Combination therapy with Axl inhibitor and immune checkpoint modulator or oncolytic virus

Abstract

An Axl inhibitor and one or more immune checkpoint (activity) modulators and/or one or more oncolytic viruses, for use in the prevention, treatment or management of cancer, wherein the Axl inhibitor and the one or more immune checkpoint (activity) modulators and/or the one or more oncolytic viruses are administered concurrently, separately or sequentially; compositions containing such components in combination; and methods of treating cancer in a patient by administering such components in combination.

Claims

1. A method of controlling cancer in a patient comprising administering to the patient in need thereof a therapeutically effective amount of an Axl inhibitor in concurrent, separate or sequential combination with a therapeutically effective amount of at least two immune checkpoint inhibitors wherein the Axl inhibitor is a compound of formula (I): ##STR00011## wherein: R.sup.1, R.sup.4 and R.sup.5 are each independently selected from the group consisting of hydrogen, alkyl, alkenyl, aryl, aralkyl, —C(O)R.sup.8, —C(O)N(R.sup.6)R.sup.7, and —C(═NR.sup.6)N(R.sup.6)R.sup.7; R.sup.2 and R.sup.3 are each independently a polycyclic heteroaryl containing more than 14 ring atoms optionally substituted by one or more substituents selected from the group consisting of oxo, thioxo, cyano, nitro, halo, haloalkyl, alkyl, optionally substituted cycloalkyl, optionally substituted cycloalkylalkyl, optionally substituted aryl, optionally substituted aralkyl, optionally substituted heteroaryl, optionally substituted heterocyclyl, —R.sup.9—OR.sup.8, —R.sup.9—O—R.sup.10—OR.sup.8, —R.sup.9—O—R.sup.10—O—R.sup.10—OR.sup.8, —R.sup.9—O—R.sup.10—CN, —R.sup.9—O—R.sup.10—C(O)OR.sup.8, —R.sup.9—O—R.sup.10—C(O)N(R.sup.6)R.sup.7, —R.sup.9—O—R.sup.10—S(O).sub.pR.sup.8 (where p is 0, 1 or 2), —R.sup.9—O—R.sup.10—N(R.sup.6)R.sup.7, —R.sup.9—O—R.sup.10—C(NR.sup.11)N(R.sup.11)H, —R.sup.9—OC(O)—R.sup.8, —R.sup.9—N(R.sup.6)R.sup.7, —R.sup.9—C(O)R.sup.8, —R.sup.9—C(O)OR.sup.8, —R.sup.9—C(O)N(R.sup.6)R.sup.7, —R.sup.9—N(R.sup.6)C(O)OR.sup.8, —R.sup.9—N(R.sup.6)C(O)R.sup.8, —R.sup.9—N(R.sup.6) S(O).sub.tR.sup.8 (where t is 1 or 2), —R.sup.9—S(O).sub.tOR.sup.8 (where t is 1 or 2), —R.sup.9—S(O).sub.pR.sup.8 (where p is 0, 1 or 2), and —R.sup.9—S(O).sub.tN(R.sup.6)R.sup.7 (where t is 1 or 2); or R.sup.2 is a polycyclic heteroaryl containing more than 14 ring atoms as described above and R.sup.3 is selected from the group consisting of aryl and heteroaryl, where the aryl and the heteroaryl are each independently optionally substituted by one or more substituents selected from the group consisting of alkyl, alkenyl, alkynyl, halo, haloalkyl, haloalkenyl, haloalkynyl, oxo, thioxo, cyano, nitro, optionally substituted aryl, optionally substituted aralkyl, optionally substituted aralkenyl, optionally substituted aralkynyl, optionally substituted cycloalkyl, optionally substituted cycloalkylalkyl, optionally substituted cycloalkylalkenyl, optionally substituted cycloalkylalkynyl, optionally substituted heterocyclyl, optionally substituted heterocyclylalkyl, optionally substituted heterocyclylalkenyl, optionally substituted heterocyclylalkynyl, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, optionally substituted heteroarylalkenyl, optionally substituted heteroarylalkynyl, —R.sup.13—OR.sup.12, —R.sup.13—OC(O)—R.sup.12, —R.sup.13—O—R.sup.14—N(R.sup.12).sub.2, —R.sup.13—N(R.sup.12)—R.sup.14—N(R.sup.12).sub.2, —R.sup.13—N(R.sup.12)—R.sup.14—N(R.sup.12).sub.2, —R.sup.13—N(R.sup.12).sub.2, —R.sup.13—C(O)OR.sup.12, —R.sup.13—C(O)N(R.sup.12).sub.2, —R.sup.13—C(O)N(R.sup.12)—R.sup.14—N(R.sup.12)R.sup.13, —R.sup.13—C(O)N(R.sup.12)—R.sup.14—OR.sup.12, —R.sup.13—N(R.sup.12)C(O)OR.sup.12, —R.sup.13—N(R.sup.12)C(O)R.sup.12, —R.sup.13—N(R.sup.12)S(O).sub.tR.sup.12 (where t is 1 or 2), —R.sup.13—S(O).sub.tOR.sup.12 (where t is 1 or 2), —R.sup.13—S(O).sub.pR.sup.12 (where p is 0, 1 or 2), and —R.sup.13—S(O).sub.tN(R.sup.12).sub.2 (where t is 1 or 2); or R.sup.3 is a polycyclic heteroaryl containing more than 14 ring atoms as described above, and R.sup.2 is selected from the group consisting of aryl and heteroaryl, where the aryl and the heteroaryl are each independently optionally substituted by one or more substituents selected from the group consisting of alkyl, alkenyl, alkynyl, halo, haloalkyl, haloalkenyl, haloalkynyl, oxo, thioxo, cyano, nitro, optionally substituted aryl, optionally substituted aralkyl, optionally substituted aralkenyl, optionally substituted aralkynyl, optionally substituted cycloalkyl, optionally substituted cycloalkylalkyl, optionally substituted cycloalkylalkenyl, optionally substituted cycloalkylalkynyl, optionally substituted heterocyclyl, optionally substituted heterocyclylalkyl, optionally substituted heterocyclylalkenyl, optionally substituted heterocyclylalkynyl, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, optionally substituted heteroarylalkenyl, optionally substituted heteroarylalkynyl, —R.sup.13—OR.sup.12, —R.sup.13—OC(O)—R.sup.12, —R.sup.13—O—R.sup.14—N(R.sup.12).sub.2, —R.sup.13—N(R.sup.12)—R.sup.14—N(R.sup.12).sub.2, —R.sup.13—N(R.sup.12)—R.sup.14—N(R.sup.12).sub.2, —R.sup.13—N(R.sup.12).sub.2, —R.sup.13—C(O)OR.sup.12, —R.sup.13—C(O)N(R.sup.12).sub.2, —R.sup.13—C(O)N(R.sup.12)—R.sup.14—N(R.sup.12)R.sup.13, —R.sup.13—C(O)N(R.sup.12)—R.sup.14—OR.sup.12, —R.sup.13—N(R.sup.12)C(O)OR.sup.12, —R.sup.13—N(R.sup.12)C(O)R.sup.12, —R.sup.13—N(R.sup.12)S(O).sub.tR.sup.12 (where t is 1 or 2), —R.sup.13—S(O).sub.tOR.sup.12 (where t is 1 or 2), —R.sup.13—S(O).sub.pR.sup.12 (where p is 0, 1 or 2), and —R.sup.13—S(O).sub.tN(R.sup.12).sub.2 (where t is 1 or 2); each R.sup.6 and R.sup.7 is independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, haloalkyl, haloalkenyl, haloalkynyl, hydroxyalkyl, optionally substituted aryl, optionally substituted aralkyl, optionally substituted aralkenyl, optionally substituted aralkynyl, optionally substituted cycloalkyl, optionally substituted cycloalkylalkyl, optionally substituted cycloalkylalkenyl, optionally substituted cycloalkylalkynyl, optionally substituted heterocyclyl, optionally substituted heterocyclylalkyl, optionally substituted heterocyclylalkenyl, optionally substituted heterocyclylalkynyl, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, optionally substituted heteroarylalkenyl, optionally substituted heteroarylalkynyl, —R.sup.10—OR.sup.8, —R.sup.10—CN, —R.sup.10—NO.sub.2, —R.sup.10—N(R.sup.8).sub.2, —R.sup.10—C(O)OR.sup.8 and —R.sup.10—C(O)N(R.sup.8).sub.2, or any R.sup.6 and R.sup.7, together with the common nitrogen to which they are both attached, form an optionally substituted N-heteroaryl or an optionally substituted N-heterocyclyl; each R.sup.8 is independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, haloalkyl, haloalkenyl, haloalkynyl, optionally substituted aryl, optionally substituted aralkyl, optionally substituted aralkenyl, optionally substituted aralkynyl, optionally substituted cycloalkyl, optionally substituted cycloalkylalkyl, optionally substituted cycloalkylalkenyl, optionally substituted cycloalkylalkynyl, optionally substituted heterocyclyl, optionally substituted heterocyclylalkyl, optionally substituted heterocyclylalkenyl, optionally substituted heterocyclylalkynyl, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, optionally substituted heteroarylalkenyl, and optionally substituted heteroarylalkynyl; each R.sup.9 is independently selected from the group consisting of a direct bond, an optionally substituted straight or branched alkylene chain, an optionally substituted straight or branched alkenylene chain and an optionally substituted straight or branched alkynylene chain; each R.sup.10 is independently selected from the group consisting of an optionally substituted straight or branched alkylene chain, an optionally substituted straight or branched alkenylene chain and an optionally substituted straight or branched alkynylene chain; each R.sup.11 is independently selected from the group consisting of hydrogen, alkyl, cyano, nitro and —OR.sup.8; each R.sup.12 is independently selected from the group consisting of hydrogen, alkyl, alkenyl, haloalkyl, optionally substituted cycloalkyl, optionally substituted cycloalkylalkyl, optionally substituted aryl, optionally substituted aralkyl, optionally substituted heterocyclyl, optionally substituted heterocyclylalkyl, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, —R.sup.10—OR.sup.8, —R.sup.10—CN, —R.sup.10—NO.sub.2, —R.sup.10—N(R.sup.8).sub.2, —R.sup.10—C(O)OR.sup.8 and —R.sup.10—C(O)N(R.sup.8).sub.2, or two R.sup.12's, together with the common nitrogen to which they are both attached, form an optionally substituted N-heterocyclyl or an optionally substituted N-heteroaryl; each R.sup.13 is independently selected from the group consisting of a direct bond, an optionally substituted straight or branched alkylene chain and an optionally substituted straight or branched alkenylene chain; and each R.sup.14 is independently selected from the group consisting of an optionally substituted straight or branched alkylene chain and an optionally substituted straight or branched alkenylene chain; as an isolated stereoisomer or mixture thereof or as a tautomer or mixture thereof, or a pharmaceutically acceptable salt or N-oxide thereof, and wherein one of the at least two immune checkpoint inhibitors is an anti-CTLA-4 antibody or an anti-PD-1 antibody; and wherein the cancer is an Axl-expressing cancer.

2. The method according to claim 1, wherein the compound of formula (I) is selected from the group consisting of: 1-(6,7-dihydro-5H-benzo[6,7]cyclohepta[1,2-c]pyridazin-3-yl)-N.sup.3-(7-(pyrrolidin-1-yl)-6,7,8,9-tetrahydro-5H-benzo[7]annulene-2-yl)-1H-1,2,4-triazole-3,5-diamine; 1-(6,7-dihydro-5H-benzo[6,7]cyclohepta[1,2-c]pyridazin-3-yl)-N.sup.3-((7-(S)-pyrrolidin-1-yl)-6,7,8,9-tetrahydro-5H-benzo[7]annulene-2-yl)-1H-1,2,4-triazole-3,5-diamine; 1-(6,7-dihydro-5H-benzo[6,7]cyclohepta[1,2-c]pyridazin-3-yl)-N.sup.3-((7-(R)-pyrrolidin-1-yl)-6,7,8,9-tetrahydro-5H-benzo[7]annulene-2-yl)-1H-1,2,4-triazole-3,5-diamine; 1-(6,7-dihydro-5H-pyrido[2′,3′:6,7]cyclohepta[1,2-c]pyridazin-3-yl)-N.sup.3-(3-fluoro-4-(4-(pyrrolidin-1-yl)piperidin-1-yl)phenyl)-1H-1,2,4-triazole-3,5-diamine; 1-(6,7-dihydro-5H-benzo[6,7]cyclohepta[1,2-c]pyridazin-3-yl)-N.sup.5-(7-(pyrrolidin-1-yl)-6,7,8,9-tetrahydro-5H-benzo[7]annulene-1-yl)-1H-1,2,4-triazole-3,5-diamine; 1-(6,7-dihydro-5H-benzo[6,7]cyclohepta[1,2-c]pyridazin-3-yl)-N.sup.5-(7-(S)-pyrrolidin-1-yl-6,7,8,9-tetrahydro-5H-benzo[7]annulene-2-yl)-1H-1,2,4-triazole-3,5-diamine; 1-(6,7-dihydro-5H-benzo[6,7]cyclohepta[1,2-c]pyridazin-3-yl)-N.sup.3-((7S)-7-(t-butoxycarbonylamino)-6,7,8,9-tetrahydro-5H-benzo[7]annulene-2-yl)-1H-1,2,4-triazole-3,5-diamine; 1-(6,7-dihydro-5H-benzo[6,7]cyclohepta[1,2-c]pyridazin-3-yl)-N.sup.3-(7-(acetamido)-6,7,8,9-tetrahydro-5H-benzo[7]annulene-2-yl)-1H-1,2,4-triazole-3,5-diamine; 1-(6,7-dihydro-5H-benzo[6,7]cyclohepta[1,2-c]pyridazin-3-yl)-N.sup.3-(7-((2R)-2-(methoxycarbonyl)pyrrolidin-1-yl)-6,7,8,9-tetrahydro-5H-benzo[7]annulene-2-yl)-1H-1,2,4-triazole-3,5-diamine; 1-(6,7-dihydro-5H-benzo[6,7]cyclohepta[1,2-c]pyridazin-3-yl)-N.sup.3-(7-(4,4-difluoropiperidin-1-yl)-6,7,8,9-tetrahydro-5H-benzo[7]annulene-2-yl)-1H-1,2,4-triazole-3,5-diamine; 1-(6,7-dihydro-5H-benzo[6,7]cyclohepta[1,2-c]pyridazin-3-yl)-N.sup.3-(7-((methoxycarbonylmethyl)(methyl)amino)-6,7,8,9-tetrahydro-5H-benzo[7]annulene-2-yl)-1H-1,2,4-triazole-3,5-diamine; 1-(6,7-dihydro-5H-benzo[6,7]cyclohepta[1,2-c]pyridazin-3-yl)-N.sup.3-(7-((2R)-2-(carboxy)pyrrolidin-1-yl)-6,7,8,9-tetrahydro-5H-benzo[7]annulene-2-yl)-1H-1,2,4-triazole-3,5-diamine; 1-(6,7-dihydro-5H-benzo[6,7]cyclohepta[1,2-c]pyridazin-3-yl)-N.sup.3-(7-(4-(ethoxycarbonyl)piperidin-1-yl)-6,7,8,9-tetrahydro-5H-benzo[7]annulene-2-yl)-1H-1,2,4-triazole-3,5-diamine; 1-(6,7-dihydro-5H-benzo[6,7]cyclohepta[1,2-c]pyridazin-3-yl)-N.sup.3-(7-(4-(carboxy)piperidin-1-yl)-6,7,8,9-tetrahydro-5H-benzo[7]annulene-2-yl)-1H-1,2,4-triazole-3,5-diamine; 1-(6,7-dihydro-5H-benzo[6,7]cyclohepta[1,2-c]pyridazin-3-yl)-N.sup.3-(7-((carb oxymethyl)(methyl)amino)-6,7,8,9-tetrahydro-5H-benzo[7]annulene-2-yl)-1H-1,2,4-triazole-3,5-diamine; 1-(6,7-dihydro-5H-benzo[6,7]cyclohepta[1,2-c]pyridazin-3-yl)-N.sup.3-(7-(4-(ethoxycarbonylmethyl)piperazin-1-yl)-6,7,8,9-tetrahydro-5H-benzo[7]annulene-2-yl)-1H-1,2,4-triazole-3,5-diamine; 1-(6,7-dihydro-5H-benzo[6,7]cyclohepta[1,2-c]pyridazin-3-yl)-N.sup.3-(7-(4-(carboxymethyl)piperazin-1-yl)-6,7,8,9-tetrahydro-5H-benzo[7]annulene-2-yl)-1H-1,2,4-triazole-3,5-diamine; 1-(6,7-dihydro-5H-benzo[6,7]cyclohepta[1,2-c]pyridazin-3-yl)-N.sup.3-(7-(pyrrolidin-1-yl)-6,7,8,9-tetrahydro-5H-benzo[7]annulene-1-yl)-1H-1,2,4-triazole-3,5-diamine; 1-(6,7-dihydro-5H-benzo[6,7]cyclohepta[1,2-c]pyridazin-3-yl)-N.sup.3-((7S)-7-amino-6,7,8,9-tetrahydro-5H-benzo[7]annulene-2-yl)-1H-1,2,4-triazole-3,5-diamine; 1-(6,7-dihydro-5H-benzo[6,7]cyclohepta[1,2-c]pyridazin-3-yl)-N.sup.3-((7s)-7-(di(cyclopropylmethyl)amino)-6,7,8,9-tetrahydro-5H-benzo[7]annulene-2-yl)-1H-1,2,4-triazole-3,5-diamine; 1-(6,7-dihydro-5H-benzo[6,7]cyclohepta[1,2-c]pyridazin-3-yl)-N.sup.3-((7S)-7-((2-methylpropyl)amino)-6,7,8,9-tetrahydro-5H-benzo[7]annulene-2-yl)-1H-1,2,4-triazole-3,5-diamine; 1-(6,7-dihydro-5H-benzo[6,7]cyclohepta[1,2-c]pyridazin-3-yl)-N.sup.3-((7S)-7-((propyl)amino)-6,7,8,9-tetrahydro-5H-benzo[7]annulene-2-yl)-1H-1,2,4-triazole-3,5-diamine; 1-(6,7-dihydro-5H-benzo[6,7]cyclohepta[1,2-c]pyridazin-3-yl)-N.sup.3-((7S)-7-(dipropylamino)-6,7,8,9-tetrahydro-5H-benzo[7]annulene-2-yl)-1H-1,2,4-triazole-3,5-diamine; 1-(6,7-dihydro-5H-benzo[6,7]cyclohepta[1,2-c]pyridazin-3-yl)-N.sup.3-((7S)-7-(diethylamino)-6,7,8,9-tetrahydro-5H-benzo[7]annulene-2-yl)-1H-1,2,4-triazole-3,5-diamine; 1-(6,7-dihydro-5H-benzo[6,7]cyclohepta[1,2-c]pyridazin-3-yl)-N.sup.3-((7S)-7-(cyclohexylamino)-6,7,8,9-tetrahydro-5H-benzo[7]annulene-2-yl)-1H-1,2,4-triazole-3,5-diamine; 1-(6,7-dihydro-5H-benzo[6,7]cyclohepta[1,2-c]pyridazin-3-yl)-N.sup.3-((7S)-7-(cyclopentylamino)-6,7,8,9-tetrahydro-5H-benzo[7]annulene-2-yl)-1H-1,2,4-triazole-3,5-diamine; 1-(6,7-dihydro-5H-benzo[6,7]cyclohepta[1,2-c]pyridazin-3-yl)-N.sup.3-((7S)-7-(1-cyclopentylethyl)amino)-6,7,8,9-tetrahydro-5H-benzo[7]annulene-2-yl)-1H-1,2,4-triazole-3,5-diamine; 1-(6,7-dihydro-5H-benzo[6,7]cyclohepta[1,2-c]pyridazin-3-yl)-N.sup.3-((7S)-7-(2-propylamino)-6,7,8,9-tetrahydro-5H-benzo[7]annulene-2-yl)-1H-1,2,4-triazole-3,5-diamine; 1-(6,7-dihydro-5H-benzo[6,7]cyclohepta[1,2-c]pyridazin-3-yl)-N.sup.3-((7S)-7-(3,3-dimethylbut-2-yl)amino)-6,7,8,9-tetrahydro-5H-benzo[7]annulene-2-yl)-1H-1,2,4-triazole-3,5-diamine; 1-(6,7-dihydro-5H-benzo[6,7]cyclohepta[1,2-c]pyridazin-3-yl)-N.sup.3-((7S)-7-((cyclohexylmethyl)amino)-6,7,8,9-tetrahydro-5H-benzo[7]annulene-2-yl)-1H-1,2,4-triazole-3,5-diamine; 1-(6,7-dihydro-5H-benzo[6,7]cyclohepta[1,2-c]pyridazin-3-yl)-N.sup.3-((7S)-7-(di(cyclohexylmethyl)amino)-6,7,8,9-tetrahydro-5H-benzo[7]annulene-2-yl)-1H-1,2,4-triazole-3,5-diamine; 1-(6,7-dihydro-5H-benzo[6,7]cyclohepta[1,2-c]pyridazin-3-yl)-N.sup.3-((7S)-7-(5-chlorothien-2-yl)methyl)amino-6,7,8,9-tetrahydro-5H-benzo[7]annulene-2-yl)-1H-1,2,4-triazole-3,5-diamine; 1-(6,7-dihydro-5H-benzo[6,7]cyclohepta[1,2-c]pyridazin-3-yl)-N.sup.3-((7S)-7-(2-carboxyphenyl)methyl)amino-6,7,8,9-tetrahydro-5H-benzo[7]annulene-2-yl)-1H-1,2,4-triazole-3,5-diamine; 1-(6,7-dihydro-5H-benzo[6,7]cyclohepta[1,2-c]pyridazin-3-yl)-N.sup.3-((7S)-7-(3-bromophenyl)methyl)amino-6,7,8,9-tetrahydro-5H-benzo[7]annulene-2-yl)-1H-1,2,4-triazole-3,5-diamine; 1-(6,7-dihydro-5H-benzo[6,7]cyclohepta[1,2-c]pyridazin-3-yl)-N.sup.3-((7S)-7-(dimethylamino)-6,7,8,9-tetrahydro-5H-benzo[7]annulene-2-yl)-1H-1,2,4-triazole-3,5-diamine; 1-(6,7-dihydro-5H-benzo[6,7]cyclohepta[1,2-c]pyridazin-3-yl)-N.sup.3-((7S)-7-(cyclobutylamino)-6,7,8,9-tetrahydro-5H-benzo[7]annulene-2-yl)-1H-1,2,4-triazole-3,5-diamine; 1-(6,7-dihydro-5H-benzo[6,7]cyclohepta[1,2-c]pyridazin-3-yl)-N.sup.3-((7S)-7-(3-pentylamino)-6,7,8,9-tetrahydro-5H-benzo[7]annulene-2-yl)-1H-1,2,4-triazole-3,5-diamine; 1-(6,7-dihydro-5H-benzo[6,7]cyclohepta[1,2-c]pyridazin-3-yl)-N.sup.3-((7S)-7-(2,2-dimethylpropyl)amino)-6,7,8,9-tetrahydro-5H-benzo[7]annulene-2-yl)-1H-1,2,4-triazole-3,5-diamine; 1-(6,7-dihydro-5H-benzo[6,7]cyclohepta[1,2-c]pyridazin-3-yl)-N.sup.3-((7S)-7-(di(cyclopentylmethyl)amino)-6,7,8,9-tetrahydro-5H-benzo[7]annulene-2-yl)-1H-1,2,4-triazole-3,5-diamine; 1-(6,7-dihydro-5H-benzo[6,7]cyclohepta[1,2-c]pyridazin-3-yl)-N.sup.3-((7S)-7-((cyclopentylmethyl)amino)-6,7,8,9-tetrahydro-5H-benzo[7]annulene-2-yl)-1H-1,2,4-triazole-3,5-diamine; 1-(6,7-dihydro-5H-benzo[6,7]cyclohepta[1,2-c]pyridazin-3-yl)-N.sup.3-((7S)-7-(di(bicyclo[2.2.1]hept-2-en-5-ylmethyl)amino)-6,7,8,9-tetrahydro-5H-benzo[7]annulene-2-yl)-1H-1,2,4-triazole-3,5-diamine; 1-(6,7-dihydro-5H-benzo[6,7]cyclohepta[1,2-c]pyridazin-3-yl)-N.sup.3-((7S)-7-((bicyclo[2.2.1]hept-2-en-5-ylmethyl)amino)-6,7,8,9-tetrahydro-5H-benzo[7]annulene-2-yl)-1H-1,2,4-triazole-3,5-diamine; 1-(6,7-dihydro-5H-benzo[6,7]cyclohepta[1,2-c]pyridazin-3-yl)-N.sup.3-((7S)-7-(3-methylbutylamino)-6,7,8,9-tetrahydro-5H-benzo[7]annulene-2-yl)-1H-1,2,4-triazole-3,5-diamine; 1-(6,7-dihydro-5H-benzo[6,7]cyclohepta[1,2-c]pyridazin-3-yl)-N.sup.3-((7S)-7-(di(3-methylbutyl)amino)-6,7,8,9-tetrahydro-5H-benzo[7]annulene-2-yl)-1H-1,2,4-triazole-3,5-diamine; 1-(6,7-dihydro-5H-benzo[6,7]cyclohepta[1,2-c]pyridazin-3-yl)-N.sup.3-((7S)-7-(2-ethylbutylamino)-6,7,8,9-tetrahydro-5H-benzo[7]annulene-2-yl)-1H-1,2,4-triazole-3,5-diamine; 1-(6,7-dihydro-5H-benzo[6,7]cyclohepta[1,2-c]pyridazin-3-yl)-N.sup.3-((7S)-7-(but-2-enylamino)-6,7,8,9-tetrahydro-5H-benzo[7]annulene-2-yl)-1H-1,2,4-triazole-3,5-diamine; 1-(6,7-dihydro-5H-benzo[6,7]cyclohepta[1,2-c]pyridazin-3-yl)-N.sup.3-((7S)-7-(butyl(but-2-enyl)amino)-6,7,8,9-tetrahydro-5H-benzo[7]annulene-2-yl)-1H-1,2,4-triazole-3,5-diamine; 1-(6,7-dihydro-5H-pyrido[2′,3′:6,7]cyclohepta[1,2-c]pyridazin-3-yl)-N.sup.5-((7S)-7-(t-butoxycarbonylamino)-6,7,8,9-tetrahydro-5H-benzo[7]annulene-2-yl)-1H-1,2,4-triazole-3,5-diamine; 1-(6,7-dihydro-5H-pyrido[2′,3′:6,7]cyclohepta[1,2-c]pyridazin-3-yl)-N.sup.3-((7S)-7-amino-6,7,8,9-tetrahydro-5H-benzo[7]annulene-2-yl)-1H-1,2,4-triazole-3,5-diamine; 1-(6,7-dihydro-5H-pyrido[2′,3′:6,7]cyclohepta[1,2-c]pyridazin-3-yl)-N.sup.3-((7 S)-7-(dimethylamino)-6,7,8,9-tetrahydro-5H-benzo[7]annulene-2-yl)-1H-1,2,4-triazole-3,5-diamine; 1-(6,7-dihydro-5H-pyrido[2′,3′:6,7]cyclohepta[1,2-c]pyridazin-3-yl)-N.sup.3-((7S)-7-(diethylamino)-6,7,8,9-tetrahydro-5H-benzo[7]annulene-2-yl)-1H-1,2,4-triazole-3,5-diamine; 1-(6,7-dihydro-5H-pyrido[2′,3′:6,7]cyclohepta[1,2-c]pyridazin-3-yl)-N.sup.3-((7S)-7-(dipropylamino)-6,7,8,9-tetrahydro-5H-benzo[7]annulene-2-yl)-1H-1,2,4-triazole-3,5-diamine; 1-(6,7-dihydro-5H-pyrido[2′,3′:6,7]cyclohepta[1,2-c]pyridazin-3-yl)-N.sup.3-((7S)-7-(di(cyclopropylmethyl)amino)-6,7,8,9-tetrahydro-5H-benzo[7]annulene-2-yl)-1H-1,2,4-triazole-3,5-diamine; 1-(6,7-dihydro-5H-pyrido[2′,3′:6,7]cyclohepta[1,2-c]pyridazin-3-yl)-N.sup.3-((7S)-7-(di(3-methylbutyl)amino)-6,7,8,9-tetrahydro-5H-benzo[7]annulene-2-yl)-1H-1,2,4-triazole-3,5-diamine; 1-(6,7-dihydro-5H-pyrido[2′,3′:6,7]cyclohepta[1,2-c]pyridazin-3-yl)-N.sup.3-((7S)-7-(cyclobutylamino)-6,7,8,9-tetrahydro-5H-benzo[7]annulene-2-yl)-1H-1,2,4-triazole-3,5-diamine; 1-(6,7-dihydro-5H-pyrido[2′,3′:6,7]cyclohepta[1,2-c]pyridazin-3-yl)-N.sup.3-((7S)-7-(cyclohexylamino)-6,7,8,9-tetrahydro-5H-benzo[7]annulene-2-yl)-1H-1,2,4-triazole-3,5-diamine; 1-(6,7-dihydro-5H-pyrido[2′,3′:6,7]cyclohepta[1,2-c]pyridazin-3-yl)-N.sup.3-((7S)-7-((methylethyl)amino)-6,7,8,9-tetrahydro-5H-benzo[7]annulene-2-yl)-1H-1,2,4-triazole-3,5-diamine; 1-(6,7-dihydro-5H-pyrido[2′,3′:6,7]cyclohepta[1,2-c]pyridazin-3-yl)-N.sup.3-((7S)-7-(cyclopentylamino)-6,7,8,9-tetrahydro-5H-benzo[7]annulene-2-yl)-1H-1,2,4-triazole-3,5-diamine; and 1-(6,7-dihydro-5H-pyrido[2′,3′:6,7]cyclohepta[1,2-c]pyridazin-3-yl)-N.sup.3-((7S)-7-(2-butylamino)-6,7,8,9-tetrahydro-5H-benzo[7]annulene-2-yl)-1H-1,2,4-triazole-3,5-diamine, or pharmaceutically acceptable salts thereof.

3. The method according to claim 1, wherein the Axl inhibitor is 1-(6,7-dihydro-5H-benzo[6,7]cyclohepta[1,2-c]pyridazin-3-yl)-N.sup.3-((7-(S)-pyrrolidin-1-yl)-6,7,8,9-tetrahydro-5H-benzo[7]annulene-2-yl)-1H-1,2,4-triazole-3,5-diamine, or a pharmaceutically acceptable salt thereof.

4. The method according to claim 1, wherein the at least two immune checkpoint inhibitors are selected from the group consisting of anti-CTLA-4 antibodies, anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-4-1BB antibodies, anti-OX-40 antibodies, anti-GITR antibodies, anti-CD27 antibodies, anti-CD40 antibodies, and anti-LAG3 antibodies.

5. The method according to claim 4, wherein the at least two immune checkpoint inhibitors are selected from the group consisting of anti-CTLA-4 antibodies and anti-PD-1 antibodies.

6. The method according to claim 5, wherein the at least two immune checkpoint inhibitors are selected from the group consisting of ipilimumab, tremelimumab, pembrolizumab, and nivolumab.

7. The method according to claim 1, wherein the at least two immune checkpoint inhibitors comprise an anti-CTLA-4 antibody and an anti-PD-1 antibody.

8. The method according to claim 1, wherein the Axl inhibitor and the at least two immune checkpoint inhibitors are administered concurrently.

9. The method according to claim 1, wherein the Axl inhibitor and the at least two immune checkpoint inhibitors are administered separately and/or sequentially.

10. The method according to claim 1, wherein the cancer is breast cancer.

11. A method of controlling cancer in a patient to whom an immune checkpoint inhibitor has been or will be administered, the method comprising administering to a patient in need thereof a therapeutically effective amount of an Axl inhibitor; wherein the Axl inhibitor is a compound of formula (I): ##STR00012## wherein: R.sup.1, R.sup.4 and R.sup.5 are each independently selected from the group consisting of hydrogen, alkyl, alkenyl, aryl, aralkyl, —C(O)R.sup.8, —C(O)N(R.sup.6)R.sup.7, and —C(═NR.sup.6)N(R.sup.6)R.sup.7; R.sup.2 and R.sup.3 are each independently a polycyclic heteroaryl containing more than 14 ring atoms optionally substituted by one or more substituents selected from the group consisting of oxo, thioxo, cyano, nitro, halo, haloalkyl, alkyl, optionally substituted cycloalkyl, optionally substituted cycloalkylalkyl, optionally substituted aryl, optionally substituted aralkyl, optionally substituted heteroaryl, optionally substituted heterocyclyl, —R.sup.9—OR.sup.8, —R.sup.9—O—R.sup.10—OR.sup.8, —R.sup.9—O—R.sup.10—O—R.sup.10—OR.sup.8, —R.sup.9—O—R.sup.10—CN, —R.sup.9—O—R.sup.10—C(O)OR.sup.8, —R.sup.9—O—R.sup.10—C(O)N(R.sup.6)R.sup.7, —R.sup.9—O—R.sup.10—S(O).sub.pR.sup.8 (where p is 0, 1 or 2), —R.sup.9—O—R.sup.10—N(R.sup.6)R.sup.7, —R.sup.9—O—R.sup.10—C(NR.sup.11)N(R.sup.11)H, —R.sup.9—OC(O)—R.sup.8, —R.sup.9—N(R.sup.6)R.sup.7, —R.sup.9—C(O)R.sup.8, —R.sup.9—C(O)OR.sup.8, —R.sup.9—C(O)N(R.sup.6)R.sup.7, —R.sup.9—N(R.sup.6)C(O)OR.sup.8, —R.sup.9—N(R.sup.6)C(O)R.sup.8, —R.sup.9—N(R.sup.6)S(O).sub.tR.sup.8 (where t is 1 or 2), —R.sup.9—S(O).sub.tOR.sup.8 (where t is 1 or 2), —R.sup.9—S(O).sub.pR.sup.8 (where p is 0, 1 or 2), and —R.sup.9—S(O).sub.tN(R.sup.6)R.sup.7 (where t is 1 or 2); or R.sup.2 is a polycyclic heteroaryl containing more than 14 ring atoms as described above and R.sup.3 is selected from the group consisting of aryl and heteroaryl, where the aryl and the heteroaryl are each independently optionally substituted by one or more substituents selected from the group consisting of alkyl, alkenyl, alkynyl, halo, haloalkyl, haloalkenyl, haloalkynyl, oxo, thioxo, cyano, nitro, optionally substituted aryl, optionally substituted aralkyl, optionally substituted aralkenyl, optionally substituted aralkynyl, optionally substituted cycloalkyl, optionally substituted cycloalkylalkyl, optionally substituted cycloalkylalkenyl, optionally substituted cycloalkylalkynyl, optionally substituted heterocyclyl, optionally substituted heterocyclylalkyl, optionally substituted heterocyclylalkenyl, optionally substituted heterocyclylalkynyl, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, optionally substituted heteroarylalkenyl, optionally substituted heteroarylalkynyl, —R.sup.13—OR.sup.12, —R.sup.13—OC(O)—R.sup.12, —R.sup.13—O—R.sup.14—N(R.sup.12).sub.2, —R.sup.13—N(R.sup.12)—R.sup.14—N(R.sup.12).sub.2, —R.sup.13—N(R.sup.12)—R.sup.14—N(R.sup.12).sub.2, —R.sup.13—N(R.sup.12).sub.2, —R.sup.13—C(O)R.sup.12, —R.sup.13—C(O)OR.sup.12, —R.sup.13—C(O)N(R.sup.12).sub.2, —R.sup.13—C(O)N(R.sup.12)—R.sup.14—N(R.sup.12)R.sup.13, —R.sup.13—C(O)N(R.sup.12)—R.sup.14—OR.sup.12, —R.sup.13—N(R.sup.12)C(O)OR.sup.12, —R.sup.13—N(R.sup.12)C(O)R.sup.12, —R.sup.13—N(R.sup.12)S(O).sub.tR.sup.12 (where t is 1 or 2), —R.sup.13—S(O).sub.tOR.sup.12 (where t is 1 or 2), —R.sup.13—S(O).sub.pR.sup.12 (where p is 0, 1 or 2), and —R.sup.13—S(O).sub.tN(R.sup.12).sub.2 (where t is 1 or 2); or R.sup.3 is a polycyclic heteroaryl containing more than 14 ring atoms as described above, and R.sup.2 is selected from the group consisting of aryl and heteroaryl, where the aryl and the heteroaryl are each independently optionally substituted by one or more substituents selected from the group consisting of alkyl, alkenyl, alkynyl, halo, haloalkyl, haloalkenyl, haloalkynyl, oxo, thioxo, cyano, nitro, optionally substituted aryl, optionally substituted aralkyl, optionally substituted aralkenyl, optionally substituted aralkynyl, optionally substituted cycloalkyl, optionally substituted cycloalkylalkyl, optionally substituted cycloalkylalkenyl, optionally substituted cycloalkylalkynyl, optionally substituted heterocyclyl, optionally substituted heterocyclylalkyl, optionally substituted heterocyclylalkenyl, optionally substituted heterocyclylalkynyl, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, optionally substituted heteroarylalkenyl, optionally substituted heteroarylalkynyl, —R.sup.13—OR.sup.12, —R.sup.13—OC(O)—R.sup.12, —R.sup.13—O—R.sup.14—N(R.sup.12).sub.2, —R.sup.13—N(R.sup.12)—R.sup.14—N(R.sup.12).sub.2, —R.sup.13—N(R.sup.12)—R.sup.14—N(R.sup.12).sub.2, —R.sup.13—N(R.sup.12).sub.2, —R.sup.13—C(O)OR.sup.12, —R.sup.13—C(O)N(R.sup.12).sub.2, —R.sup.13—C(O)N(R.sup.12)—R.sup.14—N(R.sup.12)R.sup.13, —R.sup.13—C(O)N(R.sup.12)—R.sup.14—OR.sup.12, —R.sup.13—N(R.sup.12)C(O)OR.sup.12, —R.sup.13—N(R.sup.12)C(O)R.sup.12, —R.sup.13—N(R.sup.12)S(O).sub.tR.sup.12 (where t is 1 or 2), —R.sup.13—S(O).sub.tOR.sup.12 (where t is 1 or 2), —R.sup.13—S(O).sub.pR.sup.12 (where p is 0, 1 or 2), and —R.sup.13—S(O).sub.tN(R.sup.12).sub.2 (where t is 1 or 2); each R.sup.6 and R.sup.7 is independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, haloalkyl, haloalkenyl, haloalkynyl, hydroxyalkyl, optionally substituted aryl, optionally substituted aralkyl, optionally substituted aralkenyl, optionally substituted aralkynyl, optionally substituted cycloalkyl, optionally substituted cycloalkylalkyl, optionally substituted cycloalkylalkenyl, optionally substituted cycloalkylalkynyl, optionally substituted heterocyclyl, optionally substituted heterocyclylalkyl, optionally substituted heterocyclylalkenyl, optionally substituted heterocyclylalkynyl, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, optionally substituted heteroarylalkenyl, optionally substituted heteroarylalkynyl, —R.sup.10—OR.sup.8, —R.sup.10—CN, —R.sup.10—NO.sub.2, —R.sup.10—N(R.sup.8).sub.2, —R.sup.10—C(O)OR.sup.8 and —R.sup.10—C(O)N(R.sup.8).sub.2, or any R.sup.6 and R.sup.7, together with the common nitrogen to which they are both attached, form an optionally substituted N-heteroaryl or an optionally substituted N-heterocyclyl; each R.sup.8 is independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, haloalkyl, haloalkenyl, haloalkynyl, optionally substituted aryl, optionally substituted aralkyl, optionally substituted aralkenyl, optionally substituted aralkynyl, optionally substituted cycloalkyl, optionally substituted cycloalkylalkyl, optionally substituted cycloalkylalkenyl, optionally substituted cycloalkylalkynyl, optionally substituted heterocyclyl, optionally substituted heterocyclylalkyl, optionally substituted heterocyclylalkenyl, optionally substituted heterocyclylalkynyl, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, optionally substituted heteroarylalkenyl, and optionally substituted heteroarylalkynyl; each R.sup.9 is independently selected from the group consisting of a direct bond, an optionally substituted straight or branched alkylene chain, an optionally substituted straight or branched alkenylene chain and an optionally substituted straight or branched alkynylene chain; each R.sup.10 is independently selected from the group consisting of an optionally substituted straight or branched alkylene chain, an optionally substituted straight or branched alkenylene chain and an optionally substituted straight or branched alkynylene chain; each R.sup.11 is independently selected from the group consisting of hydrogen, alkyl, cyano, nitro and —OR.sup.8; each R.sup.12 is independently selected from the group consisting of hydrogen, alkyl, alkenyl, haloalkyl, optionally substituted cycloalkyl, optionally substituted cycloalkylalkyl, optionally substituted aryl, optionally substituted aralkyl, optionally substituted heterocyclyl, optionally substituted heterocyclylalkyl, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, —R.sup.10—OR.sup.8, —R.sup.10—CN, —R.sup.10—NO.sub.2, —R.sup.10—N(R.sup.8).sub.2, —R.sup.10—C(O)OR.sup.8 and —R.sup.10—C(O)N(R.sup.8).sub.2, or two R.sup.12's, together with the common nitrogen to which they are both attached, form an optionally substituted N-heterocyclyl or an optionally substituted N-heteroaryl; each R.sup.13 is independently selected from the group consisting of a direct bond, an optionally substituted straight or branched alkylene chain and an optionally substituted straight or branched alkenylene chain; and each R.sup.14 is independently selected from the group consisting of an optionally substituted straight or branched alkylene chain and an optionally substituted straight or branched alkenylene chain; as an isolated stereoisomer or mixture thereof or as a tautomer or mixture thereof, or a pharmaceutically acceptable salt or N-oxide thereof, wherein the immune checkpoint inhibitor is an anti-CTLA-4 antibody or an anti-PD-1 antibody; and wherein cancer is an Axl-expressing cancer.

12. The method according to claim 1, further comprising administering to the patient in need thereof a therapeutically effective amount of one or more oncolytic viruses in concurrent, separate or sequential combination.

13. The method according to claim 12, wherein the one or more oncolytic viruses are selected from reovirus, Newcastle disease virus, adenovirus, herpes virus, polio virus, mumps virus, measles virus, influenza virus, vaccinia virus, rhabdovirus, vesicular stomatitis virus, and derivatives and variants thereof.

14. The method according to claim 11, wherein an oncolytic virus has also been or will also be administered.

15. The method of claim 1, wherein said controlling is delaying spread of cancer.

16. The method of claim 1, wherein said controlling is minimizing spread of cancer.

17. The method of claim 11, wherein said controlling is delaying spread of cancer.

18. The method of claim 11, wherein said controlling is minimizing spread of cancer.

Description

FIGURES

(1) FIG. 1 shows the post-immune response tumour recurrence and metastasis in the mammary adenocarcinoma 4T1-Luc/Balb/C syngeneic mouse model. The top image shows control shRNA and the bottom image shows shAXL. Detected images are shown in the adjacent Table.

(2) FIGS. 2A-B show body weight changes (BWC) in Balb/e mice carrying 4T1 orthotopic tumors treated with vehicle, 50 mg/kg BGD324 Did or 10 mg/kg CTLA-4/PD-1 (each) in combinations or alone as indicated over the course of 46 (a) or (b) 104 days. Means±SEM are plotted, n=4 (vehicle), 5 (BGB324), 9 (CTLA4/PD1+/−BGB324 and CTLA4-BGB324). The sudden drop in BWC in Group D at day 38 is due to euthanisation of mice.

(3) FIGS. 3A-C show transformed survival curves of Balb/e mice carrying 4T orthotopic tumors treated with vehicle, BGB324 or CTLA-4/PD-1 alone or in combinations as indicated for 46 (a) or 104 (b and c) days. Endpoints for survival were set to the day when the tumor reached 500 mm.sup.3. Significance by Mantel-Cox; *p<0.05; *p<0.01; ***p<0.001; ****p<0.0001; ns: not significant.

(4) FIG. 4 shows tumor volumes at day 28 after treatment initiation of all tumors presented in FIGS. 3 (a-c).

(5) FIG. 5 shows the combined transformed survival curves for two separate mouse experiments of Balb/c mice carrying 4T1 orthotopic tumors treated with vehicle, CTLA-4/PD-1 or CTLA-4/PD-1+BGB324. The studies combined in the survival curves are presented in FIG. 2 (Report 153-SR-502P1MS6.2_Ver2) and in Report 102-SR-324; data not presented individually here). Significance by Mantel-Cox.

(6) FIG. 6 shows enhanced tumor infiltration of anti-tumorigenic Cytotoxic T cells (CTLs) in Balb/c mice carrying 4T1 orthotopic tumors treated with CTLA-4/PD-1+BGB324 compared to CTLA-4/PD1-alone. Tumors were analysed at day 11 after treatment initiation as described in legends to FIG. 1.

(7) FIG. 7 shows enhanced presence of anti-tumorigenic Natural Killer cells, macrophages and Neutrophiles in spleens of Balb/c mice carrying 4T1 orthotopic tumors treated with CTLA-4/PD-1-1BGB324 compared to CTLA-4/PD-1 alone. Tumors were analysed at day 11 after treatment initiation as described in legends to FIG. 1. Significance by one-way ANOVA.

(8) FIG. 8 shows reduced presence of pro-tumorgenic Myelo Derived Suppressor Cells (mMDSCs) in spleens of Balb/c mice carrying 4T1 orthotopic tumors treated with CTLA-4/PD-1+BGB324 compared to CTLA-4/PD-1 alone. Tumors were analysed at day 11 after treatment initiation as described in legends to FIG. 1.

(9) FIG. 9 shows body weight changes (BWC) C57Bl/6 mice carrying subcutaneous Lewis Lung tumors treated with vehicle, 50 mg/kg BGB324 Bid or 10 mg/kg BGB324 or 10 mg/kg PD-1/PD-L1 (each) alone or in combinations as indicated over the course of 21 days. Means±SEM are plotted, n=10 for all groups.

(10) FIG. 10 shows transformed survival curves of C57Bl/6 mice carrying subcutaneous Lewis Lung tumors treated with vehicle, BGB324 Bid or PD-1/PD-L alone or in combinations as indicated. Endpoints for survival were set to the day when the tumor reached 500 mm.sup.3. Significance by Mantel-Cox test.

(11) FIG. 11 shows average tumor volumes for C57Bl/6 mice carrying subcutaneous Lewis Lung tumors treated with vehicle, BGB324 or PD-1/PD-L1 alone or in combinations as indicated. Means±SEM are plotted, n=10 for all groups. Significance by Two-way ANOVA; p<0.0001.

(12) FIG. 12 shows tumor volumes at day 21 after treatment initiation for all mice presented in FIG. 11. Significance by Mann Whitney test; **p<0.01.

(13) FIG. 13 shows enhanced tumor infiltration of anti-tumorigenic Cytotoxic T cells (CTLs) and Natural Killer cells (NK) in C57Bl/6 mice carrying subcutaneous Lewis Lung tumors treated with PD-1/PD-L1+BGB324 compared to PD-1/PD-L1 alone Tumors were analysed at day 21 after treatment initiation as described in legends to FIG. 9. Significance by one-way ANOVA. **p<0.01; ***p<0.01

(14) FIG. 14 shows reduced presence of pro-tumorgenic Myelo Derived Suppressor Cells (mMDSCs) in C57Bl/6 mice carrying subcutaneous Lewis Lung tumors treated with PD-1/PD-L1+BGB324 compared to PD-1/PD-L1 alone. Tumors were analysed at day 21 after treatment initiation as described in legends to FIG. 9.

(15) FIG. 15 shows a typical survival curve for mice treated with oncolytic virus, oncolytic virus/immune checkpoint (activity) modulator, and oncolytic virus/immune checkpoint (activity) modulator/anti-Axl.

EXAMPLES

(16) Compounds of formula (I) utilised in the combination therapies of the invention can be made using organic synthesis techniques known to those skilled in the art, as well as by the methods described in PCT Published Patent Application No. WO 2008/083367. Specific examples of the compounds of formula (I) can be found in this publication.

(17) Alternatively, certain compounds of formula (I), as defined above, can be made by the methods disclosed in PCT Published Patent Application No. WO 2010/083465.

Biological Examples

(18) The following biological examples are provided by way of illustration, not limitation. In the following biological examples, 1-(6,7-dihydro-5H-benzo[6,7]cyclohepta[1,2-c]pyridazin-3-yl)-N.sup.3-((7-(S)-pyrrolidin-1-yl)-6,7,8,9-tetrahydro-5H-benzo[7]annulene-2-yl)-1H-1,2,4-triazole-3,5-diamine, which is a compound of formula (I), as defined above, and which is designated in the following examples and the Figures as “BGB324”, “Compound A” or “Cpd A” or “Cmpd A”, was assayed for its ability to prevent, treat or manage cancer in combination with immune checkpoint (activity) modulators.

ABBREVIATIONS

(19) TKI Tyrosine Kinase Inhibitor

(20) CTLA-4 Cytotoxic T-Lymphocyte Antigen 4

(21) PD-1 Programmed Death Receptor-1

(22) Qd Once a day

(23) PO Per Orally

(24) FBS Fetal Bovine Serum

(25) SOP Standard Operating Procedures

(26) SC Subcutaneous

Experimental Procedures

(27) Materials

(28) Experimental Animals for Syngeneic 4T1 and 4T1-Luc Model:

(29) Species/Strain: Balb/c ABomTac Source: Taconic Farms Sex: Female Weight: 20-30 grams on the day of implantation Age: at least 6 weeks old on the day of randomization Animal Identification: Cage number and ear notching
Experimental Animals for Syngeneic Lewis Lung Model: Species/Strain: C57BL/6JOlaHsd Source: Harlan Laboratories Sex: Female Weight: 20-30 grams on the day of implantation Age: at least 6 weeks old on the day of randomization Animal Identification: Cage number and ear notching
Cells and Materials: RPMI-1640 (Sigma, Cat. # R8758) supplemented with 10% fetal bovine serum (FBS), L-glutamine (4 mM), streptomycin (5 μg/ml) and penicillin (5 U/ml). BD Matrigel™ Basement Membrane Matrix Growth Factor Reduced, BD Bioscience, Cat. #354230, Lot #2229975. 0.25% Trypsin-EDTA, Sigma, Cat. # SLBD8049.
Drugs
Vehicle: 0.5% HPMC/0.1% Tween 80 vehicle for BGB324. Sterile PBS for immune checkpoint inhibitors.
IgG: InVivoMAb Polyclonal Armenian Hamster IgG, BE0091, BioXCell, Isotype control for IP injections.
BGB324: Manufacturer: Almac Group, N Ireland, batch Q1080. BGB324 powder was stored at room temperature. The BGB324 dosage given was well below the MTD in mice (023-TR-324). The BGB324 dosage administered (50 mg/kg Bid) is expected to result in a plasma concentration in mice (micro molar range) that is comparable to the one achieved in humans after administration of BGB324 (micromolar range). BGB324 was diluted in vehicle to 5 mg/ml dosing solution and administered to the mice immediately. BGB324 solution was freshly prepared every day.
Anti-mCTLA-4 (CD152) monoclonal antibody (CTAA-4): Syrian Hamster IgG, clone 9H10 (BioXCell, Cat. # BE0131).
Anti-mPD-1 monoclonal antibody (PD-1): rat IgG2a, clone RMP1-14 (BioXCell, Cat. # BE0146).
Anti-mPD-L1 monoclonal antibody (PD-L1): rat IgG2b, clone 10F.9G2 (BioXCell, Cat # BE0101).
Methods
Cell Culture
4T1 mammary adenocarcinoma cells and Non Small Cell Lung Cancer (NSCLC) Lewis Lung (LL2) were propagated at sub-confluence and split on a regular basis every 3.sup.rd day.
After trypsinization, the cells were washed once in RPMI/FBS (7 min at 1200 rpm) and re-suspended at 4×10.sup.6 cells/ml (4T1 cells) or 2.5×10.sup.6 per ml (LL2 cells) in a mixture of serum-free RPMI medium and Matrigel (1:1).
Subcutaneous Tumor Inoculation
Each animal was weighed before cell implantation. Injection of cells was performed after anesthetizing of mice by inhalation of sevoflurane. Anesthesia was induced with 8% sevoflurane and maintained at 4%. During injection, the mouse was placed on a heating pad. For implantation of 4T1 tumors: under a suitable depth of unconsciousness, each mouse was shaved and skin surrounding the region was washed with Chlorhexidine (1 mg/ml) with use of sterile gauze. Cells were injected into the 4.sup.th mammary gland on the right side with 0.05 ml cell suspension comprising approximately 2×10.sup.5 4T1 cells in serum-free RPMI medium/Matrigel (1:1). The mice were kept under surveillance until regaining of consciousness. For implantation of LL2 tumors: under a suitable depth of unconsciousness, animals were shaved, and skin surrounding the region was washed with Chlorhexidine (1 mg/ml) with use of sterile gauze. Injection was subcutaneous with one tumor per mouse with 0.1 ml of approximately 2.5×10.sup.5 LLC cells in serum-free RPMI medium/Matrigel (1:1).
Assignment of Experimental Groups
Before commencement of treatment, the animals were weighted and the tumor volume was measured twice a week. Since the tumor volume can affect the effectiveness of any given treatment, the mice were randomized into the groups using a Latin square method. Randomization was based on the tumor volume to ensure that each animal had the same probability of being assigned to a given treatment to reduce systematic error and that treatment groups were comparable at the baseline. When the average tumor volume reached 50-100 mm.sup.3, animals were randomized into treatment groups.
Dosing Procedure
Dosing administration was 10 ml/kg PO (BGB324 and 0.5% HPMC/0.1% Tween 80) and the dosing schedule was twice a day (Bid) on a 5 days on, 2 days off schedule. Dosing administration was 10 ml/kg IP (IgG, anti-mPD-1, anti-mPD-L1 and anti-mCTLA-4) by a 30-gauge needle. For the 4T1 model, dosing schedule was 4 times with CTLA4 and PD1 on days 0, 2, 4 and 6. For the Lewis Lung model, dosing schedule was 4 times with PD1 and PD-L1 on days 4, 8, 14 and 18 on days 0, 2, 4 and 6, respectively.
Clinical Observations
Animals were weighed prior to dosing, together with tumor growth measurements, and prior to euthanasia. At the time of routine monitoring, animals were checked for any effects of tumor growth or treatments on normal behavior, such as mobility, dehydration, body weight gain/loss, eye matting and any other abnormal effect. Death and observed clinical signs were recorded. Non-fasted body weights were recorded every day.
Tumor Measurements and Endpoint
Tumor measurements: Tumor size was measure twice a week in two dimensions using a caliper, and the tumor volume was calculated using the formula: V=0.5 a×b.sup.2[mm.sup.3], where a and b are the long and short diameter of the tumor, respectively (see Attachment 6 for raw data).
Endpoint: Mice were euthanized with CO.sub.2. Tumors and spleens were snap frozen in liquid nitrogen and stored in a −80° C. freezer, and/or fixed in 4% formaldehyde, transferred to 70% ethanol after 24 h and stored at 4° C. and/or subjected to tissue dissociation for analysis of immune cell infiltration. Liver and lungs were fixed in 4% formaldehyde, transferred to 70% ethanol after 24 h and stored at 4° C. for further evaluation.
Statistical Analysis
The tumor volume of 500 mm.sup.3 was used as an endpoint for the survival analysis. The Kaplan-Meier survival plots were generated using the software program PRISM (GraphPad) and the survival curves were compared using a log-rank (Mantel-Cox) test. Figures were generated using software PRISM (GraphPad). For individual time points, tumor volume values of different treatment groups were compared with other groups and significance was determined by one-way ANOVA or two-tailed unpaired t-test using software PRISM (GraphPad). Differences between the groups were considered significant when P<0.05. Figures were generated using software PRISM (GraphPad).
Results
AXL is Necessary for Evation of Anti-Tumorgenic Immune Response
An initial robust anti-tumor immune response to the 4T1.sup.Luc cell luciferase/GFP neo-antigens resulted in complete tumor regression (FIG. 1). After 4 weeks, tumor immune escape with robust regrowth of the primary tumor and multiorgan metastasis was detectable in all control animals. In contrast, AXL knockdown completely blocked post-immune response tumor regrowth and metastasis. This indicates that AXL is required for tumor immune escape in this model.
BGB324 Potentiates the Effect of Immune Checkpoint Inhibiting Therapy and Enhances the Presence of Pro-Tumorgenic CTLs and NK Cells in Syngeneic Mouse Models of Breast and Lung Cancer.
Body Weight Changes
Body weight changes in 4T1 implanted Balb/c mice as a result of treatment with vehicle, BGB324 or CTLA-4/PD-1 alone or in combination over the course of 104 days were recorded; the results are shown in FIG. 2. In general, a drop in body weight >20% would indicate treatment toxicity and should lead to termination of the treatment and culling of the mouse. None of the treatment groups showed a reduction in body weight that could indicate treatment toxicity.
Body weight changes in Lewis Lung implanted C57Bl/6 mice as a result of treatment with vehicle, BGB324 or PD-1/PD-L1 alone or in combinations over the course of 21 days were recorded; the results are shown in FIG. 9. In the triple combination group treated with PD-1/PD-L1+BGB324 a drop in body weight was observed at day 21. For 2 mice this was so severe that the study was terminated and tissue collected for analysis.
Effects of BGB324 on Survival and Tumor Volume Changes
For 4T1 tumor bearing Balb/C mice, durable tumor clearance was observed in 23% of BGB324+CTLA-4/PD-L1 versus 5.6% CTLA4/PD1. Complete tumor clearance was observed in 22% of BGB324+CTLA4 treated mice versus zero for CTLA4 (FIGS. 3-5). Following treatment with BGB324+CTLA4/PD1 or BGB324+CTLA4, metastases were abrogated in responders (Tables 1 and 2).

(30) TABLE-US-00005 TABLE 1 Number of metastasis detected in lung, liver and spleen in mice treated with CTLA-4 or CTLA-4 + BGB324. CTLA-4 CTLA-4 + BGB324 Lung Liver Spleen Lung Liver Spleen Non- 6/8 5/8 5/8 6/7 5/7 4/7 responders P = P = P = 0.02 0.09 0.19 Responders na na na 0/2 0/2 0/2

(31) TABLE-US-00006 TABLE 2 Number of metastasis detected in lung, liver and spleen in mice treated with CTL-A4/PD-1 or CTLA-4/PD-1 + BGB324 CTLA-4/PD-1 CTLA-4/PD-1 + BGB324 Lung Liver Spleen Lung Liver Spleen Non- 11/15 10/15 12/15 14/16  9/16 10/16 responders P = P = P = 0.0009 0.006 0.102 Responders 0/1 0/1 0/1 3/8 0/8 1/8
Tables 1 and 2 show metastasis detected in Balb/c mice carrying 4T1 orthotopic tumors treated with vehicle, BGB324 or CTLA-4/PD-1 alone or in combinations as indicated for the transformed survival study presented in FIG. 3c (Table 1) and FIG. 5 (Table 2). Significance by unpaired two-tailed Student t-test.
For Lewis Lung tumor bearing C57BI/6 mice, BGB324 significantly prolonged survival and reduced tumor burden when combined with PD-1/PD-L1 compared to PD-1/PD-L1 alone (FIGS. 10-12). Metastasis was not observed for any treatment groups in this model (data not shown).
Effect of BGB324 on Immune Cell Sub-Populations
BGB324 in combination with immune checkpoint inhibitors enhanced CTL tumor infiltration in both the 4T1 and Lewis Lung model (FIGS. 6 and 13 respectively). In the Lewis lung model, also enhanced presence of NK cells (FIG. 13) was observed. For the 4T1 model, enhanced presence of NK cells, macrophages and PMN neuotrophiles were found in the spleen (FIG. 7). In addition, a reduction in the presence of pro-tumorigenic mMDSC were observed in both models (FIGS. 8 and 14). These findings support that BGB324 can mediate its anti-tumorgenic effect (FIGS. 3, 4, 5 and Tables 1 and 2; FIGS. 10-12) by enhancing the presence of tumor killing immune cells such as CTLs and NK cells and by reducing the presence of pro-tumorigenic neutrophils such as mMDSC.

CONCLUSION

(32) Targeting AXL signaling represents a unique opportunity to address multiple tumor immune suppression mechanisms. Our results in breast and lung cancer mouse models support combining the clinical-stage AXL inhibitor, BGB324, with cancer immune checkpoint inhibitors to improve treatment of human cancers.

REFERENCES

(33) Chen L, 2014. Rejection of metastatic 4T1 breast cancer by attenuation of Treg cells in combination with immune stimulation. Mol Ther. 2007 December; 15(12):2194-202. Epub 2007 Oct. 30. PubMed PMID: 17968355. Chen L, et al. 2014. Metastasis is regulated via microRNA-200/ZEB1 axis control of tumour cell PD-L1 expression and intratumoral immunosuppression. Nat Commun. 2014 Oct. 28; 5:5241. doi: 10.1038/ncomms6241. Gjerdrum C, et al. 2010. Axl is an essential epithelial-to-mesenchymal transition-induced regulator of breast cancer metastasis and patient survival. Proc Natl Acad Sci USA. 2010 Jan. 19;107(3):1124-9. doi: 10.1073/pnas.0909333107. Epub 2009 Dec. 28. Grosso J F, et al. 2013. CTLA-4 blockade in tumor models: an overview of preclinical and translational research. Cancer Immun. 2013; 13:5. Epub 2013 Jan. 22. Review. PubMed PMID: 23390376. Kyi C, et al. 2014. Checkpoint blocking antibodies in cancer immunotherapy. FEBS Lett. 2014 Jan. 21; 588(2):368-76. doi: 10.1016/j.febslet.2013.10.015. Epub 2013 Oct. 23. Review. Lou Y, et al 2014. Association of epithelial-mesenchymal transition status with PD1/PDL1 expression and a distinct immunophenotype in non-small cell lung cancer: Implications for immunotherapy biomarkers. J Clin Oncol 32:5s, 2014 (suppl; abstr 3018). Lu J, et al. 2014. Clinical evaluation of compounds targeting PD-1/PD-L1 pathway for cancer immunotherapy. J Oncol Pharm Pract. Epub ahead of print] PubMed PMID: 24917416. Paolino M, et al. 2014. The E3 ligase Cbl-b and TAM receptors regulate cancer metastasis via natural killer cells. Nature. 2014 Mar. 27; 507(7493):508-12. doi: 10.1038/nature12998. Rothlin C V, et al. 2007. TAM receptors are pleiotropic inhibitors of the innate immune response. Cell. 2007 Dec. 14; 131(6):1124-36.
Effect of Axl Inhibitor on Immune Checkpoint Inhibitor Efficacy
Background
Signaling via the Axl receptor tyrosine kinase is a key suppressor of anti-tumour innate immune response. Axl is expressed on several cells associated with the suppressive tumpour immune microenvironment including natural killer cells, dendritic cells and tumour-associated macrophages. Axl is also an important regulator of tumour plasticity related to epithelial-to-mesenchymal transition (EMT) that contributes to anti-tumour evasion. Hence Axl signaling contributes uniquely to tumour intrinsic and microenvironmental immune suppression in tumours. It was therefore evaluated whether blocking Axl signaling with BGB324, a selective clinical-stage small molecule checkpoint blockade in sygeneic cancer mouse models that display limited immunogenicity.
Axl, EMT and Immune Suppression
Axl is a down-regulator of the innate immune response upon activation of the adaptive immune system. Axl mediates M1 to Mr polarisation (Chiu, K. C., et al. (2015). Oral Oncol). High Axl expression on tumour associated macrophages in human primary breast cancer (Ye, X., et al. (2010). Oncogene). TAM inhibition in NK cells reduces metastasis from melanoma and mammary carcinoma (Paolino, M., et al. (2014). Nature). The Axl ligand Gas6 is upregulated by tumour infiltrating macrophages and contributes to tumour growth and metastasis (Loges, S., et al. (2010). Blood). High EMT score correlates with immunosuppressive phenotype (Lou, Y., et al. (2014). J Clin Oncol suppl; abstr 3018). PD-L1 expression correlates with mesenchymal phenptype (Chen, L., et al. (2014). Nat Commun). The EMT transcription factor Snail induces immunosuppression leading to increased metastasis and confers resistance yo cytotoxic T cell attack (Kudo-Saito, C., et al. (2009). Cancer Cell). EMT increases autophagy flux, and autophagy inhibition sensitises EMTed cells to cytotoxic T cell lysis (Akalay, I., et al. (2013) Cancer Res).
Axl Inhibition in Post-Immune Response Tumour Recurrence and Metastasis in the Mammary Adenocarcinoma 4T1/Balb/C Syngeneic Mouse Model
BalbC mice were orthotopically implanted with 1×106 4T1-GFPLuc cells infected with the mouse Axl-targeting shRNA (4T1-GFPLuc-shmAxl2; shAXL) or negative control human-specific shRNA (4T1-GFP-Luc-shAxl279; control shRNA) cells. Tumour growth and metastasis spread was monitored every week by bioluminescent imaging. After 9 weeks, organs were excised and imaged ex vivo for occurrence of metastasis. The results are shown in FIG. 1. An initial robust immune response induced tumours regression followed by tumour immune evasion with regrowth of the primary tumour and widespread metastasis in mice implanted with 4T1-GFP-Luc control shRNA cells. Axl knock down suppressed regrowth at the primary site and abolished metastasis (Gjerdrum, C., et al. (2010). Proc Natl Acad Sci USA). This indicates that Axl contributes to immune evasion.
Axl Inhibition Potentiates the Effect of Immune Checkpoint Inhibitors in the Mammary Adenocarcinoma 4T1/Balb/C Syngeneic Mouse Model
BalbC mice were orthotopically implanted with 4×105 4T1 cells. Treatment was initiated when average tumour volume reached 100 mm.sup.3. Animals were treated with anti-CTLA4 and anti-PD1 as indicated at 10 mg/kg of each (4 doses every 2nd day, IP). BGB324 was administered at 50 mg/kg twice a day (oral gavage). Vehicle groups were injected with control IgG. Transformed survival curves are shown. The day each individual tumour reached 500 mm.sup.3 was used as an endpoint. Complete tumour clearance was observed in 23% of the anti-CTLA4/anti-PD1+BGB324 treated mice versus 5.6% for the anti-CTLA4/anti-PD1 treated mice. Complete tumour clearance was observed in 22% of the anti-CTLA4+BGB324 treated mice versus zero for the anti-CTLA4 treated mice. BalbC mice that displayed complete clearance of the tumour were re-injected orthotopically with 4T1 cells at day 105 after the first cell injection. Subsequent tumour growth was not observed in any of these mice (from anti-CTLA4/PD-1 group, n=1; from anti-CTLA4/PD-1+BGB324, n=4) indicating that these mice were immune towards subsequent 4T1 cell exposure. The results are shown in FIG. 5.
Axl Inhibition Treatment Enhances the Number of CTLs in Tumours and Spleens
For CTL (cytotoxic T-lymphocyte) analysis in tumors: BalbC mice were orthotopically implanted with 4×105 4T1 cells. Treatment (anti-CTLA4+anti-PD1 at 10 mg/kg of each, 3 doses every 2nd day, IP; BGB324 at 50 mg/kg twice a day, oral gavage; Vehicle control IgG at 20 mg/kg, 3 doses every 2nd day, IP) was initiated when average tumour volume reached 500 mm.sup.3. Tumours were harvested 5 days after treatment initiation (n=5 for all groups), dissociated using MACS Tumor Dissociation Kit, stained for markers of CTLs and analysed on a BD Fortessa Cell Analyser. Anti-CTLA4/PD1 treatment enhanced infiltration of CTLs in 4T1 tumors when compared to Vehicle or BGB324 treated mice. Treatment with BGB324 further enhanced tumour infiltration of CTLs. The results are shown in FIG. 6.
For CTL analysis in spleen: BalbC mice were orthotopically implanted with 4×105 4T1 cells. Treatment (anti-CTLA4+anti-PD at 10 mg/kg of each, 4 doses every 2nd day, IP; BGB324 as above) was initiated when average tumor volume reached 100 mm.sup.3. Mice were culled 43 days after treatment initiation. Spleens were dissociated using MACS Tumor Dissociation Kit and stained for markers of CTLs. Responders (top two points in CTLA4; top two points in CTLA4/PD1; top three points in CTLA4/PD1/BGB324) in all groups had a higher number of CTLs in the spleen compared to nonresponders (remaining points). BGB324 further enhanced the number of CTLs compared to treatment with immune check point inhibitors alone.
Tumours Escaping Checkpoint Blockade Show an Increased Mesenchymal Phenotype
BalbC were orthotopically implanted with 4×105 4T1 cells, and treatment was initiated when average tumour volume reached 100 mm.sup.3. Animals were treated with anti-CTLA4+anti-PD1 as indicated at 10 mg/kg of each (4 doses every 2nd day, IP). BGB324 was administered at 50 mg/kg twice a day (oral gavage). Control groups were injected with control IgG. Tumours were harvested from non-responders (i.e. tumours that had escaped treatment inhibition and reached 1500 mm.sup.3) and responders (i.e. tumours that responded to treatment and remained below 500 mm.sup.3 until termination of experiment) and processed by IHC for evaluation of known EMT markers.
Tumours treated with checkpoint inhibitors alone or in combination with BGB324 that did not respond to the treatment, displayed stronger staining for Axl and Vimentin compared tumours from the control groups (vehicle, BGB324 alone). However, in a responding tumour treated with BGB324+anti-CTLA4/anti-PD-1 weaker Axl- and Vimentin staining were observed.
This suggests that EMT is involved in the immune evasion of tumors escaping checkpoint inhibition, and that targeting Axl may inhibit EMT mediated immune evasion.

CONCLUSION

(34) This data therefore shows that Axl inhibition (particularly by BGB324) represents an unique opportunity to target anti-tumour immune suppressive mechanisms and supports clinical translation of Axl inhibition in combination with cancer immunotherapy in human cancers.
Effect of Anti-Axl, Oncolytic Virus and Immune Checkpoint Inhibitor Combination Therapy on Metastasis
Tumours are implanted into female Balb/c mice and mice are injected when tumours reach ˜50-100 mm.sup.3. Tumour volume is monitored by caliper measurement and defined by V(mm.sup.3)=π/6×W2×L, where W and L are the width and the length of the tumour, respectively. Mice are injected with 2×10.sup.8 plaque forming units (pfu) of Oncolytic Virus (OV) through the tail vein starting at day 0. For the anti-Immune Checkpoint Inhibitor (ICI) group, 100 μg of anti-ICI antibody is injected intraperitoneally (IP) at day 4 after virus injection, with treatments consisting of 3 doses each 3 days apart. For the combination group, anti-Axl is administered along with the OV at day 0, then 100 μg of anti-ICI antibody is administered at day 4. Anti-Axl treatment is either monoclonal anti-Axl antibody or a small molecule Axl inhibitor. The anti-Axl antibody is administered IP at doses of 30 mg/kg body weight, twice weekly. The small molecule inhibitor is administered at 50 mg/kg in 0.5% (w/w) HPMC/0.1% (w/w) Tween 80 twice daily by oral gavage. For the Kaplan-Meier survival curve, end point is established at a tumour volume ≥750 mm.sup.3. A typical result is shown in FIG. 15.