aSMA and Podoplanin) and Sirius red staining for collagens I and III in mice treated as indicated.

[0023] Fig. 9 a- Kaplan-Meier analysis showing the Pdx1-Cre, LSL-Kras, and 4-HRP-positive controls starting with treatment with vehicle control (n=10), Ab7326 mlgG1 (n=10), UCB-554 (n=10), or Ab7326 mlgG1 in combination with UCB-554 (n=11) as indicated. G12D / + , LSL-Trp53 R172H / + (KPC) Survival rate of mice.

[0024] Fig. 9 b-Box plot showing tumor volume measured by high-resolution ultrasound on day 7 after the start of treatment 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 depicts the interquartile range, and the line depicts the minimum and maximum observed values. The solid horizontal bar represents the median, and the dashed horizontal line represents the mean volume.

[0025] Fig. 9 c—The graph shows tumor burden over time as measured by high-resolution ultrasound in individual KPC mice treated with vehicle control (n=10) or Ab7326 mlgG1 in combination with UCB-554 (n=11).

[0026] Fig.10 a- Kaplan-Meier analysis showing the Pdx1-Cre, LSL-Kras, and 4-HRP-positive controls from the start of treatment with vehicle control (n=10), Ab7326mIgG1 (n=19), selumetinib (n=17), or Ab7326 mIgG1 in combination with selumetinib (n=19) as indicated. G12D / + , LSL-Trp53 R172H / + (KPC) mice survival rate. Data are included Figure 7 Combined dataset of the data shown.

[0027] Fig.10b-Box plot showing tumor volume measured by high-resolution ultrasound on day 7 after the start of treatment 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 depicts the interquartile range, and the line depicts the minimum and maximum observed values. The solid horizontal bar represents the median, and the dashed horizontal line represents the mean volume.

[0028] Fig.10 c—The graph shows tumor burden over time measured by high-resolution ultrasound in individual KPC mice treated with vehicle control (n=10) or Ab7326 mlgG1 in combination with selumetinib (n=10).

[0029] A brief description of the table

[0030] Table 1 - This table shows the relationship between G12D+ Grem1 is upregulated in KPC tumors compared to mouse pancreatic ductal cells and in all pancreatic mouse models combined.

[0031] Table 2 - This table shows the dose and schedule of a given treatment, the number of mice per treatment, and the median survival in each treatment cohort.

[0032] Table 3 - This table shows the dose and schedule of a given treatment, the number of mice in each group, and the time points of sampling.

[0033] Table 4 - This table shows the dose and schedule of a given treatment, the number of mice per treatment, and the median survival in each treatment cohort. *Indicates historical data used.

[0034] Table 5 - This table compares survival statistics between treatment cohorts (log-rank analysis).Mice treated with the combination of Ab7326 mlgG1 and selumetinib showed a significant improvement in survival compared to selumetinib alone.

[0035] Table 6 - This table shows the survival data and censoring for individual mice that received treatment.

[0036] Table 7 - This table shows the individual mouse samples analyzed.

[0037] Table 8 - This table shows the dose and schedule of a given treatment, the number of mice per treatment, and the median survival in each treatment cohort.

[0038] Table 9 - Summary of survival statistics for KPC mice treated with the combination of Ab7326 and UCB-554.

[0039] Table 10 - Summary of survival statistics for KPC mice treated with the combination of Ab7326 and selumetinib. DETAILED DESCRIPTION OF THE INVENTION

[0041] It should be understood that different applications of the disclosed products and methods can be customized according to specific needs in the art.It should also be understood that the terminology used herein is for the purpose of describing specific embodiments of the present invention only and is not intended to be limiting.

[0042] Furthermore, as used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context 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, etc.

[0043] All publications, patents and patent applications cited supra or infra are hereby incorporated by reference in their entirety.

[0044] Combination therapy using an anti-GREM1 antagonist and an inhibitor of Ras-Raf-MEK-ERK signaling

[0045] The present invention provides an anti-GREM1 antagonist for use in a method of treating or preventing cancer in combination with a Ras-Raf-MEK-ERK signaling inhibitor (such as a MEK or ERK inhibitor). The cancer is typically a cancer with overexpression of GREM1 and / or a cancer associated with dysregulation of the Ras-Raf-MEK-ERK signaling pathway. Additionally or alternatively, the cancer may be a cancer that exhibits dysregulation of the Ras-Raf-MEK-ERK pathway after exposure to / treatment with an anti-GREM1 antagonist. Specifically, the cancer may be a cancer in which Ras-Raf-MEK-ERK pathway signaling is induced / upregulated after treatment with an anti-GREM1 antagonist described herein. In the absence of anti-GREM1 antagonist treatment, such a cancer may not show dysregulation of the Ras-Raf-MEK-ERK pathway. In other words, prior to treatment with an anti-GREM1 antagonist described herein, the cancer may exhibit normal Ras-Raf-MEK-ERK pathway signaling relative to a reference sample or reference value. Additionally or alternatively, the cancer may be characterized as comprising a mutation in a RAS or RAF gene.

[0046] The present invention further provides a Ras-Raf-MEK-ERK signalling inhibitor for use in a method for treating or preventing cancer, wherein the method comprises administering an anti-GREM1 antagonist alone, sequentially or simultaneously.

[0047] Ras-Raf-MEK-ERK signaling inhibitors

[0048] Ras-Raf-MEK-ERK signaling inhibitors can be any inhibitors targeting the Ras-Raf-MEK-ERK mitogen-activated protein kinase (MAPK) signal transduction cascade (which may also be 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 (such as EGF / EGFR) at the cell surface results in the recruitment of the small GTPase Ras, which activates the serine threonine 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-specific kinases that phosphorylate Erk1 and Erk2 (MAPK), causing them to translocate to the nucleus and activate multiple transcription factors. Therefore, MEK inhibitors generally block the phosphorylation of Erk1 / Erk2 by MEK1 / MEK2 and downstream signaling pathways. ERK inhibitors can also prevent the phosphorylation of Erk1 / Erk2 and / or inhibit the catalytic activity of Erk1 / Erk2.

[0049] The inventors of the present invention have determined that KRAS signaling is upregulated in a KPC mouse model following exposure to an anti-GREM1 antagonist ( Figure 4 In addition, in the KPC mouse model, genes involved in the Ras-Raf-MEK-ERK pathway, such as Mapk1, Nras, and Myc, were upregulated after exposure to the anti-GREM1 antibody Ab7326 ( Figure 3 ). The inventors of the present invention have further demonstrated that the combination of an anti-GREM1 antibody and a MEK inhibitor has significant anti-tumor activity in this model. Therefore, combination therapy comprising an anti-GREM1 antagonist in combination with a Ras-Raf-MEK-ERK signaling inhibitor may be used to treat cancers associated with GREM1 overexpression, Ras-Raf-MEK-ERK pathway dysregulation, and / or Ras and Raf gene mutations.

[0050] In addition, 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) can be used to treat or prevent a cancer in which the Ras-Raf-MEK-ERK pathway is dysregulated following early exposure to / treatment with an anti-GREM1 antagonist. In particular, the cancer may be a cancer in which Ras-Raf-MEK-ERK pathway signaling is induced / upregulated following treatment with an anti-GREM1 antagonist as described herein. In the absence of anti-GREM1 antagonist treatment, such a cancer may not show dysregulation of the Ras-Raf-MEK-ERK pathway. In other words, prior to treatment with an anti-GREM1 antagonist as described herein, the cancer may exhibit normal Ras-Raf-MEK-ERK pathway signaling relative to a reference sample or reference value.

[0051] Disregulation refers to the upregulation or downregulation of the Ras-Raf-MEK-ERK pathway compared to a reference sample or reference level. The Ras-Raf-MEK-ERK pathway can be upregulated or downregulated at the mRNA or protein level. For example, the cancer can be a cancer in which genes / proteins in the Ras-Raf-MEK-ERK pathway are shown to be upregulated after exposure to an anti-GREM1 antagonist of the present invention. Alternatively or additionally, the cancer can be a cancer that shows increased phosphorylation of Map2k1, Map2k2, Erk1 and / or Erk2 in response to treatment with an anti-GREM1 antagonist of the present invention. The cancer can 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, colorectal cancer, breast cancer, and prostate cancer. In a preferred embodiment, the cancer is pancreatic cancer.

[0052] The Ras-Raf-MEK-ERK signaling inhibitor used herein includes pharmaceutically acceptable salts and solvates thereof.

[0053] The inhibitor used in the present invention can target any protein in the Ras-Raf-MEK-ERK pathway (classical MAPK cascade). Therefore, the inhibitor targets at least one component of the Ras-Raf-MEK-ERK pathway to inhibit Ras-Raf-MEK-ERK signal transduction. The inhibition of Ras-Raf-MEK-ERK signal transduction generally involves the reduction of the activity of at least one component of the pathway. Therefore, the inhibitor can be a direct or indirect inhibitor of any node (usually 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 Ras-Raf-MEK-ERK signal transduction inhibitor can be a RAF inhibitor, such as sorafenib, vemurafenib, dabrafenib or conafenib. In another aspect, the Ras-Raf-MEK-ERK signal transduction inhibitor can be a MEK inhibitor. Exemplary MEK inhibitors are described in detail below.In yet another aspect, the Ras-Raf-MEK-ERK signaling inhibitor can be an ERK inhibitor, such as uritinib (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), temuterkib (LY32 14996; 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, ravoxertinib (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-indazole-5-yl) -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 uritinib. In a particularly preferred embodiment of the invention, the ERK inhibitor is uritinib 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.

[0054] In a preferred aspect of the present invention, Ras-Raf-MEK-ERK signaling inhibitor is a MEK inhibitor. The MEK inhibitor used in combination with an anti-GREM1 antagonist can be an allosteric MEK inhibitor and / or a MEK1 and / or MEK2 inhibitor. The term "allosteric MEK inhibitor" means an inhibitor that binds to an allosteric site outside the ATP binding pocket of MEK. Allosteric MEK inhibitors (also known as type III MEK inhibitors) show high selectivity and generally play a role by reducing the movement in the activation loop of MEK1 / 2. Other MEK inhibitors include type I and type II MEK inhibitors, which compete for ATP binding and occupy the catalytic site of MEK (type I) or bind to adjacent sites in the ATP binding pocket (type II). All three types of MEK inhibitors are considered to be used in the method of the present invention. That is, type I, type II or type III MEK inhibitors can be used in the method of the present invention.

[0055] 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-3,4,6,7-tetrahydropyrido[4,3-d]pyrimidin-1(2H)-yl}phenyl)acetamide.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)azetidin-1-yl]methanone), bimetinib (MEK162; 5-((4-bromo-2-fluorophenyl)amino)-4-fluoro-N-(2-hydroxyethoxy)-1-methyl-1H-benzo[d]imidazole-6-carboxamide), CI-1040 (PD184352), modametinib (PD0325901; N-[(2R)-2,3- dihydroxypropyl]-3,4-difluoro-2-[(2-fluoro-4-iodophenyl)amino]benzamide), 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), rivatinib (RDEA119 / Bay86-9766; 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′-methyl-sulfonamide), RO5068760, pimasetinib (AS703026; N-[(2S)-2,3-dihydroxypropyl]-3-(2-fluoro-4-iodoanilino)pyridine-4-carboxamide), AZD8330 (2-( 2-((2-fluoro-4-iodophenyl)amino)-N-(2-hydroxyethoxy)-1,5-dimethyl-6-oxopyridine-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-oxoxazinane-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-difluoro-benzamide), PD318088 (5-bromo-N-(2,3-dihydroxypropoxy)-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]butane dicarbonitrile) or SL327.

[0056] In a preferred embodiment of the present invention, the MEK inhibitor is selumetinib (AZD6244) or trametinib (GSK-1120212). In a particularly preferred embodiment of the present 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).

[0057] In a preferred embodiment of the present invention, the MEK inhibitor belongs to the class of thieno[2,3-b]pyridine derivatives substituted with a substituted anilino moiety at position 2. Exemplary MEK inhibitors belonging to this class include:

[0058] -N-(2-fluoro-4-iodophenyl)-3-(morpholin-4-ylcarbonyl)thieno[2,3-b]pyridin-2-amine;

[0059] -N-(2-fluoro-4-iodophenyl)-3-[(4-methylpiperazin-1-yl)carbonyl]thieno[2,3-b]pyridin-2-amine;

[0060] -[2-[((2-fluoro-4-iodophenyl)amino]thieno[2,3-b]pyridin-3-yl](pyrrolidin-1-yl)methanone;

[0061] -(1-{2-[(2-fluoro-4-iodophenyl)amino]thieno[2,3-b]pyridine-3-carbonyl}piperidin-4-yl)-carbamic acid tert-butyl ester;

[0062] -2-[((2-fluoro-4-iodophenyl)amino]thieno[2,3-b]pyridine-3-carboxylic acid (4-aminopiperidin-1-yl)amide dihydrochloride;

[0063] -tert-butyl (1-{2-[(2-fluoro-4-iodophenyl)amino]thieno[2,3-b]pyridine-3-carbonyl}azetidin-3-ylmethyl)carbamate;

[0064] -[3-(Aminomethyl)azetidin-1-yl]-{2-[(2-fluoro-4-iodophenyl)amino]thieno[2,3-b]pyridin-3-yl}-methanone;

[0065] -tert-butyl (1-{2-[(2-fluoro-4-iodophenyl)amino]thieno[2,3-b]pyridine-3-carbonyl}-(3R)-pyrrolidin-3-yl)carbamate;

[0066] -tert-butyl (1-{2-[(2-fluoro-4-iodophenyl)amino]thieno[2,3-b]pyridine-3-carbonyl}-(3S)-pyrrolidin-3-yl)carbamate;

[0067] -(1-{2-[(2-fluoro-4-iodophenyl)amino]thieno[2,3-b]pyridine-3-carbonyl}azetidin-3-yl)-carbamic acid tert-butyl ester;

[0068] -[(3R)-3-aminopyrrolidin-1-yl]-{2-[(2-fluoro-4-iodophenyl)amino]thieno[2,3-b]pyridin-3-yl}-methanone;

[0069] -[(3S)-3-aminopyrrolidin-1-yl]-{2-[(2-fluoro-4-iodophenyl)amino]thieno[2,3-b]pyridin-3-yl}-methanone;

[0070] -(3-aminoazetidin-1-yl)-{2-[(2-fluoro-4-iodophenyl)amino]thieno[2,3-b]pyridin-3-yl]-methanone;

[0071] -{2-[((2-fluoro-4-iodophenyl)amino]thieno[2,3-b]pyridin-3-yl}-(3-hydroxyazetidin-1-yl)-methanone;

[0072] -{2-[((2-fluoro-4-iodophenyl)amino]thieno[2,3-b]pyridin-3-yl}-[(3R)-3-hydroxypyrrolidin-1-yl]-methanone;

[0073] -{2-[((2-fluoro-4-iodophenyl)amino]thieno[2,3-b]pyridin-3-yl}-[(3S)-3-hydroxypyrrolidin-1-yl]-methanone;

[0074] -{2-[((2-fluoro-4-iodophenyl)amino]thieno[2,3-b]pyridin-3-yl}-[2-(hydroxymethyl)-piperidin-1-yl]-methanone;

[0075] -{2-[((2-fluoro-4-iodophenyl)amino]thieno[2,3-b]pyridin-3-yl}-[(3S)-3-(hydroxymethyl)-morpholin-4-yl]-methanone;

[0076] -tert-butyl 4-{2-[(2-fluoro-4-iodophenyl)amino]thieno[2,3-b]pyridine-3-carbonyl}-(3R)-3-(hydroxymethyl)piperazine-1-carboxylate;

[0077] -2-[4({2-[(2-fluoro-4-iodophenyl)amino]thieno[2,3-b]pyridin-3-yl}carbonyl)piperazin-1-yl]ethanol;

[0078] -3-((1,4-diazepan-1-ylcarbonyl)-N-(2-fluoro-4-iodophenyl)thieno[2,3-b]pyridin-2-amine;

[0079] -N-(2-fluoro-4-iodophenyl)-3-(piperazin-1-ylcarbonyl)thieno[2,3-b]pyridin-2-amine;

[0080] -[4-({2-[(2-fluoro-4-iodophenyl)amino]thieno[2,3-b]pyridin-3-yl}carbonyl)-piperazin-1-yl]acetic acid ethyl ester;

[0081] -[4-({2-[(2-fluoro-4-iodophenyl)amino]thieno[2,3-b]pyridin-3-yl}carbonyl)piperazin-1-yl]acetic acid;

[0082] -{2-[((2-fluoro-4-iodophenyl)amino]thieno[2,3-b]pyridin-3-yl}-[(2R)-2-(methoxymethyl)-pyrrolidin-1-yl]-methanone (methone); and

[0083] -{2-[((2-Fluoro-4-iodophenyl)amino]thieno[2,3-b]pyridin-3-yl}-[(2R)-2-(hydroxymethyl)-piperazin-1-yl]-methanone.

[0084] Methods for preparing these MEK inhibitors are disclosed in WO 2007 / 088345, which is incorporated herein by reference in its entirety.

[0085] Preferred MEK inhibitors 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 are represented by compounds of formula (II) and pharmaceutically acceptable salts, solvates and N-oxides thereof:

[0086]

[0087] in

[0088] R 12 represents halogen;

[0089] R 3 ConnR b R c ;and

[0090] R b and R c Together with the nitrogen atom to which they are both attached, it represents azetidin-1-yl, pyrrolidin-1-yl, piperidin-1-yl, morpholin-4-yl, thiomorpholin-4-yl, piperazin-1-yl, homopiperidin-1-yl, homomorpholin-4-yl or homopiperazin-1-yl, any of which may be optionally substituted by one or more substituents selected from C 1-6 Alkyl, C 1-6 Alkoxy, hydroxy, hydroxy (C 1-6 ) alkyl (akyl), amino-(C 1-6 )alkyl, (amino)(hydroxy)(C 1-6 ) alkyl, halogen, oxo, C 2-6 Alkyl (akyl) carbonyl, carboxyl, C 2-6 Alkoxycarbonyl, di(C 1-6 ) alkyl (alhyl) hydrazine carbonyl, amino, C 1-6 Alkylamino, di(C 1-6 ) alkyl-amino, 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, carboxyl (C 1-6 ) alkyl, C 2-6 Alkoxycarbonyl (C 1-6 ) alkyl, C 2-6 Alkoxycarbonylamino and C 2-6 Alkoxycarbonylamino-(C1-6)alkyl.

[0091] In one embodiment, R 12 In another embodiment, R 12 It's iodine.

[0092] Processes for preparing compounds of formula (II) are disclosed in WO 2007 / 088345, which is incorporated herein by reference in its entirety.

[0093] In a preferred embodiment of the present invention, the MEK inhibitor is a compound of formula (II) or a pharmaceutically acceptable salt, solvate or N-oxide thereof. In a particularly preferred embodiment of the present invention, the MEK inhibitor is a compound of formula (II) or a pharmaceutically acceptable salt, solvate or N-oxide thereof and the cancer to be treated is pancreatic cancer, preferably pancreatic ductal adenocarcinoma (PDAC).

[0094] In a preferred embodiment of the present invention, the MEK inhibitor is WX-554 (UCB-554). In a particularly preferred embodiment of the present invention, the MEK inhibitor is WX-554 (UCB-554) and the cancer to be treated is pancreatic cancer, preferably pancreatic ductal adenocarcinoma (PDAC).

[0095] 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 depicted here:

[0096]

[0097] Methods for preparing WX-554 are disclosed in WO 2007 / 088345, which is incorporated herein by reference in its entirety.

[0098] The above-mentioned MEK inhibitors belonging to the class of thieno[2,3-b]pyridine derivatives substituted at the 2-position with a substituted anilino moiety are potent and selective MEK inhibitors having a binding affinity (IC) of 50 μM or less, usually 20 μM or less, often 5 μM or less, typically 1 μM or less, suitably 500 nM or less, ideally 100 nM or less and preferably 20 nM or less for human MEK1 and / or MEK2 enzymes. 50 ) (The skilled person will understand that the smaller the IC 50 Numbers indicate more active compounds). The MEK inhibitors for use in the present invention may have at least a 10-fold selective affinity, typically at least a 20-fold selective affinity, suitably at least a 50-fold selective affinity and ideally at least a 100-fold selective affinity for human MEK1 and / or MEK2 enzymes relative to other human kinases.

[0099] The activity of a MEK inhibitor can be determined using a variety of methods, such as an in vitro MEK assay. An exemplary in vitro MEK assay is described in WO 2007 / 088345, which is incorporated herein by reference in its entirety.

[0100] It is also contemplated that a combination therapy comprising a GREM1 antagonist as defined herein and a Ras-Raf-MEK-ERK signaling inhibitor (such as a MEK inhibitor or an ERK inhibitor) may be used to treat or prevent a cancer that is typically targeted with a Ras-Raf-MEK-ERK signaling inhibitor. The cancer may be a cancer or tumor previously described for treatment with a Ras-Raf-MEK-ERK signaling inhibitor. Such cancers include, but are not limited to, pancreatic cancer, multiple myeloma, colorectal cancer, lung cancer, and melanoma.

[0101] The cancer may be a cancer that responds to treatment with a Ras-Raf-MEK-ERK signaling inhibitor and / or a cancer that is considered for treatment with a Ras-Raf-MEK-ERK signaling inhibitor. Alternatively, the cancer may be a cancer that responds poorly, unresponsively, or refractory to treatment with a Ras-Raf-MEK-ERK signaling inhibitor. The cancer may have been previously described as being unsuitable for treatment with a Ras-Raf-MEK-ERK signaling inhibitor. In some cases, the cancer may initially respond to treatment with a Ras-Raf-MEK-ERK signaling inhibitor, but develops resistance to a Ras-Raf-MEK-ERK signaling inhibitor. Responsiveness may be measured in any manner. For example, responsiveness to treatment may be assessed by measuring tumor size before and after treatment using X-rays, CT or MRI scans or by measuring tumor markers. Blood tests may also be used to determine organ function to assess responsiveness to treatment. An exemplary blood marker for monitoring responsiveness in pancreatic cancer is CA19-9. Those skilled in the art know how to measure the responsiveness of a cancer to treatment.

[0102] cancer

[0103] In the context of a combination therapy comprising an anti-GREM1 antagonist and a Ras-Raf-MEK-ERK signaling inhibitor (such as a MEK or ERK inhibitor), the cancer can be any cancer or tumor. Specifically, in the context of a combination therapy comprising an anti-GREM1 antagonist and a Ras-Raf-MEK-ERK signaling inhibitor, the cancer can be any cancer or tumor with stroma (typically a desmoplastic stroma). The cancer can be any cancer or tumor initiated by GREM1. The cancer can be any cancer in which stromal and / or epithelial GREM1 overexpression is observed. The cancer or tumor can have stromal GREM1 overexpression, but no epithelial GREM1 overexpression. The cancer or tumor can have epithelial GREM1 overexpression, but no stromal GREM1 overexpression. In a preferred embodiment, the cancer or tumor has overexpression of GREM1 in the desmoplastic stroma. The cancer or tumor can be any cancer or tumor suitable for targeting with a GREM1 antagonist, MEK inhibitor and / or ERK inhibitor, including any cancer or tumor known in the art to be suitable for targeting with such an agent, and any cancer or tumor known in the art to be suitable for treatment with such an agent.

[0104] The cancer or tumor may be a solid tumor. The solid tumor may have a desmoplastic stroma.

[0105] Anti-GREM1 antagonists have previously been demonstrated to be effective in treating a variety of cancers in WO 2019 / 243801, filed on June 18, 2019, the disclosure of which is incorporated herein by reference in its entirety.

[0106] Particularly preferred cancers that may 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, spleen cancer, bone resident cancer, melanoma and osteosarcoma. The cancer that may be treated may be intestinal cancer, colon cancer or rectal cancer. The cancer to be treated may be a disseminated cancer, such as a metastatic cancer. A disseminated cancer should be understood as a cancer that has spread from its original site of origin in the body. For example, a disseminated cancer may be a cancer that originated in the pancreas, bone marrow, colon, prostate or breast tissue of a patient but has spread to, for example, the patient's liver or lungs.

[0107] Combination therapy including a GREM1 antagonist and an inhibitor of Ras-Raf-MEK-ERK signaling may also be used to prevent the spread of cancer.

[0108] Combination therapy comprising a GREM1 antagonist and an inhibitor of Ras-Raf-MEK-ERK signaling may be used to prevent cancer-associated polyposis.

[0109] Grading systems are used in cancer biology and medicine to classify cancer cells according to their lack of cell differentiation. This reflects the degree of difference between cancer cells and healthy cells found in the tissue of origin of cancer cells in morphology. The grading system can be used to indicate the speed at which a particular cancer may be expected to grow. The commonly used cancer grades are (G)X grades and 1 to 4 grades. GX indicates that the cancer grade cannot be assessed. G1 (low grade) cancer cells have a morphology similar to normal healthy cells (i.e. they are well differentiated) and are expected to grow slowly and are unlikely 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 poorly differentiated) and are expected to grow faster than G1 and G2 cells. G4 (high grade) cancer cells are undifferentiated (also known as anaplastic) and are expected to have the highest proliferation capacity.

[0110] Cancer grading is different from cancer staging, which can indicate how cancer is likely to spread. A common cancer staging system has five stages, namely Stage 0: cancer cells are in situ (i.e., located in their normal tissues); Stage I: cancer is confined to one part of the body; Stage II: cancer is locally advanced; Stage III: cancer is also locally advanced (whether a cancer is designated as Stage II or Stage III can depend on the specific type of cancer); and Stage IV: cancer has typically metastasized, or spread to other organs or throughout the body.

[0111] Those skilled in the art know how to determine the grade and / or stage of a cancer. In one embodiment, the present invention relates to the use of an anti-GREM1 antagonist in combination with a Ras-Raf-MEK-ERK signaling inhibitor for the treatment and / or prevention of an 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 cancer of stage II or above. The established cancer may be a stage III or stage IV cancer. In one embodiment, the established cancer is a stage IV cancer that has metastasized. In one embodiment, the established cancer is an established pancreatic cancer.

[0112] In addition to the specifically exemplified use in the treatment and prevention of pancreatic cancer, the inventors envision that the therapeutic effects of GREM1 antagonists in combination with Ras-Raf-MEK-ERK signaling inhibitors shown in the Examples will also be applicable to the treatment of other cancers with corresponding characteristics described herein.

[0113] In particular, it is envisioned that a combination comprising a GREM1 antagonist will be useful for preventing or treating a cancer in which the Ras-Raf-MEK-ERK pathway is dysregulated in response to treatment / exposure with an anti-GREM1 antagonist described herein. In particular, the cancer may be a cancer in which Ras-Raf-MEK-ERK signaling is induced / upregulated following treatment with an anti-GREM1 antagonist described herein. In the absence of anti-GREM1 antagonist treatment, such a cancer may not exhibit dysregulation of the Ras-Raf-MEK-ERK pathway. In other words, prior to treatment with an anti-GREM1 antagonist described herein, the cancer may exhibit normal Ras-Raf-MEK-ERK signaling relative to a reference sample or reference value.

[0114] It is also envisioned that combinations comprising GREM1 antagonists will be useful for preventing or treating cancers in which there is mesenchymal and / or epithelial GREM1 overexpression and this overexpression promotes malignant cell growth. Such cancers include pancreatic cancer, bladder cancer, lung cancer, gastric cancer, duodenal cancer, esophageal cancer, head and neck cancer, glioma, endometrial cancer, liver cancer, spleen cancer, bone resident cancer and osteosarcoma.

[0115] Antagonists of the present invention are used in combination with Ras-Raf-MEK-ERK signaling inhibitors (such as MEK inhibitors or ERK inhibitors) to treat or prevent cancer. Preventing cancer can include preventing a subject from being diagnosed with cancer or delaying the onset of cancer. Preventing cancer can also include preventing cancer recurrence or reoccurrence in subjects who have previously been diagnosed with cancer. Preventing cancer can also include increasing the survival of subjects who have not yet been diagnosed with cancer or have previously been diagnosed with cancer.

[0116] Treating cancer can improve one or more symptoms of cancer, induce or prolong the remission of cancer, or delay the recurrence or reoccurrence of cancer. Treatment can cure, alleviate or partially inhibit cancer. It may lead to a reduction in the severity of disease symptoms, or an increase in the frequency or duration of the asymptomatic phase. Treating cancer can also include preventing cancer (e.g., a determined cancer) from its primary site in the patient's body to one or more secondary sites in the patient's body. Therefore, treating cancer may include preventing the spread or metastasis of existing cancer. In a preferred embodiment, the antagonists of the present invention are combined with Ras-Raf-MEK-ERK signaling inhibitors (such as MEK inhibitors or ERK inhibitors) for the treatment or prevention of pancreatic cancer. Treatment of pancreatic cancer may result in a reduction in the size of the primary tumor, such as measured by CT or endoscopic ultrasound. Such a reduction can be beneficial for Whipple surgery (pancreaticoduodenectomy) for resection of tumors from the head of the pancreas. The inventors have demonstrated that anti-GREM1 antagonist treatment combined with a MEK inhibitor can increase the survival of the KPC mouse model. Therefore, the use of anti-GREM1 antagonists and MEK inhibitors to treat pancreatic cancer may also improve patient survival. It is anticipated that the use of ERK inhibitors to treat cancer, particularly pancreatic cancer, will produce similar favorable results.

[0117] Pancreatic cancer

[0118] The present invention is preferably directed to the treatment or prevention of pancreatic cancer. As described in more detail in the Examples, the inventors of the present invention have demonstrated 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 In addition, the inventors have demonstrated that KRAS signaling is upregulated in a KPC mouse model following exposure to an anti-GREM1 antagonist ( Figure 4 ), and genes involved in the Ras-Raf-MEK-ERK signaling pathway, such as Mapk1, Nras, and Myc, were upregulated in the KPC mouse model after exposure to the anti-GREM1 antibody Ab7326 ( Figure 3 The inventors of the present invention have also demonstrated that a combination therapy comprising an anti-GREM1 antagonist and a MEK inhibitor significantly increases survival, slows tumor growth, and promotes tumor shrinkage in a KPC mouse model. KPC mice develop tumors that are histologically and pathologically similar to human pancreatic tumors and mimic human pancreatic cancer.

[0119] Thus, in one embodiment, the pancreatic cancer is a pancreatic cancer characterized by having GREM1 overexpression. In another embodiment, the pancreatic cancer can be characterized as having an exocrine tumor or a neuroendocrine tumor. Pancreatic neuroendocrine cancer (also known as islet cell tumor) occurs in the endocrine glands of the pancreas. A particularly preferred form of pancreatic cancer is exocrine pancreatic cancer, such as pancreatic ductal adenocarcinoma (PDAC). It has been described that PDAC accounts for 90% of all pancreatic cancers (Feldmann et al. J Hepatobiliary Pancreat Surg. 2007; 14 (3): 224-32). Pancreatic cancer can be any cancer that occurs in the inner wall of the pancreatic duct.

[0120] It has been reported that the Ras-Raf-MEK-ERK signaling pathway plays 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 enhanced cell proliferation, motility, and survival, but targeting KRAS signaling remains challenging, and clinical trials using MEK inhibitors have largely failed to achieve 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 for treatment is a pancreatic cancer described herein with a dysregulated Ras-Raf-MEK-ERK pathway and / or a mutation in a Ras (such as KRAS, NRAS and / or HRAS) or Raf gene (such as ARAF, BRAF and / or CRAF). For example, the pancreatic cancer may be a pancreatic cancer with a KRAS G12D or KRAS G12R Alternatively, the pancreatic cancer may be a pancreatic cancer tumor with a BRAF mutation / replacement. V600E Mutated / permuted pancreatic cancer tumors.

[0121] In another embodiment, the pancreatic cancer is a pancreatic cancer in which the Ras-Raf-MEK-ERK pathway is dysregulated in response to treatment / exposure with an anti-GREM1 antagonist described herein. Specifically, the pancreatic cancer may be a pancreatic cancer in which Ras-Raf-MEK-ERK signaling is induced / upregulated after treatment with an anti-GREM1 antagonist. In the absence of anti-GREM1 antagonist treatment, such a pancreatic cancer may not show dysregulation of the Ras-Raf-MEK-ERK pathway. In other words, prior to treatment with an anti-GREM1 antagonist described herein, the pancreatic cancer may exhibit normal Ras-Raf-MEK-ERK pathway signaling relative to a reference sample or reference value.

[0122] Other exocrine pancreatic cancers include squamous cell carcinoma, which develops in the pancreatic ducts; adenosquamous carcinoma; signet ring cell carcinoma; and colloid carcinoma, which usually develops from intraductal papillary mucinous neoplasms.

[0123] A preferred pancreatic cancer type for treatment may be resistant to one or more known anticancer agents (such as chemotherapy). The pancreatic cancer may be diffuse pancreatic cancer. The pancreatic cancer may be metastatic pancreatic cancer. Metastatic cancer should be understood as a cancer that spreads from its initial origin site in vivo. Therefore, metastatic pancreatic cancer means a cancer that starts from the pancreas and spreads to other organs (such as lungs, liver, bones and brain). Usually, metastatic pancreatic cancer means a cancer that starts from the pancreas and spreads to lungs and / or liver. The pancreatic cancer may also be a recurrent pancreatic cancer. In other words, the pancreatic cancer to be treated by the method of the present invention includes pancreatic cancer that recurs after a few months or even a few years after early treatment (such as chemotherapy, radiotherapy or curative surgery). As further described below, a preferred pancreatic cancer type for treatment may be resistant to one or more known anticancer agents (such as chemotherapeutic agents).

[0124] In certain aspects, the pancreatic cancer can be a pancreatic cancer that responds to treatment with a Ras-Raf-MEK-ERK signaling inhibitor, for example, a pancreatic cancer that responds to treatment with a MEK or ERK inhibitor described herein. The pancreatic cancer can alternatively be a pancreatic cancer that responds poorly, unresponsively, or refractory to treatment with a Ras-Raf-MEK-ERK signaling inhibitor, for example, a pancreatic cancer that responds poorly, unresponsively, or refractory to treatment with a MEK or ERK inhibitor described herein. The pancreatic cancer may have been previously considered unsuitable for treatment with a Ras-Raf-MEK-ERK signaling inhibitor, for example, a pancreatic cancer that was previously considered unsuitable for treatment with a MEK or ERK inhibitor described herein. In some cases, the pancreatic cancer may initially respond to treatment with a Ras-Raf-MEK-ERK signaling inhibitor, but develops resistance to a Ras-Raf-MEK-ERK signaling inhibitor. For example, the pancreatic cancer may initially respond to treatment with a MEK or ERK inhibitor, but develop resistance to the MEK or ERK inhibitors described herein.

[0125] Lung cancer

[0126] In another 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 may be advanced NSCLC, such as stage III or stage IV NSCLC. The NSCLC may be squamous cell carcinoma, adenocarcinoma or large cell carcinoma. The cancer may be small cell lung cancer. The lung cancer may be unresectable. The lung cancer may be non-angiogenic and / or may show vascular co-option. The lung cancer may include alveolar and / or interstitial tumor growth. The lung cancer may be primary lung cancer or any secondary cancer that has spread to the lungs, such as breast cancer or pancreatic cancer. The lung cancer may be diffuse lung cancer. The lung cancer may be metastatic lung cancer. The lung cancer may be characterized by lung cancer with overexpression of GREM1. The lung cancer may also be recurrent lung cancer. In other words, the lung cancer to be treated by the method of the present invention includes lung cancer that recurs months or even years after early treatment (such as chemotherapy, radiotherapy or curative surgery). A preferred type of lung cancer for treatment may be resistant to one or more known anticancer agents (such as chemotherapeutic agents) as further described below.

[0127] In certain aspects, the lung cancer may be a lung cancer that responds to treatment with a Ras-Raf-MEK-ERK signaling inhibitor, for example, a lung cancer that responds to treatment with a MEK or ERK inhibitor described herein. The lung cancer may alternatively be a lung cancer that responds poorly, unresponsively, or refractory to treatment with a Ras-Raf-MEK-ERK signaling inhibitor, for example, a lung cancer that responds poorly, unresponsively, or refractory to treatment with a MEK or ERK inhibitor described herein. The lung cancer may have been previously considered unsuitable for treatment with a Ras-Raf-MEK-ERK signaling inhibitor, for example, a lung cancer that was previously considered unsuitable for treatment with a MEK or ERK inhibitor described herein. In some cases, the lung cancer may initially respond to treatment with a Ras-Raf-MEK-ERK signaling inhibitor, but develops resistance to a Ras-Raf-MEK-ERK signaling inhibitor. For example, the lung cancer may initially respond to treatment with a MEK or ERK inhibitor, but develops resistance to a MEK or ERK inhibitor described herein.

[0128] In another aspect, the lung cancer for treatment is a lung cancer with a Ras-Raf-MEK-ERK pathway disorder and / or a mutation in Ras (such as KRAS, NRAS and / or HRAS) or a Raf gene (such as ARAF, BRAF and / or CRAF) as described herein. For example, the lung cancer for treatment can be a non-small cell lung cancer with a MAPK / ERK pathway disorder and / or a mutation in Ras (such as KRAS, NRAS and / or HRAS) or a Raf gene (such as ARAF, BRAF and / or CRAF) as described herein.

[0129] In another embodiment, the lung cancer is a lung cancer in which the Ras-Raf-MEK-ERK signaling pathway is dysregulated in response to treatment / exposure with an anti-GREM1 antagonist described herein. Specifically, the lung cancer may be a lung cancer in which Ras-Raf-MEK-ERK signaling is induced / upregulated after treatment with an anti-GREM1 antagonist. In the absence of anti-GREM1 antagonist treatment, such a lung cancer may not show dysregulation of the Ras-Raf-MEK-ERK pathway. In other words, prior to treatment with an anti-GREM1 antagonist described herein, the lung cancer may exhibit normal Ras-Raf-MEK-ERK pathway signaling relative to a reference sample or reference value.

[0130] Melanoma

[0131] In another aspect, the present invention relates to the treatment or prevention of melanoma. The melanoma may be an epidermal / skin melanoma, such as a superficial spreading melanoma, a nodular melanoma, a lentigo maligna melanoma, or an acral lentigo melanoma. The skin melanoma may be an amelanotic melanoma, a nevus-like melanoma, a spitz-like melanoma, or a desmoplastic melanoma. In certain aspects, the melanoma may be an eye / ocular melanoma, such as a conjunctival melanoma, a choroidal melanoma, or an iris melanoma. In other aspects, the melanoma may be a mucosal melanoma.

[0132] The melanoma may be a metastatic melanoma. The melanoma may be a melanoma characterized by overexpression of GREM1. The melanoma may also be a recurrent melanoma. In other words, melanomas to be treated by the methods of the present invention include melanomas that recur months or even years after early treatment (such as chemotherapy, radiotherapy, or curative surgery). A preferred melanoma type for treatment may be resistant to one or more known anticancer agents (such as chemotherapeutic agents) as further described below.

[0133] In certain aspects, the melanoma may be a melanoma that is responsive to treatment with a Ras-Raf-MEK-ERK signaling inhibitor, for example, a melanoma that is responsive to treatment with a MEK or ERK inhibitor described herein. The melanoma may alternatively be a melanoma that is poorly responsive, unresponsive, or refractory to treatment with a Ras-Raf-MEK-ERK signaling inhibitor, for example, a melanoma that is poorly responsive, unresponsive, or refractory to treatment with a Ras-Raf-MEK-ERK inhibitor described herein. The melanoma may have been previously considered unsuitable for treatment with a Ras-Raf-MEK-ERK signaling inhibitor, for example, a melanoma that was previously considered unsuitable for treatment with a MEK or ERK inhibitor described herein. In some cases, the melanoma may initially respond to treatment with a Ras-Raf-MEK-ERK signaling inhibitor, but develops resistance to a Ras-Raf-MEK-ERK signaling inhibitor. For example, the melanoma may initially respond to treatment with a MEK or ERK inhibitor, but develop resistance to the MEK or ERK inhibitors described herein.

[0134] In another aspect, the melanoma used for treatment is a melanoma described herein with dysregulation of the MAPK / ERK pathway and / or mutations in Ras (such as KRAS, NRAS and / or HRAS) or Raf genes (such as ARAF, BRAF and / or CRAF).

[0135] In another embodiment, the melanoma is a melanoma in which the MAPK / ERK pathway is dysregulated in response to treatment / exposure with an anti-GREM1 antagonist described herein. Specifically, the melanoma may be a melanoma in which MAPK / ERK signaling is induced / upregulated after treatment with an anti-GREM1 antagonist. In the absence of anti-GREM1 antagonist treatment, such a melanoma may not show dysregulation of the MAPK / ERK pathway. In other words, prior to treatment with an anti-GREM1 antagonist described herein, the melanoma may exhibit normal MAPK / ERK pathway signaling relative to a reference sample or reference value.

[0136] Colorectal cancer

[0137] The present invention relates in one aspect to the prevention or treatment of colorectal cancer. As background, the intestinal mucosa is a complex ecosystem, and the epithelium has an interdependent relationship with its microenvironment, particularly the underlying stroma. Stromal-epithelial crosstalk is closely related to regulating homeostasis in vivo and is dynamically altered in intestinal regeneration and cancer. Cell signaling networks are effector pathways of intercompartmental crosstalk and control epithelial cell fate decisions, but can be co-opted and disrupted by the tumor microenvironment in colorectal cancer.

[0138] Current chemotherapy management of colorectal cancer has not changed substantially in the past 20 years and is primarily based on the use of combination cytotoxic agents (such as the FOLFOX and FOLFIRI regimens, http: / / www.cancerresearchuk.org / about-cancer / cancer-in-general / treatment / cancer-drugs / drugs) to combat the proliferating tumor epithelium and the potential for resistance to these epithelial-targeting agents. Identifying new treatments for colorectal cancer is now more important than ever.

[0139] Thus, the cancer or tumor for treatment is colorectal cancer or a colorectal tumor. A particularly preferred form of colorectal cancer for treatment is a colorectal cancer characterized by having GREM1 overexpression in stromal cells (i.e., stromal GREM1 overexpression). The stromal cells may be cancer-associated fibroblasts. Colorectal cancers with stromal GREM1 overexpression may not show epithelial GREM1 overexpression. Colorectal cancers with stromal GREM1 overexpression may include stromal Foxl1 overexpression. A particularly suitable form of colorectal cancer for treatment is colorectal cancer as a stromal subtype of colorectal cancer, also described as CMS4 (Guinney et al., Nat Med 2015). Any other subtype of colorectal cancer may also be treated, including any of CMS1, CMS2 and CMS3 as described by Guinney et al. above. The colorectal cancer described herein may be a proximal colorectal cancer (or a proximal colorectal tumor). The proximal colon is the region of the large intestine upstream of the splenic flexure, i.e. the cecum, ascending colon and transverse colon. Cancer or tumors in this region are also referred to as right-sided cancers or tumors. The present invention may be directed to treating right-sided colorectal cancer or right-sided colorectal tumors.

[0140] The colorectal cancer may be a distal colorectal cancer (or a distal colorectal tumor). The distal colon is the region of the large intestine downstream of the splenic flexure, i.e. the descending colon, sigmoid colon and rectum. Cancers or tumors in this region are also referred to as left-sided cancers or tumors. The present invention may relate to treating left-sided colorectal cancer or left-sided colorectal tumors. The cancer with stromal overexpression of GREM1 may preferably be a sporadic cancer. The sporadic cancer may be caused by somatic mutations. The sporadic cancer may be caused by carcinogens. Sporadic cancer is not caused by inherited gene mutations. The sporadic cancer may cause stromal overexpression of GREM1. The proliferation of the sporadic cancer may depend on stromal overexpression of GREM1 in the cancer.

[0141] At least three single nucleotide polymorphisms (SNPs) close to GREM1 are independently associated with colorectal cancer (CRC) risk in Northern European whites and possibly other ethnic groups (Tomlinson et al., PLos Genet, 2011). There is a direct link with GREM1 expression, and other SNPs may have similar effects. In addition, two common SNPs near BMP2, two SNPs near BMP4, and one SNP near BMP7 affect the expression of BMP ligands and affect CRC risk. Therefore, the cancer may contain one or more of the above SNPs.

[0142] Another type of cancer or tumor to be treated according to the present invention is a cancer or tumor that exhibits overexpression of GREM1 in epithelial cells. Overexpression of GREM1 in epithelial cells may cause cancer. The proliferation of cancer may be dependent on epithelial overexpression of GREM1. Therefore, the cancer may have an epithelial origin. The cancer may be colorectal cancer or duodenal cancer. The cancer may be GREM1-induced. GREM1-induced means that a mutagenic event that enhances the activity or expression of GREM1 is the cause of the cancer. Such a cancer may be due to an inherited gene mutation. Therefore, the cancer may be a familial cancer (see below).

[0143] One preferred type of colorectal cancer for treatment may be resistant to one or more known anti-cancer agents, such as chemotherapeutic agents, as described further below.

[0144] The colorectal cancer may be a disseminated colorectal cancer. The colorectal cancer may be a metastatic colorectal cancer. The colorectal cancer may be a metastatic colorectal cancer of the lung. The colorectal cancer may be a metastatic colorectal cancer of the liver. The colorectal cancer may be a metastatic colorectal cancer of the bone.

[0145] The colorectal cancer may be characterized by stromal overexpression of Foxl1. The colorectal cancer may be characterized by stromal overexpression of one or more Wnt ligands. For example, the colorectal cancer may be characterized by stromal overexpression of Wnt5A and / or Wnt2B. If a colorectal cancer has stromal overexpression of Foxl1 and / or a Wnt ligand (e.g., Wnt5A or Wnt2B), the colorectal cancer may be particularly suitable for prevention or treatment using a GREM1 antagonist.

[0146] In certain aspects, the colorectal cancer may be a colorectal cancer that is responsive to treatment with a Ras-Raf-MEK-ERK signaling inhibitor, e.g., a colorectal cancer that is responsive to treatment with a MEK or ERK inhibitor described herein. The colorectal cancer may alternatively be a colorectal cancer that is poorly responsive, unresponsive, or refractory to treatment with a Ras-Raf-MEK-ERK signaling inhibitor, e.g., a colorectal cancer that is poorly responsive, unresponsive, or refractory to treatment with a MEK or ERK inhibitor described herein. The colorectal cancer may have been previously considered unsuitable for treatment with a Ras-Raf-MEK-ERK signaling inhibitor, e.g., a colorectal cancer that was previously considered unsuitable for treatment with a MEK or ERK inhibitor described herein. In some cases, the colorectal cancer may have initially responded to treatment with a Ras-Raf-MEK-ERK signaling inhibitor, but developed resistance to the Ras-Raf-MEK-ERK signaling inhibitor. For example, the colorectal cancer may initially respond to treatment with a MEK or ERK inhibitor described herein, but develop resistance to the MEK or ERK inhibitor.

[0147] In another aspect, the colorectal cancer for treatment is a colorectal cancer described herein having a dysregulated Ras-Raf-MEK-ERK pathway and / or a mutation in a Ras (such as KRAS, NRAS and / or HRAS) or Raf gene (such as ARAF, BRAF and / or CRAF). For example, the colorectal cancer may be a KRAS / BRAF mutated colorectal cancer. The KRAS mutated colorectal cancer may be characterized as having any of the following KRAS mutations: G12D, G12V, G12C, G12A, G13D, Q61H, Q61L, Q61R, A146T and / or A146V. The BRAF mutated colorectal cancer may be a BRAF V600E mutation.

[0148] In another embodiment, the colorectal cancer is a colorectal 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 colorectal cancer may be a colorectal cancer in which Ras-Raf-MEK-ERK signaling is induced / upregulated following treatment with an anti-GREM1 antagonist. In the absence of anti-GREM1 antagonist treatment, such a colorectal cancer may not show dysregulation of the Ras-Raf-MEK-ERK pathway. In other words, prior to treatment with an anti-GREM1 antagonist as described herein, the colorectal cancer may exhibit normal Ras-Raf-MEK-ERK pathway signaling relative to a reference sample or reference value.

[0149] Familial cancer

[0150] Familial cancers include cancers caused by one or more mutations in the gene encoding GREM1 or any other mutation affecting GREM1 gene expression. The autosomal dominant disorder Hereditary Mixed Polyposis Syndrome (HMPS) is caused by a 40 kb duplication upstream of GREM1 that results in a shift in pathological compartment expression from a restricted mesenchymal gradient to ectopic GREM1 gene expression throughout the epithelium.

[0151] The subject to be treated with an anti-GREM1 antagonist may have been previously identified as being at risk for developing a familial cancer. For example, the subject may be identified as being at risk based on the subject's family history and / or because the subject carries a genetic mutation known to cause or increase the risk of a familial cancer.

[0152] The familial cancer may be Lynch syndrome, which is also known as hereditary non-polyposis colorectal cancer (HNPCC). The familial cancer may be familial adenomatous polyposis (FAP).

[0153] Patients or subjects with 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 a Ras-Raf-MEK-ERK signaling inhibitor may be FAP. An anti-GREM1 antagonist in combination with a Ras-Raf-MEK-ERK signaling inhibitor may be administered prophylactically to a subject who previously had FAP, for example to prevent recurrence. An anti-GREM1 antagonist in combination with a Ras-Raf-MEK-ERK signaling inhibitor may be administered prophylactically to a subject who has not previously had FAP but has previously been determined to be at risk of developing FAP. A subject may have been determined to be at risk of developing FAP because the subject has been found to carry a deleterious mutation in his Apc gene.

[0154] In certain aspects, the familial cancer may be a familial cancer that responds to treatment with a Ras-Raf-MEK-ERK signaling inhibitor, for example, a familial cancer that responds to treatment with a MEK or ERK inhibitor described herein. The familial cancer may alternatively be a familial cancer that responds poorly, unresponsively, or refractory to treatment with a Ras-Raf-MEK-ERK signaling inhibitor, for example, a familial cancer that responds poorly, unresponsively, or refractory to treatment with a MEK or ERK inhibitor described herein. The familial cancer may have been previously considered unsuitable for treatment with a Ras-Raf-MEK-ERK signaling inhibitor, for example, the familial cancer may have been previously considered unsuitable for treatment with a MEK or ERK inhibitor described herein. In some cases, the familial cancer may initially respond to treatment with a Ras-Raf-MEK-ERK signaling inhibitor, but develops resistance to a Ras-Raf-MEK-ERK signaling inhibitor. For example, the familial cancer may initially respond to treatment with a MEK or ERK inhibitor described herein, but develop resistance to the MEK or ERK inhibitor.

[0155] In another aspect, the familial cancer used for treatment is a familial cancer described herein with dysregulation of the Ras-Raf-MEK-ERK pathway and / or mutations in Ras (such as KRAS, NRAS and / or HRAS) or Raf genes (such as ARAF, BRAF and / or CRAF).

[0156] 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 an anti-GREM1 antagonist described herein. Specifically, the familial cancer may be a familial cancer in which Ras-Raf-MEK-ERK signaling is induced / upregulated following treatment with an anti-GREM1 antagonist. In the absence of anti-GREM1 antagonist treatment, such a familial cancer may not show dysregulation of the Ras-Raf-MEK-ERK pathway. In other words, prior to treatment with an anti-GREM1 antagonist described herein, the familial cancer may exhibit normal Ras-Raf-MEK-ERK pathway signaling relative to a reference sample or reference value.

[0157] Multiple Myeloma

[0158] The present invention relates to the treatment or prevention of multiple myeloma in another aspect. Multiple myeloma (MM) is a blood system malignancy characterized by the clonal proliferation of plasma cells (PC) in the bone marrow (BM). It is well known that BM supports tumor growth in MM, and bidirectional signaling between tumor cells and BM is essential for the continued growth, spread and survival of MM PC. Cellular and non-cellular BM components have different effects on the growth and spread of MM PC. Although recent studies have identified BM components that play a role in disease progression, and therapies targeting these have been developed, the standard of care treatment for MM still relies mainly on targeting tumor cells themselves. Although such therapy is effective for prolonging patient survival, since BM plays an important role in the growth, spread, survival and resistance of MM cells, more effective therapies targeting this important aspect of the disease are needed. In fact, MM is a basically incurable disease, and disease recurrence is a key problem faced by the effective treatment of the disease.

[0159] Therefore, the present invention also relates to the treatment or prevention of multiple myeloma. Multiple myeloma typically comprises the presence of more than one plasma cell mass in the bone marrow. Therefore, multiple myeloma is typically associated with abnormal proliferation of plasma cells in the bone marrow. A particularly preferred form of multiple myeloma for treatment is characterized by having overexpression of GREM1 in the bone marrow. Therefore, the multiple myeloma may comprise interstitial GREM1 overexpression. The interstitial GREM1 overexpression may be present in the compact bone compartment of the skeleton. The interstitial GREM1 overexpression may reflect an increase in the number of interstitial cells, or an increase in the expression level of GREM1 in existing interstitial cells expressing GREM1. The bone marrow may comprise osteochondral reticular (OCR) stem cells. Interstitial cells overexpressing GREM1 may comprise OCR stem cells. A preferred type of multiple myeloma for treatment may be resistant to one or more known anticancer agents (such as chemotherapeutic agents) as further described below.

[0160] In certain aspects, the multiple myeloma may be a multiple myeloma that responds to treatment with a Ras-Raf-MEK-ERK signaling inhibitor, such as a multiple myeloma that responds to treatment with a MEK or ERK inhibitor described herein. The multiple myeloma may alternatively be a multiple myeloma that responds poorly, unresponsively, or refractory to treatment with a Ras-Raf-MEK-ERK signaling inhibitor, such as a multiple myeloma that responds poorly, unresponsively, or refractory to treatment with a MEK or ERK inhibitor described herein. The multiple myeloma may have been previously considered unsuitable for treatment with a Ras-Raf-MEK-ERK signaling inhibitor, such as a multiple myeloma that was previously considered unsuitable for treatment with a MEK or ERK inhibitor. In some cases, the multiple myeloma may initially respond to treatment with a Ras-Raf-MEK-ERK signaling inhibitor, but develops resistance to a Ras-Raf-MEK-ERK signaling inhibitor. For example, the multiple myeloma may initially respond to treatment with a MEK or ERK inhibitor described herein, but develop resistance to the MEK or ERK inhibitor.

[0161] In another aspect, the multiple myeloma used for treatment is a multiple myeloma described herein with dysregulation of the Ras-Raf-MEK-ERK pathway and / or mutations in Ras (such as KRAS, NRAS and / or HRAS) or Raf genes (such as ARAF, BRAF and / or CRAF).

[0162] 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 described herein. Specifically, 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. In the absence of anti-GREM1 antagonist treatment, such multiple myeloma may not show dysregulation of the Ras-Raf-MEK-ERK pathway. In other words, prior to treatment with an anti-GREM1 antagonist described herein, the multiple myeloma may exhibit normal Ras-Raf-MEK-ERK pathway signaling relative to a reference sample or reference value.

[0163] Breast cancer

[0164] In another aspect, the present invention relates to the treatment or prevention of breast cancer. The breast cancer may be invasive breast cancer, such as 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 angiosarcoma of the breast. The breast cancer may be ductal carcinoma in situ or lobular carcinoma in situ.

[0165] The present invention provides for the treatment and prevention of breast cancer by administering an anti-GREM1 antagonist in combination with a Ras-Raf-MEK-ERK signaling inhibitor. The breast cancer may comprise interstitial GREM1 overexpression. Interstitial breast cells overexpressing GREM1 may comprise interstitial fibroblasts, also described herein as cancer-associated fibroblasts. The breast cancer may also be recurrent breast cancer. In other words, breast cancer to be treated by the methods of the present invention includes breast cancer that recurs months or even years after early treatment (such as chemotherapy, radiotherapy, or curative surgery). A preferred type of breast cancer for treatment may be resistant to one or more known anticancer agents (such as chemotherapeutic agents) as further described below. The breast cancer may be diffuse breast cancer. The breast cancer may be metastatic breast cancer. The breast cancer may be metastatic breast cancer of the lung. The breast cancer may be metastatic breast cancer of the liver. The breast cancer may be metastatic breast cancer of the bone.

[0166] In certain aspects, the breast cancer may be a breast cancer that responds to treatment with a Ras-Raf-MEK-ERK signaling inhibitor, such as a breast cancer that responds to treatment with a MEK or ERK inhibitor described herein. The breast cancer may alternatively be a breast cancer that responds poorly, unresponsively, or refractory to treatment with a Ras-Raf-MEK-ERK signaling inhibitor, such as a breast cancer that responds poorly, unresponsively, or refractory to treatment with a MEK or ERK inhibitor described herein. The breast cancer may have been previously considered unsuitable for treatment with a Ras-Raf-MEK-ERK signaling inhibitor, such as a breast cancer that was previously considered unsuitable for treatment with a MEK or ERK inhibitor described herein. In some cases, the breast cancer may initially respond to treatment with a Ras-Raf-MEK-ERK signaling inhibitor, but develops resistance to a Ras-Raf-MEK-ERK signaling inhibitor. For example, the breast cancer may initially respond to treatment with a MEK or ERK inhibitor described herein, but develops resistance to a MEK or ERK inhibitor.

[0167] 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 described herein. Specifically, 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. In the absence of anti-GREM1 antagonist treatment, such a breast cancer may not show dysregulation of the Ras-Raf-MEK-ERK pathway. In other words, prior to treatment with an anti-GREM1 antagonist described herein, the breast cancer may exhibit normal Ras-Raf-MEK-ERK pathway signaling relative to a reference sample or reference value.

[0168] Prostate cancer

[0169] In another 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.

[0170] The prostate cancer can be a prostate cancer characterized by overexpression of GREM1. The prostate cancer can also be a recurrent prostate cancer. In other words, the prostate cancer to be treated by the method of the present invention is included in the prostate cancer that recurs after a few months or even a few years of early treatment (such as chemotherapy, radiotherapy or curative surgery). A preferred prostate cancer type for the treatment of may be resistant to one or more known anticancer agents (such as chemotherapeutic agents) as further described below.

[0171] The present invention further provides for treating and preventing prostate cancer by administering an anti-GREM1 antagonist in combination with a Ras-Raf-MEK-ERK signaling inhibitor. The prostate cancer may be diffuse prostate cancer. The prostate cancer may be metastatic prostate cancer. The prostate cancer may be metastatic prostate cancer of the lung. The prostate cancer may be metastatic prostate cancer of the liver. The prostate cancer may be metastatic prostate cancer of the bone.

[0172] In certain aspects, the prostate cancer can be a prostate cancer that responds to treatment with a Ras-Raf-MEK-ERK signaling inhibitor, such as a prostate cancer that responds to treatment with a MEK or ERK inhibitor described herein. The prostate cancer can alternatively be a prostate cancer that responds poorly, unresponsively, or refractory to treatment with a Ras-Raf-MEK-ERK signaling inhibitor, such as a prostate cancer that responds poorly, unresponsively, or refractory to treatment with a MEK or ERK inhibitor described herein. The prostate cancer may have been previously considered unsuitable for treatment with a Ras-Raf-MEK-ERK signaling inhibitor, such as a prostate cancer that was previously considered unsuitable for treatment with a MEK or ERK inhibitor described herein. In some cases, the prostate cancer may initially respond to treatment with a Ras-Raf-MEK-ERK signaling inhibitor, but develops resistance to a Ras-Raf-MEK-ERK signaling inhibitor. For example, the prostate cancer may initially respond to treatment with a MEK or ERK inhibitor described herein, but develops resistance to a MEK or ERK inhibitor.

[0173] In another embodiment, the prostate cancer is a prostate cancer in which the Ras-Raf-MEK-ERK pathway is dysregulated in response to treatment / exposure with an anti-GREM1 antagonist described herein. Specifically, the prostate cancer may be a prostate cancer in which Ras-Raf-MEK-ERK signaling is induced / upregulated after treatment with an anti-GREM1 antagonist. In the absence of anti-GREM1 antagonist treatment, such a prostate cancer may not show dysregulation of the Ras-Raf-MEK-ERK pathway. In other words, prior to treatment with an anti-GREM1 antagonist described herein, the prostate cancer may exhibit normal Ras-Raf-MEK-ERK pathway signaling relative to a reference sample or reference value.

[0174] Bladder cancer

[0175] In another 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 originate 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 an adenocarcinoma. The bladder cancer may be a high-grade T1 tumor that grows from the bladder lining into the lamina propria. The bladder cancer may be a superficial cancer or an invasive bladder cancer. The bladder cancer may be a recurrent bladder cancer. The term recurrent bladder cancer as used herein refers to bladder cancer that has come back after treatment, such as surgical treatment.

[0176] The present invention further provides for the treatment and prevention of bladder cancer by administering an anti-GREM1 antagonist in combination with a Ras-Raf-MEK-ERK signaling inhibitor. The bladder cancer may be diffuse bladder cancer. The bladder cancer may be metastatic bladder cancer. The bladder cancer may be metastatic bladder cancer of the lung. The bladder cancer may be metastatic bladder cancer of the liver. The bladder cancer may be metastatic bladder cancer of the bone. The bladder cancer may be a bladder cancer characterized by overexpression of GREM1. The bladder cancer may also be a recurrent bladder cancer. In other words, bladder cancer to be treated by the methods of the present invention includes bladder cancer that recurs months or even years after early treatment (such as chemotherapy, radiotherapy, or curative surgery). A preferred type of bladder cancer for treatment may be resistant to one or more known anticancer agents (such as chemotherapeutic agents) as further described below.

[0177] In certain aspects, the bladder cancer may be a bladder cancer that responds to treatment with a Ras-Raf-MEK-ERK signaling inhibitor, such as a bladder cancer that responds to treatment with a MEK or ERK inhibitor described herein. The bladder cancer may alternatively be a bladder cancer that responds poorly, unresponsively, or refractory to treatment with a Ras-Raf-MEK-ERK signaling inhibitor, such as a bladder cancer that responds poorly, unresponsively, or refractory to treatment with a MEK or ERK inhibitor. The bladder cancer may have been previously considered unsuitable for treatment with a Ras-Raf-MEK-ERK signaling inhibitor, such as a bladder cancer that was previously considered unsuitable for treatment with a MEK or ERK inhibitor described herein. In some cases, the bladder cancer may initially respond to treatment with a Ras-Raf-MEK-ERK signaling inhibitor, but develops resistance to a Ras-Raf-MEK-ERK signaling inhibitor. For example, the bladder cancer may initially respond to treatment with a MEK or ERK inhibitor described herein, but develops resistance to a MEK or ERK inhibitor.

[0178] 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 described herein. Specifically, the bladder cancer may be a bladder cancer in which Ras-Raf-MEK-ERK signaling is induced / upregulated after treatment with an anti-GREM1 antagonist. In the absence of anti-GREM1 antagonist treatment, such bladder cancer may not show dysregulation of the Ras-Raf-MEK-ERK pathway. In other words, prior to treatment with an anti-GREM1 antagonist described herein, the bladder cancer may exhibit normal Ras-Raf-MEK-ERK pathway signaling relative to a reference sample or reference value.

[0179] Ovarian cancer

[0180] In another aspect, the 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 as a marginal ovarian tumor. The ovarian cancer may be characterized as a germ cell ovarian tumor. The ovarian cancer may be a clear cell ovarian cancer. The cancer may be a serous ovarian cancer. The ovarian cancer may be a mucinous ovarian cancer. The ovarian cancer may be an endometrioid cancer.

[0181] The ovarian cancer may be an ovarian cancer characterized by overexpression of GREM1. The ovarian cancer may also be a recurrent ovarian cancer. In other words, the ovarian cancer to be treated by the methods of the present invention includes ovarian cancer that recurs months or even years after early treatment (such as chemotherapy, radiotherapy, or curative surgery). A preferred type of ovarian cancer for treatment may be resistant to one or more known anticancer agents (such as chemotherapeutic agents) as further described below.

[0182] The present invention further provides for treating and preventing ovarian cancer by administering an anti-GREM1 antagonist in combination with a Ras-Raf-MEK-ERK signaling inhibitor. The ovarian cancer may be diffuse ovarian cancer. The ovarian cancer may be metastatic ovarian cancer. The ovarian cancer may be metastatic ovarian cancer of the lung. The ovarian cancer may be metastatic ovarian cancer of the liver. The ovarian cancer may be metastatic ovarian cancer of the bone.

[0183] In certain aspects, the ovarian cancer may be an ovarian cancer that responds to treatment with a Ras-Raf-MEK-ERK signaling inhibitor, such as an ovarian cancer that responds to treatment with a MEK or ERK inhibitor described herein. The ovarian cancer may alternatively be an ovarian cancer that responds poorly, unresponsively, or refractory to treatment with a Ras-Raf-MEK-ERK signaling inhibitor, such as an ovarian cancer that responds poorly, unresponsively, or refractory to treatment with a MEK or ERK inhibitor described herein. The ovarian cancer may have been previously considered unsuitable for treatment with a Ras-Raf-MEK-ERK signaling inhibitor, such as an ovarian cancer that was previously considered unsuitable for treatment with a MEK or ERK inhibitor described herein. In some cases, the ovarian cancer may initially respond to treatment with a Ras-Raf-MEK-ERK signaling inhibitor, but develops resistance to a Ras-Raf-MEK-ERK signaling inhibitor. For example, the ovarian cancer may initially respond to treatment with a MEK or ERK inhibitor described herein, but develops resistance to a MEK or ERK inhibitor.

[0184] 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 an anti-GREM1 antagonist described herein. Specifically, the ovarian cancer may be an ovarian cancer in which Ras-Raf-MEK-ERK signaling is induced / upregulated after treatment with an anti-GREM1 antagonist. In the absence of anti-GREM1 antagonist treatment, such ovarian cancer may not show dysregulation of the Ras-Raf-MEK-ERK pathway. In other words, prior to treatment with an anti-GREM1 antagonist described herein, the ovarian cancer may exhibit normal Ras-Raf-MEK-ERK pathway signaling relative to a reference sample or reference value.

[0185] Mesenchyme and epithelium

[0186] The cancers prevented or treated using the combination of a GREM1 antagonist and a MAPK / ERK signaling inhibitor described herein may comprise stromal and / or epithelial GREM1 overexpression.

[0187] As used herein, the term "stromal cells" or "stroma" refers to the structural and / or connecting parts of a tissue or organ.

[0188] Interstitial tissue is mainly composed of extracellular matrix containing connective tissue cells. The extracellular matrix is ​​mainly composed of ground substance (a porous hydrated gel, mainly made of proteoglycan aggregates) and connective tissue fibers. Three types of fibers are commonly found in the interstitium: collagen type I fibers, elastic fibers, and reticular (collagen type III) fibers. Fibroblasts and pericytes are among the most common interstitial cell types.

[0189] In the context of cancer or tumors (e.g., initiated in the epithelium of a tissue or organ), the stroma of the tissue or organ can aid cancer growth and progression. The stroma associated with a cancer or tumor can be a desmoplastic stroma caused by the growth of fibrous or connective tissue surrounding the cancer or tumor.

[0190] Overexpression of GREM1 can be observed in any part of the stroma / any stroma cell. The stroma cell may be a fibroblast or a fibroblast-like supporting cell. The stroma cell may be a fibroblast or a fibroblast-like supporting cell isolated from the desmoplastic stroma of any of the above cancers or tumors (such as from the pancreas, colon or rectum in colorectal cancer, or bone marrow in multiple myeloma). The stroma cell may be a cancer-associated fibroblast.

[0191] The term "epithelium" used herein means a cell derived from the outer or inner lining of a tissue or organ. In terms of the colon, the intestinal epithelium is a cell layer that forms the luminal surface or lining of the small and large intestines of the gastrointestinal tract. It is composed of simple columnar epithelium. "Upper barrier" is an intestinal epithelial monolayer columnar cell composed of four types of intestinal epithelial cells: absorptive enterocytes, goblet cells, Paneth cells and enteroendocrine cells. The upper barrier feature is similar in the small intestine and large intestine. The main difference is the protuberance and projection (annular folds, villi and microvilli) present in the duodenum, jejunum and ileum, which allows to increase the absorption surface. This is not observed in the colon, and the colon shows a flat surface on the contrary. In the mucosal projections known as villi, there is a bend called the Lieberkühn crypt, which is a distinct glandular invagination. The cell in which epithelial GREM1 overexpression is observed can be any epithelial cell, such as any intestinal epithelial cell.

[0192] While not being bound by theory, the inventors speculate that overexpression of GREM1 in the epithelium and / or stroma may promote stem / progenitor cell phenotypes (increase the number of stem / progenitor cells), promote epithelial stem cell behavior and drive cancer progression and / or resistance to chemotherapeutic agents. Therefore, combination therapies comprising a GREM1 antagonist and a Ras-Raf-MEK-ERK signaling inhibitor used in accordance with the present invention may prevent the induction of an abnormal cancer stem / progenitor cell phenotype, reduce epithelial stem cell behavior and / or reduce the number of stem / progenitor cells in the epithelium of a tissue or organ of a subject in which cancer is to be prevented or treated. The ability of GREM1 antagonists to affect stem cell behavior can be clinically determined by evaluating known epithelial and cancer stem cell markers.

[0193] Overexpression of GREM1 in stroma and / or epithelium can be determined by any means. Overexpression of GREM1 is usually determined by comparing with marker levels (i.e., basal expression levels) in normal cells of the same tissue type. The expression is usually normalized relative to the expression levels of other genes, preferably including one or more housekeeping genes. GREM1 can also be classified as showing overexpression or underexpression in a threshold percentage of a cancer patient population. The overexpression of each patient in the population can be 2 higher than the geometric mean. At least 10%, more preferably at least 15% or more patients in the population may show such overexpression.

[0194] GREM1 stroma overexpression means stroma GREM1 levels that are higher than the levels of matched normal tissue. For example, stroma GREM1 levels may be at least twice the levels of matched normal tissue.

[0195] In the case where GREM1 is overexpressed, its amount can be increased by any amount relative to the basal amount. For example, a GREM1-induced cancer such as HMPS can contain thousands of times upregulation of epithelial GREM1, while no GREM1 expression 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 the organ. The technician can evaluate the presence of overexpression in the stroma or epithelium compared to the level of GREM1 in normal cells of the same type.

[0196] The determined amount can be the amount of mRNA. Therefore, the cancer may include overexpression of GREM1 mRNA. Compared with normal cells of the same tissue type, the cancer may include an increased amount of GREM1 mRNA. The mRNA may be increased in any amount. The amount of mRNA can be measured using quantitative reverse transcription polymerase chain reaction (qRT-PCR) such as real-time qRT-PCR, quantitative gene determination (Affymetrix / Thermo Fisher), by Northern blotting or using microarrays, RNA sequencing. Preferably, mRNA expression is determined by comparing the gene expression of the sample with the expression level distribution of a specific gene in a reference sample composed of a tumor of the gene diploid. Using RNAseq, a z-score can be derived by the expectation maximization (RSEM) algorithm (cBioportal for Cancer Genomics, www.cbioportal.org; Gao et al., 2013 and Serami et al. 2012). Preferably, a z-score that is 2 standard deviations higher or lower than the mean of the reference set is regarded as overexpression or underexpression, respectively.

[0197] The amount measured can be the amount of protein. The cancer can comprise overexpression of GREM1 protein, such as compared to normal cells of the same tissue type. The protein can be increased by any amount. The amount of protein can be measured using immunohistochemistry, Western blotting, mass spectrometry or fluorescence activated cell sorting (FACS), including by using the anti-GREM1 antibodies of the present invention. The threshold for determining expression can vary depending on the technique used and can be verified based on the immunohistochemical score.

[0198] Thus, the use of a GREM1 antagonist in combination with a Ras-Raf-MEK-ERK signaling inhibitor as described herein for treating or preventing cancer in a patient can 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 a Ras-Raf-MEK-ERK signaling inhibitor, and thereby treating or preventing the cancer. The amount of GREM1 can be the amount of mRNA or protein, and the overexpression can be any of the overexpressions discussed above.

[0199] The above measurements can be performed in any suitable sample from the patient. The measurements can be performed in a cancer or tumor biopsy sample obtained from the patient. The stroma and / or epithelium (stromal cells and / or epithelial cells) can be separated from the biopsy sample. 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, gastric tissue, duodenal tissue, esophageal tissue, bone marrow or colorectal tissue. Any of the methods discussed above can be performed on a cancer biopsy sample. Such a method can also be performed on cancer cells circulating in the patient's blood. The RNA method can be performed on urine or blood exosomes.

[0200] Cancers associated with dysregulation of the Ras-Raf-MEK-ERK signaling pathway

[0201] As noted above, the inventors have demonstrated that KRAS signaling in the KPC mouse model is upregulated after exposure to an anti-GREM1 antagonist (Ab7326) according to the present invention. In addition, genes involved in the Ras-Raf-MEK-ERK pathway (such as Mapk1, Nras, and Myc) are upregulated in the KPC mouse model after exposure to Ab7326. Therefore, a combination therapy targeting the Gremlin-1 and Ras-Raf-MEK-ERK pathways would be advantageous.

[0202] The present invention encompasses the use of anti-GREM1 antagonists in combination with Ras-Raf-MEK-ERK signaling inhibitors for the treatment or prevention of cancers associated with dysregulation of the Ras-Raf-MEK-ERK pathway and / or cancers containing Ras or Raf gene mutations. The Ras gene can be, for example, KRAS, NRAS and / or HRAS. The Raf gene can be, for example, ARAF, BRAF and / or CRAF. Cancers associated with dysregulation of the MAPK / ERK pathway and / or cancers containing Ras or Raf gene mutations can include, for example, colorectal 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, spleen cancer, bone resident cancer and osteosarcoma.

[0203] The present invention also includes a combination therapy comprising an anti-GREM1 antagonist as defined herein and a Ras-Raf-MEK-ERK signaling inhibitor for use in the treatment or prevention of a cancer in which the Ras-Raf-MEK-ERK pathway is dysregulated following early exposure / treatment with an anti-GREM1 antagonist. In particular, the cancer may be a cancer in which Ras-Raf-MEK-ERK pathway signaling is induced / upregulated following treatment with an anti-GREM1 antagonist as described herein. In the absence of anti-GREM1 antagonist treatment, such a cancer may not show dysregulation of the Ras-Raf-MEK-ERK pathway. In other words, prior to treatment with an anti-GREM1 antagonist as described herein, the cancer may exhibit normal Ras-Raf-MEK-ERK pathway signaling relative to a reference sample or reference value.

[0204] In a preferred embodiment, the present invention relates 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 a Ras-Raf-MEK-ERK signaling inhibitor for the treatment or prevention of pancreatic cancer in which the Ras-Raf-MEK-ERK pathway is dysregulated after early exposure / treatment with an anti-GREM1 antagonist. Alternatively or additionally, the pancreatic cancer may be characterized by constitutive activation of the classical MAPK pathway. In many cases, pancreatic cancer involves gain-of-function mutations in KRAS (which encodes RAS). Typical gain-of-function mutations include G12D or G12R substitutions and are found in pancreatic ductal adenocarcinoma. In other cases, pancreatic cancer involves gain-of-function mutations in BRAF (which encodes B-RAF). A typical gain-of-function mutation is found at position V600E, resulting in constitutive kinase activity of RAF.

[0205] As indicated above, the invention may also relate to the treatment or prevention of KRAS / BRAF mutated colorectal cancer.In another aspect, the invention relates to BRAF mutated melanoma, such as a melanoma carrying a V600E mutation in BRAF.

[0206] GREM1

[0207] The term GREM1 or Gremlin-1 used in the context of a protein in the present invention refers to a protein generally having an amino acid sequence as shown in UniProt entry O60565 (SEQ ID NO: 1), i.e. human GREM1. The terms GREM1 and Gremlin-1 may also refer to a Gremlin-1 polypeptide, which:

[0208] (a) comprises or consists of the amino acid sequence of SEQ ID NO: 1 with or without an N-terminal signal peptide, i.e. may comprise or consist of the mature peptide sequence as shown in SEQ ID NO: 21; or

[0209] (b) is 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 an N-terminal signal peptide (as shown in SEQ ID NO: 21), which retains the activity of Gremlin-1, such as the amino acid sequence of SEQ ID NO: 20.

[0210] (c) variants thereof, such variants typically having 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 have about 60% to about 99% identity to SEQ ID NO: 1, suitably about 80% to about 99% identity to SEQ ID NO: 1, more suitably about 90% to about 99% identity to SEQ ID NO: 1, and most suitably about 95% to about 99% identity to SEQ ID NO: 1. Variants are further described below.

[0211] As discussed further below, residue numbers are generally referenced based on the sequence of SEQ ID NO: 1. However, a skilled person can easily extrapolate residue numbers to derivative or variant sequences as discussed above. Where residue numbers are referenced, the present invention also includes those residues on the variant or derivative sequence.

[0212] The GREM1 or Gremlin-1 nucleic acid sequence may comprise or consist of a sequence of SEQ ID NO: 36 or SEQ ID NO: 37 or a variant 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.

[0213] Antagonists

[0214] 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 to any extent. 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 GREM1 function or activity. By measuring the GREM1 function or activity in cells in the presence and absence of an anti-GREM1 antagonist, it can be determined to what extent an anti-GREM1 antagonist reduces the function or activity of GREM1. The cell can be a normal cell or a cancer cell. The cell can be a cancer cell as described above. They can be a pancreatic cancer cell. The pancreatic cancer cell can be present in a KPC mouse model as described in the Examples. Therefore, in vivo determination of the activity of GREM1 antagonists in combination therapy for pancreatic cancer can be performed in a mouse model. More generally, GREM1 antagonists that are shown to reduce the function or activity of GREM1 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.

[0215] The antagonist may reduce GREM1 function by any means. It may directly or indirectly increase or decrease the activity or amount of any molecule that affects GREM1 function. It may reduce the amount of GREM1 at the mRNA or protein level. It may increase the degradation of GREM1. It may reduce the function of GREM1 by inhibitory modification. It may reduce the transcription of molecules that enhance GREM1 function. It may use agents such as zinc finger nucleases to disrupt the DNA encoding GREM1 or molecules that enhance GREM1 function.

[0216] The antagonist can be an agent that interacts with Gremlin-1. An agent that interacts with Gremlin-1 is typically an agent that binds Gremlin-1. An agent that interacts with Gremlin-1 can modulate Gremlin-1. Inhibitory modulators can have an effect on any function of Gremlin-1, but typically reduce the binding of Gremlin-1 to BMP (BMP 2 / 4 / 7). The antagonist can be a BMP-7 mimetic molecule. Gremlin-1 is a negative regulator of BMP, so reduced binding increases signaling through BMP. Activating modulators can increase the binding of Gremlin-1 to BMP.

[0217] BMP binding and signaling can be detected by any method known in the art.

[0218] The antagonist may act by binding to the active site of GREM1, or may act allosterically by binding to a different site. The antagonist may act by binding to a modulator or ligand of GREM1, thereby reducing the activation of GREM1. The antagonist may be reversible or irreversible.

[0219] 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).

[0220] The antagonist of GREM1 can be an oligonucleotide that specifically hybridizes to an mRNA encoding GREM1 or an mRNA encoding a molecule that enhances GREM1 activity. The antagonist of GREM1 can be a polynucleotide encoding any molecule that reduces GREM1 function. For example, the GREM1 antagonist can be a polynucleotide encoding an anti-GREM1 antibody described herein.

[0221] An antagonist of GREM1 can be an antibody that specifically binds to any target molecule (usually a protein) thereby directly or indirectly reducing GREM1 function. The antagonist can be an antibody that specifically binds to GREM1. In this regard, the antibody can reduce GREM1 function by allosteric inactivation or by blocking the interaction between its target and a ligand required for activity.

[0222] The distance between the residue and the antagonist (usually less than 100 μm) is determined by any suitable method known in the art, such as by x-ray crystallography. or less than ), the interaction of the antagonist with the protein residues can be determined. Regions of Gremlin-1 that can be targeted by therapeutic agents may include amino acids Asp92-Leu99, Arg116-His130, Ser137-Ser142, Cys176-Cys178. These are the ones that are mutated on the surface of Gremlin-1. Within.

[0223] Antibody antagonists

[0224] The term "antibody" as used herein includes whole antibodies and any antigen-binding fragments (i.e., "antigen-binding portions") or single chains thereof. Antibodies are glycoproteins comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or antigen-binding portions thereof. Each heavy chain consists of a heavy chain variable region (abbreviated herein as HCVR or V H ) and a heavy chain constant region. Each light chain comprises a light chain variable region (abbreviated herein as LCVR or V L ) and a light chain constant region. The variable regions of the heavy and light chains contain binding domains that interact with antigens. H and V L The regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FRs).

[0225] 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.

[0226] The antibody used according to the present invention can be a monoclonal antibody or a polyclonal antibody, and will generally be a monoclonal antibody. The antibody used according to the present invention can be a chimeric antibody, a CDR-grafted antibody, a nanobody, a human or humanized antibody or any antigen binding portion thereof. In order to produce monoclonal antibodies and polyclonal antibodies, experimental animals are generally non-human mammals such as goats, rabbits, rats or mice, but the antibody can also be produced in other species.

[0227] Polyclonal antibodies can be produced by conventional methods, such as immunizing a suitable animal with an antigen of interest. Blood can then be taken from the animal and the IgG fraction purified.

[0228] When it is necessary to immunize an animal, antibodies against Gremlin-1 can be obtained by administering the polypeptide to an animal (e.g., a non-human animal) using a well-known and conventional protocol (see, e.g., Handbook of Experimental Immunology, DM Weir (ed.), Vol. 4, Blackwell Scientific Publishers, Oxford, England, 1986). Many warm-blooded animals, such as rabbits, mice, rats, sheep, cattle, camels, or pigs can be immunized. However, mice, rabbits, pigs, and rats are generally the most suitable.

[0229] Monoclonal antibodies can be prepared by any method known in the art, such as the hybridoma technique (Kohler and 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, pp. 77-96, Alan R Liss, Inc., 1985).

[0230] Antibodies used according to the present invention can also be produced using the single lymphocyte antibody method by cloning and expressing immunoglobulin variable region cDNAs produced by a single lymphocyte selected for the production of specific antibodies, for example by the methods described in Babcook, J. et al., 1996, Proc. Natl. Acad. Sci. USA 93(15):7843-78481, WO92 / 02551, WO2004 / 051268 and WO2004 / 106377.

[0231] The antibodies may also be produced using various phage display methods known in the art, including those disclosed in Brinkman et al. (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) and WO 90 / 02809; WO 91 / 10737; WO 92 / 01047; WO 92 / 18619; WO 93 / 11236; WO 95 / 15982; WO 95 / 20401; and US Pat. Nos. 5,698,426; 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.

[0232] Fully human antibodies are antibodies in which the variable and constant regions of the heavy and light chains, if present, are all from human origin, or are substantially identical to human sequences, but not necessarily from the same antibody. Examples of fully human antibodies may include antibodies, for example, produced by the above-mentioned phage display method and antibodies produced by mice, wherein murine immunoglobulin variable regions and optional constant region genes have been replaced by their human counterparts, for example, as generally described in EP 0546073, U5,545,806, US 5,569,825, US 5,625,126, US 5,633,425, US 5,661,016, US 5,770,429, EP 0438474, and EP0463151.

[0233] Alternatively, the antibodies used according to the present invention can be produced by a method comprising the steps of: immunizing a non-human mammal with a Gremlin-1 immunogen; obtaining an antibody preparation from the mammal; and deriving therefrom a monoclonal antibody that recognizes Gremlin-1.

[0234] The antibody molecule used according to the present invention can include a complete antibody molecule with 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 retains the ability to selectively bind to an antigen. It has been confirmed that the antigen-binding function of an antibody can be performed by a fragment of a full-length antibody. Antibodies and their fragments and antigen-binding portions can 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, divalent, trivalent or tetravalent antibodies, Bis-scFv, diabody, triabody, tetrabody, and an epitope-binding fragment 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 creating and making these antibody fragments are well known in the art (see, e.g., 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 Applications WO 2005 / 003169, WO 2005 / 003170 and WO2005 / 003171 and Fab-dAb fragments described in International Patent Application WO2009 / 040562. Multivalent antibodies may contain multiple specificities, or may be monospecific (see, e.g., WO 92 / 22853 and WO 05 / 113605). These antibody fragments may be obtained using conventional techniques known to those skilled in the art, and the fragments may be screened for use in the same manner as intact antibodies.

[0235] If present, the constant region domain of the antibody molecule can be selected taking into account the intended function of the antibody molecule and 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. Specifically, when the antibody molecule is intended for therapeutic use and antibody effector function is required, human IgG constant region domains, especially constant region domains of 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.

[0236] The antibodies used in accordance with the present invention can be prepared, expressed, created or isolated by recombinant means, such as (a) antibodies isolated from transgenic or transchromosomal animals (e.g., mice) for the immunoglobulin genes of interest or hybridomas prepared therefrom, (b) antibodies isolated from host cells transformed to express the antibodies of interest (e.g., from transfectomas), (c) antibodies isolated from recombinant combinatorial antibody libraries, and (d) antibodies prepared, expressed, created or isolated by any other means involving splicing of immunoglobulin gene sequences to other DNA sequences.

[0237] The antibody used according to the present invention can be a human antibody or a humanized antibody. The term "human antibody" used herein is intended to include antibodies with variable regions, wherein the framework region and the CDR region are all derived from human germline immunoglobulin sequences. In addition, if the antibody contains a constant region, the constant region is also derived from human germline immunoglobulin sequences. The human antibody described herein can include amino acid residues not encoded by human germline immunoglobulin sequences (for example, mutations introduced by random or site-specific in vitro mutagenesis or by somatic mutations in vivo). However, the term "human antibody" used herein is not intended to include such antibodies: wherein the CDR sequences derived from the germline of another mammalian species (such as mice) have been transplanted to human framework sequences.

[0238] Such human antibodies can be human monoclonal antibodies. Such human monoclonal antibodies can be produced by hybridomas comprising B cells obtained from transgenic non-human animals (e.g., transgenic mice) having a genome comprising human heavy chain transgenes and light chain transgenes fused to immortalized cells.

[0239] Human antibodies can be prepared by immunizing human lymphocytes in vitro and then transforming the lymphocytes with Epstein-Barr (EB) virus.

[0240] The term "human antibody derivative" refers to any modified form of a human antibody, for example, a conjugate of the antibody with another agent or antibody.

[0241] The term "humanized antibody" is intended to refer to a CDR-grafted antibody molecule in which CDR sequences derived from the germline of another mammalian species (such as a mouse) have been grafted onto human framework sequences. Additional framework region modifications may be made within the human framework sequences.

[0242] The term "CDR-grafted antibody molecule" as used herein refers to an antibody molecule in which the heavy chain and / or light chain contains one or more CDRs (including one or more modified CDRs, if desired) from a donor antibody (e.g., a murine or rat monoclonal antibody) that are grafted into the heavy chain and / or light chain variable region framework of an acceptor antibody (e.g., a human antibody). For review, see Vaughan et al., Nature Biotechnology, 16 , 535-539, 1998. In one embodiment, instead of the entire CDR being transferred, only one or more specificity determining residues from any one of the above CDRs are transferred to the human antibody framework (see, for example, Kashmiri et al., 2005, Methods, 36, 25-34). In one embodiment, only the specificity determining residues from one or more CDRs described above are transferred to the human antibody framework. In another embodiment, only the specificity determining residues from each of the above CDRs are transferred to the human antibody framework.

[0243] When transplanting CDR or specificity determining residues, considering the category / type of the donor antibody from which CDR is derived, including mouse, primate and human framework regions, any suitable acceptor variable region framework sequence can be used. Suitably, CDR-transplanted antibodies according to the present invention have variable domains, which comprise human acceptor framework regions and one or more of the above-mentioned CDR or specificity determining residues. Therefore, a kind of neutralization CDR-transplanted antibody is provided in one embodiment, wherein the variable domains comprise human acceptor framework regions and non-human donor CDR.

[0244] 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 sequences can be obtained, for example, at http: / / www.vbase2.org / (see Retter et al., Nucl. Acids Res. (2005) 33 (Suppl. 1), D671-D674).

[0245] In the CDR-grafted antibodies described herein, the acceptor heavy and light chains do not necessarily need to be derived from the same antibody, and may, if desired, comprise composite chains having framework regions derived from different chains.

[0246] In addition, in CDR-grafted antibodies described herein, framework regions do not need to have sequences identical to receptor antibodies. For example, for the receptor chain category or type, uncommon residues can be changed to residues that occur more often. Alternatively, the selected residues in the receptor framework regions can be changed so that they correspond to the residues found at the same position in the donor antibody (see Reichmann et al., 1998, Nature, 332, 323-324). Such changes should be kept at a minimum to restore the affinity of the donor antibody. A scheme for selecting residues that may need to be changed in the receptor framework regions is proposed in WO 91 / 09967.

[0247] It will also be appreciated by those skilled in the art that antibodies may undergo a variety of post-translational modifications. The type and extent of these modifications generally depend on the host cell line and culture conditions used to express the antibody. Such modifications may include changes in glycosylation, methionine oxidation, diketopiperazine formation, aspartic acid isomerization, and asparagine deamidation. A common modification is the loss of carboxyl terminal basic residues (such as lysine or arginine) due to the action of carboxypeptidases (as described in Harris, RJ. Journal of Chromatography 705: 129-134, 1995).

[0248] In one embodiment, the antibody heavy chain comprises a CH1 domain, and the antibody light chain comprises a CL domain (κ or λ).

[0249] Biomolecules (such as antibodies or fragments) contain acidic and / or basic functional groups, thereby giving the molecule a net positive or negative charge. The amount of "observed" charge in general will depend on the absolute amino acid sequence of the entity, the local environment of the charged groups in the three-dimensional 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, an 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 pI of the antibody is 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.

[0250] In order to characterize the preferred Gremlin-1 epitope, the inventors have crystallized human Gremlin-1 alone and in complex with an antibody (Fab fragment) called Ab 7326. Crystallization of Gremlin-1 enabled the putative residues in the BMP binding site to be determined. In addition, by crystallizing with the allosteric inhibitory antibody Ab 7326, the residues in the antibody epitope could be determined. Antibodies that bind to this epitope have particular potential as therapeutic agents for the treatment of diseases associated with Gremlin-1.

[0251] It has been determined that the preferred Ab 7326 antibody described herein binds 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), wherein Lys126(147), Lys127(148), Phe128(149), Thr129(150), Thr130(151), Arg148(169), Lys153(174) and Gln154(175) are present on one Gremlin-1 monomer and Ile110(131) is present on a second Gremlin-1 monomer. The numbering outside the brackets is based on the structure file (and it matches the numbering of mouse Gremlin-2 based on the structural alignment). The numbers in brackets represent the residues based on the UniProt entry O60565 of SEQ ID NO: 1. The Gremlin-1-Ab7326 Fab complex was analyzed using NCONT These epitope residues were identified.

[0252] Thus, the antibodies described herein can bind to an epitope comprising at least one residue selected from Ile131, Lys147, Lys148, Phe149, Thr150, Thr151, Arg169, Lys174, and Gln175 (residue numbering is based on SEQ ID NO: 1). The antibodies described herein can bind to an epitope comprising 2, 3, 4, 5, 6, 7, 8, or all 9 of these residues (preferably at least 5 residues).

[0253] The antibodies described herein may also recognize an epitope in which Ile131 is present on the Gremlin-1 monomer differently from other residues.

[0254] Although these residues are provided for a specific sequence of human Gremlin-1, the skilled person can easily extrapolate the positions of these residues to other corresponding Gremlin sequences (e.g., mouse) using conventional techniques. Therefore, the present invention also provides antibodies that bind to epitopes comprising corresponding residues within these other Gremlin sequences.

[0255] To screen for antibodies that bind to a specific epitope, a conventional cross-blocking assay, such as that described in Harlow and Lane's Antibodies (Cold Spring Harbor Press, Cold Spring Harb., NY) can be performed. Other methods include alanine scanning mutants, peptide blotting (Reineke (2004) Methods Mol Biol 248:443-63) or peptide cleavage analysis. In addition, methods such as epitope excision, epitope extraction, and chemical modification of the antigen can be used (Tomer (2000) Protein Science 9:487-496). Such methods are well known in the art.

[0256] Antibody epitopes can also be determined by x-ray crystallographic analysis. Thus, antibodies of the present disclosure can be evaluated by x-ray crystallographic analysis of antibodies that bind to Gremlin-1. Specifically, by determining the residues on Gremlin-1 that are in the antibody paratope, The epitope can be determined in this way.

[0257] Thus, the antibodies described herein can bind to an epitope on Gremlin-1 comprising at least one residue selected from Trp93, Phe117, Tyr119, Phe125, Tyr126, and Phe138, wherein the residue numbering is according to SEQ ID NO: 1. Further described herein is an antibody that binds to an epitope comprising all of Trp93, Phe117, Tyr119, Phe125, Tyr126, and Phe138. Additionally described is an antibody that binds to an epitope comprising 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 (Gremlin-1 dimer binds to BMP dimer).

[0258] If the antibody paratope is determined by x-ray crystallography to be at the residue If the antibody is within 100,000, the antibody can bind to the above-mentioned Gremlin-1 residues.

[0259] An antibody that binds to an epitope disclosed herein may comprise at least one, at least two or all three heavy chain CDR sequences (HCDR1 / HCDR2 / HCDR3, respectively) of SEQ ID NOs: 4 to 6. These are the HCDR1 / HCDR2 / HCDR3 sequences of the Ab 7326 antibody of the Examples determined using the Kabat method.

[0260] The Kabat and Chothia methods (and other techniques) for determining CDR sequences are well known in the art. Any appropriate method can be used to determine CDR sequences, and in the present invention, although Kabat is generally used, other techniques can also be used. In this example, SEQ ID NO:3 represents the Ab7326HCDR1 sequence determined using the Chothia and Kabat definitions of the combination.

[0261] The antibody used according to the invention may comprise at least one, at least two or all three light chain CDR sequences (LCDR1 / LCDR2 / LCDR3, respectively) of SEQ ID NOs: 7 to 9. These are the LCDR1 / LCDR2 / LCDR3 sequences of Ab 7326 obtained using the Kabat method.

[0262] The antibody preferably comprises at least the HCDR3 sequence of SEQ ID NO:6.

[0263] 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.

[0264] 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 a HCVR / LVCR pair of SEQ ID NO: 10 / 11 or 12 / 13).

[0265] The antibody may comprise the following heavy chain (H-chain) sequence:

[0266] SEQ ID NO: 14 Mouse full length IgG1 heavy chain variant 1, or

[0267] SEQ ID NO:28 Mouse full length IgG1 heavy chain variant 2, or

[0268] SEQ ID NO: 30 Human full length IgG1 heavy chain variant 1, or

[0269] SEQ ID NO: 16 Human full length IgG1 heavy chain variant 2, or

[0270] SEQ ID NO: 22 Human full length IgG4P heavy chain variant 1, or

[0271] SEQ ID NO: 34 Human full length IgG4P heavy chain variant 2, or

[0272] SEQ ID NO: 18 Fab heavy chain variant 1, or

[0273] SEQ ID NO:32Fab heavy chain variant 2.

[0274] The antibody may comprise the following light chain (L-chain) sequence:

[0275] SEQ ID NO: 15 Mouse full length IgG1 light chain variant 1, or

[0276] SEQ ID NO:29 Mouse full length IgG1 light chain variant 2, or

[0277] SEQ ID NO: 31 Human full length IgG1 light chain variant 1, or

[0278] SEQ ID NO: 17 Human full length IgG1 light chain variant 2, or

[0279] SEQ ID NO: 23 Human full-length IgG4P light chain variant 1, or

[0280] SEQ ID NO: 35 Human full-length IgG4P light chain variant 2, or

[0281] SEQ ID NO:19Fab light chain variant 1, or

[0282] SEQ ID NO:33Fab light chain variant 2.

[0283] In one embodiment, the antibody comprises the following heavy chain / light chain sequence pair:

[0284] SEQ ID NO:14 / 15 mouse full length IgG1 variant 1, or

[0285] SEQ ID NO:28 / 29 Mouse full length IgG1 variant 2, or

[0286] SEQ ID NO:30 / 31 human full length IgG1 variant 1, or

[0287] SEQ ID NO: 16 / 17 human full length IgG1 variant 2, or

[0288] SEQ ID NO:22 / 23 human full length IgG4P variant 1, or

[0289] SEQ ID NO:34 / 35 human full length IgG4P variant 2, or

[0290] SEQ ID NO:18 / 19 Fab light chain variant 1, or

[0291] SEQ ID NO:32 / 33 Fab light chain variant 2.

[0292] Variant forms of the corresponding sequences can be interchanged. For example, the antibody can comprise the following heavy chain / light chain sequence pairs:

[0293] SEQ ID NO: 14 / 29 mouse full length IgG1 heavy chain variant 1 / light chain variant 2, or

[0294] SEQ ID NO:28 / 15 mouse full length IgG1 heavy chain variant 2 / light chain variant 1, or

[0295] SEQ ID NO:30 / 17 human full length IgG1 heavy chain variant 1 / light chain variant 2, or

[0296] SEQ ID NO: 16 / 31 human full length IgG1 heavy chain variant 2 / light chain variant 1, or

[0297] SEQ ID NO: 22 / 35 human full-length IgG4P heavy chain variant 1 / light chain variant 2, or

[0298] SEQ ID NO: 34 / 23 human full-length IgG4P heavy chain variant 2 / light chain variant 1, or

[0299] SEQ ID NO: 18 / 33 Fab heavy chain variant 1 / light chain variant 2, or

[0300] SEQ ID NO:32 / 19Fab heavy chain variant 2 / light chain variant 1.

[0301] The antibody may be a chimeric antibody, a human antibody or a humanized antibody.

[0302] The antibody may alternatively be or may comprise a variant of one of the above-mentioned specific sequences.The following description of antibody variants also applies to the selection of GREM1 polypeptide variants as described above.

[0303] For example, the variant may be a substitution, deletion or addition variant of any of the above amino acid sequences.

[0304] Variant antibodies can include 1, 2, 3, 4, 5, up to 10, up to 20 or more (usually up to 50) amino acid substitutions and / or deletions from the specific sequences discussed above. "Deletion" variants can include the deletion of a single amino acid, the deletion of a small group of amino acids (such as 2, 3, 4 or 5 amino acids) or the deletion of a larger amino acid region (such as the deletion of a specific amino acid domain or other features). "Replacement" variants generally involve replacing one or more amino acids with the same number of amino acids and performing conservative amino acid substitutions. For example, an amino acid can be replaced by an alternative amino acid with 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 of the properties of the 20 main amino acids that can be used to select suitable substituents are as follows:

[0305]

[0306] "Derivatives" or "variants" generally include those in which the amino acids appearing in the sequence are not naturally occurring amino acids but structural analogs thereof. The amino acids used in the sequence may also be derivatized or modified, such as labeled, as long as the function of the antibody is not significantly adversely affected.

[0307] Derivatives and variants as described above may be prepared during antibody synthesis or by post-production modification, or when the antibody is in recombinant form, using site-directed mutagenesis, random mutagenesis or known techniques for enzymatic cleavage and / or nucleic acid ligation.

[0308] Variant antibodies may comprise amino acid sequences having greater than about 60% or greater than about 70% (e.g., 75 or 80%, typically greater than about 85%, for example greater than about 90 or 95%) amino acid identity with the amino acid sequences disclosed herein (particularly HCVR / LCVR sequences and H- and L-chain sequences). In addition, the antibodies may be variants having greater than about 60% or greater than about 70% (e.g., 75 or 80%, typically greater than about 85%, for example greater than about 90 or 95%) amino acid identity with the HCVR / LCVR sequences and H- and L-chain sequences disclosed herein, while retaining the precise CDRs disclosed for these sequences. Variants may retain at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the HCVR / LCVR sequences and H- and L-chain sequences disclosed herein (in some cases, while retaining the precise CDRs).

[0309] 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 seen over the entire length of the relevant SEQ ID NO sequence or a portion of the sequence, such as in about 20, 30, 50, 75, 100, 150, 200 or more amino acids, depending on the size of the full-length polypeptide.

[0310] With respect to amino acid sequences, "sequence identity" means sequences that have a specified value when assessed using ClustalW (Thompson et al., 1994, supra) with the following parameters:

[0311] Pairwise alignment parameters—method: accurate, matrix: PAM, gap opening penalty: 10.00, gap extension penalty: 0.10;

[0312] Multiple alignment parameters - Matrix: PAM, Gap Open Penalty: 10.00, Delayed Percent Identity: 30, Penalty End Gaps: On, Gap Separation Distance: 0, Negative Matrix: No, Gap Extension Penalty: 0.20, Residue-Specific Gap Penalty: On, Hydrophilic Gap Penalty: On, Hydrophilic Residues: GPSNDQEKR. Sequence identities at specific residues are intended to include identical residues that have been simply derivatized.

[0313] Thus provided are antibodies having specific sequences and variants that maintain the function or activity of these chains.

[0314] The antibody may compete for binding to Gremlin-1 with those defined above for H-chain / L-chain, HCVR / LCVR or CDR sequences, or bind to the same epitope as them. Specifically, the antibody may compete for binding to Gremlin-1 with an antibody comprising a HCDR1 / HCDR2 / HCDR3 / LCDR1 / LCDR2 / LCDR3 sequence combination of SEQ ID NO: 4 / 5 / 6 / 7 / 8 / 9, or bind to the same epitope as them. The antibody may compete for binding to Gremlin-1 with an antibody comprising a HCVR and LCVR sequence pair of SEQ ID NO: 10 / 11 or 12 / 13, or a full-length chain of SEQ ID NO: 14 / 15 or 16 / 17, or bind to the same epitope as them.

[0315] The term "epitope" is the region of an antigen to which an antibody binds. An epitope can be defined as structural or functional. Functional epitopes are typically subsets of structural epitopes and have those residues that contribute directly to the affinity of the interaction. An epitope can also be conformational, i.e., composed of nonlinear amino acids. In certain embodiments, an epitope can include a determinant, which is a chemically active surface grouping of a molecule (such as an amino acid, a sugar side chain, a phosphoryl group, or a sulfonyl group), and in certain embodiments, can have specific three-dimensional structural features and / or specific charge characteristics.

[0316] By using conventional methods known in the art, it can be easily determined whether an antibody binds to the same epitope as a reference antibody, or competes with the reference antibody for binding. 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 allowed to bind to a protein or peptide under saturation conditions. Next, the ability of the test antibody to bind to the protein or peptide is evaluated. If the test antibody is able to 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 is unable to 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.

[0317] To determine whether an antibody competes with a reference antibody for binding, the above binding method is performed in two directions. In the first direction, the reference antibody is allowed to bind to the protein / peptide under saturation conditions, and then the binding of the test antibody to the protein / peptide molecule is evaluated. In the second direction, the test antibody is allowed to bind to the protein / peptide under saturation conditions, and then the binding of the reference antibody to the protein / peptide is evaluated. If in both directions, only the first (saturated) antibody is able to bind to the protein / peptide, it is concluded that the test antibody and the reference antibody compete for binding to the protein / peptide. The skilled person will understand that an antibody that competes for binding with a reference antibody does not necessarily bind to the same epitope as the reference antibody, but may sterically block the binding of the reference antibody by binding to overlapping or adjacent epitopes.

[0318] If two antibodies each competitively inhibit (block) the binding of another antibody to the antigen, the two antibodies bind to the same or overlapping epitopes. That is, as measured in a competitive binding assay, a 1, 5, 10, 20 or 100-fold excess of one antibody will inhibit the binding of the other antibody by at least 50%, 75%, 90% or even 99% (see, e.g., Junghans et al., Cancer Res, 1990:50:1495-1502). Alternatively, if substantially all amino acid mutations in the antigen that reduce or eliminate the binding of one antibody reduce or eliminate the binding of the other antibody, the two antibodies have the same epitope. If certain amino acid mutations that reduce or eliminate the binding of one antibody reduce or eliminate the binding of another antibody, the two antibodies have overlapping epitopes.

[0319] 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 actually due to binding to the same epitope as the reference antibody or whether steric blocking (or another phenomenon) is causing the observed lack of binding. Such 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.

[0320] The antibodies can be tested for binding to Gremlin-1 by, for example, standard ELISA or Western blotting. ELISA assays can also be used to screen hybridomas that show positive reactivity with the target protein. The binding selectivity of the antibodies can also be determined by monitoring the binding of the antibodies to cells expressing the target protein, for example, by flow cytometry. Thus, the screening method can include the step of determining antibodies that are able to bind to Gremlin-1 by performing an ELISA or Western blotting or by flow cytometry.

[0321] The antibody can selectively (or specifically) recognize Gremlin-1. When an antibody or other compound binds with preferential or high affinity to a protein for which it is selective, but does not substantially bind or binds with low affinity to other proteins, the antibody or other compound "selectively binds" or "selectively recognizes" the protein. The selectivity of the antibody can be further studied by determining whether the antibody binds to other related proteins as discussed above or whether it discriminates between them. The antibodies used according to the present invention generally recognize human Gremlin-1.

[0322] The antibodies may also have cross-reactivity with related proteins or human Gremlin-1 and Gremlin-1 from other species.

[0323] By specific (or selective), it should be understood that the antibody is combined with the protein of interest, and there is no significant cross-reactivity with any other molecule. Cross-reactivity can be assessed by any suitable method described herein. If the intensity of antibody binding to another molecule is at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 100% of the intensity of the protein of interest, it can be considered that the cross-reactivity of the antibody is significant. The intensity of specific (or selective) antibody binding to another molecule can be about 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25% or 20% of the intensity of the protein of interest. An antibody may bind another molecule less than about 20%, less than about 15%, less than about 10%, or less than about 5%, less than about 2%, or less than about 1% as strongly as it binds the protein of interest.

[0324] Anti-gremlin antibodies have been described previously, for example WO 2014 / 159010A1 (Regeneron) describes anti-gremlin antibodies that inhibit Gremlin-1 activity with binding affinities K in the range of 625 pM to 270 nM at 25°C. D Ciuclan et al. (2013) described a -10 The binding affinity K of M D Anti-Gremlin-1 monoclonal antibody.

[0325] The anti-Gremlin-1 antibodies described herein (as well as WO / 2018 / 115017 filed on December 19, 2017 and WO2019 / 243801 filed on June 18, 2019, both of which are incorporated herein by reference in their entirety) are allosteric inhibitors of Gremlin-1 activity and bind to a novel epitope as described above that is distal to the BMP binding site. The antibodies bind to Gremlin-1 with extremely high binding affinity with a Kd value of <100 pM. Thus, the antibodies are significantly improved over currently available antibodies and are expected to be particularly useful for treating Gremlin-1-mediated diseases.

[0326] Thus, antibodies suitable for use with the present invention may have high affinity binding to (human) Gremlin-1. The antibodies may have a dissociation constant (K) of less than <1 nM and preferably <500 pM. D In one embodiment, the antibody has a dissociation constant (K) of less than 200 pM. D In one embodiment, the antibody has a dissociation constant (K) of less than 100 pM. D The binding affinity of an antibody to its target antigen can be determined using a variety of methods, such as surface plasmon resonance assays, saturation assays, or immunoassays such as ELISA or RIA, which are well known to those skilled in the art. An exemplary method for determining binding affinity is by using a CM5 sensor chip in a BIAcore TM Surface plasmon resonance analysis on a Biacore 2000 instrument (Biacore AB, Freiburg, Germany) as described by Krinner et al. (2007) Mol. Immunol. February; 44(5): 916-25. (Epub 2006 May 11).

[0327] The antibodies used according to the 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.

[0328] Specific functional assays that can be used to screen antibodies for their ability to inhibit Gremlin 1 include SMAD phosphorylation assays and Hek Id1 reporter gene assays. Typically, inhibitory antibodies will restore SMAD phosphorylation and / or restore BMP signaling in a Hek Id1 reporter gene assay. SMAD phosphorylation may be restored to at least 80%, 90%, or 100% compared to a BMP control. Inhibitory antibodies may have an IC of less than 10 nM, preferably less than 5 nM, in a Hek Id1 reporter gene assay. 50 .

[0329] Once a suitable antibody is determined and selected, the amino acid sequence of the antibody can be determined by methods known in the art. The gene encoding the antibody can be cloned using degenerate primers. The antibody can be recombinantly produced by conventional methods.

[0330] The present disclosure also provides isolated DNA sequences encoding the heavy and / or light chain variable regions (or full-length H-chains and L-chains) of the antibody molecules newly described herein.

[0331] A variant polynucleotide may comprise 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 of any nucleic acid sequence given in the sequence listing (including GREM1 and anti-GREM1 antibody nucleic acid sequences). Typically, the variant has 1-20, 1-50, 1-75 or 1-100 substitutions and / or deletions.

[0332] Suitable variants can have at least about 70% homology with the polynucleotide of any nucleotide sequence disclosed herein, usually with at least about 80% or 90% homology and more appropriately at least about 95%, 97% or 99% homology. Variant can retain at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity. Variant usually 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 usually exist at least relative to the coding region of the polynucleotide. The method of measuring homology is well known in the art, and it will be appreciated by those skilled in the art that in the context of the present invention, homology is calculated based on nucleic acid identity. Such homology can exist in a region of at least about 15, at least about 30, for example at least about 40, 60, 100, 200 or more consecutive nucleotides (depending on the length). Such homology can exist over the entire length of the unmodified polynucleotide sequence.

[0333] 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, pp. 387-395).

[0334] PILEUP and BLAST algorithms can also be used to calculate homology or align sequences (usually with their default settings), for example as described in Altschul SF (1993) J Mol Evol 36:290-300; Altschul, S, F et al. (1990) J Mol Biol 215:403-10.

[0335] Software for performing BLAST analyses is publicly available through the National Centre for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ). The algorithm involves first determining high scoring sequence pairs (HSPs) by determining short words of length W in the query sequence that match or satisfy some positive threshold score T when aligned with a word of the same length in the database sequence. T refers to the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word samples serve as seeds for initiating searches to find HSPs containing them. The word samples are extended in both directions along each sequence until the cumulative alignment score can be increased. Extension of the word samples in each direction is stopped when: the cumulative alignment score reaches zero or below due to the accumulation of one or more negatively scored residue alignments; 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 alignments (B), an expectation (E) of 10, M=5, N=4, and a comparison of both strands.

[0336] The BLAST algorithm performs a statistical analysis of the similarity between two sequences; see, e.g., Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5787. One measure of similarity provided by the BLAST algorithm is the smallest 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 first sequence is considered similar to a second sequence if the smallest sum probability of one sequence compared to the other sequence is less than about 1, typically less than about 0.1, suitably less than about 0.01, and most suitably less than about 0.001. For example, the smallest sum probability can be in the range of about 1 to about 0.001, typically about 0.01 to about 0.001.

[0337] The homologues may differ from the sequences of the relevant polynucleotides by less than about 3, 5, 10, 15, 20 or more mutations (each of which may be a substitution, deletion or insertion). For example, the homologues may differ by 3 to 50 mutations, typically 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 consecutive nucleotides of the homologues.

[0338] In one embodiment, variant sequences can be different from the specific sequences given in the sequence table due to the redundancy of the genetic code. The DNA code has 4 kinds of main nucleic acid residues (A, T, C and G), and uses these "spelling out" three-letter codons, which represent the amino acid of the protein encoded in the gene of the organism. The linear sequence of the codons along the DNA molecule is translated into the linear sequence of the amino acid in the protein encoded by those genes. The code is highly degenerate, with 61 codons encoding 20 kinds of natural amino acids, and 3 codons representing "stop" signals. Therefore, most amino acids are encoded by more than one codon-in fact, several amino acids are encoded by four or more different codons. Therefore, the variant polynucleotide of the present invention can encode the same polypeptide sequence as another polynucleotide of the present invention, but can have different nucleotide sequences, because different codons are used to encode the same amino acid.

[0339] The DNA sequence may comprise synthetic DNA, such as DNA produced by chemical processing, cDNA, genomic DNA, or any combination thereof.

[0340] The DNA sequence encoding the antibody molecules described herein can be obtained by methods well known to those skilled in the art. For example, a DNA sequence encoding part or all of the heavy and light chains of an antibody can be synthesized from a determined DNA sequence or based on the corresponding amino acid sequence as needed.

[0341] The general methods for constructing vectors, transfection methods and culture methods are well known to those skilled in the art. In this regard, reference is made to "Current Protocols in Molecular Biology", 1999, FM Ausubel (ed.), Maniatis Manual published by Wiley Interscience, New York and Cold Spring Harbor Publishing.

[0342] Nucleic acid antagonists

[0343] Polynucleotides (such as nucleic acids) are polymers comprising two or more nucleotides. The nucleotides may be naturally occurring or artificial. Nucleotides generally contain a nucleobase, a sugar and at least one linking group, such as a phosphate, 2'O-methyl, 2'methoxy-ethyl, phosphoramidate, methylphosphonate or thiophosphate group. The nucleobase is generally heterocyclic. Nucleobases include, but are not limited to, purines and pyrimidines, and more specifically include adenine (A), guanine (G), thymine (T), uracil (U) and cytosine (C). The sugar is generally a pentose. Nucleotide sugars include, but are not limited to, ribose and deoxyribose. The nucleotides are generally ribonucleotides or deoxyribonucleotides. The nucleotides generally contain monophosphates, diphosphates or triphosphates. Phosphate can be attached to the 5' or 3' side of the nucleotide.

[0344] 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 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 (dA 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 nucleotide is preferably selected from AMP, TMP, GMP, UMP, dAMP, dTMP, dGMP or dCMP.

[0345] The nucleotides may contain additional modifications. Specifically, suitable modified nucleotides include, but are not limited to, 2'aminopyrimidines (such as 2'-aminocytidine and 2'-aminouridine), 2'-hydroxypurines (such as 2'-fluoropyrimidines (such as 2'-fluorocytidine and 2'fluorouridine), hydroxypyrimidines (such as 5'-α-P-borouridine), 2'-O-methyl nucleotides (such as 2'-O-methyladenosine, 2'-O-methylguanosine, 2'-O-methylcytidine and 2'-O-methyluridine), 4'-thiopyrimidines (such as 4'-thiouridine and 4'-thiocytidine), and nucleotides have modifications of nucleobases (such as 5-pentynyl-2'-deoxyuridine, 5-(3-aminopropyl)-uridine and 1,6-diaminohexyl-N-5-carbamoylmethyluridine).

[0346] The nucleotides in the polynucleotide can be connected to each other in any way. The nucleotides can be connected by phosphate, 2'O-methyl, 2'methoxy-ethyl, phosphoramidate, methylphosphonate or thiophosphate bonds. The nucleotides are usually connected by their sugar and phosphate groups, as in nucleic acids. The nucleotides can be connected by their core bases, as in pyrimidine dimers.

[0347] The GREM1 antagonist may be a polynucleotide encoding an anti-GREM1 antibody described herein.

[0348] The polynucleotide can be a nucleic acid, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). The polynucleotide can 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 can be single-stranded or double-stranded.

[0349] The polynucleotide sequence can be cloned into any suitable expression vector. In the expression vector, the polynucleotide sequence encoding the construct is usually operably linked to a control sequence that can provide a host cell with expression of the encoding sequence. Such an expression vector can be used to express the construct.

[0350] In one embodiment, the anti-GREM1 antagonist is a polynucleotide encoding an anti-GREM1 antibody described herein. The polynucleotide may be provided for use in gene therapy. The polynucleotide may be provided in any suitable vector capable of providing for expression of an anti-GREM1 antibody in vivo.

[0351] The polynucleotide encoding the anti-GREM1 antibody can be a DNA sequence. The DNA sequence can be provided in any suitable vector (e.g., an expression vector) for administration to a subject in need thereof. For example, the DNA sequence can be administered to a subject in an expression vector capable of providing in vivo expression of the anti-GREM1 antibody. The expression vector can be a viral expression vector, such as 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, wherein the DNA sequence encodes 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, wherein the AAV comprises a DNA sequence encoding the anti-GREM1 antibody described herein.

[0352] The polynucleotide encoding the anti-GREM1 antibody can be an RNA sequence. The RNA sequence can be administered to a subject in need thereof in any suitable carrier. The RNA sequence can be a messenger RNA (mRNA) sequence. The mRNA sequence can be administered to a subject in need thereof in a stabilized form. For example, the mRNA sequence can be provided in a lipid nanoparticle (LNP) composition. The LNP composition can include any suitable LNP capable of encapsulating the mRNA sequence to provide increased stability of the mRNA sequence. Therefore, 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, wherein the mRNA sequence encodes the anti-GREM1 antibody described herein. In one embodiment, the anti-GREM1 antagonist is an 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, wherein the LNP composition comprises mRNA encoding an anti-GREM1 antibody described herein.

[0353] The term "operably linked" refers to a juxtaposition in which 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 linked 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 may be introduced into a vector.

[0354] The expression vector can then be introduced into a suitable host cell. Thus, a construct can be generated by inserting a polynucleotide sequence encoding a 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.

[0355] Nucleic acid-based GREM1 antagonists can reduce the expression of GREM1. Antisense and RNA interference (RNAi) technologies for knocking down protein expression are well known in the art, and standard methods can be used to knock down the expression of target molecules. Both antisense and siRNA technologies interfere with mRNA. Antisense oligonucleotides interfere with mRNA by binding (hybridizing) to a portion of mRNA. Therefore, antisense oligonucleotides are designed to be complementary to mRNA (although the oligonucleotides do not have to be 100% complementary as discussed below). In other words, antisense oligonucleotides can be part of a cDNA. In addition, the oligonucleotide sequence may not be 100% identical to the cDNA sequence. This is also discussed below. RNAi includes the use of double-stranded RNA that can bind to mRNA and inhibit protein expression, such as small interfering RNA (siRNA) or small hairpin RNA (shRNA).

[0356] Thus, the antagonist may be an oligonucleotide that specifically hybridizes to an mRNA encoding GREM1, such as 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 when the oligonucleotide hybridizes to the target sequence with preferential or high affinity, but does not substantially hybridize, does not hybridize, or hybridizes with only low affinity to other sequences. More preferably, the oligonucleotide hybridizes to the target sequence with a high affinity. m T than other nucleic acids m The conditions for hybridization are at least 5°C, at least 10°C, at least 20°C, at least 30°C or at least 40°C higher. 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 conditions may be stringent conditions described in the art.

[0357] Oligonucleotide is a short nucleotide polymer, which usually has 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 nucleotides. The oligonucleotide length used can be 20 to 25 nucleotides, and more preferably the length is 21 or 22 nucleotides. The nucleotide can be naturally occurring or artificial. The nucleotide can be any of the above-mentioned ones.

[0358] A GREM1 antagonist can be an antibody that binds to GREM1, typically specifically. An antibody "specifically binds" a protein when it binds to it preferentially or with high affinity, but does not substantially bind, does not bind, or only binds to other proteins with low affinity. For example, an antibody "specifically binds" a target molecule when it binds to it preferentially or with high affinity, but does not substantially bind, does not bind, or only binds to other human proteins with low affinity.

[0359] If the antibody is present at 1x 10 -7 M or less, more preferably 5 x 10 -8 M or less, more preferably 1 x 10 -8 M or lower or more preferably 5 x 10 -9 If the antibody binds with a Kd of 1 x 10 -6 M or more, more preferably 1 x 10 -5 M or more, more preferably 1 x 10 -4 M or more, more preferably 1 x 10 -3 M or higher, even more preferably 1 x 10 -2 If the antibody binds with a Kd of M or higher, then the antibody binds with low affinity.

[0360] The antibody can 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 can be a complete immunoglobulin molecule or a fragment thereof such as a Fab, F(ab')2 or Fv fragment.

[0361] patient

[0362] Any patient may be treated according to the present invention. The patient is typically a human. However, the patient may be another mammal, such as a commercially raised 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.

[0363] Pharmaceutical compositions, dosages and dosage regimens

[0364] GREM1 antagonists for use in the methods of the present invention can be provided in pharmaceutical compositions. Ras-Raf-MEK-ERK signaling inhibitors (such as MEK inhibitors or ERK inhibitors described herein) for use in the methods of the present invention can also be provided as part of the same pharmaceutical composition or in a separate pharmaceutical composition. For example, a GREM1 antagonist for use in the methods of the present invention can be provided in a pharmaceutical composition together with a MEK inhibitor or ERK inhibitor described herein or other inhibitors of Ras-Raf-MEK-ERK signaling. The pharmaceutical compositions are generally sterile and generally include a pharmaceutically acceptable carrier and / or adjuvant. In addition to the therapeutically active ingredients, these compositions may also include 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 should not interfere with the efficacy of the active ingredient. The pharmaceutical carrier or diluent can be, for example, an isotonic solution.

[0365] As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc. that are physiologically compatible. 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.

[0366] The carrier can be suitable for parenteral administration, such as intravenous, intramuscular, intradermal, intraocular, intraperitoneal, subcutaneous, spinal or other parenteral administration routes, such as by injection or infusion. Alternatively, the carrier can be suitable for non-parenteral administration, such as topical, epidermal or mucosal administration routes. The carrier can be suitable for oral administration. Depending on the route of administration, the modulator can be wrapped in a material to protect the compound from the effects of acids and other natural conditions that may inactivate the compound. For example, solid oral forms may contain, together with the active substance: diluents, such as lactose, dextrose, sucrose, cellulose, corn starch or potato starch; lubricants, such as silicon dioxide, talc, stearic acid, magnesium stearate or calcium stearate and / or polyethylene glycol; binders, such as starch, gum arabic, gelatin, methylcellulose, carboxymethylcellulose or polyvinylpyrrolidone; deagglomerants, such as starch, alginic acid, alginates or sodium starch glycolate; effervescent mixtures; dyes; sweeteners; wetting agents, such as lecithin, polysorbate, lauryl sulfate; and, in general, nontoxic and pharmacologically inactive substances used in pharmaceutical preparations. Such pharmaceutical preparations can be manufactured in a known manner, for example by means of mixing, granulation, tableting, sugar coating or film coating processes.

[0367] Other oral formulations include commonly used excipients such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. These compositions are in the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain 10% to 95% of active ingredients, preferably 25% to 70%. In the case where the pharmaceutical composition is lyophilized, the lyophilized material can be reconstituted, such as a suspension, before administration. Reconstitution is preferably carried out in a buffer.

[0368] Capsules, tablets, and pills for oral administration to an individual can be provided with enteric coatings including, for example, Eudragit "S", Eudragit "L", cellulose acetate, cellulose acetate phthalate, or hydroxypropyl methylcellulose.

[0369] Liquid dispersions for oral administration may be syrups, emulsions or suspensions. The syrups may contain, for example, sucrose or sucrose with glycerol and / or mannitol and / or sorbitol as carriers.

[0370] Suspensions and emulsions may contain, for example, natural gums, agar, sodium alginate, pectin, methylcellulose, carboxymethylcellulose or polyvinyl alcohol as carriers. Suspensions or solutions for intramuscular injection may contain, together with the active substance, a pharmaceutically acceptable carrier such as sterile water, olive oil, ethyl oleate, glycols such as propylene glycol, and, if necessary, an appropriate amount of lidocaine hydrochloride.

[0371] Solutions for intravenous administration or infusion may contain, for example, sterile water as a carrier, or preferably they may be in the form of sterile, aqueous, isotonic saline solutions.

[0372] 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%.

[0373] The polynucleotide or oligonucleotide inhibitor can be a naked nucleotide sequence, or combined with a cationic lipid, polymer or targeting system. They can be delivered by any available technology. For example, the polynucleotide or oligonucleotide can be introduced by needle injection, preferably intradermal, subcutaneous or intramuscular injection. Alternatively, a delivery device such as particle-mediated gene delivery can be used to directly deliver the polynucleotide or oligonucleotide through the skin. The polynucleotide or oligonucleotide can be topically applied to the skin or mucosal surface, for example, by intranasal, oral or rectal administration.

[0374] The uptake of polynucleotide or oligonucleotide constructs can be enhanced by several known transfection techniques, such as those including the use of transfection agents. Examples of these agents include cationic agents such as calcium phosphate and DEAE-dextran, and lipofectants such as lipofectam and transfectam. The dosage of the polynucleotide or oligonucleotide to be administered can be varied.

[0375] The pharmaceutical composition of the present invention may include one or more pharmaceutically acceptable salts. "Pharmaceutically acceptable salts" means salts that retain the desired biological activity of the parent compound and do not produce any undesirable toxicological effects. Examples of such salts include acid addition salts and base addition salts.

[0376] Pharmaceutically acceptable carriers include aqueous carriers or diluents. Examples of suitable aqueous carriers that can be used in the pharmaceutical composition of the present invention include water, buffered water and saline. Examples of other carriers include ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.) and their suitable mixtures, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. In many cases, it is desirable to include isotonic agents in the composition, for example, sugar, polyols (such as mannitol, sorbitol) or sodium chloride.

[0377] 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 for high drug concentration.

[0378] The pharmaceutical compositions of the present invention may contain additional active ingredients.

[0379] Kits comprising the combination therapies described herein and instructions for use are also within the scope of the present disclosure.The kits may further contain one or more additional agents, such as additional therapeutic or prophylactic agents discussed herein.

[0380] The antagonists described herein, or formulations or compositions thereof, may be administered for prophylactic and / or therapeutic treatments.

[0381] In therapeutic applications, a compound is administered to a subject already suffering from an obstacle or condition as described above in an amount sufficient to cure, alleviate or partially inhibit the condition or one or more symptoms thereof. Such therapeutic treatment may result in a reduction in the severity of disease symptoms, or an increase in the frequency or duration of the asymptomatic phase. An amount sufficient to achieve this purpose is defined as a "therapeutically effective amount".

[0382] In preventive applications, a subject at risk of a disorder or condition as described above is administered an amount of the formulation sufficient to prevent or alleviate the subsequent effects of the condition or one or more of its symptoms. An amount sufficient to achieve this purpose is defined as a "prophylactically effective amount". The effective amount for each purpose will depend on the severity of the disease or injury and the weight and overall condition of the subject.

[0383] The administration subject can be a human or a non-human animal. The term "non-human animal" includes all vertebrates, for example, mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc. It is usually administered to humans.

[0384] The antagonists, Ras-Raf-MEK-ERK signaling inhibitors (e.g., MEK or ERK inhibitors), or pharmaceutical compositions of the present invention may be administered by one or more routes of administration using one or more of a variety of methods known in the art. The skilled artisan will appreciate that the route and / or mode of administration will vary with the desired results. 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 by injection or infusion. The phrase "parenteral administration" as used herein refers to a mode of administration other than enteral and topical administration, typically by injection. Alternatively, the antibodies / modulators or pharmaceutical compositions of the present invention may be administered by non-parenteral routes, such as topical, epidermal or mucosal routes of administration. The antibodies / modulators or pharmaceutical compositions of the present invention may be used for oral administration.

[0385] The appropriate dosage of the antibody / modulator or pharmaceutical composition of the present invention can be determined by skilled medical practitioners. The actual dosage level of the active ingredient in the pharmaceutical composition of the present invention can be changed to obtain the amount of the active ingredient that effectively achieves the desired therapeutic response for a specific patient, composition and mode of administration without toxicity to the patient. The dosage level selected will depend on a variety of pharmacokinetic factors, including the activity of the specific composition of the present invention adopted, the route of administration, the time of administration, the excretion rate of the specific compound being adopted, the duration of treatment, other drugs, compounds and / or materials used in combination with the specific composition used, the age, sex, weight, condition, overall health and previous medical history of the patient treated, and similar factors well-known in the medical field.

[0386] According to the above, for the patient to be treated, a suitable dosage can be, for example, in the range of about 0.01 μg / kg to about 1000 mg / kg body weight, typically about 0.1 μg / kg to about 100 mg / kg body weight. For example, a suitable dosage can be about 1 μg / kg to about 10 mg / kg body weight per day, or about 10 μg / kg to about 5 mg / kg body weight per day.

[0387] The dosage regimen can be adjusted to provide the best desired response (e.g., therapeutic response). For example, a single dose can be administered, several divided doses can be administered over time, or the dosage can be proportionally reduced or increased as indicated by the urgency of the treatment situation. The dosage can be provided as multiple doses, for example, administered at fixed intervals, for example, 2, 3 or 4 doses are administered per hour. Multiple doses can be administered by the same or different routes, to the same or different locations. Alternatively, the dosage can also be by sustained release formulations, in which case, less frequent administration is required. Dosage and frequency can depend on the half-life of the antagonist in the patient and the desired treatment duration.

[0388] 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.

[0389] Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject 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.

[0390] As noted above, the modulators / antibodies or pharmaceutical compositions of the invention may be co-administered with one or more other therapeutic agents.

[0391] Examples of the above-mentioned techniques and protocols can be found in: Remington's Pharmaceutical Sciences, 20th edition, 2000, published by Lippincott, Williams and Wilkins.

[0392] The combined administration of two or more agents can be achieved in a variety of different ways. Both can be administered together in a single composition, or they can be administered as part of a combination therapy in a separate composition. For example, one can be administered before or alone, after or in sequence, or in parallel or simultaneously with the other. For example, the anti-GREM1 antagonist can be administered before or alone, after or in sequence, or in parallel or simultaneously with the Ras-Raf-MERK-ERK signaling inhibitor. For example, the anti-GREM1 antagonist can be administered before or alone, after or in sequence, or in parallel or simultaneously with the MEK inhibitor described herein. In another aspect of the invention, the anti-GREM1 antagonist can be administered before or alone, after or in sequence, or in parallel or simultaneously with the ERK inhibitor described herein.

[0393] Compositions and kits

[0394] In addition, a composition or kit comprising an anti-GREM1 antagonist and a Ras-Raf-MEK-ERK signaling inhibitor is provided. The anti-GREM1 antagonist can be any anti-GREM1 antagonist described herein. The Ras-Raf-MEK-ERK signaling inhibitor can be any Ras-Raf-MEK-ERK signaling inhibitor 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 may 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 uritinib.

[0395] A bispecific antibody combining an anti-GREM1 specificity and one of the other above-mentioned specificities may be provided in a composition or kit described herein.The anti-GREM1 antagonist in any of the above-mentioned compositions and kits may preferably be an anti-GREM1 antibody.

[0396] Other treatment combinations

[0397] The combination therapy of the present invention as described above can be used / administered in combination with another therapeutic composition for treatment, for example as an adjuvant therapy. The other therapeutic composition or treatment can be one or more of those discussed herein, and can be administered simultaneously or sequentially with the composition of the present invention.

[0398] GREM1 antagonists have particular use in combination therapies because they can be used to sensitize cancers or tumors to other anticancer agents, such as radiation therapy or surgery. In the absence of a GREM1 antagonist, the cancer may be resistant to other anticancer agents or cancer therapies.

[0399] Therefore, the anti-GREM1 antagonist combined with the Ras-Raf-MEK-ERK signaling inhibitor can be further used in combination with any other cancer therapy or any other cancer therapeutic agent. The other cancer therapy can be selected from any known therapy for related cancers, such as any known therapy for pancreatic cancer. The other cancer therapy can be radiotherapy. For example, Van Cutsem (and others) described suitable radiotherapy treatment in Annals of Oncology, 2014. Volume 25, Issue 3. The radiotherapy can be performed before cancer surgery or after cancer surgery. The radiotherapy can be adjuvant radiotherapy. The radiotherapy can be combined with the Ras-Raf-MEK-ERK signaling inhibitor described herein. For example, a combination therapy comprising a GREM1 antagonist and selumetinib (AZD6244) can be used in combination with radiotherapy. In addition, a combination therapy comprising a GREM1 antagonist and trametinib (GSK-1120212) can be used in combination with radiotherapy. Alternatively, a combination therapy comprising a GREM1 antagonist and WX-554 (UCB-554) can be used in combination with radiotherapy. A combination therapy comprising a GREM1 antagonist and any MEK inhibitor described herein can be used in combination with radiotherapy. Alternatively, a combination therapy comprising a GREM1 antagonist and any ERK inhibitor described herein can be used in combination with radiotherapy. For example, a combination therapy comprising a GREM1 antagonist and Uritinib can be used in combination with radiotherapy.

[0400] The cancer may be resistant to radiation therapy when not administered with a combination therapy described herein comprising a GREM1 antagonist.

[0401] As part of the above aspects, the present invention provides an anti-GREM1 antagonist according to the present invention in combination with a Ras-Raf-MEK-ERK signaling inhibitor for use in a method of treating and / or preventing cancer (such as pancreatic cancer), wherein the method further comprises administering an additional anti-cancer agent, either alone, sequentially or simultaneously. In a preferred embodiment, the present invention provides an anti-GREM1 antagonist according to the present invention in combination with one or more MEK inhibitors (such as selumetinib (AZD6244), trametinib (GSK-1120212) and / or WX-554 (UCB-554)), for use in a method of treating and / or preventing cancer (such as pancreatic cancer), wherein the method further comprises administering an additional anti-cancer agent, either alone, sequentially or simultaneously.

[0402] 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 may provide an anti-GREM1 antagonist in combination with the MEK inhibitor selumetinib (AZD6244) according to the present invention for use in a method of treating and / or preventing cancer, such as pancreatic cancer, wherein the method further comprises administering, alone, sequentially or simultaneously, an additional MEK inhibitor, such as trametinib (GSK-1120212), cobimetinib (GDC-0973), bimetinib (MEK162), CI-1040 (PD184352), modametinib (PD0325901), TAK733, refactinib (RDEA119 / Bay 86-9766), RO-5126766, RO5068760, pimasetinib (AS703026), AZD8330, GDC-0623, RO-4987655, WX-554 (UCB-554), HL-085, ARRY-300, ClnQ-03, G-573, PD184161, PD318088, PD98059, U0126, or SL327.

[0403] In an alternative embodiment, the present invention may provide an anti-GREM1 antagonist according to the present invention in combination with the MEK inhibitor trametinib (GSK-1120212) for use in a method of treating and / or preventing cancer (such as pancreatic cancer), wherein the method further comprises administering, alone, sequentially or simultaneously, an additional MEK inhibitor such as selumetinib (AZD6244), cobimetinib (GDC-0973), bimetinib (MEK162), CI-1040 (PD184352), modametinib (PD0325901), TAK733, rivatinib (RDEA119 / Bay 86-9766), RO-5126766, RO5068760, pimasetinib (AS703026), AZD8330, GDC-0623, RO-4987655, WX-554 (UCB-554), HL-085, ARRY-300, ClnQ-03, G-573, PD184161, PD318088, PD98059, U0126, or SL327.

[0404] In yet another alternative embodiment, the present invention may provide an anti-GREM1 antagonist according to the present invention in combination with a MEK inhibitor WX-554 (UCB-554) for use in a method of treating and / or preventing cancer (such as pancreatic cancer), wherein the method further comprises administering, alone, sequentially or simultaneously, an additional MEK inhibitor, such as trametinib (GSK-1120212), selumetinib (AZD6244), cobimetinib (GDC-0973), bimetinib (MEK162), CI-1040 (PD184352), modametinib (PD0325901), TAK733, rivatinib (RDEA119 / Bay 86-9766), RO-5126766, RO5068760, pimatitinib (AS703026), AZD8330, GDC-0623, RO-4987655, HL-085, ARRY-300, ClnQ-03, G-573, PD184161, PD318088, PD98059, U0126, or SL327.

[0405] In yet another alternative embodiment, the present invention may provide an anti-GREM1 antagonist according to the present invention in combination with the ERK inhibitor uritinib for use in a method for treating and / or preventing cancer (such as pancreatic cancer), wherein the method further comprises administering an additional ERK inhibitor, such as uritinib, temuterkib, JSI-1187, ravoxertinib, ASN007 or MK-8353, separately, sequentially or simultaneously.

[0406] The present invention may further provide an anti-GREMl antagonist in combination with a MEK inhibitor and further in combination with an ERK inhibitor described herein.

[0407] Detection and diagnosis

[0408] Based on the correlation between interstitial GREM1 and cancer, combined with the finding that genes associated with the Ras-Raf-MEK-ERK pathway are upregulated after treatment with a GREM1 antagonist, the present invention also provides additional methods for predicting the responsiveness of cancer to treatment. Such methods may include measuring Ras-Raf-MEK-ERK pathway signaling, interstitial expression and / or epithelial expression of GREM1 in samples obtained from patients.

[0409] The present invention provides a method for determining whether a patient having or suspected of having cancer or at risk of developing cancer is likely to respond to a combination treatment with a GREM1 antagonist and a Ras-Raf-MEK-ERK signaling inhibitor, the method comprising measuring the mesenchymal and / or epithelial expression of GREM1 and Ras-Raf-MEK-ERK signaling in the patient, and thereby predicting whether the patient is likely to respond to treatment with the combination.

[0410] The present invention further provides a method for determining whether a patient having or suspected of having cancer or at risk of developing cancer is likely to respond to a combination treatment with an anti-GREM1 antagonist and a Ras-Raf-MEK-ERK signaling inhibitor, the method comprising measuring Ras-Raf-MEK-ERK pathway signaling in a patient after treatment / exposure with an anti-GREM1 antagonist, and thereby predicting whether the patient is likely to respond to treatment with the combination. For example, upregulated / induced Ras-Raf-MEK-ERK signaling in response to anti-GREM1 antagonist treatment can indicate that the patient is a good candidate for a combination therapy according to the present invention. The present invention further provides a method for determining whether a patient having or suspected of having pancreatic cancer or at risk of developing pancreatic cancer is likely to respond to a combination treatment with an anti-GREM1 antagonist and a Ras-Raf-MEK-ERK signaling inhibitor, the method comprising measuring Ras-Raf-MEK-ERK pathway signaling in a patient after treatment / exposure with an anti-GREM1 antagonist, and thereby predicting whether the patient is likely to respond to treatment with the combination. For example, upregulated Ras-Raf-MEK-ERK signaling in response to anti-GREM1 antagonist treatment can indicate that the patient is a good candidate for a combination therapy according to the present invention. The present invention further provides a method for determining whether a patient having or suspected of having pancreatic cancer or at risk of developing pancreatic cancer is likely to respond to a combination treatment with a GREM1 antagonist and a Ras-Raf-MEK-ERK signaling inhibitor, the method comprising measuring the mesenchymal and / or epithelial expression of GREM1 in the patient and determining whether the patient has a RAS gene or RAF gene mutation, and thereby predicting whether the patient is likely to respond to treatment with the combination.

[0411] The predicted responsiveness of an individual to a given therapy means that the individual is expected to gain a benefit or a sufficient degree of benefit from receiving the therapy. The predicted non-responsiveness of an individual to therapy means that the individual is not expected to gain a benefit or a sufficient degree of benefit from receiving the therapy. The method for predicting a response can be performed before administering a combination therapy of a GREM1 antagonist and a Ras-Raf-MEK-ERK signaling inhibitor. The prediction can then be considered when selecting or recommending a suitable treatment for the individual. Alternatively, the method can be performed after treatment with the therapy and used to monitor and predict the individual's response to treatment. Typically, the method is used to predict whether the individual will have an initial response to treatment using the therapy, i.e., whether the individual will respond when receiving the therapy for the first time. In some cases, the method is used to predict secondary non-responsiveness, i.e., whether an individual who initially responds to treatment will stop responding to treatment later or will respond poorly to treatment.

[0412] In some cases, overexpression of GREM1 as compared to a reference sample or reference level indicates that the individual will respond to therapy using a combination therapy described herein. Disregulation of Ras-Raf-MEK-ERK signaling, such as upregulation of Nras, Mapk1 and / or Myc as compared to a reference sample or reference level may also indicate that the individual will respond to a combination therapy described herein. Ras-Raf-MEK-ERK signaling can be measured by examining phosphorylation levels, for example, using phosphorylation site-specific antibodies, such as phosphorylation-specific antibodies to MEK and ERK. For example, increased phosphorylation of Map2k1 / Map2k2 / Erk1 / Erk2 in response to treatment with an anti-GREM1 antagonist of the present invention as compared to a reference sample or reference level indicates that the individual will respond to therapy using a combination therapy described herein. A combination therapy comprising a GREM1 antagonist in combination with a Ras-Raf-MEK-ERK signaling inhibitor (such as a MEK inhibitor or an ERK inhibitor) can then be selected or recommended, and can then be further administered to the individual.

[0413] In other cases, a reduced or normal GREM1 level compared to a reference sample or reference level indicates that the individual will not respond to therapy with a GREM1 antagonist. Normal or reduced Ras-Raf-MEK-ERK signaling compared to a reference sample or reference level can indicate that the individual will not respond to therapy with a Ras-Raf-MEK-ERK signaling inhibitor. A combination therapy comprising a GREM1 antagonist and a Ras-Raf-MEK-ERK signaling inhibitor is then administered to the individual. In addition, a therapeutic treatment other than the combination therapy described herein can be selected or recommended to treat the individual and can then be further administered to the individual.

[0414] In other cases, a mutation in a RAS gene (such as KRAS, NRAS and / or HRAS) or a RAF gene (such as ARAF, BRAF and / or CRAF) indicates that the individual will respond to therapy using a combination therapy described herein. A combination therapy comprising a GREM1 antagonist in combination with a Ras-Raf-MEK-ERK signaling inhibitor may then be selected or recommended and may then be further administered to the individual.

[0415] In all aspects of the invention, an individual suffering from cancer (e.g., pancreatic cancer) or an individual suspected of suffering from the disease or condition and / or an individual at risk of suffering from the disease or condition may be selected for treatment or determination. For example, the individual may not have been formally diagnosed, but may be suspected of suffering from the disease or condition due to the presence of one or more symptoms. An individual may be considered to be at risk of suffering from cancer if he or she has one or more risk factors associated with cancer and / or one or more tendencies that increase his or her susceptibility to cancer. Risk factors associated with pancreatic cancer may include familial pancreatitis caused by mutations in the PRSS1 gene and inherited genetic mutations, such as one or more mutations in the GREM1 encoding gene or any other mutation that affects GREM1 gene expression.

[0416] The following examples illustrate the invention. Example

[0417] Materials and methods

[0418] Anti-gremlin-1 antibody - Ab7326 mIgG1-APP.4405.IgG.mFc

[0419] Vehicle - Phosphate buffered saline pH 7.4 (provided by Beatson Institute)

[0420] Selumetinib (AZD6244) - provided by the Beatson Institute (purchased from SYNthesis Pharm) in 0.5% HPMC + 0.1% Tween-80.

[0421] UCB-554 MEK inhibitor (UCB1366554-000, batch ID B10476792, provided by UCB) in 10% DMSO + 90% 1% (w / v) methylcellulose. Administered at 10 mg / kg in 125 μl, once every 2 days orally

[0422] The vehicle in Example 5 was 125 μl of 10% DMSO + 90% of 1% (w / v) methylcellulose, administered orally once every 2 days (po, q2d)

[0423] Genetically modified mice

[0424] LSL has been described previously. K ras G12D / + Tr p 53 R172H / + ;Pdx1- C re (KPC) mice (Hingorani et al., 2005). Mice were generated by crossing mice with Pdx1-Cre and conditional LSL-KrasG12D or LSL-Trp53R172H alleles (strains 01XJ6 and 01XL9, mouse models of the Human Cancer Consortium [Mouse Models of Human Cancer Consortium, MMHCC], NCI-Frederick, Frederick, MD, USA). Mice with mixed backgrounds were bred internally at the CRUK Beatson Institute and maintained in conventional cages with environmental enrichment, with free access to standard food and water. Genotyping was performed by Transnetyx (Cordoba, TN, USA). Mice of both sexes were recruited for the study. All animal experiments were performed under the permission of the UK Home Office and approved by the Animal Welfare and Ethical Review Committee of the University of Glasgow.

[0425] Treatment of Examples 3 and 4

[0426] Mice were monitored 3 times a week until pancreatic cancer was diagnosed by abdominal palpation and confirmed by ultrasound imaging. Mice were randomly assigned to treatment groups and administered as follows: 30 mg / kg Ab7326mIgG1 subcutaneously twice a week; or 30 mg / kg Ab7326mIgG1 subcutaneously twice a week and 25 mg / kg selumetinib orally (po, bid) twice a day. Historical data of mice treated orally with 25 mg / kg selumetinib twice a day are provided. Each group has ≥12 mice. Mice were monitored daily and killed 7 days after the start of treatment (4 mice per group) or when the ethical endpoint was reached (≥8 mice per group, symptoms included abdominal distension, cachexia, intermittent arched back or decreased activity, erect hair, mild diarrhea, anemia). The survival of these mice from the start of treatment was statistically evaluated by Kaplan-Meier and log-rank analysis.

[0427] Ultrasound imaging

[0428] The VisualSonics Vevo 3100 preclinical imaging platform (FUJIFILM VisualSonics, Toronto, Canada) was used for high-resolution ultrasound imaging to confirm tumor diagnosis and monitor tumor progression weekly. Anesthesia was induced and maintained with a mixture of isoflurane and medical air. Tumor volume was calculated and plotted longitudinally for each mouse weekly.

[0429] sampling

[0430] Mice were sacrificed according to the protocol 1 in accordance with institutional guidelines. Tumor burden was assessed postmortem by gross pathology and histology. Organs were removed and fixed in 10% buffered formalin. At sacrifice, terminal blood samples were collected by cardiac puncture and processed for serum when possible. The majority of the tumor was fixed in 10% buffered formalin for FFPE processing, and any remaining portions were harvested to RNA preparation and / or snap freezing were performed in Sigma-Aldrich. Fixed tissues were embedded in paraffin and 5 μm sections were mounted on poly-L-lysine slides for IHC analysis.

[0431] Histology and immunohistochemistry

[0432] As previously described, formalin-fixed, paraffin-embedded tissues were stained with hematoxylin and eosin and picrosirius red. Immunohistochemistry was performed using standard protocols. In brief, formalin-fixed paraffin-embedded sections were deparaffinized and rehydrated through xylene and graded alcohol series. Endogenous peroxidase activity was inactivated by treatment with hydrogen peroxide, followed by antigen retrieval with citrate buffer. The sections were blocked in serum and then incubated with primary antibodies. The sections were incubated in secondary antibodies for 30 minutes and stained with 3,3′-diaminobenzidine tetrahydrochloride. The primary antibodies used were anti-α-SMA (1A4, Sigma-Aldrich) 1:20,000) and anti-podoplanin (RTD4E10, AbCam 1:250).

[0433] Treatment of Example 5

[0434] Mice were monitored 3 times per week until pancreatic cancer was diagnosed by abdominal palpation and confirmed by ultrasound imaging. Mice were randomly assigned to treatment groups (n=9-11 per group) and administered: 125 μl vehicle, orally once every 2 days; 30 mg / kg Ab7326 mIgG1 subcutaneously twice a week; 25 mg / kg selumetinib orally twice a day; 30 mg / kg Ab7326 mIgG1 subcutaneously twice a week and 25 mg / kg selumetinib orally twice a day; UCB-554 10 mg / kg in 125 μl, orally once every 2 days; or 30 mg / kg Ab7326 mIgG1 subcutaneously twice a week and UCB-554 10 mg / kg in 125 μl, orally once every 2 days.

[0435] Mice were monitored daily and sacrificed when ethical endpoints were reached.

[0436] Statistics of Example 5

[0437] The survival of these mice from the start of treatment was statistically evaluated using Cox's proportional hazards model to calculate hazard ratios. Tumor volume data at day 7 were analyzed using a linear model fitted to the randomized design, with the treated tumor volume at day 0 used as a covariate to improve the accuracy of the estimate.

[0438] Example 1: Confirmation of Grem1 mRNA expression in pancreatic cancer

[0439] To investigate whether gremlin-1 may play a role in pancreatic cancer, Grem1 mRNA expression was determined in a cohort of human pancreatic ductal adenocarcinoma (PDAC) patients as determined by RNAseq analysis. This confirmed that Grem1 mRNA is expressed in human PDAC and that high expression is significantly associated with poor prognosis ( Figure 1The results are consistent with a published study reporting an association between Grem1 expression and poor outcome in pancreatic cancer (Yu et al., 2018).

[0440] To determine whether grem1 mRNA is expressed in the KPC genetically engineered pancreatic cancer mouse model, gene expression was examined in KPC tumors (and other primary pancreatic cancer models) and compared to controls, normal pancreatic ductal epithelium expressing KrasG12D. The results showed that grem1 mRNA expression was elevated in KPC tumors (and other murine pancreatic tumors) compared to controls (Table 1).

[0441] Table 1

[0442]

[0443] Example 2: Changes in gene expression in the KPC mouse model following treatment with Ab7326 mIgG1.

[0444] KPC mice with confirmed pancreatic tumors were established as described in Materials and Methods. Mice were included in groups for treatment with anti-gremlin antibody Ab7326 mIgG1 or vehicle control as shown in Table 2.

[0445] Mice were sacrificed at the ethical endpoint, and primary tumor tissues were harvested for RNA extraction was performed using Sigma-Aldrich. RNA samples were interrogated using Nanostring nCounter gene expression technology using a panel of approximately 750 probes.

[0446] Table 2

[0447]

[0448] Analysis of gene expression changes in the KPC mouse model after exposure to Ab7326 mIgG1 revealed upregulation of the MAPK / ERK (MEK) pathway and its canonical members.

[0449] The typical members of the MAPK / ERK (MEK) pathway, Mapk1, Nras, and Myc, were upregulated ( Figure 3 Due to insufficient power (3 samples per group, i.e., Ab7326 mIgG1-exposed and control), these genes lost their statistical significance after multiple hypothesis correction (FDR < 5%). However, the box plots depicting the differences in gene expression distribution of these three genes clearly show upregulated expression when Ab7326 mIgG1 is compared to the control group ( Figure 3 ).

[0450] Pathway enrichment analysis confirmed this finding ( Figure 4 Downregulation of KRAS signaling was enriched in the downregulated gene set (p=0.03), while upregulation of KRAS signaling was associated with the upregulated differentially expressed gene set (p=0.01). Although these enriched pathways were no longer statistically significant after multiple hypothesis correction, the consistent pattern between upregulated and downregulated effects was striking.

[0451] Furthermore, when KPC mice were exposed to Ab7326 mIgG1, proliferation signaling was increased (G2M, E2F, and MYC targets). This is consistent with the downstream effects of MAPK / ERK (MEK) pathway activation. Figure 4 In , log10(p) scores are shown, and only pathways with p<0.05 were retained. No multiple hypothesis correction was performed. However, the MYC_target, E2F_target, and G2M_checkpoint pathways remained significant after this correction (FDR<5%).

[0452] The findings support the potential combination of Ab7326 mIgG1 with MEK inhibitors for the treatment of pancreatic cancer. In addition, Ab7326 mIgG1 exposure may make pancreatic cancer more sensitive to this targeted therapy.

[0453] Example 3: Treatment with a combination of Ab7326 mIgG1 and selumetinib results in slower tumor growth compared to treatment with Ab7326 mIgG1 as a single agent

[0454] LSL-Kras has been described previously G12D / + ;LSL-Trp53 R172H / + ; Pdx1-Cre (KPC) mice (Hingorani et al., 2005). These mice develop tumors that are histologically and pathologically similar to human pancreatic tumors and are extremely aggressive, often metastatic, and highly chemoresistant, thus again mimicking human pancreatic cancer. A group of KPC mice was established and the mice were monitored until they developed pancreatic cancer that was detectable by palpation. The breeding strategy required to generate these mice and the experimental design are as follows Figure 2 shown.

[0455] Mice were monitored by palpation at least weekly until pancreatic tumors were detectable. At this time, high-resolution ultrasound imaging was used to confirm the presence of pancreatic cancer and mice were included in groups for treatment with anti-Gremlin1 antibody Ab7326 mIgG1 or Ab7326 mIgG1 + selumetinib combination (see Tables 3 and 4 for details).

[0456] Table 3

[0457]

[0458] Table 4

[0459]

[0460] Four mice in each group received follow-up ultrasound scans after 1 week and were then sacrificed to generate samples for pharmacodynamic evaluation (Table 3). Gross pathological analysis did not reveal any differences in tumor burden or phenotype under different experimental conditions. Analysis of 3D tumor imaging by ultrasound revealed that tumor growth was slowed in mice treated with the Ab7326 mIgG1 + selumetinib combination ( Figure 5 ).

[0461] Embodiment 4: Treatment with the combination of Ab7326 mIgG1 and selumetinib resulted in slower tumor growth and tumor shrinkage and prolonged responses compared to treatment with Ab7326 mIgG1 alone, and resulted in a significant increase in median survival compared to treatment with selumetinib and Ab7326 mIgG1 as single agents.

[0462] For the surviving group (Table 4), high-resolution ultrasound imaging was performed weekly during treatment to monitor tumor burden in each individual mouse treated. Mice continued on study and were closely monitored until ethical endpoints were reached, at which time they were sacrificed according to institutional guidelines. Clinical features presented included abdominal distension, poor body condition (suggestive of cachexia), decreased activity, and occasional jaundice.

[0463] Analysis of 3D tumor imaging by ultrasound revealed slower tumor growth, tumor shrinkage, and prolonged responses in several mice treated with the combination of Ab7326 mIgG1 and selumetinib ( Figure 6 In addition, we observed that mice treated with the combination of Ab7326 mIgG1 and selumetinib (33 days) showed a significant increase in median survival compared to mice treated with selumetinib alone (17 days, log rank, p = 0.050), and compared to mice treated with Ab7326 alone (19 days, for individual mouse data, see Figure 7 , Table 5 and Table 6). Serum samples from both the pharmacodynamic evaluation and the survival study were sent to UCB for analysis (Table 7).

[0464] Table 5

[0465]

[0466] Table 6

[0467]

[0468]

[0469] Table 7

[0470]

[0471]

[0472] After sacrificing the experimental mice, gross pathological examination did not reveal any differences in tumor burden or phenotype under different experimental conditions. Immunohistochemistry (IHC) analysis was performed on formalin-fixed paraffin-embedded tumor tissue to examine the tumor microenvironment to assess the number of α-SMA-positive and podoplanin-positive tumor-associated fibroblasts or any changes in the quality or quantity of collagen I and III (measured by picrosirius red staining). Staining was scored using HALO digital software, however, there was no significant effect of any of the experimental regimens on any of these parameters ( Figure 8 ).

[0473] result

[0474] In summary, the results presented herein demonstrate that MAPK / ERK pathway genes are upregulated following exposure to Ab7326 in the KPC mouse model. Furthermore, Ab7326 mIgG1 can be safely administered to KPC mice in combination with selumetinib. A significant improvement in survival was observed when mice were treated with a combination of Ab7326 mIgG1 and selumetinib compared to treatment with selumetinib alone (see Figure 7 , Table 5 and individual mouse data in Table 6). In addition, tumor shrinkage was observed in several mice treated with the combination of Ab7326 mIgG1 and selumetinib, and the response in these mice was prolonged. This suggests that the combination of Ab7326 mIgG1 and selumetinib represents a very promising pancreatic cancer treatment.

[0475] Example 5: Treatment of KPC mice with Ab7326 mlgG1 in combination with selumetinib or UCB-554 MEK inhibitor resulted in slower tumor growth and prolonged survival compared to treatment with single agents.

[0476] To determine whether the extended survival observed with the combination of Ab7326 and selumetinib in Example 3 is applicable to other MEK inhibitors, and to further improve the robustness of the study by increasing the number of KPC mice in each group, a study comparing Ab7326 in combination with the MEK inhibitor selumetinib or the MEK inhibitor UCB-554 was performed. KPC mice with confirmed pancreatic tumors were established as described in Materials and Methods, enrolled in groups for treatment as shown in Table 8, and tumor growth was monitored by ultrasound and ethical survival endpoints.

[0477] Table 8

[0478]

[0479] Treatment with the MEK inhibitor UCB-554 resulted in a modest improvement in survival of KPC mice compared to vehicle-only controls or Ab7326 treatment ( Fig. 9 a and Table 8 for median survival). However, combined treatment with Ab7326 and UCB-554 resulted in a further improvement in the survival of KPC mice compared to UCB-554 alone ( Fig. 9 a and Table 8 median survival). A Cox proportional hazard model was fitted to the survival data, and the hazard ratio (HR) was estimated for comparison of different treatment groups (Table 9). The hazard ratio of Ab7326+UCB-554 relative to vehicle control (HR=3.57, p=0.005) or Ab7326 alone (HR=4.38, p=0.001) indicated that the combination statistically significantly improved survival. The hazard ratio of Ab7326+UCB-554 relative to UCB-554 alone was also greater than 1 (HR=1.75), indicating that the combination improved survival over MEK inhibition alone, but did not reach statistical significance (p=0.211).

[0480] Table 9 - Summary of survival statistics of KPC mice treated with the combination of Ab7326 and UCB-554

[0481]

[0482] The volume of pancreatic tumors in control or treated KPC mice was measured over time using 3D tumor imaging by ultrasound. Comparison of tumor volume at day 7 after the start of treatment showed that, consistent with the survival data, tumor volume was reduced in the UCB-554 treated group compared to vehicle or Ab7326 alone. Fig. 9 b) Furthermore, the combination of Ab7326 + UCB-554 had a statistically significant reduction in tumor volume at day 7 compared to UCB-554 alone (p=0.040).

[0483] Tumor volumes were measured over time in this group of mice until the ethical endpoint was reached, indicating that the combination treatment of Ab7326 and UCB-554 resulted in tumor shrinkage or reduced tumor growth in a subgroup of mice compared to vehicle controls ( Fig. 9 c).

[0484] The KPC mouse survival data collected in the selumetinib MEK inhibitor study presented in Example 3 (Table 4) were combined with the data from the Example 5 cohort (Table 8) to improve the robustness of the analysis by including a larger number of animals. Fig.10 As shown in a and Table 10, the combination of Ab7326 and selumetinib significantly improved the survival of KPC mice compared to vehicle alone (HR=5.16, p<0.001) or Ab7326 treatment (HR=3.82, p<0.001). In addition, the combination of Ab7326 and selumetinib significantly improved survival compared to selumetinib treatment alone, with a hazard ratio of 2.92 (p=0.006), indicating that in this pancreatic cancer model, there is a significant beneficial effect of combining gremlin-1 inhibition with MEK pathway inhibition.

[0485] Table 10 - Summary of survival statistics of KPC mice treated with the combination of Ab7326 and selumetinib

[0486]

[0487] Comparison of tumor volumes at day 7 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 alone treatment ( Fig.10 b).

[0488] Tumor volumes were measured over time in mice in this cohort until ethical endpoints were reached, indicating that combination treatment with Ab7326 and selumetinib resulted in tumor shrinkage or reduced tumor growth in a subset of mice compared to vehicle controls ( Fig.10 c).

[0489] Taken together, these data demonstrate that in the KPC murine model of pancreatic cancer, anti-gremlin-1 antibody Ab7326 administered in combination with MEK inhibitors such as selumetinib or UCB-554 resulted in reduced tumor growth and increased survival compared to treatment with a single agent. This suggests that simultaneous targeting of gremlin-1 and MEK pharmacological pathways may produce additive or synergistic effects in the treatment of pancreatic cancer.

[0490] aspect

[0491] 1. An anti-GREM1 antagonist for use in a method of treating or preventing cancer, wherein the method further comprises administering a Ras-Raf-MEK-ERK signaling inhibitor.

[0492] 2. The anti-GREM1 antagonist for use according to aspect 1, wherein the cancer is a solid cancer.

[0493] 3. An anti-GREM1 antagonist for use according to aspect 1 or aspect 2, wherein the cancer has stromal GREM1 overexpression.

[0494] 4. An 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.

[0495] 5. An anti-GREMl antagonist for use according to any one of the preceding aspects, wherein the cancer shows dysregulation of the Ras-Raf-MEK-ERK pathway after exposure to the anti-GREMl antagonist.

[0496] 6. An anti-GREM1 antagonist for use according to any one of the preceding aspects, wherein the cancer contains a mutation in a Ras gene, optionally KRAS, NRAS and / or HRAS.

[0497] 7. An anti-GREMl antagonist for use according to any one of the preceding aspects, wherein the cancer contains a mutation in a Raf gene, optionally ARAF, BRAF and / or CRAF.

[0498] 8. An 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 a Ras-Raf-MEK-ERK signalling inhibitor.

[0499] 9. An 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, spleen cancer, bone resident cancer, melanoma and osteosarcoma.

[0500] 10. The anti-GREM1 antagonist for use according to aspect 9, wherein the cancer is pancreatic cancer.

[0501] 11. An anti-GREM1 antagonist for use according to aspect 10, wherein the pancreatic cancer is exocrine pancreatic cancer.

[0502] 12. An anti-GREM1 antagonist for use according to aspect 10 or aspect 11, wherein the pancreatic cancer is pancreatic ductal adenocarcinoma (PDAC).

[0503] 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.

[0504] 14. An anti-GREM1 antagonist for use according to aspect 9, wherein the cancer is lung cancer; optionally wherein the cancer is non-small cell lung cancer (NSCLC).

[0505] 15. An anti-GREM1 antagonist for use according to aspect 9, wherein the cancer is melanoma; optionally a BRAF-mutated melanoma.

[0506] 16. An anti-GREM1 antagonist for use according to any one of the preceding aspects, wherein the cancer has epithelial GREM1 overexpression.

[0507] 17. An anti-GREM1 antagonist for use according to aspect 16, wherein the cancer is a GREM1 driven cancer.

[0508] 18. An anti-GREM1 antagonist for use according to any one of the preceding aspects, wherein the cancer is a disseminated cancer.

[0509] 19. An anti-GREM1 antagonist for use according to any one of the preceding aspects, wherein the cancer is an established cancer.

[0510] 20. An 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).

[0511] 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, the epitope comprising at least one residue selected from Ile131, Lys147, Lys148, Phe149, Thr150, Thr151, Arg169, Lys174 and Gln175, wherein the residues are numbered according to SEQ ID NO: 1.

[0512] 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.

[0513] 23. The 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.

[0514] 24. An anti-GREM1 antagonist for use according to aspect 20, wherein the antagonist is an anti-Gremlin-1 antibody comprising a heavy chain complementary determining region (HCDR) sequence contained within the heavy chain variable region (HCVR) of SEQ ID NO: 10 or 12 and / or a light chain complementary determining region (LCDR) sequence contained within the light chain variable region (LCVR) of SEQ ID NO: 11 or 13.

[0515] 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 the group consisting of SEQ ID NOs: 3, 4, 5 and 6 and / or at least one LCDR sequence selected from the group consisting of SEQ ID NOs: 7, 8 and 9.

[0516] 26. The anti-GREM1 antagonist for use according to aspect 25, wherein the anti-Gremlin-1 antibody comprises the HCDR3 sequence of SEQ ID NO: 6.

[0517] 27. An anti-GREM1 antagonist for use according to aspect 25 or 26, wherein the anti-Gremlin-1 antibody comprises a HCDR1 / HCDR2 / HCDR3 sequence combination selected from SEQ ID NO: 4 / 5 / 6 or selected from SEQ ID NO: 3 / 5 / 6 and / or a LCDR1 / LCDR2 / LCDR3 sequence combination selected from SEQ ID NO: 7 / 8 / 9.

[0518] 28. An anti-GREM1 antagonist for use according to any one of aspects 25 to 27, wherein the anti-Gremlin-1 antibody comprises the HCDR1 / HCDR2 / HCDR3 / LCDR1 / LCDR2 / LCDR3 sequence combination of SEQ ID NO: 4 / 5 / 6 / 7 / 8 / 9 or SEQ ID NO: 3 / 5 / 6 / 7 / 8 / 9.

[0519] 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 having at least 95% identity thereto.

[0520] 30. An anti-GREMl antagonist for use according to aspect 29, wherein the anti-Gremlin-1 antibody comprises the HCVR and LCVR sequence pair of SEQ ID NO: 10 / 11 or 12 / 13 or a sequence having at least 95% identity thereto.

[0521] 31. An anti-GREM1 antagonist for use according to aspect 30, wherein the anti-Gremlin-1 antibody comprises a 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.

[0522] 32. An anti-GREM1 antagonist for use according to aspects 29-31, 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 having at least 95% identity thereto.

[0523] 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 NO: 14 / 15, 16 / 17, 18 / 19, 22 / 23, 28 / 29 or 30 / 31, 32 / 33, 34 / 35, or a sequence having at least 95% identity thereto.

[0524] 34. An anti-GREM1 antagonist for use according to aspect 33, wherein the HCDR1 / HCDR2 / HCDR3 / LCDR1 / LCDR2 / LCDR3 sequence of the antibody consists 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.

[0525] 35. An anti-GREMl antagonist for use according to aspect 20, wherein the antagonist is an antibody that competes with the antibody as defined in any one of aspects 22-32 for binding to Gremlin-1.

[0526] 36. An anti-GREMl antagonist for use according to aspect 20, wherein the antagonist is an antibody that binds to the same epitope on Gremlin-1 as the antibody as defined in any one of aspects 22-32.

[0527] 37. An anti-GREMl 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.

[0528] 38. An anti-GREMl 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', a F(ab')2, a Fv, a single domain antibody or a scFv.

[0529] 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 carrying the polynucleotide.

[0530] 40. An anti-GREMl antagonist for use according to any one of aspects 20-38, wherein the antagonist antibody is comprised in a pharmaceutical composition further comprising a pharmaceutically acceptable adjuvant and / or carrier.

[0531] 41. An anti-GREM1 antagonist for use according to any one of the preceding aspects, wherein the Ras-Raf-MEK-ERK signalling inhibitor downregulates Mapk1, Nras, Kras and / or Myc signalling.

[0532] 42. An anti-GREMl antagonist for use according to any one of the preceding aspects, wherein the Ras-Raf-MEK-ERK signalling inhibitor is a MEK inhibitor; optionally an allosteric MEK inhibitor.

[0533] 43. An anti-GREM1 antagonist for use according to aspect 42, wherein the MEK inhibitor is a MEK1 and / or MEK2 inhibitor.

[0534] 44. An anti-GREM1 antagonist for use according to aspect 42 or aspect 43, wherein the MEK inhibitor is selumetinib (AZD6244), trametinib (GSK-1120212), cobimetinib (GDC-0973), bimetinib (MEK162), CI-1040 (PD184352), modametinib (PD0325901), TAK733, rivatinib (RDEA119 / B ay86-9766), RO-5126766, RO5068760, pimasetinib (AS703026), AZD8330, GDC-0623, RO-4987655, WX-554 (UCB-554), HL-085, ARRY-300, ClnQ-03, G-573, PD184161, PD318088, PD98059, U0126, or SL327.

[0535] 45. The anti-GREM1 antagonist for use according to aspect 42, wherein the MEK inhibitor is selected from:

[0536] (a) N-(2-fluoro-4-iodophenyl)-3-(morpholin-4-ylcarbonyl)thieno[2,3-b]pyridin-2-amine;

[0537] (b) N-(2-fluoro-4-iodophenyl)-3-[(4-methylpiperazin-1-yl)carbonyl]thieno[2,3-b]pyridin-2-amine;

[0538] (c) [2-[((2-Fluoro-4-iodophenyl)amino]thieno[2,3-b]pyridin-3-yl](pyrrolidin-1-yl)methanone;

[0539] (d) (1-{2-[(2-fluoro-4-iodophenyl)amino]thieno[2,3-b]pyridine-3-carbonyl}piperidin-4-yl)-carbamic acid tert-butyl ester;

[0540] (e) 2-[((2-Fluoro-4-iodophenyl)amino]thieno[2,3-b]pyridine-3-carboxylic acid (4-aminopiperidin-1-yl)amide dihydrochloride;

[0541] (f) tert-butyl (1-{2-[(2-fluoro-4-iodophenyl)amino]thieno[2,3-b]pyridine-3-carbonyl}azetidin-3-ylmethyl)carbamate;

[0542] (g) [3-((Aminomethyl)azetidin-1-yl]-{2-[(2-fluoro-4-iodophenyl)amino]thieno[2,3-b]pyridin-3-yl}-methanone;

[0543] (h) tert-butyl (1-{2-[(2-fluoro-4-iodophenyl)amino]thieno[2,3-b]pyridine-3-carbonyl}-(3R)-pyrrolidin-3-yl)carbamate;

[0544] (i) tert-butyl (1-{2-[(2-fluoro-4-iodophenyl)amino]thieno[2,3-b]pyridine-3-carbonyl}-(3S)-pyrrolidin-3-yl)carbamate;

[0545] (j) (1-{2-[(2-fluoro-4-iodophenyl)amino]thieno[2,3-b]pyridine-3-carbonyl}azetidin-3-yl)-carbamic acid tert-butyl ester;

[0546] (k) [(3R)-3-aminopyrrolidin-1-yl]-{2-[(2-fluoro-4-iodophenyl)amino]thieno[2,3-b]pyridin-3-yl}-methanone;

[0547] (l) [(3S)-3-aminopyrrolidin-1-yl]-{2-[(2-fluoro-4-iodophenyl)amino]thieno[2,3-b]pyridin-3-yl}-methanone;

[0548] (m)(3-aminoazetidin-1-yl)-{2-[(2-fluoro-4-iodophenyl)amino]thieno[2,3-b]pyridin-3-yl]-methanone;

[0549] (n) {2-[((2-Fluoro-4-iodophenyl)amino]thieno[2,3-b]pyridin-3-yl}-(3-hydroxyazetidin-1-yl)-methanone;

[0550] (o) {2-[((2-Fluoro-4-iodophenyl)amino]thieno[2,3-b]pyridin-3-yl}-[(3R)-3-hydroxypyrrolidin-1-yl]-methanone;

[0551] (p) {2-[((2-Fluoro-4-iodophenyl)amino]thieno[2,3-b]pyridin-3-yl}-[(3S)-3-hydroxypyrrolidin-1-yl]-methanone;

[0552] (q) {2-[((2-Fluoro-4-iodophenyl)amino]thieno[2,3-b]pyridin-3-yl}-[2-(hydroxymethyl)-piperidin-1-yl]-methanone;

[0553] (r) {2-[((2-Fluoro-4-iodophenyl)amino]thieno[2,3-b]pyridin-3-yl}-[(3S)-3-(hydroxymethyl)-morpholin-4-yl]-methanone;

[0554] (s) tert-butyl 4-{2-[(2-fluoro-4-iodophenyl)amino]thieno[2,3-b]pyridine-3-carbonyl}-(3R)-3-(hydroxymethyl)piperazine-1-carboxylate;

[0555] (t) 2-[4({2-[(2-fluoro-4-iodophenyl)amino]thieno[2,3-b]pyridin-3-yl}carbonyl)piperazin-1-yl]ethanol;

[0556] (u) 3-((1,4-diazepan-1-ylcarbonyl)-N-(2-fluoro-4-iodophenyl)thieno[2,3-b]pyridin-2-amine;

[0557] (v) N-(2-fluoro-4-iodophenyl)-3-(piperazin-1-ylcarbonyl)thieno[2,3-b]pyridin-2-amine;

[0558] (w) ethyl [4-({2-[(2-fluoro-4-iodophenyl)amino]thieno[2,3-b]pyridin-3-yl}carbonyl)-piperazin-1-yl]acetate;

[0559] (x) [4-({2-[(2-fluoro-4-iodophenyl)amino]thieno[2,3-b]pyridin-3-yl}carbonyl)piperazin-1-yl]acetic acid;

[0560] (y) {2-[((2-fluoro-4-iodophenyl)amino]thieno[2,3-b]pyridin-3-yl}-[(2R)-2-(methoxymethyl)-pyrrolidin-1-yl]-methanone (methone); and

[0561] (z) {2-[((2-Fluoro-4-iodophenyl)amino]thieno[2,3-b]pyridin-3-yl}-[(2R)-2-(hydroxymethyl)-piperazin-1-yl]-methanone.

[0562] 46. ​​The anti-GREM1 antagonist for use according to aspect 42, wherein the MEK inhibitor is represented by formula (II), and pharmaceutically acceptable salts, solvates and N-oxides thereof:

[0563]

[0564] in

[0565] R 12 represents halogen;

[0566] R 3 ConnR b R c ;and

[0567] R b and R c Together with the nitrogen atom to which they are both attached, it represents azetidin-1-yl, pyrrolidin-1-yl, piperidin-1-yl, morpholin-4-yl, thiomorpholin-4-yl, piperazin-1-yl, homopiperidin-1-yl, homomorpholin-4-yl or homopiperazin-1-yl, any of which may be optionally substituted by one or more substituents selected from C 1-6 Alkyl, C 1-6 Alkoxy, hydroxy, hydroxy (C 1-6 ) alkyl (akyl), amino-(C 1-6 )alkyl, (amino)(hydroxy)(C 1-6 ) alkyl, halogen, oxo, C 2-6 Alkyl (akyl) carbonyl, carboxyl, C 2-6 Alkoxycarbonyl, di(C 1-6 ) alkyl (alhyl) hydrazine carbonyl, amino, C 1-6 Alkylamino, di(C 1-6 ) alkyl-amino, 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, carboxyl (C 1-6 ) alkyl, C 2-6 Alkoxycarbonyl (C 1-6 ) alkyl, C 2-6 Alkoxycarbonylamino and C 2-6 Alkoxycarbonylamino-(C1-6)alkyl.

[0568] 47. The anti-GREM1 antagonist for use according to claim 46, wherein R 12 Represents bromine or iodine.

[0569] 48. An anti-GREM1 antagonist for use according to aspect 44, wherein the MEK inhibitor is selumetinib (AZD6244).

[0570] 49. An anti-GREM1 antagonist for use according to aspect 44, wherein the MEK inhibitor is trametinib (GSK-1120212).

[0571] 50. The anti-GREM1 antagonist for use according to aspect 44, wherein the MEK inhibitor is WX-554 (UCB-554).

[0572] 51. An anti-GREMl antagonist for use according to aspect 44, wherein the MEK inhibitor is selumetinib (AZD6244) and the cancer is pancreatic cancer; optionally PDAC.

[0573] 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.

[0574] 53. An anti-GREMl antagonist for use according to aspect 44, wherein the MEK inhibitor is WX-554 (UCB-554) and the cancer is pancreatic cancer; optionally PDAC.

[0575] 54. An anti-GREM1 antagonist for use according to any one of aspects 1 to 41, wherein the Ras-Raf-MEK-ERK signalling inhibitor is an ERK inhibitor.

[0576] 55. The anti-GREM1 antagonist for use according to aspect 54, wherein the ERK inhibitor is uritinib, temuterkib, JSI-1187, ravoxertinib, ASN007 or MK-8353.

[0577] 56. An anti-GREM1 antagonist for use according to aspect 54 or 55, wherein the ERK inhibitor is uritinib.

[0578] 57. An anti-GREMl antagonist for use according to aspect 56, wherein the ERK inhibitor is uritinib and the cancer is pancreatic cancer; optionally PDAC.

[0579] 58. An anti-GREMl antagonist for use according to any one of the preceding aspects, wherein the method further comprises administering an additional anti-cancer agent.

[0580] 59. A Ras-Raf-MEK-ERK signalling inhibitor for use in a method of treating or preventing cancer, wherein the method further comprises administering an anti-GREM1 antagonist.

[0581] 60. A Ras-Raf-MEK-ERK signalling inhibitor for use according to aspect 59, wherein the cancer, the antagonist and / or the method is as defined in any one of aspects 1-58.

[0582] 61. A Ras-Raf-MEK-ERK signalling inhibitor for use according to aspect 59 or 60, wherein the Ras-Raf-MEK-ERK signalling inhibitor is a MEK inhibitor.

[0583] 62. A Ras-Raf-MEK-ERK signalling inhibitor for use according to aspect 61, wherein the MEK inhibitor is selumetinib (AZD6244).

[0584] 63. A Ras-Raf-MEK-ERK signalling inhibitor for use according to aspect 61, wherein the MEK inhibitor is trametinib (GSK-1120212).

[0585] 64. The Ras-Raf-MEK-ERK signalling inhibitor for use according to aspect 61, wherein the MEK inhibitor is WX-554 (UCB-554).

[0586] 65. A Ras-Raf-MEK-ERK signalling inhibitor for use according to aspect 59 or 60, wherein the Ras-Raf-MEK-ERK signalling inhibitor is an ERK inhibitor.

[0587] 66. A Ras-Raf-MEK-ERK signalling inhibitor for use according to aspect 65, wherein the ERK inhibitor is uritinib.

[0588] 67. A method of treating cancer comprising administering to a subject in need thereof a therapeutically effective amount of an anti-GREMl antagonist in combination with a therapeutically effective amount of a Ras-Raf-MEK-ERK signaling inhibitor.

[0589] 68. The method according to aspect 67, wherein the cancer, the antagonist and / or the method is as defined in any one of aspects 1-66.

[0590] 69. A composition or kit comprising an anti-GREMl antagonist and an inhibitor of Ras-Raf-MEK-ERK signaling.

[0591] 70. A composition or kit according to aspect 69, wherein the anti-GREM1 antagonist is as defined in any one of aspects 20 to 39.

[0592] 71. A composition or kit according to aspect 69 or 70, wherein the Ras-Raf-MEK-ERK signalling inhibitor is as defined in any one of aspects 41 to 57.

[0593] 72. A method for determining whether a patient having or suspected of having cancer or at risk of developing cancer is likely to respond to a combination treatment with a GREM1 antagonist and a Ras-Raf-MEK-ERK signaling inhibitor, the method comprising measuring mesenchymal expression of GREM1, epithelial expression of GREM1 and / or Ras-Raf-MEK-ERK pathway signaling in the patient, and thereby predicting whether the patient is likely to respond to treatment with the combination.

[0594] 73. A method for determining whether a patient having or suspected of having cancer or at risk of developing cancer is likely to respond to a combination treatment with a GREM1 antagonist and a Ras-Raf-MEK-ERK signaling inhibitor, the method comprising measuring the mesenchymal expression of GREM1 and / or the epithelial expression of GREM1 in the patient, and determining whether the patient has a RAS gene or RAF gene mutation, and thereby predicting whether the patient is likely to respond to treatment with the combination.

[0595] 74. A method for determining whether a patient having or suspected of having cancer or at risk of developing cancer is likely to respond to a combination treatment with a GREM1 antagonist and a Ras-Raf-MEK-ERK signaling inhibitor, the method comprising measuring Ras-Raf-MEK-ERK pathway signaling in the patient in response to anti-GREM1 antagonist treatment, and thereby predicting whether the patient is likely to respond to treatment with the combination.

[0596] Sequence Listing

[0597] SEQ ID NO: 1 (human Gremlin-1; Uniprot ID: O60565)

[0598] MSRTAYTVGALLLLLGTLLPAAEGKKKGSQGAIPPPDKAQHNDSEQTQSPQQPGSRNRGRGQGRGTAMPGEEVLESSQEALHVTERKYLKRDWCKTQPLKQTIHEEGCNSRTIINRFCYGQCNSFYIPRHIRKEEGSFQSCSFCKPKKFTTMMVTLNCPELQPPTKKKRVTRVKQCRCISIDLD

[0599] SEQ ID NO: 2 (human truncated Gremlin-1 used in crystallography with N-terminal tag)

[0600] MGSSHHHHHHSSGENLYFQGSAMPGEEVLESSQEALHVTERKYLKRDWCKTQPLKQTIHEEGCNSRTIINRFCYGQCNSFYIPRHIRKEEGSFQSCSFCKPKKFTTMMVTLNCPELQPPTKKKRVTRVKQCRCISIDLD

[0601] SEQ ID NO: 3 (Kabat and Chothia combined with Ab7326 HCDR1)

[0602] GYTFTDYYMH

[0603] SEQ ID NO:4(Ab7326 HCDR1 Kabat)

[0604] D Y

[0605] SEQ ID NO:5(Ab7326 HCDR2 Kabat)

[0606] LVDPEDGETIYAEKFQG

[0607] SEQ ID NO:6(Ab7326 HCDR3 Kabat)

[0608] DARGSGSYYPNHFDY

[0609] SEQ ID NO:7(Ab7326 LCDR1 Kabat)

[0610] KSSQSVLYSSNNKNYLA

[0611] SEQ ID NO:8(Ab7326 LCDR2 Kabat)

[0612] WASTRES

[0613] SEQ ID NO:9(Ab7326 LCDR3 Kabat)

[0614] QQYYDTPT

[0615] SEQ ID NO: 10 (Ab7326 heavy chain variable region variant 1)

[0616] QVQLVESGAEVKKPGATVKISCKVSGYTFTDYYMHWVQQAPGKGLEWMGLVDPEDGETIYAEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCATDARGSGSYYPNHFDYWGQGTLVTVSS

[0617] SEQ ID NO: 11 (Ab7326 light chain variable region variant 1)

[0618] DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTINSLQAEDVAVYFCQQYYDTPTFGQGTRLEIK

[0619] SEQ ID NO: 12 (Ab7326 heavy chain variable region variant 2)

[0620] QVQLVQSGAEVKKPGATVKISCKVSGYTFTDYYMHWVQQAPGKGLEWMGLVDPEDGETIYAEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCATDARGSGSYYPNHFDYWGQGTLVTVSS

[0621] SEQ ID NO: 13 (Ab7326 light chain variable region variant 2)

[0622] DIVMTQTPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTINSLQAEDVAVYFCQQYYDTPTFGQGTRLEIK

[0623] SEQ ID NO: 14 (mouse full-length IgG1 heavy chain variant 1)

[0624] QVQLVESGAEVKKPGATVKISCKVSGYTFTDYYMHWVQQAPGKGLEWMGLVDPEDGETIYAEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCATDARGSGSYYPNHFDYWGQGTLVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK

[0625] SEQ ID NO:15 (Mouse full-length IgG1 light chain variant 1)

[0626] DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTINSLQAEDVAVYFCQQYYDTPTFGQGTRLEIKRTDAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

[0627] SEQ ID NO:16 (Human full-length IgG1 heavy chain variant 2)

[0628] QVQLVQSGAEVKKPGATVKISCKVSGYTFTDYYMHWVQQAPGKGLEWMGLVDPEDGETIYAEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCATDARGSGSYYPNHFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0629] SEQ ID NO:17 (Human full-length IgG1 light chain variant 2)

[0630] DIVMTQTPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTINSLQAEDVAVYFCQQYYDTPTFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0631] SEQ ID NO:18 (Fab heavy chain variant 1)

[0632] QVQLVESGAEVKKPGATVKISCKVSGYTFTDYYMHWVQQAPGKGLEWMGLVDPEDGETIYAEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCATDARGSGSYYPNHFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC

[0633] SEQ ID NO:19 (Fab light chain variant 1)

[0634] DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTINSLQAEDVAVYFCQQYYDTPTFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0635] SEQ ID NO:20 (Human truncated Gremlin-1 used in crystallography, without N-terminal tag)

[0636] AMPGEEVLESSQEALHVTERKYLKRDWCKTQPLKQTIHEEGCNSRTIINRFCYGQCNSFYIPRHIRKEEGSFQSCSFCKPKKFTTMMVTLNCPELQPPTKKKRVTRVKQCRCISIDLD

[0637] SEQ ID NO:21 (Mature Gremlin-1 sequence of SEQ ID NO:1, lacking the signal peptide of amino acids 1-21)

[0638] KKKGSQGAIPPPDKAQHNDSEQTQSPQQPGSRNRGRGQGRGTAMPGEEVLESSQEALHVTERKYLKRDWCKTQPLKQTIHEEGCNSRTIINRFCYGQCNSFYIPRHIRKEEGSFQSCSFCKPKKFTTMMVTLNCPELQPPTKKKRVTRVKQCRCISIDLD

[0639] SEQ ID NO:22 (Human IgG4P heavy chain variant 1)

[0640] QVQLVESGAEVKKPGATVKISCKVSGYTFTDYYMHWVQQAPGKGLEWMGLVDPEDGETIYAEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCATDARGSGSYYPNHFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0641] SEQ ID NO:23 (Human IgG4P light chain variant 1)

[0642] DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTINSLQAEDVAVYFCQQYYDTPTFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0643] SEQ ID NO:24 (Human IgG1 heavy chain DNA variant 1)

[0644]

[0645] SEQ ID NO:25 (Human IgG1 light chain DNA variant 1)

[0646] gacattgtgatgacccagtcccccgattcgcttgcggtgtccctgggagaacgggccaccattaactgcaagagctcacagtccgtcctgtattcatcgaacaacaagaattacctcgcatggtatcagcagaagcctggacagcctcccaagctgctcatctactgggctagcacccgcgaatccggggtgccggatagattctccggatcgggttcgggcactgacttcactctgactatcaactcactgcaagccgaggatgtcgcggtgtacttctgtcagcagtactacgacaccccgacctttggacaaggcaccagactggagattaagcgtacggtggccgctccctccgtgttcatcttcccaccctccgacgagcagctgaagtccggcaccgcctccgtcgtgtgcctgctgaacaacttctacccccgcgaggccaaggtgcagtggaaggtggacaacgccctgcagtccggcaactcccaggaatccgtcaccgagcaggactccaaggacagcacctactccctgtcctccaccctgaccctgtccaaggccgactacgagaagcacaaggtgtacgcctgcgaagtgacccaccagggcctgtccagccccgtgaccaagtccttcaaccggggcgagtgc

[0647] SEQ ID NO:26 (Human IgG4 P heavy chain DNA variant 1)

[0648]

[0649] SEQ ID NO:27 (Human IgG4 P light chain DNA variant 1)

[0650] gacattgtgatgacccagtcccccgattcgcttgcggtgtccctgggagaacgggccaccattaactgcaagagctcacagtccgtcctgtattcatcgaacaacaagaattacctcgcatggtatcagcagaagcctggacagcctcccaagctgctcatctactgggctagcacccgcgaatccggggtgccggatagattctccggatcgggttcgggcactgacttcactctgactatcaactcactgcaagccgaggatgtcgcggtgtacttctgtcagcagtactacgacaccccgacctttggacaaggcaccagactggagattaagcgtacggtggccgctccctccgtgttcatcttcccaccctccgacgagcagctgaagtccggcaccgcctccgtcgtgtgcctgctgaacaacttctacccccgcgaggccaaggtgcagtggaaggtggacaacgccctgcagtccggcaactcccaggaatccgtcaccgagcaggactccaaggacagcacctactccctgtcctccaccctgaccctgtccaaggccgactacgagaagcacaaggtgtacgcctgcgaagtgacccaccagggcctgtccagccccgtgaccaagtccttcaaccggggcgagtgc

[0651] SEQ ID NO:28 (Mouse full-length IgG1 heavy chain variant 2)

[0652] QVQLVQSGAEVKKPGATVKISCKVSGYTFTDYYMHWVQQAPGKGLEWMGLVDPEDGETIYAEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCATDARGSGSYYPNHFDYWGQGTLVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK

[0653] SEQ ID NO:29 (Mouse full-length IgG1 light chain variant 2)

[0654] DIVMTQTPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTINSLQAEDVAVYFCQQYYDTPTFGQGTRLEIKRTDAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

[0655] SEQ ID NO:30 (Human full-length IgG1 heavy chain variant 1)

[0656] QVQLVESGAEVKKPGATVKISCKVSGYTFTDYYMHWVQQAPGKGLEWMGLVDPEDGETIYAEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCATDARGSGSYYPNHFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0657] SEQ ID NO:31 (Human full-length IgG1 light chain variant 1)

[0658] DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTINSLQAEDVAVYFCQQYYDTPTFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0659] SEQ ID NO:32 (Fab heavy chain variant 2)

[0660] QVQLVQSGAEVKKPGATVKISCKVSGYTFTDYYMHWVQQAPGKGLEWMGLVDPEDGETIYAEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCATDARGSGSYYPNHFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC

[0661] SEQ ID NO:33 (Fab light chain variant 2)

[0662] DIVMTQTPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTINSLQAEDVAVYFCQQYYDTPTFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0663] SEQ ID NO:34 (human IgG4 P heavy chain variant 2)

[0664] QVQLVQSGAEVKKPGATVKISCKVSGYTFTDYYMHWVQQAPGKGLEWMGLVDPEDGETIYAEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCATDARGSGSYYPNHFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0665] SEQ ID NO:35 (Human IgG4 P light chain variant 2)

[0666] DIVMTQTPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTINSLQAEDVAVYFCQQYYDTPTFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0667] SEQ ID NO:36 (Human Gremlin-1; full sequence)

[0668]

[0669] SEQ ID NO:37 (Human Gremlin-1; Coding Sequence)

[0670] Atgagccgcacagcctacacggtgggagccctgcttctcctcttggggaccctgctgccggctgctgaagggaaaaagaaagggtcccaaggtgccatccccccgccagacaaggcccagcacaatgactcagagcagactcagtcgccccagcagcctggctccaggaaccgggggcggggccaagggcggggcactgccatgcccggggaggaggtgctggagtccagccaagaggccctgcatgtgacggagcgcaaatacctgaagcgagactggtgcaaaacccagccgcttaagcagaccatccacgaggaaggctgcaacagtcgcaccatcatcaaccgcttctgttacggccagtgcaactctttctacatccccaggcacatccggaaggaggaaggttcctttcagtcctgctccttctgcaagcccaagaaattcactaccatgatggtcacactcaactgccctgaactacagccacctaccaagaagaagagagtcacacgtgtgaagcagtgtcgttgcatatccatcgatttggattaa

[0671] References

[0672] Hingorani, S.R., L. Wang, A.S. Multani, C. Combs, T.B. Deramaudt, R.H. 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.

[0673] Kobayashi, Y., S. O. 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.

[0674] 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.

[0675] 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.

[0676] 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. An anti-GREM1 antagonist for use in a method of treating or preventing cancer, wherein the method further comprises administering a Ras-Raf-MEK-ERK signaling inhibitor.

2. The anti-GREM1 antagonist for use according to claim 1, wherein: (a) the cancer is a solid cancer; (b) the cancer has stromal GREM1 overexpression; (c) the cancer is a cancer associated with dysregulation of the Ras-Raf-MEK-ERK pathway; (d) the cancer exhibits dysregulation of the Ras-Raf-MEK-ERK pathway after exposure to an anti-GREM1 antagonist; (e) the cancer contains a mutation in a Ras gene, optionally KRAS, NRAS and / or HRAS; (f) the cancer contains a mutation in a Raf gene, optionally ARAF, BRAF and / or CRAF; (g) the cancer is a cancer that is poorly responsive, unresponsive or refractory to treatment with a Ras-Raf-MEK-ERK signaling inhibitor; and / or (h) 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, spleen cancer, bone resident cancer, melanoma and osteosarcoma.

3. The anti-GREM1 antagonist for use according to claim 2(h), wherein: (a) the cancer is pancreatic cancer; optionally wherein: (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; optionally colorectal cancer with a KRAS / BRAF mutation; (c) the cancer is lung cancer; optionally wherein the cancer is non-small cell lung cancer (NSCLC); or (d) the cancer is melanoma; optionally a BRAF-mutated melanoma.

4. An anti-GREM1 antagonist for use according to any one of the preceding claims, wherein: (a) the cancer has epithelial GREM1 overexpression; optionally wherein the cancer is a GREM1 driven cancer; (b) the cancer is a disseminated cancer; and / or (c) the cancer is an established cancer.

5. An anti-GREMl antagonist for use according to any one of the preceding claims, 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).

6. The anti-GREM1 antagonist for use according to claim 5, wherein: (a) the antagonist is an antibody that binds to an epitope on Gremlin-1, the epitope 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; optionally wherein: (i) the antibody binds to an epitope comprising all of Ile131, Lys147, Lys148, Phe149, Thr150, Thr151, Arg169, Lys174 and Gln175; and / or (ii) Lys147, Lys148, Phe149, Thr150, Thr151, Arg169, Lys174, and Gln175 are located on the same Gremlin-1 monomer, and Ile131 is located on the second Gremlin-1 monomer; (b) the antagonist is an anti-Gremlin-1 antibody comprising a heavy chain complementary determining region (HCDR) sequence contained in a heavy chain variable region (HCVR) of SEQ ID NO: 10 or 12 and / or a light chain complementary determining region (LCDR) sequence contained in a light chain variable region (LCVR) of SEQ ID NO: 11 or 13; or (c) 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; optionally wherein: (i) the anti-Gremlin-1 antibody comprises the HCDR3 sequence of SEQ ID NO: 6; (ii) the anti-Gremlin-1 antibody comprises a HCDR1 / HCDR2 / HCDR3 sequence combination selected from SEQ ID NO: 4 / 5 / 6 or selected from SEQ ID NO: 3 / 5 / 6 and / or a LCDR1 / LCDR2 / LCDR3 sequence combination selected from SEQ ID NO: 7 / 8 / 9; (iii) the anti-Gremlin-1 antibody comprises the HCDR1 / HCDR2 / HCDR3 / LCDR1 / LCDR2 / LCDR3 sequence combination of SEQ ID NO: 4 / 5 / 6 / 7 / 8 / 9 or SEQ ID NO: 3 / 5 / 6 / 7 / 8 / 9; and / or (iv) 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 having at least 95% identity thereto.

7. The anti-GREM1 antagonist for use according to claim 6(c)(iv), wherein: (a) the anti-Gremlin-1 antibody comprises a HCVR and LCVR sequence pair of SEQ ID NO: 10 / 11 or 12 / 13 or a sequence having at least 95% identity thereto; optionally wherein the anti-Gremlin-1 antibody comprises a 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 have at least 95% identity to SEQ ID NO: 10, 11, 12 and / or 13, respectively; and / or (b) 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 having at least 95% identity thereto.

8. An anti-GREM1 antagonist for use according to claim 7(b), wherein the anti-Gremlin-1 antibody comprises a heavy chain and a light chain pair of SEQ ID NO: 14 / 15, 16 / 17, 18 / 19, 22 / 23, 28 / 29 or 30 / 31, 32 / 33, 34 / 35, or a sequence having at least 95% identity thereto; optionally wherein the HCDR1 / HCDR2 / HCDR3 / LCDR1 / LCDR2 / LCDR3 sequence of the antibody consists 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 have at least 95% identity to SEQ ID NO: 14, 15, 16 and / or 17, respectively.

9. The anti-GREM1 antagonist for use according to claim 5, wherein: (a) the antagonist is an antibody that competes with the antibody as defined in any one of claims 6(a)(i)-7 for binding to Gremlin-1; or (b) The antagonist is an antibody that binds to the same epitope on Gremlin-1 as the antibody defined in any one of claims 6(a)(i)-7.

10. An anti-GREM1 antagonist for use according to any one of claims 5 to 9, wherein: (a) the antagonist antibody is a chimeric antibody, a human antibody or a humanized antibody; and / or (b) The antagonist antibody is Fab, modified Fab, Fab', modified Fab', F(ab')2, Fv, single domain antibody or scFv.

11. The anti-GREM1 antagonist for use according to claim 5, wherein the antagonist is a polynucleotide encoding an antibody as defined in any one of claims 6 to 10, or an expression vector carrying the polynucleotide.

12. The anti-GREMl antagonist for use according to any one of claims 5 to 10, wherein the antagonist antibody is contained in a pharmaceutical composition further comprising a pharmaceutically acceptable adjuvant and / or carrier.

13. An anti-GREM1 antagonist for use according to any one of the preceding claims, wherein: (a) the Ras-Raf-MEK-ERK signaling inhibitor downregulates Mapk1, Nras, Kras and / or Myc signaling; and / or (b) The Ras-Raf-MEK-ERK signaling inhibitor is a MEK inhibitor; optionally an allosteric MEK inhibitor.

14. The anti-GREM1 antagonist for use according to claim 13(b), wherein: (a) the MEK inhibitor is a MEK1 and / or MEK2 inhibitor; and / or (b) The MEK inhibitor is selumetinib (AZD6244), trametinib (GSK-1120212), cobimetinib (GDC-0973), bimetinib (MEK162), CI-1040 (PD184352), modametinib (PD0325901), TAK733, rifatinib (RDEA119 / Bay 86-9766), RO-5126766, RO5068760, pimasetinib (AS703026), AZD8330, GDC-0623, RO-4987655, WX-554 (UCB-554), HL-085, ARRY-300, ClnQ-03, G-573, PD184161, PD318088, PD98059, U0126 or SL327; optionally wherein: (i) the MEK inhibitor is selumetinib (AZD6244); (ii) the MEK inhibitor is trametinib (GSK-1120212); (iii) the MEK inhibitor is WX-554 (UCB-554); (iv) the MEK inhibitor is selumetinib (AZD6244) and the cancer is pancreatic cancer; optionally PDAC; or (v) the MEK inhibitor is trametinib (GSK-1120212) and the cancer is pancreatic cancer; optionally PDAC; (vi) the MEK inhibitor is WX-554 (UCB-554) and the cancer is pancreatic cancer; optionally PDAC.

15. The anti-GREM1 antagonist for use according to claim 13(b), wherein the MEK inhibitor is selected from: (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) tert-butyl (1-{2-[(2-fluoro-4-iodophenyl)amino]thieno[2,3-b]pyridine-3-carbonyl}azetidin-3-ylmethyl)carbamate; (g) [3-((Aminomethyl)azetidin-1-yl]-{2-[(2-fluoro-4-iodophenyl)amino]thieno[2,3-b]pyridin-3-yl}-methanone; (h) tert-butyl (1-{2-[(2-fluoro-4-iodophenyl)amino]thieno[2,3-b]pyridine-3-carbonyl}-(3R)-pyrrolidin-3-yl)carbamate; (i) tert-butyl (1-{2-[(2-fluoro-4-iodophenyl)amino]thieno[2,3-b]pyridine-3-carbonyl}-(3S)-pyrrolidin-3-yl)carbamate; (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) tert-butyl 4-{2-[(2-fluoro-4-iodophenyl)amino]thieno[2,3-b]pyridine-3-carbonyl}-(3R)-3-(hydroxymethyl)piperazine-1-carboxylate; (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]-methanone (methone); and (z) {2-[((2-Fluoro-4-iodophenyl)amino]thieno[2,3-b]pyridin-3-yl}-[(2R)-2-(hydroxymethyl)-piperazin-1-yl]-methanone.

16. The anti-GREM1 antagonist for use according to claim 13(b), wherein the MEK inhibitor is represented by formula (II), and pharmaceutically acceptable salts, solvates and N-oxides thereof: in R 12 represents halogen; R 3 ConnR b R c ;and R b and R c Together with the nitrogen atom to which they are both attached, it represents azetidin-1-yl, pyrrolidin-1-yl, piperidin-1-yl, morpholin-4-yl, thiomorpholin-4-yl, piperazin-1-yl, homopiperidin-1-yl, homomorpholin-4-yl or homopiperazin-1-yl, any of which may be optionally substituted by one or more substituents selected from C 1-6 Alkyl, C 1-6 Alkoxy, hydroxy, hydroxy (C 1-6 ) alkyl (akyl), amino-(C 1-6 )alkyl, (amino)(hydroxy)(C 1-6 ) alkyl, halogen, oxo, C 2-6 Alkyl (akyl) carbonyl, carboxyl, C 2-6 Alkoxycarbonyl, di(C 1-6 ) alkyl (alhyl) hydrazine carbonyl, amino, C 1-6 Alkylamino, di(C 1-6 ) alkyl-amino, 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, carboxyl (C 1-6 ) alkyl, C 2-6 Alkoxycarbonyl (C 1-6 ) alkyl, C 2-6 Alkoxycarbonylamino and C 2-6 Alkoxycarbonylamino-(C1-6)alkyl; Optionally, R 12 Represents bromine or iodine.

17. An anti-GREM1 antagonist for use according to any one of claims 1 to 13(a), wherein the Ras-Raf-MEK-ERK signaling inhibitor is an ERK inhibitor; optionally wherein: (a) the ERK inhibitor is uritinib, temuterkib, JSI-1187, ravoxertinib ASN007 or MK-8353; and / or (b) the ERK inhibitor is uritinib; further optionally wherein the ERK inhibitor is uritinib and the cancer is pancreatic cancer; optionally PDAC.

18. An anti-GREMl antagonist for use according to any one of the preceding claims, wherein the method further comprises administering an additional anti-cancer agent.

19. A Ras-Raf-MEK-ERK signalling inhibitor for use in a method for treating or preventing cancer, wherein the method comprises further administering an anti-GREM1 antagonist.

20. A Ras-Raf-MEK-ERK signalling inhibitor for use according to claim 19, wherein the cancer, the antagonist and / or the method are as defined in any one of claims 1-18.

21. A Ras-Raf-MEK-ERK signaling inhibitor for use according to claim 19 or claim 20, wherein: (a) the Ras-Raf-MEK-ERK signaling inhibitor is a MEK inhibitor; optionally wherein: (i) the inhibitor is selumetinib (AZD6244); (ii) the inhibitor is trametinib (GSK-1120212); or (iii) the inhibitor is WX-554 (UCB-554); or (b) the Ras-Raf-MEK-ERK signaling inhibitor is an ERK inhibitor; optionally wherein the ERK inhibitor is uritinib.

22. A method of treating cancer comprising administering to a subject in need thereof a therapeutically effective amount of an anti-GREMl antagonist in combination with a therapeutically effective amount of a Ras-Raf-MEK-ERK signaling inhibitor.

23. The method of claim 22, wherein the cancer, the antagonist and / or the method are as defined in any one of claims 1-21.

24. A composition or kit comprising an anti-GREM1 antagonist and an inhibitor of Ras-Raf-MEK-ERK signaling.

25. The composition or kit according to claim 24, wherein: (a) the anti-GREM1 antagonist is as defined in any one of claims 5 to 11; and / or (b) The Ras-Raf-MEK-ERK signaling inhibitor is as defined in any one of claims 13(a) to 17.

26. To determine: (a) a method of determining whether a patient having or suspected of having or being at risk of developing cancer is likely to respond to a combination treatment with a GREM1 antagonist and a Ras-Raf-MEK-ERK signalling inhibitor, the method comprising measuring mesenchymal expression of GREM1, epithelial expression of GREM1 and / or Ras-Raf-MEK-ERK pathway signalling in the patient, and thereby predicting whether the patient is likely to respond to treatment with the combination; or (b) a method for determining whether a patient having or suspected of having cancer or at risk of developing cancer is likely to respond to a combination treatment with a GREM1 antagonist and a Ras-Raf-MEK-ERK signaling inhibitor, the method comprising measuring the mesenchymal expression of GREM1 and / or the epithelial expression of GREM1 in the patient, and determining whether the patient has a RAS gene or RAF gene mutation, and thereby predicting whether the patient is likely to respond to treatment with the combination; or (c) A method for determining whether a patient having or suspected of having cancer or at risk of developing cancer is likely to respond to a combination treatment with a GREM1 antagonist and a Ras-Raf-MEK-ERK signaling inhibitor, the method comprising measuring Ras-Raf-MEK-ERK signaling pathway signaling in the patient in response to anti-GREM1 antagonist treatment, and thereby predicting whether the patient is likely to respond to treatment with the combination.

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