WO2018187355A1 - Met kinase inhibitors and uses therefor - Google Patents

Abstract

Provided are compositions and methods for inhibition of cancer metastasis, such as metastasis associated with kidney cancer. The compositions comprise one or more c-MET kinase inhibitors and one or more inhibitors of Angiopoietin/Tie-2 axis, such as trebananib The method comprises administering to an individual in need of treatment one or more c-MET kinase inhibitors and one or more inhibitors of Angiopoietin/Tie-2 axis, such as trebananib.

Description

MET KINASE INHIBITORS AND USES THEREFOR

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional application no.

62/480,901, filed on April 3, 2017, the disclosure of which is incorporated herein by reference.

BACKGROUND OF THE DISCLOSURE

[0002] Kidney cancer remains among the top ten most diagnosed cancers in both men and women, with >62,000 new cases of kidney cancer and >14,000 deaths anticipated in 2016. Of those cases, approximately 15% are expected to develop aggressive metastatic disease (Jonasch et al. BMJ 2014; 349:g479). Clear cell renal cell carcinoma (ccRCC) is the most commonly diagnosed (70%) form of kidney cancer and metastatic disease remains the leading cause of cancer related deaths with common ccRCC metastases to the lungs, bones and lymph nodes resulting in poor prognosis among ccRCC patients (Cai et al., Exp Ther Med 2013;6(6): 1489-93).

[0003] RTKIs (receptor tyrosine kinase inhibitors) remain the main treatment for ccRCC both in the first-line setting and subsequent therapies. Recently, two RTKIs, cabozantinib and lenvatinib, have been approved in RTKIs resistant disease. Disappointingly, to date the use of RTKIs has not shown clinical benefit in the adjuvant setting (Grenga et al., J. Immunother Cancer 2015; 3 :52).

[0004] Poor prognosis and high rate of metastases is predicted for patients with high tumor associated macrophage (TAM) infiltrates (Kovaleva et al. Anal Cell Pathol (Amst) 2016; 2016:9307549). The angiopoietin/Tie-2 axis plays a central role in the recruitment of TAMs which significantly contribute to the maturation/disruption of tumor vasculature (Coffelt et al. Cancer Res 2010; 70(13):5270-80). While the regulation of angiogenesis through the Ang-Tie-2 pathway has been well studied, there is a lack of understanding of the role this pathway plays in metastatic disease.

[0005] Successfully treating cancer patients with metastatic disease remains one of the most daunting tasks for clinicians treating kidney cancers such as renal cell carcinomas. SUMMARY OF THE DISCLOSURE

[0006] The present disclosure provides compositions and methods for treatment of cancer metastasis. As described herein, it was observed that when used in combination with inhibitors of Angiopoietin/Tie-2 axis, the c-MET kinase inhibitors (also referred to herein as MET kinase inhibitors) significantly inhibit cancer metastases. For example, it was observed that the combination of Angiopoietin/Tie-2 axis inhibitors and c-MET kinase inhibitors reduced infiltrating M2 type macrophages, and prolonged survival in a highly metastatic animal model.

[0007] In one aspect, the present invention provides compositions comprising one or more compounds having the structure of Formula I and further comprising one or more inhibitors of the angiopoietin/Tie-2 axis. In specific embodiment, the compound of Formula I has the structure of Formula II. Inhibitors of angiopoietin/Tie-2 axis include molecules that inhibit the binding of angiopoietin 1 and/or angiopoietin 2 to their receptor Tie-2. An example of such an inhibitor is trebananib (also known as AMG 386 or 2XCon4C). In one embodiment, the composition comprises a compound of Formula I (such as a compound of Formula II) and trebananib.

[0008] In one aspect, this disclosure provides methods for treatment of cancer. The method comprises administering to an individual in need of treatment one or more inhibitors of the angiopoietin/Tie-2 axis and one or more inhibitors of MET kinase. For example, the method can comprise administering to an individual in need of treatment a MET kinase inhibitor having the structure of Formula I (e.g., Formula II) and trebananib. The inhibitor of the angiopoietin/Tie-2 axis and the MET kinase inhibitor may be administered as part of the same composition or as different composition. If administered as different compositions, they can be administered concurrently or sequentially, and by the same route or different routes, over the same administration periods or different administration periods.

[0009] The administration of inhibitors of the angiopoietin/Tie-2 axis and MET kinase may be used as an adjuvant therapeutic approach in conjunction with other therapies. For example, administration of the present composition(s) may be accompanied by other treatment approaches including, but not limited to, surgery, radiation and the like. Moreover, removal of primary tumor may be carried out in addition to treatment regimens with inhibitors of the angiopoietin/Tie-2 axis and MET kinase. The additional therapeutic approaches may be carried out prior to, concurrently, or after cessation of administration of the present compositions.

[0010] As further detailed in Examples 1 and 2, the results show that the combination of trebananib and a c-MET inhibitor alters tumor microenvironment, inhibits the spread of metastases, reduces infiltrating M2 type macrophages, and prolongs survival in a highly metastatic renal cell carcinoma model, indicating a use for this combination therapy in treating patients with cancer, such as kidney cancer.

BRIEF DESCRIPTION OF THE FIGURES

[0011] For a fuller understanding of the nature and objects of the disclosure, reference should be made to the following detailed description taken in conjunction with the accompanying figures.

[0012] Figure 1 shows trebananib combination with a MET kinase inhibitor (Formula

II compound) results in moderate inhibition of primary tumor growth as compared to single agent treatments in multiple models of ccRCC. (A) Endpoint tumor weight showing significance between trebananib single treatment and vehicle cohorts. (B-C) Representative images of CD31 staining in the RENCA model indicate that treatment with MET kinase inhibitor and trebananib alone do not significantly inhibit vasculature while combination treatment appears to slightly reduce the vasculature compared to the vehicle cohort. (D) Tumor growth curve and endpoint tumor weight of sunitinib sensitive mice in the

combination cohort showed a significant reduction in growth compared to single agent trebananib alone, while the trebananib cohort had an increase in tumor growth. (E) Mice treated first with sunitinib until the time of resistance - defined as the point which the tumor doubled the size at the start of sunitinib treatment - showed no growth inhibition in either single agent or combination treatments. (F) RP-R-02 parental tumors in SCID mice showed no significant difference in primary tumor growth across the cohorts. Results are given as mean + SEM. *p-value = <0.05, **p-value = <0.005, ***p-value = <0.001, ****p-value = 0.0001.

[0013] Figure 2 shows combination treatment of trebananib and MET kinase inhibitor

(Formula II, also referred to herein as Compound A) results in significant inhibition of metastases in the metastatic ccRCC PDX model, RP-R-02LM. (A) (left) Tumor growth curves show no significant difference across the cohorts, (right) Spider graphs show the growth of individual mice in each cohort, again no significant difference across cohorts. (B) End-point tumor weights show no significant difference in tumor size. (C) Representative H&E staining of the lungs show a striking reduction in the presence of lung metastases in the trebananib and combination groups. (D-E) Blinded analysis of the total lung macro- and micro-metastases showed a significant reduction in the presence of metastases in the combination group as compared to all other groups. Results are given as mean + SEM. *p- value = <0.05, **p-value = <0.005, ***p-value = <0.001, ****p-value = O.0001. [0014] Figure 3 shows survival studies with RP-R-02LM reveal significant increases in survival and increased presence of epithelial marker e-cadherin. (A) Kaplan-Meier survival curve showing a significant increase in the survival of the combination treatment, compared to the vehicle cohort. (B) Kaplan-Meier survival curve revealing a significant increase in the survival rates of mice in the combination group as compared to all other groups in the study. Lungs from each mouse at the time of euthanasia as compared to the days on treatment, weight of primary tumor at time of extraction (3 months post-implantation). (C) Lung and tumor images from each mouse at the time of euthanasia as compared to the days on treatment and Western blot analysis of epithelial marker e-cadherin. (D) Western blot densitometry analysis shows a significant increase in the presence of e-cadherin in the combination group compared to the MET kinase inhibitor group. (E-F) qRT-PCR expression shows that, when normalized to the vehicle group, there is a trend of increased e-cadherin expression in the trebananib and combination groups while there is a significant decrease of Snail expression in the combination cohort. Results are given as mean + SEM. *p-value = <0.05, **p-value = <0.005, ***p-value = <0.001, ****p-value = O.0001. The MET kinase inhibitor is Compound A (Formula II), and AMG386 is trebananib.

[0015] Figure 4 shows single agent MET kinase inhibitor, but not combination treatment, alters downstream constituents of the MET signaling pathway in tumors. (A) Western blot results for pSTAT3, pAKT, pERKl/2, and GAPDH relative to mice in the four treatment groups and their days on treatment. (B) Densitometry data for the protein expression of p-ERKl/2 shows a decrease in the MET kinase inhibitor group and a loss of this inhibition in the combination group. (C) Densitometry data for p-AKT shows a significant decrease of expression in the MET kinase inhibitor group and loss of this inhibition in the combination group. (D) pSTAT3 shows an overall trend of decreased pSTAT3 expression in each of the treatment groups when compared to the vehicle. (E)

Schematic of downstream constituents in the MET kinase pathway. Results are given as mean + SEM. *p-value = <0.05, **p-value = <0.005, ***p-value = <0.001, ****p-value =

0.0001.

[0016] Figure 5 shows treatment of RP-R-02LM tumors with trebananib and MET kinase inhibitor results in increased pericyte coverage of the tumor vasculature. (A)

Immunofluorescent staining for CD31, NG2, and DAPI reveals a trend of increased blood vessel coverage by pericytes in the MET kinase inhibitor and combination groups. (B) Blinded assessment of the positive areas for NG2 co-verifies the IF staining in Figure 5 A, revealing a significant increase in pericyte coverage in the combination group. (C) Western blot analysis and densitometry data (D) show a significant increase in the presence of pericyte marker NG2 in the trebananib and combination groups as compared to the vehicle and MET kinase inhibitor treatment groups. (E) Confocal images of vehicle and combination treatment groups suggest more functional and stable vasculature in the combination group (left). Co- localization analysis reveals a statistically significant, p<0.0001, increase in NG2⁺ CD31⁺ co- localized cells in the combination cohort (right).

[0017] Figure 6 shows concomitant inhibition of the angiopoietin-Tie-2 axis and the

MET kinase pathway yield significant alterations in pro-tumor macrophages. (A) Single stain and merge of Tie-2⁺ M2-like, tumor promoting macrophages (CD206⁺F4/80⁺) reveals a significant reduction in the presence of M2-like tumor associated macrophages in the combination treatment cohort. Tie-2⁺CD206⁺ Co-localization indicates the potential for trebananib to directly impact the function and presence of TAMs in the tumor

microenvironment. Pearson coefficient: vehicle cohort = 0.986; combination cohort = 0.98. (B) Blinded quantitation of the immunofluorescent staining reveals a statistically significant reduction in TAMs (F4/80⁺CD206⁺) in the tumor microenvironment. Tie-2 is expressed on multiple cell types within the tumor and thus is not significantly altered in this instance.

[0018] Figure 7 shows the combination of MET kinase inhibitor and trebananib decreases hypoxia. Immunohistochemical staining was carried out for CA9 in renal cell carcinoma cells. Results are shown in (A) for control (vehicle), MET kinase inhibitor (Formula II, also referred to herein as Compound A), trebananib (AMG386) and the combination of the MET kinase inhibitor and trebananib (Combination). The results are expressed as optical density in (B) for the various groups.

[0019] Figure 8 shows that the combination of cMET inhibitor and trebananib decreases epithelial mesenchymal transition or EMT by increasing the epithelial

differentiation marker E-cadherin (increased E-cadherin). Immunohistochemical staining is shown for the same groups as in Figure 7. The results are expressed as optical density in (B) for the various groups.

[0020] Figure 9 shows that the combination of cMET inhibitor and trebananib decreases EMT by decreasing the EMT mark vimentin. Immunohistochemical staining is shown for the same groups as in Figure 7. The results are expressed as optical density in (B) for the various groups.

[0021] Figure 10 shows that trebananib decreases TI2 phosphorylation in RENCA tumors. Western blot analysis for individual mice shows target modulation by trebananib by the inhibition of TI2 phosphorylation in RENC A tumors treated with either with trebananib or the combination with the c-MET inhibitor.

DETAILED DESCRIPTION OF THE DISCLOSURE

[0022] The present disclosure provides compositions and methods for treatment of cancers, such as kidney cancers. For example, the present compositions and methods are used to treat renal cell carcinomas.

[0023] It is considered that the present compositions and methods aid in inhibiting the spread of metastases, and prolong survival.

[0024] Definitions

[0025] As used herein, unless otherwise stated, the term "group" refers to a chemical entity that has one terminus that can be covalently bonded to other chemical species.

Examples of groups include, but are not limited to:

[0026] As used herein, unless otherwise stated, the term "moiety" refers to a chemical entity that has two or more termini that can be covalently bonded to other chemical species. Examples of moieties include, but are not limited to:

[0027] As used herein, unless otherwise indicated, the term "halogen" refers to fluorine, chlorine, bromine, or iodine, and the terms "halo" or "halide" means fluoro group (- F), chloro group (-C1), bromo group (-Br), and iodo group (-1).

[0028] As used herein, unless otherwise indicated, the term "aliphatic" refers to branched or unbranched hydrocarbon groups that, optionally, contain one or more degrees of unsaturation. Degrees of unsaturation include, but are not limited to, alkenyl groups/moieties, alkynyl groups/moieties, and cyclic aliphatic groups/moieties. For example, aliphatic groups are saturated alkyl groups/moieties. The aliphatic group can be a Ci to C12 aliphatic group, a Ci to C9 aliphatic group, a Ci to C₆ aliphatic group, or a Ci to C3 aliphatic group, including all integer numbers of carbons and ranges of numbers of carbons therebetween. The aliphatic group can be unsubstituted or substituted with one or more substituent. Examples of substituents include halides (-F, -CI, -Br, and -I), additional aliphatic groups (e.g., alkenes, alkynes), aryl groups, alkoxides, carboxylates, carboxylic acids, ether groups, and the like, and combinations thereof. [0029] As used herein, unless otherwise indicated, the term "alkyl" refers to branched or unbranched saturated hydrocarbon groups. Examples of alkyl groups include, but are not limited to, methyl groups, ethyl groups, propyl groups, butyl groups, isopropyl groups, tert- butyl groups, and the like. For example, the alkyl group can be a Ci to C12 alkyl group, a Ci to C9 alkyl group, a Ci to C₆ alkyl group, or a Ci to C3 alkyl group, including all integer numbers of carbons and ranges of numbers of carbons therebetween. The alkyl group can be unsubstituted or substituted with one or more substituent. Examples of substituents include halides (-F, -CI, -Br, and -I), aliphatic groups (e.g., alkyl groups, alkenyl groups, and alkynl groups), aryl groups, alkoxide groups, carboxylate groups, carboxylic acids, ether groups, and the like, and combinations thereof.

R

[0030] As used herein, unless otherwise indicated, "aminoalkyl" refers to a M R group where each R is selected independently from the group consisting of hydrogen atom, substituted or unsubstituted Ci to C12 alkyl chain, Ci to C9 alkyl chain, Ci to C₆ alkyl chain, or Ci to C3 alkyl chain, including all integer numbers of carbons and ranges of numbers of carbons therebetween, substituted or unsubstituted phenyl, substituted or unsubstituted heteroaryl, substituted carbonyl, substituted sulfonyl, haloalkyl, and substituted or unsubstituted benzyl groups. S^_R

[0031] As used herein, unless otherwise indicated, "thioalkyl" refers to a

group, where R is selected from a substituted or unsubstituted Ci to C12 alkyl chain, Ci to C9 alkyl chain, Ci to C₆ alkyl chain, or Ci to C3 alkyl chain, including all integer numbers of carbons and ranges of numbers of carbons therebetween, substituted or unsubstituted phenyl, substituted or unsubstituted heteroaryl, substituted carbonyl, substituted sulfonyl, haloalkyl, and substituted or unsubstituted benzyl groups.

[0032] As used herein, unless otherwise indicated, the term "alkyloxy" refers to -OR groups, where R is an alkyl group as defined herein. Examples of alkyloxy groups include, but are not limited to, methoxy groups, ethoxy groups, n-propoxy groups, i-propoxy groups, n-butoxy groups, i-butoxy groups, s-butoxy groups, and the like.

[0033] As used herein, the term "Angiopoietin 1," "Angl," or "human Angl" refers to the polypeptide human angiopoietin 1, a ligand of the human Tie-2 receptor. An "Angl inhibitor" refers to an Angl -specific binding agent that specifically binds to human Angl and/or human Tie-2 thereby inhibiting specific binding of Angl to the human Tie-2 receptor. [0034] As used herein, the term "Angiopoietin 2," "Ang2," or "human Ang2" refers to the polypeptide also called angiopoietin 2 set forth, for example, in Figure 6 (SEQ ID NO: 6) of U.S. Patent No. 6, 166,185 (hereby incorporated by reference) ("Tie-2 ligand-2") (see also, National Center for Biotechnology Information (NCBI) Accession No. AAI26203) as well as related native (i.e., wild-type) polypeptides such as allelic variants or mature forms of the polypeptide (absent the signal peptide), or splice variants (isoforms).

[0035] As used herein, the term "angiopoietin/Tie-2 axis inhibitor" means any molecule that binds to Angl and/or Ang2, thereby preventing the interaction of the angiopoietins with their target Tie-2 receptor. Such inhibitors may be small molecules or antigen binding proteins (e.g., antibodies). These inhibitors are also referred to herein as Ang/Tie-2 axis inhibitors. An example of an Ang/Tie-2 axis inhibitor is trebananib, which has the sequence of SEQ ID NO: 1.

MDKTHTCPPC PAPELLGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSRDELT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH EALHNHYTQK SLSLSPGKGG GGGAQQEECE WDPWTCEHMG SGSATGGSGS TASSGSGSAT HQEECEWDPW TCEHMLE (SEQ ID NO: l) [0036] The term "antibody" refers to isolated forms of both glycosylated and non- glycosylated immunoglobulins of any isotype or subclass, including: 1) human (e.g., CDR- grafted), humanized, and chimeric antibodies; and 2) monospecific or multi-specific antibodies, monoclonal, polyclonal, irrespective of whether such antibodies are produced, in whole or in part, via immunization, through recombinant technology, by way of in vitro synthetic means, or otherwise. Thus, the term "antibody" is inclusive of those that are prepared, expressed, created or isolated by recombinant means, such as (a) antibodies isolated from an animal (e.g., a mouse) that is transgenic for human immunoglobulin genes or a hybridoma prepared therefrom; (b) antibodies isolated from a host cell transfected to express the antibody (e.g., from a transfectoma); (c) antibodies isolated from a recombinant, combinatorial antibody library; and (d) antibodies prepared, expressed, created or isolated by any other means that involve splicing of immunoglobulin gene sequences to other DNA sequences. Antibodies may be monoclonal antibodies, such as humanized or fully-human monoclonal antibodies. Typically, antibodies will be IgG class antibodies (e.g., IgGl or IgG2).

[0037] The term "antigen binding polypeptide" refers to a molecule that comprises a polypeptide wherein the polypeptide specifically binds to a target. Exemplary binding polypeptides include: antibodies, peptibodies, avimers, Fc-soluble receptor fusion ligand trap (e.g., an Fc-soluble Tie2 fusion), CovX-bodies (see, WO 2008/056346), or specifically binding peptides (such as those obtained from screening a peptide library). A binding polypeptide of the present invention includes those that bind to a single epitope as well as multispecific binding polypeptides that bind to two epitopes (bispecific), three (trispecific), four (tetraspecific), or more epitopes.

[0038] The term "Fc" in the context of an antibody or peptibody is typically a fully human Fc, and may be any of the immunoglobulins (e.g., IgG, such as IgGl or IgG2). Fc molecules that are partially human or obtained from non-human species are also included herein.

[0039] The term "Fc-peptide fusion" refers to a peptide that is covalently bonded, directly or indirectly, to an Fc. Exemplary Fc-peptide fusion molecules include a peptibody such as those disclosed in WO 03/057134 (hereby incorporated by reference) as well as an Fc covalently bonded, directly or indirectly, to an Ang2 specific binding fragment of the Tie2 receptor.

[0040] The term "human antibody" refers to an antibody in which both the constant and framework regions consist of fully or substantially all human sequences.

[0041] The term "humanized antibody" refers to an antibody in which all or substantially all of the constant region is derived from or corresponds to human

immunoglobulins, while all or part of one or more variable regions is derived from another species, for example a mouse.

[0042] The term "monoclonal antibody" or "monoclonal antibody composition" as used herein refers to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope. The term "human monoclonal antibody" refers to antibodies displaying a single binding specificity which have variable and constant regions derived from human germline immunoglobulin sequences. The term "monoclonal" is not limited to any particular method for making an antibody.

[0043] The terms "peptide," "polypeptide," or "protein" are used interchangeably throughout and refer to a molecule comprising two or more amino acid residues joined to each other by peptide bonds. The terms "polypeptide", "peptide" and "protein" are also inclusive of modifications including, but not limited to, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP- ribosylation. [0044] The term "peptibody" refers to a specific binding agent that is a molecule comprising an antibody Fc domain attached to at least one peptide. The production of peptibodies is generally described in PCT publication WO 00/24782 (published May 4, 2000 and incorporated herein by reference). Exemplary peptides may be generated by any of the methods set forth therein, such as carried in a peptide library (e.g., a phage display library), generated by chemical synthesis, derived by digestion of proteins, or generated using recombinant DNA techniques.

[0045] The term "specifically binds" refers to the ability of, e.g., a specific binding agent of the present invention, under specific binding conditions, to bind a target molecule such that its affinity is at least 10 times as great as the average affinity of the same specific binding agent to a collection of random peptides or polypeptides. In some embodiments, the specific binding agent binds a target molecule such that its affinity is 50, 100, 250, 500, or 1000 times as great as the average affinity of the same specific binding agent to a collection of random peptides or polypeptides. A specific binding agent need not bind exclusively to a single target molecule but may specifically bind to a non-target molecule due to similarity in structural conformation between the target and non-target (e.g., paralogs or orthologs). Those of skill will recognize that specific binding to a molecule having the same function in a different species of animal (i.e., ortholog) or to a molecule having a substantially similar epitope as the target molecule (e.g., a paralog) is within the scope of the term "specific binding" which is determined relative to a statistically valid sampling of unique non-targets (e.g., random polypeptides). Thus, a specific binding agent of the invention may specifically bind to more than one distinct species of target molecule, such as specifically binding to both Ang2 and Angl . Solid-phase ELISA immunoassays can be used to determine specific binding. Generally, specific binding proceeds with an association constant of at least about 1 x 10⁷ M^"1, and often at least 1 x 10⁸ M^"1, 1 x 10⁹ M^"1, or, 1 x 10¹⁰ M^"1.

[0046] Compounds and Compositions

[0047] The present disclosure provides compositions comprising one or more compounds having the structure of Formula I and one or more compounds that are Ang/Tie-2 axis inhibitors. Formula I is shown below:

where R¹, R², and R³ are independently selected from the group consisting of hydrogen, halide (e.g., fluorine, bromine, etc.), aliphatic group (e.g., alkyl groups such as, for example, methyl, ethyl, propyl, isopropyl, etc.), amine group, aminoalkyl group (e.g., aminomethyl, aminoethyl, etc.), thiol group, thioalkyl group (e.g., thiomethyl, thioethyl, etc.), sulfate group, cycloalkyl group (e.g., cyclopropane), hydroxyl group, and alkyloxy group (e.g., methoxy, ethoxy, etc.). R⁴ is independently selected from the group consisting of hydrogen, halide (e.g., fluorine, bromine, etc.), aliphatic group (e.g., alkyl groups such as, for example, methyl, ethyl, propyl, isopropyl, etc.), amine group, aminoalkyl group (e.g., aminomethyl, aminoethyl, etc.), thiol group, thioalkyl group (e.g., thiomethyl, thioethyl, etc.), sulfate group, cycloalkyl group (e.g., cyclopropane), hydroxyl group, alkyloxy group (e.g., methoxy, ethoxy, etc.), and acyl group (e.g., acetyl, formyl, alkyl ester, alkylated amide, formamide, etc.

[0048] For example, the composition can comprise a compound having the structure of Formula I where R¹, R², and R³ are independently the same or different halides and R⁴ is an acyl group, such as an acetyl group. In another example, the compound has the structure of Formula II (Compound A):

[0049] This disclosure provides compositions comprising one or more compounds of

Formula I and further comprise Ang/Tie-2 axis inhibitors. For example, the compositions may comprise one or more compounds of Formula I and one or more Ang/Tie-2 axis inhibitors (e.g., a small molecule or antigen binding peptide (e.g., an antibody). In one instance, the composition comprises one or more compounds of Formula I and trebananib. A composition may comprise a compound having the structure of Formula II and Ang/Tie-2 axis inhibitor (e.g., a small molecule or antigen binding peptide (e.g., an antibody). For example, a composition may comprise a compound having the structure of Formula II and trebananib (also referred to herein as AMG386). In certain embodiments, the Ang/Tie-2 axis inhibitor is a polypeptide comprising SEQ ID NO: 1.

[0050] The the compositions of the present invention may contain one or more compounds of Formula I (e.g., Formula II) and one or more Ang/Tie-2 axis inhibitor (e.g., trebananib) as the only active agents. For example, a composition may have the one or more compounds having the structure of Formula I as the only agents that inhibit MET kinase and one or more Ang/Tie-2 axis inhibitors as the only active agents against the Ang/Tie axis. In one embodiment, a composition may have the compound of Formula II as the only agent that inhibit MET kinase and one or more Ang/Tie-2 axis inhibitors as the only active agents against the Ang/Tie axis. In one embodiment, a composition may have the compound of Formula II as the only agent that inhibit MET kinase and trebananib as the only active agents against the Ang/Tie axis. In one embodiment, a composition may have the one or more compounds having the structure of Formula I as the only agents that inhibit MET kinase and trebananib as the only active agents against the Ang/Tie axis.

[0051] The one or more compounds of Formula I may be the only active agents present in the composition (i.e., may be the only compounds other than excipients, or may be the only c-MET kinase inhibitors). For example, a composition may have a compound of Formula II as the only agent that is a MET kinase inhibitor. For example, a composition may have a compound of Formula I (such as a compound of Formula II) as the only agent that inhibits MET kinase, and trebananib as the only active agent that inhibits the Ang/Tie-2 axis.

[0052] The present disclosure provides: [1] compositions comprising one or more compounds having the structure of Formula I and one or more Ang/Tie-2 axis inhibitors; [2] pharmaceutical compositions comprising one or more compounds having the structure of Formula I and one or more Ang/Tie-2 axis inhibitors. The pharmaceutical compositions may comprise suitable pharmaceutically acceptable carriers, excipients, stabilizers, or a combination thereof. Examples of pharmaceutically acceptable carriers, excipients, and stabilizers can be found in Remington: The Science and Practice of Pharmacy (2005) 21st Edition, Philadelphia, PA. Lippincott Williams & Wilkins. For example, suitable carriers, excipients and stabilizers which are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as, for example, acetate, Tris, phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives such as, for example, octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as, for example, methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol; amino acids such as, for example, glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as, for example, EDTA;

tonicifiers such as, for example, trehalose and sodium chloride; sugars such as, for example, sucrose, mannitol, trehalose or sorbitol; surfactant such as, for example, polysorbate; salt- forming counter-ions such as, for example, sodium; and/or non-ionic surfactants such as, for example, Tween or polyethylene glycol (PEG). The pharmaceutical compositions may comprise other therapeutic agents. The present compositions can be provided as single doses or in multiple doses covering the entire or partial treatment regimen. The compositions can be provided in liquid, solid, semi-solid, gel, aerosolized, vaporized, or any other form from which it can be delivered to an individual.

[0053] In one aspect, the present disclosure provides uses of compounds described herein (e.g., compounds of Formula I (e.g., Formula II) and Ang/Tie-2 axis inhibitors (e.g., trebananib) and compositions comprising same. For example, the compositions can be used for inhibiting Ang/Tie-2 and/or MET kinase. The present compounds or compositions comprising the compounds can be used for treatment of cancers (e.g., kidney cancers) associated with Ang/Tie-2, MET kinase, and the kidneys. For example, the present compounds are used for treatment of kidney cancer; such as, for example, clear cell renal cell carcinoma (ccRCC).

[0054] Pharmaceutical compositions comprising or consisting essentially of the present compounds can be administered to an individual in need of treatment. Clinicians will be able to assess individuals who are in need of being treated for these conditions. The present compositions can be used in combination with other diagnostic approaches and/or therapeutic approaches for the conditions. The additional therapeutic approaches can be carried out sequentially or concurrently with the treatment involving the present

compositions. The term "treatment" refers to reduction in one or more symptoms or features associated with the presence of the particular condition being treated. Treatment does not necessarily mean complete remission, nor does it preclude recurrence or relapses. For example, the present disclosure provides a method for reducing or inhibiting metastases, reducing the size of a tumor, arresting the growth of a tumor, reducing the rate of growth of a tumor, and/or reducing any other symptom that is associated with an individual being afflicted with the tumor - all of which are considered as "treatment" - comprising

administering to an individual in need of treatment, a therapeutically effective amount of a composition comprising the inhibitors as described herein.

[0055] Administration of present compositions, separately or in combination, as described herein can be carried out using any suitable route of administration known in the art. For example, the compositions may be administered via intravenous, intramuscular, intraperitoneal, intracerobrospinal, subcutaneous, intra-articular, intrasynovial, oral, topical, or inhalation routes. The compositions may be administered parenterally or enterically. The compositions may be introduced as a single administration or as multiple administrations or may be introduced in a continuous manner over a period of time. For example, the administration(s) can be a pre-specified number of administrations or daily, weekly or monthly administrations, which may be continuous or intermittent, as may be clinically needed and/or therapeutically indicated.

[0056] Methods of Treatment

[0057] The present compositions can be used in the treatment of cancers, such as kidney cancers. For example, the compounds and compositions of the present invention can be used in the treatment of renal cell carcinomas such as ccRCC. The compounds and compositions of the present invention are particularly useful for inhibiting metastases associated with cancers. For example, the compounds and compositions of the present invention can be administered to reduce metastases associated with renal cell carcinomas.

[0058] Administration of the compounds and compositions of the present invention can be carried out in conjunction with other treatment modalities. The present compositions may be administered in conjunction with removal or regression of the primary or secondary tumors. The tumor(s) may be removed by surgical or other ablation methods and the administration of the present compositions can be carried out prior to, concurrently or after such removal or regression. Other modalities used in the treatment of cancers, such as radiation and the like may also be used in conjunction with the present methods.

[0059] Determination of suitable dosages of the present compounds and compositions are within the purview of those skilled in the art. For example, a clinician can determine a suitable dosage. An example of suitable dosages of the present compounds and compositions of the present invention include 10 to 100 mg/kg for MET kinase inhibitor and 1 to 20 mg/kg for trebananib. The dosage used in the animal experiments was 5.6 mg/kg subcutaneous injections of trebananib twice a week and 30 mg/kg MET kinase inhibitor by oral gavage 5 days a week. Based on the above, suitable dosages for a human can be determined by one skilled in the art.

[0060] The steps of the methods described in the various embodiments and examples disclosed herein are sufficient to carry out the methods of the present invention.

[0061] The present compositions and methods can be used in any mammal, including humans, domestic animals, farm animals and the like. An individual in need of treatment is typically an individual who has been diagnosed with cancer such as kidney cancer. A clinician is well-qualified to identify individuals in need of treatment.

[0062] In one instance, the disclosure provides a composition comprising or consistin essentially of one or more compounds having the following structure:

wherein R¹, R², and R³ are independently selected from the group consisting of hydrogen, halide, aliphatic group, amine group, aminoalkyl group, thiol group, thioalkyl group, sulfate group, cycloalkyl group, hydroxyl group, and alkyloxy group, and R⁴ is selected from the group consisting of hydrogen, halide, alkyl group, aliphatic group, amine group, aminoalkyl, thiol group, thioalkyl group, sulfate group, cycloalkyl group, hydroxyl group, alkyloxy group, and acyl group, wherein the one or more compounds have MET kinase inhibitor activity, and one or more Ang/Tie-2 axis inhibitors. In certain embodiments, the Ang/Tie-2 axis inhibitor is a polypeptide comprising SEQ ID NO: 1.

[0063] In one instance, the disclosure provides a composition comprising or consisting essentially of the compound of Formula I having the structure:

[0064] In one instance, the composition comprises or consists essentially of trebananib and one or more compounds having the following structure:

(Formula I) wherein R¹, R², and R³ are independently selected from the group consisting of hydrogen, halide, aliphatic group, amine group, aminoalkyl group, thiol group, thioalkyl group, sulfate group, cycloalkyl group, hydroxyl group, and alkyloxy group, and R⁴ is selected from the group consisting of hydrogen, halide, alkyl group, aliphatic group, amine group, aminoalkyl, thiol group, thioalkyl group, sulfate group, cycloalkyl group, hydroxyl group, alkyloxy group, and acyl group, wherein the one or more compounds of Formula I have MET kinase inhibitor activity.

[0065] In one instance, the composition comprises or consists essentially of trebananib and a compound having the structure:

(Formula II) [0066] The compound of Formula I or II, and the Ang/Tie-2 axis inhibitors may be present in a pharmaceutical composition.

[0067] The one or more compounds of Formula I and the one or more Ang/Tie-2 axis inhibitors may be present in the same composition or may be present as separate

compositions in the form of a kit for administration in conjunction with each other. The kit may further comprise administration guidelines, storage recommendations and the like.

[0068] In one instance, this disclosure provides the compound(s) of Formula I and/or

II, or pharmaceutical compositions comprising such compounds for the inhibition of the metastasis of a cancer. The metastasis may be a metastases associated with kidney cancer.

[0069] The following examples are presented to illustrate the present disclosure. They are not intended to limiting in any matter

EXAMPLE 1

[0070] The following is an example of combining inhibitors of both the MET and

Ang/Tie-2 pathways as treatment for metastatic renal cell carcinoma.

MATERIALS AND METHODS

[0071] Cell lines: The murine renal cell carcinoma cell line RENCA was initially purchased from American Type Culture Collection (National Cancer Institute) and stably tagged with a luciferase reporter. Cells were cultured in RPMI 1640 (Corning) with 10% fetal bovine serum (Corning) and 1% Pen/Strep (LifeTechnologies) and incubated in 37°C in an incubator containing 5% CO2. Non-confluent cells were harvested using 0.25% Trypsin (Corning) and suspended in matrigel (Corning) and DPBS (Gibco) in a 1 : 1 ratio, ΙΟμΙ containing lxlO⁴ cells was injected under the renal capsule. Mice were serially imaged using a bioluminescent IVIS imaging machine.

[0072] Mouse Studies

[0073] All procedures were approved and performed in strict accordance with the

Institutional Animal Care and use Committee (IACUC) at Roswell Park Cancer Institute and with the NIH Guide for the Care and Use of Laboratory Animals guidelines.

[0074] In Vivo tumor growth (RENCA). Five- to six- week old Balb/c mice

(National Cancer Institute) were kept in a temperature controlled room on a 12/12-hour light /dark schedule with food and water ad libitum. Non-confluent RENCA-Luc cells were harvested using 0.25% Trypsin (Corning) and suspended in matrigel (Corning) and DPBS (Gibco) in a 1 : 1 ratio, ΙΟμΙ containing lxlO⁴ cells was injected under the renal capsule. Animals were randomly distributed into four groups: vehicle (soy bean oil), Trebananib (AMG 386), MET kinase inhibitor (Formula II), or combination. Mouse tumors were serially imaged using a bioluminescent IVIS imaging machine.

[0075] Xenograft models: Wild type male ICR Severe Combined Immune-deficient (SCID) mice, ages 6-8 weeks, were purchased from Charles River and housed in a sterile, pathogen-free facility and maintained in a temperature controlled room under a 12-hour light/dark schedule with food and water ad libitum. Upon arrival and acclimation mice were implanted orthotopically (mm² tumor piece) or subcutaneously (~lmm² tumor piece) with RP-R-01, RP-R-02, or RP-R02LM, which were established from the skin metastasis of a patient with sporadic ccRCC which developed sunitinib resistance and skin metastasis from a patient with hereditary VHL syndrome ccRCC, respectively.

[0076] Trebananib and MET kinase inhibitor treatment. Mice in the vehicle group were given soy bean oil daily (5d/week) by oral gavage. Mice in the treatment groups were treated with 5.6 mg/kg of trebananib (AMG 386), twice a week i.p., and/or 30mg/kg of MET kinase inhibitor, 5 days a week by oral gavage. In the sunitinib resistant studies, RP-R-01 and RP-R-02, mice were treated with 40mg/kg of sunitinib for three weeks, or until tumor progression. Following this, the mice were treated with trebananib, MET kinase inhibitor, or a combination of both.

[0077] Histologic and Immunohistochemical Analysis

[0078] Tumor tissue specimen from each treatment group were fixed for 24 hours in formalin, paraffin-embedded and sectioned (5μπι). Slide sections were deparaffinized and rehydrated via gradient alcohol washes. Antigen unmasking was performed by boiling slides in sodium citrate buffer (pH=6.0). Slides were subsequently incubated in hydrogen peroxide to reduce endogenous activity. For probing of the tissue for the proteins of interest, tissue sections were blocked in 2.5% horse serum (Vector Laboratories), and incubated overnight in primary antibodies against CD31 (1 : 100, Dianova). Following primary antibody incubation, slides were incubated with horseradish-peroxidase (HRP)-conjugated anti-rat antibody according to the manufacturer's protocol (Vector Laboratories). Following this slides were subjected to enzymatic development in diaminobenzidine (DAB). Sections were then dehydrated and mounted with cytoseal 60 (Thermo Scientific). Stained sections were imaged under bright field (IHC) using the Zeiss Axio microscope. The positive fields were calculated in a blinded fashion by analyzing four random 20x fields per tissue and quantified using Image J software.

[0079] Western Blot Analysis [0080] Tumors from each treatment group were lysed in RTPA buffer (Sigma-

Aldrich) containing protease and phosphatase inhibitor cocktails (Pierce). Protein

concentrations were assessed using a standard BSA assay (Bio-Rad). 50ug of protein from each sample was subjected to electrophoresis on 10% SDS-polyacrylamide gels (Bio-Rad) and transferred onto nitrocellulose membranes. Proteins of interest were detected with the following primary antibodies: E-cadherin (1 : 1000; Cell Signaling Technology), NG2 - Chondroitin Sulfate Proteoglycan (1 : 1000; EMD Millipore), phosphor-p44/42 MAPK (Erkl/2) (Thr202/Tyr204) (1 : 1000; Cell Signaling Technology), phosphor-AKT (Ser473) (1 : 1000; Cell Signaling Technology), GAPDH (1 : 1000; Cell Signaling Technology).

Following incubation with primary antibody the membranes were probed with an HRP- conjugated secondary antibodies (Bio-Rad) and exposed to chemiluminescence per the manufacturer's instructions (Thermo Fisher Scientific) and exposed to film.

[0081] Quantitative real-time PCR

[0082] Total RNA was isolated from tumor samples according to manufacturer's instructions using TRIzol (Life Technologies) and measured using nano-drop technology.

Quantitative RT-PCR was performed with e-cadherin and snail primers (IDT-Technologies).

Samples were denatured at 95°C for 10 seconds, annealed at 60°C for 30 seconds, and extended at 72°C for 1 minute using the Applied Biosystems 7900HT fast real time PCR system (Applied Biosystems). Sequence Detection Systems software v2.3 was used to identify the cycle threshold (G) values and to generate gene expression curves. Data were normalized to GAPDH expression and fold change was calculated.

[0083] Immunoflourescence Staining

[0084] Tissue sections were prepared as described previously and stained for anti-rat

CD31 (Dianova) at a 1 :50 dilution and anti-rabbit NG2 at a 1 :50 dilution overnight at 4°C. Following the primary incubation, slides were incubated with conjugated secondary antibodies - anti -rabbit FITC and anti-rat Cy3, co-stained with DAPI and mounted using

Vectashield immunofluorescence mounting reagent. Analysis of the NG2 CD31 co-staining was performed in a blinded manner utilizing ImageJ software.

[0085] Confocal Microscopy

[0086] CD31 and NG2 co-staining was performed on thicker sections of tumor tissue

(μπι) as described above. The Indiana Center for Biological Microscopy at IUPUI assisted in acquiring confocal images of the co-stained sections.

[0087] Statistical Analysis [0088] All statistical analyses were performed using GraphPad Prism7 software for

Windows. Analysis of survival was conducted using the Kaplan-Meier method. Differences in treatment group survivals were assessed with the log-rank test. All other statistical analyses in this study were performed between experimental groups using the Student' s T test with Welch's correction. A p-value <0.05 was considered to be statistically significant.

[0089] RESULTS

[0090] Combined inhibition of Angl/Ang2 and MET significantly reduces spontaneous lung metastases in the patient derived xenograft model RP-R-02LM. To assess the efficacy of combining Angl/Ang2 inhibition and MET kinase inhibition on primary tumor growth, luciferase tagged syngeneic orthotopic murine model of renal cell carcinoma RENCA-Luc and patient derived xenograft (PDX) models RP-R-01 and RP-R-02 was used. These PDX models have been detailed previously as maintaining their original clear cell morphology, FHZ-negative status, human Alu positive status, and containing common ccRCC gene mutations(Adelaiye et al., Mol Cancer Ther 2015, 14(2): 13-22). Preliminary experiments assessed the primary tumor growth of RENCA-Luc, RP-R-01, and RP-R-02 tumors. When the tumors reached a detectable size, approximately l-3xl0⁵ average radiance for RENCA or 50mm² for the subcutaneous PDX models, mice were randomized into four groups: vehicle (soy bean oil), trebananib, MET kinase inhibitor, and combination. Mice were treated with 5.6 mg/kg subcutaneous injections of trebananib twice per week and/or 30 mg/kg MET kinase inhibitor by oral gavage 5 days per week. In the RENCA model, a significant decrease was observed in final tumor weights in the trebananib treatment group but no significant advantage when combined with the MET kinase inhibitor (Figure 1 A). The angiopoietin-Tie-2 pathway is required for vascular stability in tumor development and progression. With this information, the vascularization of our model was examined. MET inhibition alone was found to significantly increase the presence of the endothelial cell marker CD31. However, combination of MET inhibition with trebananib reversed the increase in CD31⁺ area, leading to a significant reduction in CD31⁺ area compared to the MET inhibitor alone (Figure IB, C). These results suggest that the angiopoietin-Tie-2 axis inhibitor, trebananib, alters the tumor microenvironment in a manner which inhibits the sprouting of new vasculature.

[0091] Clinically efficacious first line therapeutics for ccRCC, such as VEGFR- targeting sunitinib, elicit anti -tumor responses in ccRCC patients. However, the majority of patients who initially respond to sunitinib develop resistance and their tumors continue to progress on treatment. Treatment of sunitinib-sensitive and sunitinib-resistant models with trebananib and MET inhibition was examined to assess the efficacy of this novel combination in comparison with the current standard of care for ccRCC patients. The results showed significant inhibition of primary tumor growth in the RP-R-01 sunitinib-sensitive

combination group (Figure ID). In the RP-R-01 sunitinib-resistant (Figure IE) and RP-R-02 non-metastatic models (Figure IF) there was no significant difference in primary tumor growth and no significant change in tumor vascularization comparing the single agents to the combination treatment in both the sunitinib sensitive and sunitinib-resistant models (Figure 1E-F)

[0092] In parallel with our initial experiments, a study was conducted using our RP- R-02LM model, which spontaneously metastasizes to the lungs when implanted

subcutaneously or orthotopically. With this model there was no significant difference in the growth of the primary tumors with any of the treatments (Figure 2A, B). While the MET kinase inhibitor restricted tumor growth in our metastatic model, the mice in this group had similar accumulation and diameter of lung metastases as the untreated cohort (Figure 2C). Strikingly, the overall number (Figure 2D) and diameter of lung metastases (Figure 2E) were significantly reduced after treatment with trebananib as compared to the vehicle.

Further, the combination group had a significant reduction in both the number and size of metastases as compared to vehicle and both single agent treatments (Figure 2C-E). These data demonstrate that inhibition of the angiopoietin-Tie-2-MET kinase axis results in significant vascular alterations to the tumor microenvironment which may hinder metastatic escape of ccRCC cells from the primary tumor.

[0093] Combination treatment hinders shedding of metastatic cells from the primary tumor. To determine the effect of combination treatment on the shedding of metastatic cells from the primary tumor, the survival and metastatic burden of mice was examined in vehicle, single agent, and combination groups. RP-R-02LM tumor pieces were implanted orthotopically, under the kidney capsule, and treatment was begun with AMG 386 at 5.6 mg/kg subcutaneously twice a week and/or 30 mg/kg MET kinase inhibitor at 4.5 weeks post-implantation - a time-point at which metastases have begun to shed from the primary tumor to the lungs. Treatment was continued for the duration of the survival of the mice. A significant increase was observed in survival of mice in the combination group as compared to the vehicle cohort (p<0.0005) while the single agent MET kinase cohort performed much worse (Figure 3A). In congruence with the initial RP-R-02LM study, mice in the combination group died from the growth of their primary tumor rather than their lung metastases, suggesting that the combination treatment is preventing shedding of metastatic cells from the primary tumor. It was observed that as in the first set of experiments, the vehicle and MET kinase inhibitor cohorts with smaller primary tumors had more lung metastases and those with larger primary tumors had significantly fewer metastases. These data indicate that dual inhibition of the MET kinase pathway and the antiopoietin-Tie-2 axis are modulating the TME in a manner which inhibits the shedding of metastatic cells from the primary tumor.

[0094] Combination treatment hinders the metastatic potential of RP-R-02LM post tumor resection. Clinically, patients with renal cancer often have their primary tumor resected yet develop aggressive and lethal metastatic disease - most commonly with metastases to the lungs. To study the efficacy of trebananib in combination with MET kinase inhibition for the treatment of ccRCC, RP-R-02LM tumors were implanted subcutaneously and began treatment five and a half weeks post-implantation, a time point at which cells from the subcutaneous tumors begin to metastasize to the lungs. The primary tumor was then removed at 3 months post-implantation while continuing to treat the mice for the duration of the study. The results showed a significant increase in survival of mice in the combination group compared to each cohort in the study (p<0.0005 combination - vehicle, p<0.005 combination - MET kinase inhibitor and p<0.05 combination - trebananib) (Figure 3B). In addition, a significant delay was observed in development of lung metastases in the combination cohort. In Figure 3C this is shown as the day of euthanasia listed below the images of the primary tumor and the metastatic laden lungs. There are strikingly less macro- metastases in the trebananib and combination cohorts as compared to the control and Formula II cohorts. These findings suggest that our combination therapy inhibits the ability of RP-R- 02LM cells to settle in the lung niche. To better understand the molecular changes taking place in the primary tumors with the trebananib and Formula II treatment combination the molecular characteristics of EMT markers and the tumor microenvironment were examined, with a focus on macrophage infiltration.

[0095] Markers of EMT are reduced by trebananib and c-MET inhibitor combined treatment. E-cadherin, which fails to be expressed in FHZ-defective models of ccRCC, is a prognostic factor for patient survival, indicating that FHZ-defective ccRCC cases are more likely to be aggressive and metastatic. During the establishment and

characterization of the RP-R-02LM model we observed changes in the expression of genes associated with metastasis, including an increase in snail expression and a decrease in E- cadherin - changes that have been associated with tumor dissemination and subsequent establishment and growth in the lung metastatic niche which have been shown to play a key role in tumor metastases in both characterization of the RP-R-02LM model as well as in various other metastatic models. Based on these findings and in congruence with our observation of extended survival in mice with orthotopic and subcutaneous RP-R-02LM tumors, the expression of E-cadherin was evaluated to elucidate the impact of combination treatment on tumor dissemination. Tumor sample analysis yielded an increase in E-cadherin and a significant decrease in snail levels in the combination treatment group (Figure 3C-F). The subcutaneous group, in which tumors were removed after only six and a half weeks of treatment, showed less of a trend of E-cadherin increase (data not shown) indicating that the impact of combination treatment on e-cadherin requires longer exposure of the primary tumor to treatment and alters the dissemination ability of the primary tumor.

[0096] A potential mechanism by which tumor dissemination and establishment of metastases in the lungs is hindered is through the alteration of EMT markers. The data presented here suggest that we are impacting the EMT of these cells and in turn inhibiting their capacity to metastasize to the lungs.

[0097] Pathway activation downstream of Tie-2 and c-MET is altered. To elucidate whether if the target was being hit in the TME of our orthotopic survival study, the effects of treatment on downstream effectors of the MET and Tie-2 signaling pathways were assessed including AKT, STAT3 and ERK via Western blot analysis (Figure 4A), followed by densitometry quantification. A decrease in p-ERK levels was observed in the MET kinase inhibitor group relative to vehicle, indicating an inhibition of the MET pathway. However, no decrease in the p-ERK levels were observed in either the trebananib alone or the combination groups (Figure 4B). This unusual finding may suggest that trebananib treatment is reversing the effect of the MET kinase inhibitor. Additionally, the MET kinase inhibitor group exhibited a significant decrease in p-AKT levels while the trebananib and combination groups were not significantly different to vehicle (Figure 4C). Finally, p-STAT3, a marker of aggressiveness and proliferation was examined, and noted that while there was an overall trend of p-STAT3 inhibition in the single agent and combination groups, there was no significant change from the vehicle (Figure 4D). Taken together these results suggest that the MET signaling pathway in the MET kinase inhibitor group can be inhibited while in some cases a resistance to this treatment in the combination group was seen. This may explain why, in the RP-R-02LM model, inhibition of tumor growth was limited to the MET kinase inhibitor treatment group (Figure 2A).

[0098] Combination treatment of the patient derived xenograft, RP-R-02LM, leads to enhanced pericyte coverage. In addition to examining the EMT markers and downstream constituents of the MET and Tie-2 pathways, there was interest in examining the mechanism by which combination treatment inhibits tumor metastases - as the expected inhibition of downstream markers in the combination treatment group was not seen. To achieve this, the presence of pericytes (NG2) co-situated with endothelial cells (CD31) was analyzed. Since the combination treatment of trebananib and MET kinase inhibitor inhibits the dissemination of RP-R-02LM cells from the primary tumor to the lungs, despite continued growth of the primary tumor, these data point to an alteration in the vasculature of the primary tumor. In the combination group there was a statistically significant increase in the presence of NG2 positive pericytes co-situated with CD31 endothelial staining (Figure 5A-B, E). This increase of pericyte expression was verified by Western blot and densitometry analysis using ImageJ software (Figure 5C-D). Co-localization analysis reveals a significant increase in NG2-CD31 localization in the combination group as compared to the control group (p=<0.0001) (Figure E). These results indicate that the combination treatment of trebananib and MET kinase inhibitor may be strengthening the vasculature in a manner that allows for more secure nutrient delivery to the tumor at the same time hindering the metastases with a less leaky system, thus explaining the continued growth of the primary tumor concurrent with decreased lung metastases.

[0099] Tumor associated macrophage presence is significantly decreased with inhibition of the angiopoietin-Tie-2-cMET axis. Tumor associated macrophages have been shown to facilitate tumor metastases via promotion of tissue remodeling, angiogenesis, and production of extra cellular matrix remodeling enzymes, such as matrix metalloproteinases. It was considered that perivascular, tumor associated macrophages, angiopoietin receptor, Tie- 2⁺ cells, which have been shown to mediate tumor metastasis, may be reduced in the tumor microenvironment of our combination group. To test this, tumors from our in vivo study of RP-R-02LM, implanted subcutaneously were stained for Tie-2, F4/80 pan-macrophage marker, and CD206 M2-like macrophage marker. In both the control and the combination treated groups there was a significant co-localization of Tie-2/CD206 (Pearson coefficient = 0.98 in both vehicle and combination cohorts) (Figure 6A), confirming the presence of Tie- 2⁺ macrophages in our tumors. In the combination treatment cohort, there was a significant decrease in the presence of TAMs (F480⁺ Tie-2⁺CD206⁺) in the tumor microenvironment (Figure 6). These data demonstrate that combination treatment of trebananib and Formula II inhibit the infiltration of M2-like TAMs into the tumor microenvironment, suggesting a potential role for these TAMs in the metastases of PDX model RP-R-02LM. In summary, these data indicate that inhibition of the angiopoietin 1/2-TIE-2 - cMET axis inhibits ccRCC lung metastases, alters pericyte coverage and vascular stability and inhibits EMT progression. A significant alteration of the tumor microenvironment with inhibition of the angiopoietin 1/2 - TIE-2 - cMET axis was also seen. M2 macrophage presence, which contributes to the progression, dissemination, and survival of the tumor, is significantly reduced in correlation with inhibition of lung metastases indicating a potential mechanism by which the

combination and trebananib treatment inhibit metastases of RP-R-02LM tumors.

[0100] DISCUSSION

[0101] In this study, the effect of the Angl/Ang2 peptide, trebananib, as a single agent and in combination with inhibition of the MET kinase pathway was assessed. This disclosure provides evidence that the combination alters both the tumor niche and the tumor microenvironment by inhibiting the shedding and establishment of metastatic cells, altering pericyte presence, and reducing the presence of metastases promoting macrophages in a potently metastatic ccRCC PDX model.

[0102] Trebananib, a recombinant peptide-Fc fusion protein which negates the receptor/ligand interaction of Angl/Ang2 with the Tie-2 receptor, has been reported to improve progression free survival (PFS) in patients with ovarian cancer in a phase III trial as compared with paclitaxel (Fujiwara et al., Ann Oncol 2016, 27(6): 1006-13). The combination of MET kinase inhibition with trebananib was tested in both murine and patient derived xenograft models of ccRCC. Although the combination treatment studies did not yield a significant difference in primary tumor growth compared to the single agent treatments, a striking inhibition of metastases, increased expression of epithelial marker e-cadherin, upregulated pericyte coverage, and reduced presence of metastasis promoting M2-like macrophages in the primary tumor of our potently metastatic ccRCC PDX model, RP-R- 02LM was discovered. These results suggest that in our models combination of MET kinase inhibition and angiopoietin inhibition may not provide an advantage in inhibition of primary tumor growth as compared with single agent treatments alone. In contrast, trebananib treatment combined with MET kinase inhibition in our metastatic PDX model, RP-R-02LM, not only decreased the presence of tumor metastases to the lungs but also significantly enhanced survival. In this highly metastatic model the need to inhibit both the

angiopoietin/Tie-2 axis and the MET axis concomitantly to significantly inhibit metastases and enhance survival was observed.

[0103] RP-R-02LM, a highly metastatic patient derived xenograft, is applicable as a clinically translatable model. The tumors, passaged in vivo since the initial patient derivation, maintain heterogeneity, clear cell morphology, VHL negative & human Alu positive status, and spontaneous metastatic phenotype rarely observed in PDX models. In the initial short term treatment study with this model, a significant decrease was noted in metastases to the lungs, indicating that the metastatic potential of these tumors was impaired. Even more striking were the subsequent survival studies performed with the RP-R-02LM model both orthotopically and subcutaneously. In the orthotopic study, we noted that when RP-R-02LM tumor pieces were implanted into the kidney and treated for at least 4 months a significant improvement in survival in the combination groups were observed. The mice in the combination group died of their primary tumor growth and not their metastatic burden while those in the vehicle and c-MET inhibition group died of metastatic burden. In a clinically relevant subcutaneous study the primary tumors were removed at 3 months post-implantation, a time which at which these tumors have already shed metastatic cells, and treatment was continued and the mice were assessed for metastatic burden. The combination group had a striking increase in survival compared to both single agent and vehicle cohorts. These findings together indicate that combining Angl/Ang2 inhibition with c-MET inhibition results in stifling of metastatic cell shedding - orthotopic study - and the establishment of these cells within their metastatic niche - subcutaneous study.

[0104] The present results indicate that increased e-cadherin expression, pericyte coverage, reduced macrophage presence, and stabilization of tumor vasculature in

combination cohort may contribute to prolonged survival and reduced lung metastases. These results further indicate that MET kinase and angiopoietin inhibition can affect the tumor microenvironment in a manner that can inhibit the metastatic phenotype of ccRCC. The present studies indicate a selective anti-metastatic effect of concomitant angiopoietinl/2 and c-MET inhibition.

[0105] In conclusion, targeting the tumor microenvironment and the tumor niche via combining a small molecule MET kinase inhibitor with the peptide inhibitor of angiopoietin 1/2, trebananib has striking biological effects. In the present metastatic patient-derived xenograft ccRCC model, RP-R-02LM, this combination suppressed both tumor cell invasiveness and metastases and enhanced survival. In addition, the combination resulted in blood vessel stabilization, reduced tumor promoting macrophage presence, increased pericyte coverage and e-cadherin expression. The combination treatment inhibited upregulation of mesenchymal markers, reduced M2-like macrophage presence and increased the pericyte coverage within the tumor, suggesting a less permissive environment for the escape of metastatic cells from the primary tumor microenvironment. Taken together, these results indicate that combining angiopoietin/ Tie-2 inhibition with MET kinase inhibition can be a clinically effective adjuvant therapy for patients with ccRCC.

EXAMPLE 2

[0106] This example demonstrates that dual inhibition of angiopoietin-TIE2 and MET alters the tumor microenvironment and prolongs survival in a metastatic model of renal cell carcinoma. As shown in Figures 7-10, dual inhibition of angiopoietin-TIE2 and MET inhibits EMT and promote tumor cell differentiation.

[0107] Although the present disclosure has been described with respect to one or more particular embodiments and/or examples, it will be understood that other embodiments and/or examples of the present disclosure may be made without departing from the scope of the present disclosure.

Claims

What is claimed is:

1. A composition comprising:

a one or more compounds having the following structure:

(Formula I) wherein R¹, R², and R³ are independently selected from the group consisting of hydrogen, halide, aliphatic group, amine group, aminoalkyl group, thiol group, thioalkyl group, sulfate group, cycloalkyl group, hydroxyl group, and alkyloxy group, and R⁴ is selected from the group consisting of hydrogen, halide, alkyl group, aliphatic group, amine group, aminoalkyl, thiol group, thioalkyl group, sulfate group, cycloalkyl group, hydroxyl group, alkyloxy group, and acyl group, wherein the one or more compounds have MET kinase inhibitor activity; and b) one or more Ang/Tie-2 axis inhibitors,

pharmaceutically acceptable carrier or excipient.

2. The composition of claim 1, wherein the compound of Formula I has the following structure:

(Formula II).

3. The composition of claim 1, wherein the Ang/Tie-2 axis inhibitor is trebananib.

4. The composition of claim 2, wherein the Ang/Tie-2 axis inhibitor is trebananib.

5. A method of inhibiting cancer metastases comprising administering to an individual in need of treatment a composition of claim 1, wherein the administration results in inhibition of metastases.

6. The method of claim 5, wherein the compound of Formula I has the structure:

7. The method of claim 5, wherein the Ang/Tie-2 axis inhibitor is trebananib.

8. The method of claim 6, wherein the Ang/Tie-2 axis inhibitor is trebananib.

9. The method of claim 5, wherein the Ang/Tie-2 axis inhibitor is a polypeptide comprising the sequence of SEQ ID NO: 1.

10. The method of claim 5, wherein said cancer is a kidney cancer.

11. The method of claim 10, wherein the kidney cancer is renal cell carcinoma.

12. The method of any one of claims 5 to 11, further comprising removing the primary tumor, prior to, concurrent with or following cessation of administration of said composition.

13. The method of any one of claims 5 to 11, further comprising treating the individual with radiation.

14. A method of inhibiting cancer metastases comprising administering to an individual in need of treatment: a) a composition comprising one or more compounds having the following structure:

wherein R¹, R², and R³ are independently selected from the group consisting of hydrogen, halide, aliphatic group, amine group, aminoalkyl group, thiol group, thioalkyl group, sulfate group, cycloalkyl group, hydroxyl group, and alkyloxy group, and R⁴ is selected from the group consisting of hydrogen, halide, alkyl group, aliphatic group, amine group, aminoalkyl, thiol group, thioalkyl group, sulfate group, cycloalkyl group, hydroxyl group, alkyloxy group, and acyl group, wherein the one or more compounds have MET kinase inhibitor activity; and

b) a composition comprising one or more Ang/Tie-2 axis inhibitors.

15. The method of claim 14, wherein the compound of Formula I has the structure:

16. The method of claim 14 or 15, wherein the Ang/Tie-2 axis inhibitor is trebananib.

17. The method of claim 14 or 15, wherein the Ang/Tie-2 axis inhibitor is a polypeptide comprising the sequence of SEQ ID NO: 1.

18. The method of claim 14 or 15, wherein the compound of Formula I and the Ang/Tie-2 axis inhibitor are administered sequentially or concurrently, by different or same routes of administration.

19. The method claim 14 or 15, further comprising removing the primary tumor, prior to, concurrent with or following cessation of administration of said compositions.

20. The method of claim 14 or 15, further comprising treating the individual with radiation.