Combination of Gremlin-1 antagonists with cytidine analogs or deoxycytidine analogs

Abstract

The present invention relates to an anti-GREM1 antagonist for use in a method of treating or preventing cancer in combination with a proliferation-dependent cytotoxic agent.

Description

Field of the Invention

[0001] The present invention relates to combination therapies for treating or preventing cancer. Specifically, the present invention relates to anti-GREM1 antagonists for use in combination with cytidine analogs or deoxycytidine analogs (e.g., gemcitabine) for treating or preventing cancer, and related compositions and kits. The present invention also relates to anti-GREM1 antagonists for use in combination with proliferation-dependent cytotoxic agents for treating or preventing pancreatic cancer, and related compositions and kits. The present invention also relates to methods for predicting whether a patient is likely to respond to combination therapy based on stromal GREM1 overexpression. Background of the Invention

[0003] Despite advances in cancer immunotherapy, gene therapy, data science, and precision medicine technologies, cancer remains a significant and complex global challenge. Setbacks associated with drug resistance and poor responses to existing therapies highlight the need to develop improved therapies for the treatment and prevention of cancer. Combination therapies that can enhance the effectiveness of existing treatments are also needed.

[0004] One such cancer that presents a significant challenge is pancreatic cancer. Pancreatic cancer is a deadly disease with an increasing incidence in recent years. Environmental risk factors associated with pancreatic cancer include smoking, pancreatitis, alcohol, obesity, infection and diet, which means that pancreatic cancer is becoming increasingly common in developed countries. Surgical resection (pancreaticoduodenectomy) combined with chemotherapy is the only long-term potential cure for pancreatic cancer. However, limited screening coupled with a lack of symptoms means that patients are often diagnosed with late, advanced disease, limiting the opportunity for surgical resection. As a result, pancreatic cancer has one of the lowest survival rates of all common cancers, with a five-year survival rate of less than 7% and a survival rate of only 5% for 10 years or more. Limited improvements in survival over the past 40 years have highlighted the urgent need for new treatments for pancreatic cancer. For unresectable tumors, the standard treatment for pancreatic cancer is a chemotherapy regimen. However, chemotherapy has side effects and cancer recurrence is common. Late diagnosis coupled with poor prognosis and limited pancreatic cancer treatment strategies warrants improved therapies. More broadly, combination therapies are urgently needed to improve responsiveness to and / or reduce side effects of chemotherapeutic drugs used to treat cancer. SUMMARY OF THE INVENTION

[0006] The inventors surprisingly showed that GREM1 antagonists can be advantageously administered in combination with chemotherapeutic agents to treat or prevent pancreatic cancer. Using the LSL-KrasG12D / +;LSL-Trp53R172H / +;Pdx1-Cre(KPC) pancreatic cancer mouse model, the inventors have demonstrated that a combination therapy comprising an anti-GREM1 antagonist and a chemotherapeutic agent can significantly improve the survival of these mice compared to vehicle controls. Without being bound by theory, the inventors hypothesize that stromal targeting using anti-GREM1 antagonists can enhance the efficacy of pancreatic cancer chemotherapy treatment because the stroma itself can confer chemoresistance to tumor cells. Based on these results, the inventors also envision that combination therapies comprising GREM1 antagonists and proliferation-dependent cytotoxic agents (e.g., gemcitabine and other cytidine analogs or deoxycytidine analogs) will have general utility in the treatment and prevention of cancer. Specifically, the inventors envision that combination therapies comprising a GREM1 antagonist and a proliferation-dependent cytotoxic agent will have general utility in treating and preventing cancers characterized by the presence of dormant stem-like cancer cells. The inventors' discovery provides a new approach to preventing and treating cancer, particularly pancreatic cancer.

[0007] Therefore, in a first aspect of the present invention, there is provided an anti-GREM1 antagonist for use in a method for treating or preventing cancer, wherein the method further comprises administering a cytidine analogue or a deoxycytidine analogue.

[0008] In another aspect of the present invention, an anti-GREM1 antagonist for use in a method of treating or preventing pancreatic cancer is provided, wherein the method further comprises administering a proliferation-dependent cytotoxic agent.

[0009] In another aspect of the present invention, a cytidine analog or a deoxycytidine analog is provided for use in a method for treating or preventing cancer, wherein the method further comprises administering an anti-GREM1 antagonist.

[0010] In another aspect of the present invention, a method of treating cancer is provided, comprising administering to a subject in need thereof a therapeutically effective amount of an anti-GREM1 antagonist in combination with a therapeutically effective amount of a cytidine analog or a deoxycytidine analog.

[0011] In another aspect of the present invention, a proliferation-dependent cytotoxic agent for use in a method for treating or preventing pancreatic cancer is provided, wherein the method further comprises administering an anti-GREM1 antagonist.

[0012] In another aspect of the invention, there is provided 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.

[0013] Another aspect of the present invention provides a composition or kit comprising an anti-GREM1 antagonist and a cytidine analog or a deoxycytidine analog.

[0014] Another aspect of the present invention provides a composition or kit comprising:

[0015] Resistance to GREM1 antagonists and mitotic inhibitors.

[0016] Another aspect of the invention provides a method for determining whether a patient having or suspected of having cancer or being at risk of developing cancer is likely to respond to a combination treatment with a GREM1 antagonist and a cytidine analog or a deoxycytidine analog, 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 the combination treatment.

[0017] Another aspect of the invention provides a method for determining whether a patient having or suspected of having pancreatic cancer or at risk of developing pancreatic cancer is likely to respond to 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 -Kaplan-Meier analysis showing survival of PDAC patients with gremlin-1 expression above or below the median as indicated. Patients with tumors expressing high levels of gremlin 1 had a significantly worse prognosis compared to patients with low expression levels. Generated by KMplotter.

[0020] Figure 2 - Schematic diagram showing (a) breeding strategy and (b) experimental design.

[0021] Figure 3 - Graphs show tumor burden (measured by high-resolution ultrasound) in individual mice treated as indicated.

[0022] Figure 4 -Kaplan-Meier analysis showed that Pdx1-Cre;LSL- KrasG12D / + ; Survival of LSL-Trp53R172H / + (KPC) mice treated with Ab7326 mIgG1 (n=7), vehicle (n=7), gemcitabine (n=5), or Ab7326 mIgG1 in combination with gemcitabine (n=6) from the beginning. Mice treated with Ab7326 mIgG1 in combination with gemcitabine showed significantly improved survival compared to vehicle controls (log rank, p=0.04).

[0023] Brief description of the form

[0024] Table 1 - Table showing the relationship with Kras G12D+ Grem1 is upregulated in KPC tumors and in all pancreatic mouse models combined compared to mouse pancreatic ductal cells

[0025] Table 2 - Table showing given treatment doses and schedules, number of mice per treatment (including censored cases), and median survival for each treatment group. Mice treated with the combination of Ab7326 mIgG1 and gemcitabine showed significantly increased median survival compared to vehicle controls

[0026] Table 3 - Table showing survival data and censoring for individual mice treated. * indicates smaller than average tumor at start of treatment. DETAILED DESCRIPTION OF THE INVENTION

[0028] It should be understood that different applications of the disclosed products and methods can be customized according to specific needs in the art.It should also be understood that the terminology used herein is only used to describe specific embodiments of the present invention and is not intended to be limiting.

[0029] Furthermore, 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, etc.

[0030] All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.

[0031] Combination therapy with an anti-GREM1 antagonist and a cytidine analog or deoxycytidine analog

[0032] The present invention provides an anti-GREM1 antagonist in combination with a cytidine analog or a deoxycytidine analog (e.g., gemcitabine) for use in a method of treating or preventing cancer. The cancer is typically a cancer with overexpression of GREM1 and / or a cancer that can be targeted with a cytidine analog or a deoxycytidine analog (e.g., gemcitabine or a derivative thereof). The cancer may additionally or alternatively be characterized as comprising dormant cancer cells (e.g., dormant stem cell-like cancer cells) and / or as a recurrent cancer.

[0033] The present invention also provides a cytidine analog or a deoxycytidine analog, such as gemcitabine or a derivative thereof, for use in a method for treating or preventing cancer, wherein the method comprises administering an anti-GREM1 antagonist alone, sequentially or simultaneously.

[0034] Cytidine analogs or deoxycytidine analogs

[0035] The present invention provides a combination therapy for the treatment or prevention of cancer using an anti-GREM1 antagonist together with a cytidine analog or a deoxycytidine analog. Cytidine analogs and deoxycytidine analogs are specific nucleoside inhibitors or antimetabolites that exert cytotoxic activity by mimicking endogenous nucleosides and interfering with the synthesis of nucleic acids. Nucleoside inhibitors or antimetabolites include analogs of physiological pyrimidine and purine nucleobases and nucleosides. Cytidine analogs and deoxycytidine analogs particularly mimic endogenous cytidine or deoxycytidine. Such compounds may also interfere with DNA methylation or alter the metabolism of physiological nucleosides. The phrase "analog" refers to a compound having structural or functional similarity to a target / compound of interest. For example, a cytidine analog or a deoxycytidine analog may have a structure similar to cytidine or deoxycytidine and / or have chemical and biological properties similar to cytidine or deoxycytidine.

[0036] The present invention contemplates the use of any cytidine analog or deoxycytidine analog in the context of treating any cancer. Exemplary cytidine analogs 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)oxacyclopentan-2-yl]pyrimidin-2-one) or derivatives thereof; azacitidine (5-azacytidine; 4-amino-1-β-D-ribofuranosyl-1,3,5-triazine-2(1H)-one) or derivatives thereof; cytosine arabinoside (Ara-C / cytosine 1-[β]-D-arabinofuranoside; 4-amino-1-[(2R,3S,4S,5R)-3 ,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]pyrimidin-2-one) or its derivatives; decitabine (5-aza-2'-deoxycytidine / 5-azadeoxycytidine; 4-amino-1-(2-deoxy-β-D-erythro-pentofuranosyl)-1,3,5-triazine-2(1H)-one; 4-amino-1-[(2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)oxolan-2-yl]-1,3,5-triazine-2-one, 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.

[0037] In some embodiments, the combination therapy provides an anti-GREM1 antagonist and one or more cytidine analogs or deoxycytidine analogs. In a preferred embodiment, the present invention provides an anti-GREM1 antagonist for use in a method of treating or preventing cancer, wherein the method further comprises administering gemcitabine. In an exemplary embodiment, the present invention provides an anti-GREM1 antagonist for use in a method of treating or preventing cancer, wherein the method further comprises administering gemcitabine in combination with troxacitabine. In a particularly preferred embodiment, the present invention provides an anti-GREM1 antagonist for use in a method of treating or preventing pancreatic cancer, wherein the method further comprises administering gemcitabine in combination with troxacitabine.

[0038] It is expected that a combination therapy comprising a GREM1 antagonist as defined herein and a cytidine analog or deoxycytidine analog as described herein can be used to treat or prevent a cancer that is normally targeted with the cytidine analog or deoxycytidine analog. The cancer may be a cancer or tumor previously described for treatment with a 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.

[0039] The cancer may be a cancer that responds to treatment with a cytidine analog or a deoxycytidine analog, and / or a cancer that is considered for treatment with a cytidine analog or a deoxycytidine analog. Alternatively, the cancer may be a cancer that responds poorly, does not respond, or is refractory to treatment with a cytidine analog or a deoxycytidine analog. The cancer may have been previously described as being unsuitable for treatment with a cytidine analog or a deoxycytidine analog. In some cases, the cancer may initially respond to treatment with a cytidine analog or a deoxycytidine analog, but develops resistance to treatment with a cytidine analog or a deoxycytidine analog. Responsiveness may be measured in any manner. For example, responsiveness to treatment may be assessed by measuring tumor size before and after treatment using an X-ray, CT, or MRI scan, or by measuring tumor markers. Blood tests that determine organ function may also be used to assess responsiveness to treatment. An exemplary blood marker for monitoring pancreatic cancer responsiveness is CA19-9. Those skilled in the art know how to measure the responsiveness of cancer to treatment.

[0040] The cancer may additionally or alternatively be characterized as comprising dormant cancer cells, such as dormant stem-like cancer cells and / or as being a recurrent cancer.

[0041] In a particularly preferred embodiment, an anti-GREMl antagonist is administered in combination with a cytidine analog or a deoxycytidine analog to treat pancreatic cancer.An exemplary pancreatic cancer is an exocrine pancreatic cancer, such as pancreatic ductal adenocarcinoma.

[0042] Gemcitabine

[0043] Gemcitabine (2'-deoxy-2',2'-difluorocytidine / 2',2'-difluoro-2'deoxycytidine) is a chemotherapeutic agent that is commonly used to treat pancreatic cancer, breast cancer, ovarian cancer, non-small cell lung cancer, bladder cancer, and other solid cancers. Gemcitabine is an antimetabolite. As described above and in more detail below, antimetabolites exert cytotoxic activity by mimicking endogenous nucleosides and interfering with the synthesis of nucleic acids. Gemcitabine is a pyrimidine nucleotide analog of cytidine. It is able to enter cells through 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 the extending DNA chain, resulting in masked chain termination, while gemcitabine diphosphate inhibits ribonucleotide reductase, resulting in reduced dCTP availability.

[0044] In the context of the present invention, a combination therapy comprising gemcitabine or a derivative thereof is considered. The phrase "derivative thereof" refers to a compound that exerts a similar therapeutic effect and is derived from a similar / precursor compound. Gemcitabine derivatives may include, for example, a stereoisomer of gemcitabine, or an ester or amide of gemcitabine.

[0045] The inventors surprisingly showed that GREM1 antagonists can be advantageously administered in combination with gemcitabine to treat or prevent pancreatic cancer in the KPC mouse model. Without being bound by theory, the inventors hypothesize that anti-GREM1 antagonists can cause dormant stem-like cancer cells to enter a more proliferative state, making them more susceptible to treatment with proliferation-dependent cytotoxic agents (e.g., gemcitabine or other cytidine or deoxycytidine analogs). The inventors also hypothesize that matrix targeting using anti-GREM1 antagonists can enhance the efficacy of pancreatic cancer chemotherapy treatment because the matrix itself can confer chemoresistance to tumor cells. Therefore, it is conceivable that combination therapies comprising GREM1 antagonists and gemcitabine or its derivatives can be used to treat or prevent a variety of cancers. It is also conceivable that combination therapies comprising GREM1 antagonists and other cytidine analogs or deoxycytidine analogs (such as those described herein) can be used to treat or prevent a variety of cancers.

[0046] In particular, it is contemplated that a combination therapy comprising a GREM1 antagonist as defined herein and gemcitabine or a derivative thereof can be used to treat or prevent cancers that are typically targeted with gemcitabine or a derivative thereof. The cancer may be a cancer or tumor previously described for treatment with gemcitabine or a derivative thereof. Such cancers include, but are not limited to, pancreatic cancer, breast cancer, ovarian cancer, non-small cell lung cancer, and bladder cancer.

[0047] Cancer can be a cancer that responds to treatment with gemcitabine or its derivatives, and / or a cancer that is considered to be treated with gemcitabine or its derivatives. Alternatively, cancer can be a cancer that is poorly responsive, unresponsive or refractory to treatment with gemcitabine or its derivatives. Cancer may have been previously described as being unsuitable for treatment with gemcitabine or its derivatives. In some cases, cancer may initially respond to treatment with gemcitabine or its derivatives, but develops resistance to gemcitabine treatment. Reactivity can be measured in any manner. For example, the responsiveness to treatment can be assessed by measuring the size of the tumor before and after treatment using X-rays, CT or MRI scans or by measuring tumor markers. Reactivity to treatment can also be assessed using blood tests that determine organ function. An exemplary blood marker for monitoring pancreatic cancer responsiveness is CA19-9. Those skilled in the art know how to measure the responsiveness of cancer to treatment.

[0048] The cancer may additionally or alternatively be characterized as comprising dormant cancer cells, such as dormant stem-like cancer cells, and / or as being a recurrent cancer.

[0049] In a particularly preferred embodiment, an anti-GREMl antagonist is administered in combination with gemcitabine or a derivative thereof to treat pancreatic cancer.An exemplary pancreatic cancer is exocrine pancreatic cancer, such as pancreatic ductal adenocarcinoma.

[0050] cancer

[0051] In the context of a combination therapy comprising an anti-GREM1 antagonist and a cytidine analog or a deoxycytidine analog (e.g., gemcitabine or a derivative thereof), the cancer can be any cancer or tumor. Specifically, in the context of a combination therapy comprising an anti-GREM1 antagonist and a cytidine analog or a deoxycytidine analog (e.g., gemcitabine or a derivative thereof), the cancer can be any cancer or tumor having a stroma (typically a desmoplastic stroma). The cancer can be any cancer or tumor initiated by GREM1. The cancer can be any cancer in which stromal and / or epithelial GREM1 overexpression is observed. The cancer or tumor can have stromal GREM1 overexpression, but no epithelial GREM1 overexpression. The cancer or tumor can have epithelial GREM1 overexpression, but no stromal GREM1 overexpression. In a preferred embodiment, the cancer or tumor has overexpression of GREM1 in the desmoplastic stroma. The cancer or tumor can be any cancer or tumor suitable for targeting with a GREM1 antagonist and / or a cytidine analog or a deoxycytidine analog, including any cancer or tumor known in the art to be suitable for targeting with such agents, and any cancer or tumor known in the art to be suitable for treatment with such agents. For example, the cancer or tumor can be any cancer or tumor suitable for targeting with gemcitabine (2'-deoxy-2', 2'-difluorocytidine) or its derivatives. The cancer or tumor can be any cancer or tumor suitable for targeting with azacitidine (5-azacytidine) or its derivatives. The cancer or tumor can be any cancer or tumor suitable for targeting with cytarabine (Ara-C / cytosine 1-[β]-D-arabinofuranoside) or its derivatives. The cancer or tumor can be any cancer or tumor suitable for targeting with decitabine (5-aza-2'-deoxycytidine / 5-azadeoxycytidine) or its derivatives. The cancer or tumor can 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 can be a solid tumor. A solid tumor can have a desmoplastic stroma.

[0052] Anti-GREM1 antagonists have previously been shown to be effective in treating a range of cancers in WO 2019 / 243801, filed on June 18, 2019, the disclosure of which is incorporated herein by reference in its entirety. Particularly preferred cancers that can be treated include colorectal cancer, multiple myeloma, pancreatic cancer, bladder cancer, breast cancer, lung cancer, gastric cancer, duodenal cancer, esophageal cancer, head and neck cancer, prostate cancer, glioma, endometrial cancer, ovarian cancer, liver cancer, spleen cancer, bone resident cancer and osteosarcoma. The treatable cancer can be intestinal cancer, colon cancer or rectal cancer. The cancer to be treated can be a disseminated cancer, such as a metastatic cancer. Disseminated cancer is understood to be a cancer that has spread from the original site of origin in the body. For example, a disseminated cancer can be a cancer that originates in the patient's bone marrow, colon, prostate or breast tissue but has spread to, for example, the patient's liver or lungs.

[0053] Combination therapies comprising a GREM1 antagonist and a cytidine analog or a deoxycytidine analog (e.g., gemcitabine or a derivative thereof) can also be used to prevent the spread of cancer. For example, a combination therapy comprising a GREM1 antagonist and gemcitabine or a derivative thereof can be used to prevent the spread of cancer. A combination therapy comprising a GREM1 antagonist and azacitidine or a derivative thereof can be used to prevent the spread of cancer. A combination therapy comprising a GREM1 antagonist and cytarabine or a derivative thereof can be used to prevent the spread of cancer. A combination therapy comprising a GREM1 antagonist and decitabine or a derivative thereof can be used to prevent the spread of cancer. A combination therapy comprising a GREM1 antagonist and troxacitabine or a derivative thereof can be used to prevent the spread of cancer.

[0054] Combination therapies comprising a GREM1 antagonist and a cytidine analog or a deoxycytidine analog (e.g., gemcitabine or a derivative thereof) can be used to prevent polyposis associated with cancer. For example, a combination therapy comprising a GREM1 antagonist and gemcitabine or a derivative thereof can be used to prevent polyposis associated with cancer. Combination therapies comprising a GREM1 antagonist and azacitidine or a derivative thereof can be used to prevent polyposis associated with cancer. Combination therapies comprising a GREM1 antagonist and cytarabine or a derivative thereof can be used to prevent polyposis associated with cancer. Combination therapies comprising a GREM1 antagonist and decitabine or a derivative thereof can be used to prevent polyposis associated with cancer. Combination therapies comprising a GREM1 antagonist and troxacitabine or a derivative thereof can be used to prevent polyposis associated with cancer.

[0055] Grading systems are used in cancer biology and medicine to classify cancer cells according to the lack of cell differentiation of cancer cells. This reflects the degree of difference between cancer cells and healthy cells in the tissue from which the cancer cells originate. Grading systems can be used to provide an indication of how fast a particular cancer may be expected to grow. The cancer grades commonly used are grades (G) X and 1 to 4. GX means that the cancer grade cannot be assessed. G1 (low-grade) cancer cells have a morphology similar to normal healthy cells (i.e., they are well differentiated), are expected to grow slowly, and are less likely to spread. G2 (intermediate) 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 than normal cells (i.e., they are poorly differentiated), and are expected to grow faster than G1 and G2 cells. G4 (high-grade) cancer cells are undifferentiated (also known as anaplastic) and are expected to have the highest proliferation capacity.

[0056] Cancer grading is different from cancer staging, which gives an indication of how the cancer is likely to spread. A common cancer staging system has five stages, namely Stage 0: cancer cells in situ (i.e., located in their normal tissue); Stage I: cancer is confined to one part of the body; Stage II: cancer is locally advanced; Stage III: cancer is also locally advanced (whether the cancer is designated as Stage II or Stage III can depend on the specific type of cancer); and Stage IV: cancer has usually metastasized, or spread, to other organs or throughout the body.

[0057] Those skilled in the art know how to determine the grade and / or stage of a cancer. In one embodiment, the present invention relates to the use of anti-GREM1 antagonists to treat and / or prevent established cancers. In one embodiment, the cancer is an established cancer. The established cancer can be a high-grade cancer, such as a G3 or G4 cancer. The established cancer can be a stage II or above cancer. The established cancer can be a stage III or stage IV cancer. In one embodiment, the established cancer is a stage IV cancer that has metastasized. In one embodiment, the established cancer is an established pancreatic cancer.

[0058] In addition to the specifically exemplified applications 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 analogs or deoxycytidine analogs as shown in the Examples will also be applicable to the treatment of other cancers having the corresponding characteristics described herein.

[0059] In particular, it is envisioned that combinations comprising GREM1 antagonists will be useful in preventing or treating cancers in which there is stromal and / or epithelial GREM1 overexpression, and such overexpression contributes to the growth of malignant cells. Such cancers include pancreatic cancer, bladder cancer, lung cancer, gastric cancer, duodenal cancer, esophageal cancer, head and neck cancer, glioma, endometrial cancer, liver cancer, spleen cancer, bone resident cancer and osteosarcoma.

[0060] Antagonists of the invention are used in combination with cytidine analogs or deoxycytidine analogs (e.g., gemcitabine or its derivatives) for the treatment or prevention of cancer. Other cytidine analogs or deoxycytidine analogs considered for use in the invention are described herein, including azacitidine or its derivatives; cytarabine or its derivatives; decitabine or its derivatives; or troxacitabine or its derivatives. Antagonists of the invention are also used in combination with proliferation-dependent cytotoxic agents for the treatment or prevention of pancreatic cancer.

[0061] Preventing cancer may include preventing a subject from being diagnosed with cancer or delaying the onset of cancer. Preventing cancer may also include preventing the recurrence or recurrence of cancer in a subject previously diagnosed with cancer. Preventing cancer may also include increasing the survival rate of a subject not diagnosed with cancer or previously diagnosed with cancer.

[0062] Cancer treatment can improve one or more symptoms of cancer, induce or prolong the remission period of cancer, or delay the recurrence or recurrence of cancer. Treatment can cure, alleviate or partially inhibit cancer. Treatment can lead to a decrease in the severity of disease symptoms, or an increase in the frequency or duration of asymptomatic periods. Cancer treatment can also include preventing cancer (e.g., established cancer) from spreading from an initial site in the patient's body to one or more secondary sites in the patient's body. Therefore, cancer treatment can include preventing the spread or metastasis of existing cancer. Pancreatic cancer treatment can lead to a decrease in primary tumor size, such as measured by CT or endoscopic ultrasound. This reduction can promote Whipple surgery (pancreaticoduodenectomy) to remove tumors from the head of the pancreas. The inventors have demonstrated that anti-GREM1 antagonists combined with gemcitabine can improve the survival rate of KPC mouse models. Therefore, treating pancreatic cancer with gemcitabine or another cytidine analog or deoxycytidine analog according to the present invention can also improve the survival rate of patients.

[0063] Pancreatic cancer

[0064] 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 demonstrated overexpression of GREM1 in human pancreatic ductal adenocarcinoma samples and demonstrated that high expression of Grem1 mRNA is significantly associated with poor prognosis ( Figure 1 ). Furthermore, the present inventors have demonstrated that combination therapy comprising an anti-GREM1 antagonist and gemcitabine increases survival in a KPC mouse model.

[0065] Therefore, in one embodiment, pancreatic cancer is characterized as pancreatic cancer with GREM1 overexpression. In another embodiment, pancreatic cancer may be characterized as having an exocrine tumor or a neuroendocrine tumor. Pancreatic neuroendocrine cancer (also known as islet cell tumor) develops in the pancreatic endocrine glands. A particularly preferred form of pancreatic cancer is exocrine pancreatic cancer, such as pancreatic ductal adenocarcinoma, which develops in the inner wall of the pancreatic duct and accounts for 90% of all pancreatic cancers (Feldmann et al., J Hepatobiliary Pancreat Surg. 2007; 14 (3): 224-32). Other exocrine pancreatic cancers include squamous cell carcinoma (which forms in the pancreatic duct); adenosquamous carcinoma; signet ring cell carcinoma; and colloid carcinoma (which usually develops from intraductal papillary mucinous tumors). The late diagnosis of pancreatic cancer combined with the poor prognosis and limited treatment strategies requires improved treatment methods.

[0066] Preferred pancreatic cancer types for treatment may be resistant to one or more known anticancer agents (e.g., chemotherapeutic agents). Pancreatic cancer may be disseminated pancreatic cancer. Pancreatic cancer may be metastatic pancreatic cancer. Metastatic cancer is understood to be a cancer that has spread from the original origin site in the body. Therefore, metastatic pancreatic cancer refers to a cancer that begins in the pancreas and spreads to other organs (e.g., lungs, liver, bones, and brain). Pancreatic cancer may also be recurrent pancreatic cancer. In other words, pancreatic cancer treated by the methods of the present invention includes pancreatic cancer that recurs months or even years after early treatment (e.g., chemotherapy, radiotherapy, or curative surgery). Preferred pancreatic cancer types for treatment may be resistant to one or more known anticancer agents (e.g., chemotherapeutic agents), as further described below.

[0067] In some aspects, pancreatic cancer can be a pancreatic cancer that responds to treatment using cytidine analogs or deoxycytidine analogs (e.g., gemcitabine or its derivatives). Pancreatic cancer can also be a pancreatic cancer that responds poorly, unresponsively, or refractory to treatment using cytidine analogs or deoxycytidine analogs (e.g., gemcitabine or its derivatives). Pancreatic cancer may have previously been considered unsuitable for treatment with cytidine analogs or deoxycytidine analogs (e.g., gemcitabine or its derivatives). In some cases, pancreatic cancer may initially respond to treatment using cytidine analogs or deoxycytidine analogs (e.g., gemcitabine or its derivatives), but develops resistance to treatment using cytidine analogs or deoxycytidine analogs, such as gemcitabine treatment.

[0068] For example, in some aspects, pancreatic cancer can be a pancreatic cancer that is responsive to the treatment of gemcitabine, azacitidine, cytarabine, decitabine, troxacitabine or any of its derivatives. Pancreatic cancer can also be a pancreatic cancer that is poorly responsive, unresponsive or refractory to the treatment of gemcitabine, azacitidine, cytarabine, decitabine, troxacitabine or any of its derivatives. Pancreatic cancer may have previously been considered unsuitable for treatment with gemcitabine, azacitidine, cytarabine, decitabine, troxacitabine or any of its derivatives. In some cases, pancreatic cancer may initially be responsive to the treatment of gemcitabine, azacitidine, cytarabine, decitabine, troxacitabine or any of its derivatives, but resistance is produced to the treatment of gemcitabine, azacitidine, cytarabine, decitabine, troxacitabine or any of its derivatives.

[0069] Lung cancer

[0070] On the other hand, the present invention relates to the treatment or prevention of lung cancer. Lung cancer is most preferably non-small cell lung cancer (NSCLC). NSCLC may be advanced NSCLC, such as stage III or stage IV NSCLC. NSCLC may be squamous cell carcinoma, adenocarcinoma or large cell carcinoma. Cancer may be small cell lung cancer. Lung cancer may be unresectable. Lung cancer may be primary lung cancer or any secondary cancer that has spread to the lungs, such as breast cancer or pancreatic cancer. Lung cancer may be disseminated lung cancer. Lung cancer may be metastatic lung cancer. Lung cancer may be characterized as lung cancer with overexpression of GREM1. Lung cancer may be recurrent lung cancer. In other words, lung cancer treated by the method of the present invention includes lung cancer that recurs months or even years after early treatment (such as chemotherapy, radiotherapy or curative surgery). Preferred lung cancer types for treatment may be resistant to one or more known anticancer agents (such as chemotherapeutic agents), as further described below.

[0071] In some aspects, lung cancer (e.g., NSCLC) may be a lung cancer that responds to treatment with a cytidine analog or a deoxycytidine analog (e.g., gemcitabine or its derivatives). Lung cancer (e.g., NSCLC) may also be a lung cancer that responds poorly, unresponsively, or refractory to treatment with a cytidine analog or a deoxycytidine analog (e.g., gemcitabine or its derivatives). Lung cancer (e.g., NSCLC) may have been previously considered unsuitable for treatment with a cytidine analog or a deoxycytidine analog (e.g., gemcitabine or its derivatives). In some cases, lung cancer (e.g., NSCLC) may initially respond to treatment with a cytidine analog or a deoxycytidine analog (e.g., gemcitabine or its derivatives), but develops resistance to treatment with a cytidine analog or a deoxycytidine analog, such as gemcitabine treatment.

[0072] For example, in some aspects, lung cancer can be a lung cancer that responds to treatment with gemcitabine, azacitidine, cytarabine, decitabine, troxacitabine, or any of its derivatives. Lung cancer can also be a lung cancer that responds poorly, unresponsively, or refractory to treatment with gemcitabine, azacitidine, cytarabine, decitabine, troxacitabine, or any of its derivatives. Lung cancer may have previously been considered unsuitable for treatment with gemcitabine, azacitidine, cytarabine, decitabine, troxacitabine, or any of its derivatives. In some cases, lung cancer may initially respond to treatment with gemcitabine, azacitidine, cytarabine, decitabine, troxacitabine, or any of its derivatives, but develops resistance to treatment with gemcitabine, azacitidine, cytarabine, decitabine, troxacitabine, or any of its derivatives.

[0073] Bladder cancer

[0074] In another aspect, the present invention relates to the treatment or prevention of bladder cancer. The bladder cancer may be transitional cell (urothelial) bladder cancer. The bladder cancer may originate from the lining of the bladder epithelium. The bladder cancer may be non-muscle invasive bladder cancer. The bladder cancer may be squamous cell bladder cancer. The bladder cancer may be an adenocarcinoma. The bladder cancer may be a high-grade T1 tumor that grows from the lining of the bladder to the lamina propria. The bladder cancer may be a superficial cancer or an invasive bladder cancer. The bladder cancer may be a recurrent bladder cancer. The term recurrent bladder cancer as used herein refers to bladder cancer that has recurred after treatment (e.g., surgical treatment).

[0075] The present invention also provides for treating and preventing 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 overexpression of GREM1. The bladder cancer may also be recurrent bladder cancer. In other words, the bladder cancer treated by the methods of the present invention includes bladder cancer that recurs months or even years after early treatment (e.g., chemotherapy, radiotherapy, or curative surgery). The preferred bladder cancer types for treatment may be resistant to one or more known anticancer agents (e.g., chemotherapeutic agents), as further described below.

[0076] In some aspects, bladder cancer can be a bladder cancer that responds to treatment with cytidine analogs or deoxycytidine analogs (e.g., gemcitabine or its derivatives). Bladder cancer can also be a bladder cancer that responds poorly, unresponsive or refractory to treatment with cytidine analogs or deoxycytidine analogs (e.g., gemcitabine or its derivatives). Bladder cancer may have previously been considered unsuitable for treatment with cytidine analogs or deoxycytidine analogs (e.g., gemcitabine or its derivatives). In some cases, bladder cancer may initially respond to treatment with cytidine analogs or deoxycytidine analogs (e.g., gemcitabine or its derivatives), but develops resistance to treatment with cytidine analogs or deoxycytidine analogs, such as gemcitabine treatment.

[0077] For example, in some aspects, bladder cancer can be a bladder cancer that responds to treatment with gemcitabine, azacitidine, cytarabine, decitabine, troxacitabine, or any of its derivatives. Bladder cancer can also be a bladder cancer that responds poorly, unresponsively, or refractory to treatment with gemcitabine, azacitidine, cytarabine, decitabine, troxacitabine, or any of its derivatives. Bladder cancer may have been previously considered unsuitable for treatment with gemcitabine, azacitidine, cytarabine, decitabine, troxacitabine, or any of its derivatives. In some cases, bladder cancer may initially respond to treatment with gemcitabine, azacitidine, cytarabine, decitabine, troxacitabine, or any of its derivatives, but develops resistance to treatment with gemcitabine, azacitidine, cytarabine, decitabine, troxacitabine, or any of its derivatives.

[0078] Ovarian cancer

[0079] In another aspect, the invention relates to the treatment or prevention of ovarian cancer. The ovarian cancer may be epithelial ovarian cancer, germ cell ovarian cancer, or germ cord-stromal ovarian cancer. The ovarian cancer may be primary peritoneal cancer. The ovarian cancer may be fallopian tube cancer. The ovarian cancer may be characterized as a borderline ovarian tumor. The ovarian cancer may be characterized as a germ cell ovarian tumor. The ovarian cancer may be a clear cell ovarian cancer. The cancer may be a serous ovarian cancer. The ovarian cancer may be a mucinous ovarian cancer. The ovarian cancer may be an endometrioid cancer.

[0080] The ovarian cancer may be an ovarian cancer characterized by overexpression of GREM1. The ovarian cancer may also be a recurrent ovarian cancer. In other words, the ovarian cancer treated by the methods of the present invention includes ovarian cancer that recurs months or even years after early treatment (e.g., chemotherapy, radiotherapy, or curative surgery). Preferred ovarian cancer types for treatment may be resistant to one or more known anticancer agents (e.g., chemotherapeutic agents), as further described below.

[0081] The present invention also provides for treating and preventing 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.

[0082] In some aspects, ovarian cancer can be an ovarian cancer that responds to treatment with cytidine analogs or deoxycytidine analogs (e.g., gemcitabine or its derivatives). Ovarian cancer can also be an ovarian cancer that responds poorly, unresponsive or refractory to treatment with cytidine analogs or deoxycytidine analogs (e.g., gemcitabine or its derivatives). Ovarian cancer may have previously been considered unsuitable for treatment with cytidine analogs or deoxycytidine analogs (e.g., gemcitabine or its derivatives). In some cases, ovarian cancer may initially respond to treatment with cytidine analogs or deoxycytidine analogs (e.g., gemcitabine or its derivatives), but develops resistance to treatment with cytidine analogs or deoxycytidine analogs, such as gemcitabine treatment.

[0083] For example, in some aspects, ovarian cancer can be an ovarian cancer that responds to treatment with gemcitabine, azacitidine, cytarabine, decitabine, troxacitabine, or any of its derivatives. Ovarian cancer can also be an ovarian cancer that responds poorly, unresponsively, or refractory to treatment with gemcitabine, azacitidine, cytarabine, decitabine, troxacitabine, or any of its derivatives. Ovarian cancer may have previously been considered unsuitable for treatment with gemcitabine, azacitidine, cytarabine, decitabine, troxacitabine, or any of its derivatives. In some cases, ovarian cancer may initially respond to treatment with gemcitabine, azacitidine, cytarabine, decitabine, troxacitabine, or any of its derivatives, but develops resistance to treatment with gemcitabine, azacitidine, cytarabine, decitabine, troxacitabine, or any of its derivatives.

[0084] Colorectal cancer

[0085] One aspect of the present invention relates to the prevention or treatment of colorectal cancer. As background, the intestinal mucosa is a complex ecosystem in which the epithelium has an interdependent relationship with its microenvironment, particularly the underlying stroma. Mesenchymal-epithelial crosstalk is closely related to regulating homeostasis and is dynamically altered in intestinal regeneration and cancer. Cell signaling networks are effector pathways of intercompartmental crosstalk and control epithelial cell fate decisions, but can be exploited and subverted by the tumor microenvironment in colorectal cancer.

[0086] The current chemotherapy management of colorectal cancer has not changed substantially over the past 20 years and is mainly based on the use of combination cytotoxic drugs (e.g. 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) against the proliferative tumor epithelium and the potential development of resistance to these epithelial-targeted drugs. The identification of new treatments for colorectal cancer is now more important than ever.

[0087] Therefore, the cancer or tumor for treatment is colorectal cancer or colorectal tumor. A particularly preferred form of colorectal cancer for treatment is colorectal cancer, characterized in that GREM1 in stromal cells is overexpressed, i.e., stromal GREM1 is overexpressed. Stromal cells may be cancer-associated fibroblasts. Colorectal cancer with stromal GREM1 overexpression may not show epithelial GREM1 overexpression. Colorectal cancer with stromal GREM1 overexpression may include stromal Foxl1 overexpression. A particularly suitable form of colorectal cancer for treatment is colorectal cancer, which is a mesenchymal subtype colorectal cancer, also known as CMS4 (Guinney et al., Nat Med 2015). Any other subtype of colorectal cancer can also be treated, including any of CMS1, CMS2 and CMS3 as described in Guinney et al. above. Colorectal cancer described herein may be proximal colorectal cancer (or proximal colorectal tumor). The proximal colon is the area of ​​the large intestine upstream of the splenic flexure, meaning the cecum, ascending colon and transverse colon. Cancer or tumors in this area are also referred to as right-sided cancers or tumors. The present invention may be directed to treating right-sided colorectal cancer or right-sided colorectal tumors.

[0088] Colorectal cancer can be distal colorectal cancer (or distal colorectal tumor). The distal colon is the area of ​​the large intestine downstream of the splenic flexure, meaning the descending colon, sigmoid colon and rectum. Cancer or tumors in this area are also referred to as left-sided cancers or tumors. The present invention may relate to treating left-sided colorectal cancer or left-sided colorectal tumors. Cancer with matrix overexpression of GREM1 may preferably be a sporadic cancer. Sporadic cancer may be caused by somatic mutations. Sporadic cancer may be caused by carcinogens. Sporadic cancer is not caused by inherited gene mutations. Sporadic cancer may result in matrix overexpression of GREM1. The proliferation of sporadic cancer may depend on matrix overexpression of GREM1 in cancer.

[0089] At least three single nucleotide polymorphisms (SNPs) close to GREM1 are independently associated with colorectal cancer (CRC) risk in Northern European whites and possibly other ethnicities (Tomlinson et al., PLos Genet, 2011). There is a direct link with GREM1 expression, and other SNPs may have similar effects. In addition, two common SNPs near BMP2, two common SNPs near BMP4, and one common SNP near BMP7 affect the expression of BMP ligands and affect CRC risk. Therefore, cancer may contain one or more of the above SNPs.

[0090] According to the present invention, another type of cancer or tumor for treatment is a cancer or tumor that exhibits overexpression of GREM1 in epithelial cells. Overexpression of GREM1 in epithelial cells may lead to cancer. The proliferation of cancer may depend on epithelial overexpression of GREM1. Therefore, the cancer may be epithelial. The cancer may be colorectal cancer or duodenal cancer. The cancer may be GREM1-induced. GREM1-induced means that a mutagenic event that enhances GREM1 activity or expression is the cause of the cancer. This cancer may be caused by an inherited gene mutation. Therefore, the cancer may be a familial cancer (see below).

[0091] As described further below, preferred types of colorectal cancer for treatment may be resistant to one or more known anti-cancer agents (eg, chemotherapeutic agents).

[0092] The colorectal cancer may be disseminated colorectal cancer. The colorectal cancer may be metastatic colorectal cancer. The colorectal cancer may be metastatic colorectal cancer of the lung. The colorectal cancer may be metastatic colorectal cancer of the liver. The colorectal cancer may be metastatic colorectal cancer of the bone.

[0093] Colorectal cancer may be characterized by stromal overexpression of Foxl1. Colorectal cancer may be characterized by stromal overexpression of one or more Wnt ligands. For example, colorectal cancer may be characterized by stromal overexpression of Wnt5A and / or Wnt2B. If a colorectal cancer has stromal overexpression of Foxl1 and / or a Wnt ligand (e.g., Wnt5A or Wnt2B), the colorectal cancer may be particularly suitable for prevention or treatment using a GREM1 antagonist.

[0094] In some aspects, colorectal cancer can be a colorectal cancer that responds to treatment using cytidine analogs or deoxycytidine analogs (e.g., gemcitabine or its derivatives). Colorectal cancer can also be a colorectal cancer that is poorly, unresponsive or refractory to treatment using cytidine analogs or deoxycytidine analogs (e.g., gemcitabine or its derivatives). Colorectal cancer may have previously been considered unsuitable for treatment with cytidine analogs or deoxycytidine analogs (e.g., gemcitabine or its derivatives). In some cases, colorectal cancer may initially respond to treatment using cytidine analogs or deoxycytidine analogs (e.g., gemcitabine or its derivatives), but resistance is produced to treatment using cytidine analogs or deoxycytidine analogs, such as gemcitabine treatment.

[0095] For example, in some aspects, the colorectal cancer can be a colorectal cancer that is responsive to the treatment of gemcitabine, azacitidine, cytarabine, decitabine, troxacitabine or any of its derivatives. The colorectal cancer can also be a colorectal cancer that is poorly responsive, unresponsive or refractory to the treatment of gemcitabine, azacitidine, cytarabine, decitabine, troxacitabine or any of its derivatives. The colorectal cancer may have previously been considered to be unsuitable for treatment with gemcitabine, azacitidine, cytarabine, decitabine, troxacitabine or any of its derivatives. In some cases, colorectal cancer may initially be responsive to the treatment of gemcitabine, azacitidine, cytarabine, decitabine, troxacitabine or any of its derivatives, but resistance is produced to the treatment of gemcitabine, azacitidine, cytarabine, decitabine, troxacitabine or any of its derivatives.

[0096] Familial cancer

[0097] Familial cancers include cancers caused by one or more mutations in the gene encoding GREM1 or any other mutation that affects GREM1 gene expression. 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 restricted mesenchymal gradient to ectopic GREM1 gene expression throughout the epithelium.

[0098] The subject receiving anti-GREM1 antagonist therapy may have previously been identified as being at risk for developing a familial cancer. For example, the subject may be identified as being at risk based on the subject's family history and / or because the subject carries a genetic mutation known to cause or increase the risk of developing a familial cancer.

[0099] A familial cancer may be Lynch syndrome, also known as hereditary nonpolyposis colorectal cancer (HNPCC). A familial cancer may be familial adenomatous polyposis (FAP).

[0100] Patients or subjects with familial adenomatous polyposis (FAP) may be particularly suitable for treatment with a combination therapy comprising an anti-GREM1 antagonist. The familial cancer treated or prevented with a combination therapy comprising an anti-GREM1 antagonist (e.g., an anti-GREM1 antibody) and gemcitabine or a derivative thereof may be FAP. An anti-GREM1 antagonist in combination with gemcitabine or a derivative thereof may be administered prophylactically to a subject who previously had FAP, for example to prevent recurrence. An anti-GREM1 antagonist in combination with gemcitabine may be administered prophylactically to a subject who did not previously have FAP but who has been previously determined to be at risk of developing FAP. The subject may have been determined to be at risk of developing FAP because the subject has been found to carry a deleterious mutation in their Apc gene.

[0101] In some aspects, familial cancer can be a familial cancer that responds to treatment using cytidine analogs or deoxycytidine analogs (e.g., gemcitabine or its derivatives). Familial cancer can alternatively be a familial cancer that responds poorly, unresponsively or refractory to treatment using cytidine analogs or deoxycytidine analogs (e.g., gemcitabine or its derivatives). Familial cancer may have been previously considered unsuitable for treatment with cytidine analogs or deoxycytidine analogs (e.g., gemcitabine or its derivatives). In some cases, familial cancer may initially respond to treatment using cytidine analogs or deoxycytidine analogs (e.g., gemcitabine or its derivatives), but develops resistance to treatment using cytidine analogs or deoxycytidine analogs, such as gemcitabine treatment.

[0102] For example, in some aspects, familial cancer can be a familial cancer that is responsive to treatment with gemcitabine, azacitidine, cytarabine, decitabine, troxacitabine, or any of its derivatives. Familial cancer can also be a familial cancer that is poorly responsive, unresponsive, or refractory to treatment with gemcitabine, azacitidine, cytarabine, decitabine, troxacitabine, or any of its derivatives. Familial cancer may have previously been considered unsuitable for treatment with gemcitabine, azacitidine, cytarabine, decitabine, troxacitabine, or any of its derivatives. In some cases, familial cancer may initially be responsive to treatment with gemcitabine, azacitidine, cytarabine, decitabine, troxacitabine, or any of its derivatives, but resistance is developed to treatment with gemcitabine, azacitidine, cytarabine, decitabine, troxacitabine, or any of its derivatives.

[0103] Multiple Myeloma

[0104] On the other hand, the present invention relates to the treatment or prevention of multiple myeloma. Multiple myeloma (MM) is a hematological malignancy characterized by the clonal proliferation of plasma cells (PCs) in the bone marrow (BM). It is well known that BM supports tumor growth in MM, wherein bidirectional signaling between tumor cells and BM is essential for the continued growth, spread and survival of MM PCs. Cellular and non-cellular BM components have different effects on the growth and spread of MM PCs. Although recent studies have identified BM components that play a role in disease progression and therapies for these components have been developed, the standard treatment of MM still relies mainly on targeting the tumor cells themselves. Although such therapies can effectively prolong patient survival, since BM plays an important role in the growth, spread, survival and drug resistance of MM cells, more effective therapies for this important aspect of the disease are needed. In fact, MM is a basically incurable disease, and disease recurrence is a key problem faced by the effective treatment of the disease.

[0105] Therefore, the present invention is also directed to the treatment or prevention of multiple myeloma. Multiple myeloma typically includes the presence of more than one plasma cell mass in the bone marrow. Therefore, multiple myeloma is typically associated with abnormal proliferation of plasma cells in the bone marrow. A particularly preferred form of multiple myeloma for treatment is characterized by overexpression of GREM1 in the bone marrow. Therefore, multiple myeloma may include stromal GREM1 overexpression. Stromal GREM1 overexpression may be present in the compact bone compartment of the bone. Stromal GREM1 overexpression may reflect an increase in the number of stromal cells, or an increase in the level of GREM1 expression in existing GREM1-expressing stromal cells. The bone marrow may contain osteochondral reticular (OCR) stem cells. Stromal cells overexpressing GREM1 may include OCR stem cells. As further described below, a preferred type of multiple myeloma for treatment may be resistant to one or more known anticancer agents (e.g., chemotherapeutic agents).

[0106] In some aspects, multiple myeloma can be a multiple myeloma that responds to treatment with cytidine analogs or deoxycytidine analogs (e.g., gemcitabine or its derivatives). Multiple myeloma can also be a multiple myeloma that is poorly responsive, unresponsive, or refractory to treatment with cytidine analogs or deoxycytidine analogs (e.g., gemcitabine or its derivatives). Multiple myeloma may have been previously considered unsuitable for treatment with cytidine analogs or deoxycytidine analogs (e.g., gemcitabine or its derivatives). In some cases, multiple myeloma may initially respond to treatment with cytidine analogs or deoxycytidine analogs (e.g., gemcitabine or its derivatives), but develops resistance to treatment with cytidine analogs or deoxycytidine analogs, such as gemcitabine treatment.

[0107] For example, in some aspects, multiple myeloma can be a multiple myeloma that is responsive to treatment with gemcitabine, azacitidine, cytarabine, decitabine, troxacitabine or any of its derivatives. Multiple myeloma can also be a multiple myeloma that is poorly responsive, unresponsive or refractory to treatment with gemcitabine, azacitidine, cytarabine, decitabine, troxacitabine or any of its derivatives. Multiple myeloma may have previously been considered unsuitable for treatment with gemcitabine, azacitidine, cytarabine, decitabine, troxacitabine or any of its derivatives. In some cases, multiple myeloma may initially be responsive to treatment with gemcitabine, azacitidine, cytarabine, decitabine, troxacitabine or any of its derivatives, but resistance is produced to treatment with gemcitabine, azacitidine, cytarabine, decitabine, troxacitabine or any of its derivatives.

[0108] Breast cancer

[0109] In another aspect, the present invention relates to the treatment or prevention of breast cancer. The breast cancer may be invasive breast cancer, such as invasive lobular breast cancer. The breast cancer may be triple negative breast cancer. The breast cancer may be inflammatory breast cancer. The breast cancer may be angiosarcoma of the breast. The breast cancer may be ductal carcinoma in situ or lobular carcinoma in situ.

[0110] The present invention treats and prevents breast cancer by administering an anti-GREM1 antagonist in combination with gemcitabine or a derivative thereof. Breast cancer may include stromal GREM1 overexpression. Stromal breast cells that overexpress GREM1 may include stromal fibroblasts, also described herein as cancer-associated fibroblasts. Breast cancer may also be recurrent breast cancer. In other words, breast cancer treated by the methods of the present invention includes breast cancer that recurs months or even years after early treatment (e.g., chemotherapy, radiotherapy, or curative surgery). Preferred types of breast cancer for treatment may be resistant to one or more known anticancer agents (e.g., chemotherapeutic agents), as further described below. Breast cancer may be disseminated breast cancer. Breast cancer may be metastatic breast cancer. Breast cancer may be metastatic breast cancer of the lung. Breast cancer may be metastatic breast cancer of the liver. Breast cancer may be metastatic breast cancer of the bone.

[0111] In some aspects, breast cancer can be a breast cancer that responds to treatment with a cytidine analog or a deoxycytidine analog (e.g., gemcitabine or its derivatives). Alternatively, breast cancer can be a breast cancer that responds poorly, unresponsively, or refractory to treatment with a cytidine analog or a deoxycytidine analog (e.g., gemcitabine or its derivatives). Breast cancer may have previously been considered unsuitable for treatment with a cytidine analog or a deoxycytidine analog (e.g., gemcitabine or its derivatives). In some cases, breast cancer may initially respond to treatment with a cytidine analog or a deoxycytidine analog (e.g., gemcitabine or its derivatives), but develops resistance to treatment with a cytidine analog or a deoxycytidine analog (e.g., gemcitabine).

[0112] For example, in some aspects, breast cancer can be a breast cancer that is responsive to treatment with gemcitabine, azacitidine, cytarabine, decitabine, troxacitabine or any of its derivatives. Breast cancer can also be a breast cancer that is poorly responsive, unresponsive or refractory to treatment with gemcitabine, azacitidine, cytarabine, decitabine, troxacitabine or any of its derivatives. Breast cancer may have been previously considered unsuitable for treatment with gemcitabine, azacitidine, cytarabine, decitabine, troxacitabine or any of its derivatives. In some cases, breast cancer may initially be responsive to treatment with gemcitabine, azacitidine, cytarabine, decitabine, troxacitabine or any of its derivatives, but develops resistance to treatment with gemcitabine, azacitidine, cytarabine, decitabine, troxacitabine or any of its derivatives.

[0113] Prostate cancer

[0114] In another aspect, the invention relates to the treatment or prevention of prostate cancer. The prostate cancer may be a prostate adenocarcinoma. The prostate cancer may be a transitional cell carcinoma or a urothelial carcinoma that has spread to the prostate.

[0115] Prostate cancer can be characterized as having GREM1 overexpression of prostate cancer. Prostate cancer can also be recurrent prostate cancer. In other words, the prostate cancer treated by the method of the present invention includes prostate cancer that recurs months or even years after early treatment (e.g., chemotherapy, radiotherapy, or curative surgery). Preferred prostate cancer types for treatment may be resistant to one or more known anticancer agents (e.g., chemotherapeutic agents), as further described below.

[0116] The present invention also provides for treating and preventing 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 metastatic prostate cancer of the lung. The prostate cancer may be metastatic prostate cancer of the liver. The prostate cancer may be metastatic prostate cancer of the bone.

[0117] In some aspects, prostate cancer can be a prostate cancer that is responsive to treatment using cytidine analogs or deoxycytidine analogs (e.g., gemcitabine or its derivatives). Prostate cancer can also be a prostate cancer that is poorly responsive, unresponsive or refractory to treatment using cytidine analogs or deoxycytidine analogs (e.g., gemcitabine or its derivatives). Prostate cancer may previously be considered unsuitable for treatment with cytidine analogs or deoxycytidine analogs (e.g., gemcitabine or its derivatives). In some cases, prostate cancer may initially be responsive to treatment using cytidine analogs or deoxycytidine analogs (e.g., gemcitabine or its derivatives), but resistance is produced to treatment using cytidine analogs or deoxycytidine analogs, such as gemcitabine treatment.

[0118] For example, in some aspects, prostate cancer can be a prostate cancer that is responsive to the treatment of gemcitabine, azacitidine, cytarabine, decitabine, troxacitabine or any of its derivatives. Prostate cancer can also be a poor, unresponsive or refractory prostate cancer to the treatment of gemcitabine, azacitidine, cytarabine, decitabine, troxacitabine or any of its derivatives. Prostate cancer may previously be considered to be unsuitable for treatment with gemcitabine, azacitidine, cytarabine, decitabine, troxacitabine or any of its derivatives. In some cases, prostate cancer may initially be responsive to the treatment of gemcitabine, azacitidine, cytarabine, decitabine, troxacitabine or any of its derivatives, but resistance is produced to the treatment of gemcitabine, azacitidine, cytarabine, decitabine, troxacitabine or any of its derivatives.

[0119] Stroma and epithelium

[0120] Cancers prevented or treated using a GREM1 antagonist in combination with gemcitabine described herein may include stromal and / or epithelial GREM1 overexpression.

[0121] As used herein, the term "stromal cells" or "stroma" refers to the structural and / or connective portion of a tissue or organ.

[0122] Matrix tissue is mainly composed of extracellular matrix containing connective tissue cells. The extracellular matrix is ​​mainly composed of ground substance (a porous hydrated gel, mainly made of proteoglycan aggregates) and connective tissue fibers. There are usually three types of fibers in the matrix: type I collagen fibers, elastic fibers and reticular fibers (type III collagen fibers). Fibroblasts and pericytes are the most common types of matrix cells.

[0123] In the case of cancer or tumors (e.g., originating from the epithelium of a tissue or organ), the stroma of the tissue or organ may contribute to the growth and progression of the cancer. The stroma associated with a cancer or tumor may be a desmoplastic stroma resulting from the growth of fibrous or connective tissue surrounding the cancer or tumor.

[0124] Overexpression of GREM1 can be observed in any part of the matrix / any matrix cell. The matrix cell can be a fibroblast or a fibroblast-like supporting cell. The matrix cell can be a fibroblast or a fibroblast-like supporting cell isolated from the desmoplastic matrix of any of the above-mentioned cancers or tumors, for example, isolated from the pancreas, colon or rectum in colorectal cancer or the bone marrow in multiple myeloma. The matrix cell can be a cancer-associated fibroblast.

[0125] The term "epithelium" as used herein refers to cells derived from the outer layer or inner lining of a tissue or organ. For the colon, the intestinal epithelium is a layer of the luminal surface or inner lining of the small and large intestines that form the gastrointestinal tract. It is composed of a single layer of columnar epithelium. The "upper barrier" is a single layer of columnar cells of the intestinal epithelium, which is composed of four types of intestinal epithelial cells: absorptive enterocytes, goblet cells, Paneth cells, and enteroendocrine cells. The upper barrier features in the small intestine and large intestine are similar. The main difference is the presence of ridges and protrusions (annular folds, villi, and microvilli) in the duodenum, jejunum, and ileum, which allow an increase in the absorption surface. This is not observed in the colon, and the surface of the colon is flat. In the mucosal protrusions known as villi, there are bends known as Lieberkuhn's crypts, which are distinct glandular invaginations. The cells observed to be overexpressed by epithelial GREM1 can be any epithelial cell, such as any intestinal epithelial cell.

[0126] While not being bound by theory, the inventors hypothesize that stromal targeting using anti-GREM1 antagonists can enhance the efficacy of pancreatic cancer chemotherapy treatment because the stroma itself can confer chemo-resistance to tumor cells. The inventors also hypothesize that overexpression of GREM1 in the epithelium and / or stroma may promote stem cell / progenitor cell phenotypes (increase the number of stem cells / progenitor cells), promote epithelial stem cell behavior and drive cancer progression and / or resistance to chemotherapeutic agents. Therefore, combination therapies comprising a GREM1 antagonist and a cytidine analog or deoxycytidine analog (e.g., gemcitabine or a derivative thereof) used in accordance with the present invention can prevent the induction of abnormal cancer stem cell / progenitor cell phenotypes, reduce epithelial stem cell behavior and / or reduce the number of stem cells / progenitor cells in the epithelium of a tissue or organ of a subject to prevent or treat cancer. The ability of GREM1 antagonists to affect stem cell behavior can be clinically determined by evaluating known epithelial and cancer stem cell markers.

[0127] Overexpression of GREM1 in the matrix and / or epithelium can be determined by any means. Overexpression of GREM1 is usually determined by comparing the marker level (i.e., basal expression level) with that in normal cells of the same tissue type. The expression is usually standardized for the expression levels of other genes (preferably including one or more housekeeping genes). GREM1 can also be classified as showing overexpression or underexpression in a threshold percentage of a cancer patient population. The overexpression of each patient in the population may be higher than 2 of the geometric mean. At least 10%, more preferably at least 15% or more of the patients in the population may show such overexpression.

[0128] Stromal overexpression of GREM1 refers to stromal GREM1 levels being higher than matched normal tissue. For example, stromal GREM1 levels may be at least two-fold higher than matched normal tissue.

[0129] When GREM1 is overexpressed, its amount can be increased by any amount relative to the substrate. For example, a GREM1-induced cancer (e.g., HMPS) may include a thousand-fold upregulation of epithelial GREM1, while no GREM1 expression is observed in normal epithelium. Sporadic cancers that include stromal GREM overexpression may include any level of stromal overexpression that exceeds the physiological GREM1 expression level in the normal stroma of the organ. A skilled person can assess whether there is overexpression in the stroma or epithelium compared to the level of GREM1 in normal cells of the same type.

[0130] The amount measured can be the amount of mRNA. Therefore, cancer can include overexpression of GREM1 mRNA. Compared with normal cells of the same tissue type, cancer can include increased GREM1 mRNA amount. mRNA can be increased by any amount. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) such as real-time qRT-PCR, quantigene determination (Affymetrix / Thermo Fisher), northern blot or microarray, RNA sequencing can be used to measure the amount of mRNA. mRNA expression is preferably determined by comparing the gene expression of the sample with the expression level distribution of a specific gene in a reference sample composed of a tumor that is diploid for the gene. RNAseq can be used to derive a z score by the expectation maximization (RSEM) algorithm (cBioportalfor Cancer Genomics, www.cbioportal.org; Gao et al., 2013 and Serami et al. 2012). A z score that is 2SD higher or lower than the mean of the reference set is preferably regarded as overexpression or underexpression, respectively.

[0131] The amount determined can be the amount of protein. Cancer can include overexpression of GREM1 protein, for example compared to normal cells of the same tissue type. The protein can be increased by any amount. The amount of protein can be measured using immunohistochemistry, Western blot, mass spectrometry or fluorescence activated cell sorting (FACS), including the use of anti-GREM1 antibodies of the present invention. The threshold for determining expression may vary depending on the technology used and can be verified based on immunohistochemistry scoring.

[0132] Thus, as described herein, the use of a GREM1 antagonist in combination with a cytidine analog or a deoxycytidine analog for treating or preventing cancer in a patient 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 a 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 described above. The GREM1 antagonist may be any GREM1 antagonist described herein.

[0133] Thus, as described herein, the use of a GREM1 antagonist in combination with gemcitabine or a derivative thereof for treating or preventing cancer in a patient 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 gemcitabine or a derivative thereof, 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 GREM1 antagonist as described herein.

[0134] The above measurements can be performed in any suitable sample from a patient. The measurements can be performed in a cancer or tumor biopsy obtained from a patient. Matrix and / or epithelium (stromal and / or epithelial cells) can be isolated from a 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, gastric tissue, duodenal tissue, esophageal tissue, bone marrow or colorectal tissue. Any of the above methods can be performed on a cancer biopsy. This method can also be performed on cancer cells circulating in the patient's blood. The RNA method can be performed on urine or blood exosomes.

[0135] Combination therapy using anti-GREM1 antagonists in combination with proliferation-dependent cytotoxic agents

[0136] In another embodiment of the invention, an anti-GREM1 antagonist in combination with a proliferation-dependent cytotoxic agent for use in a method of treating or preventing pancreatic cancer is provided. The method may comprise administering the proliferation-dependent cytotoxic agent separately, sequentially or simultaneously.

[0137] The present invention also provides a proliferation-dependent cytotoxic agent for use in a method of treating or preventing pancreatic cancer, wherein the method comprises administering an anti-GREM1 antagonist alone, sequentially or simultaneously.

[0138] In one embodiment, pancreatic cancer is a pancreatic cancer characterized by having GREM1 overexpression. In another embodiment, pancreatic cancer may be characterized by having an exocrine tumor or a neuroendocrine tumor. Pancreatic neuroendocrine cancer (also known as islet cell tumor) develops in the endocrine glands of the pancreas. A particularly preferred form of pancreatic cancer is exocrine pancreatic cancer, such as pancreatic ductal adenocarcinoma. Other exocrine pancreatic cancers include squamous cell carcinomas formed in the pancreatic duct; adenosquamous carcinomas; signet ring cell carcinomas; and colloid carcinomas, which usually develop from intraductal papillary mucinous tumors.

[0139] Preferred pancreatic cancer types for treatment may be resistant to one or more known anticancer agents (e.g., chemotherapeutic agents). Pancreatic cancer may be disseminated pancreatic cancer. Pancreatic cancer may be metastatic pancreatic cancer. Metastatic cancer is understood to be a cancer that has spread from the original site of origin in the body. Therefore, metastatic pancreatic cancer refers to a cancer that begins in the pancreas and spreads to other organs (e.g., lungs, liver, bones, and brain). Pancreatic cancer may also be recurrent pancreatic cancer. In other words, pancreatic cancer treated by the methods of the present invention includes pancreatic cancer that recurs months or even years after early treatment (e.g., chemotherapy, radiotherapy, or curative surgery).

[0140] In some aspects, the pancreatic cancer may be a pancreatic cancer that responds to treatment with a proliferation-dependent cytotoxic agent. The pancreatic cancer may be a pancreatic cancer that responds poorly, does not respond, or is 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 initially respond to treatment with a proliferation-dependent cytotoxic agent, but develops resistance to treatment with a proliferation-dependent cytotoxic agent.

[0141] As described herein, the use of a GREM1 antagonist in combination with a proliferation-dependent cytotoxic agent for treating or preventing pancreatic cancer in a patient can include (a) measuring the amount of GREM1 in the cancer, and (b) if the pancreatic cancer comprises overexpression of GREM1, administering to the patient a GREM1 antagonist in combination with a proliferation-dependent cytotoxic agent, thereby treating or preventing pancreatic cancer. The amount of GREM1 can be the amount of mRNA or protein, and the overexpression can be any overexpression discussed above. The GREM1 antagonist can be any GREM1 antagonist described herein. The proliferation-dependent cytotoxic agent can be any proliferation-dependent cytotoxic agent described herein.

[0142] Proliferation-dependent cytotoxic agents

[0143] In the present invention, in the context of treating or preventing pancreatic cancer, the term "proliferation-dependent cytotoxic agent" describes any cytotoxic agent that targets proliferating cells. In other words, highly proliferative cell populations, including rapidly dividing cancer cells, show greater sensitivity to such drugs. Without being bound by theory, the inventors hypothesize that anti-GREM1 antagonists are able to drive dormant stem-like cancer cells into a more proliferative state, making them more susceptible to treatment with proliferation-dependent cytotoxic agents. Thus, in one aspect, the proliferation-dependent cytotoxic agents used in the methods of the present invention are cytotoxic to proliferating stem-like cancer cells.

[0144] In one embodiment, the proliferation-dependent cytotoxic agent is a nucleoside inhibitor or antimetabolite. Nucleoside inhibitors or antimetabolites exert cytotoxic activity by mimicking endogenous nucleosides and interfering with the synthesis of nucleic acids. For example, nucleoside inhibitors or antimetabolites include analogs of physiological pyrimidine and purine nucleobases and nucleosides. Such compounds may also interfere with DNA methylation or change the metabolism of physiological nucleosides. Nucleoside inhibitors or antimetabolites may also mediate enzyme inhibition and interfere with the synthesis of nucleic acids. Therefore, nucleoside inhibitors or antimetabolites are generally described as cell cycle specific.

[0145] Examples of nucleoside inhibitors or antimetabolites include folic acid antagonists, such as methotrexate and pemetrexed; pyrimidine antagonists, such as 5-fluorouracil, fucitabine, capecitabine, cytarabine and gemcitabine; purine antagonists, such as 6-mercaptopurine and 6-thioguanine; and adenosine deaminase inhibitors, such as cladribine, fludarabine and valerate. In a preferred embodiment in the context of pancreatic cancer, the proliferation-dependent cytotoxic agent is selected from gemcitabine and capecitabine.

[0146] In a preferred embodiment, the nucleoside inhibitor or antimetabolite is a cytidine analog or a deoxycytidine analog. As mentioned above, a cytidine analog or a deoxycytidine analog simulates endogenous cytidine or deoxycytidine. Such compounds can also interfere with DNA methylation or change the metabolism of physiological nucleosides. Therefore, cytidine analogs and deoxycytidine analogs are widely used in anticancer therapy.

[0147] Exemplary cytidine analogs or deoxycytidine analogs for treating pancreatic cancer include gemcitabine (2'-deoxy-2',2'-difluorocytidine) or a derivative thereof; azacitidine (5-azacytidine) or a derivative thereof; cytarabine (Ara-C / cytosine 1-[β]-D-arabinofuranoside) or a derivative thereof; decitabine (5-aza-2'-deoxycytidine / 5-azadeoxycytidine) 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.

[0148] In some embodiments, the combination therapy provides an anti-GREM1 antagonist together with one or more cytidine analogs or deoxycytidine analogs. In a preferred embodiment, the invention provides an anti-GREM1 antagonist for use in a method of treating or preventing pancreatic cancer, wherein the method further comprises administering gemcitabine. In an exemplary embodiment, the invention provides an anti-GREM1 antagonist for use in a method of treating or preventing pancreatic cancer, wherein the method further comprises administering gemcitabine in combination with troxacitabine. An exemplary pancreatic cancer is an exocrine pancreatic cancer, such as pancreatic ductal adenocarcinoma.

[0149] In another embodiment, the proliferation-dependent cytotoxic agent is a mitotic inhibitor. The term mitotic inhibitor as used herein refers to an inhibitor that inhibits mitosis by destroying microtubules, thereby preventing cell division. For example, a mitotic inhibitor can target tubulin, thereby impairing the normal function of the mitotic spindle, resulting in the destruction of microtubule polymerization. In one embodiment of the invention, a mitotic inhibitor is a microtubule stabilizing drug. Such drugs can inhibit cell division by destroying microtubule-dependent signaling events, stimulating tubulin formation and / or increasing cell microtubule density.

[0150] Examples of mitotic inhibitors and / or microtubule stabilizing drugs include cabazitaxel, docetaxel, paclitaxel, vinblastine, vincristine, vinorelbine, and nab-paclitaxel (Abraxane). In a preferred embodiment in the context of pancreatic cancer, the mitotic inhibitor and / or microtubule stabilizing drug is selected from nab-paclitaxel and paclitaxel.

[0151] In another embodiment of the invention, the proliferation-dependent cytotoxic agent may include 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.

[0152] In a preferred embodiment of the invention, the proliferation-dependent cytotoxic agent is gemcitabine and the cancer is pancreatic cancer.

[0153] GREM1

[0154] In the present invention, the term GREM1 or Gremlin-1 used in the context of a protein refers to a protein generally having an amino acid sequence as shown in the human GREM1 UniProt entry O60565 (SEQ ID NO: 1). The terms GREM1 and Gremlin-1 may also refer to a Gremlin-1 polypeptide, which:

[0155] (a) comprising or consisting of the amino acid sequence of SEQ ID NO: 1 with or without an N-terminal signal peptide, that is, may comprise or consist of the mature peptide sequence as shown in SEQ ID NO: 21; or

[0156] (b) is a derivative having one or more amino acid substitutions, modifications, deletions or insertions relative to the amino acid sequence of SEQ ID NO: 1 with or without an N-terminal signal peptide (as shown in SEQ ID NO: 21), which retains the activity of Gremlin-1 (e.g., the amino acid sequence of SEQ ID NO: 20).

[0157] (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, suitably retain about 80% to about 99% identity to SEQ ID NO: 1, more suitably retain about 90% to about 99% identity to SEQ ID NO: 1, and most suitably retain about 95% to about 99% identity to SEQ ID NO: 1. Variants are further described below.

[0158] As discussed further below, residue numbering is generally based on sequence reference to SEQ ID NO: 1. However, one skilled in the art can readily extrapolate residue numbering to derivative or variant sequences as described above. When residue numbers are referenced, the invention also includes those residues on variant or derivative sequences.

[0159] The GREM1 or Gremlin-1 nucleic acid sequence may comprise or consist of a sequence of SEQ ID NO: 36 or SEQ ID NO: 37 or a variant thereof. Variant nucleic acid sequences are further described below. The GREM1 or Gremlin-1 nucleic acid sequence may comprise or consist of any GREM1 transcript variant. Examples of GREM1 transcript variants are transcript 1 (NCBI: NM_013372.6; ENSEMBL: ENST00000560677.5); transcript 2: NCBI: NM_001191323.1; ENSEMBL: ENST00000560830.1); transcript 3: NCBI: NM_001191322.1; ENSEMBL: ENST00000622074.1. The sequences provided by the above accession numbers as of June 18, 2018 are incorporated herein by reference.

[0160] Antagonists

[0161] 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 in 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 GREM1 function or activity. 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 an 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. Pancreatic cancer cells can be present in a KPC mouse model as described in the examples. Therefore, in vivo determination of the activity of GREM1 antagonists in combination therapy for pancreatic cancer can be performed in a mouse model. More generally, GREM1 antagonists that are shown in any manner to reduce GREM1 function or activity can then be assayed in vitro or in vivo for their ability to prevent or reduce proliferation of cancer cells (eg, pancreatic cancer cells) or to prevent, reduce or eliminate cancer or tumors.

[0162] An antagonist may reduce GREM1 function by any means. It may directly or indirectly increase or decrease the activity or amount of any molecule that affects GREM1 function. It may reduce the amount of GREM1 at the mRNA or protein level. It may increase the degradation of GREM1. It may reduce the function of GREM1 by inhibitory modification. It may reduce the transcription of a molecule that enhances GREM1 function. It may use agents such as zinc finger nucleases to disrupt the DNA encoding GREM1 or molecules that enhance GREM1 function.

[0163] Antagonists can be agents that interact with Gremlin-1. Agents that interact with Gremlin-1 are typically agents that bind to Gremlin-1. Agents that interact with Gremlin-1 can modulate Gremlin-1. Inhibitory modulators can affect any function of Gremlin-1, but typically reduce the binding of Gremlin-1 to BMP (BMP2 / 4 / 7). Antagonists can be BMP-7 mimetic molecules. Gremlin-1 is a negative regulator of BMP, so reduced binding increases signaling through BMP. Activating modulators can increase the binding of Gremlin-1 to BMP.

[0164] BMP binding and signaling can be detected by any method known in the art.

[0165] Antagonists can act by binding to the active site of GREM1, or allosterically by binding at a different site. Antagonists can act by binding to modulators or ligands of GREM1, thereby reducing the activation of GREM1. Antagonists can be reversible or irreversible.

[0166] The GREM1 antagonist may 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).

[0167] A GREM1 antagonist can be an oligonucleotide that specifically hybridizes to an mRNA encoding GREM1 or an mRNA encoding a molecule that enhances GREM1 activity. A GREM1 antagonist can be a polynucleotide that encodes any molecule that reduces GREM1 function. For example, a GREM1 antagonist can be a polynucleotide that encodes an anti-GREM1 antibody described herein.

[0168] GREM1 antagonists can be antibodies that specifically bind to any target molecule (usually a protein) to directly or indirectly reduce GREM1 function. Antagonists can be antibodies that specifically bind to GREM1. In this regard, antibodies can reduce GREM1 function by allosteric inactivation or by blocking the interaction between its target and a ligand required for activity.

[0169] The interaction of the antagonist with the protein residues can be determined by any suitable method known in the art, such as the distance between the residue and the agent as determined by x-ray crystallography (usually less than 1000 m / s). or less than The regions of Gremlin-1 that may be targeted by therapeutic agents may include amino acids Asp92-Leu99, Arg116-His130, Ser137-Ser142, Cys176-Cys178. within the range.

[0170] Antibody antagonists

[0171] The term "antibody" as used herein includes whole antibodies and any antigen-binding fragments (i.e., "antigen-binding portions") or single chains thereof. An antibody is a glycoprotein or antigen-binding portion thereof that comprises at least two heavy chains (H) and two light chains (L) interconnected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as HCVR or V H ) and a heavy chain constant region. Each light chain consists of a light chain variable region (abbreviated herein as LCVR or V L The variable regions of the heavy and light chains contain the binding domains that interact with the antigen. H and V L The regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FRs).

[0172] 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.

[0173] The antibody used according to the present invention can be a monoclonal antibody or a polyclonal antibody, and is typically a monoclonal antibody. The antibody used according to the present invention can be a chimeric antibody, an antibody transplanted with CDR, a nanobody, a human or humanized antibody, or an antigen binding portion thereof of any one. In order to produce monoclonal antibodies and polyclonal antibodies, experimental animals are typically non-human mammals, such as goats, rabbits, rats or mice, but antibodies can also be produced in other species.

[0174] Polyclonal antibodies can be produced by conventional methods, such as immunizing a suitable animal with an antigen of interest. Blood can then be taken from the animal and the IgG fraction purified.

[0175] When it is necessary to immunize an animal, antibodies against Gremlin-1 can be obtained by administering the polypeptide to an animal (e.g., a non-human animal) using a well-known conventional protocol, for example, see Handbook of Experimental Immunology, DM Weir (ed.), Vol4, Blackwell Scientific Publishers, Oxford, England, 1986). Many warm-blooded animals, such as rabbits, mice, rats, sheep, cattle, camels or pigs can be immunized. However, mice, rabbits, pigs and rats are usually the most suitable.

[0176] Monoclonal antibodies can be prepared by any method known in the art, such as hybridoma technology (Kohler & Milstein, 1975, Nature, 256: 495-497), trioma technology, human B cell hybridoma technology (Kozbor et al., 1983, Immunology Today, 4: 72) and EBV-hybridoma technology (Cole et al., Monoclonal Antibodies and Cancer Therapy, pp77-96, Alan R Liss, Inc., 1985).

[0177] Antibodies used according to the present invention can also be produced using the single lymphocyte antibody method by cloning and expressing immunoglobulin variable region cDNAs produced from single lymphocytes, which are selected for producing specific antibodies by the methods described in, for example, Babcook, J. et al., 1996, Proc. Natl. Acad. Sci. USA 93(15):7843-78481; WO92 / 02551; WO2004 / 051268 and WO2004 / 106377.

[0178] Antibodies can also be generated using various phage display methods known in the art, including Brinkman et al. (J. Immunol. Methods, 1995, 182: 41-50), Ames et al. (J. Immunol. Methods, 1995, 184: 177-186), Kettleborough et al. (Eur. J. Immunol. 1994, 24: 952-958), Persic et al. (Gene, 1997 187 9-18), Burton et al. (Advances in Immunology, 1994, 57: 191-280) and WO 90 / 02809; WO 91 / 10737; WO 92 / 01047; WO 92 / 18619; WO 93 / 11236; WO 95 / 15982; WO 95 / 20401; and the methods disclosed in US Pat. Nos. 5,698,426, 5,223,409, 5,403,484, 5,580,717, 5,427,908, 5,750,753, 5,821,047, 5,571,698, 5,427,908, 5,516,637, 5,780,225, 5,658,727, 5,733,743, and 5,969,108.

[0179] Fully human antibodies are heavy and light chain variable regions and constant regions (if present) that are all human origin, or substantially identical to human origin sequences, but not necessarily those antibodies from the same antibody. Examples of fully human antibodies can include antibodies, for example, produced by the above-mentioned phage display method and antibodies produced by mice, wherein murine immunoglobulin variable regions and optionally constant region genes have been replaced by their human counterparts, such as described in general terms in EP0546073, U5,545,806, US5,569,825, US5,625,126, US5,633,425, US5,661,016, US5,770,429, EP0438474 and EP0463151.

[0180] Alternatively, the antibody used according to the present invention can be produced by a method comprising immunizing a non-human mammal with a Gremlin-1 immunogen; obtaining an antibody preparation from the mammal; and deriving a monoclonal antibody recognizing Gremlin-1 therefrom.

[0181] The antibody molecule used according to the present invention can include a complete antibody molecule or its fragment or antigen binding portion thereof having a full-length heavy chain and a light chain. The term "antigen binding portion" of an antibody refers to one or more fragments of an antibody that retains the ability to selectively bind to an antigen. It has been shown that the antigen binding function of an antibody can be achieved by a fragment of a full-length antibody. Antibodies and their fragments and antigen binding portions can be, but are not limited to, Fab, modified Fab, Fab', modified Fab', F(ab')2, Fv, single domain antibodies (e.g., VH or VL or VHH), scFv, bivalent, trivalent or tetravalent antibodies, Bis-scFv, double antibodies, three antibodies, four antibodies and the epitope binding fragments of any of the above antibodies (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 producing and manufacturing these antibody fragments are well known in the art (e.g., see Verma et al., 1998, Journal of Immunological Methods, 216, 165-181). Other antibody fragments used in the present invention include Fab and Fab' fragments described in International Patent Applications WO2005 / 003169, WO2005 / 003170 and WO2005 / 003171 and Fab-dAb fragments described in International Patent Applications WO2009 / 040562. Multivalent antibodies can contain multiple specificities, or can be monospecific (e.g., see WO92 / 22853 and WO05 / 113605). These antibody fragments can be obtained using conventional techniques known to those skilled in the art, and the utility of these fragments can be screened in the same manner as intact antibodies.

[0182] The constant region domain of the antibody molecule (if present) can be selected according to the expected 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. Specifically, when the antibody molecule is used for therapeutic purposes and antibody effector functions are required, human IgG constant region domains, especially IgG1 and IgG3 isotypes can be used. Alternatively, when the antibody molecule is used for therapeutic purposes and antibody effector functions are not required, IgG2 and IgG4 isotypes can be used.

[0183] The antibodies used in accordance with the present invention may be prepared, expressed, generated or isolated by recombinant means, such as (a) antibodies isolated from an animal (e.g., a mouse) that is transgenic or transchromosomal for the immunoglobulin gene of interest or a hybridoma prepared therefrom, (b) antibodies isolated from a host cell transformed to express the antibody of interest (e.g., from a transfectoma), (c) antibodies isolated from a recombinant combinatorial antibody library, and (d) antibodies prepared, expressed, generated or isolated by any other means involving splicing of immunoglobulin gene sequences to other DNA sequences.

[0184] The antibody used according to the present invention can be a human antibody or a humanized antibody. The term "human antibody" used herein is intended to include antibodies with variable regions, wherein the framework region and the CDR region are all derived from human germline immunoglobulin sequences. In addition, if the antibody comprises a constant region, the constant region is also derived from human germline immunoglobulin sequences. Human antibodies as described herein may include amino acid residues that are not encoded by human germline immunoglobulin sequences (for example, mutations introduced by random or site-specific mutagenesis in vitro or somatic mutations in vivo). However, the term "human antibody" used herein is not intended to include antibodies that will be transplanted to human framework sequences from CDR sequences derived from another mammalian species (for example, mouse) germline.

[0185] Such human antibodies may be human monoclonal antibodies. Such human monoclonal antibodies may be produced by hybridomas comprising B cells obtained from transgenic non-human animals (e.g., transgenic mice) having a genome comprising human heavy chain transgenes and light chain transgenes fused with immortalized cells.

[0186] Human antibodies can be prepared by immunizing human lymphocytes in vitro and then transforming the lymphocytes with Epstein-Barr virus.

[0187] The term "human antibody derivative" refers to any modified form of a human antibody, such as a conjugate of the antibody with another agent or antibody.

[0188] The term "humanized antibody" is intended to refer to a CDR-grafted antibody molecule in which CDR sequences derived from the germline of another mammalian species (e.g., a mouse) have been grafted onto human framework sequences. Additional framework region modifications may be made within the human framework sequences.

[0189] As used herein, the term "CDR-grafted antibody molecule" refers to an antibody molecule in which the heavy chain and / or light chain comprises one or more CDRs (including one or more modified CDRs, if desired) from a donor antibody (e.g., a murine or rat monoclonal antibody) that are grafted into the heavy chain and / or light chain variable region framework of a recipient antibody (e.g., a human antibody). For review, see Vaughan et al., Nature Biotechnology, 16, 535-539, 1998. In one embodiment, instead of transferring the entire CDR, only one or more specificity determining residues in any of the above CDRs are transferred to the human antibody framework (see, for example, Kashmiri et al., 2005, Methods, 36, 25-34). In one embodiment, only the specificity determining residues in one or more of the above CDRs are transferred to the human antibody framework. In another embodiment, only the specificity determining residues in each of the above CDRs are transferred to the human antibody framework.

[0190] When transplanting CDR or specificity determining residues, any suitable acceptor variable region framework sequence can be used in the case of the category / type of the donor antibody from which CDR is derived, including mouse, primate and human framework regions. Suitably, CDR-transplanted antibodies according to the present invention have variable domains, which comprise human acceptor framework regions and above-mentioned one or more CDR or specificity determining residues. Therefore, in one embodiment, a neutralization CDR-transplanted antibody is provided, wherein the variable domains comprise human acceptor framework regions and non-human donor CDRs.

[0191] Examples of human frameworks that can be used in the present invention are KOL, NEWM, REI, EU, TUR, TEI, LAY, and POM (Kabat et al., supra). For example, KOL and NEWM can be used for the heavy chain, REI can be used for the light chain, and EU, LAY, and POM can be used for both the heavy and light chains. Alternatively, human germline sequences can be used; these sequences are available at http: / / www.vbase2.org / (see Retter et al., Nucl. Acids Res. (2005) 33 (Supplement 1), D671-D674).

[0192] In the CDR-grafted antibodies described herein, the acceptor heavy and light chains do not necessarily need to be from the same antibody, and may comprise composite chains having framework regions derived from different chains if desired.

[0193] In addition, in CDR transplanted antibodies described herein, the framework region does not need to have a sequence identical to that of the receptor antibody. For example, uncommon residues can be changed to more common residues of the receptor chain class or type. Alternatively, the selected residues in the receptor framework region can be changed to correspond to the residues in the same position in the donor antibody (see Reichmann et al., 1998, Nature, 332, 323-324). Such changes should be maintained at the minimum required for restoring the affinity of the donor antibody. The scheme of selecting the receptor framework region residues that may need to be changed is set forth in WO91 / 09967.

[0194] It will also be appreciated by those skilled in the art that antibodies may undergo various post-translational modifications. The type and extent of these modifications generally depend on the host cell line and culture conditions used to express the antibody. Such modifications may include changes in glycosylation, methionine oxidation, diketopiperazine formation, aspartic acid isomerization, and asparagine deamidation. Common modifications are due to the action of carboxypeptidases resulting in the loss of carboxyl terminal basic residues (e.g., lysine or arginine) (as described in Harris, RJ. Journal of Chromatography 705: 129-134, 1995).

[0195] In one embodiment, the antibody heavy chain comprises a CH1 domain and the antibody light chain comprises a CL domain (κ or λ).

[0196] Biomolecules (e.g., antibodies or fragments) contain acidic and / or basic functional groups, thereby imparting a net positive or negative charge to the molecule. The total "observed" charge will depend 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 when a particular molecule or surface has no net charge. In one embodiment, an antibody or fragment according to the present disclosure has an isoelectric point (pi) of at least 7. In one embodiment, an antibody or fragment has an isoelectric point of at least 8 (e.g., 8.5, 8.6, 8.7, 8.8, or 9). In one embodiment, the pI of an antibody is 8. Programs such as **ExPASY http: / / www.expasy.ch / tools / pi_tool.html (see Walker, The Proteomics Protocols Handbook, Humana Press (2005), 571-607) can be used to predict the isoelectric point of an antibody or fragment.

[0197] To characterize the preferred Gremlin-1 epitope, the inventors crystallized human Gremlin-1 alone and in complex with an antibody called Ab7326 (Fab fragment). Crystallization of Gremlin-1 allowed the identification of putative residues in the BMP binding site. In addition, crystallization with Ab 7326 (an allosteric inhibitory antibody) allowed the identification of residues in the antibody epitope. Antibodies that bind to this epitope have particular potential as therapeutic agents in treating diseases associated with Gremlin-1.

[0198] The preferred antibody Ab 7326 described herein has been identified as binding to the following residues of Gremlin-1: Ile110(131), Lys126(147), Lys127(148), Phe128(149), Thr129(150), Thr130(151), Arg148(169), Lys153(174) and Gln154(175), wherein Lys126(147), Lys127(148), Phe128(149), Thr129(150), Thr130(151), Arg148(169), Lys153(174) and Gln154(175) are present on one Gremlin-1 monomer and Ile110(131) is present on a second Gremlin-1 monomer. Numbering outside brackets is based on the structure file (matching the numbering of mouse Gremlin-2 based on the structural alignment). Numbers inside brackets represent residues based on UniProt entry O60565 of SEQ ID NO: 1. These epitope residues are Identified from the Gremlin-1-Ab 7326Fab complex using NCONT analysis.

[0199] 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 of these residues (preferably at least 5 residues).

[0200] The antibodies described herein may also recognize an epitope in which Ile131 is present on the Gremlin-1 monomer differently from other residues.

[0201] Although these residues are provided for a specific sequence of human Gremlin-1, the skilled person can clearly extrapolate the positions of these residues to other corresponding Gremlin sequences (e.g., mouse) using conventional techniques. Therefore, the present invention also provides antibodies that bind to epitopes comprising corresponding residues within these other Gremlin sequences.

[0202] To screen for antibodies that bind to a specific epitope, a conventional cross-blocking assay, such as that described in Antibodies, Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harb., NY) can be performed. Other methods include alanine scanning mutants, peptide blotting (Reineke (2004) Methods Mol Biol 248: 443-63) or peptide cleavage analysis. In addition, methods such as epitope excision, epitope extraction, and antigen chemical modification (Tomer (2000) Protein Science 9: 487-496) can be used. Such methods are well known in the art.

[0203] Antibody epitopes can also be determined by x-ray crystallography. Thus, antibodies of the present disclosure can be evaluated by performing x-ray crystallography on antibodies that bind to Gremlin-1. Specifically, the antibody epitopes can be determined by determining the residues on Gremlin-1 that are in the antibody paratope. Epitopes can be identified in this way by using residues within the

[0204] Thus, the antibodies described herein can bind to an epitope on Gremlin-1 comprising at least one residue selected from Trp93, Phe117, Tyr119, Phe125, Tyr126, and Phe138, wherein the residue numbering is according to SEQ ID NO: 1. Further described herein is an antibody that binds to an epitope comprising all of Trp93, Phe117, Tyr119, Phe125, Tyr126, and Phe138. In addition, an antibody that binds to an epitope comprising the following residues is also described: 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, while Ile131 is located on another monomer of Gremlin-1 (Gremlin-1 dimer binds to BMP dimer).

[0205] If the antibody paratope is at the Gremlin-1 residue range (determined by x-ray crystallography), then the antibody can bind to the above-mentioned Gremlin-1 residues.

[0206] An antibody that binds to an epitope disclosed herein may comprise at least one, at least two or all three heavy chain CDR sequences (HCDR1 / HCDR2 / HCDR3, respectively) of SEQ ID NOs: 4 to 6. These are the HCDR1 / HCDR2 / HCDR3 sequences of the Ab7326 antibody of the Examples determined using the Kabat method.

[0207] The Kabat and Chothia methods for determining CDR sequences are well known in the art (as well as other techniques). Any suitable method may be used to determine CDR sequences, and although Kabat is generally used in the present invention, other techniques may also be used. In this example, SEQ ID NO: 3 represents the Ab7326 HCDR1 sequence determined using the combined Chothia and Kabat definitions.

[0208] The antibodies used according to the invention may comprise at least one, at least two or all three light chain CDR sequences (LCDR1 / LCDR2 / LCDR3 respectively) of SEQ ID NOs: 7 to 9. These are the LCDR1 / LCDR2 / LCDR3 sequences of Ab 7326 determined using the Kabat method.

[0209] The antibody preferably comprises at least one HCDR3 sequence of SEQ ID NO:6.

[0210] 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 of SEQ ID NOs: 4 to 6 (HCDR1 / HCDR2 / HCDR3, respectively) and all three light chain CDR sequences of SEQ ID NOs: 7 to 9 (LCDR1 / LCDR2 / LCDR3, respectively). The antibody may be a chimeric antibody, a human antibody or a humanized antibody.

[0211] The antibody may comprise a heavy chain variable region (HCVR) sequence of SEQ ID NO: 10 or 12 (HCVR of Ab7326 variants 1 and 2). The antibody may comprise a light chain variable region (LCVR) sequence of SEQ ID NO: 11 or 13 (LCVR of Ab 7326 variants 1 and 2). The antibody preferably comprises a heavy chain variable region sequence of SEQ ID NO: 10 or 12 and a light chain variable region sequence of SEQ ID NO: 11 or 13 (particularly a HCVR / LVCR pair of SEQ ID NO: 10 / 11 or 12 / 13).

[0212] The antibody may comprise the following heavy chain (H-chain) sequence

[0213] SEQ ID NO: 14 Mouse full length IgG1 heavy chain variant 1, or

[0214] SEQ ID NO: 28 Mouse full length IgG1 heavy chain variant 2, or

[0215] SEQ ID NO: 30 Human full length IgG1 heavy chain variant 1, or

[0216] SEQ ID NO: 16 Human full length IgG1 heavy chain variant 2, or

[0217] SEQ ID NO: 22 Human full length IgG4P heavy chain variant 1, or

[0218] SEQ ID NO: 34 Human full length IgG4P heavy chain variant 2, or

[0219] SEQ ID NO: 18 Fab heavy chain variant 1, or

[0220] SEQ ID NO: 32 Fab heavy chain variant 2.

[0221] The antibody may comprise the following light chain (L-chain) sequence

[0222] SEQ ID NO: 15 Mouse full length IgG1 light chain variant 1, or

[0223] SEQ ID NO: 29 Mouse full length IgG1 light chain variant 2, or

[0224] SEQ ID NO: 31 Human full length IgG1 light chain variant 1, or

[0225] SEQ ID NO: 17 Human full length IgG1 light chain variant 2, or

[0226] SEQ ID NO: 23 Human full-length IgG4P light chain variant 1, or

[0227] SEQ ID NO: 35 Human full length IgG4P light chain variant 2, or

[0228] SEQ ID NO: 19 Fab light chain variant 1, or

[0229] SEQ ID NO: 33 Fab light chain variant 2.

[0230] In one example, the antibody comprises the following heavy chain / light chain sequence pair:

[0231] SEQ ID NO: 14 / 15 Mouse full length IgG1 variant 1, or

[0232] SEQ ID NO: 28 / 29 Mouse full length IgG1 variant 2, or

[0233] SEQ ID NO: 30 / 31 Human full length IgG1 variant 1, or

[0234] SEQ ID NO: 16 / 17 human full length IgG1 variant 2, or

[0235] SEQ ID NO: 22 / 23 Human full length IgG4P variant 1, or

[0236] SEQ ID NO: 34 / 35 Human full length IgG4P variant 2, or

[0237] SEQ ID NO: 18 / 19 Fab light chain variant 1, or

[0238] SEQ ID NO: 32 / 33 Fab light chain variant 2.

[0239] Variant forms of the corresponding sequences can be interchanged. For example, an antibody may comprise the following heavy chain / light chain sequence pairs:

[0240] SEQ ID NO: 14 / 29 mouse full length IgG1 heavy chain variant 1 / light chain variant 2, or

[0241] SEQ ID NO: 28 / 15 mouse full length IgG1 heavy chain variant 2 / light chain variant 1, or

[0242] SEQ ID NO: 30 / 17 human full length IgG1 heavy chain variant 1 / light chain variant 2, or

[0243] SEQ ID NO: 16 / 31 human full length IgG1 heavy chain variant 2 / light chain variant 1, or

[0244] SEQ ID NO: 22 / 35 human full length IgG4P heavy chain variant 1 / light chain variant 2, or

[0245] SEQ ID NO: 34 / 23 human full length IgG4P heavy chain variant 2 / light chain variant 1, or

[0246] SEQ ID NO: 18 / 33 Fab heavy chain variant 1 / light chain variant 2, or

[0247] SEQ ID NO: 32 / 19 Fab heavy chain variant 2 / light chain variant 1.

[0248] The antibody may be a chimeric antibody, a human antibody or a humanized antibody.

[0249] The antibody may alternatively be or may comprise a variant of one of the above-specified sequences.The following description of antibody variants also applies to the selection of GREM1 polypeptide variants as described above.

[0250] For example, the variant may be a substitution, deletion or addition variant of any of the amino acid sequences described above.

[0251] Variant antibodies may comprise 1, 2, 3, 4, 5, up to 10, up to 20 or more (usually up to 50) amino acid substitutions and / or deletions from the above-mentioned specific sequences. "Deletion" variants may include deletions of individual amino acids, deletions of small groups of amino acids (e.g., 2, 3, 4 or 5 amino acids), or deletions of larger amino acid regions (e.g., deletions of specific amino acid domains or other features). "Substitution" variants typically involve replacing one or more amino acids with the same number of amino acids and performing conservative amino acid substitutions. For example, an amino acid may be substituted 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 major amino acids that can be used to select suitable substituents are as follows:

[0252]

[0253] "Derivatives" or "variants" generally include those in which the amino acids appearing in the sequence are not naturally occurring amino acids but structural analogs thereof. The amino acids used in the sequence may also be derivatized or modified, such as labeled, as long as the function of the antibody is not significantly adversely affected.

[0254] Such derivatives and variants may be prepared during antibody synthesis or by post-production modification, or when the antibody is in recombinant form, using known techniques for site-directed mutagenesis, random mutagenesis or enzymatic cleavage and / or nucleic acid ligation.

[0255] Variant antibodies 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 HCVR / LCVR sequences and H and L chain sequences). In addition, the antibody may be a variant that has more than about 60% or more than about 70% amino acid identity, such as 75% or 80% amino acid identity, typically more than about 85% amino acid identity, such as more than about 90% or 95% amino acid identity with the HCVR / LCVR sequences and H and L chain sequences disclosed herein, while retaining the precise CDRs disclosed for these sequences. The variant may retain at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the HCVR / LCVR sequences and H and L chain sequences disclosed herein (while retaining the precise CDRs in some cases).

[0256] 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. Such amino acid identity levels can be seen over the entire length of the relevant SEQ ID NO sequence or 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.

[0257] With respect to amino acid sequences, "sequence identity" refers to sequences having the stated value when assessed using ClustalW (Thompson et al., 1994, supra) using the following parameters:

[0258] Pairwise alignment parameters—method: accurate, matrix: PAM, gap opening penalty: 10.00, gap extension penalty: 0.10;

[0259] Multiple alignment parameters - Matrix: PAM, Gap Open Penalty: 10.00, Delayed % Identity: 30, Penalize End Gaps: on, Gap Separation Distance: 0, Negative Matrix: on, Gap Extension Penalty: 0.20, Residue Specific Gap Penalty: on, Hydrophilic Gap Penalty: on, Hydrophilic Residues: GPSNDQEKR. Sequence identities for specific residues are intended to include identical residues that are derivatized only.

[0260] Thus provided are antibodies having specific sequences and variants which retain the function or activity of these chains.

[0261] The antibody may compete for binding to Gremlin-1 or bind to the same epitope as those defined above for H chain / L chain, HCVR / LCVR or CDR sequences. Specifically, the antibody may compete for binding to Gremlin-1 with an antibody comprising a HCDR1 / HCDR2 / HCDR3 / LCDR1 / LCDR2 / LCDR3 sequence combination of SEQ ID NO: 4 / 5 / 6 / 7 / 8 / 9, or bind to the same epitope as the antibody. The antibody may compete for binding to Gremlin-1 with an antibody comprising a HCVR and LCVR sequence pair of SEQ ID NO: 10 / 11 or 12 / 13, or a full-length chain of SEQ ID NO: 14 / 15 or 16 / 17, or bind to the same epitope as the antibody.

[0262] The term "epitope" is the region of an antigen to which an antibody binds. An epitope can be defined as a structural epitope or a functional epitope. Functional epitopes are typically a subset of structural epitopes and have residues that directly contribute to the affinity of the interaction. An epitope can also be a conformational epitope, i.e., comprise nonlinear amino acids. In certain embodiments, an epitope can include a determinant, which is a chemically active surface group of a molecule, such as an amino acid, a sugar side chain, a phosphoryl group, or a sulfonyl group, and in certain embodiments, can have specific three-dimensional structural features and / or specific charge characteristics.

[0263] It can be easily determined whether an antibody binds to the same epitope as a reference antibody or competes for binding with a reference antibody by using conventional 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 allowed to bind to a protein or peptide under saturation conditions. Next, the ability of the test antibody to bind to the protein or peptide is evaluated. If the test antibody is able to bind to the protein or peptide after saturation binding with the reference antibody, it can be concluded that the epitope bound by the test antibody is different from that bound by the reference antibody. On the other hand, if the test antibody is unable to bind to the protein or peptide after saturation binding with the reference antibody, the test antibody may bind to the same epitope as the epitope bound by the reference antibody of the present invention.

[0264] To determine whether an antibody competes with a reference antibody for binding, the above binding method is performed in two directions. In the first direction, the reference antibody is allowed to bind to the protein / peptide under saturation conditions, and then the binding of the test antibody to the protein / peptide molecule is evaluated. In the second direction, the test antibody is allowed to bind to the protein / peptide under saturation conditions, and then the binding of the reference antibody to the protein / peptide is evaluated. If in both directions, only the first (saturated) antibody is able to bind to the protein / peptide, it is concluded that the test antibody and the reference antibody compete for binding to the protein / peptide. Those skilled in the art will understand that an antibody that competes for binding with a reference antibody may not necessarily bind to the same epitope as the reference antibody, but may spatially block the binding of the reference antibody by binding to overlapping or adjacent epitopes.

[0265] If two antibodies each competitively inhibit (block) the binding of another antibody to the antigen, the two antibodies bind to the same or overlapping epitopes. That is, as measured in a competitive binding assay, 1, 5, 10, 20 or 100 times excess of one antibody inhibits the binding of another antibody by at least 50%, 75%, 90% or even 99% (see, e.g., Junghans et al., Cancer Res, 1990:50:1495-1502). Alternatively, if substantially all amino acid mutations that reduce or eliminate the binding of one antibody in the antigen reduce or eliminate the binding of another antibody, the two antibodies have the same epitope. If some amino acid mutations that reduce or eliminate the binding of one antibody reduce or eliminate the binding of another antibody, the two antibodies have overlapping epitopes.

[0266] Additional routine experiments (e.g., peptide mutations and binding analysis) can then be performed to confirm whether the observed lack of binding of the test antibody is actually due to binding to the same epitope as the reference antibody, or whether steric blocking (or another phenomenon) is the cause of the observed lack of binding. Such experiments can be performed using ELISA, RIA, surface plasmon resonance, flow cytometry, or any other quantitative or qualitative antibody binding assay available in the art.

[0267] The binding of the antibody to Gremlin-1 can be tested, for example, by standard ELISA or Western blot. ELISA assays can also be used to screen hybridomas that show a positive reaction 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. Therefore, the screening method can include a step of identifying antibodies capable of binding to Gremlin-1 by performing ELISA or Western blot or by flow cytometry.

[0268] The antibody can selectively (or specifically) recognize Gremlin-1. An antibody or other compound "selectively binds" or "selectively recognizes" a protein when it binds preferentially or with high affinity to the protein to which it selectively binds but does not substantially bind or binds with low affinity to other proteins. The selectivity of the antibody can be further studied by determining whether the antibody binds to other related proteins as described above or whether it discriminates between them. The antibodies used according to the present invention generally recognize human Gremlin-1.

[0269] Antibodies may also be cross-reactive to related proteins, or to human Gremlin-1 and Gremlin-1 from other species.

[0270] Specificity (or selectivity) will be understood as antibody and protein of interest binding, and no significant cross reactivity with any other molecule.Cross reactivity can be assessed by any suitable method as described herein.If the binding strength of antibody to another molecule is at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 100% of the binding strength of the protein of interest, then it can be considered that the cross reactivity of the antibody is significant.The binding strength of specific (or selectivity) antibody to another molecule may be lower than about 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25% or 20% of the binding strength of the protein of interest. The antibody may bind to the other molecule with less than about 20%, less than about 15%, less than about 10%, or less than about 5%, less than about 2%, or less than about 1% of its binding to the protein of interest.

[0271] Anti-Gremlin antibodies have been described previously, for example, WO2014 / 159010A1 (Regeneron) describes an anti-Gremlin antibody that inhibits Gremlin-1 activity with a binding affinity of K D The values ​​ranged from 625 pM to 270 nM at 25 °C. Ciuclan et al. (2013) described an anti-Gremlin-1 monoclonal antibody with a binding affinity K D 5.6x10 -10 M.

[0272] The anti-Gremlin-1 antibodies described herein (as well as WO / 2018 / 115017 filed on December 19, 2017 and WO2019 / 243801 filed on June 18, 2019, both of which are incorporated herein by reference in their entirety) are allosteric inhibitors of Gremlin-1 activity and bind to a new epitope that is away from the BMP binding site as described above. These antibodies have extremely high binding affinity to Gremlin-1, with Kd values ​​of <100 pM. Therefore, these antibodies are significantly improved over currently available antibodies and are expected to be particularly useful for treating Gremlin-1-mediated diseases.

[0273] Therefore, antibodies suitable for use in the present invention may have high affinity binding to (human) Gremlin-1. The antibodies may have a dissociation constant (K) of less than <1 nM, preferably <500 pM. D In one example, the antibody has a dissociation constant (K) of less than 200 pM. D In one example, the antibody has a dissociation constant (K) of less than 100 pM. D As is well known to those skilled in the art, a variety of methods can be used to determine the binding affinity of an antibody to its target antigen, such as surface plasmon resonance assays, saturation assays, or immunoassays such as ELISA or RIA. An exemplary method for determining binding affinity is to use a CM5 sensor chip in a BIAcore TM Surface plasmon resonance analysis was performed on a Biacore 2000 instrument (Biacore AB, Freiburg, Germany) as described by Krinner et al. (2007) Mol. Immunol. February; 44(5):916-25 (Epub 2006 May 11)).

[0274] The antibodies used according to the invention are typically inhibitory antibodies. Gremlin-1 negatively regulates BMP-2, 4 and 7, so inhibition of Gremlin-1 results in increased signaling through BMPs.

[0275] Specific functional assays that can be used to screen whether an antibody is able to inhibit Gremlin-1 include SMAD phosphorylation assays and Hek Id1 reporter gene assays. Typically, the inhibitory antibody restores SMAD phosphorylation and / or restores BMP signaling in the Hek Id1 reporter gene assay. SMAD phosphorylation may be restored by at least 80%, 90%, or 100% compared to a BMP control. In the Hek Id1 reporter gene assay, the inhibitory antibody may have an IC of less than 10 nM, preferably less than 5 nM. 50 .

[0276] 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 recombinantly produced by conventional methods.

[0277] The present disclosure also provides isolated DNA sequences encoding the heavy and / or light chain variable regions (or full-length H-chains and L-chains) of the antibody molecules newly described herein.

[0278] Variant polynucleotides may comprise 1, 2, 3, 4, 5, up to 10, up to 20, up to 30, up to 40, up to 50, up to 75 or more nucleic acid substitutions and / or deletions in any nucleic acid sequence given 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.

[0279] Suitable variants can be at least about 70% homologous to the polynucleotides of any one of the nucleic acid sequences disclosed herein, usually at least about 80 or 90% and more preferably at least about 95%, 97% or 99% homologous thereto. Variants can maintain at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity. Variants generally maintain about 60%-about 99% identity, about 80%-about 99% identity, about 90%-about 99% identity or about 95%-about 99% identity. These levels of homology and identity generally exist at least relative to the coding region of the polynucleotide. The method of measuring homology is well known in the art, and those skilled in the art will appreciate that homology is calculated based on nucleic acid identity in this article. This homology can be present in at least about 15, at least about 30, for example, at least about 40, 60, 100, 200 or more continuous nucleotides (depending on the length) region. Such homology may exist over the entire length of the unmodified polynucleotide sequence.

[0280] 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 under its default settings) (Devereux et al. (1984) Nucleic Acids Research 12, p387-395).

[0281] The PILEUP and BLAST algorithms can also be used to calculate homology or align sequences (usually under their default settings), as described, for example, in Altschul SF (1993) J Mol Evol 36:290-300; Altschul, S, F et al. (1990) J Mol Biol 215:403-10.

[0282] Software for performing BLAST analysis is publicly available through 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 match or satisfy a certain positive threshold score T when aligned with a word of the same length in the database sequence. T is called the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits serve as seeds for initiating searches to find HSPs containing them. As long as the cumulative alignment score can be increased, the word hits are extended in both directions along each sequence. The extension of the word hits in each direction is stopped when: the cumulative alignment score becomes zero or lower due to the accumulation of one or more negatively scored residue alignments; or 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), alignments (B) of 50, expectation (E) of 10, M=5, N=4, and a comparison of both strands.

[0283] The BLAST algorithm performs a statistical analysis of the similarity between two sequences; see, e.g., Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5787. One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability that a match between two nucleotide or amino acid sequences will occur by chance. For example, a sequence is considered similar to another sequence if the smallest sum probability in a comparison of a first sequence to a second sequence is less than about 1, typically less than about 0.1, suitably less than about 0.01, and most suitably less than about 0.001. For example, the smallest sum probability can be in the range of about 1 to about 0.001, typically about 0.01 to about 0.001.

[0284] Homologues can differ from sequences in related polynucleotides by less than about 3, 5, 10, 15, 20 or more mutations (each of which can be a substitution, deletion or insertion). For example, homologues can differ by 3-50 mutations, typically 3-20 mutations. These mutations can be measured over a region of at least 30, e.g., at least about 40, 60 or 100 or more consecutive nucleotides of a homologue.

[0285] In one embodiment, due to the redundancy of the genetic code, the variant sequence may be different from the specific sequence given in the sequence table. The DNA code has 4 main nucleic acid residues (A, T, C and G), and uses them to "spell" three letter codons, which represent the amino acids of the proteins encoded in the genes of the organism. The linear sequence of codons along the DNA molecule is translated into a linear sequence of amino acids in the proteins encoded by these genes. The code is highly degenerate, with 61 codons encoding 20 natural amino acids and 3 codons representing "stop" signals. Therefore, most amino acids are encoded by more than one codon-actually several are encoded by four or more different codons. Therefore, the variant polynucleotide of the present invention can encode the same polypeptide sequence as another polynucleotide of the present invention, but because different codons are used to encode the same amino acids, different nucleic acid sequences can be provided.

[0286] The DNA sequence may comprise synthetic DNA (eg, produced by chemical processing), cDNA, genomic DNA, or any combination thereof.

[0287] The DNA sequences encoding the antibody molecules described herein can be obtained by methods well known to those skilled in the art. For example, the DNA sequences encoding part or all of the heavy and light chains of the antibodies can be synthesized from a determined DNA sequence or based on the corresponding amino acid sequence as needed.

[0288] The general methods for constructing vectors, transfection methods and culture methods are well known to those skilled in the art. In this regard, reference is made to "Current Protocols in Molecular Biology", 1999, FM Ausubel (ed), Wiley Interscience, New York and the Maniatis Manual published by Cold Spring Harbor Publishing.

[0289] Nucleic acid antagonists

[0290] Polynucleotides, such as nucleic acids, are polymers comprising two or more nucleotides. Nucleotides may be naturally occurring or artificial. Nucleotides typically comprise a nucleobase, a sugar and at least one linking group, such as a phosphate, 2'O-methyl, 2'methoxyethyl, phosphoramidate, methylphosphonate or thiophosphate group. Nucleobases are typically heterocyclic. Nucleobases include, but are not limited to, purines and pyrimidines, more specifically adenine (A), guanine (G), thymine (T), uracil (U) and cytosine (C). Sugars are typically pentoses. Nucleotide sugars include, but are not limited to, ribose and deoxyribose. Nucleotides are typically ribonucleotides or deoxyribonucleotides. Nucleotides typically comprise monophosphates, diphosphates or triphosphates. Phosphates may be attached to the 5' or 3' side of a nucleotide.

[0291] Nucleotides include, but are not limited to, adenosine monophosphate (AMP), adenosine diphosphate (ADP), adenosine triphosphate (ATP), guanosine monophosphate (GMP), guanosine diphosphate (GDP), guanosine triphosphate (GTP), thymidine monophosphate (TMP), thymidine diphosphate (TDP), thymidine triphosphate (TTP), uridine monophosphate (UMP), uridine diphosphate (UDP), uridine triphosphate (UTP), cytidine monophosphate (CMP), cytidine diphosphate (CDP), cytidine triphosphate (CTP), 5-methylcytidine monophosphate, 5-methylcytidine diphosphate, 5-methylcytidine triphosphate, 5-hydroxymethylcytidine monophosphate, 5-hydroxymethylcytidine diphosphate, 5-hydroxymethylcytidine triphosphate, cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP), deoxyadenosine monophosphate (dAMP), deoxyadenosine diphosphate (d deoxycytidine triphosphate (dCTP), 5-methyl-2'-deoxycytidine monophosphate, 5-methyl-2'-deoxycytidine diphosphate, 5-methyl-2'-deoxycytidine triphosphate, 5-hydroxymethyl-2'-deoxycytidine monophosphate, 5-hydroxymethyl-2'-deoxycytidine diphosphate, and 5-hydroxymethyl-2'-deoxycytidine triphosphate. The nucleotide is preferably selected from AMP, TMP, GMP, UMP, dAMP, dTMP, dGMP or dCMP.

[0292] Nucleotides may contain additional modifications. Specifically, suitable modified nucleotides include, but are not limited to, 2'aminopyrimidines (e.g., 2'-aminocytidine and 2'-aminouridine), 2'-hydroxypurines (e.g., 2'-fluoropyrimidines (e.g., 2'-fluorocytidine and 2'-fluorouridine), hydroxypyrimidines (e.g., 5'-α-P-borane uridine), 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 with nucleobase modifications (e.g., 5-pentynyl-2'-deoxyuridine, 5-(3-aminopropyl)-uridine, and 1,6-diaminohexyl-N-5-carbamoylmethyluridine).

[0293] The nucleotides in a polynucleotide can be linked to each other in any manner. The nucleotides can be linked by phosphate, 2'O-methyl, 2'methoxyethyl, phosphoramidate, methylphosphonate or thiophosphate bonds. The nucleotides are usually linked by their sugar and phosphate groups, as in nucleic acids. The nucleotides can be linked by their core bases, as in pyrimidine dimers.

[0294] The GREM1 antagonist may be a polynucleotide encoding an anti-GREM1 antibody described herein.

[0295] A polynucleotide can be a nucleic acid, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). A polynucleotide can be any synthetic nucleic acid known in the art, such as peptide nucleic acid (PNA), glycerol nucleic acid (GNA), threose nucleic acid (TNA), locked nucleic acid (LNA), morpholino nucleic acid or other synthetic polymers with nucleotide side chains. A polynucleotide can be single-stranded or double-stranded.

[0296] The polynucleotide sequence can be cloned into any suitable expression vector. In the expression vector, the polynucleotide sequence encoding the construct is usually operably linked to a control sequence that can provide a host cell with expression of the encoding sequence. Such expression vectors can be used to express the construct.

[0297] In one embodiment, the anti-GREM1 antagonist is a polynucleotide encoding an anti-GREM1 antibody as described herein. The polynucleotide can be used for gene therapy. The polynucleotide can be provided in any suitable vector capable of providing in vivo expression of the anti-GREM1 antibody.

[0298] The polynucleotide encoding the anti-GREM1 antibody can be a DNA sequence. The DNA sequence can be provided in any suitable vector, such as an expression vector, for administration to a subject in need. For example, the DNA sequence can be administered to a subject in an expression vector that is capable of providing in vivo expression of the anti-GREM1 antibody. The expression vector can be a viral expression vector, such as an adeno-associated virus (AAV) vector. In one embodiment, the anti-GREM1 antagonist is a DNA sequence encoding an anti-GREM1 antibody as described herein. In one embodiment, the anti-GREM1 antagonist is a DNA sequence for gene therapy, wherein the DNA sequence encodes an anti-GREM1 antibody as described herein. In one embodiment, the anti-GREM1 antagonist is an AAV comprising a DNA sequence encoding an anti-GREM1 antibody as described herein. In one embodiment, the anti-GREM1 antagonist is an AAV for gene therapy, wherein the AAV comprises a DNA sequence encoding an anti-GREM1 antibody as described herein.

[0299] The polynucleotide encoding the anti-GREM1 antibody can be an RNA sequence. The RNA sequence can be administered to a subject in need in any suitable vector. The RNA sequence can be a messenger RNA (mRNA) sequence. The mRNA sequence can be administered to a subject in need in a stable form. For example, the mRNA sequence can be provided in a lipid nanoparticle (LNP) composition. The LNP composition can include any suitable LNP capable of encapsulating the mRNA sequence to provide increased stability of the mRNA sequence. Therefore, in one embodiment, the anti-GREM1 antagonist is a stabilized mRNA sequence encoding the anti-GREM1 antibody described herein. In one embodiment, the anti-GREM1 antagonist is a stabilized mRNA sequence for gene therapy, wherein the mRNA sequence encodes the anti-GREM1 antibody described herein. In one embodiment, the anti-GREM1 antagonist is a LNP composition comprising an mRNA encoding an anti-GREM1 antibody described herein. In one embodiment, the anti-GREM1 antagonist is a LNP composition for gene therapy, wherein the LNP composition comprises an mRNA encoding an anti-GREM1 antibody described herein.

[0300] The term "operably linked" refers to a juxtaposition wherein the described components are in a relationship permitting them to function in their intended manner. A control sequence "operably linked" to a coding sequence is linked in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences. Multiple copies of the same or different polynucleotides may be introduced into a vector.

[0301] The expression vector can then be introduced into a suitable host cell.Thus, the construct can be produced by inserting a construct encoding a polynucleotide sequence into an expression vector, introducing the vector into a compatible bacterial host cell and culturing the host cell under conditions that result in expression of the polynucleotide sequence.

[0302] Nucleic acid-based GREM1 antagonists can reduce the expression of GREM1. Antisense and RNA interference (RNAi) technologies for knocking down protein expression are well known in the art, and standard methods can be used to knock down the expression of molecules of interest. Both antisense and siRNA technologies interfere with mRNA. Antisense oligonucleotides interfere with mRNA by binding (hybridizing) to a portion of mRNA. Therefore, antisense oligonucleotides are designed to be complementary to mRNA (although oligonucleotides do not have to be 100% complementary, as described below). In other words, antisense oligonucleotides can be part of a cDNA. Similarly, the oligonucleotide sequence may not be 100% identical to the cDNA sequence. This will also be 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.

[0303] Thus, the antagonist may be an oligonucleotide that specifically hybridizes to an mRNA encoding GREM1 (e.g., a coding sequence of SEQ ID NO: 36 or SEQ ID NO: 37, or a variant thereof). An oligonucleotide "specifically hybridizes" to a target sequence when the oligonucleotide preferentially or highly hybridizes to the target sequence but does not substantially hybridize, does not hybridize, or hybridizes with only low affinity to other sequences. More preferably, the oligonucleotide hybridizes to the target sequence with a high affinity. m T m The conditions for hybridization are at least 5°C, at least 10°C, at least 20°C, at least 30°C or at least 40°C. 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-lnterscience, New York (1995)). The hybridization conditions may be stringent conditions described in the art.

[0304] Oligonucleotide is a short nucleotide polymer, which usually has 50 or less nucleotides, such as 40 or less, 30 or less, 22 or less, 21 or less, 20 or less, 10 or less or 5 or less nucleotides. The oligonucleotide length used can be 20 to 25 nucleotides, more preferably 21 or 22 nucleotides. Nucleotide can be naturally occurring or artificial. Nucleotide can be any of the above.

[0305] A GREM1 antagonist can be an antibody that binds to GREM1, typically specifically. An antibody "specifically binds" a protein when it binds to the protein with a preferential or high affinity, but does not substantially bind, does not bind, or only binds to other proteins with a low affinity. For example, an antibody "specifically binds" a target molecule when it binds to the target molecule with a preferential or high affinity, but does not substantially bind, does not bind, or only binds to other human proteins with a low affinity.

[0306] If the antibody binds with a Kd of 1x10-7 M or less, more preferably 5x10-8 M or less, more preferably 1x10-8 M or less, or more preferably 5x10-9 M or less, then the antibody binds with preferential or high affinity. If the antibody binds with a Kd of 1x10-6 M or more, more preferably 1x10-5 M or more, more preferably 1x10-4 M or more, more preferably 1x10-3 M or more, even more preferably 1x10-2 M or more, then the antibody binds with low affinity.

[0307] The antibody can be, for example, a monoclonal antibody, a polyclonal antibody, a single-chain antibody, a chimeric antibody, a bispecific antibody, a CDR-grafted antibody or a humanized antibody. The antibody can be a complete immunoglobulin molecule or a fragment thereof, such as a Fab, F(ab')2 or Fv fragment.

[0308] patient

[0309] Any patient can be treated according to the present invention. The patient is typically a human. However, the patient can be another mammal, such as 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.

[0310] Pharmaceutical compositions, dosages and dosage regimens

[0311] The GREM1 antagonists of the present invention can be provided in pharmaceutical compositions. Cytidine analogs or deoxycytidine analogs can be provided as part of the same pharmaceutical composition or in a separate pharmaceutical composition. For example, gemcitabine or a derivative thereof can be provided as part of the same pharmaceutical composition or in a separate pharmaceutical composition. For example, azacitidine, cytarabine, decitabine, troxacitabine or a derivative thereof can be provided as part of the same pharmaceutical composition or in a separate pharmaceutical composition. Proliferation-dependent cytotoxic agents can also be provided as part of the same pharmaceutical composition or in a separate pharmaceutical composition. Pharmaceutical compositions are generally sterile and generally include a pharmaceutically acceptable carrier and / or adjuvant. These compositions, in addition to the therapeutically active ingredients, may also include pharmaceutically acceptable excipients, carriers, diluents, buffers, stabilizers or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The pharmaceutical carrier or diluent can be, for example, an isotonic solution.

[0312] As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc. that are physiologically compatible. The precise nature of the carrier or other material may depend on the route of administration, for example oral, intravenous, cutaneous or subcutaneous, nasal, intramuscular and intraperitoneal.

[0313] The carrier may be suitable for parenteral administration, such as intravenous, intramuscular, intradermal, intraocular, intraperitoneal, subcutaneous, spinal or other parenteral administration routes, such as by injection or infusion. Alternatively, the carrier may be suitable for non-parenteral administration, such as topical, epidermal or mucosal administration routes. The carrier may be suitable for oral administration. Depending on the route of administration, the modulator may be encapsulated in a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound. For example, solid oral forms may contain diluents such as lactose, glucose, sucrose, cellulose, corn starch or potato starch together with the active substance; lubricants such as silicon dioxide, talc, stearic acid, magnesium stearate or calcium stearate and / or polyethylene glycol; binders such as starch, gum arabic, gelatin, methylcellulose, carboxymethylcellulose or polyvinylpyrrolidone; disintegrators such as starch, alginic acid, alginates or sodium starch glycolate; effervescent mixtures; dyes; sweeteners; wetting agents such as lecithin, polysorbate, lauryl sulfate; and, in general, nontoxic and pharmacologically inactive substances used in pharmaceutical preparations. Such pharmaceutical preparations can be prepared in a known manner, for example by mixing, granulating, tabletting, sugar coating or film coating processes.

[0314] Other oral formulations include commonly used excipients, such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. These compositions are in the form of solutions, suspensions, tablets, pills, capsules, sustained-release formulations or powders, and contain 10% to 95% of active ingredients, preferably 25% to 70%. When the pharmaceutical composition is lyophilized, the lyophilized material can be reconstituted before administration, such as a suspension. Reconstitution is preferably carried out in a buffer.

[0315] Capsules, tablets, and pills for oral administration to a subject may be provided with enteric coatings comprising, for example, Eudragit "S", Eudragit "L", cellulose acetate, cellulose acetate phthalate, or hydroxypropyl methylcellulose.

[0316] Liquid dispersions for oral administration may be syrups, emulsions or suspensions. Syrups may contain as carriers, for example, sucrose or sucrose with glycerol and / or mannitol and / or sorbitol.

[0317] Suspensions and emulsions may contain, as carriers, for example, natural gums, agar, sodium alginate, pectin, methylcellulose, carboxymethylcellulose or polyvinyl alcohol. Suspensions or solutions for intramuscular injection may contain, together with the active substance, a pharmaceutically acceptable carrier such as sterile water, olive oil, ethyl oleate, glycols such as propylene glycol, and, if necessary, an appropriate amount of lidocaine hydrochloride.

[0318] Solutions for intravenous administration or infusion may contain, for example, sterile water as a carrier, or preferably they may be in the form of sterile, aqueous, isotonic saline solutions.

[0319] 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%.

[0320] Polynucleotide or oligonucleotide inhibitor can be naked nucleotide sequence, or can be combined with cationic lipid, polymer or targeting system.They can be delivered by any available technology.For example, polynucleotide or oligonucleotide can be introduced by needle injection, preferably intradermal, subcutaneous or intramuscular injection.Or, delivery device (such as particle-mediated gene delivery) can be used to deliver polynucleotide or oligonucleotide directly through skin.Polynucleotide or oligonucleotide can be topically applied to skin, or by for example intranasal, oral or rectal administration to mucosal surface.

[0321] The absorption of polynucleotide or oligonucleotide constructs can be enhanced by several known transfection techniques, such as those involving the use of transfection agents. Examples of these agents include cationic agents, such as calcium phosphate and DEAE-dextran and lipofectants, such as lipofectam and transfectam. The dosage of the polynucleotide or oligonucleotide to be administered can be varied.

[0322] The pharmaceutical composition of the present invention may contain one or more pharmaceutically acceptable salts. "Pharmaceutically acceptable salts" refers to salts that retain the desired biological activity of the parent compound and do not impart any undesirable toxicological effects. Examples of these salts include acid addition salts and base addition salts.

[0323] Pharmaceutically acceptable carriers include aqueous carriers or diluents. Examples of suitable aqueous carriers that can be used for pharmaceutical compositions of the present invention include water, buffered water, and saline. Examples of other carriers include ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.), and suitable mixtures thereof, vegetable oils (e.g., olive oil), and injectable organic esters (e.g., ethyl oleate). In many cases, it is desirable to include isotonic agents such as sugars, polyols such as mannitol, sorbitol, or sodium chloride in the compositions.

[0324] 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.

[0325] The pharmaceutical compositions of the present invention may contain additional active ingredients.

[0326] Also within the scope of the present disclosure are kits comprising the combination therapies described herein and instructions for use.The kits may further contain one or more additional agents, such as additional therapeutic or prophylactic agents discussed herein.

[0327] The antagonists described herein, or formulations or compositions thereof, may be administered for prophylactic and / or therapeutic treatments.

[0328] In therapeutic applications, the compound is administered to a subject already suffering from a disorder or condition as described above in an amount sufficient to cure, alleviate, or partially arrest the condition or one or more symptoms thereof. 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 purpose is defined as a "therapeutically effective amount".

[0329] In preventive applications, the formulation is administered to a subject at risk of having a disorder or condition as described above 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 achieve this purpose is defined as a "prophylactically effective amount". The effective amount for each purpose will depend on the severity of the disease or injury and the weight and general state of the subject.

[0330] The subject of administration can be a human or non-human animal. The term "non-human animal" includes all vertebrates, such as mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc. Administration to humans is typical.

[0331] Antagonists, proliferation-dependent cytotoxic agents (e.g., gemcitabine, cytidine analogs or deoxycytidine analogs) or pharmaceutical compositions of the present invention can be administered by 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 of administration and / or mode will vary depending on the desired result. Examples of routes of administration of the compounds or pharmaceutical compositions of the present invention include intravenous, intramuscular, intradermal, intraocular, intraperitoneal, subcutaneous, spinal or other parenteral routes of administration, such as by injection or infusion. The phrase "parenteral administration" as used herein refers to a mode of administration other than enteral and topical administration, typically by injection. Alternatively, the antibodies / modulators or pharmaceutical compositions of the present invention can be administered by non-parenteral routes, such as local, epidermal or mucosal routes of administration. The antibodies / modulators or pharmaceutical compositions of the present invention can be used for oral administration.

[0332] The antibody / modulator or pharmaceutical composition of the present invention of suitable dosage can be determined by a skilled physician. The actual dosage level of the active ingredient in the pharmaceutical composition of the present invention can be changed to obtain the amount of the active ingredient that is effective to achieve the desired therapeutic response of a specific patient, composition and mode of administration and is non-toxic to the patient. The dosage level selected will depend on a variety of pharmacokinetic factors, including the activity of the specific composition of the present invention used, the route of administration, the time of administration, the excretion rate of the specific compound used, the duration of treatment, other drugs, compounds and / or materials used in combination with the specific composition used, 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.

[0333] For example, depending on the above-mentioned circumstances, a suitable dosage may be, for example, about 0.01 μg / kg to about 1000 mg / kg body weight, typically about 0.1 μg / kg to about 100 mg / kg body weight, which is the body weight of the patient to be treated. For example, a suitable dosage may be about 1 μg / kg to about 10 mg / kg body weight / day or about 10 μg / kg to about 5 mg / kg body weight / day.

[0334] The dosage regimen can be adjusted to provide the best desired response (e.g., therapeutic response). For example, a single dose can be administered, multiple divided doses can be administered over time, or the dose can be proportionally reduced or increased according to the urgency of the treatment situation. The dosage can be provided as multiple doses, for example, taken at fixed intervals, such as 2, 3, or 4 doses per hour. Multiple doses can be administered by the same or different routes and administered to the same or different locations. Alternatively, the dosage can be administered by a sustained release formulation, in which case less frequency of administration is required. The dosage and frequency can vary depending on the half-life of the antagonist in the patient and the desired duration of treatment.

[0335] 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.

[0336] Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.

[0337] As noted above, the modulators / antibodies or pharmaceutical compositions of the invention may be co-administered with one or more other therapeutic agents.

[0338] Examples of the above techniques and protocols can be found in Remington's Pharmaceutical Sciences, 20th Edition, 2000, pub. Lippincott, Williams & Wilkins.

[0339] The combined administration of two or more agents can be achieved in a variety of different ways. Both can be administered together in a single composition, or can be administered as part of a combined therapy in a separate composition. For example, one agent can be administered before another agent or administered alone, after or sequentially, or in parallel or simultaneously with it. The anti-GREM1 antagonist can be administered before or administered alone, after or sequentially, or in parallel or simultaneously with the cytidine analog or deoxycytidine analog described herein. For example, the anti-GREM1 antagonist can be administered before or administered alone, after or sequentially, or in parallel or simultaneously with gemcitabine or its derivatives. The anti-GREM1 antagonist can be administered before or administered alone, after or sequentially, or in parallel or simultaneously with gemcitabine, azacitidine, cytarabine, decitabine, troxacitabine or its derivatives, or administered alone, after or sequentially, or in parallel or simultaneously with it. Alternatively, in the case of pancreatic cancer, the anti-GREM1 antagonist can be administered before or administered alone, after or sequentially, or in parallel or simultaneously with the proliferation-dependent cytotoxic agent.

[0340] Other treatment combinations

[0341] The combination therapy of the present invention as described above can be used / administered in combination with another therapeutic therapeutic composition, for example as an adjuvant therapy. Other therapeutic compositions or methods of treatment can be one or more of those discussed herein, and can be administered simultaneously or sequentially with the compositions of the present invention.

[0342] As described above, GREM1 antagonists have particular utility in combination therapies because they can be used to sensitize a cancer or tumor to another anticancer agent, such as radiation therapy or surgery. In the absence of GREM1 antagonists, the cancer may be resistant to other anticancer agents or cancer therapies.

[0343] Therefore, the anti-GREM1 antagonist combined with gemcitabine or its derivatives can also be used in combination with any other cancer therapy or any other cancer therapeutic agent. In addition, the anti-GREM1 antagonist combined with the proliferation-dependent cytotoxic agent can also be used in combination with any other cancer therapy or any other therapeutic agent for the treatment of pancreatic cancer. Other cancer therapies can be selected from any known therapy for related cancers, such as any known therapy for pancreatic cancer. Other cancer therapies can be radiotherapy. Suitable radiotherapy treatments are described, for example, in Van Cutsem (et al.) Annals of Oncology, 2014. Vol 25, Issue 3. Radiotherapy can be performed before or after cancer surgery. Radiotherapy can be adjuvant radiotherapy. Radiotherapy can be combined with chemotherapy, for example, in combination with gemcitabine or proliferation-dependent cytotoxic agents described herein. For example, a combination therapy comprising a GREM1 antagonist and gemcitabine or its derivatives can be used in combination with radiotherapy. In addition, in the case of pancreatic cancer, a combination therapy comprising a GREM1 antagonist and a proliferation-dependent cytotoxic agent can be used in combination with radiotherapy.

[0344] Another therapeutic agent for cancer, such as another chemotherapeutic agent, can be selected from any known therapeutic agent for the relevant cancer, including any known chemotherapeutic agent or combination of chemotherapeutic agents for the relevant cancer. For example, a combination therapy comprising a GREM1 antagonist and gemcitabine can be used in combination with one or more of nab-paclitaxel, paclitaxel, oxaliplatin, folinic acid, irinotecan, fluorouracil, FOLFIRINOX or FOLFOX, particularly in the treatment of pancreatic cancer. For example, an anti-GREM1 antagonist used according to the present invention can be administered in combination with gemcitabine and another cytidine analog or deoxycytidine analog.

[0345] In addition, the combination therapy comprising a GREM1 antagonist and a proliferation-dependent cytotoxic agent can be used in combination with another chemotherapeutic agent (e.g., gemcitabine, albumin-bound paclitaxel, paclitaxel, oxaliplatin, folinic acid, irinotecan, fluorouracil, FOLFIRINOX or FOLFOX). Any combination described herein is considered in the context of compositions and kits for treating cancer or pancreatic cancer. In a preferred embodiment, the anti-GREM1 antagonist used in accordance with the present invention can be administered in combination with gemcitabine and troxacitabine. In a particularly preferred embodiment, the anti-GREM1 antagonist used in the method for treating pancreatic cancer can be administered in combination with gemcitabine and troxacitabine. When not administered with a combination therapy comprising a GREM1 antagonist described herein, the cancer can be resistant to radiotherapy or one or more chemotherapeutic agents (e.g., one of the above-mentioned chemotherapeutic agents).

[0346] As part of the above aspects, the present invention provides an anti-GREM1 antagonist according to the present invention in combination with gemcitabine or a derivative thereof or a proliferation-dependent cytotoxic agent for use in a method for treating and / or preventing cancer (e.g. pancreatic cancer), wherein the method further comprises administering an additional anti-cancer agent separately, continuously or simultaneously.

[0347] Compositions and kits

[0348] Also provided are compositions or kits comprising an anti-GREM1 antagonist and a cytidine analog or a deoxycytidine analog. The cytidine analog or deoxycytidine analog described herein may be one or more of gemcitabine, azacitidine, cytarabine, decitabine, or troxacitabine. In one embodiment, a composition or kit comprising an anti-GREM1 antagonist and gemcitabine or a derivative thereof is provided. The present invention also includes a composition or kit comprising an anti-GREM1 antagonist and capecitabine or a derivative thereof. The derivative of gemcitabine may be any derivative as described above. The anti-GREM1 antagonist may be any anti-GREM1 antagonist described herein. The composition or kit may be suitable for treating pancreatic cancer.

[0349] Compositions or kits comprising an anti-GREM1 antagonist and a mitotic inhibitor are also provided. The composition or kit may comprise an anti-GREM1 antagonist and one or more mitotic inhibitors selected from the group consisting of: cabazitaxel, docetaxel, paclitaxel, vinblastine, vincristine, vinorelbine and nab-paclitaxel. In a preferred embodiment in the context of pancreatic cancer, the mitotic inhibitor and / or microtubule stabilizing drug is selected from the group consisting of nab-paclitaxel and paclitaxel, particularly as part of a composition or kit for treating pancreatic cancer. A preferred combination comprises an anti-GREM1 antagonist and paclitaxel or nab-paclitaxel.

[0350] A bispecific antibody combining an anti-GREM1 specificity and one of the other specificities described above may be provided in a composition or kit as described herein.The anti-GREM1 antagonist in any of the compositions and kits described above may preferably be an anti-GREM1 antibody.

[0351] Detection and diagnosis

[0352] Based on the correlation between stromal GREM1 and cancer, the present invention also provides additional means for predicting the responsiveness of cancer to treatment.

[0353] The present invention provides a method for determining whether a patient having or suspected of having cancer or being at risk of developing cancer is likely to respond to a combination treatment using a GREM1 antagonist and a cytidine analog or a deoxycytidine analog (e.g., gemcitabine or a derivative thereof), 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 the combination treatment.

[0354] The present invention also provides a method for determining whether a patient having or suspected of having pancreatic cancer or at risk of developing pancreatic cancer is likely to respond to a combination treatment with a GREM1 antagonist and a 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 the combination treatment.

[0355] The predicted individual responsiveness to a given therapy means that the individual is expected to benefit from receiving the therapy or a sufficient degree of benefit. The predicted individual non-responsiveness to therapy means that the individual cannot be expected to benefit from receiving the therapy or a sufficient degree of benefit. The method of predicting the response can be performed before administering a combination therapy using a GREM1 antagonist and a cytidine analog or a deoxycytidine analog (e.g., gemcitabine or its derivatives) or a GREM1 antagonist and a proliferation-dependent cytotoxic agent. The prediction can then be considered when selecting or recommending a suitable treatment for the individual. Alternatively, the method can be performed after treatment with therapy and used to monitor and predict the individual's response to treatment. Typically, the method is used to predict whether an individual will have a primary response to treatment using therapy, i.e., whether the individual will respond when first receiving treatment. In some cases, the method is used to predict secondary non-responsiveness, i.e., whether an individual who initially responds to treatment will stop responding to treatment later or will respond poorly to treatment.

[0356] In some cases, overexpression of GREM1 compared to a reference sample or reference level indicates that the individual will respond to therapy using a combination therapy described herein. A combination therapy comprising a GREM1 antagonist in combination with a cytidine analog or a deoxycytidine analog as described herein can then be selected or recommended, which can then be further administered to the individual. A combination therapy comprising a GREM1 antagonist in combination with gemcitabine or a derivative thereof can then be selected or recommended, which can then be further administered to the individual. Similarly, in the case of pancreatic cancer, a therapy comprising a GREM1 antagonist used in combination with a proliferation-dependent cytotoxic agent can be selected based on overexpression of GREM1.

[0357] In other cases, a reduced or normal GREM1 level compared to a reference sample or reference level indicates that the individual will not respond to therapy with a GREM1 antagonist. 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. In addition, a therapeutic treatment other than the combination therapy described herein may be selected or recommended to treat the individual, which may then be further administered to the individual.

[0358] In all aspects of the invention, an individual with cancer (e.g., pancreatic cancer) or an individual suspected of having a disease or condition and / or an individual at risk of developing a disease or condition may be selected for treatment or identification. For example, an individual may not have been formally diagnosed, but may be suspected of having the disease or condition due to the presence of one or more symptoms. An individual may be considered to be at risk of developing cancer if they have one or more risk factors associated with cancer and / or one or more tendencies that increase their susceptibility to cancer. Risk factors associated with pancreatic cancer may include familial pancreatitis and inherited gene mutations caused by PRSS1 gene mutations, such as one or more mutations in the GREM1 encoding gene, or any other mutation that affects GREM1 gene expression.

[0359] The following examples illustrate the invention. Example

[0360] Materials and methods

[0361] Test antibodies and drugs Anti-gremlin-1 antibody - Ab7326 mIgG1-APP.4405.IgG.mFc - Lot No. - PB4682

[0362] Vehicle - phosphate buffered saline, pH 7.4 (provided by Beatson Institute)

[0363] Gemcitabine - (provided by the Beatson Institute, purchased from LC Labs, Woburn, MA, USA) in phosphate buffered saline, pH 7.4.

[0364] Genetically modified mice

[0365] LSL- K ras G12D / +;Tr p 53 R172H / +; Pdx1- C re(KPC) mice have been described previously (Hingorani et al., 2005). Mice were generated by crossing mice carrying Pdx1-Cre and conditional LSL-KrasG12D or LSL-Trp53R172H alleles (strains 01XJ6 and 01XL9, Mouse Models of Human Cancer Consortium [MMHCC], NCI-Frederick, Frederick, MD, USA). Mice of mixed background were bred in-house at the CRUK Beatson Institute and maintained in conventional cages in an enriched environment with free access to standard food and water. Genotyping was performed by Transnetyx (Cordoba, TN, USA). Mice of both sexes were recruited to the study. All animal experiments were performed under a UK Home Office license and approved by the University of Glasgow Animal Welfare and Ethical Review Committee.

[0366] treat

[0367] Mice were monitored 3 times per week until pancreatic cancer was diagnosed by abdominal palpation and confirmed by ultrasound imaging. Mice were randomly assigned to treatment groups and given the following doses: 30 mg / kg Ab7326 mIgG1 subcutaneously twice a week; 100 mg / kg gemcitabine intraperitoneally twice a week; 30 mg / kg Ab7326 mIgG1 subcutaneously twice a week and 100 mg / kg gemcitabine intraperitoneally twice a week; or PBS vehicle control twice a week. There were ≥5 mice in each group. Mice were monitored daily and killed when ethical endpoints were reached (symptoms included abdominal distension, cachexia, intermittent hunchback or decreased activity, erect hair, mild diarrhea, anemia). Statistical evaluation of survival from the start of treatment was performed by Kaplan-Meier and Log-Rank analysis.

[0368] Ultrasound imaging

[0369] The VisualSonics Vevo 3100 preclinical imaging platform (FUJIFILM VisualSonics, Toronto, Canada) was used for high-resolution ultrasound imaging to confirm tumor diagnosis and monitor tumor progression weekly. Anesthesia was induced and maintained with a mixture of isoflurane and medical air. Tumor volume was calculated and plotted longitudinally for each mouse weekly.

[0370] sampling

[0371] Mice were culled according to the schedule 1 method according to institutional guidelines. Postmortem tumor burden was assessed by gross pathology and histology. Organs were removed and fixed in 10% buffered formalin. Terminal blood samples were collected whenever possible. The majority of tumors were fixed in 10% buffered formalin for FFPE processing, and the remainder was harvested to (Sigma-Aldrich) for RNA preparation and / or snap freezing. Fixed tissues were embedded in paraffin and 5 μm sections were placed on poly-L-lysine slides for IHC analysis.

[0372] Histology and immunohistochemistry

[0373] H&E and picrochrome staining were performed on formalin-fixed, paraffin-embedded tissues as described previously. Immunohistochemistry was performed using standard protocols. Briefly, formalin-fixed, paraffin-embedded sections were dewaxed 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 in serum and then incubated with primary antibodies. Sections were incubated in secondary antibodies for 30 min and visualized by staining with 3,3'-diaminobenzidine tetrahydrochloride. The 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).

[0374] Example 1: Confirmation of Grem1 mRNA expression in pancreatic cancer

[0375] To investigate whether gremlin-1 might play a role in pancreatic cancer, Grem1 mRNA expression as measured 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 that high expression is significantly associated with poor prognosis ( Figure 1 The results are consistent with a published study that reported an association between Grem1 expression and poor prognosis in pancreatic cancer (Yu et al., 2018).

[0376] 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 primary pancreatic cancer models) and compared to normal pancreatic duct epithelium expressing the control KrasG12D. The results showed that grem1 mRNA expression was elevated in KPC tumors (and other mouse pancreatic tumors) compared to controls (Table 1).

[0377] Table 1

[0378]

[0379]

[0380] Example 2: Treatment with a combination of Ab7326 mIgG1 and gemcitabine results in improved survival in the KPC mouse model compared to treatment with vehicle control or Ab7326 mIgG1 and gemcitabine as single agents

[0381] In accordance with local requirements, a pilot study was conducted in healthy littermate mice to ensure tolerability of the dose and schedule provided. No adverse effects were observed.

[0382] Next, the effectiveness of gremlin-1 inhibition as a potential treatment for pancreatic cancer was tested in the KPC model. KPC mice develop tumors that resemble human pancreatic tumors both histologically and pathologically. In addition, these tumors are extremely aggressive, often metastatic, and highly chemoresistant, again mimicking human pancreatic cancer. A cohort of KPC mice was established and the mice were monitored until they developed pancreatic cancer that was detectable by palpation. The breeding strategy and experimental design required to generate these mice are described in detail in the literature. Figure 2 shown.

[0383] Mice were monitored at least once a week by palpation until pancreatic tumors were detectable. At this point, high-resolution ultrasound imaging was used to confirm the presence of pancreatic cancer, and mice were included in cohorts for treatment with anti-Gremlin1 antibody Ab7326 mIgG1, gemcitabine standard chemotherapy, Ab7326mIgG1 + gemcitabine combination, or vehicle control (see Table 2 for details). High-resolution ultrasound imaging was performed once a week during treatment to monitor the tumor burden of each individual mouse during treatment. Mice continued to be studied and closely monitored until ethical endpoints were reached, at which time they were sacrificed according to institutional guidelines. Clinical features of manifestation included abdominal distension, decreased ability to regulate body (indicating cachexia), decreased activity, and occasional jaundice.

[0384] Table 2

[0385]

[0386]

[0387] Analysis of ultrasound 3D tumor imaging showed no tumor shrinkage or stagnation in any treatment group ( Figure 3 However, the median survival of mice treated with the combination of Ab7326 mIgG1 and gemcitabine (37 days) was significantly increased compared to vehicle controls (20 days) (log rank, p = 0.037, see Figure 4 , data for individual mice in Tables 2 and 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) alone. The benefit of gemcitabine monotherapy was minimal, which is consistent with recent findings. (Olive et al., 2009; Frese et al., 2012; Provenzano et al., 2012).

[0388] Table 3

[0389]

[0390]

[0391] After the experimental mice were sacrificed, gross pathological examination did not reveal any differences in tumor burden or phenotype between the different experimental conditions. Immunohistochemical (IHC) analysis was performed on formalin-fixed paraffin-embedded tumor tissues to assess tumor cell proliferation (Ki67) and apoptosis (cleaved caspase 3). We also performed IHC-based analysis of the tumor microenvironment to assess any changes in the number of alpha-SMA-positive tumor-associated fibroblasts or the quality or quantity of collagens I and III (measured by picric acid red staining). Staining did not reveal any obvious effects on these parameters in any of the tested regimens (data not shown).

[0392] result

[0393] In conclusion, Gremlin-1 represents an effective target for the treatment of pancreatic cancer, and the results presented here suggest that Ab7326mIgG1 can be safely used in KPC mice as a single agent or in combination with gemcitabine. When mice were treated with a combination of Ab7326 mIgG1 and gemcitabine, a significant improvement in survival was observed compared to vehicle controls ( Figure 4 , 9Table 2, and data for individual mice in Table 3).

[0394] Aspects of the Invention

[0395] 1. An anti-GREM1 antagonist for use in a method of treating or preventing cancer, wherein the method further comprises administering a cytidine analog or a deoxycytidine analog.

[0396] 2. An anti-GREM1 antagonist for use according to aspect 1, wherein the cancer is a solid cancer.

[0397] 3. An anti-GREM1 antagonist for use according to aspect 1 or aspect 2, wherein the cancer has stromal GREM1 overexpression

[0398] 4. An anti-GREM1 antagonist for use according to any preceding aspect, wherein the cancer is a metastatic cancer.

[0399] 5. An anti-GREM1 antagonist for use according to any preceding aspect, wherein the cancer comprises dormant cancer cells, optionally dormant stem-like cancer cells.

[0400] 6. An anti-GREM1 antagonist for use according to any preceding aspect, wherein the cancer is a recurrent cancer and / or wherein the method is for preventing cancer recurrence.

[0401] 7. An anti-GREM1 antagonist for use according to any of the preceding aspects, wherein the cancer is a cancer that is poorly responsive, unresponsive or refractory to treatment with a cytidine analogue or a deoxycytidine analogue.

[0402] 8. An anti-GREM1 antagonist for use according to aspect 7, wherein the cancer is a cancer that is poorly responsive, unresponsive or refractory to treatment with gemcitabine or a derivative thereof.

[0403] 9. An anti-GREM1 antagonist for use according to any 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, spleen cancer, bone resident cancer and osteosarcoma.

[0404] 10. An anti-GREM1 antagonist for use according to aspect 9, wherein the cancer is pancreatic cancer.

[0405] 11. An anti-GREM1 antagonist for use according to aspect 10, wherein the pancreatic cancer is exocrine pancreatic cancer.

[0406] 12. An anti-GREM1 antagonist for use according to aspect 10 or aspect 11, wherein the pancreatic cancer is pancreatic ductal adenocarcinoma (PDAC).

[0407] 13. An anti-GREM1 antagonist for use according to aspect 9, wherein the cancer is lung cancer.

[0408] 14. An anti-GREM1 antagonist for use according to aspect 13, wherein the cancer is non-small cell lung cancer.

[0409] 15. An anti-GREM1 antagonist for use according to aspect 9, wherein the cancer is bladder cancer.

[0410] 16. An anti-GREM1 antagonist for use according to aspect 9, wherein the cancer is breast cancer.

[0411] 17. An anti-GREM1 antagonist for use according to aspect 9, wherein the cancer is ovarian cancer.

[0412] 18. An anti-GREM1 antagonist for use according to any preceding aspect, wherein the cancer has epithelial GREM1 overexpression.

[0413] 19. An anti-GREM1 antagonist for use according to aspect 18, wherein the cancer is a GREM1 driven cancer.

[0414] 20. An anti-GREM1 antagonist for use according to any preceding aspect, wherein the cancer is a disseminated cancer.

[0415] 21. An anti-GREM1 antagonist for use according to any preceding aspect, wherein the cancer is an established cancer.

[0416] 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.

[0417] 23. An anti-GREM1 antagonist for use according to any one of aspects 1 to 21, wherein the cytidine analogue or deoxycytidine analogue is azacitidine or a derivative thereof.

[0418] 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.

[0419] 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.

[0420] 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.

[0421] 27. An anti-GREMl antagonist for use in a method of treating or preventing pancreatic cancer, wherein the method further comprises administering a proliferation-dependent cytotoxic agent.

[0422] 28. An anti-GREMl antagonist for use according to aspect 27, wherein the pancreatic cancer is exocrine pancreatic cancer.

[0423] 29. An anti-GREMl antagonist for use according to aspect 27 or aspect 28, wherein the pancreatic cancer is pancreatic ductal adenocarcinoma (PDAC).

[0424] 30. An anti-GREMl antagonist for use according to any one of aspects 27 to 29, wherein the pancreatic cancer is metastatic pancreatic cancer.

[0425] 31. An anti-GREMl antagonist for use according to any one of aspects 27 to 30, wherein the pancreatic cancer is recurrent pancreatic cancer.

[0426] 32. An anti-GREMl antagonist for use according to any one of aspects 27 to 31, wherein the pancreatic cancer is a pancreatic cancer that is poorly responsive, unresponsive or refractory to treatment with a proliferation-dependent cytotoxic agent.

[0427] 33. An anti-GREMl antagonist for use according to any one of aspects 27 to 32, wherein the proliferation-dependent cytotoxic agent is a nucleoside inhibitor or an antimetabolite.

[0428] 34. An anti-GREMl 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.

[0429] 35. An anti-GREMl antagonist for use according to any one of aspects 27 to 32, wherein the proliferation-dependent cytotoxic agent is a mitotic inhibitor.

[0430] 36. An anti-GREM1 antagonist for use according to aspect 35, wherein the mitotic inhibitor is a microtubule stabilizing drug.

[0431] 37. An anti-GREM1 antagonist for use according to aspect 35 or 36, wherein the mitotic inhibitor and / or microtubule stabilizing drug is selected from nab-paclitaxel and paclitaxel.

[0432] 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, wherein the proliferation-dependent cytotoxic agent is FOLFIRINOX or FOLFOX.

[0433] 39. An anti-GREM1 antagonist for use according to any one of aspects 27 to 32, wherein the proliferation-dependent cytotoxic agent is:

[0434] (a) gemcitabine or its derivatives;

[0435] (b) azacitidine or a derivative thereof;

[0436] (c) cytarabine or its derivatives;

[0437] (d) decitabine or its derivatives;

[0438] (e) troxacitabine or a derivative thereof; or

[0439] (f) Capecitabine.

[0440] 40. An anti-GREM1 antagonist for use according to any preceding aspect, 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).

[0441] 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.

[0442] 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.

[0443] 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.

[0444] 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 a heavy chain variable region (HCVR) of SEQ ID NO: 10 or 12 and / or a light chain complementarity determining region (LCDR) sequence contained within a light chain variable region (LCVR) of SEQ ID NO: 11 or 13.

[0445] 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 the group consisting of SEQ ID NOs: 3, 4, 5 and 6 and / or at least one LCDR sequence selected from the group consisting of SEQ ID NOs: 7, 8 and 9.

[0446] 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.

[0447] 47. An anti-GREM1 antagonist for use according to aspect 45 or 46, wherein the anti-Gremlin-1 antibody comprises a HCDR1 / HCDR2 / HCDR3 sequence combination selected from SEQ ID NO: 4 / 5 / 6 or SEQ ID NO: 3 / 5 / 6 and / or a LCDR1 / LCDR2 / LCDR3 sequence combination selected from SEQ ID NO: 7 / 8 / 9.

[0448] 48. An anti-GREMl antagonist for use according to any one of aspects 45 to 47, wherein the anti-Gremlin-1 antibody comprises the HCDR1 / HCDR2 / HCDR3 / LCDR1 / LCDR2 / LCDR3 sequence combination of SEQ ID NO: 4 / 5 / 6 / 7 / 8 / 9 or SEQ ID NO: 3 / 5 / 6 / 7 / 8 / 9.

[0449] 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.

[0450] 50. An anti-GREMl antagonist for use according to aspect 49, wherein the anti-Gremlin-1 antibody comprises the HCVR and LCVR sequence pair of SEQ ID NO: 10 / 11 or 12 / 13, or a sequence at least 95% identical thereto.

[0451] 51. An anti-GREM1 antagonist for use according to aspect 50, wherein the anti-Gremlin-1 antibody comprises a HCDR1 / HCDR2 / HCDR3 / LCDR1 / LCDR2 / LCDR3 sequence consisting of SEQ ID NO: 4 / 5 / 6 / 7 / 8 / 9 or SEQ ID NO: 3 / 5 / 6 / 7 / 8 / 9, and the remainder of the HCVR and LCVR are at least 95% identical to SEQ ID NO: 10, 11, 12 and / or 13, respectively.

[0452] 52. An anti-GREMl antagonist for use according to aspects 49-51, 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.

[0453] 53. An anti-GREMl antagonist for use according to aspect 52, wherein the anti-Gremlin-1 antibody comprises a heavy and light chain pair of SEQ ID NO: 14 / 15, 16 / 17, 18 / 19, 22 / 23, 28 / 29 or 30 / 31, 32 / 33, 34 / 35, or a sequence at least 95% identical thereto.

[0454] 54. An anti-GREM1 antagonist for use according to aspect 53, wherein the HCDR1 / HCDR2 / HCDR3 / LCDR1 / LCDR2 / LCDR3 sequence of the antibody consists of SEQ ID NO: 4 / 5 / 6 / 7 / 8 / 9 or SEQ ID NO: 3 / 5 / 6 / 7 / 8 / 9, and the remainder of the heavy and light chains are at least 95% identical to SEQ ID NO: 14, 15, 16 and / or 17, respectively.

[0455] 55. An anti-GREM1 antagonist for use according to aspect 40, wherein the antagonist is an antibody that competes with the antibody defined in any one of aspects 42 to 52 for binding to Gremlin-1.

[0456] 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 the antibody defined in any one of aspects 42 to 52.

[0457] 57. An anti-GREMl antagonist for use according to any one of aspects 40 to 56, wherein the antagonist antibody is a chimeric, human or humanized antibody.

[0458] 58. An anti-GREM1 antagonist for use according to any one of aspects 40 to 57, wherein the antagonist antibody is a Fab, modified Fab, Fab', modified Fab', F(ab')2, Fv, single domain antibody or scFv.

[0459] 59. An anti-GREM1 antagonist for use according to aspect 40, wherein the antagonist is a polynucleotide encoding the antibody as defined in any one of aspects 41 to 58, or an expression vector carrying the polynucleotide.

[0460] 60. An anti-GREMl 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 pharmaceutically acceptable adjuvant and / or carrier.

[0461] 61. An anti-GREMl antagonist for use according to any preceding aspect, wherein the method further comprises administering an additional anti-cancer agent.

[0462] 62. An anti-GREMl antagonist for use according to aspect 61, wherein the method comprises administering gemcitabine in combination with another cytidine analogue or a deoxycytidine analogue.

[0463] 63. An anti-GREMl antagonist for use according to aspect 61 or 62, wherein the method comprises administering gemcitabine in combination with troxacitabine.

[0464] 64. A cytidine analog or a deoxycytidine analog for use in a method of treating or preventing cancer, wherein the method further comprises administering an anti-GREMl antagonist.

[0465] 65. A cytidine analogue or a deoxycytidine analogue for use according to aspect 64, wherein the cancer, the antagonist, the cytidine analogue or a deoxycytidine analogue and / or the method are as defined in any one of aspects 1-26 and 40 to 63.

[0466] 66. A method of treating cancer comprising administering to a subject in need thereof a therapeutically effective amount of an anti-GREMl antagonist in combination with a therapeutically effective amount of a cytidine analog or a deoxycytidine analog.

[0467] 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 1-26 and 40 to 63.

[0468] 68. A proliferation-dependent cytotoxic agent for use in a method of treating or preventing pancreatic cancer, wherein the method further comprises administering an anti-GREMl antagonist.

[0469] 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.

[0470] 70. A method of treating pancreatic cancer comprising administering a therapeutically effective amount of an anti-GREMl antagonist in combination with a therapeutically effective amount of a proliferation-dependent cytotoxic agent.

[0471] 71. The method according to aspect 70, wherein the proliferation-dependent cytotoxic agent, the pancreatic cancer, the antagonist and / or the method are as defined in any one of aspects 27 to 63.

[0472] 72. A composition or kit comprising an anti-GREMl antagonist and a cytidine analog or a deoxycytidine analog.

[0473] 73. A 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.

[0474] 74. A composition or kit comprising:

[0475] Resistance to GREM1 antagonists and mitotic inhibitors.

[0476] 75. A composition or kit according to aspect 74, wherein the mitotic inhibitor is as defined in any one of aspects 36 or 37.

[0477] 76. A 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.

[0478] 77. A method for determining whether a patient having or suspected of having or being at risk of developing cancer is likely to respond to combination treatment with a GREM1 antagonist and a cytidine analog or a deoxycytidine analog, 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.

[0479] 78. A method for determining whether a patient having or suspected of having or being 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.

[0480] Sequence Listing

[0481] SEQ ID NO: 1 (human Gremlin-1; Uniprot ID: O60565)

[0482] MSRTAYTVGALLLLLGTLLPAAEGKKKGSQGAIPPPDKAQHNDSEQTQSPQQPGSRNRGRGQGRGTAMPGEEVLESSQEALHVTERKYLKRDWCKTQPLKQTIHEEGCNSRTIINRFCYGQCNSFYIPRHIRKEEGSFQSCSFCKPKKFTTMMVTLNCPELQPPTKKKRVTRVKQCRCISIDLD

[0483] SEQ ID NO: 2 (human truncated Gremlin-1 with N-terminal tag for crystallography)

[0484] MGSSHHHHHHSSGENLYFQGSAMPGEEVLESSQEALHVTERKYLKRDWCKTQPLKQTIHEEGCNSRTIINRFCYGQCNSFYIPRHIRKEEGSFQSCSFCKPKKFTTMMVTLNCPELQPPTKKKRVTRVKQCRISIDLD

[0485] SEQ ID NO: 3 (Ab7326 HCDR1, combined Kabat and Chothia)

[0486] GYTFTDYYMH

[0487] SEQ ID NO: 4(Ab7326 HCDR1 Kabat)

[0488] D Y

[0489] SEQ ID NO: 5(Ab7326 HCDR2 Kabat)

[0490] LVDPEDGETIYAEKFQG

[0491] SEQ ID NO: 6(Ab7326 HCDR3 Kabat)

[0492] DARGSGSYYPNHFDY

[0493] SEQ ID NO: 7(Ab7326 LCDR1 Kabat)

[0494] KSSQSVLYSSNNKNYLA

[0495] SEQ ID NO: 8(Ab7326 LCDR2 Kabat)

[0496] WASTRES

[0497] SEQ ID NO: 9(Ab7326 LCDR3 Kabat)

[0498] QQYYDTPT

[0499] SEQ ID NO: 10 (Ab7326 heavy chain variable region variant 1)

[0500] QVQLVESGAEVKKPGATVKISCKVSGYTFTDYYMHWVQQAPGKGLEWMGLVDPEDGETIYAEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCATDARGSGSYYPNHFDYWGQGTLVTVSS

[0501] SEQ ID NO: 11 (Ab7326 light chain variable region variant 1)

[0502] DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTINSLQAEDVAVYFCQQYYDTPTFGQGTRLEIK

[0503] SEQ ID NO: 12 (Ab7326 heavy chain variable region variant 2)

[0504] QVQLVQSGAEVKKPGATVKISCKVSGYTFTDYYMHWVQQAPGKGLEWMGLVDPEDGETIYAEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCATDARGSGSYYPNHFDYWGQGTLVTVSS

[0505] SEQ ID NO: 13 (Ab7326 light chain variable region variant 2)

[0506] DIVMTQTPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTINSLQAEDVAVYFCQQYYDTPTFGQGTRLEIK

[0507] SEQ ID NO: 14 (mouse full-length IgG1 heavy chain variant 1)

[0508] QVQLVESGAEVKKPGATVKISCKVSGYTFTDYYMHWVQQAPGKGLEWMGLVDPEDGETIYAEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCATDARGSGSYYPNHFDYWGQGTLVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK

[0509] SEQ ID NO:15 (Mouse full-length IgG1 light chain variant 1)

[0510] DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTINSLQAEDVAVYFCQQYYDTPTFGQGTRLEIKRTDAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

[0511] SEQ ID NO:16 (Human full-length IgG1 heavy chain variant 2)

[0512] QVQLVQSGAEVKKPGATVKISCKVSGYTFTDYYMHWVQQAPGKGLEWMGLVDPEDGETIYAEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCATDARGSGSYYPNHFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0513] SEQ ID NO:17 (Human full-length IgG1 light chain variant 2)

[0514] DIVMTQTPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTINSLQAEDVAVYFCQQYYDTPTFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0515] SEQ ID NO:18 (Fab heavy chain variant 1)

[0516] QVQLVESGAEVKKPGATVKISCKVSGYTFTDYYMHWVQQAPGKGLEWMGLVDPEDGETIYAEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCATDARGSGSYYPNHFDY WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC

[0517] SEQ ID NO: 19 (Fab light chain variant 1)

[0518] DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTINSLQAEDVAVYFCQQYYDTPTFGQGTRL EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0519] SEQ ID NO: 20 (human truncated Gremlin-1 without N-terminal tag for crystallography)

[0520] AMPGEEVLESSQEALHVTERKYLKRDWCKTQPLKQTIHEEGCNSRTIINRFCYGQCNSFYIPRHIRKEEGSFQSCSFCKPKKFTTMMVTLNCPELQPPTKKKRVTRVKQCRISIDLD

[0521] SEQ ID NO:21 (the mature Gremlin-1 sequence of SEQ ID NO:1 lacking the signal peptide of amino acids 1-21)

[0522] KKKGSQGAIPPPDKAQHNDSEQTQSPQQPGSRNRGRGQGRGTAMPGEEVLESSQEALHVTERKYLKRDWCKTQPLKQTIHEEGCNSRTIINRFCYGQCNSFYIPRHIRKEEGSFQSCSFCKPKKFTTMMVTLNCPELQPPTKKKRVTRVKQCRCISIDLDSEQ ID NO:22 (Human IgG4P heavy chain variant 1)

[0523] QVQLVESGAEVKKPGATVKISCKVSGYTFTDYYMHWVQQAPGKGLEWMGLVDPEDGETIYAEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCATDARGSGSYYPNHFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0524] SEQ ID NO:23 (Human IgG4P light chain variant 1)

[0525] DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTINSLQAEDVAVYFCQQYYDTPTFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0526] SEQ ID NO: 24 (human IgG1 heavy chain DNA variant 1)

[0527]

[0528] SEQ ID NO:25 (Human IgG1 light chain DNA variant 1)

[0529] gacattgtgatgacccagtcccccgattcgcttgcggtgtccctgggagaacgggccaccattaactgcaagagctcacagtccgtcctgtattcatcgaacaacaagaattacctcgcatggtatcagcagaagcctggacagcctcccaagctgctcatctactgggctagcacccgcgaatccggggtgccggatagattctccggatcgggttcgggcactgacttcactctgactatcaactcactgcaagccgaggatgtcgcggtgtacttctgtcagcagtactacgacaccccgacctttggacaaggcaccagactggagattaagcgtacggtggccgctccctccgtgttcatcttcccaccctccgacgagcagctgaagtccggcaccgcctccgtcgtgtgcctgctgaacaacttctacccccgcgaggccaaggtgcagtggaaggtggacaacgccctgcagtccggcaactcccaggaatccgtcaccgagcaggactccaaggacagcacctactccctgtcctccaccctgaccctgtccaaggccgactacgagaagcacaaggtgtacgcctgcgaagtgacccaccagggcctgtccagccccgtgaccaagtccttcaaccggggcgagtgc

[0530] SEQ ID NO:26 (Human IgG4P heavy chain DNA variant 1)

[0531]

[0532] SEQ ID NO:27 (Human IgG4P light chain DNA variant 1)

[0533] gacattgtgatgacccagtcccccgattcgcttgcggtgtccctgggagaacgggccaccattaactgcaagagctcacagtccgtcctgtattcatcgaacaacaagaattacctcgcatggtatcagcagaagcctggacagcctcccaagctgctcatctactgggctagcacccgcgaatccggggtgccggatagattctccggatcgggttcgggcactgacttcactctgactatcaactcactgcaagccgaggatgtcgcggtgtacttctgtcagcagtactacgacaccccgacctttggacaaggcaccagactggagattaagcgtacggtggccgctccctccgtgttcatcttcccaccctccgacgagcagctgaagtccggcaccgcctccgtcgtgtgcctgctgaacaacttctacccccgcgaggccaaggtgcagtggaaggtggacaacgccctgcagtccggcaactcccaggaatccgtcaccgagcaggactccaaggacagcacctactccctgtcctccaccctgaccctgtccaaggccgactacgagaagcacaaggtgtacgcctgcgaagtgacccaccagggcctgtccagccccgtgaccaagtccttcaaccggggcgagtgc

[0534] SEQ ID NO:28 (Mouse full-length IgG1 heavy chain variant 2)

[0535] QVQLVQSGAEVKKPGATVKISCKVSGYTFTDYYMHWVQQAPGKGLEWMGLVDPEDGETIYAEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCATDARGSGSYYPNHFDYWGQGTLVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK

[0536] SEQ ID NO:29 (Mouse full-length IgG1 light chain variant 2)

[0537] DIVMTQTPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTINSLQAEDVAVYFCQQYYDTPTFGQGTRLEIKRTDAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

[0538] SEQ ID NO:30 (Human full-length IgG1 heavy chain variant 1)

[0539] QVQLVESGAEVKKPGATVKISCKVSGYTFTDYYMHWVQQAPGKGLEWMGLVDPEDGETIYAEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCATDARGSGSYYPNHFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0540] SEQ ID NO:31 (Human full-length IgG1 light chain variant 1)

[0541] DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTINSLQAEDVAVYFCQQYYDTPTFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0542] SEQ ID NO:32 (Fab heavy chain variant 2)

[0543] QVQLVQSGAEVKKPGATVKISCKVSGYTFTDYYMHWVQQAPGKGLEWMGLVDPEDGETIYAEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCATDARGSGSYYPNHFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC

[0544] SEQ ID NO:33 (Fab light chain variant 2)

[0545] DIVMTQTPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTINSLQAEDVAVYFCQQYYDTPTFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0546] SEQ ID NO:34 (Human IgG4 P heavy chain variant 2)

[0547] QVQLVQSGAEVKKPGATVKISCKVSGYTFTDYYMHWVQQAPGKGLEWMGLVDPEDGETIYAEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCATDARGSGSYYPNHFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0548] SEQ ID NO:35 (Human IgG4 P light chain variant 2)

[0549] DIVMTQTPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTINSLQAEDVAVYFCQQYYDTPTFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0550] SEQ ID NO:36 (Human Gremlin-1; full-length sequence)

[0551]

[0552] SEQ ID NO:37 (Human Gremlin-1; Coding Sequence)

[0553] atgagccgcacagcctacacggtgggagccctgcttctcctcttggggaccctgctgccggctgctgaagggaaaaagaaagggtcccaaggtgccatccccccgccagacaaggcccagcacaatgactcagagcagactcagtcgccccagcagcctggctccaggaaccgggggcggggccaagggcggggcactgccatgcccggggaggaggtgctggagtccagccaagaggccctgcatgtgacggagcgcaaatacctgaagcgagactggtgcaaaacccagccgcttaagcagaccatccacgaggaaggctgcaacagtcgcaccatcatcaaccgcttctgttacggccagtgcaactctttctacatccccaggcacatccggaaggaggaaggttcctttcagtcctgctccttctgcaagcccaagaaattcactaccatgatggtcacactcaactgccctgaactacagccacctaccaagaagaagagagtcacacgtgtgaagcagtgtcgttgcatatccatcgatttggattaa

Claims

1. An anti-GREM1 antagonist for use in a method of treating or preventing cancer, wherein the method further comprises administering a cytidine analog or a deoxycytidine analog.

2. The anti-GREM1 antagonist for use according to claim 1, wherein: (a) the cancer is a solid cancer; (b) cancers have stromal GREM1 overexpression; (c) the cancer is metastatic; (d) the cancer comprises dormant cancer cells, optionally dormant stem-like cancer cells; (e) the cancer is a recurrent cancer and / or wherein the method is used to prevent cancer recurrence; (f) the cancer is a cancer that is poorly responsive, unresponsive or refractory to treatment with a cytidine analog or a deoxycytidine analog; optionally, wherein the cancer is a cancer that is poorly responsive, unresponsive or refractory to treatment with gemcitabine or a derivative thereof; and / or (g) The cancer is selected from the group consisting of colorectal cancer, multiple myeloma, pancreatic cancer, bladder cancer, breast cancer, lung cancer, gastric cancer, duodenal cancer, esophageal cancer, head and neck cancer, prostate cancer, glioma, endometrial cancer, ovarian cancer, liver cancer, spleen cancer, bone resident cancer and osteosarcoma.

3. The anti-GREM1 antagonist for use according to claim 2(g), wherein: (a) the cancer is pancreatic cancer; optionally, wherein: (i) the pancreatic cancer is exocrine pancreatic cancer; and / or (ii) pancreatic cancer is pancreatic ductal adenocarcinoma (PDAC); (b) the cancer is lung cancer; optionally, wherein the cancer is non-small cell lung cancer; (c) the cancer is bladder cancer; (d) the cancer is breast cancer; or (e) The cancer is ovarian cancer.

4. An anti-GREM1 antagonist for use according to any one of the preceding claims, wherein: (a)(i) the cancer has epithelial GREM1 overexpression; optionally, wherein the cancer is a GREM1 driven cancer; (ii) the cancer is disseminated; and / or (iii) the cancer is an established cancer; and / or (b)(i) the cytidine analog or deoxycytidine analog is gemcitabine or a derivative thereof; (ii) the cytidine analog or deoxycytidine analog is azacitidine or a derivative thereof; (iii) the cytidine analog or deoxycytidine analog is cytarabine or a derivative thereof; (iv) the cytidine analog or deoxycytidine analog is decitabine or a derivative thereof; or (v) The cytidine analog or deoxycytidine analog is troxacitabine or a derivative thereof.

5. An anti-GREM1 antagonist for use in a method of treating or preventing pancreatic cancer, wherein the method further comprises administering a proliferation-dependent cytotoxic agent.

6. The anti-GREM1 antagonist for use according to claim 5, wherein: (a) Pancreatic cancer is exocrine pancreatic cancer; (b) pancreatic cancer is pancreatic ductal adenocarcinoma (PDAC); (c) pancreatic cancer is metastatic pancreatic cancer; (d) the pancreatic cancer is recurrent pancreatic cancer; and / or (e) Pancreatic cancer is pancreatic cancer that is poorly responsive, unresponsive, or refractory to treatment with proliferation-dependent cytotoxic agents.

7. The anti-GREM1 antagonist for use according to claim 5 or 6, wherein: (a) the proliferation-dependent cytotoxic agent is a nucleoside inhibitor or an antimetabolite; (b) the proliferation-dependent cytotoxic agent is a cytidine analog or a deoxycytidine analog; (c) the proliferation-dependent cytotoxic agent is a mitotic inhibitor; optionally, wherein: (i) the mitotic inhibitor is a microtubule stabilizing drug; and / or (ii) a mitotic inhibitor and / or microtubule stabilizing drug selected from nab-paclitaxel and paclitaxel; (d) the proliferation-dependent cytotoxic agent comprises one or more of oxaliplatin, folinic acid, irinotecan and fluorouracil; optionally, wherein the proliferation-dependent cytotoxic agent is FOLFIRINOX or FOLFOX; or (e) The proliferation-dependent cytotoxic agent is: (i) gemcitabine or its derivatives; (ii) azacitidine or a derivative thereof; (iii) cytarabine or its derivatives; (iv) decitabine or its derivatives; (v) troxacitabine or a derivative thereof; or (vi) Capecitabine.

8. An anti-GREM1 antagonist for use according to any one of the preceding claims, wherein the antagonist is a peptide, a protein, an antibody, a polynucleotide, an oligonucleotide, an antisense RNA, a small interfering RNA (siRNA), a small molecule inhibitor or a small hairpin RNA (shRNA).

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

10. The anti-GREM1 antagonist for use according to claim 9(c)(iv), wherein: (a) an anti-Gremlin-1 antibody comprises a HCVR and LCVR sequence pair of SEQ ID NO: 10 / 11 or 12 / 13, or a sequence at least 95% identical thereto; optionally, wherein the anti-Gremlin-1 antibody comprises a HCDR1 / HCDR2 / HCDR3 / LCDR1 / LCDR2 / LCDR3 sequence consisting of SEQ ID NO: 4 / 5 / 6 / 7 / 8 / 9 or SEQ ID NO: 3 / 5 / 6 / 7 / 8 / 9, and the remainder of the HCVR and LCVR are at least 95% identical to SEQ ID NO: 10, 11, 12 and / or 13, respectively; and / or (b) the anti-Gremlin-1 antibody comprises the heavy chain of SEQ ID NO: 14, 16, 18, 22, 28, 30, 32 or 34 and / or the light chain of SEQ ID NO: 15, 17, 19, 23, 29, 31, 33 or 35, or a sequence at least 95% identical thereto.

11. The anti-GREM1 antagonist for use according to claim 10(b), wherein the anti-Gremlin-1 antibody comprises a heavy chain and a light chain pair of SEQ ID NO: 14 / 15, 16 / 17, 18 / 19, 22 / 23, 28 / 29 or 30 / 31, 32 / 33, 34 / 35, or a sequence at least 95% identical thereto; optionally, wherein the HCDR1 / HCDR2 / HCDR3 / LCDR1 / LCDR2 / LCDR3 sequence of the antibody consists of SEQ ID NO: 4 / 5 / 6 / 7 / 8 / 9 or SEQ ID NO: 3 / 5 / 6 / 7 / 8 / 9, and the remainder of the heavy chain and light chain are at least 95% identical to SEQ ID NO: 14, 15, 16 and / or 17, respectively.

12. The anti-GREM1 antagonist for use according to claim 8, wherein: (a) the antagonist is an antibody that competes with the antibody as defined in any one of claims 9(a)(i)-10 for binding to Gremlin-1; or (b) The antagonist is an antibody that binds to the same epitope on Gremlin-1 as the antibody defined in any one of claims 9(a)(i)-10.

13. An anti-GREM1 antagonist for use according to any one of claims 8 to 12, wherein: (a) the antagonist antibody is a chimeric antibody, a human antibody or a humanized antibody; and / or (b) The antagonist antibody is Fab, modified Fab, Fab', modified Fab', F(ab')2, Fv, single domain antibody or scFv.

14. The anti-GREM1 antagonist for use according to claim 8, wherein the antagonist is a polynucleotide encoding the antibody as defined in any one of claims 9 to 13, or an expression vector carrying the polynucleotide.

15. An anti-GREMl antagonist for use according to any one of claims 8 to 13, wherein the antagonist antibody is contained in a pharmaceutical composition further comprising a pharmaceutically acceptable adjuvant and / or carrier.

16. An anti-GREM1 antagonist for use according to any one of the preceding claims, wherein the method further comprises administering an additional anti-cancer agent; optionally, wherein: (a) the method comprises administering gemcitabine in combination with another cytidine analog or a deoxycytidine analog; and / or (b) The method comprises administering gemcitabine in combination with troxacitabine.

17. A cytidine analog or deoxycytidine analog for use in a method of treating or preventing cancer, wherein the method further comprises administering an anti-GREM1 antagonist; optionally, wherein the cancer, the antagonist, the cytidine analog or deoxycytidine analog and / or the method are as defined in any one of claims 1-4 and 8 to 16.

18. 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 analog or a deoxycytidine analog; optionally, wherein the cancer, the antagonist, the cytidine analog or deoxycytidine analog and / or the method is as defined in any one of claims 1-4 and 8 to 16.

19. A proliferation-dependent cytotoxic agent for use in a method of treating or preventing pancreatic cancer, wherein the method further comprises administering an anti-GREM1 antagonist; optionally, wherein the proliferation-dependent cytotoxic agent, the pancreatic cancer, the antagonist and / or the method are as defined in any one of claims 5 to 16.

20. 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; optionally, wherein the proliferation-dependent cytotoxic agent, the pancreatic cancer, the antagonist and / or the method are as defined in any one of claims 5 to 16.

21. A composition or kit comprising an anti-GREMl antagonist and a cytidine analog or a deoxycytidine analog; optionally, wherein the cytidine analog or the deoxycytidine analog is as defined in claim 4(b).

22. A composition or kit comprising: Resistance to GREM1 antagonists and mitotic inhibitors.

23. A composition or kit according to claim 22, wherein the mitotic inhibitor is as defined in any one of claims 7(c)(i) or 7(c)(ii).

24. A composition or kit according to any one of claims 21 to 23, wherein the anti-GREMl antagonist is as defined in any one of claims 8 to 14.

25. A method for determining: (a) whether a patient having or suspected of having cancer or being at risk of developing cancer is likely to respond to a combination treatment with a GREM1 antagonist and a cytidine analog or a deoxycytidine analog, 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; or (b) Whether a patient having or suspected of having pancreatic cancer or being at risk of developing pancreatic cancer is likely to respond to a combination treatment with a GREM1 antagonist and a proliferation-dependent cytotoxic agent, wherein the method comprises measuring stromal and / or epithelial expression of GREM1 in the patient, thereby predicting whether the patient is likely to respond to the combination treatment.

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