Combination of a Gremlin-1 antagonist and a cytidine or deoxycytidine analogue

Abstract

The present invention relates to anti-GREM1 antagonists for use in combination with proliferation-dependent cytotoxic agents in methods of treating or preventing cancer.

Description

[Technical field]

[0001] The present invention relates to combination therapy for treating or preventing cancer.In particular, the present invention relates to anti-GREM1 antagonists for use in methods for treating or preventing cancer, in combination with cytidine analogs such as gemcitabine or deoxycytidine analogs, and related compositions and kits.The present invention also relates to anti-GREM1 antagonists for use in methods for treating or preventing pancreatic cancer, in combination with proliferation-dependent cytotoxic agents, and related compositions and kits.The present invention also relates to a method for predicting whether a patient is likely to respond to combination therapy based on stromal GREM1 overexpression. [Background technology]

[0002] Cancer remains a significant and complex global challenge, despite technological advances in cancer immunotherapy, gene therapy, data science and precision medicine. Drug resistance and failure to respond to existing therapies highlight the need to develop improved therapies for the treatment and prevention of cancer. Combination therapies that can improve the efficacy of existing treatments are also needed.

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

[0004] The inventors have surprisingly shown that GREM1 antagonists can be advantageously administered in combination with chemotherapeutic agents for the treatment or prevention of pancreatic cancer. Using the LSL-KrasG12D / +;LSL-Trp53R172H / +;Pdx1-Cre (KPC) mouse model of pancreatic cancer, the inventors have demonstrated that combination therapy comprising an anti-GREM1 antagonist and a chemotherapeutic agent significantly improves the survival of these mice compared to vehicle controls. Without being bound by theory, the inventors hypothesize that stroma targeting with an anti-GREM1 antagonist may enhance the efficacy of chemotherapy against pancreatic cancer because the stroma itself can confer chemotherapy resistance to tumor cells. Based on these results, the inventors also envision that combination therapy comprising a GREM1 antagonist and a proliferation-dependent cytotoxic agent, such as gemcitabine and other cytidine or deoxycytidine analogs, is generally useful in the treatment and prevention of cancer. In particular, the inventors envision that combination therapy comprising a GREM1 antagonist and a proliferation-dependent cytotoxic agent will be generally useful in the treatment and prevention of cancers characterized by the presence of dormant stem-like cancer cells. The inventors' findings provide a new approach to the prevention and treatment of cancer, particularly pancreatic cancer.

[0005] Thus, in a first aspect of the invention there is provided an anti-GREM1 antagonist for use in a method of treating or preventing cancer, the method further comprising administering a cytidine analogue or a deoxycytidine analogue.

[0006] In a further aspect of the invention there is provided an anti-GREM1 antagonist for use in a method of treating or preventing pancreatic cancer, the method further comprising administering a proliferation-dependent cytotoxic agent.

[0007] In another aspect of the invention there is provided a cytidine analogue or deoxycytidine analogue for use in a method of treating or preventing cancer, the method further comprising administering an anti-GREM1 antagonist.

[0008] In yet another aspect of the present invention, there is provided a method of treating cancer comprising administering to a subject in need thereof a therapeutically effective amount of an anti-GREM1 antagonist in combination with a therapeutically effective amount of a cytidine analogue or a deoxycytidine analogue.

[0009] In a further aspect of the invention there is provided a proliferation-dependent cytotoxic agent for use in a method of treating or preventing pancreatic cancer, the method further comprising administering an anti-GREM1 antagonist.

[0010] In yet another aspect of the invention, a method of treating pancreatic cancer is provided, comprising administering a therapeutically effective amount of an anti-GREM1 antagonist in combination with a therapeutically effective amount of a proliferation-dependent cytotoxic agent.

[0011] In another aspect of the invention, there is provided a composition or kit comprising an anti-GREM1 antagonist and a cytidine analog or a deoxycytidine analog.

[0012] In yet another aspect of the invention, there is provided a composition or kit comprising an anti-GREM1 antagonist and an anti-mitotic agent.

[0013] In a further aspect of the present invention, there is provided a method for determining whether a patient suffering from or suspected of suffering from cancer, or at risk of developing cancer, is likely to respond to combination treatment with a GREM1 antagonist and a cytidine analogue or a deoxycytidine analogue, the method comprising measuring stromal and / or epithelial expression of GREM1 in the patient, thereby predicting whether the patient is likely to respond to combination treatment.

[0014] In yet another aspect of the present invention, there is provided a method for determining whether a patient suffering from or suspected of suffering from pancreatic cancer, or at risk of developing pancreatic cancer, is likely to respond to combined treatment with a GREM1 antagonist and a proliferation-dependent cytotoxic agent, the method comprising measuring stromal and / or epithelial expression of GREM1 in the patient, thereby predicting whether the patient is likely to respond to combined treatment. [Brief description of the drawings]

[0015] [Figure 1] Kaplan-Meier analysis showing survival of PDAC patients with Gremlin-1 expression above or below the indicated median values. Patients with tumors expressing high levels of Gremlin-1 have a significantly worse prognosis compared to patients with low expression. Generated from KMplotter. [Diagram 2] Schematic diagram showing (a) breeding strategy and (b) experimental design. [Diagram 3] Graph showing tumor burden as measured by high resolution ultrasound in individual mice during treatment as indicated. [Figure 4] Kaplan-Meier analysis showing survival of Pdx1-Cre;LSL-KrasG12D / +;LSL-Trp53R172H / + (KPC) mice from the start of treatment with Ab7326 mIgG1 (n=7), vehicle (n=7), gemcitabine (n=5), or Ab7326 mIgG1 in combination with gemcitabine (n=6), as indicated. Mice treated with the combination of Ab7326 mIgG1 and gemcitabine show a significant increase in survival compared to vehicle controls (log rank, p=0.04). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] Brief explanation of the table Table 1-Kras G12D+Table showing upregulated grem1 in KPC tumors and all pancreatic mouse models combined compared to mouse pancreatic duct cells. Table 2 - A table showing the dose and schedule of treatment given, the number of mice on each treatment (including censored cases), and the median survival in each treatment cohort. Mice treated with the combination of Ab7326 mIgG1 and gemcitabine show a significant increase in median survival compared to vehicle controls. Table 3 - Table showing survival data and censoring for individual mice on the indicated treatments. * indicates smaller than the mean tumor at the start of treatment.

[0017] Detailed Description of the Invention It is to be understood that different applications of the disclosed products and methods can be tailored to the particular needs of the art, and that the terminology used herein is for the purpose of describing particular embodiments of the invention only, and is not intended to be limiting.

[0018] Further, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to an "inhibitor" includes two or more such inhibitors or reference to an "oligonucleotide" includes two or more such oligonucleotides, and so forth.

[0019] All publications, patents, and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.

[0020] Combination therapy with anti-GREM1 antagonists and cytidine or deoxycytidine analogues The present invention provides an anti-GREM1 antagonist in combination with a cytidine analogue such as gemcitabine or a deoxycytidine analogue for use in a method for treating or preventing cancer. The cancer is typically a cancer with GREM1 overexpression and / or a cancer that can be targeted with a cytidine analogue such as gemcitabine or a derivative thereof or a deoxycytidine analogue. The cancer can additionally or alternatively be characterized as comprising dormant cancer cells, e.g., dormant stem-like cancer cells, and / or as a recurrent cancer.

[0021] The present invention further provides a cytidine analogue, such as gemcitabine or a derivative thereof, or a deoxycytidine analogue, for use in a method of treating or preventing cancer, the method comprising separate, sequential or simultaneous administration of an anti-GREM1 antagonist.

[0022] Cytidine or deoxycytidine analogues The present invention provides a combination therapy using anti-GREM1 antagonists with cytidine or deoxycytidine analogs to treat or prevent cancer. Cytidine and deoxycytidine analogs are specific nucleoside inhibitors or antimetabolites that mimic endogenous nucleosides and exert cytotoxic activity by interfering with nucleic acid synthesis. Nucleoside inhibitors or antimetabolites include analogs of physiological pyrimidine and purine nucleobases and nucleosides. In particular, cytidine and deoxycytidine analogs mimic endogenous cytidine or deoxycytidine. Such compounds can also interfere with DNA methylation or modify the metabolism of physiological nucleosides. "Analog(s)" refers to compounds that are structurally or functionally similar to the intended target / compound. For example, a cytidine analog or deoxycytidine analog may have structural similarity to cytidine or deoxycytidine and / or similar chemical and biological properties to cytidine or deoxycytidine.

[0023] In the context of treating any cancer, the present invention encompasses the use of any cytidine or deoxycytidine analog. Exemplary cytidine or deoxycytidine analogs for use in the present invention include gemcitabine (2'-deoxy-2',2'-difluorocytidine; 4-amino-1-(2-deoxy-2,2-difluoro-β-D-erythro-pentofuranosyl)pyrimidin-2(1H)-one; 4-amino-1-[(2R,4R,5R)-3,3-difluoro-4-hydroxy-5-( ... -(hydroxymethyl)oxolan-2-yl]pyrimidin-2-one), or a derivative thereof; azacytidine (5-azacytidine; 4-amino-1-beta-D-ribofuranosyl-1,3,5-triazin-2(1H)-one), or a derivative thereof; cytarabine (Ara-C / cytosine 1-[beta]-D-arabinofuranoside; 4-amino-1-[(2R,3S,4S,5R)-3, decitabine (5-aza-2'-deoxycytidine / 5-azadeoxycytidine; 4-amino-1-(2-deoxy-β-D-erythro-pentofuranosyl)-1,3,5-triazin-2(1H)-one; 4-amino-1-[(2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)oxolan-2-yl]-1,3,5-triazin-2-one, or a derivative thereof; and troxacitabine (troxatyl / 4-amino-1-[(2S)-2-(hydroxymethyl)-1,3-dioxolan-4-yl]pyrimidin-2-one), or a derivative thereof. In a preferred embodiment, the deoxycytidine analog is gemcitabine.

[0024] In some embodiments, the combination therapy provides an anti-GREM1 antagonist together with one or more cytidine or deoxycytidine analogues. In a preferred embodiment, the present invention provides an anti-GREM1 antagonist for use in a method for treating or preventing cancer, the method further comprising administering gemcitabine. In an exemplary embodiment, the present invention provides an anti-GREM1 antagonist for use in a method for treating or preventing cancer, the method further comprising administering gemcitabine in combination with troxacitabine. In a particularly preferred embodiment, the present invention provides an anti-GREM1 antagonist for use in a method for treating or preventing pancreatic cancer, the method further comprising administering gemcitabine in combination with troxacitabine.

[0025] It is contemplated that the combination therapy comprising the GREM1 antagonist defined herein and the cytidine analog or deoxycytidine analog described herein can be used to treat or prevent cancers that are typically targeted by said cytidine analog or deoxycytidine analog.Cancer can be the cancer or tumor previously described for treatment by cytidine analog or deoxycytidine analog.Such cancers include, but are not limited to, pancreatic cancer, breast cancer, ovarian cancer, non-small cell lung cancer and bladder cancer.

[0026] The cancer may be a cancer that is responsive to treatment with cytidine or deoxycytidine analogs and / or a cancer that is intended to be treated with cytidine or deoxycytidine analogs. Alternatively, the cancer may be a cancer that is poorly responsive, non-responsive, or refractory to treatment with cytidine or deoxycytidine analogs. The cancer may be one that has been previously described as not suitable for treatment with cytidine or deoxycytidine analogs. In some cases, the cancer may be initially responsive to treatment with cytidine or deoxycytidine analogs, but may show resistance to treatment with cytidine or deoxycytidine analogs. Responsiveness may be measured by any means. For example, responsiveness to treatment may be evaluated by measuring tumor size before and after treatment using X-ray, CT or MRI scan, or by measuring tumor markers. Blood tests to determine organ function may also be used to evaluate responsiveness to treatment. One exemplary blood marker for monitoring the responsiveness of pancreatic cancer is CA19-9. Those skilled in the art know how to measure the responsiveness of a cancer to a treatment.

[0027] The cancer may additionally or alternatively be characterized as comprising dormant cancer cells, e.g., dormant stem-like cancer cells, and / or as being a recurrent cancer.

[0028] In a particularly preferred embodiment, the anti-GREM1 antagonist is administered in combination with a cytidine analog or a deoxycytidine analog for the treatment of pancreatic cancer. One exemplary pancreatic cancer is exocrine pancreatic cancer, such as pancreatic ductal adenocarcinoma.

[0029] Gemcitabine Gemcitabine (2'-deoxy-2',2'-difluorocytidine / 2',2'-difluoro 2'deoxycytidine) is a chemotherapeutic agent typically used to treat pancreatic, breast, ovarian, non-small cell lung, bladder, and other solid cancers. Gemcitabine is an antimetabolite. As mentioned above and described in more detail below, antimetabolites mimic endogenous nucleosides and exert their cytotoxic activity by interfering with the synthesis of nucleic acids. Gemcitabine is a pyrimidine nucleotide analog of cytidine. It can enter cells via nucleotide transporters on the cell membrane, and then undergo a series of phosphorylation reactions to form gemcitabine diphosphate and gemcitabine triphosphate. Gemcitabine triphosphate competes with endogenous deoxycytidine triphosphate (dCTP) for incorporation into growing DNA strands, resulting in masked chain termination, while gemcitabine diphosphate inhibits ribonucleotide reductase, resulting in reduced availability of dCTP.

[0030] In the context of the present invention, combination therapy comprising gemcitabine or its derivatives is contemplated. The term "derivatives thereof" refers to compounds that exert similar therapeutic effects and are derived from similar compounds / precursor compounds. Gemcitabine derivatives include, for example, stereoisomers of gemcitabine, or esters or amides of gemcitabine.

[0031] The inventors have surprisingly shown that GREM1 antagonists can be advantageously administered in combination with gemcitabine to treat or prevent pancreatic cancer in KPC mouse models. Without being bound by theory, the inventors hypothesize that anti-GREM1 antagonists can drive dormant stem-like cancer cells into a more proliferative state and increase their sensitivity to treatment with proliferation-dependent cytotoxic agents such as gemcitabine or other cytidine or deoxycytidine analogues. The inventors also hypothesize that stroma targeting using anti-GREM1 antagonists can enhance the efficacy of chemotherapy treatment for pancreatic cancer, since the stroma itself can confer chemotherapy resistance on tumor cells. Therefore, it is envisioned that combination therapy comprising GREM1 antagonists and gemcitabine or its derivatives can be used to treat or prevent a wide range of cancers. It is also contemplated that combination therapies including GREM1 antagonists, such as those described herein, and other cytidine or deoxycytidine analogs can be used to treat or prevent a wide range of cancers.

[0032] In particular, it is contemplated that the combination therapy comprising the GREM1 antagonist defined herein and gemcitabine or its derivatives can be used for the treatment or prevention of cancers that are typically targeted by gemcitabine or its derivatives.Cancer can be the cancer or tumor previously described for treatment with gemcitabine or its derivatives.Such cancers include, but are not limited to, pancreatic cancer, breast cancer, ovarian cancer, non-small cell lung cancer and bladder cancer.

[0033] The cancer may be a cancer that is responsive to treatment with gemcitabine or its derivatives and / or a cancer that is intended to be treated with gemcitabine or its derivatives. Alternatively, the cancer may be a cancer that is poorly responsive, non-responsive, or refractory to treatment with gemcitabine or its derivatives. The cancer may be previously described as not suitable for treatment with gemcitabine or its derivatives. In some cases, the cancer may be initially responsive to treatment with gemcitabine or its derivatives, but develop resistance to treatment with gemcitabine. Responsiveness can be measured by any means. For example, responsiveness to treatment can be evaluated by measuring tumor size before and after treatment using X-ray, CT or MRI scan, or by measuring tumor markers. Blood tests to determine organ function can also be used to evaluate responsiveness to treatment. One exemplary blood marker for monitoring responsiveness of pancreatic cancer is CA19-9. Those skilled in the art know how to measure the responsiveness of cancer to treatment.

[0034] The cancer may additionally or alternatively be characterized as comprising dormant cancer cells, e.g., dormant stem-like cancer cells, and / or as being a recurrent cancer.

[0035] In a particularly preferred embodiment, the anti-GREM1 antagonist is administered in combination with gemcitabine or a derivative thereof for the treatment of pancreatic cancer. One exemplary pancreatic cancer is exocrine pancreatic cancer, such as pancreatic ductal adenocarcinoma.

[0036] cancer In the context of a combination therapy comprising an anti-GREM1 antagonist and a cytidine analogue such as gemcitabine or a derivative thereof or a deoxycytidine analogue, the cancer may be any cancer or tumor. In particular, in the context of a combination therapy comprising an anti-GREM1 antagonist and a cytidine analogue such as gemcitabine or a derivative thereof or a deoxycytidine analogue, the cancer may be any cancer or tumor with a stroma, typically a detumorized stroma. The cancer may be any cancer or tumor that is GREM1-inducible. The cancer may be any cancer in which overexpression of stromal and / or epithelial GREM1 is observed. The cancer or tumor may have stromal GREM1 overexpression and no epithelial GREM1 overexpression. The cancer or tumor may have epithelial GREM1 overexpression and no stromal GREM1 overexpression. In a preferred embodiment, the cancer or tumor has overexpression of GREM1 in detumorized stroma. The cancer or tumor may be any cancer or tumor suitable for targeting with a GREM1 antagonist and / or a cytidine or deoxycytidine analog, including any cancer or tumor known in the art to be suitable for targeting with such an agent(s) and any cancer or tumor known in the art to be suitable for treatment with such an agent(s). For example, the cancer or tumor may be any cancer or tumor suitable for targeting with gemcitabine (2'-deoxy-2',2'-difluorocytidine) or its derivatives. The cancer or tumor may be any cancer or tumor suitable for targeting with azacytidine (5-azacytidine) or its derivatives. The cancer or tumor may be any cancer or tumor suitable for targeting with cytarabine (Ara-C / cytosine 1-[beta]-D-arabinofuranoside) or its derivatives. The cancer or tumor may be any cancer or tumor suitable for targeting with decitabine (5-aza-2'-deoxycytidine / 5-azadeoxycytidine) or a derivative thereof. The cancer or tumor may be any cancer or tumor suitable for targeting with troxacitabine (troxatyl / 4-amino-1-[(2S)-2-(hydroxymethyl)-1,3-dioxolan-4-yl]pyrimidin-2-one) or a derivative thereof. The cancer or tumor may be a solid tumor.Solid tumors may have a hypoplasmic stroma.

[0037] Anti-GREM1 antagonists have previously been shown to be effective in treating various cancers in International Publication No. WO2019 / 243801, filed June 18, 2019, the disclosure of which is incorporated herein by reference in its entirety. 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, splenic cancer, bone resident cancer, and osteosarcoma. The cancer that may be treated may be intestinal cancer, colon cancer, or rectal cancer. The cancer to be treated may be disseminated cancer, e.g., metastatic cancer. Disseminated cancer should be understood as cancer that has spread from its original site of origin in the body. For example, disseminated cancer may originate from the patient's bone marrow, colon, prostate, or breast tissue and metastasize to the patient's liver or lung, etc.

[0038] Also, combination therapy comprising a GREM1 antagonist and a cytidine analog such as gemcitabine or a derivative thereof or a deoxycytidine analog may be used to prevent cancer dissemination. For example, combination therapy comprising a GREM1 antagonist and gemcitabine or a derivative thereof may be used to prevent cancer dissemination. Combination therapy comprising a GREM1 antagonist and azacitidine or a derivative thereof may be used to prevent cancer dissemination. Combination therapy comprising a GREM1 antagonist and cytarabine or a derivative thereof may be used to prevent cancer dissemination. Combination therapy comprising a GREM1 antagonist and decitabine or a derivative thereof may be used to prevent cancer dissemination. Combination therapy comprising a GREM1 antagonist and troxacitabine or a derivative thereof may be used to prevent cancer dissemination.

[0039] Combination therapy comprising a GREM1 antagonist and a cytidine analogue such as gemcitabine or its derivatives or a deoxycytidine analogue can be used to prevent polyposis associated with cancer. For example, combination therapy comprising a GREM1 antagonist and gemcitabine or its derivatives can be used to prevent polyposis associated with cancer. Combination therapy comprising a GREM1 antagonist and azacitidine or its derivatives can be used to prevent polyposis associated with cancer. Combination therapy comprising a GREM1 antagonist and cytarabine or its derivatives can be used to prevent polyposis associated with cancer. Combination therapy comprising a GREM1 antagonist and decitabine or its derivatives can be used to prevent polyposis associated with cancer. Combination therapy comprising a GREM1 antagonist and troxacitabine or its derivatives can be used to prevent polyposis associated with cancer.

[0040] Grading systems are used in cancer biology and medicine to classify cancer cells with respect to their lack of cellular differentiation. This reflects how different the morphology of cancer cells is from healthy cells found in the tissue in which they originate. Grading systems can be used as an indication of how fast a particular cancer will grow. Typically used cancer grades are grades (G)X and 1-4. GX indicates that the cancer grade cannot be assessed. G1 (low grade) cancer cells have a similar morphology to normal, healthy cells (i.e., well differentiated) and are expected to grow slowly and not likely to spread. G2 (intermediate grade) cancer cells are moderately differentiated; i.e., they look more abnormal and are expected to grow slightly faster than G1 cells. G3 (high grade) cancer cells have a very different morphology compared to normal cells (i.e., they are less differentiated) and are expected to grow faster than G1 and G2 cells. G4 (high-grade) cancer cells are undifferentiated (also called anaplastic) and are predicted to have the highest proliferation potential.

[0041] Cancer grade is different from cancer staging, which gives an indication of how the cancer will spread. A common cancer staging system has five stages: Stage 0: cancer cells are in situ (located in normal tissue); Stage I: cancer is limited to one part of the body; Stage II: cancer is locally advanced; Stage III: cancer is more locally advanced (whether it is designated as stage II or stage III may depend on the particular type of cancer); Stage IV: cancer has often metastasized, or spread to other organs or the whole body.

[0042] The skilled artisan knows how to determine the grade and / or stage of cancer. In one embodiment, the present invention relates to the use of anti-GREM1 antagonists for the treatment and / or prevention of established cancer. In one embodiment, the cancer is an established cancer. The established cancer may be a high-grade cancer, such as a G3 or G4 cancer. The established cancer may be a stage II or higher cancer. The established cancer may be a stage III or IV cancer. In one embodiment, the established cancer is a metastatic stage IV cancer. In one embodiment, the established cancer is an established pancreatic cancer.

[0043] In addition to the specifically exemplified uses in the treatment and prevention of pancreatic cancer, the inventors envision that the therapeutic effects of GREM1 antagonists in combination with gemcitabine and other cytidine or deoxycytidine analogs, as exemplified in the Examples, are also applicable to the treatment of other cancers having corresponding properties as described herein.

[0044] In particular, it is envisaged that combinations comprising GREM1 antagonists will be useful for the prevention or treatment of cancers in which there is stromal and / or epithelial overexpression of GREM1 and this overexpression contributes to the proliferation of malignant cells, including pancreatic cancer, bladder cancer, lung cancer, gastric cancer, duodenal cancer, esophageal cancer, head and neck cancer, glioma, endometrial cancer, liver cancer, splenic cancer, bone resident cancer and osteosarcoma.

[0045] The antagonists of the present invention are used in combination with a cytidine analog or deoxycytidine analog, such as gemcitabine or a derivative thereof, to treat or prevent cancer. Other cytidine analogs or deoxycytidine analogs contemplated for use in the present invention are described herein and include azacitidine or a derivative thereof; cytarabine or a derivative thereof; decitabine or a derivative thereof; or troxacitabine or a derivative thereof. The antagonists of the present invention are also used in combination with a proliferation-dependent cytotoxic agent to treat or prevent pancreatic cancer.

[0046] Preventing cancer also includes preventing a subject from being diagnosed with cancer or delaying the onset of cancer. Preventing cancer can also include preventing the relapse or recurrence of cancer in a subject previously diagnosed with cancer. Preventing cancer additionally includes increasing the survival of a subject who has not been diagnosed with cancer or who has previously been diagnosed with cancer.

[0047] Treating cancer can improve one or more symptoms of cancer, induce or prolong remission of cancer, or delay relapse or recurrence of cancer. Treating cancer can cure, alleviate, or partially halt cancer. It may result in a decrease in the severity of disease symptoms, or an increase in the frequency or duration of symptom-free periods. Treating cancer can also include preventing the spread of cancer (e.g., established cancer) from a site of origin in the patient's body to one or more secondary sites in the patient's body. Thus, treating cancer can include preventing dissemination or metastasis of an existing cancer. Treating pancreatic cancer can result in a reduction in primary tumor size, as assayed, for example, by CT or endoscopic ultrasound. Such a reduction may facilitate Whipple (pancreaticoduodenectomy), which removes tumors from the head of the pancreas. The present inventors have demonstrated that treatment with an anti-GREM1 antagonist in combination with gemcitabine can increase survival in a KPC mouse model. Thus, treatment of pancreatic cancer with gemcitabine, or another cytidine analog or deoxycytidine analog according to the invention, can also improve patient survival.

[0048] Pancreatic cancer The present invention is preferably directed to the treatment or prevention of pancreatic cancer.As described in more detail in the examples, the inventors have confirmed the overexpression of GREM1 in human pancreatic ductal adenocarcinoma samples, and have demonstrated that high expression of GREM1 mRNA is significantly associated with poor prognosis (Figure 1).Furthermore, the inventors have demonstrated that combination therapy comprising anti-GREM1 antagonist and gemcitabine increases survival in KPC mouse model.

[0049] Thus, in one embodiment, the pancreatic cancer is a pancreatic cancer characterized by having overexpression of GREM1. In another embodiment, the pancreatic cancer can be characterized as having an exocrine tumor or a neuroendocrine tumor. Pancreatic neuroendocrine cancer (also known as pancreatic islet cell tumor) arises in the endocrine glands of the pancreas. Particularly preferred forms of pancreatic cancer are exocrine pancreatic cancers, such as pancreatic ductal adenocarcinoma, which arises in the lining of the pancreatic duct and accounts for 90% of all pancreatic carcinomas (Feldmann et al. J Hepatobiliary Pancreat Surg. 2007; 14(3): 224-32). Other exocrine pancreatic cancers include squamous cell carcinomas that form in the pancreatic duct; adenosquamous carcinomas; signet ring cell carcinomas; and colloid carcinomas that typically arise from intraductal papillary mucinous neoplasms. Improved treatments are needed for pancreatic cancer, due to late diagnosis, poor prognosis, and limited treatment strategies.

[0050] A preferred type of pancreatic cancer to be treated may be resistant to one or more known anti-cancer agents (e.g., chemotherapeutic agents). The pancreatic cancer may be disseminated pancreatic cancer. The pancreatic cancer may be metastatic pancreatic cancer. Metastatic cancer should be understood as cancer that has spread from its original site of origin in the living body. Thus, metastatic pancreatic cancer refers to cancer that starts in the pancreas and spreads to other organs such as the lung, liver, bone, brain, etc. The pancreatic cancer may also be recurrent pancreatic cancer. In other words, pancreatic cancer to be treated by the method of the present invention includes pancreatic cancer that has recurred after months or even years after previous treatment such as chemotherapy, radiotherapy, or radical surgery. A preferred type of pancreatic cancer to be treated may be resistant to one or more known anti-cancer agents (e.g., chemotherapeutic agents), as further described below.

[0051] In some aspects, the pancreatic cancer may be a pancreatic cancer that is responsive to treatment with a cytidine analog or deoxycytidine analog, such as gemcitabine or its derivatives. Alternatively, the pancreatic cancer may be a pancreatic cancer that is poorly responsive, non-responsive, or refractory to treatment with a cytidine analog or deoxycytidine analog, such as gemcitabine or its derivatives. The pancreatic cancer may be one that was previously considered unsuitable for treatment with a cytidine analog or deoxycytidine analog, such as gemcitabine or its derivatives. In some cases, the pancreatic cancer may be initially responsive to treatment with a cytidine analog or deoxycytidine analog, such as gemcitabine or its derivatives, but develop resistance to treatment with a cytidine analog or deoxycytidine analog, such as gemcitabine treatment.

[0052] For example, in some aspects, the pancreatic cancer may be a pancreatic cancer that is responsive to treatment with gemcitabine, azacitidine, cytarabine, decitabine, troxacitabine, or any derivative thereof. The pancreatic cancer may be a pancreatic cancer that is poorly responsive, non-responsive, or refractory to treatment with gemcitabine, azacitidine, cytarabine, decitabine, troxacitabine, or any derivative thereof. The pancreatic cancer may be one that was previously considered unsuitable for treatment with gemcitabine, azacitidine, cytarabine, decitabine, troxacitabine, or any derivative thereof. In some cases, pancreatic cancer may be initially responsive to treatment with gemcitabine, azacytidine, cytarabine, decitabine, troxacitabine, or any derivatives thereof, but may develop resistance to treatment with gemcitabine, azacytidine, cytarabine, decitabine, troxacitabine, or any derivatives thereof.

[0053] lung cancer In a further aspect, the present invention relates to the treatment or prevention of lung cancer. The lung cancer is most preferably non-small cell lung cancer (NSCLC). The NSCLC can be advanced NSCLC, such as stage III or stage IV NSCLC. The NSCLC can be squamous cell carcinoma, adenocarcinoma or large cell carcinoma. The cancer can be small cell lung cancer. The lung cancer can be unresectable. The lung cancer can be primary lung cancer or any secondary cancer that has spread to the lung, such as breast cancer or pancreatic cancer. The lung cancer can be disseminated lung cancer. The lung cancer can be metastatic lung cancer. The lung cancer can be lung cancer characterized by having overexpression of GREM1. The lung cancer can be recurrent lung cancer. In other words, the lung cancer to be treated by the method of the present invention includes lung cancer that has recurred after months or even years after previous treatment, such as chemotherapy, radiotherapy or radical surgery. The preferred type of lung cancer to be treated can be resistant to one or more known anti-cancer agents (e.g., chemotherapeutic agents), as further described below.

[0054] In some embodiments, lung cancer such as NSCLC can be a lung cancer that is responsive to treatment with cytidine analogs or deoxycytidine analogs, such as gemcitabine or its derivatives. Alternatively, lung cancer such as NSCLC can be a lung cancer that is poorly responsive, non-responsive, or refractory to treatment with cytidine analogs or deoxycytidine analogs, such as gemcitabine or its derivatives. Lung cancer such as NSCLC can be one that was previously considered unsuitable for treatment with cytidine analogs or deoxycytidine analogs, such as gemcitabine or its derivatives. In some cases, lung cancer such as NSCLC can be initially responsive to treatment with cytidine analogs or deoxycytidine analogs, such as gemcitabine or its derivatives, but develop resistance to treatment with cytidine analogs or deoxycytidine analogs, such as gemcitabine treatment.

[0055] For example, in some aspects, the lung cancer may be a lung cancer that is responsive to treatment with gemcitabine, azacitidine, cytarabine, decitabine, troxacitabine, or any derivative thereof. Alternatively, the lung cancer may be a lung cancer that is poorly responsive, non-responsive, or refractory to treatment with gemcitabine, azacitidine, cytarabine, decitabine, troxacitabine, or any derivative thereof. The lung cancer may be one that was previously considered unsuitable for treatment with gemcitabine, azacitidine, cytarabine, decitabine, troxacitabine, or any derivative thereof. In some cases, the lung cancer may be initially responsive to treatment with gemcitabine, azacytidine, cytarabine, decitabine, troxacitabine, or any derivative thereof, but may develop resistance to treatment with gemcitabine, azacytidine, cytarabine, decitabine, troxacitabine, or any derivative thereof.

[0056] Bladder cancer In a further aspect, the present invention relates to the treatment or prevention of bladder cancer. The bladder cancer may be transitional cell (urothelial) bladder cancer. The bladder cancer may arise from the epithelial lining of the bladder. The bladder cancer may be non-muscle invasive bladder cancer. The bladder cancer may be squamous cell bladder cancer. The bladder cancer may be adenocarcinoma. The bladder cancer may be a high-grade T1 tumor that has grown from the lining of the bladder into the lamina propria of the bladder. The bladder cancer may be superficial or invasive bladder cancer. The bladder cancer may be recurrent bladder cancer. The term recurrent bladder cancer as used herein refers to bladder cancer that has recurred after treatment, such as surgical treatment.

[0057] The present invention further provides for the treatment and prevention of bladder cancer by administering an anti-GREM1 antagonist in combination with gemcitabine or a derivative thereof. The bladder cancer may be disseminated bladder cancer. The bladder cancer may be metastatic bladder cancer. The bladder cancer may be lung metastatic bladder cancer. The bladder cancer may be liver metastatic bladder cancer. The bladder cancer may be bone metastatic bladder cancer. The bladder cancer may be bladder cancer characterized by having overexpression of GREM1. The bladder cancer may also be recurrent bladder cancer. In other words, the bladder cancer to be treated by the method of the present invention includes bladder cancer that has recurred after months or even years following previous treatments such as chemotherapy, radiotherapy or radical surgery. The preferred types of bladder cancer to be treated may be resistant to one or more known anticancer agents (e.g., chemotherapeutic agents), as further described below.

[0058] In some aspects, the bladder cancer may be a bladder cancer that is responsive to treatment with a cytidine analog or deoxycytidine analog, such as gemcitabine or its derivatives. Alternatively, the bladder cancer may be a bladder cancer that is poorly responsive, non-responsive, or refractory to treatment with a cytidine analog or deoxycytidine analog, such as gemcitabine or its derivatives. The bladder cancer may be one that was previously considered unsuitable for treatment with a cytidine analog or deoxycytidine analog, such as gemcitabine or its derivatives. In some cases, the bladder cancer may be initially responsive to treatment with a cytidine analog or deoxycytidine analog, such as gemcitabine or its derivatives, but develop resistance to treatment with a cytidine analog or deoxycytidine analog, such as gemcitabine or its derivatives.

[0059] For example, in some aspects, the bladder cancer may be a bladder cancer that is responsive to treatment with gemcitabine, azacitidine, cytarabine, decitabine, troxacitabine, or any derivative thereof. Alternatively, the bladder cancer may be a bladder cancer that is poorly responsive, non-responsive, or refractory to treatment with gemcitabine, azacitidine, cytarabine, decitabine, troxacitabine, or any derivative thereof. The bladder cancer may be one that was previously considered unsuitable for treatment with gemcitabine, azacitidine, cytarabine, decitabine, troxacitabine, or any derivative thereof. In some cases, bladder cancer may be initially responsive to treatment with gemcitabine, azacitidine, cytarabine, decitabine, troxacitabine, or any derivatives thereof, but may develop resistance to treatment with gemcitabine, azacitidine, cytarabine, decitabine, troxacitabine, or any derivatives thereof.

[0060] Ovarian cancer In a further aspect, the present invention relates to the treatment or prevention of ovarian cancer. The ovarian cancer may be epithelial ovarian cancer, germ cell ovarian cancer or sex cord stromal ovarian cancer. The ovarian cancer may be primary peritoneal cancer. The ovarian cancer may be fallopian tube cancer. The ovarian cancer may be characterized by borderline ovarian tumors. The ovarian cancer may be characterized by germ cell ovarian tumors. The ovarian cancer may be clear cell ovarian cancer. The ovarian cancer may be serous ovarian cancer. The ovarian cancer may be mucinous ovarian cancer. The ovarian cancer may be endometrial cancer.

[0061] The ovarian cancer may be characterized by having overexpression of GREM1.The ovarian cancer may also be recurrent ovarian cancer.In other words, the ovarian cancer to be treated by the method of the present invention includes ovarian cancer that recurs after several months or even years after previous treatment such as chemotherapy, radiotherapy or radical surgery.The preferred type of ovarian cancer to be treated may be resistant to one or more known anti-cancer agents (e.g., chemotherapeutic agents), as described further below.

[0062] The present invention further provides for the treatment and prevention of ovarian cancer by administering an anti-GREM1 antagonist in combination with gemcitabine or a derivative thereof. The ovarian cancer may be disseminated ovarian cancer. The ovarian cancer may be metastatic ovarian cancer. The ovarian cancer may be lung metastatic ovarian cancer. The ovarian cancer may be liver metastatic ovarian cancer. The ovarian cancer may be bone metastatic ovarian cancer.

[0063] In some aspects, the ovarian cancer may be an ovarian cancer that is responsive to treatment with a cytidine analog or deoxycytidine analog, such as gemcitabine or its derivatives. Alternatively, the ovarian cancer may be an ovarian cancer that is poorly responsive, non-responsive, or refractory to treatment with a cytidine analog or deoxycytidine analog, such as gemcitabine or its derivatives. The ovarian cancer may be one that was previously considered unsuitable for treatment with a cytidine analog or deoxycytidine analog, such as gemcitabine or its derivatives. In some cases, the ovarian cancer may be initially responsive to treatment with a cytidine analog or deoxycytidine analog, such as gemcitabine or its derivatives, but develop resistance to treatment with a cytidine analog or deoxycytidine analog, such as gemcitabine treatment.

[0064] For example, in some aspects, the ovarian cancer may be an ovarian cancer that is responsive to treatment with gemcitabine, azacitidine, cytarabine, decitabine, troxacitabine, or any derivative thereof. The ovarian cancer may be an ovarian cancer that is poorly responsive, non-responsive, or refractory to treatment with gemcitabine, azacitidine, cytarabine, decitabine, troxacitabine, or any derivative thereof. The ovarian cancer may be one that was previously considered unsuitable for treatment with gemcitabine, azacitidine, cytarabine, decitabine, troxacitabine, or any derivative thereof. In some cases, ovarian cancer may be initially responsive to treatment with gemcitabine, azacytidine, cytarabine, decitabine, troxacitabine, or any derivatives thereof, but may develop resistance to treatment with gemcitabine, azacytidine, cytarabine, decitabine, troxacitabine, or any derivatives thereof.

[0065] Colorectal cancer The present invention relates in one aspect to the prevention or treatment of colorectal cancer. By way of background, the intestinal mucosa is a complex ecosystem in which the epithelium is in an interdependent relationship with its microenvironment, particularly the underlying stroma. Mesenchymal-epithelial crosstalk is deeply involved in controlling homeostasis and is dynamically altered during intestinal regeneration and cancer. Cell signaling networks, which are effector pathways of intercompartmental crosstalk and control epithelial cell fate decisions, can be coupled and disrupted by the tumor microenvironment in colorectal cancer.

[0066] Current chemotherapy management of colorectal cancer has not changed substantially in the past 20 years and is primarily based on combination cytotoxic agents directed against the proliferating tumour epithelium (such as FOLFOX (combination therapy containing folinic acid, fluorouracil and oxaliplatin) and FOLFIRI (combination therapy containing folinic acid, fluorouracil and irinotecan) regimens, http: / / www.cancerresearchuk.org / about-cancer / cancer-in-general / treatment / cancer-drugs / drugs), with the potential for resistance to these epithelium-targeted agents to develop. Identifying novel treatments for use in colorectal cancer has never been more important.

[0067] Thus, the cancer or tumor to be treated is a colon cancer or a colon tumor. A particularly preferred form of colon cancer to be treated is a colon cancer characterized by overexpression of GREM1 in stromal cells, i.e., stromal GREM1 overexpression. The stromal cells may be cancer-associated fibroblasts. A colon cancer with stromal GREM1 overexpression may not show epithelial GREM1 overexpression. A colon cancer with stromal GREM1 overexpression may include stromal Foxl1 overexpression. A particularly suitable form of colon cancer to be treated is a colon cancer of mesenchymal subtype, also described as CMS4 (Guinney et al, Nat Med 2015). Any other subtype of colon cancer may also be treated, including any of CMS1, CMS2 and CMS3, as described in Guinney et al. supra. The colon cancer described herein may be a proximal colon cancer (or proximal colon tumor). The proximal colon is the region of the large intestine upstream from the splenic flexure, and refers to the cecum, ascending colon, and transverse colon. Cancers or tumors in this region are also called right-sided cancers or tumors. The present invention may relate to the treatment of right-sided colon cancer or right-sided colon tumors.

[0068] The colon cancer may be a distal colon cancer (or a distal colon tumor). Distal colon is the region of the colon downstream from the splenic flexure, meaning the descending colon, the sigmoid colon and the rectum. Cancers or tumors in this region are also called left-sided cancers or tumors. The present invention may relate to the treatment of left-sided colon cancers or tumors. The cancer with stromal overexpression of GREM1 may preferably be a sporadic cancer. Sporadic cancers may be caused by somatic mutations. Sporadic cancers may be caused by carcinogens. Sporadic cancers are not due to inherited genetic mutations. Sporadic cancers may cause stromal overexpression of GREM1. The growth of sporadic cancers may depend on stromal overexpression of GREM1 in the cancer.

[0069] At least three single nucleotide polymorphisms (SNPs) near GREM1 are independently associated with risk of colorectal cancer (CRC) in Northern European Caucasians and possibly other ethnicities (Tomlinson et al, PLos Genet, 2011). There is a direct link with GREM1 expression, and other SNPs are likely to have similar effects. In addition, two common SNPs near BMP2, two near BMP4, and one near BMP7 affect expression of BMP ligands and thus CRC risk. Thus, cancers may contain one or more of the above SNPs.

[0070] Further types of cancer or tumors amenable to treatment according to the invention are those that show overexpression of GREM1 in epithelial cells. Overexpression of GREM1 in epithelial cells may cause cancer. Cancer growth may depend on overexpression of GREM1 in the epithelium. Thus, the cancer may be of epithelial origin. The cancer may be colon cancer or duodenal cancer. The cancer may be GREM1-induced. By GREM1-induced, it is meant that the cancer is caused by a mutagenic event that enhances the activity or expression of GREM1. Such cancers may result from inherited genetic mutations. Thus, the cancer may be familial (see below).

[0071] Preferred types of colon cancer to be treated may be resistant to one or more known anti-cancer agents (eg, chemotherapeutic agents), as described further below.

[0072] The colorectal cancer can be disseminated colorectal cancer. The colorectal cancer can be metastatic colorectal cancer. The colorectal cancer can be lung metastatic colorectal cancer. The colorectal cancer can be liver metastatic colorectal cancer. The colorectal cancer can be bone metastatic colorectal cancer.

[0073] Colon cancer can be characterized by stromal overexpression of Foxl1. Colon cancer can be characterized by stromal overexpression of one or more Wnt ligands. For example, colon cancer can be characterized by stromal overexpression of Wnt5A and / or Wnt2B. When colon cancer has stromal overexpression of Foxl1 and / or Wnt ligand, for example, Wnt5A or Wnt2B, the colon cancer is particularly suitable for prevention or treatment with GREM1 antagonist.

[0074] In some aspects, the colon cancer may be a colon cancer that is responsive to treatment with a cytidine analog or deoxycytidine analog, such as gemcitabine or its derivatives. Alternatively, the colon cancer may be a colon cancer that is poorly responsive, non-responsive, or refractory to treatment with a cytidine analog or deoxycytidine analog, such as gemcitabine or its derivatives. The colon cancer may be one that was previously considered unsuitable for treatment with a cytidine analog or deoxycytidine analog, such as gemcitabine or its derivatives. In some cases, the colon cancer may be initially responsive to treatment with a cytidine analog or deoxycytidine analog, such as gemcitabine or its derivatives, but develop resistance to treatment with a cytidine analog or deoxycytidine analog, such as gemcitabine treatment.

[0075] For example, in some aspects, the colon cancer may be a colon cancer that is responsive to treatment with gemcitabine, azacitidine, cytarabine, decitabine, troxacitabine, or any derivative thereof. The colon cancer may be a colon cancer that is poorly responsive, non-responsive, or refractory to treatment with gemcitabine, azacitidine, cytarabine, decitabine, troxacitabine, or any derivative thereof. The colon cancer may be one that was previously considered unsuitable for treatment with gemcitabine, azacitidine, cytarabine, decitabine, troxacitabine, or any derivative thereof. In some cases, colorectal cancer may be initially responsive to treatment with gemcitabine, azacytidine, cytarabine, decitabine, troxacitabine, or any derivatives thereof, but may develop resistance to treatment with gemcitabine, azacytidine, cytarabine, decitabine, troxacitabine, or any derivatives thereof.

[0076] Familial cancer Familial cancers include those resulting from a mutation or mutations in the gene encoding GREM1 or any other mutation affecting expression of the GREM1 gene. Autosomal dominant mixed polyposis syndrome (HMPS) is caused by a 40 kb duplication upstream of GREM1, resulting in a shift in pathological compartment expression from a mesenchymal-restricted gradient to ectopic GREM1 gene expression throughout the epithelium.

[0077] A subject to be treated with an anti-GREM1 antagonist may have previously been determined to be at risk for developing a familial cancer. For example, the subject may have been determined to be at risk based on family history and / or because the subject has a mutation in a gene known to cause or increase the risk of developing a familial cancer.

[0078] The familial cancer may be Lynch syndrome, also called hereditary nonpolyposis colorectal cancer (HNPCC). The familial cancer may be familial adenomatous polyposis (FAP).

[0079] Patients or subjects suffering from familial adenomatous polyposis (FAP) may be particularly suitable for treatment with combination therapy comprising anti-GREM1 antagonists. The familial cancer to be treated or prevented with combination therapy comprising anti-GREM1 antagonists (e.g., anti-GREM1 antibodies) and gemcitabine or its derivatives may be FAP. Subjects who have previously suffered from FAP may be administered prophylactically with anti-GREM1 antagonists in combination with gemcitabine or its derivatives, for example to prevent recurrence. Subjects who have not previously suffered from FAP but have previously been determined to be at risk for developing FAP may be administered prophylactically with anti-GREM1 antagonists in combination with gemcitabine. The subject is determined to be at risk for developing FAP because the subject is found to have a fulminant mutation in the Apc gene.

[0080] In some aspects, the familial cancer may be a familial cancer that is responsive to treatment with a cytidine analog or deoxycytidine analog, such as gemcitabine or its derivatives. Alternatively, the familial cancer may be a familial cancer that is poorly responsive, non-responsive, or refractory to treatment with a cytidine analog or deoxycytidine analog, such as gemcitabine or its derivatives. The familial cancer may be one that was previously considered unsuitable for treatment with a cytidine analog or deoxycytidine analog, such as gemcitabine or its derivatives. In some cases, the familial cancer may be initially responsive to treatment with a cytidine analog or deoxycytidine analog, such as gemcitabine or its derivatives, but develop resistance to treatment with a cytidine analog or deoxycytidine analog, such as gemcitabine treatment.

[0081] For example, in some aspects, the familial cancer may be a familial cancer that is responsive to treatment with gemcitabine, azacitidine, cytarabine, decitabine, troxacitabine, or any derivative thereof. The familial cancer may be a familial cancer that is poorly responsive, non-responsive, or refractory to treatment with gemcitabine, azacitidine, cytarabine, decitabine, troxacitabine, or any derivative thereof. The familial cancer may be one that was previously considered unsuitable for treatment with gemcitabine, azacitidine, cytarabine, decitabine, troxacitabine, or any derivative thereof. In some cases, the familial cancer may be initially responsive to treatment with gemcitabine, azacytidine, cytarabine, decitabine, troxacitabine, or any of their derivatives, but may develop resistance to treatment with gemcitabine, azacytidine, cytarabine, decitabine, troxacitabine, or any of their derivatives.

[0082] Multiple myeloma In another aspect, the present invention relates to the treatment or prevention of multiple myeloma. Multiple myeloma (MM) is a hematological malignancy characterized by clonal proliferation of plasma cells (PC) within the bone marrow (BM). It is well known that BM supports MM tumor growth, and bidirectional signaling between tumor cells and BM is essential for the continued proliferation, proliferation, and survival of MM PC. Cellular and non-cellular BM components differentially affect the proliferation and proliferation of MMPC. Although recent studies have identified components of BM that play a role in disease progression and developed treatments that target these, standard treatments in MM still rely primarily on targeting the tumor cells themselves. Although such therapies are effective in prolonging patient survival, the large role played by BM in MM cell proliferation, proliferation, survival, and drug resistance necessitates more effective therapies that target key aspects of the disease. Indeed, MM is largely incurable, and disease relapse is a major problem faced in effectively treating the disease.

[0083] Therefore, the present invention is also directed to the treatment or prevention of multiple myeloma. Multiple myeloma typically includes the presence of multiple plasma cell masses in the bone marrow. Thus, multiple myeloma is typically associated with abnormal proliferation of plasma cells in the bone marrow. Particularly preferred forms of multiple myeloma to be treated are characterized by having overexpression of GREM1 in the bone marrow. Thus, multiple myeloma may include overexpression of stromal GREM1. Stromal GREM1 overexpression may be present in the dense matter compartment of bone. Stromal GREM1 overexpression may reflect an increase in the number of stromal cells or an increase in the expression level of GREM1 in existing GREM1-expressing stromal cells. Bone marrow may include osteochondral (OCR) stem cells. Stromal cells that overexpress GREM1 may include OCR stem cells. Preferred types of multiple myeloma to be treated may be resistant to one or more known anti-cancer agents (e.g., chemotherapeutic agents), as further described below.

[0084] In some aspects, the multiple myeloma can be a multiple myeloma that is responsive to treatment with a cytidine analog or deoxycytidine analog, such as gemcitabine or its derivatives. Alternatively, the multiple myeloma can be a multiple myeloma that is poorly responsive, non-responsive, or refractory to treatment with a cytidine analog or deoxycytidine analog, such as gemcitabine or its derivatives. The multiple myeloma can be one that was previously considered unsuitable for treatment with a cytidine analog or deoxycytidine analog, such as gemcitabine or its derivatives. In some cases, the multiple myeloma can be initially responsive to treatment with a cytidine analog or deoxycytidine analog, such as gemcitabine or its derivatives, but develop resistance to treatment with a cytidine analog or deoxycytidine analog, such as gemcitabine treatment.

[0085] For example, in some aspects, the multiple myeloma may be a multiple myeloma that is responsive to treatment with gemcitabine, azacitidine, cytarabine, decitabine, troxacitabine, or any derivative thereof. The multiple myeloma may be a multiple myeloma that is poorly responsive, non-responsive, or refractory to treatment with gemcitabine, azacitidine, cytarabine, decitabine, troxacitabine, or any derivative thereof. The multiple myeloma may be one that was previously considered unsuitable for treatment with gemcitabine, azacitidine, cytarabine, decitabine, troxacitabine, or any derivative thereof. In some cases, multiple myeloma may be initially responsive to treatment with gemcitabine, azacytidine, cytarabine, decitabine, troxacitabine, or any derivatives thereof, but may develop resistance to treatment with gemcitabine, azacytidine, cytarabine, decitabine, troxacitabine, or any derivatives thereof.

[0086] Breast cancer In another aspect, the present invention relates to the treatment or prevention of breast cancer. The breast cancer may be invasive breast cancer, e.g., invasive lobular breast cancer. The breast cancer may be triple-negative breast cancer. The breast cancer may be inflammatory breast cancer. The breast cancer may be breast angiosarcoma. The breast cancer may be in situ ductal carcinoma or in situ lobular carcinoma.

[0087] The present invention provides for the treatment and prevention of breast cancer by administering an anti-GREM1 antagonist in combination with gemcitabine or a derivative thereof. The breast cancer may comprise stromal GREM1 overexpression. The stromal breast cells overexpressing GREM1 may comprise stromal fibroblasts, also described herein as cancer-associated fibroblasts. The breast cancer may also be recurrent breast cancer. In other words, the breast cancer to be treated by the method of the present invention includes breast cancer that has recurred after months or even years following previous treatments such as chemotherapy, radiotherapy or curative surgery. The preferred types of breast cancer to be treated may be resistant to one or more known anti-cancer agents (e.g., chemotherapeutic agents), as further described below. The breast cancer may be disseminated breast cancer. The breast cancer may be metastatic breast cancer. The breast cancer may be lung metastatic breast cancer. The breast cancer may be liver metastatic breast cancer. The breast cancer may be bone metastatic breast cancer.

[0088] In some aspects, the breast cancer may be a breast cancer that is responsive to treatment with a cytidine analog or deoxycytidine analog, such as gemcitabine or its derivatives. Alternatively, the breast cancer may be a breast cancer that is poorly responsive, non-responsive, or refractory to treatment with a cytidine analog or deoxycytidine analog, such as gemcitabine or its derivatives. The breast cancer may be one that was previously considered unsuitable for treatment with a cytidine analog or deoxycytidine analog, such as gemcitabine or its derivatives. In some cases, the breast cancer may be initially responsive to treatment with a cytidine analog or deoxycytidine analog, such as gemcitabine or its derivatives, but develop resistance to treatment with a cytidine analog or deoxycytidine analog, such as gemcitabine treatment.

[0089] For example, in some aspects, the breast cancer may be a breast cancer that is responsive to treatment with gemcitabine, azacitidine, cytarabine, decitabine, troxacitabine, or any derivative thereof. Alternatively, the breast cancer may be a breast cancer that is poorly responsive, non-responsive, or refractory to treatment with gemcitabine, azacitidine, cytarabine, decitabine, troxacitabine, or any derivative thereof. The breast cancer may be one that was previously considered unsuitable for treatment with gemcitabine, azacitidine, cytarabine, decitabine, troxacitabine, or any derivative thereof. In some cases, breast cancer may be initially responsive to treatment with gemcitabine, azacytidine, cytarabine, decitabine, troxacitabine, or any derivative thereof, but may develop resistance to treatment with gemcitabine, azacytidine, cytarabine, decitabine, troxacitabine, or any derivative thereof.

[0090] Prostate cancer In a further aspect, the present invention relates to the treatment or prevention of prostate cancer. The prostate cancer may be prostate adenocarcinoma. The prostate cancer may be transitional cell carcinoma or urothelial carcinoma that has spread to the prostate.

[0091] Prostate cancer can be characterized by having overexpression of GREM1.Prostate cancer can also be recurrent prostate cancer.In other words, the prostate cancer to be treated by the method of the present invention includes prostate cancer that recurs after several months or even years after previous treatment such as chemotherapy, radiotherapy or radical surgery.The preferred type of prostate cancer to be treated can be resistant to one or more known anti-cancer agents (e.g., chemotherapeutic agents), as described further below.

[0092] The present invention further provides for the treatment and prevention of prostate cancer by administering an anti-GREM1 antagonist in combination with gemcitabine or a derivative thereof. The prostate cancer may be disseminated prostate cancer. The prostate cancer may be metastatic prostate cancer. The prostate cancer may be lung metastatic prostate cancer. The prostate cancer may be liver metastatic prostate cancer. The prostate cancer may be bone metastatic prostate cancer.

[0093] In some aspects, the prostate cancer can be a prostate cancer that is responsive to treatment with cytidine analogs or deoxycytidine analogs, such as gemcitabine or its derivatives. Alternatively, the prostate cancer can be a prostate cancer that is poorly responsive, non-responsive, or refractory to treatment with cytidine analogs or deoxycytidine analogs, such as gemcitabine or its derivatives. The prostate cancer can be a prostate cancer that was previously considered unsuitable for treatment with cytidine analogs or deoxycytidine analogs, such as gemcitabine or its derivatives. In some cases, the prostate cancer is initially responsive to treatment with cytidine analogs or deoxycytidine analogs, such as gemcitabine or its derivatives, but can develop resistance to treatment with cytidine analogs or deoxycytidine analogs, such as gemcitabine treatment.

[0094] For example, in some aspects, the prostate cancer may be a prostate cancer that is responsive to treatment with gemcitabine, azacitidine, cytarabine, decitabine, troxacitabine, or any derivative thereof. Alternatively, the prostate cancer may be a prostate cancer that is poorly responsive, non-responsive, or refractory to treatment with gemcitabine, azacitidine, cytarabine, decitabine, troxacitabine, or any derivative thereof. The prostate cancer may be one that was previously considered unsuitable for treatment with gemcitabine, azacitidine, cytarabine, decitabine, troxacitabine, or any derivative thereof. In some cases, prostate cancer may be initially responsive to treatment with gemcitabine, azacytidine, cytarabine, decitabine, troxacitabine, or any derivatives thereof, but may develop resistance to treatment with gemcitabine, azacytidine, cytarabine, decitabine, troxacitabine, or any derivatives thereof.

[0095] Stroma and epithelium The cancers described herein for prevention or treatment with a GREM1 antagonist in combination with gemcitabine may involve stromal and / or epithelial overexpression of GREM1.

[0096] As used herein, the term "stromal cell(s)" or "stroma" refers to the structural and / or connective parts of a tissue or organ.

[0097] Stromal tissue consists of an extracellular matrix that contains primarily connective tissue cells. The extracellular matrix is ​​composed primarily of ground substance, a porous hydrated gel made of proteoglycan aggregates, and connective tissue fibers. Three types of fibers are commonly found within the interstitium: type I collagen fibers, elastic fibers, and reticular fibers (type III collagen). Fibroblasts and pericytes are the most common types of interstitial cells.

[0098] In the case of cancer or tumors (e.g., arising from the epithelium of a tissue or organ), the stroma of the tissue or organ may aid in the growth or progression of the cancer. Stroma associated with cancer or tumors may be decellularized stroma caused by the proliferation of fibrous or connective tissue surrounding the cancer or tumor.

[0099] Overexpression of GREM1 can be observed in any part of stroma / any stromal cell. Stromal cells can be fibroblasts or fibroblast-like supporting cells. Stromal cells can be fibroblasts or fibroblast-like supporting cells isolated from the detumorized stroma of any of the above cancers or tumors, for example, from pancreas, colon or rectum in colorectal cancer, or bone marrow in multiple myeloma. Stromal cells can be cancer-associated fibroblasts.

[0100] The term "epithelium" as used herein refers to cells originating from the outer or inner lining of a tissue or organ. In the context of the large intestine, the intestinal epithelium is the layer of cells that forms the luminal surface or lining of both the small and large intestines of the digestive tract. It is composed of a simple columnar epithelium. The "upper barrier" is an intestinal epithelial monolayer of columnar cells consisting of four types of intestinal epithelial cells: absorptive enterocytes, goblet cells, Paneth cells and enteroendocrine cells. The properties of the upper barrier are similar in the small and large intestines. The main difference is established by the presence of ridges or projections (circular folds, villi, microvilli) in the duodenum, jejunum and ileum that allow an increase in the absorption area. This is not observed in the colon, which instead shows a flat surface. Among the projections of the mucosa called villi are inflexions called crypts of Lieberkuhn, which are distinct glandular ductal invasions. The cells in which overexpression of epithelial GREM1 is observed can be any epithelial cell, such as any intestinal epithelial cell.

[0101] Without being bound by theory, the inventors hypothesize that stroma targeting with anti-GREM1 antagonists can enhance the efficacy of chemotherapy treatment for pancreatic cancer, since the stroma itself can confer chemotherapy resistance to tumor cells. The inventors also hypothesize that overexpression of GREM1 in epithelium and / or stroma promotes stem cell / progenitor cell phenotype (increases stem cell / progenitor cell number), promotes epithelial stem cell behavior, and promotes cancer progression and / or resistance to chemotherapeutic agents. Thus, combination therapy comprising a GREM1 antagonist and a cytidine analogue or deoxycytidine analogue, such as gemcitabine or its derivatives, used according to the present invention can prevent the induction of abnormal cancer stem cell / progenitor cell phenotype, reduce epithelial stem cell behavior, and / or reduce the number of stem cells / progenitor cells in the epithelium of the subject's tissue or organ in which cancer is to be prevented or treated. The ability of GREM1 antagonists to affect stem cell behavior can be clinically assayed by evaluation of known epithelial stem cell and cancer stem cell markers.

[0102] Overexpression of GREM1 in stroma and / or epithelium can be determined by any method. Overexpression of GREM1 is typically determined by comparison with the level of the marker in normal cells of the same tissue type, i.e., basal expression level. Expression is typically normalized to the expression levels of other genes, preferably one or more housekeeping genes. GREM1 can also be classified as showing overexpression or underexpression in a threshold percentage of a cancer patient population. Overexpression in each patient in the population may be higher than 2 from the geometric mean. At least 10% of the patients in the population, more preferably at least 15% or more, may show such overexpression.

[0103] GREM1 stromal overexpression refers to stromal GREM1 levels that are higher than the levels in matched normal tissue, for example, stromal GREM1 levels may be at least two-fold higher than the levels in matched normal tissue.

[0104] When GREM1 is overexpressed, its amount can be increased to any amount compared to basal state.For example, GREM1-induced cancers such as HMPS can contain epithelial GREM1 upregulation of several thousand times, while no expression of GREM1 is observed in normal epithelium.Sporadic cancers containing stromal GREM overexpression can contain any level of stromal overexpression that exceeds the physiological GREM1 expression level in the normal stroma of an organ.Those skilled in the art can evaluate the presence of overexpression in stroma or epithelium by comparing it with the level of GREM1 in normal cells of the same type.

[0105] The amount determined may be the amount of mRNA. Thus, the cancer may include overexpression of GREM1 mRNA. The cancer may include an increased amount of GREM1 mRNA compared to normal cells of the same tissue type. The mRNA may be increased by any amount. The amount of mRNA may be measured using quantitative reverse transcription polymerase chain reaction (qRT-PCR), e.g., real-time qRT-PCR, QuantiGene assay (Affymetrix / Thermo Fisher), by Northern blotting, or by microarray, RNA sequencing. The mRNA expression is preferably determined by comparing the gene expression of the sample to the distribution of expression levels of a particular gene across a reference sample composed of tumors that are diploid for that gene. The z-score may be derived using the RNAseq by expectation maximisation (RSEM) algorithm (cBioportal for Cancer Genomics, www.cbioportal.org; Gao et al, 2013 and Serami eta al 2012). A z-score 2SD above or below the mean of the reference set is preferably considered as overexpression or underexpression, respectively.

[0106] The amount determined may be the amount of protein. The cancer may include overexpression of GREM1 protein compared to normal cells of the same tissue type. The protein may be increased by any amount. The amount of protein may be measured using immunohistochemistry, Western blotting, mass spectrometry or fluorescence activated cell sorting (FACS), including the use of the anti-GREM1 antibody of the present invention. The threshold for determining expression may differ between the techniques used and may be verified against the immunohistochemistry score.

[0107] Thus, the use of a GREM1 antagonist in combination with a cytidine analog or deoxycytidine analog to treat or prevent cancer in a patient as described herein may include (a) measuring the amount of GREM1 in the cancer, and (b) if the cancer comprises overexpression of GREM1, administering to the patient a GREM1 antagonist in combination with a cytidine analog or deoxycytidine analog as described herein, thereby treating or preventing the cancer. The amount of GREM1 may be the amount of mRNA or protein, and the overexpression may be any of the overexpressions discussed above. The GREM1 antagonist may be any of the GREM1 antagonists described herein.

[0108] Thus, the use of a GREM1 antagonist in combination with gemcitabine or a derivative thereof to treat or prevent cancer in a patient as described herein may include (a) measuring the amount of GREM1 in the cancer, and (b) if the cancer comprises overexpression of GREM1, administering a GREM1 antagonist in combination with gemcitabine or a derivative thereof to the patient, thereby treating or preventing the cancer. The amount of GREM1 may be the amount of mRNA or protein, and the overexpression may be any of the overexpressions described above. The GREM1 antagonist may be any of the GREM1 antagonists described herein.

[0109] The above measurements can be performed on any suitable sample from a patient. Measurements can be performed on a cancer or tumor biopsy obtained from a patient. Stroma and / or epithelium (stromal and / or epithelial cells) can be isolated from the biopsy. The biopsy tissue can be formalin-fixed, paraffin-embedded (FFPE) tissue or fresh tissue. The tissue can be pancreatic tissue, bladder tissue, lung tissue, endometrial tissue, breast tissue, stomach tissue, duodenal tissue, esophageal tissue, bone marrow or colon tissue. Any of the methods discussed above can be performed on a cancer biopsy. Such methods can also be performed on cancer cells circulating in the patient's blood. RNA methods can be performed on exosomes in urine or blood.

[0110] Combination therapy using anti-GREM1 antagonists combined with proliferation-dependent cytotoxic agents In a further embodiment of the invention, there is provided an anti-GREM1 antagonist in combination with a proliferation-dependent cytotoxic agent for use in a method of treating or preventing pancreatic cancer. The method may comprise separate, sequential or simultaneous administration of the proliferation-dependent cytotoxic agent.

[0111] The invention also provides a proliferation-dependent cytotoxic agent for use in a method of treating or preventing pancreatic cancer, the method comprising the separate, sequential or simultaneous administration of an anti-GREM1 antagonist.

[0112] In one embodiment, the pancreatic cancer is a pancreatic cancer characterized by having overexpression of GREM1. In another embodiment, the pancreatic cancer can be characterized by having an exocrine tumor or a neuroendocrine tumor. Pancreatic neuroendocrine cancer (also known as pancreatic islet cell tumor) arises in the endocrine glands of the pancreas. Particularly preferred forms of pancreatic cancer are exocrine pancreatic cancers, such as pancreatic ductal adenocarcinoma. Other exocrine pancreatic cancers include squamous cell carcinomas that form in the pancreatic duct; adenosquamous cell carcinomas; signet ring cell carcinomas; and colloid carcinomas that typically arise from intraductal papillary mucinous neoplasms.

[0113] The preferred type of pancreatic cancer to be treated may be resistant to one or more known anti-cancer agents (such as chemotherapeutic agents). The pancreatic cancer may be disseminated pancreatic cancer. The pancreatic cancer may be metastatic pancreatic cancer. Metastatic cancer should be understood as cancer that spreads from its original site of origin in the living body. Thus, metastatic pancreatic cancer refers to cancer that starts from the pancreas and metastasizes to other organs, such as the lung, liver, bone and brain. The pancreatic cancer may also be recurrent pancreatic cancer. In other words, the pancreatic cancer to be treated by the method of the present invention includes pancreatic cancer that has recurred after months or even years after previous treatments, such as chemotherapy, radiotherapy or radical surgery.

[0114] In some aspects, the pancreatic cancer may be a pancreatic cancer that is responsive to treatment with a proliferation-dependent cytotoxic agent. The pancreatic cancer may be a pancreatic cancer that is poorly responsive, non-responsive, or refractory to treatment with a proliferation-dependent cytotoxic agent. The pancreatic cancer may not be suitable for treatment with a proliferation-dependent cytotoxic agent. In some cases, the pancreatic cancer may be initially responsive to treatment with a proliferation-dependent cytotoxic agent, but develop resistance to treatment with a proliferation-dependent cytotoxic agent.

[0115] The use of a GREM1 antagonist in combination with a proliferation-dependent cytotoxic agent to treat or prevent pancreatic cancer in a patient as described herein may include (a) measuring the amount of GREM1 in the cancer, and (b) administering a GREM1 antagonist to the patient in combination with a proliferation-dependent cytotoxic agent if the pancreatic cancer comprises overexpression of GREM1, thereby treating or preventing pancreatic cancer. The amount of GREM1 may be the amount of mRNA or protein, and the overexpression may be any of the overexpressions described above. The GREM1 antagonist may be any of the GREM1 antagonists described herein. The proliferation-dependent cytotoxic agent may be any of the proliferation-dependent cytotoxic agents described herein.

[0116] Growth-dependent cytotoxic agents In the context of treating or preventing pancreatic cancer, the term proliferation-dependent cytotoxic agent used in the present invention refers to any cytotoxic agent that targets proliferating cells. In other words, highly proliferating cell populations, including rapidly dividing cancer cells, show increased sensitivity to such agents. Without being bound by theory, the inventors hypothesize that anti-GREM1 antagonists can drive dormant stem-like cancer cells into a more proliferative state, making them more sensitive to treatment with proliferation-dependent cytotoxic agents. Thus, in one aspect, the proliferation-dependent cytotoxic agent used in the method of the present invention is cytotoxic to proliferating stem-like cancer cells.

[0117] In one embodiment, the proliferation-dependent cytotoxic agent is a nucleoside inhibitor or antimetabolite. Nucleoside inhibitors or antimetabolites mimic endogenous nucleosides and exert cytotoxic activity by interfering with the synthesis of nucleic acid. For example, nucleoside inhibitors or antimetabolites include analogs of physiological pyrimidine and purine nucleobases and nucleosides. Such compounds can also interfere with DNA methylation or modify the metabolism of physiological nucleosides. Nucleoside inhibitors or antimetabolites can mediate enzyme inhibition and interfere with the synthesis of nucleic acid. Thus, nucleoside inhibitors or antimetabolites are typically described as cell cycle specific.

[0118] Examples of nucleoside inhibitors or antimetabolites include folate antagonists, such as methotrexate and pemetrexed; pyrimidine antagonists, such as 5-fluorouracil, foxuridine, capecitabine, cytarabine and gemcitabine; purine antagonists, such as 6-mercaptopurine and 6-thioguanine; and adenosine deaminase inhibitors, such as cladribine, fludarabine and pentostatin. In a preferred embodiment in the context of pancreatic cancer, the proliferation-dependent cytotoxic agent is selected from gemcitabine and capecitabine.

[0119] In a preferred embodiment, the nucleoside inhibitor or antimetabolite is a cytidine analog or a deoxycytidine analog.As mentioned above, cytidine analog or deoxycytidine analog mimics endogenous cytidine or deoxycytidine.Such compounds may also interfere with DNA methylation or modify physiological nucleoside metabolism.As a result, cytidine analog and deoxycytidine analog are widely used in anticancer drug therapy.

[0120] Examples of cytidine analogs or deoxycytidine analogs used in the treatment of pancreatic cancer include gemcitabine (2'-deoxy-2',2'-difluorocytidine) or its derivatives; azacytidine (5-azacytidine) or its derivatives; cytarabine (Ara-C / cytosine 1-[beta]-D-arabinofuranoside) or its derivatives; decitabine (5-aza-2'-deoxycytidine / 5-azadeoxycytidine) or its derivatives; and troxacitabine (troxatyl / 4-amino-1-[(2S)-2-(hydroxymethyl)-1,3-dioxolan-4-yl]pyrimidin-2-one) or its derivatives. In a preferred embodiment, the deoxycytidine analog is gemcitabine.

[0121] In some embodiments, the combination therapy provides an anti-GREM1 antagonist with one or more cytidine or deoxycytidine analogues. In a preferred embodiment, the present invention provides an anti-GREM1 antagonist for use in a method for treating or preventing pancreatic cancer, the method further comprising administering gemcitabine. In an exemplary embodiment, the present invention provides an anti-GREM1 antagonist for use in a method for treating or preventing pancreatic cancer, the method further comprises administering gemcitabine in combination with troxacitabine. One exemplary pancreatic cancer is exocrine pancreatic cancer, such as pancreatic ductal adenocarcinoma.

[0122] In another embodiment, the proliferation-dependent cytotoxic agent is a mitotic inhibitor. The term mitotic inhibitor as used herein refers to an inhibitor that blocks cell division by inhibiting mitosis through the disruption of microtubules. For example, mitotic inhibitors can target tubulin, thus impairing the normal function of the mitotic spindle and resulting in disruption of microtubule polymerization. In one embodiment of the present invention, the mitotic inhibitor is a microtubule-stabilizing drug. Such drugs can inhibit cell division by inhibiting microtubule-dependent signaling events, stimulating tubulin formation, and / or increasing the density of cellular microtubules.

[0123] Examples of mitotic inhibitors and / or microtubule stabilizing agents include cabazitaxel, docetaxel, paclitaxel, vinblastine, vincristine, vinorelbine and abraxane. In a preferred embodiment in the context of pancreatic cancer, the mitotic inhibitors and / or microtubule stabilizing agents are selected from abraxane and paclitaxel.

[0124] In further embodiments of the invention, the proliferation-dependent cytotoxic agent may comprise one or more of oxaliplatin, folinic acid, irinotecan, and fluorouracil. In the context of pancreatic cancer, the proliferation-dependent cytotoxic agent may be FOLFIRINOX or FOLFOX.

[0125] In a preferred embodiment of the invention, the proliferation-dependent cytotoxic agent is gemcitabine and the cancer is pancreatic cancer.

[0126] GREM1 The term GREM1 or Gremlin-1 as used herein in the context of a protein typically refers to human GREM1, a protein having the amino acid sequence shown in UniProt entry O60565 (SEQ ID NO: 1). The terms GREM1 and Gremlin-1 may also refer to the Gremlin-1 polypeptide, (a) comprising or consisting of the amino acid sequence of SEQ ID NO:1, with or without the N-terminal signal peptide, i.e. comprising or consisting of the mature peptide sequence shown in SEQ ID NO:21; or (b) a derivative having one or more amino acid substitutions, modifications, deletions, or insertions relative to the amino acid sequence of SEQ ID NO:1, with or without the N-terminal signal peptide (shown in SEQ ID NO:21), which retains the activity of Gremlin-1, such as the amino acid sequence of SEQ ID NO:20; (c) variants thereof, such variants typically retain at least about 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94% or 95% identity (or even about 96%, 97%, 98% or 99% identity) to SEQ ID NO: 1 (or SEQ ID NO: 20 or 21). In other words, such variants may retain about 60% to about 99% identity to SEQ ID NO: 1, preferably about 80% to about 99% identity to SEQ ID NO: 1, more preferably about 90% to about 99% identity to SEQ ID NO: 1, and most preferably about 95% to about 99% identity to SEQ ID NO: 1. Variants are further described below.

[0127] As discussed further below, residue numbers are typically cited based on the sequence of SEQ ID NO: 1. However, the residue numbering can be easily extrapolated by one of skill in the art to derivative or variant sequences, as discussed above. When residue numbers are cited, the present invention also encompasses those residues on the variant or derivative sequences.

[0128] The GREM1 or Gremlin-1 nucleic acid sequence may comprise or consist of the sequence of SEQ ID NO: 36 or SEQ ID NO: 37 or variants thereof. Variant nucleic acid sequences are further described below. The GREM1 or Gremlin-1 nucleic acid sequence may comprise or consist of any GREM1 transcript variant. Examples of GREM1 transcript variants are transcript 1 (NCBI: NM_013372.6; ENSEMBL: ENST00000560677.5); transcript 2: NCBI: NM_001191323.1; ENSEMBL: ENST00000560830.1); transcript 3: NCBI: NM_001191322.1; ENSEMBL: ENST00000622074.1. The sequences available under the above accession numbers as of June 18, 2018 are incorporated herein by reference.

[0129] Antagonist An anti-GREM1 antagonist is any molecule that reduces the function or activity of GREM1. An anti-GREM1 antagonist can reduce the function or activity of GREM1 by any amount. An anti-GREM1 antagonist can reduce the function or activity of GREM1 by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%, or can prevent any function or activity of GREM1. The extent to which an anti-GREM1 antagonist reduces the function or activity of GREM1 can be determined by measuring the function or activity of GREM1 in cells in the presence and absence of the anti-GREM1 antagonist. The cells can be normal cells or cancer cells. The cells can be cancer cells as described above. They can be pancreatic cancer cells. The pancreatic cancer cells can be in the KPC mouse model described in the examples. Thus, in vivo assays for the activity of GREM1 antagonists in combination therapy in pancreatic cancer can be performed in mouse models. More generally, GREM1 antagonists shown to reduce GREM1 function or activity by any means can then be assayed in vitro or in vivo for their ability to prevent or reduce the proliferation of cancer cells, such as pancreatic cancer cells, or to prevent, reduce or eliminate cancer or tumors.

[0130] Antagonists may reduce GREM1 function by any means. They may increase or decrease the activity or amount of any molecule that directly or indirectly affects GREM1 function. They may reduce the amount of GREM1 at the mRNA or protein level. They may increase the degradation of GREM1. They may reduce GREM1 function by inhibitory modifications. They may reduce transcription of molecules that enhance GREM1 function. They may destroy DNA encoding GREM1 or molecules that enhance GREM1 function using agents such as zinc finger nucleases.

[0131] The antagonist can be an agent that interacts with Gremlin-1. An agent that interacts with Gremlin-1 is typically an agent that binds to Gremlin-1. An agent that interacts with Gremlin-1 can modulate Gremlin-1. An inhibitory regulator can affect any of the functions of Gremlin-1, but typically reduces the binding of Gremlin-1 to BMPs (BMP 2 / 4 / 7). The antagonist can be a BMP-7 mimetic molecule. Gremlin-1 is a negative regulator of BMPs, so reduced binding increases signaling through BMPs. Activation of the regulator can increase the binding of Gremlin-1 to BMPs.

[0132] BMP binding and signaling can be detected by any method known in the art.

[0133] Antagonists can act by binding to the active site of GREM1 or allosterically by binding to a different site. Antagonists can act by binding to a modulator or ligand of GREM1, thereby decreasing activation of GREM1. Antagonists can be reversible or irreversible.

[0134] 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).

[0135] The GREM1 antagonist may be an oligonucleotide that specifically hybridizes to the mRNA encoding GREM1 or to the mRNA encoding a molecule that enhances the activity of GREM1. The GREM1 antagonist may be a polynucleotide encoding any molecule that reduces GREM1 function. For example, the GREM1 antagonist may be a polynucleotide encoding an anti-GREM1 antibody described herein.

[0136] The antagonist of GREM1 can be an antibody that specifically binds to any target molecule (typically a protein) so as to directly or indirectly reduce the function of GREM1. The antagonist can be an antibody that specifically binds to GREM1. In this embodiment, the antibody can reduce GREM1 function by allosteric inactivation or by blocking the interaction between its target and a ligand required for activity.

[0137] The interaction of the antagonist agent with the protein residue can be determined by any suitable method known in the art, such as the distance between the residue and the agent (typically less than 6 Å, or less than 4 Å) as determined by X-ray crystallography. Regions of Gremlin-1 that may be targeted by a therapeutic agent may include amino acids Asp92-Leu99, Arg116-His130, Ser137-Ser142, Cys176-Cys178, which are within 6 Å of the amino acids that are mutated on the surface of Gremlin-1.

[0138] Antibody antagonists The term "antibody" as referred to herein includes whole antibodies, any antigen-binding fragment (i.e., "antigen-binding portion"), or single chains thereof. An antibody refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains linked by disulfide bonds, or an antigen-binding portion thereof. Each heavy chain contains a heavy chain variable region (referred to herein as HCVR or V H Each light chain comprises a light chain variable region (abbreviated herein as LCVR or V L The heavy and light chain variable regions contain a binding domain that interacts with an antigen. H and V L The regions can be further subdivided into regions of hypervariability called complementarity determining regions (CDRs) and regions of high conserved activity called framework regions (FRs).

[0139] 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.

[0140] The antibody used according to the present invention may be a monoclonal or polyclonal antibody, typically a monoclonal antibody. The antibody used according to the present invention may be a chimeric antibody, a CDR-grafted antibody, a nanobody, a human antibody or a humanized antibody, or any antigen-binding portion thereof. For the production of monoclonal and polyclonal antibodies, the experimental animal is usually a non-human mammal, such as a goat, rabbit, rat or mouse, although the antibody can also be bred in other species.

[0141] Polyclonal antibodies can be produced in a routine manner, such as by immunizing a suitable animal with the antigen of interest, followed by blood removal from the animal and purification of the IgG fraction.

[0142] Antibodies against Gremlin-1 can be obtained by administering the polypeptide to an animal, e.g., a non-human animal, using well-known and routine protocols, if immunization of the animal is required, see, e.g., Handbook of Experimental Immunology, DM Weir (ed.), Vol 4, Blackwell Scientific Publishers, Oxford, England, 1986. Many warm-blooded animals can be immunized, such as rabbits, mice, rats, sheep, cattle, camels or pigs. Generally, however, mice, rabbits, pigs and rats are most suitable.

[0143] Monoclonal antibodies can be prepared by any method known in the art, such as the hybridoma technique (Kohler & Milstein, 1975, Nature, 256:495-497), the trioma technique, the human B cell hybridoma technique (Kozbor et al., 1983, Immunology Today, 4:72), and the EBV hybridoma technique (Cole et al., Monoclonal Antibodies and Cancer Therapy, pp77-96, Alan R Liss, Inc., 1985).

[0144] Antibodies to be used in accordance with the present invention can also be produced using a single lymphocyte antibody technique, for example, by cloning and expressing immunoglobulin variable region cDNA generated from a single lymphocyte selected to produce a particular antibody, by methods described by Babcook, J. et al., 1996, Proc. Natl. Acad. Sci. USA 93(15): 7843-7848l; International Publication No. WO92 / 02551; International Publication No. WO2004 / 051268 and International Publication No. WO2004 / 106377.

[0145] Antibodies can also be generated using various phage display methods known in the art, see, for example, Brinkman et al. (in J. Immunol. Methods, 1995, 182: 41-50), Ames et al. (J. Immunol. Methods, 1995, 184: 177-186), Kettleborough et al. (Eur. J. Immunol. 1994, 24: 952-958), Persic et al. (Gene, 1997 187 9-18), Burton et al. (Advances in Immunology, 1994, 57:191-280), as well as International Publication Nos. WO 90 / 02809; International Publication No. WO 91 / 10737; International Publication No. WO 92 / 01047; International Publication No. WO 92 / 18619; International Publication No. WO 93 / 11236; International Publication No. WO 95 / 15982; International Publication No. WO 95 / 20401; and U.S. Patent Nos. 5,698,426 and 5,223,409. ;5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969,108.

[0146] A fully human antibody is one in which the variable and constant regions (if present) of both the heavy and light chains are all of human origin or are substantially identical to sequences of human origin, although not necessarily derived from the same antibody. Examples of fully human antibodies include, for example, antibodies produced by the phage display methods described above, and antibodies produced in mice in which the mouse immunoglobulin variable region genes, and optionally the constant region genes, have been replaced with their human counterparts, such as those generally described in EP 0546073, U.S. Patent No. 5,545,806, U.S. Patent No. 5,569,825, U.S. Patent No. 5,625,126, U.S. Patent No. 5,633,425, U.S. Patent No. 5,661,016, U.S. Patent No. 5,770,429, EP 0438474, and EP 0463151.

[0147] Alternatively, antibodies for use in accordance with the present invention may be produced by a method comprising immunizing a non-human mammal with a Gremlin-1 immunogen; obtaining an antibody preparation from said mammal; and inducing therefrom a monoclonal antibody which recognizes Gremlin-1.

[0148] The antibody molecule used according to the present invention may comprise a complete antibody molecule having full-length heavy and light chains, or a fragment or antigen-binding portion thereof. The term "antigen-binding portion" of an antibody refers to one or more fragments of an antibody that retain the ability to selectively bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Antibodies and fragments and antigen-binding portions thereof may be, but are not limited to, Fab, modified Fab, Fab', modified Fab', F(ab')2, Fv, single domain antibodies (e.g., VH or VL or VHH), scFv, bivalent, trivalent or tetravalent antibodies, Bis-scFv, diabodies, triabodies, tetrabodies, and epitope-binding fragments of any of the above (see, e.g., Holliger and Hudson, 2005, Nature Biotech. 23(9):1126-1136; Adair and Lawson, 2005, Drug Design Reviews - Online 2(3), 209-217). Methods for generating and producing these antibody fragments are well known in the art (see, for example, Verma et al., 1998, Journal of Immunological Methods, 216, 165-181). Other antibody fragments for use in the present invention include Fab and Fab' fragments described in International Patent Application Publication Nos. WO2005 / 003169, WO2005 / 003170 and WO2005 / 003171, and Fab-dAb fragments described in International Patent Application Publication No. WO2009 / 040562. Multivalent antibodies may contain multiple specificities or may be monospecific (see, for example, International Patent Application Publication Nos. WO92 / 22853 and WO05 / 113605). These antibody fragments may be obtained using conventional techniques known to those of skill in the art and may be screened for utility in the same manner as intact antibodies.

[0149] The constant region domain of the antibody molecule, if present, can be selected taking into account the proposed function of the antibody molecule, in particular the effector function that may be required. For example, the constant region domain can be a human IgA, IgD, IgE, IgG or IgM domain. In particular, when the antibody molecule is intended for therapeutic use and antibody effector function is required, human IgG constant region domains, in particular IgG1 and IgG3 isotypes, can be used. Alternatively, when the antibody molecule is intended for therapeutic purposes and antibody effector function is not required, IgG2 and IgG4 isotypes can be used.

[0150] Antibodies used in accordance with the present invention may be prepared, expressed, created or isolated by recombinant means, such as (a) antibodies isolated from animals (e.g., mice) that are transgenic or transchromosomal for the immunoglobulin genes of interest or hybridomas prepared therefrom; (b) antibodies isolated from host cells, e.g., transfectomas, that have been transformed to express the antibody of interest; (c) antibodies isolated from recombinant, combinatorial antibody libraries; and (d) antibodies prepared, expressed, created or isolated by other means involving splicing of immunoglobulin gene sequences into other DNA sequences.

[0151] The antibody used according to the present invention may be a human antibody or a humanized antibody. The term "human antibody" as used herein is intended to include antibodies having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region is also derived from a human germline immunoglobulin sequence. The human antibodies described herein may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by in vitro random or site-specific mutagenesis or in vivo somatic mutation). However, the term "human antibody" as used herein is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

[0152] Such human antibodies can be human monoclonal antibodies, which can be produced by hybridomas comprising B cells obtained from a transgenic non-human animal, e.g., a transgenic mouse, whose genome comprises human heavy chain and light chain transgenes fused to an immortalized cell.

[0153] Human antibodies can be prepared by in vitro immunization of human lymphocytes, followed by transformation of the lymphocytes with Epstein-Barr virus.

[0154] The term "human antibody derivatives" refers to any modified form of human antibodies, such as conjugates of the antibodies and other agents or antibodies.

[0155] The term "humanized antibody" is intended to refer to CDR-grafted antibody molecules in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences within which further modifications of the framework regions may be made.

[0156] As used herein, the term "CDR-grafted antibody molecule" refers to an antibody molecule in which the heavy and / or light chains comprise one or more CDRs (optionally including one or more modified CDRs) from a donor antibody (e.g., a mouse or rat monoclonal antibody) grafted onto the heavy and / or light chain variable region framework of an acceptor antibody (e.g., a human antibody). For a review, see Vaughan et al, Nature Biotechnology, 16, 535-539, 1998. In one embodiment, instead of transferring the entire CDR, only one or more of the specificity-determining residues from any one of the CDRs described herein above are transferred to the human antibody framework (see, e.g., Kashmiri et al., 2005, Methods, 36, 25-34). In one embodiment, only the specificity-determining residues from one or more of the CDRs described herein above are transferred to the human antibody framework. In another embodiment, only the specificity-determining residues from each CDR described herein above are introduced into the human antibody framework.

[0157] Where CDRs or specificity determining residues are grafted, any suitable acceptor variable region framework sequence may be used, including murine, primate and human framework regions, taking into account the class / type of the donor antibody from which the CDRs are derived. Suitably, a CDR-grafted antibody according to the invention has a variable domain comprising human acceptor framework regions as well as one or more of the above CDRs or specificity determining residues. Thus, in one embodiment, a neutralising CDR-grafted antibody is provided, the variable domain of which comprises human acceptor framework regions and a non-human donor CDR.

[0158] Examples of human frameworks that can be used in the present invention are KOL, NEWM, REI, EU, TUR, TEI, LAY and POM (Kabat et al., supra). For example, KOL and NEWM can be used for the heavy chain, REI can be used for the light chain, and EU, LAY and POM can be used for both the heavy and light chains. Alternatively, human germline sequences can be used. These are available, for example, at http: / / www.vbase2.org / (see Retter et al, Nucl. Acids Res. (2005) 33 (supplement 1), D671-D674).

[0159] In the CDR-grafted antibodies described herein, the acceptor heavy and light chains are not necessarily from the same antibody and may, if desired, comprise composite chains having framework regions from different chains.

[0160] Also, in the CDR-grafted antibodies described herein, the framework regions need not have the exact same sequence as those of the acceptor antibody. For example, unusual residues can be changed to residues that occur more frequently for that acceptor chain class or type. Alternatively, selected residues in the acceptor framework regions can be changed to correspond to residues found at the same positions in the donor antibody (see Reichmann et al., 1998, Nature, 332, 323-324). Such changes should be the minimum necessary to restore the affinity of the donor antibody. Protocols for selecting acceptor framework region residues that may require change are described in International Publication No. WO 91 / 09967.

[0161] Those skilled in the art will also appreciate that antibodies may undergo various post-translational modifications. The type and extent of these modifications often depend on the host cell line used to express the antibody, as well as the culture conditions. Such modifications may include changes in glycosylation, methionine oxidation, diketopiperazine formation, aspartic acid isomerization, and asparagine deamidation. A frequent modification is the loss of basic residues (e.g., lysine or arginine) at the carboxy terminus by the action of carboxypeptidases (described in Harris, RJ. Journal of Chromatography 705:129-134, 1995).

[0162] In one embodiment, the antibody heavy chain comprises a CH1 domain and the antibody light chain comprises a CL domain, either kappa or lambda.

[0163] Biological molecules such as antibodies or fragments thereof contain acidic and / or basic functional groups, thereby conferring a net positive or negative charge to the molecule. The amount of overall "observed" charge depends on the absolute amino acid sequence of the entity, the local environment of the charged groups in the 3D structure, and the environmental conditions of the molecule. The isoelectric point (pI) is the pH at which a particular molecule or surface has no net charge. In one embodiment, the antibody or fragment according to the present disclosure has an isoelectric point (pI) of at least 7. In one embodiment, the antibody or fragment has an isoelectric point of at least 8, such as 8.5, 8.6, 8.7, 8.8 or 9. In one embodiment, the antibody has a pI of 8. ** Programs such as ExPASY http: / / www.expasy.ch / tools / pi_tool.html (see Walker, The Proteomics Protocols Handbook, Humana Press (2005), 571-607) can be used to predict the isoelectric point of an antibody or fragment.

[0164] To characterize the preferred Gremlin-1 epitope, we crystallized human Gremlin-1 alone and in complex with an antibody (Fab fragment) called Ab 7326. Crystallization of Gremlin-1 allowed us to determine the putative residues of the BMP binding site. Furthermore, crystallization with Ab 7326, an allosteric inhibitor antibody, allowed us to determine the residues of the antibody epitope. Antibodies that bind to this epitope are particularly promising as therapeutic agents in the treatment of diseases associated with Gremlin-1.

[0165] Preferred Abs as described herein The 7326 antibody was confirmed to bind to the following residues of Gremlin-1: Ile110(131), Lys126(147), Lys127(148), Phe128(149), Thr129(150), Thr130(151), Arg148(169), Lys153(174), and Gln154(175), with Lys126(147), Lys127(148), Phe128(149), Thr129(150), Thr130(151), Arg148(169), Lys153(174), and Gln154(175) present on one Gremlin-1 monomer and Ile110(131) present on a second Gremlin-1 monomer. Numbering not in parentheses is based on the structure file and (consistent with mouse Gremlin-2 numbering based on structural alignment). Numbers in parentheses represent residues based on UniProt entry O60565 of SEQ ID NO: 1. These epitope residues were identified using 4 Å NCONT analysis from the Gremlin-1-Ab 7326 Fab complex.

[0166] Thus, the antibodies described herein may bind to an epitope comprising at least one residue selected from Ile131, Lys147, Lys148, Phe149, Thr150, Thr151, Arg169, Lys174 and Gln175 (residue numbering based on SEQ ID NO: 1). The antibodies described herein may bind to an epitope comprising 2, 3, 4, 5, 6, 7, 8 or all 9 residues (preferably at least 5 residues) of these residues.

[0167] The antibodies described herein may also recognize an epitope present on the Gremlin-1 monomer where Ile131 is distinct from other residues.

[0168] Although these residues are provided for a particular sequence of human Gremlin-1, one of skill in the art can readily extrapolate the positions of these residues to other corresponding Gremlin sequences (e.g., mouse) using routine techniques. Thus, antibodies that bind epitopes that include corresponding residues in these other Gremlin sequences are also provided by the invention.

[0169] To screen for antibodies that bind to a specific epitope, routine cross-blocking assays such as those described in Antibodies, Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harb., NY) can be performed. Other methods include alanine scanning mutants, peptide blots (Reineke (2004) Methods Mol Biol 248:443-63), or peptide truncation analysis. In addition, methods such as epitope excision, epitope extraction, and chemical modification of antigens can be employed (Tomer (2000) Protein Science 9: 487-496). Such methods are well known in the art.

[0170] The epitope of an antibody can also be determined by X-ray crystallography. Thus, the antibodies of the present disclosure can be evaluated by X-ray crystallography of the antibody bound to Gremlin-1. The epitope can be identified in this way, particularly by determining the residues on Gremlin-1 that are within 4 Å of the antibody paratope residues.

[0171] Thus, the antibodies described herein can bind to an epitope on Gremlin-1 that includes at least one residue selected from Trp93, Phe117, Tyr119, Phe125, Tyr126, and Phe138, where residue numbering is according to SEQ ID NO: 1. Further described herein are antibodies that bind to an epitope that includes all of Trp93, Phe117, Tyr119, Phe125, Tyr126, and Phe138. Further described are antibodies that bind to an epitope that includes the following residues: Ile131, Lys147, Lys148, Phe149, Thr150, Thr151, Arg169, Lys174, and Gln175. Preferably, Lys147, Lys148, Phe149, Thr150, Thr151, Arg169, Lys174 and Gln175 are located on one monomer of Gremlin-1 and Ile131 is located on the other monomer of Gremlin-1 (the Gremlin-1 dimer binds to the BMP dimer).

[0172] An antibody is capable of binding to a Gremlin-1 residue if the paratope of the antibody is within 4 Å of said Gremlin-1 residue as determined by x-ray crystallography.

[0173] Antibodies that bind to the epitopes disclosed herein may comprise at least one, at least two, or all three of the heavy chain CDR sequences of SEQ ID NOs: 4-6 (HCDR1 / HCDR2 / HCDR3, respectively), which are the HCDR1 / HCDR2 / HCDR3 sequences of the Ab 7326 antibody of the Examples, as determined using the Kabat method.

[0174] The Kabat and Chothia methods (as well as other techniques) for determining CDR sequences are well known in the art. CDR sequences can be determined using any suitable method, and in the present invention, Kabat is typically employed, although other techniques can be used as well. In this example, SEQ ID NO:3 shows the Ab 7326 HCDR1 sequence determined using the combined definition of Chothia & Kabat.

[0175] Antibodies for use according to the invention may comprise at least one, at least two, or all three light chain CDR sequences of SEQ ID NOs: 7-9 (LCDR1 / LCDR2 / LCDR3, respectively), which are the LCDR1 / LCDR2 / LCDR3 sequences of Ab 7326 using the Kabat method.

[0176] The antibody preferably comprises at least the HCDR3 sequence of SEQ ID NO:6.

[0177] 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.

[0178] The antibody may comprise a heavy chain variable region (HCVR) sequence of SEQ ID NO: 10 or 12 (HCVR of Ab 7326 variants 1 and 2). The antibody may comprise a light chain variable region (LCVR) sequence of SEQ ID NO: 11 or 13 (LCVR of Ab 7326 variants 1 and 2). The antibody preferably comprises a heavy chain variable region sequence of SEQ ID NO: 10 or 12 and a light chain variable region sequence of SEQ ID NO: 11 or 13 (particularly the HCVR / LVCR pair of SEQ ID NO: 10 / 11 or 12 / 13).

[0179] The antibody is SEQ ID NO: 14 Mouse full length IgG1 heavy chain variant 1, or SEQ ID NO: 28 Mouse full length IgG1 heavy chain variant 2, or SEQ ID NO: 30 Human full length IgG1 heavy chain variant 1, or SEQ ID NO: 16 human full length IgG1 heavy chain variant 2, or SEQ ID NO: 22 Human full length IgG4P heavy chain variant 1, or SEQ ID NO: 34 human full length IgG4P heavy chain variant 2, or SEQ ID NO: 18 Fab heavy chain variant 1, or SEQ ID NO: 32 Fab heavy chain variant 2 It may comprise the heavy chain (H chain) sequence of

[0180] The antibody is SEQ ID NO: 15 mouse full length IgG1 light chain variant 1, or SEQ ID NO: 29 Mouse full length IgG1 light chain variant 2, or SEQ ID NO: 31 human full length IgG1 light chain variant 1, or SEQ ID NO: 17 human full length IgG1 light chain variant 2, or SEQ ID NO: 23 human full length IgG4P light chain variant 1, or SEQ ID NO: 35 human full length IgG4P light chain variant 2, or SEQ ID NO: 19 Fab light chain variant 1, or SEQ ID NO: 33 Fab light chain variant 2 It may comprise a light chain (L chain) sequence of

[0181] In one example, the antibody is SEQ ID NO: 14 / 15 Mouse full length IgG1 variant 1, or SEQ ID NO: 28 / 29 Mouse full length IgG1 variant 2, or SEQ ID NO: 30 / 31 human full length IgG1 variant 1, or SEQ ID NO: 16 / 17 human full length IgG1 variant 2, or SEQ ID NO: 22 / 23 human full length IgG4P variant 1, or SEQ ID NO: 34 / 35 human full-length IgG4P variant 2, or SEQ ID NO: 18 / 19 Fab light chain variant 1, or SEQ ID NO: 32 / 33 Fab light chain variant 2 The heavy chain / light chain sequence pair is

[0182] Variant forms of the corresponding sequences may be exchanged. For example, an antibody may SEQ ID NO: 14 / 29 Mouse full length IgG1 heavy chain variant 1 / light chain variant 2, or SEQ ID NO: 28 / 15 mouse full length IgG1 heavy chain variant 2 / light chain variant 1, or SEQ ID NO: 30 / 17 human full length IgG1 heavy chain variant 1 / light chain variant 2, or SEQ ID NO: 16 / 31 human full length IgG1 heavy chain variant 2 / light chain variant 1, or SEQ ID NO: 22 / 35 human full length IgG4P heavy chain variant 1 / light chain variant 2, or SEQ ID NO: 34 / 23 human full length IgG4P heavy chain variant 2 / light chain variant 1, or SEQ ID NO: 18 / 33 Fab heavy chain variant 1 / light chain variant 2, or SEQ ID NO: 32 / 19 Fab heavy chain variant 2 / light chain variant 1 The heavy chain / light chain sequence pair may include:

[0183] The antibody can be a chimeric antibody, a human antibody, or a humanized antibody.

[0184] The antibody may alternatively be or comprise a variant of one of the specific sequences described above. The following discussion of antibody variants is also applicable to the selection of GREM1 polypeptide variants described above.

[0185] For example, the variants can be substitution, deletion or addition variants of any of the above amino acid sequences.

[0186] A variant antibody may include 1, 2, 3, 4, 5, up to 10, up to 20 or more (typically up to 50) amino acid substitutions and / or deletions from the specific sequences discussed above. "Deletion" variants may include deletion of individual amino acids, deletion of small groups of amino acids such as 2, 3, 4 or 5 amino acids, or deletion of larger regions of amino acids such as deletion of specific amino acid domains or other properties. "Substitution" variants typically involve the replacement of one or more amino acids with the same number of amino acids, and making conservative amino acid substitutions. For example, an amino acid may be replaced with an alternative amino acid having similar properties, such as another basic amino acid, another acidic amino acid, another neutral amino acid, another charged amino acid, another hydrophilic amino acid, another hydrophobic amino acid, another polar amino acid, another aromatic amino acid, or another aliphatic amino acid. Some properties of the 20 main amino acids that can be used to select appropriate replacements are as follows: [Table A]

[0187] "Derivatives" or "variants" generally include those in which an amino acid that appears in the sequence instead of a naturally occurring amino acid is its structural analogue. The amino acids used in the sequence may be derivatized or modified, e.g., labeled, as long as the function of the antibody is not significantly adversely affected.

[0188] The above derivatives and variants can be prepared by modification during synthesis or after production of the antibody, or when the antibody is in recombinant form, using known techniques of site-directed mutagenesis, random mutagenesis, or enzymatic cleavage and / or ligation of nucleic acids.

[0189] A variant antibody may have an amino acid sequence that has more than about 60%, or more than about 70%, such as 75% or 80%, typically more than about 85%, such as more than about 90% or 95% amino acid identity with the amino acid sequences disclosed herein (particularly the HCVR / LCVR sequences, and the H-chain and L-chain sequences). Furthermore, the antibody may be a variant that has more than about 60%, or more than about 70%, such as 75% or 80%, typically more than about 85%, such as more than about 90% or 95% amino acid identity with the HCVR / LCVR sequences and the H-chain and L-chain sequences disclosed herein, while retaining the exact CDRs disclosed for these sequences. A variant may retain at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the HCVR / LCVR sequences and the H-chain and L-chain sequences disclosed herein (in some cases, while retaining the exact CDRs).

[0190] Variants typically retain about 60% to about 99% identity, about 80% to about 99% identity, about 90% to about 99% identity, or about 95% to about 99% identity. This level of amino acid identity can be found over the entire length of the sequence of the relevant SEQ ID NO, or over a portion of the sequence, for example, over about 20, 30, 50, 75, 100, 150, 200 or more amino acids, depending on the size of the full-length polypeptide.

[0191] In the context of amino acid sequences, "sequence identity" refers to sequences having the specified values ​​when assessed using ClustalW (Thompson et al., 1994, supra) with the following parameters: pairwise alignment parameters - method: exact, matrix: PAM, gap opening penalty: 10.00, gap extension penalty: 0.10; multiple alignment parameters - matrix: PAM, gap opening penalty: 10.00, delay % identity: 30, end gap penalty: on, gap separation distance: 0, negative matrix: none, gap extension penalty: 0.20, residue specific gap penalty: on, hydrophilic gap penalty: on, hydrophilic residues: GPSNDQEKR. Sequence identity at a particular residue is intended to include identical residues that have only been derivatized.

[0192] Thus, antibodies with specific sequences and variants that maintain the function or activity of these chains are provided.

[0193] The antibody may compete for binding to Gremlin-1 or bind to the same epitope as those defined above in terms of H / L chain, HCVR / LCVR or CDR sequences. In particular, the antibody may compete for binding to Gremlin-1 or bind to the same epitope as an antibody comprising the HCDR1 / HCDR2 / HCDR3 / LCDR1 / LCDR2 / LCDR3 sequence combination of SEQ ID NO: 4 / 5 / 6 / 7 / 8 / 9. The antibody may compete for binding to Gremlin-1 or bind to the same epitope as an antibody comprising the HCVR and LCVR sequence pairs of SEQ ID NO: 10 / 11 or 12 / 13, or the full length chains of SEQ ID NO: 14 / 15 or 16 / 17.

[0194] An "epitope" is a region of an antigen to which an antibody binds. Epitopes may be defined as structural or functional. Functional epitopes are generally a subset of structural epitopes and have residues that directly contribute to the affinity of the interaction. Epitopes may also be conformational, i.e., composed of non-linear amino acids. In certain embodiments, epitopes may include determinants that are chemically active surface groups of molecules such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and in certain embodiments may have specific three-dimensional structural characteristics, and / or specific charge characteristics.

[0195] Whether an antibody binds to the same epitope as a reference antibody or competes for binding can be easily determined using routine methods known in the art. For example, to determine whether a test antibody binds to the same epitope as a reference antibody of the present invention, the reference antibody is bound to a protein or peptide under saturating conditions. The ability of the test antibody to bind to the protein or peptide is then evaluated. If the test antibody can bind to the protein or peptide after saturation binding with the reference antibody, it can be concluded that the test antibody binds to a different epitope than the reference antibody. On the other hand, if the test antibody cannot bind to the protein or peptide after saturation binding with the reference antibody, the test antibody may bind to the same epitope as the epitope bound by the reference antibody of the present invention.

[0196] To determine whether an antibody competes with a reference antibody for binding, the above-mentioned binding method is carried out in two directions. In the first direction, the reference antibody is bound to the protein / peptide under saturating conditions, and then the binding of the test antibody to the protein / peptide molecule is evaluated. In the second direction, the test antibody is bound to the protein / peptide under saturating conditions, and then the binding of the reference antibody to the protein / peptide is evaluated. If in both directions only the first (saturating) antibody can bind to the protein / peptide, it is concluded that the test antibody and the reference antibody compete for binding to the protein / peptide. As will be understood by those skilled in the art, an antibody that competes with a reference antibody for binding does not necessarily bind to the same epitope as the reference antibody, but can sterically block the binding of the reference antibody by binding to an overlapping or adjacent epitope.

[0197] Two antibodies bind to the same or overlapping epitopes if each competitively inhibits (blocks) the binding of the other antibody to the antigen. That is, a 1-fold, 5-fold, 10-fold, 20-fold, or 100-fold excess of one antibody inhibits the binding of the other by at least 50%, 75%, 90%, or 99%, as measured by competitive binding assays (see, e.g., Junghans et al., Cancer Res, 1990:50:1495-1502). Alternatively, two antibodies have the same epitope if essentially all amino acid mutations in the antigen that reduce or eliminate binding of one antibody reduce or eliminate binding of the other antibody. Two antibodies have overlapping epitopes if some amino acid mutations that reduce or eliminate binding of one antibody reduce or eliminate binding of the other antibody.

[0198] Additional routine experiments (e.g., peptide mutations and binding analysis) can then be performed to confirm whether the observed lack of binding of the test antibody is indeed due to binding to the same epitope as the reference antibody, or whether the observed lack of binding is due to steric blocking (or another phenomenon). These types of experiments can be performed using ELISA, RIA, surface plasmon resonance, flow cytometry, or any other quantitative or qualitative antibody binding assay available in the art.

[0199] Antibodies can be tested for binding to Gremlin-1, for example, by standard ELISA or Western blotting. ELISA assays can also be used to screen for hybridomas that show positive reactivity with the target protein. The binding selectivity of the antibody can also be determined by monitoring the binding of the antibody to cells expressing the target protein, for example, by flow cytometry. Thus, screening methods can include identifying antibodies capable of binding to Gremlin-1 by ELISA or Western blot, or by flow cytometry.

[0200] The antibody selectively (or specifically) recognizes Gremlin-1. An antibody or other compound "selectively binds" or "selectively recognizes" a protein if it binds preferentially or with high affinity to that protein and does not substantially bind or binds with low affinity to other proteins. The selectivity of an antibody can be further studied by determining whether the antibody binds to or discriminates between other related proteins as discussed above. Antibodies used in accordance with the present invention typically recognize human Gremlin-1.

[0201] The antibodies may also be cross-reactive to related proteins, or to human Gremlin-1 and Gremlin-1 from other species.

[0202] Specific (or selective) means that the antibody binds to the protein of interest and does not exhibit significant cross-reactivity with any other molecule. Cross-reactivity can be assessed by any suitable method described herein. The cross-reactivity of an antibody can be considered significant if it binds to another molecule at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 100% as strongly as the antibody binds to the protein of interest. An antibody that is specific (or selective) can bind to another molecule less than about 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25% or 20% as strongly as it binds to the protein of interest. An antibody may bind to other molecules with less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 2%, or less than about 1% of the strength with which it binds to the protein of interest.

[0203] Anti-Gremlin antibodies have been previously reported; for example, International Publication No. WO2014 / 159010A1 (Regeneron) describes an anti-Gremlin antibody that inhibits Gremlin-1 activity, with a binding affinity of K D The binding affinity K ranges from 625 pM to 270 nM at 25 °C. Ciuclan et al. (2013) D is 5.6×10 -10 We describe an anti-Gremlin-1 monoclonal antibody, M.

[0204] The anti-gremlin-1 antibody described herein (as well as in International Publication Nos. WO2018 / 115017, filed December 19, 2017, and WO2019 / 243801, filed June 18, 2019, both of which are incorporated herein by reference in their entireties) is an allosteric inhibitor of gremlin-1 activity and binds to a novel epitope as described above, distal to the BMP binding site. The antibody binds to gremlin-1 with very high affinity, with a Kd value of less than 100 pM. Thus, the antibody is a significant improvement over currently available antibodies and is expected to be particularly useful in treating gremlin-1 mediated diseases.

[0205] Thus, antibodies suitable for use in the present invention are likely to have high affinity binding to (human) Gremlin-1. The antibodies should have a dissociation constant (K D In one example, the antibody can have a dissociation constant (K D In one example, the antibody has a dissociation constant (K D ). Various methods can be used to determine the binding affinity of an antibody to a target antigen, as is well known to those skilled in the art, such as surface plasmon resonance assays, saturation assays, or immunoassays such as ELISA or RIA. An exemplary method for determining binding affinity is by surface plasmon resonance on a BIAcore™ 2000 instrument (Biacore AB, Freiburg, Germany) using a CM5 sensor chip, as described in Krinner et al., (2007) Mol. Immunol. February; 44 (5):916-25 (Epub 2006 May 11)).

[0206] Antibodies used in accordance with the present invention are typically inhibitory antibodies. Gremlin-1 negatively regulates BMP-2, 4 and 7, so inhibition of Gremlin-1 results in increased signaling through BMPs.

[0207] Particular functional assays that can be used to screen whether an antibody can inhibit Gremlin 1 include SMAD phosphorylation assays and Hek Id1 reporter gene assays. Typically, inhibitory antibodies restore SMAD phosphorylation and / or restore BMP signaling in a Hek Id1 reporter gene assay. SMAD phosphorylation can be restored to at least 80%, 90% or 100% compared to a BMP control. In the Hek Id1 reporter gene assay, inhibitory antibodies demonstrate an IC 50 may be less than 10 nM, preferably less than 5 nM.

[0208] Once a suitable antibody is identified and selected, the amino acid sequence of the antibody can be identified by methods known in the art. The gene encoding the antibody can be cloned using degenerate primers. The antibody can be produced recombinantly by routine methods.

[0209] The present disclosure also provides isolated DNA sequences encoding the heavy and / or light chain variable region(s) (or full length H and L chains) of the antibody molecules newly described herein.

[0210] Variant polynucleotides can contain 1, 2, 3, 4, 5, up to 10, up to 20, up to 30, up to 40, up to 50, up to 75 or more nucleic acid substitutions and / or deletions from any of the nucleic acid sequences shown in the Sequence Listing (including GREM1 and anti-GREM1 antibody nucleic acid sequences). Typically, variants have 1-20, 1-50, 1-75 or 1-100 substitutions and / or deletions.

[0211] A suitable variant may be at least about 70%, typically at least about 80 or 90%, more preferably at least about 95%, 97% or 99% homologous to any one of the polynucleotides of the nucleic acid sequences disclosed herein. A variant may retain at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity. A variant typically retains about 60% to about 99% identity, about 80% to about 99% identity, about 90% to about 99% identity, or about 95% to about 99% identity. Homology and identity at these levels generally exist at least with respect to the coding region of the polynucleotide. Methods for measuring homology are well known in the art, and the skilled artisan will understand that in the present context, homology is calculated based on nucleic acid identity. Such homology may exist over a region of at least about 15, at least about 30, for example, at least about 40, 60, 100, 200 or more contiguous nucleotides (depending on the length). Such homology may exist over the entire length of the unmodified polynucleotide sequence.

[0212] Methods for measuring polynucleotide homology or identity are known in the art. For example, the UWGCG package provides the BESTFIT program, which can be used to calculate homology (e.g., used on its default settings) (Devereux et al (1984) Nucleic Acids Research 12, p387-395).

[0213] The PILEUP and BLAST algorithms can also be used (typically on their default settings) to calculate homology or align sequences, e.g., as described in Altschul SF (1993) J Mol Evol 36:290-300; Altschul, S, F et al (1990) J Mol Biol 215:403-10.

[0214] Software for performing BLAST analysis is publicly available from the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ). The algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that, when aligned with words of the same length in the database sequences, match or meet some positive threshold score T. T is called the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds to initiate searches to find HSPs containing the word. The word hits are extended in both directions along each sequence for as long as the cumulative alignment score increases. The extension of the word hits in each direction is stopped when the accumulation of one or more negatively scoring residue alignments causes the cumulative alignment score to fall below zero, or when the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLAST program uses as defaults a wordlength (W) of 11, the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1992) Proc. Natl. Acad. Sci. USA 89:10915-10919) of 50, alignment (B) of 10, expectation (E) of 10, M=5, N=4, and a comparison of both strands.

[0215] The BLAST algorithm performs a statistical analysis of the similarity between two sequences; see, for example, Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5787. One measure of similarity provided by the BLAST algorithm is the minimum sum probability (P(N)), which provides an indication of the probability that a match between two nucleotide or amino acid sequences would occur by chance. For example, a sequence is considered to be similar to another sequence if the minimum sum probability in a comparison of a first sequence to a second sequence is less than about 1, typically less than about 0.1, preferably less than about 0.01, and most preferably less than about 0.001. For example, the minimum sum probability can be in the range of about 1 to about 0.001, and often about 0.01 to about 0.001.

[0216] A homologue may differ from a sequence in the related polynucleotide by less than about 3, 5, 10, 15, 20 or more mutations, each of which may be a substitution, deletion or insertion. For example, a homologue may differ by 3 to 50 mutations, often by 3 to 20 mutations. These mutations may be measured over a region of at least 30, e.g., at least about 40, 60 or 100 or more contiguous nucleotides of the homologue.

[0217] In one embodiment, variant sequences may differ from the specific sequences shown in the sequence listing due to redundancy in the genetic code. There are four main nucleic acid residues in the DNA code (A, T, C, and G) that are used to "spell" three-letter codons that represent amino acids encoded in an organism's genes for proteins. The linear sequence of codons along a DNA molecule is translated into the linear sequence of amino acids in the protein(s) encoded by those genes. The code is highly degenerate, with 61 codons encoding the 20 natural amino acids and three codons representing "stop" signals. Thus, most amino acids are encoded by more than one codon, and in fact some amino acids are encoded by four or more different codons. Thus, a variant polynucleotide of the invention may encode the same polypeptide sequence as another polynucleotide of the invention, but may have a different nucleic acid sequence due to the use of different codons to encode the same amino acids.

[0218] The DNA sequence may include, for example, synthetic DNA produced by chemical processes, cDNA, genomic DNA, or a combination thereof.

[0219] DNA sequences encoding the antibody molecules described herein can be obtained by methods well known to those skilled in the art. For example, DNA sequences encoding part or all of the heavy and light chains of the antibodies can be synthesized, if desired, from determined DNA sequences or based on the corresponding amino acid sequences.

[0220] General methods by which vectors can be constructed, transfection methods and culture methods are well known to those skilled in the art, see in this regard "Current Protocols in Molecular Biology", 1999, FM Ausubel (ed), Wiley Interscience, New York and the Maniatis Manual produced by Cold Spring Harbor Publishing.

[0221] Nucleic Acid Antagonists A polynucleotide, such as a nucleic acid, is a polymer that includes two or more nucleotides. The nucleotides can be naturally occurring or artificial. A nucleotide typically contains a nucleobase, a sugar, and at least one linking group, such as a phosphate group, a 2'O-methyl group, a 2'methoxy-ethyl group, a phosphoramidate group, a methylphosphonate group, or a phosphorothioate group. The nucleobase is typically a heterocycle. Nucleobases include, but are not limited to, purines and pyrimidines, more specifically adenine (A), guanine (G), thymine (T), uracil (U), and cytosine (C). The sugar is typically a pentose sugar. Nucleotide sugars include, but are not limited to, ribose and deoxyribose. The nucleotides are typically ribonucleotides or deoxyribonucleotides. The nucleotides typically contain monophosphates, diphosphates, or triphosphates. The phosphate can be attached to the 5' or 3' side of the nucleotide.

[0222] Nucleotides include, but are not limited to, adenosine monophosphate (AMP), adenosine diphosphate (ADP), adenosine triphosphate (ATP), guanosine monophosphate (GMP), guanosine diphosphate (GDP), guanosine triphosphate (GTP), thymidine monophosphate (TMP), thymidine diphosphate (TDP), thymidine triphosphate (TTP), uridine monophosphate (UMP), uridine diphosphate (UDP), uridine triphosphate (UTP), cytidine monophosphate (CMP), cytidine triphosphate (CTMP), cytidine monophosphate (CTMP), cytidine diphosphate (CTMP), cytidine triphosphate (CTMP), cytidine monophosphate (CTMP), cytidine triphosphate (CTMP), cytidine monophosphate (CTMP), cytidine monophosphate (CTMP), cytidine diphosphate (CTMP), cytidine triphosphate (CTMP), cytidine mono ... ), cytidine diphosphate (CDP), cytidine triphosphate (CTP), 5-methylcytidine monophosphate, 5-methylcytidine diphosphate, 5-methylcytidine triphosphate, 5-hydroxymethylcytidine monophosphate, 5-hydroxymethylcytidine diphosphate, 5-hydroxymethylcytidine triphosphate, cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP), deoxyadenosine monophosphate (dAMP), deoxyadenosine diphosphate (dADP) ), deoxyadenosine triphosphate (dATP), deoxyguanosine monophosphate (dGMP), deoxyguanosine diphosphate (dGDP), deoxyguanosine triphosphate (dGTP), deoxythymidine monophosphate (dTMP), deoxythymidine diphosphate (dTDP), deoxythymidine triphosphate (dTTP), deoxyuridine monophosphate (dUMP), deoxyuridine diphosphate (dUDP), deoxyuridine triphosphate (dUTP), deoxycytidine The nucleotides include 5-methyl-2'-deoxycytidine monophosphate (dCMP), deoxycytidine diphosphate (dCDP) and deoxycytidine triphosphate (dCTP), 5-methyl-2'-deoxycytidine monophosphate, 5-methyl-2'-deoxycytidine diphosphate, 5-methyl-2'-deoxycytidine triphosphate, 5-hydroxymethyl-2'-deoxycytidine monophosphate, 5-hydroxymethyl-2'-deoxycytidine diphosphate and 5-hydroxymethyl-2'-deoxycytidine triphosphate. The nucleotides are preferably selected from AMP, TMP, GMP, UMP, dAMP, dTMP, dGMP or dCMP.

[0223] Nucleotides may contain additional modifications. In particular, suitable modified nucleotides include, but are not limited to, 2'aminopyrimidines (e.g., 2'-aminocytidine and 2'-aminouridine), 2'-hydroxyl purines (e.g., 2'-fluoropyrimidines (e.g., 2'-fluorocytidine and 2'fluorouridine), hydroxylpyrimidines (e.g., 5'-α-P-boranouridine), 2'-O-methyl nucleotides (e.g., 2'-O-methyladenosine, 2'-O-methylguanosine, 2'-O-methylcytidine and 2'-O-methyluridine), 4'-thiopyrimidines (e.g., 4'-thiouridine and 4'-thiocytidine), and nucleotides having nucleobase modifications (e.g., 5-pentynyl-2'-deoxyuridine, 5-(3-aminopropyl)-uridine and 1,6-diaminohexyl-N-5-carbamoylmethyluridine).

[0224] The nucleotides in a polynucleotide can be linked together in any way. They can be linked by phosphate, 2'O-methyl, 2'methoxy-ethyl, phosphoramidate, methylphosphonate or phosphorothioate linkages. Nucleotides are typically linked by their sugar and phosphate groups as in nucleic acids. Nucleotides can be connected through their nucleobases as in pyrimidine dimers.

[0225] The GREM1 antagonist may be a polynucleotide encoding an anti-GREM1 antibody described herein.

[0226] The polynucleotide may be a nucleic acid, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). The polynucleotide may be any synthetic nucleic acid known in the art, such as peptide nucleic acid (PNA), glycerol nucleic acid (GNA), threose nucleic acid (TNA), locked nucleic acid (LNA), morpholino nucleic acid, or other synthetic polymers with nucleotide side chains. The polynucleotide may be single-stranded or double-stranded.

[0227] The polynucleotide sequence can be cloned into any suitable expression vector. In the expression vector, the polynucleotide sequence encoding the construct is typically operably linked to a control sequence capable of providing the expression of the coding sequence by the host cell. Such an expression vector can be used to express the construct.

[0228] In one embodiment, the anti-GREM1 antagonist is a polynucleotide encoding the anti-GREM1 antibody described herein. The polynucleotide can be provided for use in gene therapy. The polynucleotide can be provided in any suitable vector that can provide the expression of the anti-GREM1 antibody in vivo.

[0229] The polynucleotide encoding the anti-GREM1 antibody may be a DNA sequence. The DNA sequence may be provided in any suitable vector, e.g., an expression vector, for administration to a subject in need thereof. For example, the DNA sequence may be administered to a subject in an expression vector capable of providing expression of the anti-GREM1 antibody in vivo. The expression vector may be a viral expression vector, e.g., an adeno-associated virus (AAV) vector. In one embodiment, the anti-GREM1 antagonist is a DNA sequence encoding the anti-GREM1 antibody described herein. In one embodiment, the anti-GREM1 antagonist is a DNA sequence for use in gene therapy, the DNA sequence encoding the anti-GREM1 antibody described herein. In one embodiment, the anti-GREM1 antagonist is an AAV comprising a DNA sequence encoding the anti-GREM1 antibody described herein. In one embodiment, the anti-GREM1 antagonist is an AAV for use in gene therapy, the AAV comprising a DNA sequence encoding the anti-GREM1 antibody described herein.

[0230] The polynucleotide encoding the anti-GREM1 antibody may be an RNA sequence. The RNA sequence may be administered to a subject in need thereof in any suitable vector. The RNA sequence may be a messenger RNA (mRNA) sequence. The mRNA sequence may be administered to a subject in need thereof in a stabilized form. For example, the mRNA sequence may be provided in a lipid nanoparticle (LNP) composition. The LNP composition may comprise any suitable LNP capable of encapsulating the mRNA sequence to provide increased stability of said mRNA sequence. Thus, in one embodiment, the anti-GREM1 antagonist is a stabilized mRNA sequence encoding the anti-GREM1 antibody described herein. In one embodiment, the anti-GREM1 antagonist is a stabilized mRNA sequence for use in gene therapy, the mRNA sequence encoding the anti-GREM1 antibody described herein. In one embodiment, the anti-GREM1 antagonist is a LNP composition comprising an mRNA encoding the anti-GREM1 antibody described herein. In one embodiment, the anti-GREM1 antagonist is a LNP composition for use in gene therapy, the LNP composition comprising an mRNA encoding the anti-GREM1 antibody described herein.

[0231] The term "operably linked" refers to a juxtaposition where the described components are in a relationship permitting them to function in their intended manner. A control sequence "operably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences. Multiple copies of the same or different polynucleotides can be introduced into the vector.

[0232] The expression vector can then be introduced into a suitable host cell. Thus, a construct can be produced by inserting a polynucleotide sequence encoding the construct into an expression vector, introducing the vector into a compatible bacterial host cell, and growing the host cell under conditions that result in expression of the polynucleotide sequence.

[0233] Nucleic acid-based GREM1 antagonists can reduce the expression of GREM1. Antisense and RNA interference (RNAi) techniques for knocking down protein expression are well known in the art, and standard methods can be employed to knock down the expression of a molecule of interest. Antisense and siRNA techniques both interfere with mRNA. Antisense oligonucleotides interfere with mRNA by binding (hybridizing) to a portion of the mRNA. Thus, antisense oligonucleotides are designed to be complementary to the mRNA (although, as described below, the oligonucleotides do not need to be 100% complementary). In other words, the antisense oligonucleotide can be part of a cDNA. Again, the oligonucleotide sequence does not need to be 100% identical to the cDNA sequence. This is also discussed below. RNAi involves the use of double-stranded RNA, such as small interfering RNA (siRNA) or small hairpin RNA (shRNA), which can bind to mRNA and inhibit protein expression.

[0234] Thus, an antagonist can be an oligonucleotide that specifically hybridizes to an mRNA encoding GREM1, e.g., the coding sequence of SEQ ID NO: 36 or SEQ ID NO: 37, or a variant thereof. An oligonucleotide "specifically hybridizes" to a target sequence if it hybridizes preferentially or with high affinity to the target sequence, but does not substantially hybridize, does not hybridize, or hybridizes only with low affinity to other sequences. More preferably, the oligonucleotide has a T-type specificity relative to other nucleic acids. m T at least 5° C., at least 10° C., at least 20° C., at least 30° C., or at least 40° C. higher than mThe hybridization condition is a stringent condition as described in the art. The conditions that allow hybridization are well known in the art (e.g., Sambrook et al., 2001, Molecular Cloning: a laboratory manual, 3rd edition, Cold Spring Harbour Laboratory Press; and Current Protocols in Molecular Biology, Chapter 2, Ausubel et al., Eds., Greene Publishing and Wiley-Interscience, New York (1995)). The hybridization condition can be a stringent condition as described in the art.

[0235] Oligonucleotides are short nucleotide polymers, typically having 50 or less nucleotides, such as 40 or less, 30 or less, 22 or less, 21 or less, 20 or less, 10 or less, or 5 or less. The oligonucleotides used may be 20-25 nucleotides in length, more preferably 21 or 22 nucleotides in length. The nucleotides may be naturally occurring or artificial. The nucleotides may be any of those described above.

[0236] The GREM1 antagonist can be an antibody that binds to GREM1, typically an antibody that specifically binds to GREM1. An antibody "specifically binds" to a protein if it binds preferentially or with high affinity to the protein, but does not substantially bind, does not bind, or only binds with low affinity to other proteins. For example, an antibody "specifically binds" to a target molecule if it binds preferentially or with high affinity to the target molecule, but does not substantially bind, does not bind, or only binds with low affinity to other human proteins.

[0237] An antibody binds preferentially or with high affinity if the Kd is 1×10 M or less, more preferably 5×10 M or less, even more preferably 1×10 M or less, and even more preferably 5×10 M or less. An antibody binds with low affinity if the Kd is 1×10 M or more, more preferably 1×10 M or more, more preferably 1×10 M or more, more preferably 1×10 M or more, and even more preferably 1×10 M or more.

[0238] The antibody may be, for example, a monoclonal antibody, a polyclonal antibody, a single chain antibody, a chimeric antibody, a bispecific antibody, a CDR-grafted antibody, or a humanized antibody. The antibody may be an intact immunoglobulin molecule or a fragment, such as a Fab, F(ab')2 or Fv fragment.

[0239] patient Any patient can be treated according to the present invention. The patient is typically a human. However, the patient may also be another mammal, for example a commercially farmed animal such as a horse, cow, sheep, fish, chicken or pig, a laboratory animal such as a mouse or rat, or a pet such as a guinea pig, hamster, rabbit, cat or dog.

[0240] Pharmaceutical Compositions, Dosages and Dosing Regimens The GREM1 antagonist of the present invention may be provided in a pharmaceutical composition. The cytidine analog or deoxycytidine analog may be provided as part of the same pharmaceutical composition or in a separate pharmaceutical composition. For example, gemcitabine or its derivatives may be provided as part of the same pharmaceutical composition or in a separate pharmaceutical composition. For example, azacitidine, cytarabine, decitabine, troxacitabine or its derivatives may be provided as part of the same pharmaceutical composition or in a separate pharmaceutical composition. The proliferation-dependent cytotoxic agent may also be provided as part of the same pharmaceutical composition or in a separate pharmaceutical composition. The pharmaceutical composition is usually sterile and typically includes a pharmaceutically acceptable carrier and / or adjuvant. These compositions may include, in addition to the therapeutically active ingredient(s), pharmaceutically acceptable excipients, carriers, diluents, buffers, stabilizers, or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The pharmaceutical carrier or diluent may be, for example, an isotonic solution.

[0241] As used herein, "pharmaceutically acceptable carrier" includes any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc. The precise nature of the carrier or other material may depend on the route of administration, e.g., oral, intravenous, cutaneous or subcutaneous, nasal, intramuscular, and intraperitoneal routes.

[0242] The carrier may be suitable for parenteral administration, e.g., intravenous, intramuscular, intradermal, intraocular, intraperitoneal, subcutaneous, spinal or other parenteral routes of administration, e.g., by injection or infusion. Alternatively, the carrier may be suitable for parenteral administration, such as topical, epidermal, or mucosal routes of administration. The carrier may be suitable for oral administration. Depending on the route of administration, the modulator may be coated in a material to protect the compound from the action of acids and other natural conditions which may inactivate the compound. For example, solid oral dosage forms may contain, together with the active substance, diluents such as lactose, dextrose, saccharose, cellulose, corn starch or potato starch; lubricants such as silica, talc, stearic acid, magnesium or calcium stearate, and / or polyethylene glycol; binders such as starch, gum arabic, gelatin, methylcellulose, carboxymethylcellulose or polyvinylpyrrolidone; disaggregating agents such as starch, alginic acid, alginates or sodium starch glycolate; effervescent mixtures; dyes; sweeteners; wetting agents such as lecithin, polysorbates, lauryl sulfate; and non-toxic and pharmacologically inactive substances generally used in pharmaceutical preparations. Such pharmaceutical preparations can be produced by known methods, such as mixing, granulating, tableting, sugar-coating or film-coating processes.

[0243] Other oral formulations include commonly employed excipients, such as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain 10%-95%, preferably 25%-70%, of the active ingredient. If the pharmaceutical composition is lyophilized, the lyophilized material can be reconstituted prior to administration, e.g., as a suspension. Reconstitution is preferably carried out in a buffer solution.

[0244] Capsules, tablets and pills for oral administration to an individual may be provided with an enteric coating comprising, for example, Eudragit "S", Eudragit "L", cellulose acetate, cellulose acetate phthalate, or hydroxypropyl methylcellulose.

[0245] Liquid dispersions for oral administration may be syrups, emulsions or suspensions. The syrups may contain as carriers, for example, saccharose or saccharose with glycerine and / or mannitol and / or sorbitol.

[0246] Suspensions and emulsions may contain as a carrier, for example, natural gum, agar, sodium alginate, pectin, methylcellulose, carboxymethylcellulose, or polyvinyl alcohol. Suspensions or solutions for intramuscular injections may contain, together with the active substance, a pharma- ceutically acceptable carrier, for example, sterile water, olive oil, ethyl oleate, glycols, for example, propylene glycol, and, if desired, an appropriate amount of lidocaine hydrochloride.

[0247] The solutions for intravenous administration or infusion may contain as carrier, for example, sterile water or preferably they may be in the form of sterile, aqueous, isotonic saline solutions.

[0248] 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%.

[0249] The polynucleotide or oligonucleotide inhibitors may be naked nucleotide sequences or may be combined with cationic lipids, polymers or targeting systems. They may be delivered by any available technique. For example, polynucleotides or oligonucleotides may be introduced by needle injection, preferably intradermally, subcutaneously or intramuscularly. Alternatively, polynucleotides or oligonucleotides may be delivered directly across the skin using a delivery device such as particle-mediated gene delivery. Polynucleotides or oligonucleotides may be administered topically to the skin or to mucosal surfaces, for example, by nasal, oral or rectal administration.

[0250] The uptake of polynucleotide or oligonucleotide constructs can be enhanced by several known transfection techniques, including those that involve the use of transfection agents. Examples of these agents include cationic agents, such as calcium phosphate, DEAE-dextran, and lipofectants, such as lipofectam and transfectam. The dosage of the polynucleotide or oligonucleotide to be administered can be modified.

[0251] The pharmaceutical composition of the present invention may contain one or more pharma-ceutically acceptable salts. "Pharmaceutically acceptable salts" refers to salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects. Examples of such salts include acid addition salts and base addition salts.

[0252] Pharmaceutically acceptable carriers include aqueous carriers or diluents. Examples of suitable aqueous carriers that can be used in the pharmaceutical compositions of the present invention include water, buffered water and saline. Examples of other carriers include ethanol, polyol (e.g., glycerol, propylene glycol, polyethylene glycol, etc.) and their appropriate mixtures, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. In many cases, it is desirable to include isotonicity agents, such as sugars, polyhydric alcohols such as mannitol, sorbitol, or sodium chloride in the composition.

[0253] Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration.

[0254] The pharmaceutical compositions of the invention may contain additional active ingredients.

[0255] Also included within the scope of this disclosure are kits that include the combination therapies described herein and instructions for use. The kits may further contain one or more additional reagents, such as additional therapeutic or prophylactic agents as discussed herein.

[0256] The antagonists described herein or formulations or compositions thereof can be administered for prophylactic and / or therapeutic treatments.

[0257] In therapeutic applications, compounds are administered to subjects already suffering from the above disorders or conditions in an amount sufficient to cure, alleviate or partially halt the condition or one or more of its symptoms. Such therapeutic treatment may result in a decrease in the severity of disease symptoms or an increase in the frequency or duration of symptom-free periods. An amount sufficient to achieve this is defined as a "therapeutically effective amount".

[0258] In prophylactic applications, the formulations are administered to a subject at risk of the above disorders or conditions in an amount sufficient to prevent or reduce the subsequent effects of the condition or one or more of its symptoms. An amount sufficient to accomplish this is defined as a "prophylactically effective amount." Effective amounts for each purpose depend on the severity of the disease or injury, as well as the weight and general condition of the subject.

[0259] The subject of administration can be a human or a non-human animal. The term "non-human animal" includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc. Administration to humans is typical.

[0260] The antagonist, proliferation-dependent cytotoxic agent, e.g., gemcitabine, cytidine analogs or deoxycytidine analogs, or pharmaceutical compositions of the present invention may be administered via one or more routes of administration using one or more of a variety of methods known in the art. As will be appreciated by those skilled in the art, the route and / or mode of administration will vary depending on the desired outcome. Examples of routes of administration of the compounds or pharmaceutical compositions of the present invention include intravenous, intramuscular, intradermal, intraocular, intraperitoneal, subcutaneous, spinal or other parenteral routes of administration, e.g., administration by injection or infusion. As used herein, the phrase "parenteral administration" refers to modes of administration other than enteral and topical administration, which are usually administration by injection. Alternatively, the antibody / modulator or pharmaceutical composition of the present invention may be administered via a parenteral route, such as a topical, epidermal or mucosal route of administration. The antibody / modulator or pharmaceutical composition of the present invention may be for oral administration.

[0261] The appropriate dosage of the antibody / modulator or pharmaceutical composition of the present invention can be determined by a skilled medical practitioner. The actual dosage level of the active ingredient in the pharmaceutical composition of the present invention can be varied to obtain an amount of the active ingredient effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without causing toxicity to the patient. The dosage level selected will depend on various pharmacokinetic factors, such as the activity of the particular composition of the present invention employed, the route of administration, the time of administration, the excretion rate of the particular compound employed, the duration of treatment, other drugs, compounds and / or materials used in combination with the particular composition employed, the age, sex, weight, condition, general health and previous medical history of the patient being treated, and similar factors well known in the medical field.

[0262] Suitable doses may range, for example, from about 0.01 μg / kg to about 1000 mg / kg, typically from about 0.1 μg / kg to about 100 mg / kg of the patient's body weight to be treated depending on the above conditions. For example, suitable dosages may be from about 1 μg / kg to about 10 mg / kg of body weight per day, or from about 10 μg / kg to about 5 mg / kg of body weight per day.

[0263] Dosage regimes can be adjusted to provide the optimum desired response (e.g., therapeutic response). For example, a single dose can be administered, several divided doses can be administered over time, or the dose can be proportionally reduced or increased according to the exigencies of the therapeutic situation. The dose can be provided as multiple doses, for example, at regular intervals, such as 2, 3 or 4 divided doses administered hourly. The multiple doses can be administered by the same or different routes, to the same or different locations. Alternatively, administration can be by sustained release formulation, which requires less frequent administration. Dosage and frequency can vary depending on the half-life of the antagonist in the patient and the desired duration of treatment.

[0264] 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.

[0265] Dosage unit form, as used herein, refers to physically discrete units suited as unitary dosages for the subjects to be treated, each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.

[0266] As noted above, the modulators / antibodies or pharmaceutical compositions of the invention may be co-administered with one or more other therapeutic agents.

[0267] Examples of the techniques and protocols mentioned above can be found in Remington's Pharmaceutical Sciences, 20th Edition, 2000, pub. Lippincott, Williams & Wilkins.

[0268] The co-administration of two or more agents can be accomplished in many different ways. They can be administered together in a single composition, or in separate compositions as part of a combination therapy. For example, one can be administered before or separately, after or consecutively, or concurrently or simultaneously with the other. The anti-GREM1 antagonist can be administered before or separately, after or consecutively, or concurrently or simultaneously with the cytidine analog or deoxycytidine analog described herein. For example, the anti-GREM1 antagonist can be administered before or separately, after or consecutively, or concurrently or simultaneously with gemcitabine or a derivative thereof. The anti-GREM1 antagonist may be administered before or separately, after or sequentially, or concurrently or simultaneously with gemcitabine, azacytidine, cytarabine, decitabine, troxacitabine, or derivatives thereof. Alternatively, in the context of pancreatic cancer, the anti-GREM1 antagonist may be administered before or separately, after or sequentially, or concurrently or simultaneously with a proliferation-dependent cytotoxic agent.

[0269] Additional therapeutic combinations The combination therapy of the present invention described above can be used / administered in combination with an additional therapeutic composition of treatment, for example, as an adjunct therapy. The other therapeutic composition or treatment can be, for example, one or more of those discussed herein, and can be administered simultaneously or sequentially with the composition of the present invention.

[0270] As discussed above, GREM1 antagonists are particularly useful in combination treatments, as they are used to sensitize cancers or tumors to additional anti-cancer drugs, such as radiation therapy or surgery, and in the absence of the GREM1 antagonist, the cancer may be resistant to other anti-cancer drugs or cancer treatments.

[0271] Thus, the anti-GREM1 antagonist in combination with gemcitabine or a derivative thereof can be further used in combination with any other cancer therapy or any other therapeutic agent for cancer. Furthermore, the anti-GREM1 antagonist in combination with a proliferation-dependent cytotoxic agent can be further used in combination with other cancer therapy or other therapeutic agent for the treatment of pancreatic cancer. The other cancer therapy can be selected from known treatments for related cancers, such as any known treatment for pancreatic cancer. The other cancer therapy can be radiation treatment. Suitable radiation therapy is described, for example, in Van Cutsem (and others) Annals of Oncology, 2014. Vol 25, Issue 3. Radiation therapy can be performed before cancer surgery or after cancer surgery. Radiation therapy can be adjuvant radiation therapy. Radiation therapy can be performed in combination with chemotherapy, for example in combination with gemcitabine or a proliferation-dependent cytotoxic agent described herein. For example, a combination therapy including a GREM1 antagonist and gemcitabine or a derivative thereof can be used in combination with radiation therapy. Additionally, in the context of pancreatic cancer, combination therapies including a GREM1 antagonist and a growth-dependent cytotoxic agent may be used in conjunction with radiation therapy.

[0272] The additional therapeutic agent for cancer, such as an additional chemotherapeutic agent, can be selected from any known therapeutic agent for related cancer, including any known chemotherapeutic agent or combination of chemotherapeutic agents for related cancer.For example, a combination therapy comprising a GREM1 antagonist and gemcitabine can be used in combination with one or more of Abraxane, paclitaxel, oxaliplatin, folinic acid, irinotecan, fluorouracil, FOLFIRINOX or FOLFOX, particularly in the treatment of pancreatic cancer.For example, the anti-GREM1 antagonist for use according to the present invention can be administered in combination with gemcitabine and an additional cytidine analog or deoxycytidine analog.

[0273] Furthermore, the combination therapy comprising a GREM1 antagonist and a proliferation-dependent cytotoxic agent can be used in combination with an additional chemotherapeutic agent, such as gemcitabine, Abraxane, paclitaxel, oxaliplatin, folinic acid, irinotecan, fluorouracil, FOLFIRINOX or FOLFOX. Any of the combinations described herein are contemplated in the context of compositions and kits for treating cancer or pancreatic cancer. In a preferred embodiment, the anti-GREM1 antagonist for use according to the present invention can be administered in combination with gemcitabine and troxacitabine. In a particularly preferred embodiment, the anti-GREM1 antagonist for use in the method for treating pancreatic cancer can be administered in combination with gemcitabine and troxacitabine. The cancer can be resistant to radiation therapy or one or more chemotherapeutic agents (such as one of the chemotherapeutic agents described above) when not administered with a combination therapy comprising a GREM1 antagonist as described herein.

[0274] As part of the above aspect, the present invention provides an anti-GREM1 antagonist in combination with gemcitabine or a derivative thereof as described in the present invention, or a proliferation-dependent cytotoxic agent, for use in a method for treating and / or preventing cancer, such as pancreatic cancer, the method further comprising separate, sequential or simultaneous administration of an additional anti-cancer agent.

[0275] Compositions and kits Further, a composition or kit is provided that includes an anti-GREM1 antagonist and a cytidine analog or a deoxycytidine analog. The cytidine analog or deoxycytidine analog described herein can be one or more of gemcitabine, azacitidine, cytarabine, decitabine or troxacitabine. In one embodiment, a composition or kit is provided that includes an anti-GREM1 antagonist and gemcitabine or a derivative thereof. Also included in the present invention is a composition or kit that includes an anti-GREM1 antagonist and capecitabine or a derivative thereof. The derivative of gemcitabine can be any derivative as described above. The anti-GREM1 antagonist can be any anti-GREM1 antagonist described herein. The composition or kit can be suitable for treating pancreatic cancer.

[0276] Also provided is a composition or kit comprising an anti-GREM1 antagonist and a mitotic inhibitor. The composition or kit may comprise an anti-GREM1 antagonist and one or more mitotic inhibitors selected from cabazitaxel, docetaxel, paclitaxel, vinblastine, vincristine, vinorelbine and abraxane. In a preferred embodiment in the context of pancreatic cancer, the mitotic inhibitor and / or microtubule stabilizing agent is selected from abraxane and paclitaxel, particularly as part of a composition or kit for the treatment of pancreatic cancer. A preferred combination comprises an anti-GREM1 antagonist and paclitaxel or abraxane.

[0277] Bispecific antibodies combining an anti-GREM1 specificity with one of the other above-mentioned specificities may be provided in a composition or kit as described herein. The anti-GREM1 antagonist in any of the above compositions and kits may preferably be an anti-GREM1 antibody.

[0278] Detection and Diagnosis Based on the correlation between stromal GREM1 and cancer, the present invention also provides additional means for predicting the responsiveness of cancer to treatment.

[0279] The present invention provides a method for determining whether a patient suffering from or suspected of suffering from cancer, or at risk of developing cancer, is likely to respond to combination treatment with a GREM1 antagonist and a cytidine analogue such as gemcitabine or a derivative thereof, or a deoxycytidine analogue, which method comprises measuring stromal and / or epithelial expression of GREM1 in the patient, thereby predicting whether the patient is likely to respond to combination treatment.

[0280] The present invention further provides a method for determining whether a patient suffering from or suspected of suffering from pancreatic cancer, or at risk of developing pancreatic cancer, is likely to respond to a combination treatment with a GREM1 antagonist and a proliferation-dependent cytotoxic agent, the method comprising measuring stromal and / or epithelial expression of GREM1 in the patient, thereby predicting whether the patient is likely to respond to the combination treatment.

[0281] A predicted responsiveness of an individual to a given therapy means that the individual is expected to benefit or to benefit to a sufficient degree from receiving the therapy. A predicted non-responsiveness of an individual to a therapy means that the individual is not expected to benefit or to benefit to a sufficient degree from receiving the therapy. The method of predicting response can be performed before administration of a combination treatment of a GREM1 antagonist and a cytidine analog or a deoxycytidine analog, such as gemcitabine or its derivatives, or a combination treatment of a GREM1 antagonist and a proliferation-dependent cytotoxic agent. The prediction can then be taken into account when selecting or recommending an appropriate treatment for the individual. Alternatively, the method is performed after a therapeutic treatment and used to monitor and predict the individual's response to the treatment. Typically, the method is for predicting whether an individual will have a primary response to the therapy, i.e., whether the individual will respond when first receiving the treatment. In some examples, the method is for predicting secondary non-responsiveness, i.e., whether an individual who initially responded to the treatment will later become unresponsive to the treatment or respond less to the treatment.

[0282] In some cases, overexpression of GREM1 compared to a reference sample or reference level indicates that the individual will respond to a combination treatment therapy as described herein.Then, a combination therapy comprising a GREM1 antagonist in combination with a cytidine analog or deoxycytidine analog as described herein can be selected or recommended, and then further administered to the individual.Then, a combination therapy comprising a GREM1 antagonist in combination with gemcitabine or its derivative can be selected or recommended, and then further administered to the individual.Similarly, in the context of pancreatic cancer, a therapy comprising the use of a GREM1 antagonist in combination with a proliferation-dependent cytotoxic agent can be selected based on the overexpression of GREM1.

[0283] In other cases, reduced or normal levels of GREM1 compared to a reference sample or reference level indicates that the individual will not respond to treatment with a GREM1 antagonist. A combination therapy comprising a GREM1 antagonist and gemcitabine (or a derivative thereof), or a GREM1 antagonist and a proliferation-dependent cytotoxic agent, is then not administered to the individual. Additionally, a therapeutic treatment other than the combination therapy described herein may be selected or recommended for treatment of the individual and then further administered to the individual.

[0284] In all aspects of the present invention, individuals with cancer (e.g., pancreatic cancer) or individuals suspected of having a disease or condition and / or individuals at risk of developing a disease or condition can be selected or identified for treatment. For example, an individual may not have been formally diagnosed, but may be suspected of having a disease or condition because one or more symptoms are present. If an individual has one or more risk factors associated with cancer and / or one or more predisposing factors that increase susceptibility to cancer, the individual may be considered at risk of developing cancer. Risk factors associated with pancreatic cancer may include inherited genetic mutations, such as familial pancreatitis caused by a mutation in the PRSS1 gene, a mutation or mutations in the gene encoding GREM1, or any other mutation that affects the expression of the GREM1 gene.

[0285] The following examples illustrate the invention. EXAMPLES

[0286] Materials and Methods Test Antibodies and Drugs Anti-Gremlin-1 Antibody - Ab7326 mIgG1 - APP.4405.IgG.mFc - Lot Number - PB 4682 Vehicle - Phosphate buffered saline pH 7.4 (provided by Beatson Institute) Gemcitabine (provided by the Beatson Institute and purchased from LC Labs, Woburn, Mass., USA) in phosphate buffered saline pH 7.4.

[0287] Genetically modified mice LSL-Kras G12D / + ;Trp53 R172H / + ;Pdx1-Cre(KPC) mice have been described previously (Hingorani et al., 2005). Mice were generated by crossing mice carrying Pdx1-Cre with conditional LSL-KrasG12D or LSL-Trp53R172H alleles (strains 01XJ6 and 01XL9, Mouse Models of Human Cancer Consortium [MMHCC], NCI-Frederick, Frederick, MD, USA). Mice on mixed backgrounds were bred in-house at the CRUK Beatson Institute and maintained in normal cages with environmental enrichment and free access to standard food and water. Genotyping was performed at Transnetyx (Cordoba, TN, USA). Mice of both sexes were included in the study. All animal experiments were performed under UK Home Office permission and approved by the University of Glasgow Animal Welfare Ethics Committee.

[0288] treatment Mice were monitored three times a week until pancreatic cancer was diagnosed by abdominal palpation and confirmed by ultrasound imaging. Mice were randomized into treatment groups and dosed with 30 mg / kg Ab7326 mIgG1 sc twice weekly; 100 mg / kg gemcitabine ip twice weekly; 30 mg / kg Ab7326 mIgG1 sc twice weekly and 100 mg / kg gemcitabine ip twice weekly; or PBS vehicle control, twice weekly. There were ≥5 mice per group. Mice were monitored daily and sacrificed when they reached ethical endpoints (symptoms included abdominal distension, cachexia, intermittent hunched back or reduced mobility, pilonidation, mild diarrhea, anemia). Statistical evaluation of survival from the start of treatment was performed by Kaplan-Meier and log-rank analysis.

[0289] Ultrasound Imaging A VisualSonics Vevo 3100 preclinical imaging platform (FUJIFILM VisualSonics, Toronto, Canada) was used for high-resolution ultrasound imaging to confirm tumor diagnosis and weekly monitoring of tumor progression. Anesthesia was induced and maintained with a mixture of isoflurane and medical air. Tumor volumes were calculated weekly for each mouse and plotted longitudinally.

[0290] sampling Mice were culled using schedule 1 methods according to institutional guidelines. Postmortem tumor burden was assessed by gross pathology and histology. Organs were removed and fixed in 10% buffered formalin. Terminal blood was collected whenever possible. The majority of tumors were fixed in 10% buffered formalin for FFPE processing, any remaining tissue was harvested in RNAlater® (Sigma-Aldrich) for RNA preparation and / or snap frozen. Fixed tissues were embedded in paraffin and 5 μm sections were placed on poly-L-lysine slides for IHC analysis.

[0291] Histology and immunohistochemistry H&E and picrosirius red staining were performed on formalin-fixed, paraffin-embedded tissues as previously described. Immunohistochemistry was performed using standard protocols. Briefly, formalin-fixed, paraffin-embedded sections were deparaffinized and rehydrated through xylene and a graded alcohol series. Endogenous peroxidase activity was inactivated by treatment with hydrogen peroxide, followed by antigen retrieval with citrate buffer. Sections were blocked with serum and incubated with primary antibodies. Sections were incubated with secondary antibodies for 30 min, and staining was visualized with 3,3'-diaminobenzidine tetrahydrochloride. Primary antibodies used were anti-Ki67 (SP6, ThermoFisher) 1:200, anti-cleaved caspase 3 (ASP175, Cell Signaling) 1:50, and anti-alpha-SMA (1A4, Sigma-Aldrich) 1:20,000).

[0292] (Example 1) Confirmation of Grem1 mRNA expression in pancreatic cancer To investigate whether Gremlin-1 plays a role in pancreatic cancer, Grem1 mRNA expression, as determined by RNAseq analysis, was determined in a cohort of human pancreatic ductal adenocarcinoma (PDAC) patients. This confirmed that Grem1 mRNA is expressed in human PDAC and high expression was significantly associated with poor prognosis (Figure 1). The results were consistent with a published study (Yu et al., 2018) that reported an association between Grem1 expression and poor prognosis in pancreatic cancer.

[0293] To determine whether grem1 mRNA is expressed in the KPC genetically engineered mouse pancreatic cancer model, gene expression was examined in KPC tumors (and other autologous pancreatic cancer models) and compared with control KrasG12D-expressing normal pancreatic ductal epithelium. The results show that grem1 mRNA expression was elevated in KPC tumors (and other mouse pancreatic tumors) compared with controls (Table 1). [Table 1]

[0294] Example 2: Treatment with the combination of Ab7326 mIgG1 and gemcitabine results in improved survival in the KPC mouse model compared to vehicle control or single agent treatment with Ab7326 mIgG1 and gemcitabine Pilot studies were performed in healthy littermate mice to confirm the tolerability of the offered doses and schedules, as per local requirements. No adverse effects were observed.

[0295] Next, the efficacy of Gremlin-1 inhibition as a potential therapeutic approach against pancreatic cancer was tested in the KPC model. KPC mice develop tumors that are histologically and pathologically similar to human pancreatic tumors. Furthermore, the tumors are highly aggressive, frequently metastasizing, and highly resistant to chemotherapy, again mimicking human pancreatic cancer. A cohort of KPC mice was established and the mice were monitored until they developed pancreatic cancer detectable by palpation. The breeding strategy and experimental design required to generate these mice are shown in Figure 2.

[0296] Mice were monitored at least weekly by palpation until pancreatic tumors were detected. At this point, high-resolution ultrasound imaging was used to confirm the presence of pancreatic cancer, and mice were enrolled into cohorts treated with anti-Gremlin 1 antibody Ab7326 mIgG1, standard of care chemotherapy gemcitabine, the combination of Ab7326 mIgG1 + gemcitabine, or vehicle control (detailed in Table 2). High-resolution ultrasound imaging was performed weekly throughout the treatment period to monitor the tumor burden of each individual mouse during treatment. Mice continued in the study and were closely monitored until the ethical endpoint was reached, at which point they were sacrificed in accordance with institutional guidelines. Clinical characteristics exhibited included abdominal distension, poor physical condition indicative of cachexia, decreased exercise capacity, and occasionally jaundice. [Table 2]

[0297] 3D tumor image analysis by ultrasound showed that no tumor shrinkage or congestion was observed in any of the treatment groups (Figure 3). However, mice treated with the combination of Ab7326 mIgG1 and gemcitabine showed a significant increase in median survival (37 days) compared to vehicle controls (20 days, log-rank, p=0.037, see Figure 4, Table 2, and individual mouse data in Table 3). Mice treated with the combination of Ab7326 mIgG1 and gemcitabine also showed increased survival compared to mice treated with either Ab7326 mIgG1 (median survival 26 days, ns) or gemcitabine (median survival 24 days, ns) as single agents. The benefit of gemcitabine monotherapy was minimal, consistent with recent studies (Olive et al., 2009; Frese et al., 2012; Provenzano et al., 2012). [Table 3]

[0298] After sacrificing the experimental mice, gross pathological examination did not show any differences in tumor burden and phenotype between the different experimental conditions. Immunohistochemical (IHC) analysis was performed on formalin-fixed, paraffin-embedded tumor tissues to evaluate tumor cell proliferation (Ki67) and apoptosis (cleaved caspase 3). We also performed an IHC-based analysis of the tumor microenvironment to evaluate changes in the number of alpha-SMA-positive tumor-associated fibroblasts or the quality or quantity of collagen I and III (as measured by picrosirius red staining). Staining showed no obvious effect on any of these parameters with any of the regimens tested (data not shown).

[0299] result In summary, Gremlin-1 represents a valid target in the treatment of pancreatic cancer, and the results presented herein indicate that Ab7326 mIgG1 can be safely administered to KPC mice as a single agent and in combination with gemcitabine. A significant improvement in survival was observed when mice were treated with the combination of Ab7326 mIgG1 and gemcitabine compared to vehicle controls (Figures 4, 9, Table 2, and individual mouse data in Table 3).

[0300] Aspects of the invention 1. An anti-GREM1 antagonist for use in a method of treating or preventing cancer, the method further comprising administering a cytidine analogue or a deoxycytidine analogue. 2. An anti-GREM1 antagonist for use according to embodiment 1, wherein the cancer is a solid cancer. 3. An anti-GREM1 antagonist for use according to embodiment 1 or embodiment 2, wherein the cancer has stromal GREM1 overexpression. 4. The anti-GREM1 antagonist for use according to any one of the preceding aspects, wherein the cancer is a metastatic cancer. 5. The anti-GREM1 antagonist for use according to any one of the preceding aspects, wherein the cancer comprises dormant cancer cells, optionally dormant stem-like cancer cells. 6. The anti-GREM1 antagonist for use according to any one of the preceding aspects, wherein the cancer is a recurrent cancer and / or said method is for preventing recurrence of cancer. 7. 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 cytidine or deoxycytidine analogue. 8. An anti-GREM1 antagonist for use according to aspect 7, wherein the cancer is a cancer that is poorly responsive, non-responsive or refractory to treatment with gemcitabine or a derivative thereof. 9. The anti-GREM1 antagonist for use according to any one of the preceding aspects, wherein the cancer is selected from colorectal cancer, multiple myeloma, pancreatic cancer, bladder cancer, breast cancer, lung cancer, gastric cancer, duodenal cancer, esophageal cancer, head and neck cancer, prostate cancer, glioma, endometrial cancer, ovarian cancer, liver cancer, splenic cancer, bone resident cancer, and osteosarcoma. 10. The anti-GREM1 antagonist for use according to aspect 9, wherein the cancer is pancreatic cancer. 11. The anti-GREM1 antagonist for use according to aspect 10, wherein the pancreatic cancer is exocrine pancreatic cancer. 12. An anti-GREM1 antagonist for use according to embodiment 10 or embodiment 11, wherein the pancreatic cancer is pancreatic ductal adenocarcinoma (PDAC). 13. The anti-GREM1 antagonist for use according to aspect 9, wherein the cancer is lung cancer. 14. An anti-GREM1 antagonist for use according to aspect 13, wherein the cancer is non-small cell lung cancer. 15. The anti-GREM1 antagonist for use according to aspect 9, wherein the cancer is bladder cancer. 16. The anti-GREM1 antagonist for use according to embodiment 9, wherein the cancer is breast cancer. 17. The anti-GREM1 antagonist for use according to embodiment 9, wherein the cancer is ovarian cancer. 18. The anti-GREM1 antagonist for use according to any one of the preceding aspects, wherein the cancer has epithelial GREM1 overexpression. 19. An anti-GREM1 antagonist for use according to aspect 18, wherein the cancer is a GREM1-induced cancer. 20. The anti-GREM1 antagonist for use according to any one of the preceding aspects, wherein the cancer is a disseminated cancer. 21. The anti-GREM1 antagonist for use according to any one of the preceding aspects, wherein the cancer is an established cancer. 22. An anti-GREM1 antagonist for use according to any one of aspects 1 to 21, wherein the cytidine analogue or deoxycytidine analogue is gemcitabine or a derivative thereof. 23. An anti-GREM1 antagonist for use according to any one of aspects 1 to 21, wherein the cytidine analogue or deoxycytidine analogue is azacytidine or a derivative thereof. 24. An anti-GREM1 antagonist for use according to any one of aspects 1 to 21, wherein the cytidine analogue or deoxycytidine analogue is cytarabine or a derivative thereof. 25. An anti-GREM1 antagonist for use according to any one of aspects 1 to 21, wherein the cytidine analogue or deoxycytidine analogue is decitabine or a derivative thereof. 26. An anti-GREM1 antagonist for use according to any one of aspects 1 to 21, wherein the cytidine analogue or deoxycytidine analogue is troxacitabine or a derivative thereof. 27. An anti-GREM1 antagonist for use in a method of treating or preventing pancreatic cancer, the method further comprising administering a proliferation-dependent cytotoxic agent. 28. An anti-GREM1 antagonist for use according to aspect 27, wherein the pancreatic cancer is exocrine pancreatic cancer. 29. An anti-GREM1 antagonist for use according to embodiment 27 or embodiment 28, wherein the pancreatic cancer is pancreatic ductal adenocarcinoma (PDAC). 30. An anti-GREM1 antagonist for use according to any one of aspects 27 to 29, wherein the pancreatic cancer is metastatic pancreatic cancer. 31. An anti-GREM1 antagonist for use according to any one of aspects 27 to 30, wherein the pancreatic cancer is recurrent pancreatic cancer. 32. An anti-GREM1 antagonist for use according to any one of aspects 27 to 31, wherein the pancreatic cancer is a pancreatic cancer that is poorly responsive, non-responsive or refractory to treatment with a proliferation-dependent cytotoxic agent. 33. An anti-GREM1 antagonist for use according to any one of aspects 27 to 32, wherein the proliferation-dependent cytotoxic agent is a nucleoside inhibitor or antimetabolite. 34. An anti-GREM1 antagonist for use according to any one of aspects 27 to 32, wherein the proliferation-dependent cytotoxic agent is a cytidine analogue or a deoxycytidine analogue. 35. An anti-GREM1 antagonist for use according to any one of aspects 27 to 32, wherein the proliferation-dependent cytotoxic agent is an antimitotic agent. 36. An anti-GREM1 antagonist for use according to embodiment 35, wherein the mitotic inhibitor is a microtubule stabilizing agent. 37. An anti-GREM1 antagonist for use according to aspect 35 or 36, wherein the mitotic inhibitor and / or microtubule stabilising agent is selected from Abraxane and Paclitaxel. 38. An anti-GREM1 antagonist for use according to any one of aspects 27 to 33, wherein the proliferation-dependent cytotoxic agent comprises one or more of oxaliplatin, folinic acid, irinotecan and fluorouracil; optionally, the proliferation-dependent cytotoxic agent is FOLFIRINOX or FOLFOX. 39. Growth-dependent cytotoxic agents (a) gemcitabine or its derivatives; (b) azacitidine or its derivatives; (c) cytarabine or its derivatives; (d) decitabine or its derivatives; (e) troxacitabine or its derivatives; or (f) Capecitabine 33. The anti-GREM1 antagonist for use according to any one of aspects 27 to 32, which is 40. The anti-GREM1 antagonist for use according to any one of the preceding aspects, wherein the antagonist is a peptide, a protein, an antibody, a polynucleotide, an oligonucleotide, an antisense RNA, a small interfering RNA (siRNA), a small molecule inhibitor, or a small hairpin RNA (shRNA). 41. An anti-GREM1 antagonist for use according to aspect 40, wherein the antagonist is an antibody that binds to an epitope on Gremlin-1 comprising at least one residue selected from Ile131, Lys147, Lys148, Phe149, Thr150, Thr151, Arg169, Lys174 and Gln175, wherein the residue numbering is according to SEQ ID NO:1. 42. An anti-GREM1 antagonist for use according to aspect 41, wherein the antibody binds to an epitope comprising all of Ile131, Lys147, Lys148, Phe149, Thr150, Thr151, Arg169, Lys174 and Gln175. 43. An anti-GREM1 antagonist for use according to aspect 41 or 42, 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. 44. An anti-GREM1 antagonist for use according to aspect 40, wherein the antagonist is an anti-Gremlin-1 antibody comprising a heavy chain complementarity determining region (HCDR) sequence contained within the heavy chain variable region (HCVR) of SEQ ID NO: 10 or 12, and / or a light chain complementarity determining region (LCDR) sequence contained within the light chain variable region (LCVR) of SEQ ID NO: 11 or 13. 45. An anti-GREM1 antagonist for use according to aspect 40, wherein the antagonist is an anti-Gremlin-1 antibody comprising at least one HCDR sequence selected from SEQ ID NOs: 3, 4, 5 and 6, and / or at least one LCDR sequence selected from SEQ ID NOs: 7, 8 and 9. 46. ​​An anti-GREM1 antagonist for use according to aspect 45, wherein the anti-Gremlin-1 antibody comprises the HCDR3 sequence of SEQ ID NO:6. 47. An anti-GREM1 antagonist for use according to aspect 45 or 46, wherein the anti-Gremlin-1 antibody comprises a combination of HCDR1 / HCDR2 / HCDR3 sequences selected from SEQ ID NO: 4 / 5 / 6 or SEQ ID NO: 3 / 5 / 6, and / or a combination of LCDR1 / LCDR2 / LCDR3 sequences selected from SEQ ID NO: 7 / 8 / 9. 48. An anti-GREM1 antagonist for use according to any one of aspects 45 to 47, wherein the anti-Gremlin-1 antibody comprises a combination of HCDR1 / HCDR2 / HCDR3 / LCDR1 / LCDR2 / LCDR3 sequences of SEQ ID NO: 4 / 5 / 6 / 7 / 8 / 9 or SEQ ID NO: 3 / 5 / 6 / 7 / 8 / 9. 49. An anti-GREM1 antagonist for use according to any one of aspects 45 to 48, wherein the anti-Gremlin-1 antibody comprises a heavy chain variable region (HCVR) sequence of SEQ ID NO: 10 or 12, and / or a light chain variable region (LCVR) sequence of SEQ ID NO: 11 or 13, or a sequence at least 95% identical thereto. 50. An anti-GREM1 antagonist for use according to aspect 49, wherein the anti-Gremlin-1 antibody comprises the HCVR and LCVR sequence pair of SEQ ID NOs: 10 / 11 or 12 / 13, or a sequence which is at least 95% identical thereto. 51. An anti-GREM1 antagonist for use according to aspect 50, wherein the anti-Gremlin-1 antibody comprises an HCDR1 / HCDR2 / HCDR3 / LCDR1 / LCDR2 / LCDR3 sequence consisting of SEQ ID NO: 4 / 5 / 6 / 7 / 8 / 9 or SEQ ID NO: 3 / 5 / 6 / 7 / 8 / 9, and the remainder of the HCVR and LCVR are at least 95% identical to SEQ ID NO: 10, 11, 12 and / or 13, respectively. 52. An anti-GREM1 antagonist for use according to aspects 49 to 51, wherein the anti-Gremlin-1 antibody comprises a heavy chain of SEQ ID NO: 14, 16, 18, 22, 28, 30, 32 or 34, and / or a light chain of SEQ ID NO: 15, 17, 19, 23, 29, 31, 33 or 35, or a sequence at least 95% identical thereto. 53. An anti-GREM1 antagonist for use according to aspect 52, wherein the anti-Gremlin-1 antibody comprises a heavy and light chain pair of SEQ ID NOs: 14 / 15, 16 / 17, 18 / 19, 22 / 23, 28 / 29 or 30 / 31, 32 / 33, 34 / 35, or a sequence which is at least 95% identical. 54. An anti-GREM1 antagonist for use according to aspect 53, wherein the HCDR1 / HCDR2 / HCDR3 / LCDR1 / LCDR2 / LCDR3 sequences of the antibody consist of SEQ ID NO: 4 / 5 / 6 / 7 / 8 / 9 or SEQ ID NO: 3 / 5 / 6 / 7 / 8 / 9, and the remainder of the heavy and light chains are at least 95% identical to SEQ ID NO: 14, 15, 16 and / or 17, respectively. 55. An anti-GREM1 antagonist for use according to aspect 40, wherein the antagonist is an antibody which competes for binding to Gremlin-1 with an antibody as defined in any one of aspects 42 to 52. 56. An anti-GREM1 antagonist for use according to aspect 40, wherein the antagonist is an antibody that binds to the same epitope on Gremlin-1 as an antibody defined in any one of aspects 42 to 52. 57. An anti-GREM1 antagonist for use according to any one of aspects 40 to 56, wherein the antagonist antibody is a chimeric antibody, a human antibody or a humanized antibody. 58. An anti-GREM1 antagonist for use according to any one of aspects 40 to 57, wherein the antagonist antibody is a Fab, a modified Fab, a Fab', a modified Fab', F(ab')2, an Fv, a single domain antibody or an scFv. 59. An anti-GREM1 antagonist for use according to aspect 40, wherein the antagonist is a polynucleotide encoding an antibody as defined in any one of aspects 41 to 58, or an expression vector comprising said polynucleotide. 60. An anti-GREM1 antagonist for use according to any one of aspects 40 to 58, wherein the antagonist antibody is comprised in a pharmaceutical composition further comprising a pharma- ceutically acceptable adjuvant and / or carrier. 61. The anti-GREM1 antagonist for use according to any one of the preceding aspects, wherein the method further comprises administering an additional anti-cancer agent. 62. An anti-GREM1 antagonist for use according to aspect 61, wherein the method comprises administering gemcitabine in combination with a further cytidine analogue or a deoxycytidine analogue. 63. An anti-GREM1 antagonist for use according to aspect 61 or 62, wherein the method comprises administering gemcitabine in combination with troxacitabine. 64. A cytidine or deoxycytidine analogue for use in a method for treating or preventing cancer, the method further comprising administering an anti-GREM1 antagonist. 65. The cytidine analogue or deoxycytidine analogue for use according to aspect 64, wherein the cancer, the antagonist, the cytidine analogue or deoxycytidine analogue and / or the method are as defined in any one of aspects 1 to 26 and 40 to 63. 66. A method for treating cancer, comprising administering to a subject in need thereof a therapeutically effective amount of an anti-GREM1 antagonist in combination with a therapeutically effective amount of a cytidine analogue or a deoxycytidine analogue. 67. The method according to aspect 66, wherein the cancer, the antagonist, the cytidine analogue or deoxycytidine analogue, and / or the method are as defined in any one of aspects 1 to 26 and 40 to 63. 68. A proliferation-dependent cytotoxic agent for use in a method of treating or preventing pancreatic cancer, the method further comprising administering an anti-GREM1 antagonist. 69. A proliferation-dependent cytotoxic agent for use according to aspect 68, wherein said proliferation-dependent cytotoxic agent, said pancreatic cancer, said antagonist and / or said method are as defined in any one of aspects 27 to 63. 70. A method of treating pancreatic cancer comprising administering a therapeutically effective amount of an anti-GREM1 antagonist in combination with a therapeutically effective amount of a proliferation-dependent cytotoxic agent. 71. The method according to aspect 70, wherein said proliferation-dependent cytotoxic agent, said pancreatic cancer, said antagonist and / or said method are as defined in any one of aspects 27 to 63. 72. A composition or kit comprising an anti-GREM1 antagonist and a cytidine analogue or a deoxycytidine analogue. 73. The composition or kit according to aspect 72, wherein the cytidine analogue or deoxycytidine analogue is as defined in any one of aspects 22 to 26. 74. A composition or kit comprising an anti-GREM1 antagonist and an anti-mitotic agent. 75. The composition or kit according to embodiment 74, wherein the mitotic inhibitor is as defined in any one of embodiments 36 or 37. 76. The composition or kit according to any one of aspects 72 to 75, wherein the anti-GREM1 antagonist is as defined in any one of aspects 40 to 59. 77. A method for determining whether a patient suffering from or suspected of suffering from cancer, or at risk of developing cancer, is likely to respond to combined treatment with a GREM1 antagonist and a cytidine analogue or a deoxycytidine analogue, comprising measuring stromal and / or epithelial expression of GREM1 in the patient, thereby predicting whether the patient is likely to respond to combined treatment. 78. A method for determining whether a patient suffering from or suspected of suffering from pancreatic cancer, or at risk of developing cancer, is likely to respond to combined treatment with a GREM1 antagonist and a proliferation-dependent cytotoxic agent, comprising measuring stromal and / or epithelial expression of GREM1 in the patient, thereby predicting whether the patient is likely to respond to combined treatment.

[0301] Sequence Listing SEQ ID NO:1 (human gremlin-1; Uniprot ID: O60565) MSRTAYTVGALLLLLGTLLPAAEGKKKGSQGAIPPPDKAQHNDSEQTQSPQQPGSRNRRGGQGRGTAMPGEEVLESSQEALHVTERKYLKRDWCKTQPLKQTIHEEGCNSRTIINRFCYGQCNSFYIPRHIRKEEGSFQSCSFCKPKKFTTMMVTLNCPELQPPTKKKRVTRVKQCRCISIDLD SEQ ID NO:2 (N-terminally tagged human truncated Gremlin-1 used for crystal structure analysis) MGSSHHHHHHSSGENLYFQGSAMPGEEVLESSQEALHVTERKYLKRDWCKTQPLKQTIHEEGCNSRTIINRFCYGQCNSFYIPRHIRKEEGSFQSCSFCKPKKFTTMMVTLNCPELQPPTKKKRVTRVKQCRCISIDLD SEQ ID NO:3 (Ab7326 HCDR1 Kabat & Chothia composite) GYTFTDYYMH SEQ ID NO:4 (Ab7326 HCDR1 Kabat) DYYMH SEQ ID NO:5 (Ab7326 HCDR2 Kabat) LVDPEDGETIYAEKFQG SEQ ID NO:6 (Ab7326 HCDR3 Kabat) DARGSGSYYPNHFDY SEQ ID NO:7 (Ab7326 LCDR1 Kabat) KSSQSVLYSSNNKNYLA SEQ ID NO:8 (Ab7326 LCDR2 Kabat) WASTRES SEQ ID NO:9 (Ab7326 LCDR3 Kabat) QQYYDTPT SEQ ID NO: 10 (Ab7326 heavy chain modified region variant 1) QVQLVESGAEVKKPGATVKISCKVSGYTFTDYYMHWVQQAPGKGLEWMGLVDPEDGETIYAEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCATDARGSGSYYPNHFDYWGQGTLVTVSS SEQ ID NO: 11 (Ab7326 light chain modified region variant 1) DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTINSLQAEDVAVYFCQQYYDTPTFGQGTRLEIK SEQ ID NO: 12 (Ab7326 heavy chain modified region variant 2) QVQLVQSGAEVKKPGATVKISCKVSGYTFTDYYMHWVQQAPGKGLEWMGLVDPEDGETIYAEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCATDARGSGSYYPNHFDYWGQGTLVTVSS SEQ ID NO: 13 (Ab7326 light chain modified region variant 2) DIVMTQTPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTINSLQAEDVAVYFCQQYYDTPTFGQGTRLEIK SEQ ID NO: 14 (Mouse full length IgG1 heavy chain variant 1) QVQLVESGAEVKKPGATVKISCKVSGYTFTDYYMHWVQQAPGKGLEWMGLVDPEDGETIYAEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCATDARGSGSYYPNHFD YWGQGTLVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPR DCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKT ISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK SEQ ID NO: 15 (Mouse full length IgG1 light chain variant 1) DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTINSLQAEDVAVYFCQQYYDTPTFGQGTRL EIKRTDAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC SEQ ID NO: 16 (human full length IgG1 heavy chain variant 2) QVQLVQSGAEVKKPGATVKISCKVSGYTFTDYYMHWVQQAPGKGLEWMGLVDPEDGETIYAEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCATDARGSGSYYPNHFDY WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 17 (human full length IgG1 light chain variant 2) DIVMTQTPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTINSLQAEDVAVYFCQQYYDTPTFGQGTRL EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 18 (Fab heavy chain variant 1) QVQLVESGAEVKKPGATVKISCKVSGYTFTDYYMHWVQQAPGKGLEWMGLVDPEDGETIYAEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCATDARGSGSYYPNHFDY WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC SEQ ID NO: 19 (Fab light chain variant 1) DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTINSLQAEDVAVYFCQQYYDTPTFGQGTRL EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO:20 (N-terminally tagged human truncated Gremlin-1 used for crystal structure analysis) AMPGEEVLESSQEALHVTERKYLKRDWCKTQPLKQTIHEEGCNSRTIINRFCYGQCNSFYIPRHIRKEEGSFQSCSFCKPKKFTTMMVTLNCPELQPPTKKKRVTRVKQCRCISIDLD SEQ ID NO:21 (mature Gremlin-1 sequence of SEQ ID NO:1 lacking the signal peptide of amino acids 1 to 21) KKKGSQGAIPPPDKAQHNDSEQTQSPQQPGSRNRRGGQGRGTAMPGEEVLESSQEALHVTERKYLKRDWCKTQPLKQTIHEEGCNSRTIINRFCYGQCNSFYIPRHIRKEEGSFQSCSFCKPKKFTTMMVTLNCPELQPPTKKKRVTRVKQCRCISIDLD SEQ ID NO: 22 (Human IgG4P heavy chain variant 1) QVQLVESGAEVKKPGATVKISCKVSGYTFTDYYMHWVQQAPGKGLEWMGLVDPEDGETIYAEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCATDARGSGSYYPNHFD YWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESK YGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEK TISKAKGQPREPQVYTLPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK SEQ ID NO: 23 (human IgG4P light chain variant 1) DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTINSLQAEDVAVYFCQQYYDTPTFGQGTRL EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 24 (Human IgG1 heavy chain DNA variant 1) SEQ ID NO: 25 (Human IgG1 kappa chain DNA variant 1) gacattgtgatgacccagtcccccgattcgcttgcggtgtccctgggagaacgggccaccattaactgcaagagctcacagtccgtcctgtattcatcgaacaacaagaattacctcgcatggtatcagcagaagcctggacagcctcccaagctgctcatctactgggctagcacccgcgaatccggggtgccggatagattctccggatcgggttcgggcactgacttcactctgactatcaactcactgcaagccgaggatgtcgcggtgtacttctgtcagcagtactacgacaccccgacctttggacaaggcaccagactggagattaagcgtacggtggccgctccctccgtgttcatcttcccaccctccgacgagcagctgaagtccggcaccgcctccgtcgtgtgcctgctgaacaacttctacccccgcgaggccaaggtgcagtggaaggtggacaacgccctgcagtccggcaactcccaggaatccgtcaccgagcaggactccaaggacagcacctactccctgtcctccaccctgaccctgtccaaggccgactacgagaagcacaaggtgtacgcctgcgaagtgacccaccagggcctgtccagccccgtgaccaagtccttcaaccggggcgagtgc SEQ ID NO: 26 (Human IgG4 P heavy chain DNA variant 1) SEQ ID NO: 27 (Human IgG4 P kappa light chain DNA variant 1) gacattgtgatgacccagtcccccgattcgcttgcggtgtccctgggagaacgggccaccattaactgcaagagctcacagtccgtcctgtattcatcgaacaacaagaattacctcgcatggtatcagcagaagcctggacagcctcccaagctgctcatctactgggctagcacccgcgaatccggggtgccggatagattctccggatcgggttcgggcactgacttcactctgactatcaactcactgcaagccgaggatgtcgcggtgtacttctgtcagcagtactacgacaccccgacctttggacaaggcaccagactggagattaagcgtacggtggccgctccctccgtgttcatcttcccaccctccgacgagcagctgaagtccggcaccgcctccgtcgtgtgcctgctgaacaacttctacccccgcgaggccaaggtgcagtggaaggtggacaacgccctgcagtccggcaactcccaggaatccgtcaccgagcaggactccaaggacagcacctactccctgtcctccaccctgaccctgtccaaggccgactacgagaagcacaaggtgtacgcctgcgaagtgacccaccagggcctgtccagccccgtgaccaagtccttcaaccggggcgagtgc SEQ ID NO: 28 (Mouse full-length IgG1 heavy chain variant 2) QVQLVQSGAEVKKPGATVKISCKVSGYTFTDYYMHWVQQAPGKGLEWMGLVDPEDGETIYAEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCATDARGSGSYYPNHFD YWGQGTLVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPR DCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKT ISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK SEQ ID NO: 29 (Mouse full length IgG1 light chain variant 2) DIVMTQTPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTINSLQAEDVAVYFCQQYYDTPTFGQGTRL EIKRTDAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC SEQ ID NO: 30 (human full length IgG1 heavy chain variant 1) QVQLVESGAEVKKPGATVKISCKVSGYTFTDYYMHWVQQAPGKGLEWMGLVDPEDGETIYAEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCATDARGSGSYYPNHFDY WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 31 (human full length IgG1 light chain variant 1) DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTINSLQAEDVAVYFCQQYYDTPTFGQGTRL EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 32 (Fab heavy chain variant 2) QVQLVQSGAEVKKPGATVKISCKVSGYTFTDYYMHWVQQAPGKGLEWMGLVDPEDGETIYAEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCATDARGSGSYYPNHFDY WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC SEQ ID NO: 33 (Fab light chain variant 2) DIVMTQTPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTINSLQAEDVAVYFCQQYYDTPTFGQGTRL EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 34 (Human IgG4P heavy chain variant 2) QVQLVQSGAEVKKPGATVKISCKVSGYTFTDYYMHWVQQAPGKGLEWMGLVDPEDGETIYAEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCATDARGSGSYYPNHFD YWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESK YGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEK TISKAKGQPREPQVYTLPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK SEQ ID NO: 35 (human IgG4P light chain variant 2) DIVMTQTPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTINSLQAEDVAVYFCQQYYDTPTFGQGTRL EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO:36 (human gremlin-1; full length) Accession number 37 (human grumlin-1; coding sequence) atgagccgcacagcctacacggtgggagccctgcttctcctcttggggaccctgctgccggctgctgaagggaaaaagaaagggtcccaaggtgccatccccccgccagacaaggcccagcacaatgactcagagcagactcagtcgccccagcagcctggctccaggaaccgggggcggggccaagggcggggcactgccatgcccggggaggaggtgctggagtccagccaagaggccctgcatgtgacggagcgcaaatacctgaagcgagactggtgcaaaacccagccgcttaagcagaccatccacgaggaaggctgcaacagtcgcaccatcatcaaccgcttctgttacggccagtgcaactctttctacatccccaggcacatccggaaggaggaaggttcctttcagtcctgctccttctgcaagcccaagaaattcactaccatgatggtcacactcaactgccctgaactacagccacctaccaagaagaagagagtcacacgtgtgaagcagtgtcgttgcatatccatcgatttggattaa

Claims

1. An agent for treating or preventing cancer, comprising an anti-GREM1 antagonist, which is used in combination with a cytidine analog or a deoxycytidine analog.

2. (a) The cancer is a solid tumor; (b) The cancer has stromal GREM1 overexpression; (c) The cancer is metastatic cancer; (d) The cancer includes dormant cancer cells, and possibly dormant stem-like cancer cells; (e) the cancer is recurrent and / or the drug is intended to prevent the recurrence of the cancer; (f) The cancer is unresponsive, unresponsive, or refractory to treatment with cytidine analogs or deoxycytidine analogs; and, in some cases, the cancer is unresponsive, unresponsive, or refractory to treatment with gemcitabine or its derivatives; (g) Cancer is selected from colorectal cancer, multiple myeloma, pancreatic cancer, bladder cancer, breast cancer, lung cancer, stomach cancer, duodenal cancer, esophageal cancer, head and neck cancer, prostate cancer, glioma, endometrial cancer, ovarian cancer, liver cancer, splenic cancer, bone cancer, and osteosarcoma, and in some cases, (i) the cancer is pancreatic cancer; and possibly exocrine pancreatic cancer and / or ductal adenocarcinoma (PDAC); (ii) The cancer is lung cancer; in some cases the cancer is non-small cell lung cancer; (iii) The cancer is bladder cancer; (iv) The cancer is breast cancer; or (e) The cancer is ovarian cancer, The drug according to claim 1.

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

4. A cytidine analog or a deoxycytidine analog is (a) gemcitabine or its derivatives; (b) Azacitidine or its derivatives; (c) Cytarabine or its derivatives; (d) Decitabine or its derivatives; or (e) Troxacitabine or its derivatives The drug according to claim 1.

5. An agent for treating or preventing pancreatic cancer, comprising an anti-GREM1 antagonist, to be used in combination with a proliferation-dependent cytotoxic agent.

6. (a) The pancreatic cancer is exocrine pancreatic cancer; (b) The pancreatic cancer is pancreatic ductal adenocarcinoma (PDAC); (c) The pancreatic cancer is metastatic pancreatic cancer; (d) The pancreatic cancer is recurrent pancreatic cancer; and / or (e) The agent according to claim 5, wherein the pancreatic cancer is pancreatic cancer that is poorly responsive, unresponsive, or refractory to treatment with a proliferation-dependent cytotoxic agent.

7. A proliferation-dependent cytotoxic agent, (a) a nucleoside inhibitor or antimetabolite; (b) a cytidine analog or a deoxycytidine analog; (c) It is a mitotic inhibitor; in some cases (i) The mitotic inhibitor is a microtubule stabilizer; and / or (ii) A mitotic inhibitor and / or microtubule stabilizer is selected from abraxane and paclitaxel; (d) comprising one or more of oxaliplatin, folinic acid, irinotecan, and fluorouracil; optionally the growth-dependent cytotoxic agent being FOLFIRINOX or FOLFOX; or (e) (i) gemcitabine or a derivative thereof; (ii) Azacitidine or a derivative thereof; (iii) Cytarabine or a derivative thereof; (iv) Decitabine or a derivative thereof; (v) Troxacitabine or a derivative thereof; or (vi) Capecitabine The drug according to claim 5.

8. The agent according to claim 1, wherein the antagonist is a peptide, protein, antibody, polynucleotide, oligonucleotide, antisense RNA, small interfering RNA (siRNA), small inhibitor, or small hairpin RNA (shRNA).

9. (a) The antagonist is an antibody that binds to an epitope on gremlin-1 containing at least one residue selected from Ile131, Lys147, Lys148, Phe149, Thr150, Thr151, Arg169, Lys174 and Glun175, wherein the residue numbering follows Sequence ID No. 1; in some cases (i) The antibody binds to an epitope containing all of Ile131, Lys147, Lys148, Phe149, Thr150, Thr151, Arg169, Lys174 and Glun175; and / or (ii) Lys147, Lys148, Phe149, Thr150, Thr151, Arg169, Lys174 and Glun175 are located on the same gremlin-1 monomer, and Ile131 is located on a second gremlin-1 monomer; (b) The antagonist is an anti-gremlin-1 antibody comprising a heavy chain complementarity-determining region (HCDR) sequence contained within the heavy chain variable region (HCVR) of SEQ ID NO: 10 or 12, and / or a light chain complementarity-determining region (LCDR) sequence contained within the light chain variable region (LCVR) of SEQ ID NO: 11 or 13; 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 (i) The anti-gremlin-1 antibody contains the HCDR3 sequence of SEQ ID NO: 6; (ii) The anti-gremlin-1 antibody comprises a combination of HCDR1 / HCDR2 / HCDR3 sequences selected from SEQ ID NOs: 4 / 5 / 6 or SEQ ID NOs: 3 / 5 / 6, and / or a combination of LCDR1 / LCDR2 / LCDR3 sequences selected from SEQ ID NOs: 7 / 8 / 9; (iii) an anti-gremlin-1 antibody comprising the combination of HCDR1 / HCDR2 / HCDR3 / LCDR1 / LCDR2 / LCDR3 sequences of SEQ ID NO: 4 / 5 / 6 / 7 / 8 / 9 or SEQ ID NO: 3 / 5 / 6 / 7 / 8 / 9; and / or (iv) The agent according to claim 8, wherein the anti-gremlin-1 antibody comprises the heavy chain variable region (HCVR) sequence of SEQ ID NO: 10 or 12, and / or the light chain variable region (LCVR) sequence of SEQ ID NO: 11 or 13, or a sequence that is at least 95% identical thereto.

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

11. The agent according to claim 10(b), wherein the anti-gremlin-1 antibody comprises a heavy-chain and light-chain pair of SEQ ID NOs. 14 / 15, 16 / 17, 18 / 19, 22 / 23, 28 / 29 or 30 / 31, 32 / 33, 34 / 35, or a sequence that is at least 95% identical thereto; optionally, the HCDR1 / HCDR2 / HCDR3 / LCDR1 / LCDR2 / LCDR3 sequence of the antibody comprises SEQ ID NOs. 4 / 5 / 6 / 7 / 8 / 9 or SEQ ID NOs. 3 / 5 / 6 / 7 / 8 / 9, and the remainder of the heavy-chain and light-chain each comprises at least 95% identity with SEQ ID NOs. 14, 15, 16 and / or 17.

12. (a) The antagonist is an antibody that competes with the antibody defined in any one of claims 9(a)(i) to 10 for binding to gremlin-1; or (b) The agent according to claim 8, wherein the antagonist is an antibody that binds to an epitope on gremlin-1, the same antibody as defined in any one of claims 9(a)(i) to 10.

13. (a) The antagonist antibody is a chimeric antibody, a human antibody, or a humanized antibody; and / or (b) Antagonist antibodies are Fab, modified Fab, Fab', modified Fab', F(ab') ２ The agent according to claim 8, which is Fv, a single-domain antibody, or scFv.

14. The agent according to claim 8, wherein the antagonist is a polynucleotide encoding an antibody as defined in claim 9, or an expression vector having the polynucleotide.

15. The agent according to claim 8, wherein the antagonist antibody is contained in a pharmaceutical composition further comprising a pharmaceutically acceptable adjuvant and / or carrier.

16. The drug is used in combination with an additional anticancer agent; if applicable (a) The drug is used in combination with gemcitabine and further cytidine analogs or deoxycytidine analogs; and / or (b) The drug according to claim 1, wherein the drug is used in combination with gemcitabine and troxacitabine.

17. An agent for treating or preventing cancer, comprising a cytidine analog or a deoxycytidine analog, wherein the agent is used in combination with an anti-GREM1 antagonist, and optionally the cancer, the antagonist, the cytidine analog or deoxycytidine analog and / or the agent are as defined in any one of claims 1 and 8 to 16.

18. An agent for treating or preventing pancreatic cancer, comprising a proliferation-dependent cytotoxic agent, wherein the agent is used in combination with an anti-GREM1 antagonist, and optionally the proliferation-dependent cytotoxic agent, the pancreatic cancer, the antagonist and / or the agent are as defined in any one of claims 2 to 16.

19. A composition or kit comprising an anti-GREM1 antagonist and a cytidine analog or a deoxycytidine analog, wherein the cytidine analog or deoxycytidine analog is as defined in claim 4.

20. (a) A composition or kit comprising an anti-GREM1 antagonist and a mitotic inhibitor, wherein the mitotic inhibitor is as defined in any one of Claim 7(c)(i) or 7(c)(ii).

21. The composition or kit according to any one of claims 19 to 20, wherein the anti-GREM1 antagonist is as defined in any one of claims 8 to 14.

22. (a) A method for determining whether a patient who has cancer, is suspected of having cancer, or is at risk of developing cancer is likely to respond to combination therapy with a GREM1 antagonist and a cytidine analog or a deoxycytidine analog, the method comprising measuring the interstitial and / or epithelial expression of GREM1 in the patient and thereby predicting whether the patient is likely to respond to combination therapy; or (b) A method for determining whether a patient who has or is suspected of having pancreatic cancer, or who is at risk of developing pancreatic cancer, is likely to respond to combination therapy with a GREM1 antagonist and a proliferation-dependent cytotoxic agent, the method comprising measuring the stromal and / or epithelial expression of GREM1 in the patient, thereby predicting whether the patient is likely to respond to combination therapy.

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