Treatment and prognosis of pancreatic cancer

Abstract

To provide a CCR1 antagonist for use in treatment of pancreatic cancer.SOLUTION: The invention provides a CCR1 antagonist, or a pharmaceutically acceptable salt, hydrate or solvate thereof, in particular a CCR1 antagonist, e.g., in combination with one or more further therapeutic agents effective as anti-tumor agents in the treatment of pancreatic cancer. Such an anti-tumour agent may be a chemotherapeutic agent selected from Gemcitabine, Fluorouracil (5-FU), Capecitabine, FOLFIRINOX (Leucovorin Calcium, Fluorouracil, Irinotecan Hydrochloride and Oxaliplatin), Nab-paclitaxel (Abraxane(R)) and combinations thereof. An immuno-oncology agent (e.g., a PD-1 inhibitor and / or a PD-L1 inhibitor) may also favorably be used with the CCR1 antagonist.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Introduction The present invention relates to the use of CCR1 antagonists in the treatment of pancreatic cancer. Also, the use of a pharmaceutical composition containing a CCR1 antagonist for the treatment of pancreatic cancer, and and the use of a CCR1 antagonist in combination with one or more other therapeutic agents. Novel methods of treatment and prognosis for cancer are also described. [Background technology]

[0002] Pancreatic cancer is an aggressive form that causes very few symptoms until the cancer is in an advanced stage. As the name suggests, pancreatic cancer is a type of cancer that develops in the tissues of the pancreas. Pancreatic cancer is currently the most common cancer diagnosed in the UK. It is the 0th most common form of pancreatic cancer, with 9,912 cases of pancreatic cancer in the UK in 2015 The survival rate for patients diagnosed with pancreatic cancer is extremely low, Fewer than 1% of people diagnosed with HIV survive beyond 10 years. In 2016 alone, 9,263 people died from pancreatic cancer in the UK in the same year, This roughly correlates with the number of people diagnosed with the condition.

[0003] Significant improvements in prevention, detection and treatment over the past 40 years have made it possible to treat most other forms of The survival rates of patients diagnosed with other cancers have changed dramatically, but the survival rates of patients diagnosed with pancreatic cancer have not. Clinical outcomes remain poor. Pancreatic ductal adenocarcinoma accounts for more than 90% of all pancreatic cancer cases. (PDAC) is the fourth most common form of cancer worldwide, with approximately 140,000 new cases reported globally. It is the leading cause of eye cancer-related deaths.

[0004] The poor clinical outcomes and low survival rates associated with pancreatic cancer are due, in part, to the This is due to the lack of effective treatment options currently available to treat the condition, for example: Gemcitabine (Gemz), the current first-line option for unresectable pancreatic cancer, ar®) + nab-paclitaxel (Abraxane®) and and / or FOLFIRINOX (fluorouracil, leucovorin, irinotecan, and oxaliplatin) is administered to individuals diagnosed with unresectable pancreatic cancer. and a chemotherapy drug that extends the lifespan of the individual by an average of only 11 months.

[0005] Therefore, there remains a need for new and improved treatment regimens for pancreatic cancer. Also, doctors are unsure how to treat patients with advanced forms of pancreatic cancer. to be able to make more informed decisions about what is best for you. New methods for patient prognosis and stratification are desirable. Summary of the Invention

[0006] According to a first aspect of the present invention there is provided a CCR1 antagonist for use in the treatment of pancreatic cancer. agonists, pharmaceutically acceptable salts, solvates or hydrates thereof are provided.

[0007] The present inventors have identified CCR1 and CCR2 expressed in pancreatic cancer cells (e.g., PDAC cells). The inventors determined that high levels of CCR1 correlated strongly with patient prognosis. We found that CI was strongly correlated with patients having the worst clinical outcomes. Furthermore, high levels of immune (e.g., macrophage) infiltration are also frequently associated with poor patient prognosis. Therefore, the present inventors have found that the prognosis of pancreatic cancer patients is determined by the We were able to identify new biomarkers and methods for

[0008] Furthermore, the inventors have demonstrated that several structurally distinct CCR1 antagonists inhibit the pancreatic Interfering with macrophage-mediated pro-invasive properties of cancer cells and was found to be effective in both inhibiting CCR1 expression and reducing CCR1 expression levels. In vivo tumor growth studies have shown that pancreatic cancer cells, in particular, are also exposed to macrophages. Sometimes, administration of CCR1 antagonists (e.g., BX-471) can induce cell death in pancreatic cancer cells (e.g., It has been confirmed that this compound significantly inhibits the proliferation of cells (e.g., PDAC cells). also demonstrated that the inclusion of CCR1 antagonists in standard of care drug therapy may improve the survival of patients with pancreatic ductal adenocarcinoma (PDC). We found that it increased the survival time of mice in a mouse model of DAC.

[0009] CCR1 antagonists for use according to the present invention are often referred to as "standard of care" When used in combination with established treatment regimens, such as chemotherapy, it has been shown to be effective in the treatment of pancreatic cancer. Such combinations are particularly effective in the treatment of rheumatoid arthritis. Such combinations are discussed in more detail below. In a preferred embodiment, gemcitabine (Gemzar®), fluorouracil (5-FU), capecitabine (Xeloda®), FOLFIRINOX and and Nab-paclitaxel (Abraxane®), CCR1 antagonists for use in the treatment of pancreatic cancer in combination with chemotherapeutic agents or a pharmaceutically acceptable salt, solvate or hydrate thereof.

[0010] The CCR1 antagonist may also or alternatively be used in combination with a cancer immunotherapeutic agent (e.g., PD -1 and / or PD-L1 inhibitors).

[0011] CCR1 antagonists may also or alternatively be used in combination with MEK inhibitors. It can be used.

[0012] The CCR1 antagonist may also or alternatively be combined with an IGF1R inhibitor. It can be used as follows.

[0013] As described above, the present inventors have identified a novel protein expressed in pancreatic cancer cells (e.g., PDAC cells). Therefore, a strong correlation was found between the level of CCR1 detected and the prognosis of the patient. The treatment, in one embodiment, comprises administering to a subject an increased level of CCR1 expression compared to a reference expression level. Then the identified subject is treated.

[0014] As mentioned above, the inventors have compared the level of immune (e.g., macrophage) infiltration with the patient's Therefore, in one embodiment, the treatment of the present invention is Increased levels of immune (e.g., macrophage) infiltration were identified compared with the infiltrate level. In some embodiments, the macrophages are M1 macrophages. In another embodiment, the macrophages are M2 macrophages, i.e., Suitably, the macrophage infiltration referred to herein refers to M1 or M2 macrophages. It is understood to be an infiltration.

[0015] Suitably, the CCR1 antagonist for any aspect of the present invention is UCB-3562 5, BX-471, AZD-4818, and J113863, or their pharmaceutically acceptable salts The compound is selected from a salt, solvate or hydrate thereof. [Brief explanation of the drawings]

[0016] [Figure 1] 1 shows ssGSEA analysis of the immune infiltrate fraction in TCGA human pancreatic cancer. Columns represent individual patients and rows represent various immune cell types. [Figure 2] Kaplan-Meier survival analysis of macrophage infiltration in the TCGA PAAD (Pancreatic Cancer) for stratification of pancreatic cancer patients. Survival of pancreatic cancer patients calculated by Immune Score versus M1 and M2-like macrophage signatures: However, no differential overall survival was calculated for pancreatic cancer patients according to immune infiltration status (Immune Score); higher overall survival was calculated for pancreatic cancer patients with low levels of M1 or M2-like macrophage signatures (p=0.01). *Low < 30th percentile < medium < 70th percentile < high. [Figure 3] We demonstrate that murine PDAC mesenchymal cells form vascular mimics in a 3D in vitro assay of invasion. In contrast to PDAC epithelial cells, which form cell clusters, PDAC mesenchymal cells form vascular mimic (VM)-like structures when grown on plates pre-coated with 100% Matrigel. Quantification of vascular structures obtained 96 hours after seeding (p=0.01). [Figure 4] mCherry-labeled PDAC epithelial and mesenchymal cells and ZsGreen-labeled primary bone marrow-derived macrophages (BMDMs) in 3D cell culture imaged over 7 days with IncuCyte Zoom. Representative images and quantification of the pro-invasive phenotype obtained at days 6 and 4 for PDAC epithelial and PDAC mesenchymal cells, respectively. Scale bar, 300 microns. [Figure 5]Shown are: A) Schematic of monocultured or mixed PDAC and BMDM cells in 2D culture and subsequent fluorescence-activated cell sorting (FACS) for downstream RNA-sequencing analysis; B) Chemokines and their receptors are among the highest differentially expressed genes in the PDAC transcriptome after extended coculture with BMDM; and C) Confirmation of RNA-sequencing data by qRT-PCR in PDAC cells after extended coculture with BMDM. [Figure 6] Schematic representation of monocultured or mixed PDAC and BMDM cells in 2D cultures labeled with CTAP (cell type-specific labeling using amino acid precursors) to identify novel protein targets that promote PDAC cell invasion (proteome). Monocultures and mixed cocultures were harvested in bulk for downstream proteome and secretome analysis. [Figure 7] The results of the proteomic approach are shown in Figure 6. CTAP-TMT (tandem mass tag) proteome reveals chemokines and their receptors as among the most highly differentially expressed genes in PDAC after extended coculture with BMDM. [Figure 8] The results of the secretome approach are shown in Figure 6. Representative inflammatory antibody arrays probed with conditioned medium (CM) (PDAC and BMDM cells in monoculture or mixed together in 2D culture) are shown. [Figure 9] Quantification of various CCL chemokines from the array shown in FIG. 8 is shown. [Figure 10] mCherry-labeled PDAC mesenchymal cells and ZsGreen-labeled BMDMs in 3D cell culture treated with various small molecule inhibitors of CCR1 imaged over 7 days with IncuCyte Zoom. Representative images and quantification of the pro-invasive phenotype acquired at day 4. Scale bar, 300 microns. Bottom: Quantification of the pro-invasive phenotype acquired at day 4. [Figure 11]Relative CCR1 expression (mRNA) in PDAC mesenchymal cells after co-culture with BMDM in the presence and absence of various CCR1 antagonists by qRT-PCR is shown, which is consistent with the data obtained in FIG. [Figure 12] Figure 1 shows CCR1 expression (protein) in PDAC cells after extended co-culture in the presence and absence of various CCR1 antagonists. [Figure 13] Quantification of the CCR1 expression (protein) data shown in FIG. 12 is shown. [Figure 14] Figure 1 shows siRNA-mediated knockdown of CCR1 prior to co-culture of mCherry-labeled PDAC mesenchymal cells with ZsGreen-labeled BMDMs in 3D cell culture imaged over 7 days with IncuCyte Zoom. Representative images of the pro-invasive phenotype were acquired on day 4. [Figure 15] Quantification of the 3D cell culture images of FIG. 14 is shown. [Figure 16] mCherry-labeled PDAC mesenchymal cells and ZsGreen-labeled BMDMs in 3D cell culture treated with various neutralizing antibodies against known and novel ligands that bind to CCR1, imaged over 7 days with IncuCyte Zoom. Representative images acquired at day 4 and quantification of the pro-invasive phenotype. Scale bar, 300 microns. [Figure 17] Two graphs showing tumor volume over time (e.g., days 7-34) are shown for: i) PDAC cells (K84) alone, ii) PDAC cells (K84) + CCR1 antagonist, BX471, iii) PDAC cells (K84) + bone marrow-derived macrophages, and iv) PDAC cells (K84) + CCR1 antagonist, BX471 + bone marrow-derived macrophages. [Figure 18]Graphs showing the survival percentage for: i) PDAC cells (K84) alone, ii) PDAC cells (K84) + CCR1 antagonist, BX471, iii) PDAC cells (K84) + bone marrow-derived macrophages, and iv) PDAC cells (K84) + CCR1 antagonist, BX471 + bone marrow-derived macrophages. [Figure 19] Figure 1 shows CCR1 expression in immune cell high / low samples in TCGA pancreatic cancer (PAAD). Each column shows the high and low split of samples for each cell type and CCR1 expression across groups. [Figure 20] FIG. 19 shows a graph depicting percent survival over time generated through Kaplan-Meier survival analysis of the data shown in FIG. [Figure 21] We show that gene ontology (GO) analysis of the most altered proteins after extended coculture reveals enrichment of chemokine-related biological processes. [Figure 22] 1 shows a graph depicting % survival over time generated through Kaplan-Meier survival analysis of the combined macrophage infiltration and CCR1 expression data of Example 1. [Figure 23] Figure 1 shows the expression of the IGF1 signaling pathway in PDAC mesenchymal cells. Total protein (30 μg) from PDAC cells was separated by SDS-PAGE gel. Expression of IGF1 downstream target genes was detected by Western blot analysis using anti-AKT, 4E-BP1, MEK, and MAPK antibodies. GAPDH was used as a loading control for all samples. [Figure 24] Shown is a heatmap plot generated using kinase set enrichment analysis (KSEA) to estimate the kinase activity observed for various kinases in samples treated with recombinant IGF1 compared to samples treated with a combination of both IGF1 and BX-471. [Figure 25]A study design is shown to determine the efficacy of the CCR1 antagonist BX-471 in treating pancreatic cancer using the KPC mouse model of PDAC, including a description of the five experimental cohorts used and a timeline showing treatment conditions and study duration. [Figure 26] Preliminary imaging of primary tumors from KPC mice (treated with either vehicle, BX-471, gemcitabine, gemcitabine + BX471, or gemcitabine + anti-PD1 + BX-471) stained for collagen IV, immune cells (H&E and CD45), and cancer cells (KRT19) is shown. [Figure 27] A graph showing % survival over time is shown for KPC mice treated with: (i) vehicle, (ii) BX-471, (iii) gemcitabine, (iv) gemcitabine + BX471 dual therapy, or (v) gemcitabine + anti-PD1 + BX-471 triple therapy. DETAILED DESCRIPTION OF THE INVENTION

[0017] CCR1 antagonists CC chemokine receptor type 1 (CCCR1) is a protein encoded by the CCR1 gene. CCR1) mediates signal transduction and recruits effector immune cells to sites of inflammation. Beta-chemo receptors, a family of receptors known to play an active role in CCR1, a member of the kine receptor family, in mediating immune responses and other chemokine family members (e.g., CCR2, CCR3 and CCR 5) Due to the active involvement of CCR1, several small molecule antagonists of CCR1, and C CR1 neutralizing antibodies have been developed and are highly effective in the treatment of autoimmune and chronic inflammatory diseases. It has been shown to be effective in

[0018] Widespread use of CCR1 antagonists in the treatment of autoimmune and inflammatory disorders Regardless, CCR1 antagonists are generally more effective at treating cancer than other forms of cancer. is considered much more challenging (evidenced by the poor survival rates among pancreatic cancer patients) It is believed to be an effective drug for the treatment of aggressive forms of solid cancer. Very few reports exist.

[0019] U.S. Patent Application No. 2017 / 0290808 describes certain CCR1 antagonists (e.g., For example, small molecule antagonists of CCR1) with PD-1 and / or PD-L1 inhibitors. Certain combinations are useful in the treatment of breast cancer metastases, e.g., triple-negative breast cancer metastases. However, US2017 / 02908 In 2018, CCR1 antagonists were shown to be effective in treating more aggressive forms of cancer, such as pancreatic cancer. There are also data that support or provide reason to infer that the drug may be used to treat , including data supporting the use of CCR1 antagonists in the treatment of primary cancers. Furthermore, pancreatic cancer cells typically produce a higher amount of Pancreatic cancer is characterized by collagen and / or extracellular matrix (ECM) , are generally considered to be structurally distinct forms of solid cancer (Weniger et al.,Cancers,2019,10(9),316). High levels of collagen and / or ECM typically found in pancreatic cancer cells This limits the access of chemotherapy agents to the pancreas, which in turn limits the effectiveness of many chemotherapy agents in treating pancreatic cancer. This means that it cannot be used effectively in

[0020] In US2009 / 0286823, certain CCR1 inhibitors were used to treat multiple myeloma and Pancreatic cancer has been reported to be useful for the treatment of pancreatic cancer and other disorders. The authors mention it in a list of cancers for which they speculate it may be useful. The compounds described are useful in the treatment of pancreatic cancer, either alone or in combination with any other therapy. No data are provided to support any suggestion that it would be effective in

[0021] In US2013 / 0280254, a specific antagonist of the CCR1 receptor It has been reported that antagonism of pancreatic cancer can suppress tumor metastasis in patients. , in a list of cancers for which the authors speculate that antagonists may be useful. The compounds described therein may be used alone or in combination with any other therapy. There is no data to support any suggestion that these drugs, combined with other drugs, may be effective in treating pancreatic cancer. It has not been done.

[0022] As noted above, the present inventors have demonstrated that CCR is a potent anti-cancer agent, particularly when used in combination with other treatments. We have found that antagonists of 1 are effective in treating pancreatic cancer.

[0023] The term "CCR1 antagonist" refers to an antagonist that inhibits the chemokine receptor CCR1 and its ligands. Agents that antagonize the interaction of any one of the two arms (e.g., small molecules or CCR1 receptors) CCR1 antagonists are generally understood to mean CCR1 antagonists. of processes triggered by the interaction of 1 with one of its ligands Suitably, the CCR1 antagonist may inhibit one or more of IL1B, C CL1, CCL3, CCL5, CCl6, CCL7, CCL9, CCL15, CCL20 and / or agents that antagonize the interaction of CCL23 with a ligand. CR1 antagonists inhibit IL1B, CCL1, CCL9 and / or CCL23 ligands and agents that antagonize the interaction of CCL9 with ligands, for example.

[0024] In certain embodiments, the CCR1 antagonist is a CCR1 neutralizing antibody. Examples of neutralizing antibodies are available from MBL International (Woburn, MA). The antibody IgG1 (clone 141-2) is a potential target for the antibody. It is understood that additional antibodies may be realized using.

[0025] The inventors have demonstrated that CCR1 antagonists have minimal adverse effects on non-cancerous cells. Fibroblasts treated with CCR1 antagonist (BX471) and The cells and macrophages appeared to be as viable as cells treated with the vehicle control. Therefore, their use in human subjects is beyond the reach of many other It is expected to be associated with fewer and less severe side effects than conventional cancer treatments.

[0026] Preferably, the CCR1 antagonist is a subunit of a CCR1 antagonist, such as those described herein below. Suitably, the CCR1 antagonist is a molecular weight compound of up to 1000 Da. Most suitably, the CCR1 antagonist is a small molecule CCR1 antagonist having a molecular weight of The compound is a small molecule CCR1 antagonist with a molecular weight of 250 Da to 550 Da. .

[0027] In certain embodiments, the CCR1 antagonist is BL-5923, UCB-3562 5, BX-471, BI-638683, BI-639667, PS-031291, M LN-3701, AZD-4818, AZD-0492, MLN-3897, CP-48 1715, F-18-CCR1, AOP-RANTES, PS-375179, J113 863, NSC-651016, and BAY-865047, BMS-817399, C-4462 and CCX-354, or pharmaceutically acceptable salts, solvates or is selected from hydrates (e.g., BL-5923, UCB-35625, BX-471 , BI-638683, BI-639667, PS-031291, MLN-3701, AZD-4818, AZD-0492, MLN-3897, CP-481715, F-1 8-CCR1, AOP-RANTES, PS-375179, J113863 and NS C-651016, or a pharmaceutically acceptable salt, solvate or hydrate thereof Suitably, the CCR1 antagonist is BL-5923, UCB-35625 , BX-471, BI-638683, BI-639667, PS-031291, AZ D-4818, AZD-0492, PS-375179, J113863 and NSC- 651016, or a pharmaceutically acceptable salt, solvate or hydrate thereof. More suitably, the CCR1 antagonist is UCB-35625, BX-471, B I-639667, AZD-4818, AZD-0492, and J113863, is selected from its pharmaceutically acceptable salts, solvates or hydrates. CCR1 antagonists include BX-471, BI-639667, and J113863. , AZD-4818 or AZD-0492 or a pharmaceutically acceptable salt thereof, solvate Even more suitably, the CCR1 antagonist is selected from the group consisting of B X-471, BI-639667, J113863 or AZD-0492 or It is selected from pharmaceutically acceptable salts, solvates or hydrates. Most suitably, CCR The antagonist is BX-471, or a pharmaceutically acceptable salt, solvate or is a hydrate.

[0028] In certain embodiments, the CCR1 antagonist is the following CCR1 antagonist: U CB-35625, BX-471 and J113863, or pharmaceutically acceptable salts thereof It is selected from one of a salt, a solvate or a hydrate.

[0029] For example, CCR1 antagonists are disclosed in WO03 / 035627 and / or US20 Piperazine-based CCR1 antagonists of the type described in 03 / 0109534 Thus, in some embodiments, the CCR1 antagonist is Compounds of formula (I): [ka] (In the formula, n is an integer selected from 1, 2, or 3; R 100 is a substituent selected from alkyl or hydroxyalkyl, R 200 is substituted with a chloro group at the 4-position and an aminocarbonyl, ureido or guanylide group at the 2-position. a phenyl group substituted with a lysinamide group), or a pharmaceutically acceptable salt, solvate or hydrate thereof.

[0030] For example, a CCR1 antagonist is BX-471 ((2R)-1- [[2-[(aminocarbonyl)amino]-4-chlorophenoxy]acetyl]-4-[ (4-fluorophenyl)methyl]-2-methylpiperazine): [ka] or a pharmaceutically acceptable salt, solvate or hydrate thereof.

[0031] For example, CCR1 antagonists include those described in WO2008 / 103126. Spirocyclic piperidine-based CCR1 antagonists. In embodiments, the CCR1 antagonist is a compound of formula (II) shown below: [ka] (In the formula, m is 1, t is 1, R 1 is a halogen, X, Y, and Z are independently a bond, —O—, or —CH—, provided that X, Y and only one of Z is a bond; R 2 is optionally one or more independently selected from hydroxyl and carboxyl substituted by the substituents 1-6 is an alkoxy, R 3 is a halogen, R 4 and R 5 is hydrogen and C 1-6 alkyl) or a pharmaceutically acceptable salt, solvate or hydrate thereof.

[0032] For example, a CCR1 antagonist is AZD-4818 ((2-[2 -chloro-5-[(2S)-3-(5-chlorospiro[benzofuran-2(3H),4' -piperidin]-1'-yl)-2-hydroxypropoxy]-4-[(methylamino) carbonyl]phenoxy]-2-methylpropanoic acid): [ka] or a pharmaceutically acceptable salt, solvate or hydrate thereof.

[0033] For example, CCR1 antagonists include 2, It is a 7-dichloro-9H-xanthen-9-yl-based CCR1 antagonist. That is, in some embodiments, the CCR1 antagonist has the formula (II) shown below: Compound I): [ka] (In the formula, R 10 and R 20 is a hydrogen atom, a halogen atom, or C 1-6 Independent of alkali atoms and selected, X is -O-, -S- or -CH2-; Q is an anion (e.g., Cl - , Br - or I - ) and R 30 is a cyclooctylmethyl group, a cyclononylmethyl group, a 1-decalylmethyl group, 2-decalylmethyl group, (1-cyclooctenyl)methyl group or (1-cyclononenyl ) a methyl group, R 40 is selected from methyl, ethyl, propyl or allyl or a pharmaceutically acceptable salt, solvate or hydrate thereof.

[0034] For example, CCR1 antagonists include 2, It is a 7-dichloro-9H-xanthen-9-yl-based CCR1 antagonist. That is, in some embodiments, the CCR1 antagonist has the formula (II) shown below: I) or a pharmaceutically acceptable salt, solvate or hydrate thereof: [ka] (In the formula, R 10 , R 20 , X, Q, R 30 and R 40 is as defined above. (This is the case.)

[0035] For example, a CCR1 antagonist is J113863 (1,4-ci) shown below. s-1-(1-cycloocten-1-ylmethyl)-4-[[(2,7-dichloro-9H -xanthen-9-yl)carbonyl]amino]-1-ethylpiperidinium iodide : [ka] or a pharmaceutically acceptable salt, solvate or hydrate thereof.

[0036] The present invention relates to all pharmaceutically acceptable forms of the CCR1 antagonists described herein. Suitable pharmaceutically acceptable salts of the CCR1 antagonists of the present invention include, for example, For example, acid addition salts of the compounds of the invention which are sufficiently basic, for example, inorganic or organic acids, e.g. For example, hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, trifluoroacetic acid, formic acid, citric acid, methane In addition, the acid addition salts of the present invention are sufficiently acidic. Suitable pharmaceutically acceptable salts of the CCR1 antagonists include alkali metal salts, e.g., sodium salts. thorium or potassium salts, alkaline earth metal salts, e.g., calcium or magnesium with an organic base that provides a sodium salt, an ammonium salt, or a pharmaceutically acceptable cation Salts such as methylamine, dimethylamine, trimethylamine, piperidine, morpholino It is a salt with amine or tris-(2-hydroxyethyl)amine.

[0037] The present invention also relates to a CCR1 antigen of the invention as defined herein which comprises one or more isotopic substitutions. For example, H includes 1H, 2H (D), and 3H (T). It can be in any isotopic form, and C can be any isotope, including 12C, 13C, and 14C. and O can be in any isotopic form, including 16O and 18O, etc.

[0038] Certain CCR1 antagonists of the present invention may be in solvated forms, such as, for example, hydrated forms, as well as in soluble forms. It is also understood that all compounds having anti-cancer activity may exist in unsolvated form. It is understood that all such solvated forms are encompassed.

[0039] It is understood that certain CCR1 antagonists may exhibit polymorphism and that the present invention provides compounds with anti-cancer activity. It is also understood to encompass all such forms having the following structure:

[0040] The CCR1 antagonists may also be degraded in the human or animal body to form the CC1 antagonists of the present invention. The prodrug may be administered in the form of a prodrug that releases an R1 antagonist. To alter the physical and / or pharmacokinetic properties of CR1 antagonists A prodrug is a compound in which a CCR1 antagonist has a property-modifying group attached thereto. Examples of prodrugs include C In vivo cleavage sites that may be formed at carboxy or hydroxy groups in the CR1 antagonist. Cleavable ester derivatives and carboxy or amino groups in CCR1 antagonists These include in vivo cleavable amide derivatives that may be formed with the group.

[0041] Treatment methods The present invention provides a method of treating pancreatic cancer in a subject in need thereof, The method comprises administering to the subject a therapeutically effective amount of a CCR1 antagonist, a pharmaceutically acceptable salt thereof, The present invention provides a method for treating a rheumatoid arthritis, comprising administering a salt, solvate, or hydrate thereof, or a pharmaceutical composition thereof, to a patient suffering from rheumatoid arthritis. Provide.

[0042] References to "treat" or "treatment" include prevention as well as the alleviation of established symptoms of a condition. It is understood that the term "treating" or "treating a condition" includes the term "treatment." "Treatment" includes (1) treating a person who may have or is suffering from a health condition, disorder, or condition; may have a predisposition to, but have not yet experienced, clinical or subclinical manifestations of, a health condition, disorder or condition Clinical manifestations of a health condition, disorder or condition that develops in humans who have not experienced or are not experiencing it (2) to prevent or delay the onset of a health condition, disorder, or condition; Inhibiting, i.e., preventing or minimizing the development of the disease or its recurrence (in the case of maintenance treatment) arrest, reduce or delay at least one clinical or subclinical symptom; or (3) To alleviate or attenuate a disease, i.e., to improve a health condition, disorder, or condition. or cause regression of at least one of its clinical or subclinical symptoms. Included.

[0043] The method of treating pancreatic cancer in a subject in need thereof according to the present invention comprises: When combined with established treatment regimens, such as those often referred to as "standard care," These combinations are particularly effective in treating pancreatic cancer when administered in combination. In a preferred embodiment, the method for treating pancreatic cancer includes administering to the subject a therapeutically effective amount of a CCR1 antagonist or a pharmaceutically acceptable salt thereof; The solvates or hydrates, or pharmaceutical compositions thereof, may be used in combination with gemcitabine (Gemzar®). Fluorouracil (5-FU), capecitabine (Xeloda®) , FOLFIRINOX and Nab-paclitaxel (Abraxane®) and administering the compound in combination with one or more chemotherapeutic agents selected from the group consisting of: will be done.

[0044] The CCR1 antagonist may also or alternatively be used in combination with a cancer immunotherapeutic agent (e.g., PD -1 and / or PD-L1 inhibitors).

[0045] CCR1 antagonists may also or alternatively be used in combination with MEK inhibitors. It can be used.

[0046] As described above, the present inventors have identified a novel protein expressed in pancreatic cancer cells (e.g., PDAC cells). Therefore, a strong correlation was found between the level of CCR1 detected and the prognosis of the patient. The method of treatment comprises, in one embodiment, administering an increased level of CCR1 expression compared to a reference expression level. The present invention is a method for treating pancreatic cancer in a subject identified as having the condition.

[0047] As mentioned above, the inventors have compared the level of immune (e.g., macrophage) infiltration with the patient's Therefore, in one embodiment, the method of treatment of the present invention comprises: , characterized by increased levels of immune (e.g., macrophage) infiltration compared to reference infiltration levels. The present invention is a method for treating pancreatic cancer in a subject.

[0048] As mentioned herein, the present inventors have also investigated the biomarkers PIM3, GSK3 B, ATK1, CDK1, CDK5, MAPK14, MTOR, MAPK3, CAMK2 A, MAP2K1, MAPK8, PRKACA, SRC, RPS6KA1, MAPK1 and We found a correlation between the levels of AKT / MAPK and PRKCA and poor patient prognosis. These kinases and genes in the pathway are generally associated with poor prognosis in multiple cancers. Therefore, in one embodiment, the method of treatment of the present invention is directed to the treatment of PIM3, GSK3 B, ATK1, CDK1, CDK5, MAPK14, MTOR, MAPK3, CAMK2 A, MAP2K1, MAPK8, PRKACA, SRC, RPS6KA1, MAPK1 and and PRKCA. The present invention is a method for treating pancreatic cancer in a subject.

[0049] A "therapeutically effective amount" is an amount that, when administered to a mammal for treating a disease, produces an effective amount of a compound that is effective for the disease. "Therapeutically effective amount" means the amount of a compound that is sufficient to effect such treatment. the compound, the disease and its severity, the age, weight, etc. of the mammal to be treated, and / or Varies depending on the size of the tumor imaged and / or scanned. The "therapeutically effective amount" varies depending on the size of the tumor being imaged and / or scanned. The physician should consider the factors outlined above (e.g., tumor size imaged and / or scanned). The "therapeutically effective amount" is properly qualified to be determined by taking into account the size of the compound. The current standard of care listed below has the best established drug regimens associated with it, They are also appropriate when the treatments are used in combination.

[0050] A "subject" according to the present invention is understood to mean a human and / or animal subject.

[0051] The term "pancreatic cancer" is understood to encompass all forms of cancer of the pancreas. That is, pancreatic cancer can be a cancer of the exocrine glands (e.g., pancreatic ductal adenocarcinoma) and / or the endocrine glands. The term "pancreatic cancer" also refers to cancers of the pancreas (e.g., pancreatic neuroendocrine tumors). Pancreatic malignancies originating elsewhere (e.g., liver, peritoneum, lung, adrenal gland, and bone) It is also understood that the term "cancer" may include cancer that has spread to other parts of the body.

[0052] Pancreatic cancer is typically classified by the size and location of the cancer in the body. Stage 1 pancreatic cancer is the earliest form of pancreatic cancer, in which cancer cells are predominantly contained within the pancreas. Describe the circumstances in which stage 1 pancreatic cancer is present. Stage 1 pancreatic cancer is often an early, localized, or It is called resectable pancreatic cancer (meaning the cancer can often be removed by surgery). Stage 2 pancreatic cancer refers to the fact that cancer cells leave the pancreas and enter the duodenum, for example. , a descriptive term used when it spreads to the tissues immediately surrounding the bile duct or pancreas Some forms of stage 2 pancreatic cancer are resectable. Stage 3 pancreatic cancer is , used when cancer cells have spread from the pancreas to the stomach, spleen, colon, or large blood vessels near the pancreas. Stage 3 pancreatic cancer is often not resectable. Stage 4 pancreatic cancer Pancreatic cancer is when cancer cells spread from the pancreas to other parts of the body, such as the lungs, liver, or peritoneum. Stage 4 pancreatic cancer is unresectable.

[0053] In certain embodiments, the pancreatic cancer is stage 3 or stage 4 pancreatic cancer, suitably , stage 4 pancreatic cancer.

[0054] In a further embodiment, the pancreatic cancer is pancreatic ductal adenocarcinoma (PDAC), suitably stage 4 pancreatic ductal adenocarcinoma (PDAC).

[0055] An effective amount of a CCR1 antagonist for use in treating pancreatic cancer is to treat, prevent or cure, slow the progression of, and / or prevent the development of, or It is understood that the amount is sufficient to reduce the associated symptoms.

[0056] An active ingredient (e.g., CCR) that is combined with one or more excipients to produce a single dosage form. The amount of a given antagonist (or antagonists) will necessarily vary depending on the individual being treated and the particular route of administration. For example, formulations intended for oral administration to humans generally contain about 5% of the total composition. Formulated with appropriate and convenient amounts of excipients, which may vary from about 98 weight percent to about 98 weight percent, e.g., 0.5 mg to 0.5 g of active agent (more suitably 0.5 to 100 mg, e.g., 1 to 30 Contains mg).

[0057] The size of the dose of a CCR1 antagonist for therapeutic (or prophylactic) purposes is well known. in accordance with accepted medical principles, taking into account the nature and severity of the condition, the age and sex of the animal or patient, These naturally vary depending on the dosage and route of administration.

[0058] When using CCR1 antagonists for the treatment of pancreatic cancer, split doses are required. If so, it is generally in the range of, for example, 0.1 mg / kg to 75 mg / kg body weight. Generally, when parenteral administration is used, Lower doses are administered. Thus, for example, for intravenous or intraperitoneal administration: For example, doses ranging from 0.1 mg / kg to 30 mg / kg body weight are commonly used. Similarly, for inhalation administration, for example, 0.05 mg / kg to 25 mg / kg body weight Doses in the range of 0.1 mg / kg or 0.25 mg / kg may be used. Oral administration (especially in tablet form) may also be appropriate. Typically, the unit dosage form contains about 0.5 mg to 0.5 g of the CCR1 antagonist.

[0059] Diagnosis for pancreatic cancer patients: In diagnosing pancreatic cancer in a subject, the expression level of CCR1 can be determined by methods known in the art. The level of CCR1 expression can be determined by any suitable means known in the art. For example, the level of CCR1 expression can be determined by This can be determined by measuring the CCR1 protein level. The label can be, for example, SDS-PAGE followed by a chromatographic analysis of the target protein. Western blot using appropriate antibodies, proteomics, membrane spotting by methods known in the art, such as arrays, or immunohistochemistry (IHC) methods. It may be measured using any suitable technique. Additionally or alternatively, the expression of CCR1 may be measured. The level can be determined by measuring the level of mRNA. For example, Northern blot, imaging mass cytometry (IMC), RNA sequencing Sequencing (RNAseq), Single-cell RNA sequencing (scRNAseq) or any method known in the art, such as quantitative RT-PCR (qRT-PCR). The assay may be performed on a biopsy taken from the subject. In each case, the level measured in the sample is compared to a reference expression level. It is possible.

[0060] Immune (e.g., macrophage) infiltration can be achieved by any suitable technique known in the art. It is understood that the level of macrophage infiltration can be determined using techniques such as A non-limiting list of possible techniques includes immunofluorescence (IF), immunohistochemistry (IHC), immunohistochemistry ... Imaging Mass Cytometry (IMC), flow cytometry, RNA sequencing RNA sequencing (RNAseq), single-cell RNA sequencing (scRNAseq) or protease This includes proteomics.

[0061] Test scores were calculated using the Immunoscore metric (as described in the Examples section herein below). (e.g., ) may be calculated by applying the immune score to the expression level data. Suitably, the immune score is , single sample gene set enrichment analysis (ssGSEA) was performed on the expression data. Calculate the normalized enrichment score (NES) using Most appropriately, immune scores are obtained from single-sample gene set enrichment analysis. The ssGSEA-based method, Strawberry Extraction in Malignant Tumors Using Expression Data, "Estimation of stromal and immune cells" d Immune cells in Malignant Tumors usin g Expression data”)(ESTIMATE,Nature Comm .,2013,4(2612),1-11) for expression data. It is calculated.

[0062] The pre-determined threshold score was generated from a reference sample set of pancreatic cancer patients. Suitably, the predetermined threshold score represents a predetermined immune score. A pre-determined reference sample set generated from a reference sample set of patients with pancreatic cancer (PDAC) in IV Represents the immune score.

[0063] The predetermined threshold score can be determined using any suitable method known in the art. Suitably, the predetermined threshold score may be derived from a reference score for pancreatic cancer patients. A reference sample set may be derived from expression data from a set of samples, e.g. Pancreatic cancer patients for whom expression data has been generated (e.g., stage IV pancreatic cancer) Non-limiting examples of suitable reference sample sets include, for example, The Cancer Genome Atlas (TCGA), or similar database sources (e.g., International Cancer Genome Consortium) rtium(ICDC), UT Southwestern Medical Center re and Queensland Centre for Medical Genom The database of pancreatic cancer patients (e.g., stage IV pancreatic cancer patients). The threshold score was taken from The Cancer Genome Atlas (TCGA). Use expression data from pancreatic cancer patients for whom it is possible (e.g., stage IV pancreatic cancer patients). Using a single sample gene set enrichment analysis (ssGSEA)-based method "Inferring stromal and immune cells in malignant tumors using expression data" (ESTIMATE) on the expression data.

[0064] Suitably, the predetermined threshold score is a threshold score based on the immune sequences generated from the reference sample set. This is the immunity score corresponding to the 25th percentile of the core.

[0065] Suitably the method comprises determining whether the test score (e.g. immune score) is generated from a reference sample set. The 25th percentile of the immune score achieved (i.e., a predetermined threshold score) This includes treating a subject when the condition is greater than the risk of death.

[0066] For example, the subject in need of treatment may be diagnosed with pancreatic cancer and have one or more of the following: The targets identified as: expression of an increased level of CCR1 compared to a reference expression level; an increased level of immune (e.g., macrophage) infiltration compared to reference infiltration levels; High levels of MYC expression and / or mutations in the MYC oncogene, SMAD4 mutation, and / or Classical Moffitt tumor RNA subtypes.

[0067] As used herein, the term "MYC" refers to a regulatory gene that falls within the MYC gene family. This is understood to mean the entire family of genes (transcription factors). MYC transcription factor family Members of this group include c-MYC, MYCN, and MYCL, and therefore References to "MYC" in the literature cover all such family members. MYC-regulated genes are involved in cell proliferation, growth, differentiation, and apoptosis. Normal MYC gene activation regulates cell cycle progression, Cell growth and division, metabolism, telomerase activity, adhesion and motility, angiogenesis and differentiation MYC affects numerous cellular processes, including mitochondrial differentiation. The mutated versions of these genes are known to be involved in the development of certain types of cancers, such as hematologic and solid tumor malignancies. are often upregulated and / or constitutively expressed in cancer (Miller et al., Clin. Cancer Res., 201 2,18(20),5546-5553). Therefore, if a patient is diagnosed with pancreatic cancer and has a high MY, Subjects identified as having C expression and / or a mutation in the MYC oncogene are those whose pancreas These patients may be more likely to respond poorly to standard treatment for cancer and therefore more likely to be may have a worse progression-free survival rate.

[0068] A key genetic alteration in pancreatic cancer is SMAD4 mutation, which is a SMAD4 transcription factor. SMAD4 is inactivated in over 50% of pancreatic cancer cases, leading to loss of protein expression. Many studies have shown that loss of SMAD4 expression is associated with pancreatic cancer in patients with pancreatic cancer. have shown a positive association with poor prognosis in patients with glaucoma (Wei et al., Transl .Oncol.2016,9(1),1-7). Therefore, if a patient is diagnosed with pancreatic cancer and undergoes SM Subjects identified with AD4 mutations have higher need and poorer progression-free survival It is a subject that may have.

[0069] Combination therapy In an important aspect of the present invention, one or more standard anti-cancer drugs for use in the treatment of pancreatic cancer are provided. CCR1 antagonists, their pharmaceutically acceptable salts, in combination with pancreatic cancer treatment regimens The present invention provides salts, solvates or hydrates thereof.

[0070] One or more standard pancreatic cancer treatment regimens are commonly used to treat pancreatic cancer. The treatment can be any appropriate treatment regimen (i.e., "standard of care" for pancreatic cancer) that is currently being considered. It will be appreciated that suitably, one or more standard pancreatic cancer treatment regimens may include surgery, More preferably, the treatment is selected from radiation therapy, chemotherapy, immunotherapy, and combinations thereof. The standard treatment regimen for pancreatic cancer consists of surgery, chemotherapy or immunotherapy and their Most suitably, standard pancreatic cancer treatment regimens include chemotherapy and a combination of chemotherapy and chemotherapy alone. and optionally one or more additional treatment regimens selected from surgery and immunotherapy. Including men.

[0071] For patients diagnosed with early stage pancreatic cancer (stage I or II), standard Common pancreatic cancer treatment regimens include surgery and, optionally, chemotherapy and immunotherapy. For example, the UK National Clinical Excellence For metastatic or otherwise not sufficiently "locally advanced," pancreatic cancer is amenable to surgical treatment. The first recommendation is that patients should be treated with surgery followed by adjuvant chemotherapy. The combination of mucitabine and capecitabine was administered for 6 cycles, and this combination therapy was tolerated. If this is not possible, gemcitabine alone is used.

[0072] For patients diagnosed with late-stage pancreatic cancer (stage III or IV), Standard pancreatic cancer treatment regimens consist of chemotherapy and, optionally, surgery and immunotherapy. For example, the UK National Institute for Clinical Excellence recommends one or more additional treatment regimens selected from: For locally advanced or metastatic pancreatic cancer, whether it is treated with chemotherapy or chemoradiotherapy For locally advanced pancreatic cancer, the first recommendation is to treat with systemic chemotherapy. combination chemotherapy (e.g., the FOLFIRINOX combination discussed below) If the combination is not tolerated, gemcitabine alone is used. Chemoradiation therapy is also used. If this is the case, capecitabine should be given. is mFOLFIRINOX, which is an initial injection / bolus of fluorouracil No, irinotecan level was 150 mg / m 2 The number of pancreatic cancers in the United States has decreased to Further treatment options recommended by US clinical practice guidelines for cancer include novel inhibitors, such as erlotinib, capecitabine, or taxanes (docetaxel, etc.) include.

[0073] For metastatic pancreatic cancer, the first recommendation is treatment with FOLFIRINOX ( It has the best survival statistics but is the least tolerable. If not, gemcitabine plus nab-paclitaxel should be given. If neither combination is tolerated, gemcitabine alone should be given. Further treatment options recommended by US clinical practice guidelines for pancreatic cancer in The treatment is also being considered with additional novel agents such as pembrolizumab, larotrectinib or entrectinib. This includes adding rectinib.

[0074] Second-line metastatic therapy is gemcitabine-based regimens (first-line FOLFIR after INOX), or oxaliplatin-based regimens (other first-line combinations (After) The combination of irinotecan + fluorouracil + folinic acid nanoliposomes This combination is listed as a second-line therapy in the European Society for Medical Oncology (ESMO) clinical practice guidelines. recommended for first-line chemotherapy (after first-line gemcitabine-based therapy). An additional treatment option recommended by the US clinical practice guidelines for capecitabine is capecitabine. Tabine or fluorouracil alone, and an additional new agent, pembrolizumab , including the addition of larotrectinib or entrectinib.

[0075] Surgery can be a surgical intervention by a doctor to remove all or part of a tumor .

[0076] Radiation therapy can be any form of treatment that utilizes ionizing radiation. Non-limiting examples of treatments include, for example, external beam radiation therapy (EBRT), stereotactic radiotherapy (STRS), ) and external beam radiation therapy.

[0077] Chemotherapy can be treatment with the administration of one or more anti-tumor (anti-cancer) drugs. Non-limiting examples of anti-tumor agents suitable for the treatment of pancreatic cancer include gemcitabine (Gemzar). (registered trademark), fluorouracil (5-FU), capecitabine (Xeloda FOLFIRINOX (leucovorin calcium, fluorouracil, irinotecan hydroxybenzoates (HCO3, HCl, and oxaliplatin) and Nab-paclitaxel (Abraxane) e (registered trademark).

[0078] Gemcitabine, 5-fluorouracil, and capecitabine are pyrimidine antagonists. (sometimes broadly classified as antimetabolites as well).

[0079] Immunotherapy (cancer immunotherapy) can be any form of treatment that harnesses the patient's immune system. Non-limiting examples of suitable immunotherapeutic treatments include, for example, monoclonal antibodies (MABs), prophylactic These treatments include those utilizing one or more of the following: vaccination, cytokines, and CAR-T cells. Suitably, the immunotherapy (cancer immunotherapy) comprises a PD-L1 inhibitor and / or a PD Non-limiting examples of suitable PD-1 inhibitors include pembrolizumab , nivolumab, IBI-308, mDX-400, BGB-108, MEDI-0680 , SHR-1210, PF-06801591, PDR-001, GB-226 and S Non-limiting examples of suitable PD-L1 inhibitors include Durbarma. AB, atezolizumab, avelumab, BMS-936559, ALN-PDL, TSR-0 42, KD-033, CA-170, STI-1014, and KY-1003. .

[0080] Suitably, the PD-1 inhibitor is selected from pembrolizumab or nivolumab. More suitably, the PD-1 inhibitor is nivolumab.

[0081] Therefore, in summary, for pancreatic cancer (e.g., stage I or II pancreatic cancer), The standard treatment regimen for pancreatic cancer involves surgery to remove part or all of the tumor. followed by gemcitabine (Gemzar®), fluorouracil (5-F U), capecitabine (Xeloda®), FOLFIRINOX, Nab-P clitaxel (Abraxane®) or a combination thereof Similarly, for stage III or IV pancreatic cancer, Standard tumor treatment regimens include gemcitabine (Gemzar®), fluorouracil (Fenriroline), and fluoxetine (Fenriroline). Rasil (5-FU), capecitabine (Xeloda®), FOLFIRINO X, Nab-paclitaxel (Abraxane®), or a combination thereof The method includes administering a chemotherapeutic agent selected from the group consisting of:

[0082] Preferred treatments according to the present invention include CCR1 antagonists (e.g., BX-471), or a pharmaceutically acceptable salt, solvate or hydrate thereof is an established treatment for pancreatic cancer. It is used in combination with selected therapies.

[0083] Considering the currently recommended standard treatment regimens, CCR1 antagonists are appropriate For this purpose, a chemotherapeutic agent selected from gemcitabine or capecitabine, particularly gemcitabine and The present inventors have demonstrated that a CCR1 antagonist is used in combination with gemcitabine. have seen particularly strong therapeutically beneficial effects in mouse models when administered intravenously.

[0084] Additionally, in certain combination therapies, CCR1 antagonists are combined with FOLFIRINOX. It can also be used in combination with oxaliplatin. It can also be used in combination with Nab-paclitaxel. or PD-L1 inhibitors.

[0085] As additional novel therapies are developed, CCR1 antagonists may also be useful in combination with these. Therefore, CCR1 antagonists such as erlotinib, capecitabine, Taxanes (such as docetaxel), pembrolizumab, larotrectinib, or entrezolidine diphosphate (ETP) It may be used in combination with cucinib.

[0086] Particularly suitable combination therapies include gemcitabine (e.g., in combination with FOLFIRINOX) ) and a CCR1 antagonist administered together with a PD-1 inhibitor. reported that a CCR1 antagonist was administered together with gemcitabine and a PD-1 inhibitor. In some cases, particularly strong therapeutic beneficial effects have been observed in mouse models. Combination treatment with an R1 antagonist, gemcitabine, and a PD-1 inhibitor is preferred. Paclitaxel may also optionally be included.

[0087] A particularly suitable combination therapy is FOLFIRINOX administered with a PD-1 inhibitor. Therefore, the CCR1 antagonist, FOL Combination therapy with FIRINOX and PD-1 inhibitors is preferred. Nab-paclitaxel is also recommended. It may also be included optionally.

[0088] For example, CCR1 antagonists have been shown to be effective against gemcitabine (Gem) in the treatment of pancreatic cancer. zar®), fluorouracil (5-FU), capecitabine (Xeloda®), FOLFIRINOX and Nab-paclitaxel (Abraxane (registered trademark). are gemcitabine (Gemzar®), nab-paclitaxel (Abrax ane®), FOLFIRINOX, or a combination thereof CCR1 antagonists (e.g., BX-471) in combination with one or more chemotherapeutic agents ) or a pharmaceutically acceptable salt, solvate or hydrate thereof. includes gemcitabine (Gemzar®) for use in the treatment of pancreatic cancer. ) and / or Nab-paclitaxel (Abraxane®) a CCR1 antagonist (e.g., BX-471), or a pharmaceutically acceptable salt thereof The present invention provides salts, solvates or hydrates thereof, most suitably for use in the treatment of pancreatic cancer. CCR1 antagonist in combination with gemcitabine (Gemzar®) for use in agonist (e.g., BX-471), or a pharmaceutically acceptable salt, solvate, or In particular, hydrates are provided.

[0089] In another aspect of the present invention, there is provided a method for treating pancreatic cancer with gemcitabine (Gem) zar®), fluorouracil (5-FU), capecitabine (Xeloda®), FOLFIRINOX and Nab-paclitaxel (Abraxane (registered trademark), and optionally a MEK inhibitor. agents, IGF1R inhibitors, PD-1 inhibitors, PD-L1 inhibitors and / or cancer immunotherapy in combination with one or more of the following agents (e.g., PD-1 and / or PD-L1 inhibitors) Also, a CCR1 antagonist (e.g., BX-471) or a pharmaceutically acceptable salt thereof Salts, solvates or hydrates are provided.

[0090] In another aspect of the present invention, there is provided a method for treating pancreatic cancer with gemcitabine (Gem) zar®), fluorouracil (5-FU), capecitabine (Xeloda®), FOLFIRINOX and Nab-paclitaxel (Abraxane (R), and optionally one or more cancer immunotherapies. CCR1 antagonists in combination with other therapeutic agents (e.g., PD-1 and / or PD-L1 inhibitors) agonist (e.g., BX-471), or a pharmaceutically acceptable salt, solvate, or In particular, hydrates are provided.

[0091] For example, chemotherapy agents may be administered simultaneously and one or more cancer immunotherapeutic agents may then be administered sequentially. is administered.

[0092] For example, the chemotherapy agent and one or more cancer immunotherapeutic agents are administered sequentially. 1. The antagonist, chemotherapeutic agent, and one or more cancer immunotherapeutic agents may be administered in any sequential order. It will be appreciated that the CCR1 antagonist may be administered first. Suitably, the CCR1 antagonist is administered first, This is followed by the sequential administration of a chemotherapy agent and one or more cancer immunotherapy agents.

[0093] In another aspect of the invention, there is provided a method for the treatment of pancreatic cancer, comprising: MEK inhibitors and / or IGF1R inhibitors, and Optionally, gemcitabine (Gemzar®), fluorouracil (5-FU ), capecitabine (Xeloda®), FOLFIRINOX and Nab- a chemotherapeutic agent selected from paclitaxel (Abraxane®) a CCR1 antagonist (e.g., BX-471), or a pharmaceutical composition thereof, in combination with A physiologically acceptable salt, solvate or hydrate thereof is provided.

[0094] In another aspect of the invention, there is provided a method for the treatment of pancreatic cancer, comprising: PD-1 or PD-L1 inhibitors, and Optionally, gemcitabine (Gemzar®), fluorouracil (5-FU ), capecitabine (Xeloda®), FOLFIRINOX and Nab- a chemotherapeutic agent selected from paclitaxel (Abraxane®) a CCR1 antagonist (e.g., BX-471), or a pharmaceutical composition thereof, in combination with A physiologically acceptable salt, solvate or hydrate thereof is provided.

[0095] In another aspect of the present invention, there is provided a method for treating pancreatic cancer comprising administering to a subject a MEK inhibitor and / or a medicament for use in the treatment of pancreatic cancer. or a CCR1 antagonist (e.g., BX-47) in combination with an IGF1R inhibitor. 1), and pharmaceutically acceptable salts, solvates or hydrates thereof.

[0096] Non-limiting examples of suitable MEK inhibitors include CI-1040 (PD184352), PD 0325901, selumetinib (AZD6244), MEK162, AZD8330, T AK-733, GDC-0623, refametinib (RDEA119, BAY86976 6), Pimasertib (AS703026), RO4987655 (CH4987655) , RO5126766, WX-554, HL-085 and combinations thereof. (Tian et al.,Molecules,2017,22(10),1551 ).

[0097] Non-limiting examples of suitable IGF1R inhibitors include dalotuzumab (and its MK-2206, ridaforolimus, MK-0752, cetuximab, irinotecan, Suplatin (in combination with one or more of etoposide and erlotinib) tumab (and figitumumab), carboplatin, paclitaxel, dexamethasone, In combination with one or more of docetaxel, prednisone, erlotinib, and everolimus combination), Gantitumab (and Gantitumab's Stan, fulvestrant, FOLFIRI, gemcitabine, panitumumab, sorafenib lincitumumab (and lincitumumab in combination with one or more of rifabutinib and erlotinib) citinib in combination with everolimus) and R1507 (Yee et al. al.,Molecular Endocrinology,2015,29(11) ,1549-1557).

[0098] In a further aspect of the present invention, there is provided a method for treating pancreatic cancer in a subject in need thereof. The following drugs for use in: MEK inhibitors IGF1R inhibitors, PD-1 or PD-L1 inhibitors, and / or Gemcitabine (Gemzar®), fluorouracil (5-FU), capecitabine Tabine (Xeloda®), FOLFIRINOX, and Nab-paclitaxel A chemotherapeutic agent selected from the group consisting of cerebrospinal fluid (Cell), cerebrospinal fluid (Abraxane®), a CCR1 antagonist (e.g., BX-471), in combination with one or more of or a pharmaceutically acceptable salt, solvate, or hydrate thereof, wherein the subject is a patient with pancreatic diagnosed with cancer, an increased level of CCR1 expression compared to a reference expression level, and / or have increased levels of immune (e.g., macrophage) infiltration compared to reference infiltration levels The identified subject CCR1 antagonist, or a pharmaceutically acceptable salt thereof, Salts, solvates or hydrates are provided.

[0099] In one embodiment, the present invention provides a method for treating pancreatic cancer in a subject in need thereof. The following drugs for use in: MEK inhibitors IGF1R inhibitors, PD-1 or PD-L1 inhibitors, and / or Gemcitabine (Gemzar®), Nab-paclitaxel (Abraxa ne®), FOLFIRINOX, or a combination thereof chemotherapy drugs a CCR1 antagonist (e.g., BX-471), in combination with one or more of or a pharmaceutically acceptable salt, solvate, or hydrate thereof, wherein the subject is a patient with pancreatic diagnosed with cancer, expression of an increased level of CCR1 compared to a reference expression level; an increased level of immune (e.g., macrophage) infiltration compared to reference infiltration levels; High levels of MYC expression and / or mutations in the MYC oncogene, SMAD4 mutation, and / or Subjects identified as having the classical Moffitt tumor RNA subtype, CCR 1 antagonist, or a pharmaceutically acceptable salt, solvate or hydrate thereof. do.

[0100] In certain embodiments, the subject is diagnosed with pancreatic cancer and is receiving the treatment of the present invention as defined hereinabove. A test score greater than a predetermined threshold score (e.g., determined by a known method) For example, the 25th percentile of the immune scores generated from the reference sample set. It is a specific target.

[0101] In yet another aspect of the present invention, a method for treating pancreatic cancer in a subject in need thereof is provided. a method of treating a subject, the method comprising administering to the subject a therapeutically effective amount of a CCR1 antagonist (e.g., For example, BX-471), or a pharmaceutically acceptable salt, solvate, or hydrate thereof, or the pharmaceutical composition may be administered in combination with other drugs, such as gemcitabine (Gemzar®), fluorouracil, (5-FU), capecitabine (Xeloda®), FOLFIRINOX and and Nab-paclitaxel (Abraxane®) In another aspect, methods are provided that include administering the compound of formula (I) in combination with a chemotherapeutic agent of formula (I).

[0102] In some embodiments, pancreatic cancer (e.g., and stage IV pancreatic cancer), wherein the method is effective in treating the subject. an amount of a CCR1 antagonist (e.g., BX-471), or a pharmaceutically acceptable salt thereof; The salt, solvate or hydrate, or pharmaceutical composition thereof, may be administered as a medicament for the treatment of gemcitabine (Gemzar). (registered trademark), Nab-paclitaxel (Abraxane®), FOLF one or more chemotherapeutic agents selected from IRINOX or combinations thereof, and Optionally, one or more cancer immunotherapeutic agents (e.g., PD-1 and / or PD-L1 inhibitors) Methods are provided that include administering the compound in combination with an anti-inflammatory drug (anti-inflammatory drug).

[0103] In yet another aspect of the present invention, a method for treating pancreatic cancer in a subject in need thereof is provided. a method of treating a subject, the method comprising administering to the subject a therapeutically effective amount of a CCR1 antagonist (e.g., For example, BX-471), or a pharmaceutically acceptable salt, solvate, or hydrate thereof, or the pharmaceutical composition, comprising a MEK inhibitor and / or an IGF1R inhibitor, and optionally The options are gemcitabine (Gemzar®), fluorouracil (5-FU), Pecitabine (Xeloda®), FOLFIRINOX and Nab-Pac in combination with one or more chemotherapeutic agents selected from taxane (Abraxane®), The method includes administering the compounds of formula (I) and formula (II) together.

[0104] In some embodiments, pancreatic cancer (e.g., and stage IV pancreatic cancer), wherein the method is effective in treating the subject. an amount of a CCR1 antagonist (e.g., BX-471), or a pharmaceutically acceptable salt thereof; The salt, solvate or hydrate, or pharmaceutical composition thereof, is administered in combination with a MEK inhibitor and / or IGF1R inhibitors, and optionally gemcitabine (Gemzar®), N ab-paclitaxel (Abraxane®), FOLFIRINOX or and administering the compound in combination with one or more chemotherapeutic agents selected from the combinations thereof. A method is provided that includes:

[0105] In a further aspect of the present invention, a method for treating pancreatic cancer in a subject in need thereof is provided. The method comprises administering to the subject a therapeutically effective amount of a CCR1 antagonist (e.g., BX-471), or a pharmaceutically acceptable salt, solvate, or hydrate thereof, or the pharmaceutical composition thereof, MEK inhibitors IGF1R inhibitors, PD-1 or PD-L1 inhibitors, and / or Gemcitabine (Gemzar®), fluorouracil (5-FU), capecitabine Tabine (Xeloda®), FOLFIRINOX, and Nab-paclitaxel A chemotherapeutic agent selected from the group consisting of cerebrospinal fluid (Cell), cerebrospinal fluid (Abraxane®), and wherein a subject in need of treatment is administered in combination with one or more of: an increased level of CCR1 expression compared to a reference expression level, and / or have increased levels of immune (e.g., macrophage) infiltration compared to reference infiltration levels Having identified the object, a method is provided.

[0106] In certain embodiments, pancreatic cancer (e.g., stromal cell carcinoma) is treated in a subject in need thereof. 1. A method for treating pancreatic cancer (stage IV pancreatic cancer), the method comprising administering to the subject a therapeutically effective amount of a CCR1 antagonist (e.g., BX-471), or a pharmaceutically acceptable salt thereof; The solvates or hydrates, or pharmaceutical compositions thereof, may be administered to the following drugs: MEK inhibitors IGF1R inhibitors, PD-1 or PD-L1 inhibitors, and / or Gemcitabine (Gemzar®), Nab-paclitaxel (Abraxa ne (registered trademark), FOLFIRINOX, or a combination thereof Chemotherapy wherein the subject in need of treatment is administered in combination with one or more of the following: expression of an increased level of CCR1 compared to a reference expression level; an increased level of immune (e.g., macrophage) infiltration compared to reference infiltration levels; High levels of MYC expression and / or mutations in the MYC oncogene, SMAD4 mutation, and / or Pairs identified as having one or more of the classical Moffitt tumor RNA subtypes A method is provided.

[0107] More suitably, a method for treating pancreatic cancer (e.g., stage 1 or stage 2 or stage 3 or stage 4 or stage 5 or stage 6 or stage 7 or stage 8 or stage 9 or stage 10 or stage 11 or stage 12 or stage 13 or stage 14 or stage 15 or stage 16 or stage 17 or stage 18 or stage 19 or stage 20 or stage 21 or stage 22 or stage 23 or stage 2 IV pancreatic cancer), the method comprising administering to the subject a therapeutically effective amount of CCR 1 antagonist (e.g., BX-471), or a pharmaceutically acceptable salt or solvate thereof The compound or hydrate, or a pharmaceutical composition thereof, is used in combination with gemcitabine (Gemzar®). )), fluorouracil (5-FU), capecitabine (Xeloda®), F OLFIRINOX and Nab-paclitaxel (Abraxane®) in combination with a chemotherapeutic agent selected from the group consisting of: MEK inhibitors, IGF1R inhibitors, and / or PD-1 or PD-L1 inhibitors and administering to the subject in combination with one or more of: an increased level of CCR1 expression compared to a reference expression level, and / or Increased levels of immune (e.g., macrophage) infiltration compared to reference infiltration levels A subject identified as having, and optionally in need of treatment, is High levels of MYC expression and / or mutations in the MYC oncogene, SMAD4 mutation, and / or Pairs identified as having one or more of the classical Moffitt tumor RNA subtypes A method is provided.

[0108] In certain embodiments, the subject is diagnosed with pancreatic cancer and is receiving the treatment of the present invention as defined hereinabove. A test score greater than a predetermined threshold score (e.g., determined by a known method) For example, the 25th percentile of the immune scores generated from the reference sample set. It is a specific target.

[0109] The combinations described herein include sequential, separate, and / or combined administration of the listed agents. It is understood that the combination may be simultaneous or simultaneous, i.e., the listed agents may be administered simultaneously. In situations where the combination includes more than two drugs, the combination Combinations include sequential, separate, and / or simultaneous administration of all of the listed agents. Alternatively, the combination may involve the sequential administration of some drugs and the simultaneous administration of others. In certain embodiments, the combinations described herein may include sequential administration. combination, where the listed drugs are administered sequentially (e.g., one after the other) In other embodiments, the combinations described herein are simultaneous combinations, Here, the listed drugs are administered together.

[0110] Pharmaceutical Composition According to another aspect of the present invention, there is provided a pharmaceutically acceptable carrier for use in the treatment of pancreatic cancer. a CCR1 antagonist as defined hereinabove, together with a diluent or carrier therefor, and a pharmaceutical composition comprising a pharmaceutically acceptable salt, hydrate or solvate thereof.

[0111] The present invention provides a CCR1 antagonist comprising: MEK inhibitors IGF1R inhibitors, PD-1 or PD-L1 inhibitors, and / or Gemcitabine (Gemzar®), fluorouracil (5-FU), capecitabine Tabine (Xeloda®), FOLFIRINOX, and Nab-paclitaxel one or more chemotherapeutic agents selected from the group consisting of cefotaxime (Cel) (Abraxane®) Pharmaceutical compositions containing the above together with additional therapeutic agents are provided.

[0112] Preferably, the pharmaceutical composition for use in the treatment of pancreatic cancer comprises gemcitabine (Gemcitabine mzar®), fluorouracil (5-FU), and Nab-paclitaxel (Abraxane®).

[0113] In some embodiments, the pharmaceutical composition comprises one or more MEK inhibitors and / or IG Contains F1R inhibitors.

[0114] The pharmaceutical compositions of the present invention can be administered orally (e.g., as tablets, lozenges, hard or soft capsules, Aqueous or oily suspensions, emulsions, dispersible powders or granules, syrups or elixirs topical use (e.g., as a cream, ointment, gel, or aqueous or oily solution) as a liquid or suspension), or by inhalation (e.g., as a fine powder or liquid aerosol). administration by insufflation (e.g., as a fine powder) or parenteral administration (e.g., intravenous) sterile aqueous or oily solutions for intravenous, subcutaneous, intramuscular, intraperitoneal or intramuscular administration; or as a suppository for rectal administration).

[0115] Depending on the choice of CCR1 antagonist, certain effective formulations are known and commercially available. are.

[0116] The pharmaceutical compositions of the present invention can be prepared in the conventional manner using conventional pharmaceutical excipients known in the art. Pharmaceutical compositions intended for oral use can therefore be obtained by the following procedures: For example, it may contain one or more coloring agents, sweeteners, flavoring agents and / or preservatives.

[0117] Prognostic methods The present invention provides a method for determining the prognosis of a subject diagnosed with pancreatic cancer, the method comprising: , measuring the level of CCR1 expression in a sample obtained from the subject; The step of comparing the level of CCR1 expression measured in step a) with a reference expression level. Tep and Based on the level of CCR1 relative to the reference expression level determined in step b), and determining the prognosis of the subject, An increased level of CCR1 expression compared to the reference expression level is unfavorable for the subject Decreased or unchanged levels of CCR1 expression compared to controls are prognostic. A method is provided that indicates a favorable prognosis for a subject.

[0118] In another aspect of the invention, there is provided a method of determining a prognosis for a subject diagnosed with pancreatic cancer, comprising: , the method comprises: determining the level of immune (e.g., macrophage) infiltration in a sample obtained from the subject; measuring; See the level of immune (e.g., macrophage) infiltration measured in step a). comparing with the level of invasion; the level of immune (e.g., macrophage) infiltration compared to the reference infiltration level determined in step b). and determining a prognosis for the subject based on the level of (phage) invasion; Increased levels of immune (e.g., macrophage) infiltration compared to reference infiltration levels are associated with A decrease or change in infiltration level compared to the reference level indicates an unfavorable prognosis for the patient. Undetectable levels of immune (e.g., macrophage) infiltration may be associated with a favorable prognosis for the subject. As shown, a method is provided.

[0119] For example, both the level of immune (e.g., macrophage) infiltration and CCR1 expression It may be measured in a sample obtained from the subject.

[0120] For example, the level of MYC expression, SMAD4 mutations, or classical Moffitt tumor R The NA subtype may also be determined as part of the prognosis.

[0121] According to a further aspect of the invention, the method for performing a prognosis comprises the steps of: ATK1, CDK1, CDK5, MAPK14, MTOR, MAPK3, CAMK2A, MAP2K1, MAPK8, PRKACA, SRC, RPS6KA1, MAPK1 and measuring the expression level of at least one biomarker selected from PRKCA Includes.

[0122] The method comprises determining the expression levels of CCR1 and at least one biomarker. determining a derived test score; and comparing the test score with a predetermined threshold score. and comparing a test score greater than a predetermined threshold to: A score below or equal to a predetermined threshold score indicating an unfavorable prognosis for the subject. Equal test scores indicate a favorable prognosis for the subject.

[0123] The term "prognosis" refers to the likely course of a medical condition (e.g., pancreatic cancer). Therefore, the term "favorable prognosis" is understood to mean that the course of pancreatic cancer is favorable for the patient. It is understood to mean that the outcome will be favorable for the patient. The term refers to a patient with pancreatic cancer who has undergone one or more standard pancreatic cancer treatment regimens (i.e. , surgery and / or chemotherapy treatment) for at least 3 months, preferably at least at least six months, more preferably at least one year, even more suitably at least two years This means that the patient is likely to survive for at least 5 years, and most preferably for at least 5 years. The term "favorable prognosis" is used within the context of the generally very poor prognosis among pancreatic cancer patients. and a high likelihood of responding favorably to one or more standard pancreatic cancer treatment regimens (i.e., have pancreatic cancer (i.e., a high chance that the cancer will not recur or progress after treatment) Point to the target.

[0124] The term "unfavorable prognosis" refers to a condition in which the course of pancreatic cancer becomes unfavorable for the subject. Appropriately, the term "unfavorable prognosis" is understood to mean that the pancreas Subjects with pancreatic cancer who have undergone one or more standard pancreatic cancer treatment regimens (i.e., surgery and and / or chemotherapy treatment) for at least 3 months (appropriately at least 6 months, More suitably for at least one year, even more suitably for at least two years, and most suitably for at least This means that the patient is unlikely to survive for at least five years. The term "low-dose pancreatic cancer" refers to a patient with a poor response to one or more standard pancreatic cancer treatment regimens. A pancreas with a high probability (i.e., a high likelihood that pancreatic cancer will recur or progress after treatment) It refers to a subject who has cancer.

[0125] Suitably, the prognostic method herein above comprises the first step of obtaining a sample from a subject. The sample may include, for example, a tumor sample obtained from the pancreas (or surrounding tissue) of a patient. The sample may be a sample of cells and / or a sample of the subject's blood. Any suitable technique known in the art, such as surgery, biopsy, or blood sample. It can be obtained using techniques.

[0126] The reference levels referred to herein above (i.e., "reference expression levels" and "reference "Infiltration level") is the CCR1 expression and and / or a reference point (or benchmark) against which the level of macrophage infiltration can be compared. The reference level refers to a control value that acts as a benchmark. For example, a reference level is used to measure the level of a patient who has not been diagnosed with pancreatic cancer. The mean level of CCR1 expression and / or the mean level of macrophage infiltration in healthy subjects level (e.g., negative control or reference level), or for subjects diagnosed with pancreatic cancer The average level of CCR1 expression and / or the average level of macrophage infiltration (e.g., For example, a positive control or reference level.

[0127] In one embodiment, the gene expression level or macrophage infiltration level in the subject is determined by: The level may be in the lower quartile, the highest quartile, or the middle two quartiles of the relevant population. Depending on whether they fall into a certain rank, they may be stratified into "high," "medium," and "low" groups, Alternatively, the gene expression level or macrophage infiltration level in the subject is determined to be: whether they fall in the lower quartile, the highest quartile, or the middle two quartiles of the relevant population. Depending on whether the patient has a high or low risk of developing a heart attack, the patient may be stratified into "high" or "low" groups.

[0128] Parts kit As noted above, the CCR1 antagonist for use according to the present invention is most preferably and in combination with one or more additional therapeutic agents. The insult may be provided together with such other therapeutic agents.

[0129] Another aspect of the invention therefore provides a kit of parts comprising the following components: A composition as defined herein, optionally with a pharmaceutically acceptable adjuvant, diluent or carrier. or a pharmaceutically acceptable salt, solvate or a hydrate; and Gemcitabine, optionally with a pharmaceutically acceptable adjuvant, diluent or carrier. (Gemzar®), fluorouracil (5-FU), capecitabine (Xel oda®), FOLFIRINOX and Nab-paclitaxel (Abra xane®), or a pharmaceutically acceptable salt, solvate or hydrate thereof; and one or more chemotherapeutic agents selected from the group consisting of: The components may be provided in forms suitable for sequential, separate and / or simultaneous administration. A parts kit is provided.

[0130] In another aspect of the invention, there is provided a kit of parts comprising the following components: A composition as defined herein, optionally with a pharmaceutically acceptable adjuvant, diluent or carrier. or a pharmaceutically acceptable salt, solvate or a hydrate; and MEK inhibitors, optionally with a pharmaceutically acceptable adjuvant, diluent or carrier and / or an IGF1R inhibitor, or a pharmaceutically acceptable salt, solvate or includes hydrates, The components may be provided in forms suitable for sequential, separate and / or simultaneous administration. A parts kit is provided.

[0131] In another aspect of the invention, there is provided a kit of parts comprising the following components: A composition as defined herein, optionally with a pharmaceutically acceptable adjuvant, diluent or carrier. or a pharmaceutically acceptable salt, solvate or a hydrate; and MEK inhibitors, optionally with a pharmaceutically acceptable adjuvant, diluent or carrier and / or an IGF1R inhibitor, or a pharmaceutically acceptable salt, solvate or is a hydrate, Gemcitabine, optionally with a pharmaceutically acceptable adjuvant, diluent or carrier. (Gemzar®), fluorouracil (5-FU), capecitabine (Xel oda®), FOLFIRINOX and Nab-paclitaxel (Abra xane®), or a pharmaceutically acceptable salt, solvate or hydrate thereof; and one or more chemotherapeutic agents selected from the group consisting of: The components may be provided in forms suitable for sequential, separate and / or simultaneous administration. A parts kit is provided.

[0132] In another aspect of the invention, there is provided a kit of parts comprising the following components: A composition as defined herein, optionally with a pharmaceutically acceptable adjuvant, diluent or carrier. or a pharmaceutically acceptable salt, solvate or a hydrate; and PD-1 and PD-2, optionally with a pharmaceutically acceptable adjuvant, diluent or carrier. and / or PD-L1 inhibitors, or pharmaceutically acceptable salts, solvates or hydrates thereof Japanese food and Gemcitabine, optionally with a pharmaceutically acceptable adjuvant, diluent or carrier. (Gemzar®), fluorouracil (5-FU), capecitabine (Xel oda®), FOLFIRINOX and Nab-paclitaxel (Abra xane®), or a pharmaceutically acceptable salt, solvate or hydrate thereof; and one or more chemotherapeutic agents selected from the group consisting of: The components may be provided in forms suitable for sequential, separate and / or simultaneous administration. A parts kit is provided.

[0133] The kit of parts is for the treatment of pancreatic cancer. Suitably, the kit of parts comprises For use in the treatment of pancreatic cancer as described herein, and / or For use in treating subjects identified as suitable for treatment by the methods described herein. This is for the purpose.

[0134] Features, integers, properties, compounds described in conjunction with specific aspects, embodiments or examples of the present invention or features may be used in conjunction with any other aspect, implementation, or feature described herein unless incompatible therewith. It is understood that the present invention is applicable to any embodiment or example. All of the features disclosed in the Abstract and drawings, and / or the All of the steps of any method or process described herein may be modified to include such features and / or Any combination, except combinations in which at least some of the steps are mutually exclusive, It can also be combined. [Example]

[0135] Example 1 - Macrophage populations as a fraction of the immune infiltrate in human pancreatic cancer ConsensusTME: Deconvolution Tools ConsensusTME is a comparative tumor microenvironment (TME) cellular enrichment study of 18 cell types. Integrate gene sets from all other current deconvolution methods for determination ConsensusTME is a deconvolution tool that Common cell type-specific gene sets used by open TME cell prediction methods This is a package for the R software environment that compiles the It allows for estimation of cell type abundance using bulk expression data of tumor samples. , including cell type-specific gene markers from independent cell estimation methods, specific for various cancer types Filter gene sets to enrich single sample gene sets We used ssGSEA to identify TME cell types and and tumor-specific enrichment scores are calculated.

[0136] To generate a consensus TME gene set, we used at least 2 We identified cell types in which signatures from different sources were present, for a total of 18 cell types. was used.

[0137] CIBERSORT(Nature Methods,2015,12,453-45 7) to extract genes from the signature matrix "LM22" used by To achieve this, we prioritized genes whose expression values ​​were less than 1.96 standard deviations below the mean for each cell type. In addition, activation and resting states for the corresponding cell types were measured. Once collapsed, other deconvolution methods (Bindea et al., Im munity,2013,39(4),782-795, Danaher et al. ,Journal for ImmunoTherapy of Cancer,201 7,5(18),1-15, Davoli et al.,Science,2017, 355(6322),2499-250,CIBERSORT,Nature Meth ods,2015,12,453-457,MCP-Counter,Genome B iology,2016,17(218),1-20, and xCell,Genome Biology, 2017, 18(220), 1-14) Once collected, a unique union of genes was created for each cell type. From ON, we curated a set of cell type-specific genes for each of the TCGA cancer types. This is done using the TIMER algorithm (Genome Biology, 2016, 17( 174),1-16), where the expression of the gene , negative correlation (Pearson's mean) with tumor purity (derived from ABSOLUTE) for the corresponding cancer type. Genes were included only if they had a correlation <0.2, p-value 0.05).

[0138] Finally, single sample gene set enrichment analysis (ssGSEA) was used to Calculate the normalized enrichment score (NES) for each cell type as described above. A general immune score for each tumor type was calculated by comparing various immune cell genes with each TCGA. The cancer types were generated by combining them into a single gene set.

[0139] The relative contribution of macrophages to the immune infiltration of tumor samples in pancreatic cancer was assessed by systemic immunoassay. To explore gene expression data from TCGA tumor samples, we used ConsensusT The immune cell gene expression signature was analyzed using ME to identify the individual tumor samples, as described above. The fragments were deconvoluted from the bulk RNA mixture.

[0140] Pan-cancer data (DNA-derived purity, leukocyte methylation, and H&E-derived lymphocyte counts) and cell-specific benchmark datasets (peripheral blood cells and tumor tissues). We collected a comprehensive benchmark dataset.

[0141] The ssGSEA analysis of the immune infiltrate fraction in TCGA pancreatic cancer is shown in Figure 1, where phage are one of the most abundant immune cell types in the immune infiltrate of pancreatic cancer patients. It can be seen that

[0142] Example 2 - TCGA PAAD (Pancreatic Cancer) Macros for the Stratification of Pancreatic Cancer Patients Kaplan-Meier survival analysis of phage invasion Kaplan-Meier survival analysis The Cancer, RNA-Seq data collected from cBioPortal Using gene expression data from the TCGA, Con Applying sensusTME to the PAAD cohort, we investigated the relative abundance of 18 immune cell types. Abundance was estimated. Data were collected from cbioportal.org (Cancer Disc overy,2014,2(5),401-404) The gene expression data from the study was from the same patient. Patients were first classified according to the abundance of macrophages. The results were stratified into "high," "medium," and "low" (high > 0.75 quartile > medium > 0.25 quartile). >low).

[0143] Kaplan-Meier survival analysis of progression-free survival based on this stratification showed that the difference in survival curves The high / medium group remained together, while the low group showed a better prognosis. The high and medium groups showed no significant differences and were combined to form two patient groups. , macrophage-low and macrophage-medium / high left.

[0144] Patients with a medium or high number of macrophage infiltrates correlated well with poor prognosis. was found.

[0145] Further KM analysis in TCGA PAAD stratifying patients by CCR1 expression demonstrated that patients with high CCR1 expression (>median) had significantly worse progression-free survival I did.

[0146] Next, we compared the Consensus TME macrophage gene signature with CCR1 expression. The combined signature was generated. ssGSEA was performed to analyze the combined signature. The enrichment of the combined signature was examined and the samples were enriched for this signature. Based on enrichment, it was again stratified into either "low" or "medium / high." demonstrated that combined signatures differentiate progression-free survival (PFS) in patients with pancreatic cancer. It has been shown that prediction is possible.

[0147] The results are shown in Figure 2.

[0148] Example 3 - PDAC Mesenchymal Cells Form Vascular Mimics in a 3D In Vitro Assay of Invasion accomplish cell culture The mouse cancer cell line used, TB32048 (hereinafter referred to as PDAC epithelial KPC mesenchymal lineages) and K8484 (hereinafter referred to as PDAC mesenchymal lineages) It was isolated from a tumor arising from a mouse (generated by David Tuveson), Supplied by Duncan Jodrell. All cell lines were heat-inactivated to 10% Dulbecco's modified Eagle's medium supplemented with fetal bovine serum, FBS, (Gibco): Nutrients Mixture F-12, grown in DMEM / F12 (Gibco), at 37°C in 5% CO All cell lines tested negative for mycoplasma. It was.

[0149] 3D in vitro model of invasion Cell matrix cultures were incubated with 1 mg / mL of Matrix Growth Factor Pre-coated with Reduced Matrigel (Becton Dickinson) This was done by seeding PDAC cells onto wells containing Matrigel. To observe cell invasion, the plate was placed on an IncuCyte Zoom (Essen B and green fluorescent protein (GFP / RFP) / photochromic The software was set to capture images every 3 hours in field (or phase contrast).

[0150] Quantification of VM-like structures To quantify the ability of PDAC cells to form vascular mimetic (VM) structures, we performed 3D in vitro studies. The angiogenesis assay was performed using an angiogenesis analysis solution provided by Essen Bioscience. The angiogenesis tool was used to visualize 3D structures in representative fields of view. This allows for automated and objective quantification of the number of branches that form once the parameters are defined. and incorporate them into all 3D in vitro structure formation experiments for robust and reproducible quantification. Applied.

[0151] The results are shown in Figure 3.

[0152] In contrast to PDAC epithelial cells that form clusters in 3D in vitro assays of invasion In contrast, PDAC mesenchymal cells formed extensive and complex tubular lattices across the entire surface area of ​​the well. The integrity of these networks remained stable over the course of the experiment. In addition, the tubular structures were suggested to represent VM (epithelial-mesenchymal transition). In other words, this suggests that the EMT state may mediate the ability to form 3D architecture. do.

[0153] Example 4 - Primary BMDMs express pro-invasive properties in pancreatic cancer cells regardless of EMT status Give a type In all subsequent macrophage co-culture assays, biologically relevant macrophages were To best represent the phage population, ZsGre was used in a 3D in vitro model of invasion. en-labeled primary BMDMs were co-cultured with mCherry-expressing PDAC epithelial cells.

[0154] bone marrow-derived macrophages Bone marrow was isolated from the femurs and tibiae of C57BL / 6 mice (10-14 weeks old). The femurs and tibias were flushed with PBS buffer and sieved through a 70 μM sieve (Greiner Bio- One) Passage the cells through a standard plastic tissue culture 100mm 2 Dish (Cor The cells were replated on a 10% FBS and 15% L929-Cell C Culture in DMEM / F12 medium supplemented with LCM The cells were cultured at 37°C under hypoxic conditions (1 BMDMs were incubated in 10% heat-inactivated fetal bovine serum (FB) at 47°C (25% O) for 3 days. S, Dulbecco's modified Eagle's medium supplemented with (Gibco): nutrient mixture F-12, D Grown and maintained in MEM / F12 (Gibco) and incubated at 37°C in 1% O I bet.

[0155] Cell co-culture The culture conditions for the PDAC and BMDM cell lines used in this study were as described above. For co-culture, mCherry-labeled PDAC cells were co-cultured with ZsGreen-labeled mCherry-labeled PDAC cells. The cells were mixed 1:1 with phage (typically primary BMDM unless otherwise noted). Cultures are seeded as subconfluent cultures in either 2D or 3D conditions. It was kept for 48 to 96 hours.

[0156] 3D in vitro assay quantification of invasion To quantify the invasive potential of PDAC cells, a 100-well platelet count was obtained from Essen Bioscience. 3D in vitro invasion assay using NeuroTrack, the software provided with the study. The method was designed by Essen Bioscience. Using software algorithms, the IncuCyteZoom platform Measure the kinetic data generated by the assay. Requires cell fixation, staining, and end-point analysis. Unlike high-content imaging systems, which require NeuroTrack measures the dynamics of cells invading from a 3D structure over time. This method was used to count the number of cells in the co-culture with mCherry-labeled PDAC cells. This allowed for the exclusion of macrophages (unlabeled and / or ZsGreen) in the culture. Once the parameters are defined, they can be quantified in all 3D in vitro invasion was applied to the assay.

[0157] NeuroTrack software measures fluorescently labeled cells. Two PDAC cell types (both endothelial and extracellular matrix metastasis) were identified, regardless of their epithelial transformation (EMT) status and the dimensions of the 3D structures. To consistently analyze the invasion of rhesus monkeys (whose nuclear labeling is H2B-mCherry), Here, BMDM represents ZsGreen-labeled cells. Only PDAC cells that invaded from the spleen were identified as fluorescent mCherry-labeled cells present in their nuclei. Data are plotted as number of nuclei counted on the y-axis and time on the x-axis. Plot.

[0158] The results are shown in Figure 4.

[0159] Instead of forming vascular networks, PDAC epithelial cells first formed their own vascular network within 24 hours of seeding. After forming each rounded, cohesive cluster, surprisingly, approximately 96 Once free, these invading cells spread In parallel studies, PDAC cells were cultured in 100% PBS-containing medium, covering the entire surface area of ​​the plate after approximately 7 days. The behavior of mesenchymal cells alone or in the presence of BMDM was also examined. In addition, they first form their VM structures within 24 hours of seeding, then become free and evolve much faster. Although the rate was slow, it was found to exhibit a pro-invasive phenotype at approximately 48 hours.

[0160] Example 5 - Novel RNA promotes PDAC cell invasion after extended co-culture with BMDM A multi-omics approach (transcriptome) to identify targets Transcriptomes of PDAC cells and macrophages after extended co-culture To elucidate the changes in the PDAC epithelium and BMDMs, we used pre-trained PDAC epithelium from co-culture with BMDMs. and mesenchymal cells were cultured in fluorescently activated cells along with monocultured PDAC cells and BMDM controls. The cells were sorted by fluorescence in situ hybridization (FACS) and subjected to RNA sequencing. Whole-genome transcriptome analysis from co-cultures of PDAC cells revealed that nearly all chemo showed upregulation of kine receptors and their receptor genes.

[0161] RNA quality control The quality of the RNA was confirmed using the Bioanalyzer / RNA nano 6000 kit (Ag It was checked using

[0162] Determination of differentially expressed genes Using non-specific filtering, we found that 90% or more of the samples had an interquartile range of less than 0.3. Genes with low expression levels (<3 on the log2 scale) were removed. Find differentially expressed genes (DEGs) after co-culture compared to monoculture in cells To achieve this, thresholds were set at a minimum fold change of 1.5 and a median false discovery rate (FDR) of 0.10. DEGs were identified in BMDM monoculture vs. coculture and other relevant comparisons. These same parameters were used to determine the sequence of unannotated transcripts. Ta.

[0163] qRT-PCR gene expression analysis Total RNA was purified using Qiazol reagent and miRNeasy mini kit (Qiagen) The cDNA was isolated and purified from cells using Hi Synthesized using the gh Capacity RNA-to-cDNA kit (ABI) qRT-PCR was performed using Power SYBR G on a TaqMan 7900 (ABI). The relative expression levels were determined using Green PCR Master Mix (ABI). The Ct values ​​were determined using the ΔΔCt method and were compared against 18S rRNA and / or GAPDH. and normalized it.

[0164] The results are shown in Figure 5.

[0165] Macrophage-mediated induction of chemokine family genes was observed in all PDAs tested. In C cells, regardless of EMT status, quantitative real-time polymerase This was confirmed by PCR (qRT-PCR).

[0166] Example 6 - Novel proteins promote PDAC cell invasion after extended co-culture with BMDM Multi-omics approach (proteome) to identify protein targets Proteome or sectioning of PDAC cells and macrophages after extended co-culture Pre-incubated PD from co-culture with BMDM to elucidate changes in the retome AC epithelial and mesenchymal cells were cultured in bulk with monocultured PDAC cells and BMDM controls. were harvested and subjected to TMT complete proteome and secretome analysis.

[0167] CTAP label For label exchange experiments, PDAC and BMDM cells were first transfected with 798 μM light isotope label. I-lysine (hereinafter referred to as light or DAP) or heavy isotope labeled [ 13 C 6、 15 N2]l-Lysine (+8 Daltons, Cambridge Isotope s) (hereinafter referred to as heavy or d-Lys-8) Culture in SILAC DMEM containing 10% dialyzed FBS for at least 10 cell doublings (1 For experiments that maintained the label, cells were metabolically labeled by proliferation for 10 days or more. First, the respective precursors were grown for at least 10 cell doublings (over 10 days): DOPA decarboxylase (DDC)-expressing cells (primary BMDM) in DAP(L) and Lyr-expressing cells (PDAC cells) in 10 mM D Combine in AP (L) and 1 mM d-lysine (H) and co-culture for 4 days (approximately 4 cells The co-cultures were grown together at a ratio that would result in equal numbers of cells at the end of the experiment. Conditioned medium (CM) was collected and flash-frozen for downstream secretome analysis. Cell pellets and lysates were collected and flash frozen for downstream proteomic analysis.

[0168] The results are shown in Figures 6 and 7.

[0169] Proteome analysis from PDAC cells in co-culture compared to monoculture revealed that PDAC cells Cells incorporate macrophage-derived chemokines after extended coculture with BMDMs. This confirms the RNA sequencing data suggesting that

[0170] Example 7 - Novel proteins promote PDAC cell invasion after extended co-culture with BMDM A multi-omics approach (secretome) to identify protein targets The rapid pro-invasive phenotypic changes observed in 3D mixed cultures are consistent with the phenotypic changes observed in these PDACs. This may be mediated by signals released from the interaction of the epithelium and BMDM cells. To identify this, conditioned medium (CM) was incubated for 48 hours. Protein extracts were harvested from the bated co-cultures and used to probe an inflammatory antibody array.

[0171] conditioned medium Conditioned media samples were collected from monocultures and cocultures of PDAC and BMDM 48 hours after seeding. Cells were isolated from culture and imaged on a mouse inflammation antibody array (Abcam) according to the manufacturer's instructions. Images were acquired and signal intensity was assessed using an Amersham Imager Analysis was performed using the 680.

[0172] The results are shown in Figures 8 and 9.

[0173] Among 40 potential candidates, the highest detectable upregulation by extended co-culture Three inflammatory signals with modulated intensities included CC motif chemokine receptor 1 (CC-C motif chemokine receptor 1). These include the chemokines CCL2, CCL3, and CCL9, which are ligands for CCR1 (CCR1). Ta.

[0174] Example 8 - CCR1 blockade inhibits macrophage-mediated pro-invasive phenotype Upregulation of CCR1 expression in PDAC cells after prolonged coculture with BMDM The modulation was confirmed by qRT-PCR and flow cytometry. Next, we investigated whether blockade of CCR1 using various small molecule inhibitors significantly reduced BMDM invasion in 3D in vitro. PDAC cells are able to confer a pro-invasive phenotype in vitro. Determine whether crosstalk between DAC cells and macrophages can potentially be disrupted I tried to.

[0175] CCR1 antagonist treatment in cell co-culture Pretreatment: PDAC cells and BMDMs were treated with CCR1 antagonists 24 hours before the start of the experiment. The cells were pretreated with sucrose, harvested, and then co-cultured for the duration of the experiment according to the previously described co-culture method. The seeds were sown.

[0176] Co-culture treatment: PDAC cells and BMDMs were co-cultured according to the previously described co-culture method. The cells were seeded at 100°C, treated with a CCR1 antagonist at the time of seeding, and incubated for the duration of the experiment. Ta.

[0177] The results are shown in Figures 10 to 13.

[0178] Three commercially available CCR1 antagonists (BX-471, J113863 and UCB3 5625, all supplied by Tocris Bioscience) was used for qRT- PCR and flow cytometry were used to control for the effects of macrophages on PDA. CCR1 expression resulting from extended co-culture confers functional pro-invasive ability to C cells It also showed a remarkable ability to blunt the induction of expression.

[0179] Example 9 - Knockdown of CCR1 by siRNA promotes macrophage-mediated infiltration blunting the sexual phenotype Gene Targeting and Expression siRNA-mediated gene knockdown was performed using SmartPool siRNA (Ther obtained using either the siRNA sequences (e.g., Fisher Scientific) or individual siRNA sequences. The resulting mixture was transfected with 15 nM siRNA and RNA iMAX (Invitrogen) Transfection was performed using an infection reagent.

[0180] siRNA knockdown of CCR1 in cell co-cultures siRNA knockdown of CCR1 was performed on PDAC cells and B cells 24 hours before the start of the experiment. MDMs were cultured, harvested, and then cultured according to the previously described co-culture method for the duration of the experiment. And so the seeds were sown.

[0181] The results are shown in Figures 14 and 15.

[0182] Knockdown of CCR1 in PDAC and BMDM cells before co-culture significantly reduced the expression of CCR1 compared with controls. In comparison with α-glucan, it suppressed the induction of CCR1 expression and partially inhibited macrophage-mediated infiltration in 3D. This result was observed using several different siRNAs targeting CCR1. It was.

[0183] Example 10 - Neutralizing antibodies against ligands that bind to CCR1 inhibit macrophage-mediated infiltration May alleviate pro-inflammatory phenotype Inflammatory chemokine receptors exhibit promiscuous ligand binding, and chemokines, in turn, bind multiple receptors. The extent to which this represents biological redundancy or individual Distinct signals triggered by different chemokines through their chemokine receptors It remains to be determined whether blockade of a single ligand can inhibit macrophage proliferation. Could this be sufficient to reduce the pro-invasive phenotype conferred to PDAC cells by by using neutralizing antibodies (all from R&D Systems) to test whether , two known ligands, CCL3 and CCL9 (human homolog CCL15), and neutralizing IL-1B, one unknown but potentially novel ligand of CCR1. All ligands were identified in a previous multi-omics high-throughput screen. were selected after being identified and validated.

[0184] Neutralizing antibody treatment in cell co-culture Pretreatment: PDAC cells and BMDMs were cultured in CCR1 ligand for 24 hours before the start of the experiment. The cells were pretreated with neutralizing antibodies, harvested, and then co-cultured for the duration of the experiment according to the previously described co-culture method. The seeds were sown.

[0185] Co-culture treatment: PDAC cells and BMDMs were co-cultured according to the previously described co-culture method. The cells were seeded with a CCR1 ligand neutralizing antibody (supplied by R&D Systems) at the time of seeding. treated and allowed to incubate for the duration of the experiment.

[0186] The results are shown in Figure 16.

[0187] Single blockade of each of the three ligands (CCL3, CCL9, and IL-1B) It showed efficacy in preventing PDAC cells from invading the structure in the presence of BMDM. Interestingly, anti-CCL9 was unique in its observed ability to induce macrophage migration. This was accompanied by a loss of ability to track PDAC cells for invasion. This suggests a clear dichotomy in how different ligands can bind to CCR1, providing justification for further investigation. Importantly, IL-1B blunts the pro-invasive phenotype of PDAC. It was also observed that

[0188] Example 11 - CCR1 antagonist, BX471, slows primary tumor growth in vivo R The subcutaneous flank injection model was performed using a genetically engineered mouse model of PDAC, the KPC mouse model. This was used as a pilot and surrogate experiment for the study. The DAC cell line was injected and the primary tumors were followed for growth.

[0189] In vivo mouse studies Pretreatment: PDAC and BMDM cells were treated with CCR1 antagonism 24 hours before the start of the experiment. ists, pre-treated with BX471 and for overnight incubation as previously described The following day, 1x1 cells were seeded with or without an equal number of primary BMDMs. 0 6 mCherry-labeled PDAC mesenchymal cells were cultured with Matrigel for 6–8 days. Male, week-old, immunocompetent C57BL / 6J mice (Charles River Laboratories, London, UK) were cultured. Starting on day 7, mice were injected into the flanks of mice (supplied from aboratories). They were measured separately using calipers.

[0190] The results are shown in Figures 17 and 18.

[0191] A reduction in primary tumor growth was observed in the group treated with the CCR1 antagonist, BX471. Importantly, BX471-treated mice showed a marked improvement in the ability of PDAC cells to combine with BMDM cells. When combined, they were observed to have prolonged overall survival.

[0192] Example 12 - High expression of CCR1 is associated with macrophage infiltration and is a predictor of progression in patients with pancreatic cancer can be used to stratify patients ConsensusTME: Deconvolution Tools The signature matrix "LM22" used by CIBERSORT To extract genes from the dataset, we selected genes whose expression values ​​were less than 1.96 standard deviations from the mean for each cell type. In addition, genes with activation for the corresponding cell type were first filtered out. and resting states were collapsed. Once other deconvolution methods (e.g., Bindea t al., Danaher et al., Davoli et al.CIBERS Collecting signature genes from the ORT, MCP-Counter and xCell This created a unique union of genes for each cell type. We have curated a set of cell type-specific genes for each of the TCGA cancer types. This is done using an approach similar to the TIMER algorithm, where the gene Expression of β-glucan negatively correlated with tumor purity (derived from ABSOLUTE) for the corresponding cancer type (P<0.05). Genes were included only if they had a correlation coefficient <0.2, p-value 0.05. Using single sample gene set enrichment analysis (ssGSEA), as described above A normalized enrichment score (NES) was calculated for each cell type. A general immune score for various immune cell genes was calculated for each TCGA cancer type. These were generated by combining them into one gene set.

[0193] ConsensusTME cell type comparison ConsensusTME cell type abundance was analyzed using TCGA pancreatic cancer as previously described Bulk tumor RNA-Seq from the cohort was generated using ss for each cell type. GSEA enrichment scores were further normalized by dividing by total immune cell abundance This allowed for better comparison of cell type scores, which resemble fractional proportions of the total immune cell infiltrate. These scores were then used to separate samples into those with high versus low immune cell fractions using a median split. was used to stratify into

[0194] Kaplan-Meier survival analysis Kaplan-Meier survival analysis using the TCGA expression data in Example 2 was previously described. I went there to be done.

[0195] Kaplan-Meier analysis of RNA-Seq data from the TCGA pancreatic cancer cohort Patients were stratified into "low" or "high" expression based on the median split. The log-rank test between the groups showed that the CCR1 low group was significantly better than the CCR1 high group. Progression-free survival was demonstrated (p=0.031).

[0196] Using immune cell estimates generated by ConsensusTME, macrophages Samples in the high immune cell, dendritic cell, and monocyte category had higher C than those in the low immune cell category. It can be seen that CD8+ T cells, B cells, and control This is seen for many other immune cell types, including immune T cells. These results show that there is a significant association between CCR1 and macrophage infiltration in the pu.

[0197] Patients with high CCR1 gene expression are less likely to progress than those with low levels Indicates survival.

[0198] The results are shown in Figures 19 and 20.

[0199] Example 13 - Gene Ontology (G) of the most changed proteins after extended co-culture O) Analysis reveals enrichment of chemokine-associated biological processes Gene Ontology (GO) analysis of the most changed proteins after extended co-culture ( Example 4) hereinabove demonstrates the enrichment of chemokine-related biological processes. reveal Gene Ontology (GO) is a graphical representation of genes or gene products organized in a graph structure. It provides a hierarchical classification system for terms. One of the main uses of GO is in gene sequencing. For example, extended enrichment analysis with BMDMs is A set of genes that are upregulated in pancreatic cancer cells under co-culture conditions is given. Once the gene set is selected, this enrichment analysis will generate a comprehensive set of gene sets using the annotations for that gene set. Over-represented GO terms, or biological processes, were found.

[0200] PANTHER Classification System(pantherdb .org) and list the names of the genes you want to analyze, one per line or separated by commas. The tool searches for MOD-specific gene names and UniProt IDs. Both GO aspects (molecular function, biological process, cellular organization) for analysis can be processed. Selection of the element (in this case, a biological process) was performed. Species selection, both mouse and human The results are displayed on the redirected PANTHER website. These results are used to identify all proteins in the genome selected in step 3. It is based on enrichment for a set of quality-coding genes.

[0201] The results are shown in Figure 21.

[0202] Example 14 - Combined macrophage and CCR1 signatures correlate with progression-free survival It can therefore be used to stratify pancreatic cancer patients. Kaplan-Meier analysis The combined gene signature was analyzed using the Consensus TME Macrophage Gene The signature was created by combining it with CCR1 to create a new gene set. ssGSEA was then used to identify novel genetic variants in the TCGA pancreatic cancer cohort. A normalized enrichment score for the child set was generated. Stratification for nature was performed as "medium / high" (x>0.25 quantile) and "low" (x<0 Macrophage signature Kaplan-Myers Squibb model to create .25 quantile categories The log-rank test was performed as previously described in the previous analysis. We found that enrichment of this signature was associated with significantly worse progression-free survival. showed (p=0.03).

[0203] To investigate the combined effects of high levels of CCR1 expression in tumors and macrophage infiltration, Consensus Combined Gene Signature of TME Macrophage and CCR1 Expression We used ssGSEA to identify patients in the pancreatic cancer TCGA cohort. Determining the enrichment of this score across It was observed that enrichment indicated a worse prognosis. This combined signature Instead of two separate Kaplan-Meier analyses, to help further stratify patients, It can be used for

[0204] The results are shown in Figure 22.

[0205] Example 17 - Treatment with BX-471 increases activation of downstream IGF1 signaling pathway genes to dull. Western blotting PDAC cell lines growing in 6-well plates were treated with recombinant IGF1, CCR1 antagonists, and nisto, BX-471 or J-113863, or recombinant IGF1 and CCR1 antigen The cells were then treated with a combination of agonists for 24 hours. Lysates were lysed in ice-cold lysis buffer containing a protease inhibitor cocktail (Roche). Protein extracts were denatured at 95°C for 5 min in a heat block and then subjected to SDS-PAG. Proteins were separated by E. The proteins were transferred to a nitrocellulose membrane and then incubated with the primary antibody. overnight at 4°C, followed by IRDye secondary antibody (Li-Cor Biosciences) The target protein was incubated with Li- Cor Odyssey Infrared Imaging System The primary antibodies used were AKT, Phospho-AKT, 4E-BP1, and Ph. ospho-4E-BP1, MEK, Phospho-MEK, MAPK, Phosph Antibodies against o-ERK1 / 2 and GAPDH (Cell Signaling) Included.

[0206] Previous reports have shown that IGF1 plays a central role in pancreatic cancer resistance to chemotherapy. In this regard, the present inventors have demonstrated that CCR1 inhibition plays a key role in We sought to determine whether recombinant I had any effect on the GF1 signaling pathway. Downstream in PDAC cell lines after treatment with GF1 and various CCR1 antagonists Expression of IGF1 signaling pathway genes was assessed using Western blotting. Activation and expression profiles of AKT and 4E-BP1 after recombinant IGF1 treatment , which was particularly blunted by inhibition of CCR1 using the antagonist BX-471, , provided the rationale for our preference for BX-471.

[0207] We further investigated how CCR1 inhibition affects the IGF1 signaling pathway. To investigate this, we investigated the progression of PDAC after treatment with recombinant IGF1 and various CCR1 antagonists. Cell lines were subjected to comprehensive and comparative proteomic and phosphoproteomic profiling. The data were analyzed in-house by a postdoctoral researcher in the laboratory. The IGF1 family genes were input into the developed gene pathway network analysis pipeline. The gene was upregulated by adding recombinant IGF1 compared to the control. However, treatment with the CCR1 antagonist, BX-471, abolished this upregulation. This severely slowed down the game.

[0208] The results are shown in Figures 23 and 24.

[0209] Example 18 - Treatment with CCR1 antagonist-based combination therapy improves PDAC in vivo , and is effective in a genetically engineered mouse model (KPC model) Rationale for in vivo studies This study demonstrates the potential of the CCR1 inhibitor gemcitabine as a novel treatment option for PDAC. The aim is to test combinations with HIV and / or immunotherapy. The authors report that small molecule CCR1 inhibitors are effective against tumor microcirculation, which has been implicated as being immunosuppressive. Intercellular communication between macrophages, a major subpopulation of the nucleus, and tumor cells PDAC is characterized by fibrosis, First, we will use CCR1 inhibitors such as BX-471 to improve the tumor microenvironment in patients. Targeting and then treating the tumor with gemcitabine and / or immunotherapy is the standard Improve care and inform future treatment options for PDAC in the clinic This ongoing proof-of-concept study uses an in vivo KPC mouse model. This allows for preclinical evaluation of the efficacy of treatment options for PDAC prior to clinical Phase I studies. This study included a clinical-grade A randomized, five-arm intervention study using KPC band A mice, as well as additional pyrolysis This includes lab experiments.

[0210] Methods: Animal model and sample processing LSL-KrasG12D / +;LSL-Trp53R17 carrying spontaneous PDAC The generation of 2H / +;Pdx-1-Cre(KPC) mice has been described previously (Hi ngorani et al.,Cancer Cell,2005,7(5),469 Genotyped KPC mice were used in the main intervention study and in additional pilot studies. (Their PC littermates do not develop tumors.) All KPC experimental groups Groups were randomized by sex and age.

[0211] PDAC tumors in KPC mice were detected by palpation and high-resolution ultrasound scans (Vevo 2100, VisualSonics) and confirmed at autopsy. Weight-stable PDAC-bearing mice were defined as those with tumors of 1 mm (by ultrasound). Clinical-grade KPC band A mice were enrolled in the main intervention study. Identification of multiple tumors, (2) large obstructed common bile ducts, (3) tumors larger than 6 mm, (4) (5) the presence of nephritis, (6) splenomegaly, (7) large cysts, or (8) visible cysts in the diaphragm KPC band B mice (preclinical grade KPC tumors), defined as containing metastatic tumors Tumor-bearing mice were used for additional initial pilot experiments. All tumors were Histopathologically, adenocarcinoma was confirmed. All mice were monitored daily for weight and condition. I turned it over.

[0212] Euthanasia was performed during a 12-hour light period. Terminal bleeding was performed by cardiac puncture under isoflurane anesthesia. Plasma was obtained by exsanguination via a venipuncture and death was confirmed by cervical dislocation. The tissues were prepared by centrifugation at 4°C for 5 minutes and then flash frozen in liquid nitrogen. Aliquots of the endothelial and tumor samples were rapidly dissected in a consistent order and placed in 10% neutral buffered saline. After fixation in NBF for 24 hours at room temperature, the specimens were transferred to 70% ethanol and then immunoblotted. Processed for histochemistry.

[0213] Main intervention study design Figure 25 shows the experimental design for the main intervention study, including the definition of the five experimental cohorts used. , as well as a timeline showing treatment conditions and study duration. CCR1 inhibitors used The anti-PD-1 antibody used was BX-471. oMab Rat anti-mouse PD-1 (CD279) (clone RMP1-14 monoclonal antibody) The antibodies were IgG2a,k).

[0214] Initial pilot experiments demonstrated collagen fragmentation and immune cell infiltration in combination (3x) therapy. Reveals increased moisture The distinction between hot, transformed (excluded, immunosuppressed) tumors and cold tumors is This simplification is based on the cytotoxic T cell landscape within the tumor. This reflects the outcome of complex interactions between the two. KPC band B mice were used as the primary study species. We first looked at the intervention groups.

[0215] A small number of KPC band B mice were treated with vehicle alone, BX-471 alone (Fig. 28A), gemcitabine, or PEG-471. citabine alone or in combination with BX-471 (Figure 28B), or gemcitabine; Treatment with either BX-471 and anti-PD1 as a combination (3x therapy) (Figure 28C) Due to regulatory restrictions, these mice were not maintained for extended survival studies. It was not possible to keep it and it was disposed of approximately 10 days after registration.

[0216] The results of the pilot experiment are shown in Figure 26. (A) shows the results of the 10 ColVI (collagen) and KRT in KPC band B tumors treated for 1 day (B) Immunofluorescence staining of tumor cells (tumor cells) in the 19-treated group treated with gemcitabine plus vehicle or gemcitabine. KPC bands when treated with either citabine or BX-471 (2x therapy) for 10 days (B) H&E staining of tumors. (C) Vehicle or gemcitabine + BX-471 + anti- in KPC band B tumors when treated for 10 days with either PD-1 (3x therapy) Immunoglobulins CD45 (immune cells), ColVI (collagen), and KRT19 (tumor cells) Fluorescent staining is shown.

[0217] Characterization of PDAC stroma as a simple biophysical obstacle to drug delivery This oversimplifies the contributions of the dozens of different cell types present in the stroma. Interestingly, preliminary analysis of band B tumors stained with antibodies against collagen VI Figure 1 shows the effect of treatment of mice with the CCR1 antagonist, BX-471, on the survival of mice compared to vehicle control. In comparison, we revealed changes in the morphology of the PDAC tumor microenvironment (Figure 28A). This fragmentation of CCR1 deposits significantly impairs the CCR1 signaling axis in mice. This suggests that this may lead to changes in biological architecture.

[0218] Another cohort of mice was treated with gemcitabine alone or in combination with BX-471. Interestingly, H&E staining builds on the previously observed stromal fragmentation phenotype. The primary tumor site showed an increase in infiltrating lymphocytes and leukocytes (Fig. 28B). The dichotomous differences in immune cell populations and fibrosis architecture were striking, which may be due to the presence of CCR1 blockade. These results suggest that the disruption improves immune cell infiltration into the tumor site.

[0219] Finally, one KPC band B mouse was run in parallel with a vehicle control, triple combination The triple therapy resulted in collagen fragmentation (as evidenced by collagen VI staining). (C) showed increased CD45+ staining and decreased KRT19 tumor cell staining. .

[0220] First results of the intervention study: combination 2x (BX-471 + GEM) or 3x (BX-471 + CCR1 blockade in GEM+anti-PD1 therapy extends survival in a mouse model of PDAC Show the effect The rationale for the combination therapy group is to enhance communication between tumor cells and macrophages. Blocking and fragmenting the biophysical density of collagen arrangements and preventing the flow of TILs into the primary tumor site This is due to CCR1 inhibition, which allows the entry of CCR1. The treatment acts to provide systemic and direct tumor killing (dual therapy). Adding an antibody that binds to PD-1 may help to demonstrate that this checkpoint molecule mediates cytotoxicity in invading cells. Boosting immune cell activity by stopping T cells from switching off The aim is to achieve the following (triple therapy).

[0221] Figure 27 shows the survival curves for mice enrolled in all five treatment groups of the main intervention study. Lines are shown. Increase in survival was 2×( BX-471 + gemcitabine) and 3 × (BX-471 + gemcitabine + anti-PD1) This was observed for both combination treatments.

[0222] Although specific embodiments of the present invention have been described herein for purposes of reference and illustration, various modifications may be made. , without departing from the scope of the present invention as defined by the appended claims. It becomes clear to those who

Claims

[Claim 1] CCR1 antagonists, or pharmaceutical compositions thereof, for use in the treatment of pancreatic cancer Acceptable salts, hydrates or solvates.

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