Methods for treating immunorefractory lung cancer

Abstract

The present disclosure provides methods for treating immunorefractory lung cancer using a RAS inhibitor. The present disclosure also provides a combination therapy for the treatment of immunorefractory lung cancer.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority to U.S. Provisional Application Serial No. 63 / 317,649, filed on March 8, 2022, the entire disclosure of which is incorporated herein by reference as if set forth in its entirety.

[0003] Sequence Listing

[0004] This application contains a sequence listing, which is submitted electronically in XML format and is incorporated herein by reference in its entirety. The XML copy was created on March 3, 2023, is named "51432-025WO2_Sequence_Listing_3_3_23" and is 1,785 bytes in size. Background Art

[0005] Cancer remains one of the most deadly threats to human health. In the United States, cancer affects nearly 1.3 million new patients each year and is the second leading cause of death after heart disease, accounting for approximately one in four deaths.

[0006] The development of immune checkpoint inhibitors (ICIs) has significantly improved the treatment of various solid tumors. However, initial or acquired resistance to ICI treatment remains an obstacle to durable anti-tumor activity in most cases. Current response biomarkers for anti-PD-1 or anti-PD-L1 treatment include tumor mutation load, expression of programmed cell death ligand-1 (PD-L1), and T cell density. ICI-induced anti-tumor immunity depends on the infiltration of lymphocytes into the tumor core, with "T cell-inflamed" tumors showing the best response. In contrast, "cold tumors" (also called immune-refractory or immune-evading tumors), which can be defined in part by the lack of T cell infiltration and low IFN-γ gene imprinting, rarely respond to immune checkpoint inhibition (Bonaventura et al., Front. Immunol. 2019). New compositions and methods for treating immune-refractory tumors are needed. Summary of the invention

[0007] The present disclosure provides compositions and uses thereof for treating immune-refractory lung cancer. The present disclosure is based at least in part on the following observations, and treating immune-refractory lung cancer with a RAS inhibitor (e.g., a RAS (on) inhibitor) compound that inhibits mutant RAS G12C protein makes the cancer sensitive to treatment with an immunotherapeutic agent. In some embodiments, the compound inhibits RAS with a carcinogenic G12C mutation. In some embodiments, the RAS inhibitor is a covalent inhibitor, for example, the covalent inhibitor is capable of forming a covalent bond with an oncogenic mutant form of RAS G12C at the G12C position. In some embodiments, treatment with a RAS inhibitor makes the cancer sensitive to treatment with an immune checkpoint inhibitor or a SHP2 inhibitor. In some embodiments, a compound or a combination of compounds described herein is administered to a subject whose previous immunotherapy treatment (such as an immunotherapy treatment using an immune checkpoint inhibitor) has failed.

[0008] In one aspect, the present disclosure provides a method of administering a RAS G12C A method of treating immune-refractory lung cancer in a subject by (turning on) an inhibitor.

[0009] In another aspect, the present disclosure provides a method for treating a subject by administering a RAS G12C Methods for (turning on) inhibitors to transform the tumor microenvironment of immune-cold lung cancer in a subject in need thereof.

[0010] In some embodiments, RAS G12C (Open) Inhibitors for triple-complex RAS G12C (Turn on) inhibitor.

[0011] In another aspect, the present disclosure provides a method of treating immune-refractory lung cancer in a subject, the method comprising administering to the subject a RAS inhibitor of Formula I:

[0012]

[0013] or a pharmaceutically acceptable salt thereof,

[0014] where the dashed lines represent zero, one, two, three, or four nonadjacent double bonds;

[0015] A is -N(H or CH 3 )C(O)-(CH 2 )-, in which the amino nitrogen is bound to -CH(R 10 )-, an optionally substituted 3- to 6-membered cycloalkylene group, an optionally substituted 3- to 6-membered heterocycloalkylene group, an optionally substituted 6-membered arylene group, or an optionally substituted 5- to 10-membered heteroarylene group;

[0016] B does not exist, is -CH(R 9)-、>C=CR 9 R 9’ or>CR 9 R 9’ , where the carbon is bonded to -N(R 11 )C(O)-, an optionally substituted 3- to 6-membered cycloalkylene group, an optionally substituted 3- to 6-membered heterocycloalkylene group, an optionally substituted 6-membered arylene group, or a 5- to 6-membered heteroarylene group;

[0017] G is an optionally substituted C 1 -C 4 Alkylene, optionally substituted C 1 -C 4 Alkenylene, optionally substituted C 1 -C 4 Heteroalkylene, -C(O)O-CH(R 6 )-, where C is bound to -C(R 7 R 8 )-、-C(O)NH-CH(R 6 )-, where C is bound to -C(R 7 R 8 )-, optionally substituted C 1 -C 4 Heteroalkylene or 3- to 8-membered heteroarylene;

[0018] L is a linker, wherein the linker is acyclic or comprises a monocyclic, fused bicyclic, fused polycyclic, bridged bicyclic, or bridged polycyclic group;

[0019] W is a crosslinking group comprising vinyl ketone, vinyl sulfone, alkynyl ketone, haloacetyl or alkynyl sulfone;

[0020] X 1 is an optionally substituted C 1 -C 2 Alkylene, NR, O or S(O) n ;

[0021] X 2 is O or NH;

[0022] X 3 N or CH;

[0023] n is 0, 1 or 2;

[0024] R is hydrogen, cyano, optionally substituted C 1 -C 4 Alkyl, optionally substituted C 2 -C 4 Alkenyl, optionally substituted C 2 -C 4 Alkynyl, C(O)R', C(O)OR', C(O)N(R')2 、S(O)R'、S(O) 2 R' or S(O) 2 N(R') 2 ;

[0025] Each R' is independently H or optionally substituted C 1 -C 4 alkyl;

[0026] Y 1 is C, CH or N;

[0027] Y 2 , Y 3 , Y 4 and Y 7 are independently C or N;

[0028] Y 5 CH, CH 2 or N;

[0029] Y 6 C(O), CH, CH 2 or N;

[0030] R 1 is cyano, optionally substituted C 1 -C 6 Alkyl, optionally substituted C 1 -C 6 heteroalkyl, optionally substituted 3 to 6 membered cycloalkyl, optionally substituted 3 to 6 membered cycloalkenyl, optionally substituted 3 to 6 membered heterocycloalkyl, optionally substituted 6 to 10 membered aryl, or optionally substituted 5 to 10 membered heteroaryl,

[0031] R 2 absent, hydrogen, optionally substituted C 1 -C 6 Alkyl, optionally substituted C 2 -C 6 Alkenyl, optionally substituted C 2 -C 6 alkynyl, optionally substituted 3- to 6-membered cycloalkyl, optionally substituted 3- to 7-membered heterocycloalkyl, optionally substituted 6-membered aryl, optionally substituted 5- or 6-membered heteroaryl; R 3 does not exist, or

[0032] R 1 and R 2 and the atoms to which they are attached form an optionally substituted 3- to 14-membered heterocycloalkyl group, or R 2 and R 3 and the atoms to which it is attached form an optionally substituted 3- to 8-membered cycloalkyl or an optionally substituted 3- to 14-membered heterocycloalkyl;

[0033] R 4 is absent, hydrogen, halogen, cyano or methyl optionally substituted with 1 to 3 halogens;

[0034] R 5 is hydrogen, C optionally substituted with halogen 1 -C 4 Alkyl, cyano, hydroxyl or C 1 -C 4 Alkoxy, cyclopropyl or cyclobutyl;

[0035] R 6 is hydrogen or methyl; R 7 is hydrogen, halogen or optionally substituted C 1 -C 3 Alkyl, or

[0036] R 6 and R 7 and the carbon atoms to which they are attached form an optionally substituted 3- to 6-membered cycloalkyl or an optionally substituted 3- to 7-membered heterocycloalkyl;

[0037] R 8 is hydrogen, halogen, hydroxy, cyano, optionally substituted C 1 -C 3 Alkoxy, optionally substituted C 1 -C 3 Alkyl, optionally substituted C 2 -C 6 Alkenyl, optionally substituted C 2 -C 6 alkynyl, optionally substituted 3 to 8 membered cycloalkyl, optionally substituted 3 to 14 membered heterocycloalkyl, optionally substituted 5 to 10 membered heteroaryl, or optionally substituted 6 to 10 membered aryl, or

[0038] R 7 and R 8 Combined with the carbon atom to which it is attached, it forms C=CR 7’ R 8’ ; C=N(OH), C=N(OC 1 -C 3 alkyl), C═O, C═S, C═NH, optionally substituted 3- to 6-membered cycloalkyl, or optionally substituted 3- to 7-membered heterocycloalkyl;

[0039] R 7a and R 8a are independently hydrogen, halogen, optionally substituted C 1 -C 3 an alkyl group, or in combination with the carbon to which it is attached to form a carbonyl group;

[0040] R 7’ is hydrogen, halogen or optionally substituted C1 -C 3 Alkyl; R 8’ is hydrogen, halogen, hydroxy, cyano, optionally substituted C 1 -C 3 Alkoxy, optionally substituted C 1 -C 3 Alkyl, optionally substituted C 2 -C 6 Alkenyl, optionally substituted C 2 -C 6 alkynyl, optionally substituted 3 to 8 membered cycloalkyl, optionally substituted 3 to 14 membered heterocycloalkyl, optionally substituted 5 to 10 membered heteroaryl, or optionally substituted 6 to 10 membered aryl, or

[0041] R 7’ and R 8’ and the carbon atoms to which they are attached form an optionally substituted 3- to 6-membered cycloalkyl or an optionally substituted 3- to 7-membered heterocycloalkyl;

[0042] R 9 is H, F, optionally substituted C 1 -C 6 Alkyl, optionally substituted C 1 -C 6 heteroalkyl, optionally substituted 3 to 6-membered cycloalkyl or optionally substituted 3 to 7-membered heterocycloalkyl, or

[0043] R 9 and L, combined with the atoms to which they are attached, form an optionally substituted 3- to 14-membered heterocycloalkyl;

[0044] R 9’ is hydrogen or optionally substituted C 1 -C 6 Alkyl, or

[0045] R 9 and R 9’ and the atoms to which they are attached form a 3- to 6-membered cycloalkyl group or a 3- to 6-membered heterocycloalkyl group;

[0046] R 10 is hydrogen, halogen, hydroxyl, C 1 -C 3 Alkoxy or C 1 -C 3 alkyl;

[0047] R 10a is hydrogen or halogen;

[0048] R 11 is hydrogen or C 1 -C 3 Alkyl; and

[0049] R21 H or C 1 -C 3 alkyl.

[0050] In another aspect, the present disclosure provides a method of transforming the tumor microenvironment of an immune-cold lung cancer in a subject in need thereof by administering to the subject a RAS inhibitor of Formula I or a subformula thereof as described herein. In some embodiments, prior to transforming the tumor microenvironment, the subject is resistant to immune checkpoint inhibitors. In some embodiments, administration of a RAS inhibitor transforms the tumor microenvironment, thereby sensitizing the cancer to treatment with an immune checkpoint inhibitor.

[0051] In each of the foregoing aspects, the method may further comprise administering a SHP2 inhibitor to the subject.

[0052] In another aspect, the present disclosure provides a method for treating a subject by administering a RAS G12C A method of treating immune-refractory lung cancer in a subject using a (turn on) inhibitor, a SHP2 inhibitor, and an immune checkpoint inhibitor.

[0053] In another aspect, the present disclosure provides a method for treating a subject by administering a RAS G12C Methods of transforming the tumor microenvironment of immune-cold lung cancer in subjects in need thereof using (on) inhibitors, SHP2 inhibitors, and immune checkpoint inhibitors.

[0054] In some embodiments of any of the methods described herein, the Ras inhibitor is a compound of Formula I:

[0055]

[0056] or a pharmaceutically acceptable salt thereof,

[0057] where the dashed lines represent zero, one, two, three, or four nonadjacent double bonds;

[0058] A is -N(H or CH 3 )C(O)-(CH 2 )-, in which the amino nitrogen is bound to -CH(R 10 )-, an optionally substituted 3- to 6-membered cycloalkylene group, an optionally substituted 3- to 6-membered heterocycloalkylene group, an optionally substituted 6-membered arylene group, or an optionally substituted 5- to 10-membered heteroarylene group;

[0059] B does not exist, is -CH(R 9 )-、>C=CR 9 R 9’ or>CR 9 R 9’ , where the carbon is bonded to -N(R 11)C(O)-, an optionally substituted 3- to 6-membered cycloalkylene group, an optionally substituted 3- to 6-membered heterocycloalkylene group, an optionally substituted 6-membered arylene group, or a 5- to 6-membered heteroarylene group;

[0060] G is an optionally substituted C 1 -C 4 Alkylene, optionally substituted C 1 -C 4 Alkenylene, optionally substituted C 1 -C 4 Heteroalkylene, -C(O)O-CH(R 6 )-, where C is bound to -C(R 7 R 8 )-、-C(O)NH-CH(R 6 )-, where C is bound to -C(R 7 R 8 )-, optionally substituted C 1 -C 4 Heteroalkylene or 3- to 8-membered heteroarylene;

[0061] L is a linker, wherein the linker is acyclic or comprises a monocyclic, fused bicyclic, fused polycyclic, bridged bicyclic, or bridged polycyclic group;

[0062] W is a crosslinking group comprising a vinyl ketone, a vinyl sulfone, an alkynyl ketone, a haloacetyl group, or an alkynyl sulfone;

[0063] X 1 is an optionally substituted C 1 -C 2 Alkylene, NR, O or S(O) n ;

[0064] X 2 is O or NH;

[0065] X 3 N or CH;

[0066] n is 0, 1 or 2;

[0067] R is hydrogen, cyano, optionally substituted C 1 -C 4 Alkyl, optionally substituted C 2 -C 4 Alkenyl, optionally substituted C 2 -C 4 Alkynyl, C(O)R', C(O)OR', C(O)N(R') 2 、S(O)R'、S(O) 2 R' or S(O) 2 N(R') 2 ;

[0068] Each R' is independently H or optionally substituted C 1 -C 4 alkyl;

[0069] Y 1 is C, CH or N;

[0070] Y 2 , Y 3 , Y 4 and Y 7 are independently C or N;

[0071] Y 5 CH, CH 2 or N;

[0072] Y 6 C(O), CH, CH 2 or N;

[0073] R 1 is cyano, optionally substituted C 1 -C 6 Alkyl, optionally substituted C 1 -C 6 heteroalkyl, optionally substituted 3 to 6 membered cycloalkyl, optionally substituted 3 to 6 membered cycloalkenyl, optionally substituted 3 to 6 membered heterocycloalkyl, optionally substituted 6 to 10 membered aryl, or optionally substituted 5 to 10 membered heteroaryl;

[0074] R 2 absent, hydrogen, optionally substituted C 1 -C 6 Alkyl, optionally substituted C 2 -C 6 Alkenyl, optionally substituted C 2 -C 6 alkynyl, optionally substituted 3- to 6-membered cycloalkyl, optionally substituted 3- to 7-membered heterocycloalkyl, optionally substituted 6-membered aryl, optionally substituted 5- or 6-membered heteroaryl; R 3 does not exist, or

[0075] R 1 and R 2 and the atoms to which they are attached form an optionally substituted 3- to 14-membered heterocycloalkyl group, or R 2 and R 3 and the atoms to which they are attached form an optionally substituted 3- to 8-membered cycloalkyl group or;

[0076] R 4 is absent, hydrogen, halogen, cyano or methyl optionally substituted with 1 to 3 halogens;

[0077] R 5 is hydrogen, C optionally substituted with halogen1 -C 4 Alkyl, cyano, hydroxyl or C 1 -C 4 Alkoxy, cyclopropyl or cyclobutyl;

[0078] R 6 is hydrogen or methyl; R 7 is hydrogen, halogen or optionally substituted C 1 -C 3 Alkyl, or

[0079] R 6 and R 7 and the carbon atoms to which they are attached form an optionally substituted 3- to 6-membered cycloalkyl or an optionally substituted 3- to 7-membered heterocycloalkyl;

[0080] R 8 is hydrogen, halogen, hydroxy, cyano, optionally substituted C 1 -C 3 Alkoxy, optionally substituted C 1 -C 3 Alkyl, optionally substituted C 2 -C 6 Alkenyl, optionally substituted C 2 -C 6 alkynyl, optionally substituted 3 to 8 membered cycloalkyl, optionally substituted 3 to 14 membered heterocycloalkyl, optionally substituted 5 to 10 membered heteroaryl, or optionally substituted 6 to 10 membered aryl, or

[0081] R 7 and R 8 Combined with the carbon atom to which it is attached, it forms C=CR 7’ R 8’ ; C=N(OH), C=N(OC 1 -C 3 alkyl), C═O, C═S, C═NH, optionally substituted 3- to 6-membered cycloalkyl, or optionally substituted 3- to 7-membered heterocycloalkyl;

[0082] R 7a and R 8a are independently hydrogen, halogen, optionally substituted C 1 -C 3 an alkyl group, or in combination with the carbon to which it is attached to form a carbonyl group;

[0083] R 7’ is hydrogen, halogen or optionally substituted C 1 -C 3 Alkyl; R 8’ is hydrogen, halogen, hydroxy, cyano, optionally substituted C 1 -C 3 Alkoxy, optionally substituted C 1-C 3 Alkyl, optionally substituted C 2 -C 6 Alkenyl, optionally substituted C 2 -C 6 alkynyl, optionally substituted 3 to 8 membered cycloalkyl, optionally substituted 3 to 14 membered heterocycloalkyl, optionally substituted 5 to 10 membered heteroaryl, or optionally substituted 6 to 10 membered aryl, or

[0084] R 7’ and R 8’ and the carbon atoms to which they are attached form an optionally substituted 3- to 6-membered cycloalkyl or an optionally substituted 3- to 7-membered heterocycloalkyl;

[0085] R 9 is H, F, optionally substituted C 1 -C 6 Alkyl, optionally substituted C 1 -C 6 heteroalkyl, optionally substituted 3 to 6-membered cycloalkyl or optionally substituted 3 to 7-membered heterocycloalkyl, or

[0086] R 9 and L, combined with the atoms to which they are attached, form an optionally substituted 3- to 14-membered heterocycloalkyl;

[0087] R 9’ is hydrogen or optionally substituted C 1 -C 6 Alkyl; or

[0088] R 9 and R 9’ and the atoms to which they are attached form a 3- to 6-membered cycloalkyl group or a 3- to 6-membered heterocycloalkyl group;

[0089] R 10 is hydrogen, halogen, hydroxyl, C 1 -C 3 Alkoxy or C 1 -C 3 alkyl;

[0090] R 10a is hydrogen or halogen;

[0091] R 11 is hydrogen or C 1 -C 3 Alkyl; and

[0092] R 21 H or C 1 -C 3 alkyl.

[0093] In some embodiments, the RAS inhibitor is a compound of Formula II:

[0094]

[0095] or a pharmaceutically acceptable salt thereof.

[0096] In some embodiments, the RAS inhibitor is a compound of formula III:

[0097]

[0098] or a pharmaceutically acceptable salt thereof.

[0099] In some embodiments, the RAS inhibitor is a compound of formula IV:

[0100]

[0101] or a pharmaceutically acceptable salt thereof.

[0102] In some embodiments, the RAS inhibitor is a compound of Formula V:

[0103]

[0104] or a pharmaceutically acceptable salt thereof.

[0105] In some embodiments, the RAS inhibitor is a compound of Formula VI:

[0106]

[0107] or a pharmaceutically acceptable salt thereof,

[0108] Where X e and X f are independently N or CH; and

[0109] R 12 is an optionally substituted C 1 -C 6 Alkyl, optionally substituted C 1 -C 6 heteroalkyl or optionally substituted 3- to 7-membered heterocycloalkyl.

[0110] In some embodiments of the compound of Formula I, R 7 Methyl or R 8 In some embodiments of the compound of Formula I, R 7 Methyl or R 8 It is methyl.

[0111] In some embodiments, the RAS inhibitor is a compound of Formula VII:

[0112]

[0113] or a pharmaceutically acceptable salt thereof,

[0114] Where R 13 is hydrogen, optionally substituted 3- to 10-membered heterocycloalkyl or optionally substituted C 1 -C 6 Heteroalkyl.

[0115] In some embodiments, R 2 is an optionally substituted C 1 -C 6 alkyl or an optionally substituted 3- to 6-membered cycloalkyl.

[0116] In some embodiments, L is acyclic. In some embodiments, L is a monocyclic ring.

[0117] In some embodiments, A is an optionally substituted 6-membered arylene. In some embodiments, A is an optionally substituted 5- to 6-membered heteroarylene. In some embodiments, A is an optionally substituted C 1 -C 4 In some embodiments, A is an optionally substituted 3- to 6-membered heteroarylene.

[0118] In some embodiments, B is -CHR 9 -. In some embodiments, R 9 is F, optionally substituted C 1 -C 6 Alkyl, optionally substituted C 1 -C 6 In some embodiments, B is an optionally substituted 6-membered arylene. In some embodiments, B is an optionally substituted 6-membered arylene.

[0119] In some embodiments, W is a crosslinking group comprising vinyl ketone, vinyl sulfone, alkynyl ketone, or alkynyl sulfone.

[0120] In some embodiments, W is a cross-linking group comprising a vinyl ketone. In some embodiments, W has the structure of Formula VIIIa:

[0121]

[0122] Where R 16a , R 16b and R 16c is independently hydrogen, -CN, halogen or -C optionally substituted with one or more substituents independently selected from the following 1 -C 3 Alkyl: -OH, -OC 1 -C 3 Alkyl, -NH 2、-NH(C 1 -C 3 Alkyl), -N(C 1 -C 3 alkyl) 2 or a 4- to 7-membered saturated heterocycloalkyl group.

[0123] In some embodiments, W is a cross-linking group comprising an alkynone. In some embodiments, W has the structure of Formula VIIIb:

[0124]

[0125] Where R 17 is hydrogen, optionally substituted with one or more substituents independently selected from the following 1 -C 3 Alkyl: -OH, -OC 1 -C 3 Alkyl, -NH 2 、-NH(C 1 -C 3 Alkyl), -N(C 1 -C 3 alkyl) 2 or a 4- to 7-membered saturated cycloalkyl group or a 4- to 7-membered saturated heterocycloalkyl group.

[0126] In some embodiments, W is a cross-linking group comprising vinyl sulfone. In some embodiments, W has the structure of Formula VIIIc:

[0127]

[0128] Where R 18a , R 18b and R 18c is independently hydrogen, -CN or -C optionally substituted with one or more substituents independently selected from the following 1 -C 3 Alkyl: -OH, -OC 1 -C 3 Alkyl, -NH 2 、-NH(C 1 -C 3 Alkyl), -N(C 1 -C 3 alkyl) 2 or a 4- to 7-membered saturated heterocycloalkyl group.

[0129] In some embodiments, W is a cross-linking group comprising an alkynyl sulfone. In some embodiments, W has the structure of Formula VIIId:

[0130]

[0131] Where R 19 is hydrogen, optionally substituted with one or more substituents independently selected from the following 1 -C 3 Alkyl: -OH, -OC 1 -C 3 Alkyl, -NH 2 、-NH(C 1 -C 3 Alkyl), -N(C 1 -C 3 alkyl) 2 or a 4- to 7-membered saturated heterocycloalkyl group or a 4- to 7-membered saturated heterocycloalkyl group.

[0132] In some embodiments, W has the structure of Formula VIIe:

[0133]

[0134] Where X e is a halogen; and

[0135] R 20 is hydrogen, optionally substituted with one or more substituents independently selected from the following 1 -C 3 Alkyl: -OH, -OC 1 -C 3 Alkyl, -NH 2 、-NH(C 1 -C 3 Alkyl), -N(C 1 -C 3 alkyl) 2 or a 4- to 7-membered saturated heterocycloalkyl group.

[0136] In some embodiments, the RAS inhibitor is a compound of Table 1 or a pharmaceutically acceptable salt thereof.

[0137] In some embodiments, the RAS inhibitor is:

[0138]

[0139] or a pharmaceutically acceptable salt thereof.

[0140] In some embodiments, the RAS inhibitor is a compound of Formula IX:

[0141]

[0142] or a pharmaceutically acceptable salt thereof, wherein

[0143] A is a 6-membered heterocycloalkyl group optionally substituted with methyl, -OH or =O;

[0144] A' is a 5-6 membered saturated heterocycloalkyl or a 5-6 membered heteroaryl, each optionally substituted with methyl, methoxy or halogen;

[0145] R 2 is methyl or halomethyl;

[0146] R 9' and R 9” Each is methyl or R 9' and R 9” Together they form an unsubstituted saturated C 3 -C 6 cycloalkyl; and

[0147] R 17 is hydrogen, optionally substituted with one or more substituents independently selected from the following 1 -C 3 Alkyl: -OH, -OC 1 -C 3 Alkyl, -NH 2 、-NH(C 1 -C 3 Alkyl), -N(C 1 -C 3 alkyl) 2 or a 4- to 7-membered saturated cycloalkyl group or a 4- to 7-membered saturated heterocycloalkyl group.

[0148] In some embodiments, the RAS inhibitor is a compound of formula X:

[0149]

[0150] or a pharmaceutically acceptable salt thereof, wherein

[0151] A is

[0152] B is CH(R 9 ), where R 9 for

[0153] L is

[0154] and

[0155] W is

[0156]

[0157] In some embodiments of any of the methods described herein, the method further comprises administering an immune checkpoint inhibitor to the subject.

[0158] In another aspect, the present disclosure provides a method of treating immune-refractory lung cancer in a subject by administering to the subject a RAS inhibitor, a SHP2 inhibitor, and an immune checkpoint inhibitor, wherein the RAS inhibitor is:

[0159]

[0160] or a pharmaceutically acceptable salt thereof, and

[0161] SHP2 inhibitors are:

[0162]

[0163] or a pharmaceutically acceptable salt thereof.

[0164] In some embodiments of the methods described herein, the subject is administered an immune checkpoint inhibitor that is a PD-1 inhibitor.

[0165] In another aspect, the disclosure provides a method of sensitizing an immune-refractory lung cancer in a subject, the method comprising administering to the subject a RAS inhibitor of Formula I or a subformula thereof described herein.

[0166] In some embodiments, the subject has previously been administered an immune checkpoint inhibitor. In some embodiments, the subject is resistant to treatment with an immune checkpoint inhibitor. In some embodiments, the subject has acquired resistance to treatment with an immune checkpoint inhibitor.

[0167] In some embodiments, administration of a RAS inhibitor sensitizes the cancer to treatment with an immune checkpoint inhibitor.

[0168] In some embodiments, the combination of inhibitors is administered simultaneously or sequentially. In some embodiments, the inhibitors are administered as a single formulation or in separate formulations.

[0169] In some embodiments, the subject has one or more tumors with low tumor mutation load. In some embodiments, the subject has one or more microsatellite stable tumors. In some embodiments, the subject has one or more tumors with low microsatellite instability. In some embodiments, the subject has one or more tumors with low tumor immune infiltration.

[0170] In some embodiments, relative to the tumor immune infiltration in the absence of RAS inhibitors or inhibitor combinations as disclosed herein, the one or more inhibitors are administered to change the tumor immune infiltration. In some embodiments, tumor immune infiltration includes antigen presenting cells, myeloid cells, or lymphoid cells. In some embodiments, relative to the tumor immune infiltration in the absence of RAS inhibitors or inhibitor combinations as disclosed herein, the one or more inhibitors are administered to change the anti-tumor immune response. In some embodiments, relative to the tumor immune infiltration in the absence of RAS inhibitors or inhibitor combinations as disclosed herein, the one or more inhibitors are administered to change the tumor microenvironment. In some embodiments, the administration of RAS inhibitors or inhibitor combinations as disclosed herein converts immune cold tumors into immune hot tumors. In some embodiments, the method reduces tumor size or inhibits tumor growth.

[0171] In some embodiments, the immune-refractory lung cancer is non-small cell lung cancer or small cell lung cancer. In some embodiments, the immune-refractory lung cancer comprises a Ras mutation. In some embodiments, the Ras mutation is K-Ras G12C, H-RasC12C, or N-Ras G12C. In some embodiments, the Ras mutation is K-Ras G12C.

[0172] In particular, it is contemplated that any limitation discussed with respect to one embodiment of the present disclosure may apply to any other embodiment of the present disclosure. In addition, any compound or composition of the present disclosure may be used in any method of the present disclosure, and any method of the present disclosure may be used to produce or utilize any compound or composition of the present disclosure. BRIEF DESCRIPTION OF THE DRAWINGS

[0173] Figure 1 To show the mouse syngeneic Lewis Lung (eLL2) KRAS WT / G12C NRAS - / - Figure 2. Tumor immunophenotype of A2 tumor. eLL2KRAS WT / G12C NRAS - / - The composition of A2 tumors was: an average of 2.37% T cells (CD8+, CD4+, and gdT cells), 0.35% B cells (CD19+), 1.38% NK cells (NKp46+), 3.35% dendritic cells (CD11c+ / MHCII 高 ), 39.72% myeloid cells (Ly6G+ and Ly6C+), 8.5% macrophages (F4 / 80+), 6.52% other CD45+ cells and 37.79% CD45- cells.

[0174] Figure 2AeLL2KRAS WT / G12C NRAS - / - Representative immunohistochemical staining of CD8+ cells in A2 tumors. Arrows show positive staining and scale bar represents 100 μm.

[0175] Figure 2B Shown is quantification of 4 tumors displaying an immune desert-type tumor microenvironment, with an average of 0.225% cytotoxic T cells infiltrating the tumor.

[0176] Figure 3A Murine isogenic eLL2 KRAS in mice treated with vehicle and isotype control is shown. WT / G12C NRAS - / - In vivo tumor cell growth in the A2 model.

[0177] Figure 3B Murine isogenic eLL2 KRAS in mice treated with Compound A and isotype control are shown. WT / G12C NRAS - / - In vivo tumor cell growth in the A2 model.

[0178] Figure 3C Murine isogenic eLL2 KRAS in mice treated with RMC-4550 and isotype control are shown. WT / G12C NRAS - / - In vivo tumor cell growth in the A2 model.

[0179] Figure 3D Shown are the murine isogenic eLL2KRAS in mice treated with Compound A, RMC-4550, and isotype control. WT / G12C NRAS - / - In vivo tumor cell growth in the A2 model.

[0180] Figure 3E Murine isogenic eLL2 KRAS in mice treated with vehicle and anti-PD-1 is shown WT / G12C NRAS - / - In vivo tumor cell growth in the A2 model.

[0181] Figure 3F Murine isogenic eLL2 KRAS in mice treated with Compound A and anti-PD-1 is shown WT / G12C NRAS - / - In vivo tumor cell growth in the A2 model.

[0182] Figure 3G Murine isogenic eLL2 KRAS in mice treated with RMC-4550 and anti-PD-1 is shownWT / G12C NRAS - / - In vivo tumor cell growth in the A2 model.

[0183] Figure 3H Murine isogenic eLL2KRAS in mice treated with Compound A, RMC-4550, and anti-PD-1 are shown. WT / G12C NRAS - / - In vivo tumor cell growth in the A2 model.

[0184] Fig. 3I Shown are the percentages of tumors that increased less than two-fold in volume over time compared to baseline following treatment with RMC-4550, Compound A, or both.

[0185] Figure 3J FIGURE 5 is a graph showing the percentage of tumors that increased less than two-fold in volume compared to baseline over time following treatment with RMC-4550, Compound A, anti-PD-1, or a combination thereof.

[0186] Figure 3K Shown is the percent change in body weight over time after tumor implantation following treatment with RMC-4550, Compound A, or both. Treatment was well tolerated based on body weight measurements.

[0187] Figure 3L Shown is the percent change in body weight over time after tumor implantation following treatment with RMC-4550, Compound A, anti-PD-1, or their combination. Treatment was well tolerated based on body weight measurements.

[0188] Figure 4A The graph shows that compound A, double combination with RMC-4550 or anti-PD-1, and triple combination significantly increased the infiltration of CD8+ T cells.

[0189] Figure 4B It was shown that compound A, double combination with RMC-4550 or anti-PD-1, and triple combination significantly increased the infiltration of CD4+ T cells.

[0190] Figure 4C Graph showing that monotherapy and combination therapy with Compound A and RMC-4550 significantly reduced Ly6G+ myeloid suppressor cells.

[0191] Figure 5A The graphic shows that the double combination of Compound A and RMC-4550 or the triple combination with anti-PD-1 increased the proportion of CD8+ T cells secreting granzyme B.

[0192] Figure 5BIt was shown that the double combination of Compound A and RMC-4550 or the triple combination with anti-PD-1 resulted in an increase in the proportion of CD107a+CD8+ T cells.

[0193] Figure 5C It is a graph showing that the double combination of Compound A and RMC-4550 or the triple combination with anti-PD-1 increases the proportion of TNFα+CD8+ T cells.

[0194] Fig. 6A Graph shows IHC quantification of T cell infiltration after 4 days of treatment with Compound A and combination or triple combination with RMC-4550, anti-PD-1 showing a significant increase in CD8+ T cells.

[0195] Figure 6B Shown is IHC quantification of T cell infiltration after 4 days of treatment with Compound A and combination or triple combination with RMC-4550, anti-PD-1 showing a significant increase in CD4+ T cells. DETAILED DESCRIPTION

[0196] The present disclosure generally relates to compositions and methods for treating immune-refractory lung cancer. The present disclosure is based at least in part on the following observations: treating immune-refractory lung cancer with a compound that inhibits mutant RAS G12C protein makes the cancer sensitive to treatment with an immunotherapeutic agent. In some embodiments, the compound inhibits RAS with a carcinogenic G12C mutation. In some embodiments, the RAS inhibitor is a covalent inhibitor, for example, the covalent inhibitor is capable of forming a covalent bond with an oncogenic mutant form of RAS G12C at the G12C position. In some embodiments, treatment with a RAS inhibitor makes the cancer sensitive to treatment with an immune checkpoint inhibitor or a SHP2 inhibitor. In some embodiments, a compound or a combination of compounds described herein is administered to a subject who has previously failed immunotherapy treatment (such as treatment with an immune checkpoint inhibitor).

[0197] Other aspects of the disclosure are described below.

[0198] General approach

[0199] The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of cell culture, molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the skill of the art.Such techniques are fully explained in the literature, such as Molecular Cloning: A Laboratory Manual, 3rd Edition (Sambrook et al., 2001) Cold Spring Harbor Press; Oligonucleotide Synthesis (P. Herdewijn, ed., 2004); Animal Cell Culture (RI Freshney, ed., 1987); Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (DM Weir and CC Blackwell, ed.); Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, 1987); Current Protocols in Molecular Biology (FM Ausubel, et al., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., 1994); Current Protocols in Immunology (JE Coligan, et al., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Manual of Clinical Laboratory Immunology (B. Detrick, N.R. Rose and J.D. Folds, 2006); Immunochemical Protocols (J. Pound, 2003); Lab Manual in Biochemistry: Immunology and Biotechnology (A. Nigam and A. Ayyagari, 2007); Immunology Methods Manual: The Comprehensive Sourcebook of Techniques (Ivan Lefkovits, 1996); Using Antibodies: A Laboratory Manual (E. Harlow and D. Lane, 1988); and other literature.

[0200] definition

[0201] In this application, unless the context indicates otherwise, (i) the term "a" means "one or more"; (ii) the term "or" is used to mean "and / or" (unless explicitly indicated to refer to only alternatives or the alternatives are mutually exclusive), although the present disclosure supports definitions referring only to alternatives and "and / or"; (iii) the terms "comprising" and "including" are understood to cover the listed components or steps presented alone or together with one or more other components or steps; and (iv) where a range is provided, the endpoints are included.

[0202] As used herein, the term "about" is used to indicate that a value includes the standard deviation of the error of the device or method used to determine the value. In certain embodiments, the term "about" refers to a range of values ​​of 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less in either direction (greater than or less than), unless otherwise stated or otherwise apparent from the context (e.g., where such numbers will exceed 100% of possible values).

[0203] As used herein, in the context of describing adjacent atoms, the term "adjacent" refers to divalent atoms that are directly connected by a covalent bond.

[0204] Whether or not explicitly mentioned, "compounds of the present invention" and similar terms as used herein refer to the Ras inhibitors described herein, including compounds of Formula I and its subformulae and compounds of Tables 1 and 2, as well as their salts (e.g., pharmaceutically acceptable salts), solvates, hydrates, stereoisomers (including atropisomers) and tautomers.

[0205] Those skilled in the art will appreciate that certain compounds described herein may exist in one or more different isomers (e.g., stereoisomers, geometric isomers, atomers, tautomers) or isotopic forms (e.g., wherein one or more atoms have been substituted with different isotopes of atoms, such as hydrogen for deuterium). Unless otherwise indicated or clear from the context, the structures shown may be understood to represent any such isomers or isotopic forms, either individually or in combination.

[0206] The compounds described herein may be asymmetric (e.g., have one or more stereocenters). Unless otherwise indicated, all stereoisomers, such as enantiomers and diastereomers, are intended. Compounds of the present disclosure containing asymmetrically substituted carbon atoms can be separated in optically active or racemic forms. Methods for preparing optically active forms from optically active starting materials are known in the art, such as by resolving racemic mixtures or by stereoselective synthesis. Many geometric isomers of olefins, C=N double bonds, etc. may also be present in the compounds described herein, and all such stable isomers are encompassed in the present disclosure. Cis and trans geometric isomers of the compounds of the present disclosure are described, and can be separated as a mixture of isomers or as separated isomeric forms.

[0207] In some embodiments, one or more compounds presented herein may exist in different tautomeric forms. As will be clear from the context, unless expressly excluded, reference to such compounds encompasses all such tautomeric forms. In some embodiments, tautomeric forms result from the exchange of a single bond with an adjacent double bond and the concomitant migration of a proton. In certain embodiments, tautomeric forms may be prototropic tautomers, which are isomeric protonation states having the same empirical formula and total charge as the reference form. Examples of moieties with prototropic tautomeric forms are keto-enol pairs, amide-imidic acid pairs, lactam-lactim pairs, amide-imidic acid pairs, enamine-imine pairs, and cyclic forms in which a proton can occupy two or more positions of a heterocyclic ring system, such as 1H-imidazole and 3H-imidazole, 1H-1,2,4-triazole, 2H-1,2,4-triazole and 4H-1,2,4-triazole, 1H-isoindole and 2H-isoindole, and 1H-pyrazole and 2H-pyrazole. In some embodiments, the tautomeric forms may be in equilibrium or sterically locked into one form by appropriate substitution. In certain embodiments, the tautomeric forms are caused by acetal interconversion.

[0208] Unless otherwise stated, structures shown herein are also meant to include compounds that differ only in the presence of one or more isotopically enriched atoms. Exemplary isotopes that can be incorporated into the compounds of the present disclosure include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, chlorine, and iodine, such as 2 H. 3 H. 11 C. 13 C. 14 C. 13 N. 15 N. 15 O. 17 O. 18 O. 32 P. 33 P.35 S. 18 F. 36 Cl, 123 I and 125 I. Isotope-labeled compounds (e.g. 3 H and 14 C-labeled) can be used in compound or substrate tissue distribution assays. 3 H) and carbon-14 (i.e., 14 C) isotopes can be useful due to their ease of preparation and detectability. In addition, heavier isotopes (such as deuterium, i.e. 2 H) substitution may provide certain therapeutic advantages due to greater metabolic stability (e.g., increased in vivo half-life or reduced dosage requirements). In some embodiments, one or more hydrogen atoms are replaced by 2 H or 3 H, or one or more carbon atoms are replaced by 13 C or 14 C-enriched carbon replacement. Positron-emitting isotopes (such as 15 O. 13 N. 11 C and 18 F) can be used for positron emission tomography (PET) studies to examine substrate receptor occupancy. The preparation of isotopically labeled compounds is known to those skilled in the art. For example, isotopically labeled compounds can generally be prepared by following procedures similar to those disclosed for the compounds of the present disclosure described herein, by substituting an isotopically labeled reagent for a non-isotopically labeled reagent.

[0209] Non-limiting examples of compounds of the present invention that may contain one or more deuterium-substituted moieties (wherein "R" at any position may be deuterium (D)) include as well as Other examples include R 1 Type part such as as well as The deuterated portion of R 1 The definition of can be found herein. Also contemplated are deuterated moieties within the substituent W in the compounds of the invention, wherein W is as defined herein (see, e.g., Formula I and its subformulae and specific examples of W described herein, such as as well as ). In addition, deuteration of available positions in any A portion of the compounds of the formulae described herein is also contemplated, such as as well as Additionally, deuterium substitution may also be performed at the linker position in the compounds of the present invention, such as

[0210] In another embodiment, silylation substitutions are also contemplated, such as the following in the linker:

[0211]

[0212] As is known in the art, many chemical entities can take a variety of different solid forms, such as amorphous forms or crystalline forms (e.g., polymorphs, hydrates, solvates). In some embodiments, the compounds of the present disclosure can be in any such form, including use in any solid form. In some embodiments, the compounds described or illustrated herein can be provided or used in the form of hydrates or solvates.

[0213] At various places in this specification, substituents of compounds of the present disclosure are disclosed in groups or in ranges. The present disclosure is specifically intended to include every individual subcombination of the members of such groups and ranges. For example, the term "C 1 -C 6 "Alkyl" specifically intends to disclose individually methyl, ethyl, C 3 Alkyl, C 4 Alkyl, C 5 Alkyl and C 6 In addition, where compounds include multiple positions where substituents are disclosed as groups or in ranges, unless otherwise indicated, the disclosure is intended to cover individual compounds and groups of compounds (e.g., classes and subclasses) containing every individual subcombination of members at each position.

[0214] The term "optionally substituted X" (e.g., "optionally substituted alkyl") is intended to be equivalent to "X, wherein X is optionally substituted" (e.g., "alkyl, wherein the alkyl is optionally substituted"). The feature "X" (e.g., alkyl) itself is not intended to be meant to be optional. As described herein, certain compounds of interest may contain one or more "optionally substituted" moieties. In general, the term "substituted", whether preceded by the term "optionally" or not, means that one or more hydrogens of the designated moiety are replaced with a suitable substituent, such as any of the substituents or groups described herein. Unless otherwise indicated, an "optionally substituted" group may have a suitable substituent at each substitutable position of the group, and when more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituent at each position may be the same or different. For example, in the term "optionally substituted C 1 -C 6 Alkyl-C 2 -C 9In the "heteroaryl" group, the alkyl portion, the heteroaryl portion, or both may be optionally substituted. Combinations of substituents envisioned by the present disclosure are preferably those that result in the formation of stable or chemically feasible compounds. As used herein, the term "stable" refers to compounds that are not substantially altered when subjected to conditions that allow the compound to be produced, detected, and in certain embodiments, recovered, purified, and used for one or more of the purposes disclosed herein.

[0215] Suitable monovalent substituents on the substitutable carbon atoms of the "optionally substituted" group may independently be deuterium; halogen; -(CH 2 ) 0-4 R o ;-(CH 2 ) 0-4 OR o ; -O(CH 2 ) 0-4 R o ;-O-(CH 2 ) 0-4 C(O)OR o ;-(CH 2 ) 0-4 CH(OR o ) 2 ;-(CH 2 ) 0- 4 SR o ;-(CH 2 ) 0-4 Ph, which can be used R o Substitution; -(CH 2 ) 0-4 O(CH 2 ) 0-1 Ph, which can be used R o Substitution; -CH=CHPh, which can be used R o Substitution; -(CH 2 ) 0-4 O(CH 2 ) 0-1 -pyridyl, which can be used with R o Substituted; 4-8 membered saturated or unsaturated heterocycloalkyl (eg pyridyl); 3-8 membered saturated or unsaturated cycloalkyl (eg cyclopropyl, cyclobutyl or cyclopentyl); -NO 2 ;-CN;-N 3 ;-(CH 2 ) 0-4 N(R o ) 2 ;-(CH 2 ) 0-4 N(R o )C(O)R o ;-N(Ro )C(S)R o ;-(CH 2 ) 0-4 N(R o )C(O)NR o 2 ;-N(R o )C(S)NR o 2 ;-(CH 2 ) 0-4 N(R o )C(O)OR o ;-N(R o )N(R o )C(O)R o ;-N(R o )N(R o )C(O)NR o 2 ;-N(R o )N(R o )C(O)OR o ;-(CH 2 ) 0-4 C(O)R o ;-C(S)R o ;-(CH 2 ) 0-4 C(O)OR o ;-(CH 2 ) 0-4 -C(O)-N(R o ) 2 ;-(CH 2 ) 0-4 -C(O)-N(R o )-S(O) 2 -R o ;-C(NCN)NR o 2 ;-(CH 2 ) 0-4 C(O)SR o ;-(CH 2 ) 0-4 C(O)OSiR o 3 ;-(CH 2 ) 0-4 OC(O)R o ;-OC(O)(CH 2 ) 0-4 SR o ;-SC(S)SR o ;-(CH 2 ) 0-4 SC(O)Ro ;-(CH 2 ) 0-4 C(O)NR o 2 ;-C(S)NR o 2 ;-C(S)SR o ;-(CH 2 ) 0- 4 OC(O)NR o 2 ;-C(O)N(OR o )R o ;-C(O)C(O)R o ;-C(O)CH 2 C(O)R o ;-C(NOR o )R o ;-(CH 2 ) 0-4 SSR o ;-(CH 2 ) 0 - 4 S(O) 2 R o ;-(CH 2 ) 0-4 S(O) 2 OR o ;-(CH 2 ) 0-4 OS(O) 2 R o ;-S(O) 2 NR o 2 ;-(CH 2 ) 0-4 S(O)R o ;-N(R o )S(O) 2 NR o 2 ;-N(R o )S(O) 2 R o ;-N(OR o )R o ;-C(NOR o )NR o 2 ;-C(NH)NR o 2 ;-P(O) 2 R o ;-P(O)R o 2 ;-P(O)(ORo ) 2 ;-OP(O)R o 2 ;-OP(O)(OR o ) 2 ;-OP(O)(OR o )R o 、-SiR o 3 ;-(C 1-4 Straight chain or branched chain alkylene)ON(R o ) 2 ; or -(C 1-4 Straight or branched chain alkylene)C(O)ON(R o ) 2 , where each R o may be substituted as defined below and are independently hydrogen, -C 1-6 Aliphatic group, -CH 2 Ph, -O(CH 2 ) 0-1 Ph, -CH 2 -(5-6 membered heteroaryl ring) or a 3-6 membered saturated, partially unsaturated or aromatic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen or sulfur, or, although defined above, two independent occurrences of R o Together with one or more of its central atoms, it forms a 3-12 membered saturated, partially unsaturated or aryl monocyclic or bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen or sulfur, which monocyclic or bicyclic ring may be substituted as defined below.

[0216] R o (or two independent occurrences of R o Suitable monovalent substituents on the ring formed by the middle atom thereof may independently be halogen, -(CH 2 ) 0-2 R l 、-(halogen R l )、-(CH 2 ) 0-2 OH, -(CH 2 ) 0-2 OR l 、-(CH 2 ) 0-2 CH(OR l ) 2 ;-O(haloR l ), -CN, -N 3 、-(CH 2 ) 0-2 C(O)R l 、-(CH 2 )0-2 C(O)OH, -(CH 2 ) 0-2 C(O)OR l 、-(CH 2 ) 0-2 SR l 、-(CH 2 ) 0-2 SH, -(CH 2 ) 0-2 NH 2 、-(CH 2 ) 0-2 NHR l 、-(CH 2 ) 0-2 NR l 2 、-NO 2 、-SiR l 3 、-OSiR l 3 、-C(O)SR l 、-(C 1-4 Straight or branched chain alkylene) C(O)OR l or -SSR l , where each R l is unsubstituted or, when preceded by "halo", is substituted only with one or more halogens, and is independently selected from C 1-4 Aliphatic group, -CH 2 Ph, -O(CH 2 ) 0-1 Ph or a 5-6 membered saturated, partially unsaturated or aromatic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen or sulfur. o Suitable divalent substituents on a saturated carbon atom of include =0 and =S.

[0217] Suitable divalent substituents on a saturated carbon atom of an "optionally substituted" group include the following substituents: =0, =S, =NNR * 2 、=NNHC(O)R * 、=NNHC(O)OR * 、=NNHS(O) 2 R * , =NR * , =NOR * 、-O(C(R * 2 )) 2-3 O-or-S(C(R * 2 )) 2-3 S-, where each independent occurrence of R* is selected from hydrogen, C which may be substituted as defined below 1-6 an aliphatic group or an unsubstituted 5-6 membered saturated, partially unsaturated or aromatic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen or sulfur. Suitable divalent substituents attached to the adjacent substitutable carbon of the "optionally substituted" group include: -O(CR * 2 ) 2-3 O-, where each independent occurrence of R * is selected from hydrogen, C which may be substituted as defined below 1-6 an aliphatic group or an unsubstituted 5-6 membered saturated, partially unsaturated or aromatic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen or sulfur.

[0218] R * Suitable substituents on the aliphatic group include halogen, -R l 、-(halogen R l ), -OH, -OR l 、-O(halogen R l )、-CN、-C(O)OH、-C(O)OR l 、-NH 2 、-NHR l 、-NR l 2 or-NO 2 , where each R l is unsubstituted or, when preceded by "halo", is substituted only with one or more halogens, and is independently C 1-4 Aliphatic group, -CH 2 Ph, -O(CH 2 ) 0 - 1 Ph or a 5-6 membered saturated, partially unsaturated or aromatic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen or sulfur.

[0219] Suitable substituents on a substitutable nitrogen of an "optionally substituted" group include or Each of these are independently hydrogen, C 1-6 an aliphatic group, an unsubstituted -OPh group or an unsubstituted 3-6 membered saturated, partially unsaturated or aromatic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen or sulfur, or, although defined above, two independent occurrences of Together with one or more of its central atoms, it forms an unsubstituted 3-12 membered saturated, partially unsaturated or aryl monocyclic or bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen or sulfur.

[0220] Suitable substituents on the aliphatic group are independently halogen, -R l 、-(halogen R l ), -OH, -OR l 、-O(halogen R l )、-CN、-C(O)OH、-C(O)OR l 、-NH 2 、-NHR l 、-NR l 2 or-NO 2 , where each R l is unsubstituted or, when preceded by "halo", is substituted only with one or more halogens, and is independently C 1-4 Aliphatic group, -CH 2 Ph, -O(CH 2 ) 0-1 Ph or a 5-6 membered saturated, partially unsaturated or aromatic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen or sulfur. Suitable divalent substituents on a saturated carbon atom of include =0 and =S.

[0221] Those skilled in the art who read this disclosure will understand that certain compounds described herein may be provided or used in any of a variety of forms, such as salt forms, protected forms, prodrug forms, ester forms, isomeric forms (e.g., optical or structural isomers), isotopic forms, and the like. In some embodiments, reference to a particular compound may refer to a particular form of that compound. In some embodiments, reference to a particular compound may refer to any form of that compound. In some embodiments, for example, a preparation of a single stereoisomer of a compound may be considered to be a different form of the compound than a racemic mixture of the compound; a particular salt of a compound may be considered to be a different form than another salt form of the compound; a preparation containing one conformational isomer ((Z) or (E)) of a double bond may be considered to be a different form than the form of the other conformational isomer ((E) or (Z)) containing the double bond; a preparation in which one or more atoms are different isotopes than those present in a reference preparation may be considered to be a different form.

[0222] As used herein, the term "administering" refers to administering a composition (e.g., a compound, or a formulation including a compound as described herein) to a subject or system. Administration also includes administering to a subject a prodrug derivative or analog of a compound or a pharmaceutically acceptable salt of a compound or composition, which can form an equivalent amount of active compound in the subject. Administration to an animal subject (e.g., to a human) can be performed by any appropriate route. For example, in some embodiments, administration can be transbronchial (including by bronchial instillation), buccal, enteral, intradermal, intraarterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, transmucosal, nasal, oral, rectal, subcutaneous, sublingual, surface, transtracheal (including by intratracheal instillation), transdermal, vaginal or vitreous.

[0223] As used herein, the term "acetyl" refers to the group -C(O)CH 3 .

[0224] As used herein, the term "alkoxy" refers to -OC 1 -C 20 Alkyl groups, wherein the alkoxy group is attached to the rest of the compound through an oxygen atom.

[0225] As used herein, the term "alkyl" refers to a saturated, straight or branched chain monovalent hydrocarbon group containing 1 to 20 (e.g., 1 to 10 or 1 to 6) carbons. In some embodiments, the alkyl group is a non-branched chain (i.e., a straight chain); in some embodiments, the alkyl group is a branched chain. Examples of alkyl groups are, but are not limited to, methyl, ethyl, n-propyl and isopropyl, n-butyl, sec-butyl, isobutyl and tert-butyl, and neopentyl.

[0226] As used herein, the term "alkylene" means a saturated divalent hydrocarbon group derived from a straight or branched chain saturated hydrocarbon by removing two hydrogen atoms, and is exemplified by methylene, ethylene, isopropylene, and the like. x -C y "Alkylene" means an alkylene group having x to y carbons. Exemplary values ​​for x are 1, 2, 3, 4, 5, and 6, and exemplary values ​​for y are 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, or 20 (e.g., C 1 -C 6 , C 1 -C 10 , C 2 -C 20 , C 2 -C 6 , C 2 -C 10 or C 2 -C 20In some embodiments, the alkylene group may be further substituted with 1, 2, 3, or 4 substituents as defined herein.

[0227] Unless otherwise specified, as used herein, the term "alkenyl" refers to a monovalent straight or branched chain group of 2 to 20 carbons (e.g., 2 to 6 or 2 to 10 carbons) containing one or more carbon-carbon double bonds and is exemplified by vinyl, 1-propenyl, 2-propenyl, 2-methyl-1-propenyl, 1-butenyl, and 2-butenyl. Alkenyl includes both cis and trans isomers. Unless otherwise specified, as used herein, the term "alkenylene" refers to a divalent straight or branched chain group of 2 to 20 carbons (e.g., 2 to 6 or 2 to 10 carbons) containing one or more carbon-carbon double bonds.

[0228] As used herein, the term "alkynyl" refers to a monovalent straight or branched chain group of 2 to 20 carbon atoms (eg, 2 to 4, 2 to 6, or 2 to 10 carbons) containing a carbon-carbon triple bond and is exemplified by ethynyl and 1-propynyl.

[0229] As used herein, the term "alkynyl sulfone" refers to a group comprising the structure: wherein R is any chemically feasible substituent described herein.

[0230] As used herein, the term "amino" refers to For example -NH 2 and -N(CH 3 ) 2 .

[0231] As used herein, the term "aminoalkyl" refers to an alkyl moiety substituted on one or more carbon atoms with one or more amino moieties.

[0232] As used herein, the term "amino acid" refers to a amino acid having a side chain, an amino group and an acid group (e.g., -CO 2 H or -SO 3 H) molecules, wherein the amino acid is linked to the parent molecular group through a side chain, an amino group or an acid group (e.g., a side chain). As used herein, the term "amino acid" broadly refers to any compound or substance that can be incorporated into a polypeptide chain, such as by forming one or more peptide bonds. In some embodiments, an amino acid has a general structure H 2NC(H)(R)-COOH. In some embodiments, the amino acid is a naturally occurring amino acid. In some embodiments, the amino acid is a synthetic amino acid; in some embodiments, the amino acid is a D-amino acid; in some embodiments, the amino acid is an L-amino acid. "Standard amino acids" refers to any of the twenty standard L-amino acids commonly found in naturally occurring peptides. Exemplary amino acids include alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, optionally substituted hydroxynorvaline, isoleucine, leucine, lysine, methionine, norvaline, ornithine, phenylalanine, proline, pyrrolysine, selenocysteine, serine, taurine, threonine, tryptophan, tyrosine, and valine.

[0233] As used herein, "amino acid substitution" refers to the substitution of a wild-type amino acid of a protein with a non-wild-type amino acid. Amino acid substitutions may be caused by genetic mutations and may alter one or more properties of a protein (e.g., may confer altered binding affinity or specificity, altered enzymatic activity, altered structure, or altered function).

[0234] As used herein, the term "aryl" refers to a monovalent monocyclic, bicyclic or polycyclic system formed by carbon atoms, wherein the ring connected to the side group is aromatic. Examples of aryl are phenyl, naphthyl, phenanthrenyl and anthracenyl. The aromatic ring can be connected to its side group at any heteroatom or carbocyclic atom that produces a stable structure, and unless otherwise specified, any one of the ring atoms can be optionally substituted.

[0235] As used herein, the term “C 0 " represents a bond. For example, the term -N(C(O)-(C 0 -C 5 The alkylene-H)- moieties include -N(C(O)-(C 0 Alkylene-H)-, the group is also represented by -N(C(O)-H)-.

[0236] As used herein, the terms "carbocycle" and "carbocyclyl" refer to a monovalent optionally substituted C 3 -C 12 Monocyclic, bicyclic or tricyclic structure, the ring structure can be a bridged ring, a condensed ring or a spirocyclic ring, wherein all rings are formed by carbon atoms and at least one ring is non-aromatic.Carbocyclic structure includes cycloalkyl, cycloalkenyl and cycloalkynyl.The example of carbocyclic radical is cyclohexyl, cyclohexenyl, cyclooctynyl, 1,2-dihydronaphthyl, 1,2,3,4-tetrahydronaphthyl, fluorenyl, indenyl, indanyl, decahydronaphthyl etc.Carbocyclic ring can be connected to its side group at any ring atom producing a stable structure, and unless otherwise specified, any one of the ring atoms can be optionally substituted.

[0237] As used herein, the term "carbonyl" refers to a C(O) group, which may also be represented as C=O.

[0238] As used herein, the term "carboxyl" refers to -CO 2 H, (C=O)(OH), COOH or C(O)OH or the unprotonated counterpart.

[0239] The term "combination therapy" refers to a method of treatment, which includes optionally administering at least two therapeutic agents to a subject in the form of one or more pharmaceutical compositions as part of a treatment regimen. For example, combination therapy may include administering a single pharmaceutical composition, which includes at least two therapeutic agents and one or more pharmaceutically acceptable carriers, excipients, diluents or surfactants. Combination therapy may include administering two or more pharmaceutical compositions, each composition including one or more therapeutic agents and one or more pharmaceutically acceptable carriers, excipients, diluents or surfactants. Two or more agents may optionally be administered simultaneously (as a single composition or as a separate composition) or sequentially (as a separate composition). The therapeutic agent may be administered in an effective amount. The therapeutic agent may be administered in a therapeutically effective amount. In some embodiments, compared to the therapeutic amount of the same therapeutic agent when used as a monotherapy, the effective amount of one or more of the therapeutic agents when used in combination therapy may be lower, for example, due to the additive or synergistic effect of combining two or more therapeutic agents.

[0240] As used herein, the term "cyano" refers to a -CN group.

[0241] As used herein, the term "cycloalkyl" refers to a monovalent saturated cyclic hydrocarbon group which may be a bridged, fused or spiro ring having three to eight ring carbons unless otherwise specified, and is exemplified by cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cycloheptyl.

[0242] As used herein, the term "cycloalkenyl" refers to a monovalent, non-aromatic, saturated cyclic hydrocarbon group which may be a bridged, fused or spiro ring having from three to eight ring carbons and containing one or more carbon-carbon double bonds unless otherwise specified.

[0243] As used herein, the term "diastereoisomers" means stereoisomers that are not mirror images of each other and are not superimposable on each other.

[0244] As used herein, the term "dosage form" refers to a physical discrete unit of a compound (e.g., a compound of the present disclosure) for administration to a subject. Each unit contains a predetermined amount of compound. In some embodiments, such an amount is a unit dosage amount (or its entire portion) suitable for administration according to a dosage regimen associated with a desired or beneficial result when administered to a relevant population (i.e., using a therapeutic dosage regimen). It is understood by those skilled in the art that the total amount of a therapeutic composition or compound administered to a particular subject is determined by one or more attending physicians and may involve the administration of multiple dosage forms.

[0245] As used herein, the term "dosage regimen" refers to a group of unit doses (usually more than one) that are usually administered to a subject separately by time periods. In some embodiments, a given therapeutic compound (e.g., a compound of the present disclosure) has a suggested dosing regimen that may involve one or more doses. In some embodiments, the dosing regimen includes multiple doses, each of which is separated from each other by a time period of the same length; in some embodiments, the dosing regimen includes multiple doses and at least two different time periods separate individual doses. In some embodiments, all doses within the dosing regimen have the same unit dose amount. In some embodiments, different doses within the dosing regimen have different amounts. In some embodiments, the dosing regimen includes a first dose of a first dose amount, followed by one or more other doses of a second dose amount different from the first dose amount. In some embodiments, the dosing regimen includes a first dose of a first dose amount, followed by one or more other doses of a second dose amount that is the same as the first dose amount. In some embodiments, the dosing regimen is associated with a desired or beneficial outcome when administered in a related population (i.e., a therapeutic dosing regimen).

[0246] Unless otherwise indicated, the term "disorder" is used in this disclosure to mean a disease, illness, or condition and is used interchangeably with the terms disease, illness, or condition.

[0247] As used herein, the term "enantiomer" means each individual optically active form of a compound of the invention having an optical purity or enantiomeric excess (as determined by methods standard in the art) of at least 80% (i.e., at least 90% of one enantiomer and at most 10% of the other enantiomer), preferably at least 90% and more preferably at least 98%.

[0248] The term "guanidino" refers to a group having the following structure: wherein each R is independently any chemically feasible substituent described herein.

[0249] As used herein, the term "guanidinoalkylalkyl" refers to an alkyl moiety substituted on one or more carbon atoms with one or more guanidino moieties.

[0250] As used herein, the term "haloacetyl" refers to an acetyl group in which at least one hydrogen has been replaced by a halogen.

[0251] As used herein, the term "haloalkyl" refers to an alkyl moiety substituted on one or more carbon atoms with one or more same or different halogen moieties.

[0252] As used herein, the term "halogen" denotes a halogen selected from bromine, chlorine, iodine or fluorine.

[0253] As used herein, the term "heteroalkyl" refers to an "alkyl" as defined herein, wherein at least one carbon atom has been replaced with a heteroatom (eg, O, N or S atom). The heteroatom may be present in the middle or at the terminal of the group.

[0254] As used herein, the term "heteroaryl" refers to a monovalent monocyclic or polycyclic structure containing at least one fully aromatic ring: that is, it contains 4n+2 π electrons in the monocyclic or polycyclic system and contains at least one ring heteroatom selected from N, O or S in the aromatic ring. Exemplary unsubstituted heteroaryl groups have 1 to 12 (e.g., 1 to 11, 1 to 10, 1 to 9, 2 to 12, 2 to 11, 2 to 10 or 2 to 9) carbons. The term "heteroaryl" includes bicyclic, tricyclic and tetracyclic groups, wherein any of the above heteroaromatic rings is fused to one or more aromatic or carbocyclic rings, such as a phenyl ring or a cyclohexane ring. Examples of heteroaryl include, but are not limited to, pyridyl, pyrazolyl, benzoxazolyl, benzimidazolyl, benzothiazolyl, imidazolyl, thiazolyl, quinolyl, tetrahydroquinolyl and 4-azaindolyl. The heteroaryl ring may be attached to its pendant group at any ring atom that results in a stable structure, and unless otherwise specified, any of the ring atoms may be optionally substituted. In some embodiments, the heteroaryl group is substituted with 1, 2, 3, or 4 substituents.

[0255] As used herein, the term "heterocycloalkyl" refers to a monovalent monocyclic, bicyclic or polycyclic system that can be a bridged ring, a fused ring or a spirocyclic ring, wherein at least one ring is non-aromatic and wherein the non-aromatic ring contains one, two, three, four heteroatoms independently selected from the group consisting of nitrogen, oxygen and sulfur. The 5-membered ring has zero to two double bonds, and the 6-membered and 7-membered rings have zero to three double bonds. Exemplary unsubstituted heterocycloalkyls have 1 to 12 (e.g., 1 to 11, 1 to 10, 1 to 9, 2 to 12, 2 to 11, 2 to 10 or 2 to 9) carbons. The term "heterocycloalkyl" also refers to a heterocyclic compound having a bridged polycyclic structure in which one or more carbons or heteroatoms bridge two non-adjacent members of a monocyclic ring, such as quinuclidine. The term "heterocycloalkyl" includes bicyclic, tricyclic and tetracyclic groups, wherein any of the above heterocycles is fused to one or more aromatic rings, carbocyclic rings, heteroaromatic rings or heterocyclic rings, such as aromatic rings, cyclohexane rings, cyclohexene rings, cyclopentane rings, cyclopentene rings, pyridine rings or pyrrolidine rings. Examples of heterocycloalkyl are pyrrolidinyl, piperidinyl, 1,2,3,4-tetrahydroquinolinyl, decahydroquinolinyl, dihydropyrrolopyridine and decahydronaphthyridine. The heterocycloalkyl ring can be connected to its side group at any ring atom that produces a stable structure, and unless otherwise specified, any of the ring atoms can be optionally substituted.

[0256] As used herein, the term "hydroxy" refers to an -OH group.

[0257] As used herein, the term "hydroxyalkyl" refers to an alkyl moiety substituted on one or more carbon atoms with one or more -OH moieties.

[0258] As used herein, the term "isomer" means any tautomer, stereoisomer, atropisomer, enantiomer or diastereomer of any compound of the present invention. It should be recognized that the compounds of the present invention may have one or more chiral centers or double bonds, and therefore exist in the form of stereoisomers (e.g., double bond isomers, i.e., geometric E / Z isomers) or diastereomers (e.g., enantiomers (i.e., (+) or (-)) or cis / trans isomers). According to the present invention, the chemical structures shown herein and therefore the compounds of the present invention include all corresponding stereoisomers (i.e., stereoisomerically pure forms, such as geometrically pure, enantiomerically pure or diastereomerically pure) as well as enantiomers and stereoisomer mixtures, such as racemates. Enantiomeric and stereoisomeric mixtures of the compounds of the present invention can usually be separated into their component enantiomers or stereoisomers by well-known methods, such as chiral phase gas chromatography, chiral phase high performance liquid chromatography, crystallization of the compound as a chiral salt complex or crystallization of the compound in a chiral solvent. Enantiomers and stereoisomers can also be obtained from stereoisomerically or enantiomerically pure intermediates, reagents and catalysts by well-known asymmetric synthesis methods.

[0259] As used interchangeably herein, the terms "immune refractory", "immune evasion" or "cold tumor" refer to tumors, cancers or patients with tumors or cancers for which existing immunotherapies (such as immune checkpoint inhibitors) have been found to be ineffective or intolerable. For example, patients with immune-refractory cancers include patients who have previously been administered immunotherapy (such as immune checkpoint inhibitors), and have found that immunotherapy is ineffective or insufficient to effectively slow or stop the progression of the disease or alleviate symptoms associated with disease progression. Immunorefractory cancers include cancers that have become resistant or insensitive to treatment with immunotherapy (e.g., the effectiveness of immunotherapy (such as immune checkpoint inhibitors) previously administered to patients decreases over time). Immunorefractory cancers can be identified by methods known to those skilled in the art or by methods described herein. For example, immune-refractory cancers may be characterized by low immune cell infiltration in one or more tumors. Low immune cell infiltration may include a decrease or absence of lymphocytes; a decrease or absence of tumor infiltrating lymphocytes (TILs); a decrease or absence of dendritic cells; a decrease or absence of myeloid cells; a decrease or absence of natural killer (NK) cells; a decrease or absence of macrophages; a decrease or absence of T cells; a decrease or absence of CD8+ T cells; a decrease or absence of CD4+ T cells; or a decrease or absence of CD4+ / CD8+ T cells. See, for example, Chen and Mellman, Nature, 541:321 (2017). In some embodiments, by contrast, a "hot tumor" refers to a tumor, cancer, or patient with the tumor or cancer that is not immune-refractory. A cancer or tumor with a low cytotoxic T cell count may be characterized as an "immune desert type". In some embodiments, a cancer or tumor with a cytotoxic T cell count of less than 1% of live cells is considered to be an "immune desert type". In some embodiments, a cancer or tumor with a cytotoxic T cell count of less than 0.5% of live cells is considered to be an "immune desert type". In some embodiments, a cancer or tumor with a cytotoxic T cell count of less than 0.25% of live cells is considered to be an "immune desert type".

[0260] As used herein, the term "inhibitor" refers to a compound that prevents a biomolecule (e.g., protein, nucleic acid) from completing or initiating a reaction. An inhibitor may, for example, inhibit a reaction by competitive, non-competitive or non-competitive means. In terms of its binding mechanism, an inhibitor may be an irreversible inhibitor or a reversible inhibitor. Exemplary inhibitors include, but are not limited to, nucleic acids, DNA, RNA, shRNA, siRNA, proteins, protein mimics, peptides, peptidomimetics, antibodies, small molecules, chemicals, analogs of mimicking enzyme binding sites, receptors or other proteins. In some embodiments, an inhibitor is a small molecule, such as a low molecular weight organic compound, such as an organic compound having a molecular weight (MW) of less than 1200 Daltons (Da). In some embodiments, MW is less than 1100Da. In some embodiments, MW is less than 1000Da. In some embodiments, MW is less than 900Da. In some embodiments, the MW of a small molecule ranges between 800Da and 1200Da. Small molecule inhibitors include cyclic and non-cyclic compounds. Small molecule inhibitors include natural products, derivatives thereof, and analogs thereof. Small molecule inhibitors may include covalent cross-linking groups capable of forming covalent cross-links, for example, with amino acid side chains of the target protein.

[0261] As used herein, the term "linker" refers to a divalent organic moiety that connects a first moiety (e.g., a macrocyclic moiety) to a second moiety (e.g., a cross-linking group). In some embodiments, the linker enables the compound to achieve an IC50 of 2uM or less in the Ras-RAF disruption assay provided herein:

[0262] The purpose of this biochemical assay is to measure the ability of a test compound to induce the formation of a ternary complex between nucleotide-loaded Ras isoforms and cyclophilin A; the resulting ternary complex disrupts BRAF RBD The binding of the construct to the cytokine receptor agonist inhibits Ras signaling through the RAF effectors.

[0263] The culture medium contained 25 mM HEPES pH 7.3, 0.002% Tween 20, 0.1% BSA, 100 mM NaCl, and 5 mM MgCl 2 Untagged cyclophilin A, His6-K-Ras-GMPPNP (or other Ras variants), and GST-BRAF RBDCombined in a 384-well assay plate at a final concentration of 25 μM, 12.5 nM, and 50 nM, respectively. The compound was present in the wells of the culture plate in a 10-point 3-fold dilution series starting at a final concentration of 30 μM. After incubation at 25°C for 3 hours, a mixture of anti-HisEu-W1024 and anti-GST allophycocyanin was added to the assay sample wells at final concentrations of 10 nM and 50 nM, respectively, and the reactants were incubated for another 1.5 hours. The TR-FRET signal was read on a microplate reader (Ex 320 nm, Em 665 / 615 nm). Compounds that promote the destruction of the Ras:RAF complex were identified as those compounds that triggered a decrease in the TR-FRET ratio relative to the DMSO control wells.

[0264] In some embodiments, the joint comprises 20 or less straight-chain atoms. In some embodiments, the joint comprises 15 or less straight-chain atoms. In some embodiments, the joint comprises 10 or less straight-chain atoms. In some embodiments, the joint has a molecular weight lower than 500g / mol. In some embodiments, the joint has a molecular weight lower than 400g / mol. In some embodiments, the joint has a molecular weight lower than 300g / mol. In some embodiments, the joint has a molecular weight lower than 200g / mol. In some embodiments, the joint has a molecular weight lower than 100g / mol. In some embodiments, the joint has a molecular weight lower than 50g / mol.

[0265] The term "mutation" as used herein indicates any modification to a nucleic acid or polypeptide that causes a nucleic acid or polypeptide to change. The term "mutation" may include, for example, point mutations, deletions or insertions of single or multiple residues in a polynucleotide, including changes produced in the protein coding region of a gene and changes in regions outside the protein coding sequence (such as, but not limited to, regulatory or promoter sequences), as well as amplification or chromosome breakage or translocation. In a specific embodiment, the mutation causes an amino acid substitution in the encoded protein.

[0266] A "patient" or "subject" is a mammal, eg, a human, mouse, rat, guinea pig, dog, cat, horse, cow, pig, or a non-human primate, such as a monkey, chimpanzee, baboon, or rhesus monkey.

[0267] The terms "prevent" or "preventing" with respect to a subject refer to preventing a disease or condition from afflicting a subject. Prevention includes prophylactic treatment. For example, prevention may include administering a compound disclosed herein to a subject before the subject develops a disease and the administration will prevent the subject from developing the disease.

[0268] As used herein, the term "pharmaceutical composition" refers to a compound, such as a compound of the present disclosure, or a pharmaceutically acceptable salt thereof, formulated together with a pharmaceutically acceptable excipient.

[0269] As used herein, "pharmaceutically acceptable excipient" refers to any inactive ingredient (e.g., a vehicle capable of suspending or dissolving an active compound) having the properties of being non-toxic and non-inflammatory in a subject. Typical excipients include, for example, anti-adhesive agents, antioxidants, binders, coating agents, compression aids, disintegrants, dyes (colorants), emollients, emulsifiers, fillers (diluents), film formers or coating agents, flavoring agents, fragrances, glidants (flow enhancers), lubricants, preservatives, printing inks, adsorbents, suspending or dispersing agents, sweeteners, or water of hydration. Excipients include, but are not limited to, butylated optionally substituted hydroxytoluene (BHT), calcium carbonate, calcium phosphate (diabase), calcium stearate, cross-linked carboxymethylcellulose, cross-linked polyvinyl pyrrolidone, citric acid, cross-linked polyvinylpyrrolidone, cysteine, ethylcellulose, gelatin, optionally substituted hydroxypropylcellulose, optionally substituted hydroxypropylmethylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methylcellulose, methylparaben, microcrystalline cellulose, polyethylene glycol, polyvinylpyrrolidone, povidone, pregelatinized starch, propylparaben, retinyl palmitate, shellac, silicon dioxide, sodium carboxymethylcellulose, sodium citrate, sodium starch glycolate, sorbitol, starch (corn), stearic acid, stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E, vitamin C, and xylitol. Those skilled in the art are familiar with a variety of agents and materials that can be used as excipients. See, e.g., Ansel et al., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems. Philadelphia: Lippincott, Williams & Wilkins, 2004; Gennaro et al., Remington: The Science and Practice of Pharmacy. Philadelphia: Lippincott, Williams & Wilkins, 2000; and Rowe, Handbook of Pharmaceutical Excipients. Chicago, Pharmaceutical Press, 2005. In some embodiments, the composition includes at least two different pharmaceutically acceptable excipients.

[0270] As used herein, the term "pharmaceutically acceptable salt" refers to salts of the compounds described herein that are suitable for contact with the tissues of humans and other animals without excessive toxicity, irritation, allergic response, etc. and in accordance with a reasonable benefit / risk ratio within the scope of reasonable medical judgment. Pharmaceutically acceptable salts are well known in the art. For example, Berge et al., J. Pharmaceutical Sciences 66: 1-19, 1977 and Pharmaceutical Salts: Properties, Selection, and Use, (P. H. Stahl and C. G. Wermuth, ed.), Wiley-VCH, 2008 describe pharmaceutically acceptable salts. The salts can be prepared in situ during the final separation and purification of the compounds described herein or separated by reacting the free base with a suitable organic acid.

[0271] The terms "RAS inhibitor" and "inhibitor of [an] RAS" are used interchangeably to refer to any inhibitor that targets (ie, selectively binds to or inhibits) a RAS protein.

[0272] As used herein, the term "RAS (on) inhibitor" refers to an inhibitor that targets (i.e., selectively binds to or inhibits) the GTP-bound active state of RAS (e.g., selectively binds to or inhibits the GDP-bound inactive state of RAS). The inhibition of the GTP-bound active state of RAS includes, for example, the inhibition of oncogenic signaling from the GTP-bound active state of RAS. In some embodiments, the RAS (on) inhibitor is an inhibitor that selectively binds to and inhibits the GTP-bound active state of RAS. In certain embodiments, the RAS (on) inhibitor may also bind to or inhibit the GDP-bound inactive state of RAS (e.g., with a lower affinity or inhibition constant compared to the GTP-bound active state of RAS). The RAS (on) inhibitor may be a three-complex RAS (on) inhibitor having a mechanism of action that requires the formation of a high-affinity three-component complex between a synthetic ligand (RAS (on) inhibitor) and the following two intracellular proteins that do not interact under normal physiological conditions: the target protein RAS of interest, and the cytoplasmic chaperone protein cyclophilin A widely expressed in cells. See, for example, WO 2021091982. The RAS inhibitors of Formula 0 and Formula I and subformulae thereof herein are triple complex RAS (on) inhibitors.

[0273] As used herein, the term "RAS (off) inhibitor" refers to an inhibitor that targets (i.e., selectively binds to or inhibits) the GDP-bound inactive state of RAS (e.g., selective for the GTP-bound active state of RAS). RAS (off) inhibitors are known in the art. Non-limiting examples of RAS (off) inhibitors include ARS-853, ARS-1620, ERAS-3490, JAB-21822, IBI351 / GFH-925, JDQ443, D-1553, GDC-6036, AMG510, and MRTX849.

[0274] The terms "RAS pathway" and "RAS / MAPK pathway" are used interchangeably herein to refer to the signal transduction cascade downstream of various cell surface growth factor receptors, in which activation of RAS (and its various isoforms and allotypes) is a central event that drives a variety of cellular effector events that determine cell proliferation, activation, differentiation, mobilization, and other functional characteristics. SHP2 delivers positive signals from growth factor receptors to the RAS activation / inactivation cycle, which is regulated by guanine nucleotide exchange factors (GEFs, such as SOS1), which load GTP onto RAS to produce functionally active GTP-bound RAS and GTP-accelerating proteins (GAPs, such as NF1), thereby facilitating signal termination by converting GTP to GDP. The GTP-bound RAS generated by this cycle delivers the necessary positive signals to a series of serine / threonine kinases, including RAFs and MAP kinases, from which other signals are sent to form various cellular effector functions.

[0275] As used herein, the term "resistant to treatment" refers to the ineffectiveness of the therapeutic agent in the treatment of the condition with the therapeutic agent or the previously effective therapeutic agent and becomes less effective over time. Resistance to treatment includes acquired resistance to treatment, which refers to the reduced efficacy of treatment over a period of time when the therapeutic agent is administered to the subject. Acquired resistance to treatment can be caused by mutations in the target protein that make the treatment ineffective or less effective. Therefore, resistance to treatment can be sustained even after the administration of the therapeutic agent is stopped. Specifically, after treatment with immune checkpoint inhibitors, cancer can develop resistance to treatment with immune checkpoint inhibitors. Such cancers are also referred to herein as "immune refractory". The measurement of reduced therapeutic efficacy will depend on the condition being treated, and such methods are known to those skilled in the art. For example, the efficacy of cancer treatment can be measured by the progression of the disease. Effective treatment can slow down or stop the progression of the disease. Cancers that are resistant to treatment with therapeutic agents (such as immune checkpoint inhibitors) may not be able to slow down or stop the progression of the disease.

[0276] As used herein, the term "stereoisomer" refers to all possible different isomeric and conformational forms that a compound (e.g., a compound of any formula described herein) may have, specifically all possible stereochemical and conformational isomeric forms of the basic molecular structure, all diastereomers, enantiomers or conformers, including atropisomers. Some compounds of the present invention may exist in different tautomeric forms, all tautomeric forms are included within the scope of the present invention.

[0277] As used herein, the term "sulfonyl" refers to -S(O) 2 -group.

[0278] A "therapeutic agent" is any substance, such as a compound or composition, that is capable of treating a disease or condition. In some embodiments, therapeutic agents that may be used in conjunction with the present disclosure include RAS inhibitors and cancer chemotherapeutic agents. Many such therapeutic agents are known in the art and disclosed herein.

[0279] The term "therapeutically effective amount" means an amount sufficient to treat the disease, disorder or illness when applied to a group suffering from or susceptible to the disease, disorder or illness according to a therapeutic dosing regimen. In some embodiments, the therapeutically effective amount is an amount that reduces the incidence or severity of one or more symptoms of the disease, disorder or illness or delays its onset. It will be understood by those of ordinary skill in the art that the term "therapeutically effective amount" does not actually require successful treatment to be achieved in a particular individual. On the contrary, the therapeutically effective amount may be an amount that provides a specific desired pharmacological response in a large number of subjects when applied to a patient in need of such treatment. It should be understood in particular that a particular subject may actually be "refractory" to the "therapeutically effective amount". In some embodiments, the therapeutically effective amount may refer to an amount measured in one or more specific tissues (e.g., tissues affected by a disease, disorder or illness) or fluids (e.g., blood, saliva, serum, sweat, tears, urine). It will be understood by those skilled in the art that in some embodiments, the therapeutically effective amount may be formulated or administered in a single dose. In some embodiments, the therapeutically effective amount may be formulated or administered in multiple doses, for example, as part of a dosing regimen.

[0280] As used herein, the term "thiocarbonyl" refers to a -C(S)- group.

[0281] The term "treatment" (as well as "treat" or "treating") refers broadly to any administration of a substance (e.g., a compound of the present disclosure) that partially or completely alleviates, ameliorates, relieves, inhibits, delays the onset of, reduces the severity of, or reduces the incidence of one or more symptoms, features, or causes of a particular disease, disorder, or condition. In some embodiments, such treatment may be administered to a subject who does not show signs of the relevant disease, disorder, or condition, or to a subject who shows only early signs of the disease, disorder, or condition. Alternatively or additionally, in some embodiments, treatment may be administered to a subject who shows one or more established signs of the relevant disease, disorder, or condition. In some embodiments, treatment may be treatment of a subject who has been diagnosed with the relevant disease, disorder, or condition. In some embodiments, treatment may be treatment of a subject who is known to have one or more susceptibility factors that are statistically associated with an increased risk of developing the relevant disease, disorder, or condition.

[0282] As used herein, the term "vinyl ketone" refers to a group comprising a carbonyl group directly attached to a carbon-carbon double bond.

[0283] As used herein, the term "vinyl sulfone" refers to a group comprising a sulfonyl group directly attached to a carbon-carbon double bond.

[0284] The term "wild-type" refers to an entity having structure or activity as found in nature in a "normal" (as opposed to mutant, diseased, altered condition, etc.) state or background. Those skilled in the art will appreciate that wild-type genes and polypeptides often exist in multiple different forms (e.g., alleles).

[0285] As used herein, the term "alkynone" refers to a group comprising the following structure: wherein R is any chemically feasible substituent described herein.

[0286] RAS inhibitors

[0287] Provided herein are compounds that inhibit RAS and their uses. Also provided are pharmaceutical compositions comprising one or more RAS inhibitor compounds or pharmaceutically acceptable salts thereof and pharmaceutically acceptable excipients. RAS inhibitor compounds can be used in methods for inhibiting RAS (e.g., in a subject or in a cell) and in methods for treating cancer as described herein. In some embodiments, the compounds of the present disclosure are or act as prodrugs, such as for administration to a cell or to a subject in need thereof.

[0288] RAS proteins (KRAS, HRAS and NRAS) play an important role in various human cancers and are therefore suitable targets for anticancer therapy. In fact, mutations in RAS proteins explain about 30% of all human cancers in the United States, many of which are fatal. Abnormal regulation of RAS proteins caused by activating mutations, overexpression or upstream activation is common in human tumors, and activating mutations in RAS are often found in human cancers. RAS converts between GDP-bound "off" and GTP-bound "on" states. The interaction between guanine nucleotide exchange factor (GEF) proteins (such as SOS1) that load RAS with GTP and GTPase activation proteins (GAP) proteins (such as NF1) that hydrolyze GTP to inactivate RAS promotes the conversion between states. In addition, protein tyrosine phosphatase-2 (SHP2) containing SH2 domains is associated with receptor signaling mechanisms and becomes active after RTK activation, and then promotes RAS activation. Mutations in RAS proteins can lock proteins in the "on" state, generating constitutively active signaling pathways, thereby making cell growth uncontrolled. For example, activating mutations at codon 12 in RAS proteins function by inhibiting both the GAP dependency and the intrinsic hydrolysis rate of GTP, significantly biasing the population of RAS mutant proteins toward the "on" (GTP-bound) state (RAS (on)), thereby causing oncogenic MAPK signaling. Notably, RAS exhibits picomolar affinity for GTP, enabling RAS to be activated even in the presence of low nucleotide concentrations. Mutations of RAS at codons 13 (e.g., G13D) and 61 (e.g., Q61K) also cause oncogenic activity in some cancers.

[0289] Oncogenic pathways such as KRAS as well as the presence of immunosuppressive cell populations such as tumor-associated macrophages (TAMs) and myeloid-derived suppressor cells (MDSCs) have emerged as biological mechanisms of T cell exclusion (Liu et al., Theranostics 2021).

[0290] KRAS mutations are predominant in lung, pancreatic, and colon cancers and indicate an immunosuppressive tumor microenvironment (TME) in these tumors (Gu et al., Cancers 2021). Oncogenic KRAS mutations promote carcinogenesis and resistance to immunotherapy by inducing several inflammatory cytokines, chemokines, and signaling pathways, mediating autocrine effects and crosstalk with the TME (Hamarsheh et al., Nat. Commun. 2020).

[0291] The RAS inhibitors described herein can sensitize immune-refractory lung cancer to immunotherapy. It is believed herein that the therapy using the RAS inhibitors described herein can change the tumor immune infiltration comprising T cells, B cells, APCs, monocytes, MDSCs, TAMs, neutrophils, other monocyte-derived cells, tumor-associated stroma, cancer stem cells or mesenchymal stem cells and enhance the anti-tumor therapeutic effect. In some embodiments, the RAS inhibitors described herein can sensitize subjects to immunotherapy (such as checkpoint inhibitor therapy).

[0292] The RAS inhibitors disclosed herein can form a high-affinity three-component complex or conjugate between a synthetic ligand and the following two intracellular proteins that do not interact under normal physiological conditions: a target protein of interest (e.g., RAS), and a cytoplasmic partner (presenting protein) (e.g., cyclophilin A) widely expressed in cells. More specifically, in some embodiments, the RAS inhibitors described herein induce new binding pockets in RAS by driving the formation of a high-affinity three-complex or conjugate between RAS proteins and widely expressed cytoplasmic partner cyclophilin A (CYPA). Without being bound by theory, the inventors believe that one way in which the inhibitory effect of RAS is affected by the compounds of the present invention and the complexes or conjugates they form is by spatial closure of the interaction site between RAS and downstream effector molecules (such as RAF), which are required for the propagation of oncogenic signals. See, for example, WO 2021 / 091982, which is incorporated herein by reference in its entirety.

[0293] Therefore, the present invention provides a compound having a structure of Formula 0 or a pharmaceutically acceptable salt thereof:

[0294]

[0295] where the dashed lines represent zero, one, two, three, or four nonadjacent double bonds;

[0296] A is -N(H or CH 3 )C(O)-(CH 2 )-, in which the amino nitrogen is bound to -CH(R 10 )-, an optionally substituted 3- to 6-membered cycloalkylene group, an optionally substituted 3- to 6-membered heterocycloalkylene group, an optionally substituted 6-membered arylene group, or an optionally substituted 5- to 10-membered heteroarylene group;

[0297] B does not exist, is -CH(R 9 )-、>C=CR 9 R 9’ or>CR 9 R 9’ , where the carbon is bonded to -N(R 11)C(O)-, an optionally substituted 3- to 6-membered cycloalkylene group, an optionally substituted 3- to 6-membered heterocycloalkylene group, an optionally substituted 6-membered arylene group, or a 5- to 6-membered heteroarylene group;

[0298] G is an optionally substituted C 1 -C 4 Alkylene, optionally substituted C 1 -C 4 Alkenylene, optionally substituted C 1 -C 4 Heteroalkylene, -C(O)O-CH(R 6 )-, where C is bound to -C(R 7 R 8 )-、-C(O)NH-CH(R 6 )-, where C is bound to -C(R 7 R 8 )-, optionally substituted C 1 -C 4 Heteroalkylene or 3- to 8-membered heteroarylene;

[0299] L does not exist or is a linker;

[0300] W is a crosslinking group comprising a vinyl ketone, a vinyl sulfone, an alkynyl ketone, a haloacetyl group, or an alkynyl sulfone;

[0301] X 1 is an optionally substituted C 1 -C 2 Alkylene, NR, O or S(O) n ;

[0302] X 2 is O or NH;

[0303] X 3 N or CH;

[0304] n is 0, 1 or 2;

[0305] R is hydrogen, cyano, optionally substituted C 1 -C 4 Alkyl, optionally substituted C 2 -C 4 Alkenyl, optionally substituted C 2 -C 4 Alkynyl, C(O)R', C(O)OR', C(O)N(R') 2 、S(O)R'、S(O) 2 R' or S(O) 2 N(R') 2 ;

[0306] Each R' is independently H or optionally substituted C1 -C 4 alkyl;

[0307] Y 1 is C, CH or N;

[0308] Y 2 , Y 3 , Y 4 and Y 7 are independently C or N;

[0309] Y 5 CH, CH 2 or N;

[0310] Y 6 C(O), CH, CH 2 or N;

[0311] R 1 is cyano, optionally substituted C 1 -C 6 Alkyl, optionally substituted C 1 -C 6 heteroalkyl, optionally substituted 3 to 6 membered cycloalkyl, optionally substituted 3 to 6 membered cycloalkenyl, optionally substituted 3 to 6 membered heterocycloalkyl, optionally substituted 6 to 10 membered aryl, or optionally substituted 5 to 10 membered heteroaryl, or

[0312] R 1 and R 2 and the atoms to which they are attached are combined to form an optionally substituted 3- to 14-membered heterocycloalkyl;

[0313] R 2 absent, hydrogen, optionally substituted C 1 -C 6 Alkyl, optionally substituted C 2 -C 6 Alkenyl, optionally substituted C 2 -C 6 alkynyl, optionally substituted 3- to 6-membered cycloalkyl, optionally substituted 3- to 7-membered heterocycloalkyl, optionally substituted 6-membered aryl, optionally substituted 5- or 6-membered heteroaryl; R 3 does not exist, or

[0314] R 2 and R 3 and the atoms to which it is attached form an optionally substituted 3- to 8-membered cycloalkyl or an optionally substituted 3- to 14-membered heterocycloalkyl;

[0315] R 4 is absent, hydrogen, halogen, cyano or methyl optionally substituted with 1 to 3 halogens;

[0316] R 5is hydrogen, C optionally substituted with halogen 1 -C 4 Alkyl, cyano, hydroxyl or C 1 -C 4 Alkoxy, cyclopropyl or cyclobutyl;

[0317] R 6 is hydrogen or methyl; R 7 is hydrogen, halogen or optionally substituted C 1 -C 3 Alkyl, or

[0318] R 6 and R 7 and the carbon atoms to which they are attached form an optionally substituted 3- to 6-membered cycloalkyl or an optionally substituted 3- to 7-membered heterocycloalkyl;

[0319] R 8 is hydrogen, halogen, hydroxy, cyano, optionally substituted C 1 -C 3 Alkoxy, optionally substituted C 1 -C 3 Alkyl, optionally substituted C 2 -C 6 Alkenyl, optionally substituted C 2 -C 6 alkynyl, optionally substituted 3 to 8 membered cycloalkyl, optionally substituted 3 to 14 membered heterocycloalkyl, optionally substituted 5 to 10 membered heteroaryl, or optionally substituted 6 to 10 membered aryl, or

[0320] R 7 and R 8 Combined with the carbon atom to which it is attached, it forms C=CR 7’ R 8’ ; C=N(OH), C=N(OC 1 -C 3 alkyl), C═O, C═S, C═NH, optionally substituted 3- to 6-membered cycloalkyl, or optionally substituted 3- to 7-membered heterocycloalkyl;

[0321] R 7a and R 8a are independently hydrogen, halogen, optionally substituted C 1 -C 3 an alkyl group, or in combination with the carbon to which it is attached to form a carbonyl group;

[0322] R 7’ is hydrogen, halogen or optionally substituted C 1 -C 3 Alkyl; R 8’ is hydrogen, halogen, hydroxy, cyano, optionally substituted C 1 -C 3Alkoxy, optionally substituted C 1 -C 3 Alkyl, optionally substituted C 2 -C 6 Alkenyl, optionally substituted C 2 -C 6 alkynyl, optionally substituted 3 to 8 membered cycloalkyl, optionally substituted 3 to 14 membered heterocycloalkyl, optionally substituted 5 to 10 membered heteroaryl, or optionally substituted 6 to 10 membered aryl, or

[0323] R 7’ and R 8’ and the carbon atoms to which they are attached form an optionally substituted 3- to 6-membered cycloalkyl or an optionally substituted 3- to 7-membered heterocycloalkyl;

[0324] R 9 is H, F, optionally substituted C 1 -C 6 Alkyl, optionally substituted C 1 -C 6 heteroalkyl, optionally substituted 3 to 6-membered cycloalkyl or optionally substituted 3 to 7-membered heterocycloalkyl, or

[0325] R 9 and L, combined with the atoms to which they are attached, form an optionally substituted 3- to 14-membered heterocycloalkyl;

[0326] R 9’ is hydrogen or optionally substituted C 1 -C 6 Alkyl; or

[0327] R 9 and R 9’ and the atoms to which they are attached form a 3- to 6-membered cycloalkyl group or a 3- to 6-membered heterocycloalkyl group;

[0328] R 10 is hydrogen, halogen, hydroxyl, C 1 -C 3 Alkoxy or C 1 -C 3 alkyl;

[0329] R 10a is hydrogen or halogen;

[0330] R 11 is hydrogen or C 1 -C 3 Alkyl; and

[0331] R 21 is hydrogen or C 1 -C 3Alkyl (eg, methyl). In some embodiments, the compound of the present invention is selected from Table 1, or a pharmaceutically acceptable salt or stereoisomer thereof. In some embodiments, the compound of the present invention is selected from Table 1, or a pharmaceutically acceptable salt or atropisomer thereof.

[0332] In some embodiments, the present disclosure provides a method of treating immune-refractory lung cancer in a subject, the method comprising administering to the subject a RAS inhibitor of Formula 0 or a pharmaceutically acceptable salt thereof. In some embodiments, the compound of Formula 0 is a compound of Table 1 or Table 2 or a pharmaceutically acceptable salt thereof.

[0333] In addition, the present invention provides a compound having a structure of Formula I or a pharmaceutically acceptable salt thereof:

[0334]

[0335] where the dashed lines represent zero, one, two, three, or four nonadjacent double bonds;

[0336] A is -N(H or CH 3 )C(O)-(CH 2 )-, in which the amino nitrogen is bound to -CH(R 10 )-, an optionally substituted 3- to 6-membered cycloalkylene group, an optionally substituted 3- to 6-membered heterocycloalkylene group, an optionally substituted 6-membered arylene group, or an optionally substituted 5- to 10-membered heteroarylene group;

[0337] B does not exist, is -CH(R 9 )-、>C=CR 9 R 9’ or>CR 9 R 9’ , where the carbon is bonded to -N(R 11 )C(O)-, an optionally substituted 3- to 6-membered cycloalkylene group, an optionally substituted 3- to 6-membered heterocycloalkylene group, an optionally substituted 6-membered arylene group, or a 5- to 6-membered heteroarylene group;

[0338] G is an optionally substituted C 1 -C 4 Alkylene, optionally substituted C 1 -C 4 Alkenylene, optionally substituted C 1 -C 4 Heteroalkylene, -C(O)O-CH(R 6 )-, where C is bound to -C(R 7 R 8 )-、-C(O)NH-CH(R 6 )-, where C is bound to -C(R 7 R 8)-, optionally substituted C 1 -C 4 Heteroalkylene or 3- to 8-membered heteroarylene;

[0339] L is a linker, wherein the linker is acyclic or comprises a monocyclic, fused bicyclic, fused polycyclic, bridged bicyclic, or bridged polycyclic group;

[0340] W is a crosslinking group comprising a vinyl ketone, a vinyl sulfone, an alkynyl ketone, a haloacetyl group, or an alkynyl sulfone;

[0341] X 1 is an optionally substituted C 1 -C 2 Alkylene, NR, O or S(O) n ;

[0342] X 2 is O or NH;

[0343] X 3 N or CH;

[0344] n is 0, 1 or 2;

[0345] R is hydrogen, cyano, optionally substituted C 1 -C 4 Alkyl, optionally substituted C 2 -C 4 Alkenyl, optionally substituted C 2 -C 4 Alkynyl, C(O)R', C(O)OR', C(O)N(R') 2 、S(O)R'、S(O) 2 R' or S(O) 2 N(R') 2 ;

[0346] Each R' is independently H or optionally substituted C 1 -C 4 alkyl;

[0347] Y 1 is C, CH or N;

[0348] Y 2 , Y 3 , Y 4 and Y 7 are independently C or N;

[0349] Y 5 CH, CH 2 or N;

[0350] Y 6 C(O), CH, CH 2 or N;

[0351] R 1 is cyano, optionally substituted C 1 -C 6 Alkyl, optionally substituted C 1 -C 6 heteroalkyl, optionally substituted 3 to 6 membered cycloalkyl, optionally substituted 3 to 6 membered cycloalkenyl, optionally substituted 3 to 6 membered heterocycloalkyl, optionally substituted 6 to 10 membered aryl, or optionally substituted 5 to 10 membered heteroaryl, or

[0352] R 1 and R 2 and the atoms to which they are attached are combined to form an optionally substituted 3- to 14-membered heterocycloalkyl;

[0353] R 2 absent, hydrogen, optionally substituted C 1 -C 6 Alkyl, optionally substituted C 2 -C 6 Alkenyl, optionally substituted C 2 -C 6 alkynyl, optionally substituted 3- to 6-membered cycloalkyl, optionally substituted 3- to 7-membered heterocycloalkyl, optionally substituted 6-membered aryl, optionally substituted 5- or 6-membered heteroaryl; R 3 does not exist, or

[0354] R 2 and R 3 and the atoms to which it is attached form an optionally substituted 3- to 8-membered cycloalkyl or an optionally substituted 3- to 14-membered heterocycloalkyl;

[0355] R 4 is absent, hydrogen, halogen, cyano or methyl optionally substituted with 1 to 3 halogens;

[0356] R 5 is hydrogen, C optionally substituted with halogen 1 -C 4 Alkyl, cyano, hydroxyl or C 1 -C 4 Alkoxy, cyclopropyl or cyclobutyl;

[0357] R 6 is hydrogen or methyl; R 7 is hydrogen, halogen or optionally substituted C 1 -C 3 Alkyl, or

[0358] R 6 and R 7 and the carbon atoms to which they are attached form an optionally substituted 3- to 6-membered cycloalkyl or an optionally substituted 3- to 7-membered heterocycloalkyl;

[0359] R 8 is hydrogen, halogen, hydroxy, cyano, optionally substituted C 1 -C 3 Alkoxy, optionally substituted C 1 -C 3 Alkyl, optionally substituted C 2 -C 6 Alkenyl, optionally substituted C 2 -C 6 alkynyl, optionally substituted 3 to 8 membered cycloalkyl, optionally substituted 3 to 14 membered heterocycloalkyl, optionally substituted 5 to 10 membered heteroaryl, or optionally substituted 6 to 10 membered aryl, or

[0360] R 7 and R 8 Combined with the carbon atom to which it is attached, it forms C=CR 7’ R 8’ ; C=N(OH), C=N(OC 1 -C 3 alkyl), C═O, C═S, C═NH, optionally substituted 3- to 6-membered cycloalkyl, or optionally substituted 3- to 7-membered heterocycloalkyl;

[0361] R 7a and R 8a are independently hydrogen, halogen, optionally substituted C 1 -C 3 an alkyl group, or in combination with the carbon to which it is attached to form a carbonyl group;

[0362] R 7’ is hydrogen, halogen or optionally substituted C 1 -C 3 Alkyl; R 8’ is hydrogen, halogen, hydroxy, cyano, optionally substituted C 1 -C 3 Alkoxy, optionally substituted C 1 -C 3 Alkyl, optionally substituted C 2 -C 6 Alkenyl, optionally substituted C 2 -C 6 alkynyl, optionally substituted 3 to 8 membered cycloalkyl, optionally substituted 3 to 14 membered heterocycloalkyl, optionally substituted 5 to 10 membered heteroaryl, or optionally substituted 6 to 10 membered aryl, or

[0363] R 7’ and R 8’ and the carbon atoms to which they are attached form an optionally substituted 3- to 6-membered cycloalkyl or an optionally substituted 3- to 7-membered heterocycloalkyl;

[0364] R 9is H, F, optionally substituted C 1 -C 6 Alkyl, optionally substituted C 1 -C 6 heteroalkyl, optionally substituted 3 to 6-membered cycloalkyl or optionally substituted 3 to 7-membered heterocycloalkyl, or

[0365] R 9 and L, combined with the atoms to which they are attached, form an optionally substituted 3- to 14-membered heterocycloalkyl;

[0366] R 9’ is hydrogen or optionally substituted C 1 -C 6 Alkyl; or

[0367] R 9 and R 9’ and the atoms to which they are attached form a 3- to 6-membered cycloalkyl group or a 3- to 6-membered heterocycloalkyl group;

[0368] R 10 is hydrogen, halogen, hydroxyl, C 1 -C 3 Alkoxy or C 1 -C 3 alkyl;

[0369] R 10a is hydrogen or halogen;

[0370] R 11 is hydrogen or C 1 -C 3 Alkyl; and

[0371] R 21 H or C 1 -C 3 alkyl.

[0372] In some embodiments, the present disclosure provides a method of treating immune-refractory lung cancer in a subject, the method comprising administering to the subject a RAS inhibitor of Formula I or a pharmaceutically acceptable salt thereof. In some embodiments, the compound of Formula 0 is a compound of Table 1 or a pharmaceutically acceptable salt thereof.

[0373] In some embodiments, the compound of the present invention is selected from Table 1, or a pharmaceutically acceptable salt or stereoisomer thereof. In some embodiments, the compound of the present invention is selected from Table 1, or a pharmaceutically acceptable salt or atropisomer thereof.

[0374] Table 1: Certain compounds of the present invention

[0375]

[0376]

[0377]

[0378]

[0379]

[0380]

[0381]

[0382]

[0383]

[0384]

[0385]

[0386]

[0387]

[0388]

[0389]

[0390]

[0391]

[0392]

[0393]

[0394]

[0395]

[0396]

[0397]

[0398]

[0399]

[0400]

[0401]

[0402]

[0403]

[0404]

[0405]

[0406]

[0407]

[0408]

[0409]

[0410]

[0411]

[0412]

[0413]

[0414]

[0415]

[0416]

[0417]

[0418]

[0419]

[0420]

[0421]

[0422]

[0423]

[0424]

[0425]

[0426]

[0427]

[0428]

[0429]

[0430]

[0431]

[0432]

[0433]

[0434]

[0435]

[0436]

[0437]

[0438]

[0439]

[0440]

[0441]

[0442]

[0443]

[0444]

[0445]

[0446]

[0447]

[0448]

[0449]

[0450]

[0451]

[0452]

[0453]

[0454]

[0455]

[0456]

[0457]

[0458]

[0459]

[0460]

[0461]

[0462]

[0463]

[0464]

[0465]

[0466]

[0467] In some embodiments, the compounds of the present invention are selected from Table 2, or a pharmaceutically acceptable salt or stereoisomer thereof. In some embodiments, the compounds of the present invention are selected from Table 2, or a pharmaceutically acceptable salt or atropisomer thereof.

[0468] Table 2. Certain compounds of the present invention

[0469]

[0470]

[0471]

[0472]

[0473]

[0474]

[0475]

[0476]

[0477]

[0478]

[0479]

[0480]

[0481]

[0482]

[0483]

[0484]

[0485]

[0486]

[0487] In some embodiments, the RAS inhibitor is selective for RAS including G12C amino acid substitutions relative to wild-type RAS or other RAS mutants. In some embodiments, the RAS inhibitor is a KRAS inhibitor, which is selective for KRAS including G12C amino acid substitutions relative to wild-type KRAS or other KRAS mutants. In some embodiments, the RAS inhibitor is a NRAS inhibitor, which is selective for NRAS including G12C amino acid substitutions relative to wild-type NRAS or other NRAS mutants. In some embodiments, the RAS inhibitor is a HRAS inhibitor, which is selective for HRAS including G12C amino acid substitutions. In some embodiments, the HRAS inhibitor is selective for HRAS including G12C amino acid substitutions relative to wild-type NRAS or other NRAS mutants. In some embodiments, the RAS inhibitor is selective for HRAS including G12C amino acid substitutions relative to wild-type NRAS or other NRAS mutants. In some embodiments, the RAS inhibitor that is selective for RAS including G12C relative to wild-type RAS or other RAS mutants is a RAS (on) inhibitor. In some embodiments, a RAS inhibitor that is selective for RAS comprising G12C relative to wild-type RAS or other RAS mutants is not a RAS (off) inhibitor.

[0488] Immune checkpoint inhibitors

[0489] The compositions and methods described herein may include immune checkpoint inhibitors (ICIs). Immune checkpoint inhibitors may be administered or formulated in combination with RAS inhibitors described herein. Immune checkpoint inhibitors may be administered or formulated in combination with RAS inhibitors and SHP2 inhibitors described herein.

[0490] Immune checkpoints refer to a series of inhibitory pathways inherent in the immune system that are critical for maintaining self-tolerance and regulating the duration and amplitude of physiological immune responses in peripheral tissues to minimize collateral tissue damage in response to pathogenic infections under normal physiological conditions. However, the expression of immune checkpoint proteins is often dysregulated by tumors as an important immune resistance and escape mechanism.

[0491] Since many immune checkpoints are initiated by ligand-receptor interactions, they can be easily blocked by antibodies or regulated by recombinant forms of ligands or receptors. Therefore, inhibition of these pathways has been used to activate therapeutic anti-tumor immunity. For example, the cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) antibody was the first of this class of immunotherapeutics to be approved by the U.S. Food and Drug Administration (FDA). Preliminary clinical study results of inhibitors of other immune checkpoint proteins, such as programmed cell death protein 1 (PD-1), suggest that there are broad and diverse opportunities to enhance anti-tumor immunity with the potential for durable clinical responses.

[0492] Activation of T cells by blocking immune checkpoints has been a major focus of therapeutic manipulation of endogenous antitumor immunity due to their ability to selectively recognize peptides derived from proteins in all cellular compartments; their ability to directly recognize and kill antigen-expressing cells (via CD8+ effector T cells; also known as cytotoxic T lymphocytes (CTLs)); and their ability to coordinate multiple immune responses (via CD4+ helper T cells), which integrates adaptive and innate effector mechanisms. Therefore, agonists of co-stimulatory receptors or antagonists of inhibitory signals (both of which cause amplification of antigen-specific T cell responses) are the agents of interest currently in clinical testing.

[0493] Table 3. Non-limiting list of immune checkpoint targets.

[0494]

[0495]

[0496] CTLA4, cytotoxic T lymphocyte-associated antigen 4; LAG3, lymphocyte activation gene 3; PD-1,

[0497] Programmed cell death protein 1; PD-L1, PD-1 ligand; TIM3, T-cell membrane protein 3; VISTA, V-domain immunoglobulin (Ig)-containing inhibitor of T-cell activation; KIR, killer IgG-like receptor.

[0498] ICIs approved or under development include but are not limited to (ipilimumab), (nivolumab), (pembrolizumab), tremelimumab, galiximab, MDX-1106, BMS-936558, MEDI4736, MPDL3280A, MEDI6469, BMS-986016, BMS-663513, PF-05082566, IPH2101, KW-0761, CDX-1127, CP-870, CP-893, GSK2831781, MSB0010718C, MK3475, CT-011, AMP-224, MDX-1105, IMP321, and MGA271, as well as many other antibodies or fusion proteins directed against the immune checkpoint proteins mentioned in Table 3. Common immune checkpoint proteins that can be targeted by ICI include, but are not limited to, B7.1, B7-H3, LAG3, CD137, KIR, CCR4, CD27, OX40, GITR, CD40, CTLA4, PD-1, and PD-L1.

[0499] In some embodiments, ICI therapy is selected from one or more of the following: anti-PD-1, anti-PD-L1, anti-CTLA-4, anti-LAG3, anti-B7.1, anti-B7H3, anti-B7H4, anti-TIM3, anti-VISTA, anti-CD137, anti-OX40, anti-CD40, anti-CD27, anti-CCR4, anti-GITR, anti-NKG2D and anti-KIR. In some embodiments, ICI therapy is an antibody (e.g., a monoclonal antibody selective for any one of the targets in Table 3). In some embodiments, ICI is an anti-PD-1 antibody. The antibody may be, for example, a humanized antibody or a fully human antibody. In some embodiments, the check point inhibitor is a fusion protein, such as an Fc-receptor fusion protein. In some embodiments, the check point inhibitor is an agent that interacts with a check point protein, such as an antibody. In some embodiments, the check point inhibitor is an agent that interacts with a ligand of a check point protein, such as an antibody. In some embodiments, the check point inhibitor is an inhibitor (e.g., an inhibitory antibody or a small molecule inhibitor) of CTLA-4 (e.g., an anti-CTLA-4 antibody or a fusion protein). In some embodiments, the check point inhibitor is an inhibitor or antagonist (e.g., an inhibitory antibody or a small molecule inhibitor) of PD-1. In some embodiments, the check point inhibitor is an inhibitor or antagonist (e.g., an inhibitory antibody or a small molecule inhibitor) of PD-L1. In some embodiments, the check point inhibitor is an inhibitor or antagonist (e.g., an inhibitory antibody or Fc fusion or a small molecule inhibitor) of PD-L2 (e.g., an inhibitory antibody or Fc fusion or a small molecule inhibitor) (e.g., a PD-L2 / Ig fusion protein). In some embodiments, the check point inhibitor is an inhibitor or antagonist (e.g., an inhibitory antibody or a small molecule inhibitor) of B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK 1, CHK2, A2aR, B-7 family ligand, or a combination thereof.In some embodiments, the checkpoint inhibitor is pembrolizumab, nivolumab, PDR001 (NVS), REGN2810 (Sanofi / Regeneron), a PD-L1 antibody such as avelumab, durvalumab, atezolizumab, pidilizumab, JNJ-63723283 (JNJ), BGB-A317 (also known as tislelizumab); BeiGe ne and Celgene), or Preusser, M. et al. (2015) Nat. Rev. Neurol., including but not limited to ipilimumab, tremelimumab, nivolumab, pembrolizumab, AMP224, AMP514 / MEDI0680, BMS936559, MED14736, MPDL3280A, MSB0010718C, BMS986016, IMP321, lirilumab, IPH2101, 1-7F9 and KW-6002.

[0500] SHP2 inhibitors

[0501] The compositions and methods described herein may include SHP2 inhibitors. SHP2 inhibitors may be administered or formulated in combination with RAS inhibitors described herein. SHP2 inhibitors may be administered or formulated in combination with RAS inhibitors and immune checkpoint inhibitors.

[0502] SHP2 is a non-receptor protein tyrosine phosphatase encoded by the PTPN11 gene that contributes to a variety of cellular functions, including proliferation, differentiation, cell cycle maintenance, and migration. SHP2 has two N-terminal Src homology 2 domains (N-SH2 and C-SH2), a catalytic domain (PTP), and a C-terminal tail. The two SH2 domains control the subcellular localization and functional regulation of SHP2. The molecule exists in an inactive, autoinhibitory conformation stabilized by a binding network involving residues from the N-SH2 and PTP domains. For example, stimulation by cytokines or growth factors acting through receptor tyrosine kinases (RTKs) exposes the catalytic site, leading to enzymatic activation of SHP2.

[0503] SHP2 participates in signal transduction through RAS-mitogen-activated protein kinase (MAPK), JAK-STAT or phosphoinositide 3-kinase-AKT pathways. Mutations in the PTPN11 gene and subsequently in SHP2 have been identified in several human developmental diseases (such as Noonan Syndrome and Leopard Syndrome) and human cancers (such as juvenile myelomonocytic leukemia, neuroblastoma, melanoma, acute myeloid leukemia, and breast cancer, lung cancer, and colon cancer). Some of these mutations destabilize the autoinhibitory conformation of SHP2 and promote the auto-activation of SHP2 or the enhanced growth factor-driven activation. Therefore, SHP2 represents a highly attractive target for the development of novel therapies for the treatment of various diseases including cancer. The combination of SHP2 inhibitors (such as RMC-4550 or SHP099) and RAS pathway inhibitors (such as MEK inhibitors) has been shown to inhibit the proliferation of various cancer cell lines (such as pancreatic cancer, lung cancer, ovarian cancer, and breast cancer) in vitro.

[0504] Comprehensive practical examples of SHP2 inhibition in this area: Chen et al. MolPharmacol.2006, 70, 562; Sarver et al., J.Med.Chem.2017, 62,1793; 2023282702, WO2023280283, WO 2023280237, WO 2023018155, WO 2023011513, WO 2022271966, WO2022271964, WO 2022271911, WO 2022259157, WO 2022242767, WO 2022241975, WO2022237676, WO 2022237367, WO 2022237178, WO 2022235822, WO 2022234409, WO2022208408, WO 2022207924, WO 2022167682, WO 2022166844, WO 2022161222, WO2022156765, WO 2022135568, WO 2022089406, WO 2022089389, WO 2022063190, WO2022043865, WO 2022042331, WO 2022033430, WO 2022017444, WO 2022007869, WO2021259077, WO 2021249449, WO 2021249057, WO 2021244659, WO 2021218755, WO2021281752, WO 2021149817, WO 2021148010, WO 2021147879, WO 2021143823, WO2021143701, WO 2021143680, WO 2021121397, WO 2021119525, WO 2021115286, WO2021110796, WO 2021088945, WO 2021073439, WO 2021061706, WO 2021061515, WO2021043077, WO 2021033153, WO 2021028362, WO 2021033153, WO 2021028362, WO2021018287, WO 2020259679, WO 2020249079, WO 2020210384, WO2020201991、WO2020181283、WO 2020177653、WO 2020165734、WO 2020165733、WO 2020165732、WO2020156243、WO 2020156242、WO 2020108590、WO 2020104635、WO 2020094104、WO2020094018、WO 2020081848、WO 2020073949、WO 2020073945、WO 2020072656、WO2020065453、WO 2020065452、WO 2020063760、WO 2020061103, WO 2020061101, WO2020033828, WO 2020033286, WO 2020022323, WO 2019233810, WO 2019213318, WO2019183367, WO 2019183364, WO 2019182960, WO 2019167000, WO 2019165073, WO2019158019, WO 2019152454, WO 2019051469, WO 2019051084, WO 2018218133, WO2018172984, WO 2018160731, WO 2018136265、WO 2018136264、WO 2018130928、WO2018129402、WO 2018081091、WO 2018057884、WO 2018013597、WO 2017216706、WO2017211303、WO 2017210134、WO 2017156397、WO 2017100279、WO 2017079723、WO2017078499、WO 2016203406、WO 2016203405、WO 2016203404、WO 2016196591、WO2016191328、WO 2015107495, WO 2015107494, WO 2015107493, WO 2014176488, WO2014113584, CN 115677661, CN 115677660, CN 115611869, CN 115521305, CN 115490697、CN 115466273、CN 115394612、CN 115304613、CN 115304612、CN 115300513、CN115197225、CN114957162、CN 114920759、CN 114716448、CN 114671879、CN 114539223、CN114524772、CN 114213417、CN 114195799、CN 114163457、CN 113896710、CN 113248521、CN113248449、CN 113135924、CN 113024508、CN 112920131、CN 112823796、CN 112409334、CN112402385、CN 112174935, 111848599, CN 111704611, CN 111393459, CN 111265529, CN110143949, CN 108113848, US 11179397, US 11044675, US 11034705, US 11033547, US11001561, US 10988466, US 10954243, US 10934302, US 10858359, or pharmaceutically acceptable salts, solvates, isomers (e.g., stereoisomers), prodrugs or tautomers thereof, each of which is incorporated herein by reference.

[0505] In some embodiments, the SHP2 inhibitor binds in the active site. In some embodiments, the SHP2 inhibitor is a mixed irreversible inhibitor. In some embodiments, the SHP2 inhibitor binds to an allosteric site, such as a non-covalent allosteric inhibitor. In some embodiments, the SHP2 inhibitor is a covalent SHP2 inhibitor, such as an inhibitor that targets a cysteine ​​residue (C333) located outside the phosphatase active site. In some embodiments, the SHP2 inhibitor is a reversible inhibitor. In some embodiments, the SHP2 inhibitor is an irreversible inhibitor. In some embodiments, the SHP2 inhibitor is SHP099. In some embodiments, the SHP2 inhibitor is TNO155, which has the structure or a pharmaceutically acceptable salt, solvate, isomer (e.g., stereoisomer), prodrug, or tautomer thereof. In some embodiments, the SHP2 inhibitor is RMC-4550, which has the structure or a pharmaceutically acceptable salt, solvate, isomer (e.g., stereoisomer), prodrug, or tautomer thereof. In some embodiments, the SHP2 inhibitor is RMC-4630, which has the structure or a pharmaceutically acceptable salt, solvate, isomer (e.g., stereoisomer), prodrug, or tautomer thereof. In some embodiments, the SHP2 inhibitor is JAB-3068, which has the structure or a pharmaceutically acceptable salt, solvate, isomer (e.g., stereoisomer), prodrug, or tautomer thereof. In some embodiments, the SHP2 inhibitor is JAB-3312. In some embodiments, the SHP2 inhibitor is RLY-1971, which has the structure Or a pharmaceutically acceptable salt, solvate, isomer (e.g., stereoisomer), prodrug, or tautomer thereof. In some embodiments, the SHP2 inhibitor is ERAS-601 or a pharmaceutically acceptable salt, solvate, isomer (e.g., stereoisomer), prodrug, or tautomer thereof. In some embodiments, the SHP2 inhibitor is BBP-398 or a pharmaceutically acceptable salt, solvate, isomer (e.g., stereoisomer), prodrug, or tautomer thereof.

[0506] The present disclosure also provides a pharmaceutical composition. The pharmaceutical composition comprises a RAS inhibitor, a SHP2 inhibitor, an immune checkpoint inhibitor or a combination thereof as an active agent, and at least one pharmaceutically acceptable excipient. A pharmaceutically acceptable excipient may be a diluent, a binder, a filler, a buffer, a pH regulator, a disintegrant, a dispersant, a preservative, a lubricant, a taste masking agent, a flavoring agent or a coloring agent. The amount and type of excipient used to form the pharmaceutical composition may be selected according to known pharmaceutical science principles.

[0507] The composition can be formulated into a variety of dosage forms and administered by several different means of delivering a therapeutically effective amount of the one or more active agents. Such compositions can be administered orally (e.g., inhaled) or parenterally as required in the form of dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants, and vehicles.

[0508] How to use

[0509] In some embodiments, the present disclosure provides a method for treating a subject with immune refractory lung cancer, the method generally comprising administering a compound or compound combination described herein to the subject. In some embodiments, a RAS inhibitor (i.e., a RAS G12C inhibitor) described herein is administered to the subject. In some embodiments, a combination of a RAS inhibitor described herein and an immune checkpoint inhibitor, a SHP2 inhibitor, or a combination thereof is administered to the subject. Suitable examples of RAS inhibitors, SHP2 inhibitors, and immune checkpoint inhibitors are described above and are incorporated by reference into this section.

[0510] In some embodiments, the present disclosure provides a method of sensitizing immune refractory cancer in a subject in need to immunotherapy, the method generally comprising administering a compound or compound combination described herein to the subject. In some embodiments, a RAS inhibitor (i.e., a RAS G12C inhibitor) described herein is administered to the subject. In some embodiments, a combination of a RAS inhibitor described herein and an immune checkpoint inhibitor, a SHP2 inhibitor, or a combination thereof is administered to the subject.

[0511] In some embodiments, the present disclosure provides a method for causing an immune cold tumor in a subject in need to be converted into an immune hot tumor, the method generally comprising administering a compound or compound combination described herein to the subject, thereby achieving treatment of a previously immune cold tumor that has been converted into an immune hot tumor. In some embodiments, a RAS inhibitor (i.e., a RAS G12C inhibitor) described herein is administered to the subject. In some embodiments, a combination of a RAS inhibitor described herein and an immune checkpoint inhibitor, a SHP2 inhibitor, or a combination thereof is administered to the subject.

[0512] In some embodiments, the present disclosure provides a method for increasing the efficacy of other cancer therapies when administered to a subject in combination. In some embodiments, a RAS inhibitor (i.e., a RAS G12C inhibitor) described herein is administered to a subject. In some embodiments, a combination of a RAS inhibitor described herein and an immune checkpoint inhibitor, a SHP2 inhibitor, or a combination thereof is administered to a subject.

[0513] In each of the above embodiments, the method of the present disclosure can alter the tumor immune infiltration comprising T cells, B cells, APCs, monocytes, MDSCs, TAMs, neutrophils, other monocyte-derived cells, tumor-associated stroma, cancer stem cells, and mesenchymal stem cells and enhance the anti-tumor therapeutic effect.

[0514] Identification of tumor type

[0515] In various embodiments, the present disclosure provides a method of treating lung cancer in a subject, the method comprising administering to the subject a RAS inhibitor or a combination of compounds described herein, wherein the subject has one or more tumors characterized as being immune-refractory, immune-evasive, immune-protected, immune "cold," microsatellite stable, microsatellite instability-low, containing low immune infiltration, containing low tumor mutational burden, or exhibiting heterogeneity.

[0516] In various embodiments, the present disclosure provides a method for treating a tumor (e.g., lung cancer) characterized as immune-evasive, immune-protective, immune "cold", microsatellite stable, low in microsatellite instability, comprising low immune infiltration, comprising low tumor mutation load, or exhibiting heterogeneity in a subject, the method comprising (i) diagnosing the subject as having an immune-refractory, immune-evasive tumor, immune-protective tumor, immune "cold" tumor, microsatellite stable tumor, low in microsatellite instability tumor, comprising low immune infiltration, comprising low tumor mutation load, or exhibiting heterogeneity, and (ii) administering to the subject a RAS inhibitor or a combination of compounds described herein. In various embodiments, the diagnosis comprises analyzing biomarkers / signatures associated with a tumor characterized as immune-refractory, immune-evasive, immune-protective, immune "cold", microsatellite stable, low in microsatellite instability, comprising low immune infiltration, comprising low tumor mutation load, or exhibiting heterogeneity. In various embodiments, the method further comprises (iii) determining whether the subject's tumor is immunoreactive, and then (iv) optionally administering immunotherapy in combination with a RAS inhibitor or compound described herein. In some embodiments, the subject has been previously diagnosed with a tumor characterized as immune evasive, immune protective, immune "cold", microsatellite stable, microsatellite instability low, containing low immune infiltration, containing low tumor mutation load, or exhibiting heterogeneity.

[0517] Also provided herein is a method for determining whether or not a subject is or is likely to respond to treatment with a RAS inhibitor or combination of compounds described herein and thus treating the subject. In various embodiments, a patient diagnosed with cancer undergoes a test, such as using the methods described herein and other methods described in the art, to identify the tumor as a cold tumor. The present disclosure provides a method for treating a subject with cancer (e.g., an immune-refractory cancer) using a RAS inhibitor or combination of compounds described herein, the method comprising obtaining a tumor sample from the subject, analyzing to determine whether the tumor is a cold tumor, and if the tumor is identified as a cold tumor, then treating the subject using a RAS inhibitor or combination of compounds described herein. The analysis used to determine whether a tumor is a cold tumor includes, but is not limited to, tumor mutation load analysis, microsatellite instability (MSI) testing, immune cells (e.g., CD4 + T cells, CD8 + T cells, NK1.1 + The degree of infiltration of NK cells, APCs, monocytes, and neutrophils into the tumor, the phenotype of immune cells (such as PD-1 + 、PD-L1 + and PD-L2 +), immune cell function (e.g., expression of IFN-γ, IL-12, IL-15, and MHCII), and the ratio of pro- and anti-inflammatory mediators in the tumor microenvironment (TME).

[0518] A variety of diagnostic tools designed to characterize tumors at the cellular and molecular levels are FDA-approved and commercially available. Examples of approved diagnostic tools include CDX, LIQUID, HEME, BRACAnalysis CDx, therascreen EGFR RGQ PCR Kit, cobase EGFR Mutation Test v2, PD-L1 IHC 22C3 pharmDx, Abbott Real-Time IDH1, MRDx BCR-ABL Test, VENTANAALK (D5F3) CDx Assay, Abbott Real-Time IDH2, Praxis Expanded RAS Panel, Oncomine Dx Targeted Tests, LeukoStrat CDx FLT3 Mutation Assay, FoundationFocus CDx BRCA Assay, Vysis CLL FISH Probe Kit, KITD816V Mutation Detection, PDGFRB FISH, cobas KRAS Mutation Test, therascreen KRAS RGQPCR Kit, FerriScan, Dako c-KIT pharmDx, INFORM Her-2 / neu, PathVysion HER-2 DNA Probe Kit, SPOT-LIGHT HER2 CISH Kit, Bond Oracle HER2 IHC System, HER2 CISHpharmDx Kit, INFORM HER2 DUALISH DNA Probe Cocktail, HercepTest, HER2 FISH pharmDx Kit, THXID BRAF Kit, Vysis ALK Breakthrough FISH Probe Kit, cobas 4800 BRAF V600 Mutation Test, VENTANA PD-L1 (SP142) Assay, therascreen FGFR RGQ RT-PCR Kit, and therascreen PIK3CA RGQ PCR Kit.

[0519] In various embodiments, subjects are screened for suitability for treatment with one or more immunotherapies described herein. In various embodiments, subjects who are not suitable for treatment with such immunotherapies (e.g., unresponsive to one or more immunotherapies or having a cancer characterized as unresponsive to one or more immunotherapies) may first be treated with a RAS inhibitor or compound combination described herein according to the methods described herein. Non-limiting examples of immunotherapies include pembrolizumab ( Merck Sharp & Dohme Corp), nivolumab ( Bristol-Myers Squibb), atezolizumab (TECE ), Avelumab Durvalumab Criteria for appropriate use of these immunotherapies are known in the art. For example, but not limited to, pembrolizumab Nivolumab and atezolizumab (TECE ) have eligibility criteria based on PD-L1 expression levels. PD-L1 expression criteria and methods for measuring it can be found at keytrudahcp.com / biomarker-testing / pd-11-expression-testing / (pembrolizumab; ) or FDA-approved pembrolizumab (KEYTRU Revised January 2020), atezolizumab (e.g. Revised May 2020) and nivolumab (eg Each of these publications is incorporated herein by reference in its entirety for all purposes. As described herein, treatment of such patients with a RAS inhibitor or a combination of compounds described herein may promote the conversion of tumors that are not suitable for treatment with immunotherapy into immunogenic tumors, which in turn will enable such tumors to be treated with immunotherapy. In various embodiments, tumors of subjects who are not suitable for immunotherapy may be monitored during treatment with a RAS inhibitor or a combination of compounds described herein to determine when the tumor becomes suitable for treatment with immunotherapy. After the tumor is suitable for treatment with immunotherapy, immunotherapy may be administered to the subject alone or in combination with a RAS inhibitor or a SHP2 inhibitor or a combination thereof.

[0520] In various embodiments, the present disclosure provides a method for treating lung cancer in a subject, the method comprising administering a RAS inhibitor or a combination of compounds described herein to a subject, wherein the subject has one or more tumors with low immune infiltration. In various embodiments, a subject having one or more lung tumors with low immune infiltration is administered to change tumor immune infiltration. In various embodiments, tumor immune infiltration comprises antigen presenting cells, myeloid cells, and lymphoid cells. In various embodiments, the antigen presenting cells in the tumor immune infiltration comprise macrophages or dendritic cells. In various embodiments, the myeloid cells in the tumor immune infiltration comprise monocytes, neutrophils, myeloid-derived suppressor cells (MDSC), and tumor-associated macrophages (TAM). In various embodiments, the TAMs in the tumor immune infiltration comprise M1 macrophages, M2 macrophages, and MARCO + Macrophages. In various embodiments, lymphoid cells in the tumor immune infiltration include T cells, B cells, NKT cells, and NK cells.

[0521] Qualitative and quantitative methods for characterizing tumor immune infiltration have been described, including but not limited to microscopic analysis, histological analysis, cytological analysis, flow cytometry, polymerase chain reaction (PCR), quantitative polymerase chain reaction (qPCR), RNA sequencing (RNA-seq), single cell RNA sequencing (scRNA-seq), next generation sequencing, whole exome sequencing, epigenetic sequencing, ATAC-seq, microarray analysis, and bulk blood cell counting or CyTOF. Biomarkers can be used alone or in combination for immune cell assessment and include cell surface markers and secreted proteins. Exemplary biomarkers for characterizing tumor immune infiltration include, but are not limited to, CD45, CD3, CD4, CD8, CD25, CD44, CD134, CD252, CD137, CD79, CD39, FOXP3, PD-1, LAG-3, TIM-1, IFN-γ, granzyme, perforin, CD11b, CD11c, Ly6C, Ly6G, CD14, CD16, CD80, MARCO, CD68, CD115, CD206, CD163, CD103c, F4 / 80, PD-L1, PD-L2, arginase, iNOS, ROS, TNF-α, TGF-β, MHC-I, MHC-II, NK1.1, NKG2D, CD244, Ki67, CD19, CD20, CCR2, CXCR3, CCR4, CCR5, CCR6, CCR7, CCR10, CCL2, CCL5, Cx3CR1 , CCL10, ICOS, CD40, CD40L, IL1α, IL1β, IL2, IL4, IL5, IL6, IL8, IL12, IL15, IL17, IL21, IL22, TCRγ / δ, TCRα / β, STAT3, ROR1c and RORγt.

[0522] Cancer stem cells (CSCs) have been described as subsets of cells that are tumorigenic and capable of self-renewal and differentiation found in solid tumors and hematological tumors. Several reports have described the importance of CSCs in the pathogenesis of various tumors, tumor recurrence after treatment, and the development of treatment resistance. Many cell surface markers can be used to distinguish CSCs in solid tumors and hematological tumors. CSC markers include, but are not limited to, CD19, CD20, CD24, CD34, CD38, CD44, CD90, CD133, aldehyde dehydrogenase 1, CEACAM-6 / CD66c, BM1-1, connexin43 / GJA1, DLL4, EpCAM / TROP1, GL1-1, GL1-2, integrin, PON1, PTEN, ALCAM / CD166, DPPIV / CD26, Lgr5, Musashi-1, A20, ABCG2, CD15, fractin, HIF-2α, L1CAM, c-MAF, nestin, podoplanin, SOX2, CD96, CD117, FLT3, AFP, CD13, CD90, NF2 / Merlin, ABCB5, NGFR, syndecan-1, endothelin, STRO-1, and PON1.

[0523] In various embodiments, the present disclosure provides a method of treating cancer in a subject, the method comprising administering to the subject a RAS inhibitor or a combination of compounds described herein, wherein the subject has one or more immune-refractory tumors. In various embodiments, the subject has one or more immune-protective tumors. In various embodiments, the subject has one or more microsatellite stable tumors. In various embodiments, the subject has one or more microsatellite low tumors. In various embodiments, the subject has one or more tumors with medium microsatellite instability. In various embodiments, the subject has one or more tumors with low tumor mutation load. In various embodiments, the subject has one or more tumors with medium tumor mutation load. In various embodiments, the subject has one or more tumors resistant to therapy. In various embodiments, the subject has one or more immune-heterogeneous tumors. In various embodiments, the subject has a genetically heterogeneous tumor. In various embodiments, the subject has one or more refractory tumors. In one or more embodiments, the subject has a tumor that develops resistance during treatment.

[0524] In various embodiments, tumor characteristics are determined from one or more biological samples from a subject suffering from cancer. In various embodiments, tumor characteristics are determined by comparing one or more biological samples from a subject suffering from cancer with one or more biological samples from one or more healthy subjects. In various embodiments, tumor characteristics are determined from one or more biological samples selected from the group consisting of: blood, cerebrospinal fluid, urine, feces, oral swabs, nasal swabs, lavage fluids, tissue biopsies, bone marrow biopsies, and tumor biopsies. In various embodiments, tumor characteristics are determined from the analysis of cells, proteins, or nucleic acids in one or more biological samples from a subject suffering from cancer. In various embodiments, tumor characteristics are determined by comparing the analysis of cells, proteins, or nucleic acids in one or more biological samples from a subject suffering from cancer with the analysis of one or more biological samples from one or more healthy subjects. In various embodiments, tumor characteristics are determined by comparing the analysis of cells, proteins, or nucleic acids in one or more biological samples from a subject suffering from cancer with the analysis of one or more biological samples from one or more subjects suffering from cancer and responding to treatment. In various embodiments, the cell is selected from the group consisting of: leukocytes, epithelial cells, mesenchymal cells, mesenchymal stem cells, stromal cells, endothelial cells, fibroblasts, cancer-associated fibroblasts (CAFs), pericytes, adipocytes, cancer stem cells, circulating tumor cells (CTCs), hematopoietic stem cells, and hematopoietic progenitor cells. In various embodiments, the protein is selected from the group consisting of: cytokines, chemokines, growth factors, signal transduction proteins, enzymes, proteases, and nucleases. In various embodiments, nucleic acids are selected from the group consisting of: DNA, ssDNA, circulating tumor DNA (ctDNA), RNA, mRNA, dsRNA, siRNA, miRNA, and lncRNA. In various embodiments, nucleic acid analysis is performed by PCR, RT-PCR, qRT-PCR, next generation sequencing (NGS), RNA-seq, ATAC-seq, exome sequencing, Southern Blot, microarray analysis, or single cell sequencing.

[0525] In various embodiments, the tumor characteristics of the subject suffering from cancer are determined by the analysis of one or more blood samples collected from the subject. In various embodiments, the tumor characteristics of the subject suffering from cancer are determined by the analysis of cells, proteins or nucleic acids in one or more blood samples collected from the subject. In various embodiments, the tumor characteristics of the subject suffering from cancer are determined by comparing the analysis of cells, proteins or nucleic acids in one or more blood samples from the subject suffering from cancer with the analysis of one or more blood samples from one or more healthy subjects. In various embodiments, the cells analyzed in one or more blood samples are leukocytes, epithelial cells, mesenchymal cells, mesenchymal stem cells, stromal cells, endothelial cells, fibroblasts, cancer-associated fibroblasts (CAF), pericytes, adipocytes, cancer stem cells, circulating tumor cells (CTC), hematopoietic stem cells and hematopoietic progenitor cells. In various embodiments, leukocytes are myeloid cells and lymphoid cells. In various embodiments, myeloid cells are monocytes, macrophages, neutrophils, granulocytes, dendritic cells, mast cells, eosinophils and basophils. In various embodiments, the lymphoid cell is a T cell, a B cell, a NK cell, a NK-T cell, or an iNK cell.

[0526] In various embodiments, analysis of cells from one or more blood samples collected from subjects suffering from cancer demonstrates increased levels of immunosuppressive cells compared to analysis of cells from one or more healthy subjects or one or more subjects suffering from cancer and responding to treatment (e.g., responding to immunotherapy (such as immune checkpoint inhibitors)). In various embodiments, the immunosuppressive cells are myeloid-derived suppressor cells (MDSCs), tumor-associated macrophages (TAMs), neutrophils, T cells, and / or cytokines. 调控 Cells and B 调控Cells. In various embodiments, MDSCs are monocytic MDSCs (M-MDSCs) and polymorphonuclear MDSCs (PMN-MDSCs). In various embodiments, TAMs are M2 TAMs. In various embodiments, the immunosuppressive cells are CAFs. In various embodiments, the level of immunosuppressive cells in one or more blood samples of a subject having one or more tumors characterized as immune-refractory, immune-protective, or immune "cold" is increased by about 5-100% (e.g., about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%, including all values ​​and ranges between these values), 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, or 50% compared to one or more blood samples collected from one or more healthy subjects or one or more subjects suffering from cancer and responding to treatment. In various embodiments, the level of immunosuppressive cells in one or more blood samples of subjects with one or more tumors characterized as immune-refractory, immune-protective or immune-"cold" is increased by about 2-100 times (e.g., about 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 times, including all values ​​and ranges between these values) compared to one or more blood samples collected from one or more healthy subjects or one or more subjects suffering from cancer and responding to treatment. In various embodiments, immunosuppressive cells are identified by analyzing cell surface protein expression. In various embodiments, analysis of cells from one or more blood samples collected from subjects having one or more tumors characterized as immune-refractory, immune-protective, or immune-"cold" demonstrates reduced levels or absence of activated pro-inflammatory immune cells (e.g., a reduction of about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%, including all values ​​and ranges between these values, relative to healthy subjects or subjects who have cancer and are responsive to treatment).

[0527] In various embodiments, analysis of cells from one or more blood samples collected from subjects with one or more tumors characterized as immune refractory, immune protective, or immune "cold" confirms that the level of activated proinflammatory immune cells is reduced or absent (e.g., reduced by about 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 times relative to healthy subjects or subjects suffering from cancer and responding to treatment, including all values ​​and ranges between these values). In various embodiments, activated proinflammatory cells are dendritic cells (DCs), macrophages, M1 macrophages, T cells, B cells, NK cells, NK-T cells, and iNK cells. In various embodiments, the frequency of proinflammatory immune cells is ≤10% (e.g., about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, or about 1%) among all white blood cells analyzed from one or more blood samples collected from a subject. In various embodiments, activated pro-inflammatory immune cells are identified by analyzing cell surface protein expression.

[0528] In various embodiments, the analysis of cells in one or more blood samples of a subject suffering from cancer is performed by analyzing cell surface proteins. In various embodiments, the cell surface proteins are selected from the group consisting of receptor tyrosine kinase (RTK), CD1c, CD2, CD3, CD4, CD5, CD8, CD9, CD10, CD11b, CD11c, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD24, TACI, CD25, CD27, CD28, CD30, CD30L, CD31, CD32, CD32b, CD34, CD33, CD38, CD39, CD40, CD40-L, CD41b, CD42a, CD42b, CD43, CD44, CD48 , CD47, CD45RA, CD45RO, CD48, CD52, CD55, CD56, CD58, CD61, CD66b, CD70, CD72, CD79, CD68, CD84, CD86, CD93, CD94, CD95, CRACC, BLAME, BCMA, CD103 , CD107, CD112, CD120a, CD120b, CD123, CD125, CD134, CD135, CD140a, CD141, CD154, CD155, CD160, CD163, CD172a, XCR1, CD203c, CD204, CD206, CD207CD226、CD244、CD267、CD268、CD269、CD355、CD358、NKG2A、NKG2B、NKG2 C、NKG2D、NKG2E、NKG2F、NKG2H、KIR2DL1、KIR2DL2、KIR2DL3、KIR2DL5A、 KIR2DL5B、KIR3DL1、KIR3DL2、KIR3DL3、KIR3DL4、KIR2DS1、KIR2DS2、KI R2DS3、KIR2DS4、KIR2DS5、DAP12、KIR3DS、NKp44、NKp46、TCR、BCR FcβεRI、MHC-I、MHC-II、IL-1R、IL-2Rα、IL-2Rβ、IL-2Rγ、IL-3Rα、CSF2 RB、IL-4R、IL-5Rα、IL-6Rα、gp130、IL-7Rα、IL-9R、IL-12Rβ1、IL-12Rβ2 、IL-13Rα1、IL-13Rα2、IL-15Rα、IL-21R、IL-23R、IL-27Rα、IL-31Rα、OS MR、CSF-1R、Fluorescence IL-15、IL-10Rα、IL-10Rβ、IL-20Rα、IL-20Rβ、IL-22Rα 1、IL-22Rα2、IL-22Rβ、IL-28RA、PD-1、PD-1H、BTLA、CTLA-4、PD-L1、PD -L2、2B4、B7-1、B7-2、B7-H1、B7-H4、B7-DC、DR3、LIGHT、LAIR、LTα1β2、L TβR、TIM-1、TIM-3、TIM-4、TIGIT、LAG-3、ICOS、ICOS-L、SLAM、SLAMF2、O X-40、OX-40L、GITR、GITRL、TL1A、HVEM、41-BB、41BB-L、TL-1A、TRAF1、T RAF2、TRAF3、TRAF5、BAFF、BAFF-R、APRIL、TRAIL、RANK、AITR、TRAMP、C CR1、CCR2、CCR3、CCR4、CCR5、CCR6、CCR7、CCR8、CCR9、CCR10、CCR11、CXC R1、CXCR2、CXCR3、CXCR4、CXCR5、CXCR6、CXCR7、CLECL9a、DC-SIGN、IGSF 4A、SIGLEC、EGFR、PDGFR、VEGFR、FAPα-SMA is also associated with FAS-L-F c,ICAM-1,ICAM-2,ICAM-3,ICAM-4,ICAM-5,PECAM-1,MICA,MICB,UL16,ULBP1,ULBP2,ILBP3,ULBP4,ULBP5,ULBP6,MULT1,RAE1α,β,γ,δ and ε,A 1 R.A. 2A R.A. 2B R and A 3 In various embodiments, the integrin is selected from the group consisting of α1, α2, αIIb, α3, α4, α5, α6, α7, α8, α9, α10, α11, αD, αE, αL, αM, αV, αX, β1, β2, β3, β4, β5, β6, β7, β8 or a combination thereof. In various embodiments, the TCR is selected from the group consisting of α, β, γ, δ, ε and ζTCR. Several methods for analyzing cell surface protein expression have been described in the literature, including flow cytometry and massive blood cell counting (CyTOF). The presence or abundance of one or more of these cell surface proteins indicates that the patient can be treated with the methods disclosed herein.

[0529] In various embodiments, the analysis of one or more blood samples collected from subjects with one or more tumors characterized as immune refractory, immune protection or immune "cold" shows high neutrophil to lymphocyte ratio (NLR). In various embodiments, the analysis of one or more blood samples collected from subjects with one or more tumors characterized as immune refractory, immune protection or immune "cold" confirms NLR ≥ 2. In various embodiments, the analysis of one or more blood samples collected from subjects with one or more tumors characterized as immune refractory, immune protection or immune "cold" confirms NLR between 2 and 10 (for example, NLR is 2, 3, 4, 5, 6, 7, 8, 9 and 10, including all values ​​and scopes between these values). In various embodiments, NLR is used to determine the prognosis of subjects suffering from cancer and having one or more tumors characterized as immune refractory, immune protection or immune "cold". In various embodiments, NLR ≥ 2 determines a poor prognosis.

[0530] In various embodiments, the cells analyzed by one or more blood samples collected from a subject suffering from cancer are circulating tumor cells (CTC). In various embodiments, compared with the analysis of one or more blood samples collected from one or more healthy subjects or one or more subjects suffering from cancer and responding to treatment, the analysis of one or more blood samples collected from a subject suffering from cancer confirms that the CTC frequency increases. In various embodiments, the frequency of circulating tumor cells in one or more blood samples of a subject with one or more tumors characterized as immune refractory, immune protective or immune "cold" is ≥3 or ≤5 CTCs per 7.5 ml of blood.

[0531] In various embodiments, the tumor characteristics of a subject suffering from cancer are determined from analysis of proteins in one or more blood samples of the subject. In various embodiments, the tumor characteristics of a subject suffering from cancer are determined by comparing analysis of proteins in one or more blood samples from a subject suffering from cancer with analysis of one or more blood samples from one or more healthy subjects or one or more subjects suffering from cancer and responding to treatment. In various embodiments, the protein is an intracellular protein or a secreted protein. In various embodiments, the protein is selected from the group consisting of: a cytokine, a chemokine, a growth factor, an enzyme, a protease, and a nuclease. In various embodiments, the cytokines and chemokines are selected from the group consisting of IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-35, IL-36, CCL1, CCL2, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9, CCL10, CCL11, CCL 12, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CXCL1, CXCL2 (MCP-1), CXCL3 (MIP-1α), CXCL4 (MIP-1β), CXCL5 (RANTES), CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCL17, IFN-α, IFN-β, IFN-γ, granzyme-B, perforin, TNF-α, TGF-β1, TGF-β2 and TGF-β3. In various embodiments, the growth factor is selected from the group consisting of: EGF, FGF, NGF, PDGF, VEGF, IGF, GMCSF, GCSF, TGF, erythropoietin, TPO, BMP, HGF, GDF, neurotrophins, MSF, SGF, GDF, G-CSF and GM-CSF. In various embodiments, the protein is a protease selected from the group consisting of: aspartic proteases, cysteine ​​proteases, metalloproteases, serine proteases or threonine proteases.In some embodiments, the protein is a protease selected from the group consisting of ADAM1, ADAM2, ADAM7, ADAM8, ADAM9, ADAM10, ADAM11, ADAM12, ADAM15, ADAM17, ADAM18, ADAM19, ADAM20, ADAM21, ADAM22, ADAM23, ADAM28, ADAM29, ADAM30, ADAM33, MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP18, MMP19, MMP20, MMP21, MMP23A, MMP23B, MMP24, MMP25, MMP26, MMP27, and MMP28. In various embodiments, the protein is an enzyme selected from the group consisting of arginase, asparaginase, kynurenine, indoleamine 2,3 dioxygenase (IDO1 and IDO2), tryptophan 2,3 dioxygenase (TDO), and IL4I1. In various embodiments, the protein is associated with apoptosis. In various embodiments, the protein associated with apoptosis is selected from the group consisting of: P53, caspase 1, caspase 2, caspase 3, caspase 4, caspase 5, caspase 6, caspase 7, caspase 8, caspase 9, caspase 10, caspase 11, caspase 12, caspase 13, caspase 14, BCL-2, BCL-XL, MCL-1, CED-9, A1, BFL1, BAX, BAK, DIVA, BCL-XS, BIK, BIM, BAD, BID and EGL-1. Several methods for analyzing proteins in blood samples have been described in the literature, including western blot and ELISA.

[0532] In various embodiments, analysis of proteins from one or more blood samples of subjects having one or more tumors characterized as immunorefractory, immunoprotective, or immunologically "cold" confirms increased levels of tumor promoting, anti-inflammatory, or immunosuppressive proteins. In various embodiments, the tumor promoting, anti-inflammatory, or immunosuppressive proteins are cell surface proteins, intracellular proteins, or secreted proteins. In various embodiments, the tumor promoting, anti-inflammatory, or immunosuppressive proteins are selected from the group consisting of: CD39, CD79, MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP18, MMP19, MMP20, MMP21, MMP23A, MMP23B , MMP24, MMP25, MMP26, MMP27, MMP28, CXCL12, GM-CSF, G-CSF, TGF-β1, TGF-β2, TGF-β3, arginase, asparaginase, kynurenase, indoleamine 2,3 dioxygenase (IDO1 and IDO2), tryptophan 2,3 dioxygenase (TDO), myeloperoxidase (MPO), neutrophil elastase (NE), and IL4I1. In various embodiments, the level of a tumor-promoting, anti-inflammatory, or immunosuppressive protein in one or more blood samples of a subject having one or more tumors characterized as immune-refractory, immune-protective, or immune-"cold" is increased by 5-100% (e.g., by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 15 ... %, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95% or about 100%, including all values ​​and ranges between these values), 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, 50% or 100%. In various embodiments, the level of a tumor-promoting, anti-inflammatory, or immunosuppressive protein is increased 2-100 fold (e.g., about 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 fold, including all values ​​and ranges in between) in one or more blood samples from a subject having one or more tumors characterized as immune-refractory, immune-protective, or immune "cold," compared to one or more blood samples collected from one or more healthy subjects or one or more subjects having cancer and responding to treatment.In various embodiments, analysis of proteins from one or more blood samples of subjects having one or more tumors characterized as immune-refractory, immune-protective, or immune-"cold"confirms reduced levels, low levels, or absence of tumor-suppressive, anti-tumor, or pro-inflammatory proteins. In various embodiments, the tumor-suppressive, anti-tumor, or pro-inflammatory protein is selected from the group consisting of IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-34, IL-35, IL-36, IL-37, IL-38, IL-39, IL-40, IL-41, IL-42, IL-43, IL-44, IL-45 4, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-35, IL-36, cell surface IL-15, CXCL2 (MCP-1), CXCL3 (MIP-1α), CXCL4 (MIP-1β), CXCL5 (RANTES), IFN-α, IFN-β, IFN-γ, granzyme-B, perforin and TNF-α. In various embodiments, the level of tumor suppressor, anti-tumor or pro-inflammatory protein in one or more blood samples of subjects with one or more tumors characterized as immune-refractory, immune-protective or immune "cold" is reduced by 5-100% (e.g., about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 150%, about 100%, about 200%, about 300%, about 400%, about 500%, about 600%, about 700%, about 800%, about 900%, about 1000%, about 1500%, about 1000%, about 2000%, about 3000%, about 4000%, about 5000%, about 6000%, about 7000%, about 8000%, about 10000%, about 20000%, about 30000%, about 40000%, about 50000%, about 60000%, about 70000%, about 80000%, about 100000%, about 200000%, about 300000%, about 300000%, about 4000 %, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95% or about 100%, including all values ​​and ranges between these values), 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, 50% or 100%. In various embodiments, the level of a tumor suppressor, anti-tumor, or pro-inflammatory protein is reduced by 2-100 fold (e.g., about 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 fold, including all values ​​and ranges in between) in one or more blood samples from a subject having one or more tumors characterized as immune-refractory, immune-protective, or immune "cold," compared to one or more blood samples collected from one or more healthy subjects or subjects having cancer and responding to treatment.Several methods have been described in the literature for analyzing blood samples for proteins, including Western blotting and ELISA.

[0533] In various embodiments, analysis of one or more blood samples from subjects with one or more tumors characterized as immune-refractory, immune-protective, or immune-"cold" demonstrates increased levels of neutrophil extracellular traps (NETs). In various embodiments, analysis of one or more blood samples from subjects with one or more tumors characterized as immune-refractory, immune-protective, or immune-"cold" demonstrates increased levels of neutrophil extracellular traps (NETs) compared to analysis of one or more blood samples from one or more healthy subjects or one or more subjects suffering from cancer and responding to treatment. In various embodiments, the level of NETs in one or more blood samples of a subject having one or more tumors characterized as immune-refractory, immune-protective, or immune "cold" is increased by 5-100% (e.g., by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%, including all values ​​and ranges between these values), 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, 50%, or 100% compared to one or more blood samples collected from one or more healthy subjects or one or more subjects having cancer and responding to treatment. In various embodiments, the level of NET in one or more blood samples of subjects with one or more tumors characterized as immune refractory, immune protective or immune "cold" is increased by 2-100 times (e.g., about 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 times, including all values ​​and ranges between these values) compared to one or more blood samples collected from one or more healthy subjects or one or more subjects suffering from cancer and responding to treatment. Several methods for analyzing NET in blood samples have been described in the literature, including Western blotting, ELISA, and flow cytometry.

[0534] In various embodiments, the tumor characteristics of a subject suffering from cancer are determined from the analysis of nucleic acids in one or more blood samples of the subject. In various embodiments, the tumor characteristics of a subject suffering from cancer are determined by comparing the analysis of nucleic acids in one or more blood samples from a subject suffering from cancer with the analysis of one or more blood samples from one or more healthy subjects or one or more subjects suffering from cancer and responding to treatment. In various embodiments, nucleic acids are selected from the group comprising: DNA, ssDNA, circulating tumor DNA (ctDNA), RNA, mRNA, dsRNA, siRNA, miRNA and lncRNA. In various embodiments, the analysis of ctDNA from one or more blood samples of a subject with one or more tumors characterized as immune refractory, immune protective or immune "cold" confirms that the level of one or more tumor mutations, tumor antigens or new antigens is low or absent. In various embodiments, the analysis of ctDNA from one or more blood samples of a subject suffering from cancer shows low tumor mutation load or no tumor mutation load. In various embodiments, analysis of ctDNA from one or more blood samples from subjects having one or more tumors characterized as immune-refractory, immune-protective, or immune "cold" confirms a tumor mutation burden of 5 to 0.001 (e.g., about 5, about 4, about 3, about 2, about 1, about 0.9, about 0.8, about 0.7, about 0.6, about 0.5, about 0.4, about 0.3, about 0.2, about 0.1, about 0.09, about 0.08, about 0.07, about 0.06, about 0.05, about 0.4, about 0.03, about 0.02, about 0.01, about 0.009, about 0.008, about 0.007, about 0.006, about 0.005, about 0.004, about 0.003, about 0.002, or 0.001, including all values ​​and ranges between these values) individual cell mutations per million base pairs. In various embodiments, nucleic acid analysis is performed by PCR, RT-PCR, qRT-PCR, next generation sequencing (NGS), RNA-seq, ATAC-seq, exome sequencing, Southern blotting, microarray analysis, or single cell sequencing.

[0535] In various embodiments, gene expression analysis of nucleic acids in one or more blood samples from subjects suffering from cancer is used to determine tumor characteristics of the subject. In various embodiments, gene expression analysis of nucleic acids in one or more blood samples from subjects having one or more tumors characterized as immune-refractory, immune-protective, or immune-"cold" demonstrates increased expression of tumor-promoting, tumor-permissive, or immune-suppressive genes compared to analysis of one or more blood samples from one or more healthy subjects or one or more subjects suffering from cancer and responding to treatment. In various embodiments, the expression of tumor-promoting, tumor-permissive, or immunosuppressive genes in one or more blood samples of a subject having one or more tumors characterized as immune-refractory, immune-protective, or immune-"cold" is increased by 5-100% (e.g., by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%, including all values ​​and ranges between these values), 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, or 50% compared to one or more blood samples collected from one or more healthy subjects or one or more subjects suffering from cancer and responding to treatment. In various embodiments, the expression of tumor promoting, tumor permissive or immunosuppressive genes in one or more blood samples of a subject having one or more tumors characterized as immune refractory, immune protective or immune "cold" is increased by 2-100 times (e.g., about 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 times, including all values ​​and ranges between these values) compared to one or more blood samples collected from one or more healthy subjects or one or more subjects suffering from cancer and responding to treatment. In various embodiments, analysis of nucleic acids in one or more blood samples of a subject having one or more tumors characterized as immune refractory, immune protective or immune "cold" confirms decreased expression of tumor suppressive, anti-tumor or pro-inflammatory genes compared to analysis of one or more blood samples from one or more healthy subjects or one or more subjects suffering from cancer and responding to treatment.In various embodiments, analysis of nucleic acids in one or more blood samples from subjects having one or more tumors characterized as immune-refractory, immune-protective, or immune "cold" demonstrates low or no expression of tumor suppressive, anti-tumor, or anti-inflammatory genes compared to analysis of one or more blood samples from one or more healthy subjects or one or more subjects having cancer and responding to treatment. In various embodiments, the expression of tumor suppressor, anti-tumor, or pro-inflammatory genes in one or more blood samples of a subject having one or more tumors characterized as immune-refractory, immune-protective, or immune "cold" is reduced by 5-100% (e.g., by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%, including all values ​​and ranges in between these values), 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, or 50% compared to healthy subjects or subjects with cancer who are responsive to therapy. In various embodiments, the expression of tumor suppressor, anti-tumor or pro-inflammatory genes in one or more blood samples of subjects with one or more tumors characterized as immune-refractory, immune-protective or immune "cold" is reduced by 2-100 fold (e.g., about 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 fold relative to healthy subjects or subjects suffering from cancer and responding to therapy, including all values ​​and ranges between these values). In various embodiments, gene expression analysis is performed by PCR, RT-PCR, qRT-PCR, next generation sequencing (NGS), RNA-seq, ATAC-seq, exome sequencing, Southern blotting, microarray analysis or single cell sequencing.

[0536] In various embodiments, the tumor characteristics of the subject suffering from cancer are determined by the analysis of one or more tumor samples collected from the subject. In various embodiments, the tumor sample is a biopsy. In various embodiments, the tumor characteristics of the subject suffering from cancer are determined by the analysis of cells, proteins or nucleic acids in one or more tumor samples collected from the subject. In various embodiments, the tumor characteristics of the subject suffering from cancer are determined by comparing the analysis of cells, proteins or nucleic acids in one or more tumor samples from the subject suffering from cancer with the analysis of tissue samples from one or more healthy subjects or one or more subjects suffering from cancer and responding to treatment. In various embodiments, the cells analyzed in one or more tumor samples are leukocytes, epithelial cells, mesenchymal cells, mesenchymal stem cells, stromal cells, endothelial cells, fibroblasts, pericytes, adipocytes and cancer stem cells. In various embodiments, leukocytes are myeloid cells and lymphoid cells. In various embodiments, myeloid cells are monocytes, macrophages, neutrophils, granulocytes, dendritic cells, mast cells, eosinophils and basophils. In various embodiments, the lymphoid cell is a T cell, a B cell, a NK cell, a NK-T cell, or an iNK cell.

[0537] In various embodiments, analysis of cells from one or more tumor samples collected from subjects with one or more tumors characterized as immune-refractory, immune-protective, or immune-"cold" confirms the presence of immunosuppressive cells. In various embodiments, analysis of one or more tumor samples collected from subjects with one or more tumors characterized as immune-refractory, immune-protective, or immune-"cold" confirms the presence of immunosuppressive cells in the tumor core. In various embodiments, analysis of cells from one or more tumor samples collected from subjects with one or more tumors characterized as immune-refractory, immune-protective, or immune-"cold" confirms increased levels of immunosuppressive cells. In various embodiments, analysis of one or more tumor samples confirms increased levels of immunosuppressive cells in the tumor core. In various embodiments, the immunosuppressive cells are myeloid-derived suppressor cells (MDSCs), tumor-associated macrophages (TAMs), neutrophils, T cells, or T cells. 调控 Cells and B 调控Cells. In various embodiments, MDSCs are monocytic MDSCs (M-MDSCs) and polymorphonuclear MDSCs (PMN-MDSCs). In various embodiments, TAMs are M2 TAMs. In various embodiments, the immunosuppressive cells are CAFs. In various embodiments, the level of immunosuppressive cells in one or more tumor samples from a subject having one or more tumors characterized as immune-refractory, immune-protective, or immune "cold" is increased by 5-100% (e.g., by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%, including all values ​​and ranges between these values), 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, or 50% compared to tissue samples from one or more healthy subjects or one or more subjects having cancer and responding to treatment. In various embodiments, the level of immunosuppressive cells in one or more tumor samples from a subject having one or more tumors characterized as immune-refractory, immune-protective, or immune "cold" is increased by 2-100 fold compared to one or more tissue samples from one or more healthy subjects or one or more subjects having cancer and responding to treatment (e.g., an increase of about 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 fold relative to healthy subjects or subjects having cancer and responding to treatment, including all values ​​and ranges in between these values).

[0538] In various embodiments, analysis of cells from one or more tumor samples collected from subjects with one or more tumors characterized as immune-refractory, immune-protective, or immune-"cold" confirms the absence of leukocytes. In various embodiments, analysis of cells from one or more tumor samples collected from subjects with one or more tumors characterized as immune-refractory, immune-protective, or immune-"cold" confirms reduced or low levels of leukocytes. In various embodiments, the frequency of leukocytes is ≤50%, ≤40%, ≤30%, ≤20%, ≤10%, or ≤5% of all cells analyzed, including all values ​​and ranges between these values.

[0539] In various embodiments, analysis of cells of one or more tumor samples collected from subjects with one or more tumors characterized as immune refractory, immune protective or immune "cold" confirms the absence of activated proinflammatory immune cells. In various embodiments, analysis of cells of one or more tumor samples collected from subjects with one or more tumors characterized as immune refractory, immune protective or immune "cold" confirms the absence of activated proinflammatory immune cells from the tumor core. In various embodiments, analysis of cells of one or more tumor samples collected from subjects with one or more tumors characterized as immune refractory, immune protective or immune "cold" confirms low or reduced levels of activated proinflammatory immune cells. In various embodiments, analysis of cells of one or more tumor samples collected from subjects with one or more tumors characterized as immune refractory, immune protective or immune "cold" confirms low or reduced levels of activated proinflammatory immune cells in the tumor core. In various embodiments, activated proinflammatory cells are dendritic cells (DC), macrophages, M1 macrophages, T cells, B cells, NK cells, NK-T cells, and iNK cells. In various embodiments, the frequency of proinflammatory immune cells is ≤50%, ≤40%, ≤30%, ≤20%, ≤10%, or ≤5% of all cells analyzed, including all values ​​and ranges therebetween.

[0540] In various embodiments, the tumor characteristics of a subject suffering from cancer are determined from an analysis of the location of immune cells in one or more tumor samples of the subject. In various embodiments, immune cells in one or more tumor samples of a subject with one or more immune-refractory, immune-protective, or immune-"cold" tumors are localized to the tumor periphery. In various embodiments, immune cells are not present in the tumor core in one or more tumor samples of a subject with one or more immune-refractory, immune-protective, or immune-"cold" tumors. In various embodiments, immune cells in the tumor core are reduced in one or more tumor samples of a subject with one or more immune-refractory, immune-protective, or immune-"cold" tumors. In various embodiments, the immune cells in the tumor core are reduced by 5-100% (e.g., by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%, including all values ​​and ranges between these values, relative to healthy subjects or subjects with cancer and responding to treatment), 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, or 50%.

[0541] In various embodiments, the analysis of the position of the stromal cells in one or more tumor samples of the subject determines the tumor characteristics of the subject suffering from cancer. In various embodiments, the stromal cells are CAFs, pericytes, adipocytes and endothelial cells. In various embodiments, the CAFs in the periphery of the tumor increase in one or more tumor samples of the subject with one or more immune refractory, immune protective or immune "cold" tumors. In various embodiments, the CAFs in the tumor core increase in one or more tumor samples of the subject with one or more immune refractory, immune protective or immune "cold" tumors. In various embodiments, the frequency of CAFs in the periphery of the tumor is increased by 5-100% (e.g., by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%, including all values ​​and ranges between these values), 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, 50% or 100% compared to one or more tissue samples from one or more healthy subjects or subjects suffering from cancer and responding to treatment. In various embodiments, the frequency of CAFs in the tumor periphery is increased by 2-100 fold (e.g., by about 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 fold, including all values ​​and ranges in between) compared to one or more tissue samples from one or more healthy subjects or one or more subjects suffering from cancer and responding to treatment. In various embodiments, the frequency of CAFs in the tumor core is increased by 5-100% (e.g., by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%, including all values ​​and ranges between these values), 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, 50%, or 100% compared to one or more tissue samples from one or more healthy subjects or one or more subjects suffering from cancer and responding to treatment.In various embodiments, the frequency of CAFs in the tumor core is increased by 2-100 fold compared to one or more healthy tissue samples (e.g., about 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 fold, including all values ​​and ranges in between, relative to healthy subjects or subjects suffering from cancer and responding to treatment).

[0542] In various embodiments, the analysis of cells in one or more tumor samples of a subject suffering from cancer is performed by analyzing cell surface proteins. In various embodiments, the cell surface proteins are selected from the group consisting of receptor tyrosine kinase (RTK), CD1c, CD2, CD3, CD4, CD5, CD8, CD9, CD10, CD11b, CD11c, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD24, TACI, CD25, CD27, CD28, CD30, CD30L, CD31, CD32, CD32b, CD34, CD33, CD38, CD39, CD40, CD40-L, CD41b, CD42a, CD42b, CD43, CD44, CD45 , CD47, CD45RA, CD45RO, CD48, CD52, CD55, CD56, CD58, CD61, CD66b, CD70, CD72, CD79, CD68, CD84, CD86, CD93, CD94, CD95, CRACC, BLAME, BCMA, CD103 , CD107, CD112, CD120a, CD120b, CD123, CD125, CD134, CD135, CD140a, CD141, CD154, CD155, CD160, CD163, CD172a, XCR1, CD203c, CD204, CD206, CD207CD226、CD244、CD267、CD268、CD269、CD355、CD358、NKG2A、NKG2B、NKG2 C、NKG2D、NKG2E、NKG2F、NKG2H、KIR2DL1、KIR2DL2、KIR2DL3、KIR2DL5A、 KIR2DL5B、KIR3DL1、KIR3DL2、KIR3DL3、KIR3DL4、KIR2DS1、KIR2DS2、KI R2DS3、KIR2DS4、KIR2DS5、DAP12、KIR3DS、NKp44、NKp46、TCR、BCR FcβcRI、MHC-I、MHC-II、IL-1R、IL-2Rα、IL-2Rβ、IL-2Rγ、IL-3Rα、CSF2 RB、IL-4R、IL-5Rα、IL-6Rα、gp130、IL-7Rα、IL-9R、IL-12Rβ1、IL-12Rβ2 、IL-13Rα1、IL-13Rα2、IL-15Rα、IL-21R、IL-23R、IL-27Rα、IL-31Rα、OS MR、CSF-1R、Fluorescence IL-15、IL-10Rα、IL-10Rβ、IL-20Rα、IL-20Rβ、IL-22Rα 1、IL-22Rα2、IL-22Rβ、IL-28RA、PD-1、PD-1H、BTLA、CTLA-4、PD-L1、PD -L2、2B4、B7-1、B7-2、B7-H1、B7-H4、B7-DC、DR3、LIGHT、LAIR、LTa1β2、L TβR、TIM-1、TIM-3、TIM-4、TIGIT、LAG-3、ICOS、ICOS-L、SLAM、SLAMF2、O X-40、OX-40L、GITR、GITRL、TL1A、HVEM、41-BB、41BB-L、TL-1A、TRAF1、T RAF2、TRAF3、TRAF5、BAFF、BAFF-R、APRIL、TRAIL、RANK、AITR、TRAMP、C CR1、CCR2、CCR3、CCR4、CCR5、CCR6、CCR7、CCR8、CCR9、CCR10、CCR11、CXC R1、CXCR2、CXCR3、CXCR4、CXCR5、CXCR6、CXCR7、CLECL9a、DC-SIGN、IGSF 4A、SIGLEC、EGFR、PDGFR、VEGFR、FAPα-SMA is also associated with FAS-L-F c,ICAM-1,ICAM-2,ICAM-3,ICAM-4,ICAM-5,PECAM-1,MICA,MICB,UL16,ULBP1,ULBP2,ILBP3,ULBP4,ULBP5,ULBP6,MULT1,RAE1α,β,γ,δ and ε,A 1 R.A. 2A R.A. 2B R and A 3 In various embodiments, the integrin is selected from the group consisting of α1, α2, αIIb, α3, α4, α5, α6, α7, α8, α9, α10, α11, αD, αE, αL, αM, αV, αX, β1, β2, β3, β4, β5, β6, β7, β8 or a combination thereof. In various embodiments, the TCR is selected from the group consisting of α, β, γ, δ, ε and ζTCR. Several methods for analyzing cell surface protein expression of tumor samples have been described in the literature, including immunohistochemistry, immunofluorescence, Western blotting, flow cytometry, and massive blood cell counting (CyTOF).

[0543] The tumor core is often described as the densely packed, central, bulky, and differentiated region of the tumor. In contrast, the tumor periphery is often described as the invasive edge of the tumor that interacts with the surrounding stroma and parenchyma.

[0544] In various embodiments, the tumor characteristics of a subject suffering from cancer are determined from analysis of proteins in one or more tumor samples of the subject. In various embodiments, the tumor characteristics of a subject suffering from cancer are determined by comparing analysis of proteins in one or more tumor samples from a subject suffering from cancer with analysis of one or more tissues from one or more healthy subjects or one or more subjects suffering from cancer and responding to treatment. In various embodiments, the protein is intracellular or extracellular. In various embodiments, the protein is selected from the group consisting of: cytokines, chemokines, growth factors, enzymes, proteases, and nucleases. In various embodiments, the cytokines and chemokines are selected from the group consisting of IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-35, IL-36, CCL1, CCL2, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9, CCL10, CCL11, CCL 12, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CXCL1, CXCL2 (MCP-1), CXCL3 (MIP-1α), CXCL4 (MIP-1β), CXCL5 (RANTES), CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCL17, IFN-α, IFN-β, IFN-γ, granzyme-B, perforin, TNF-α, TGF-β1, TGF-β2 and TGF-β3. In various embodiments, the growth factor is selected from the group consisting of: EGF, FGF, NGF, PDGF, VEGF, IGF, GMCSF, GCSF, TGF, erythropoietin, TPO, BMP, HGF, GDF, neurotrophins, MSF, SGF, GDF, G-CSF and GM-CSF. In various embodiments, the protein is a protease selected from the group consisting of: aspartic proteases, cysteine ​​proteases, metalloproteases, serine proteases or threonine proteases.In some embodiments, the protein is a protease selected from the group consisting of ADAM1, ADAM2, ADAM7, ADAM8, ADAM9, ADAM10, ADAM11, ADAM12, ADAM15, ADAM17, ADAM18, ADAM19, ADAM20, ADAM21, ADAM22, ADAM23, ADAM28, ADAM29, ADAM30, ADAM33, MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP18, MMP19, MMP20, MMP21, MMP23A, MMP23B, MMP24, MMP25, MMP26, MMP27, and MMP28. In various embodiments, the protein is an enzyme selected from the group consisting of arginase, asparaginase, kynurenine, indoleamine 2,3 dioxygenase (IDO1 and IDO2), tryptophan 2,3 dioxygenase (TDO), and IL4I1. In various embodiments, the protein is associated with apoptosis. In various embodiments, the protein associated with apoptosis is selected from the group consisting of: P53, caspase 1, caspase 2, caspase 3, caspase 4, caspase 5, caspase 6, caspase 7, caspase 8, caspase 9, caspase 10, caspase 11, caspase 12, caspase 13, caspase 14, BCL-2, BCL-XL, MCL-1, CED-9, A1, BFL1, BAX, BAK, DIVA, BCL-XS, BIK, BIM, BAD, BID and EGL-1. Several methods for analyzing proteins in tumor samples have been described in the literature, including immunohistochemistry, immunofluorescence, Western blotting and ELISA.

[0545] In various embodiments, analysis of proteins in one or more tumor samples of subjects having one or more tumors characterized as immune-refractory, immune-protective, or immune-"cold" confirms increased levels of proteins associated with tumor progression, anti-inflammatory activity, or immunosuppression. In various embodiments, the proteins associated with tumor progression, anti-inflammatory activity, or immunosuppression are cell surface proteins, intracellular proteins, or secreted proteins. In various embodiments, the proteins associated with tumor progression, anti-inflammatory activity, or immunosuppression are selected from the group consisting of: CD39, CD47, CD79, CD140a, CD163, CD206, FOXP3, FAP, PD-1, PD-L1, PD-L2, CSF-1R, A 1 R.A. 2A R.A. 2BR.A. 3 R, TIM-1, TIM-3, TIM-4, TIGIT, CSFR, SIGLEC, MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11 , MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP18, MMP19, MMP20, MMP21, MMP23A, MMP23B, MMP2 4, MMP25, MMP26, MMP27, MMP28, CXCL12, GM-CSF, G-CSF, FAP, TGF-β1, TGF-β2, TGF-β3, arginase, asparaginase, kynurenase, indoleamine 2,3 dioxygenase (IDO1 and IDO2), tryptophan 2,3 dioxygenase (TDO), myeloperoxidase (MPO), neutrophil elastase (NE) and IL4I1. In various embodiments, the level of a protein associated with tumor progression, anti-inflammatory activity or immunosuppression in one or more tumor samples of a subject having one or more tumors characterized as immune-refractory, immune-protective or immune-"cold" is increased by 5-100% (e.g., by about 5%, about 10%, about 15%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 110%, about 120%, about 130%, about 140%, about 150%, about 160%, about 170%, about 180%, about 190%, about 200%, about 210%, about 220%, about 230%, about 240%, about 250%, about 260%, about 270%, about 280%, about 290%, about 300%, about 310%, about 320%, about 330%, about 340%, about 350%, about 360%, about 370%, about 380%, about 390%, about 400%, about 410%, about 420%, about 430%, about 440%, about 450%, about 460%, about 470%, about 480%, about 490%, about 5 %, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95% or about 100%, including all values ​​and ranges between these values), 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, 50% or 100%. In various embodiments, the level of a protein associated with tumor progression, anti-inflammatory activity, or immunosuppression is increased 2-100 fold in one or more tumor samples from a subject having one or more tumors characterized as immune-refractory, immune-protective, or immune-"cold" compared to one or more tissue samples from one or more healthy subjects or subjects having cancer and responding to treatment (e.g., about 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 fold relative to healthy subjects or subjects having cancer and responding to treatment, including all values ​​and ranges in between these values).

[0546] In various embodiments, analysis of proteins from one or more tumor samples from subjects having one or more tumors characterized as immune-refractory, immune-protective, or immune-"cold"confirms reduced levels, low levels, or absence of proteins associated with tumor growth inhibition, anti-tumor activity, or pro-inflammatory activity. In various embodiments, the protein associated with tumor growth inhibition, anti-tumor activity or pro-inflammatory activity is selected from the group consisting of: CD44, CD56, CD103c, CD69, KG2A, NKG2B, NKG2C, NKG2D, NKG2E, NKG2F, NKG2H, ICOS, ICOS-L, SLAM, SLAMF2, OX-40, OX-40L, GITR, GITRL, TL-1A, HVEM, 41-BB, 41BB-L, TRAF1, TRAF2, TRAF3, TRAF5, BAFF, BAFF-R, APRIL, TRAIL, RANK, AITR, TRAMP, IL-1α, IL-1β, IL-2, IL-3, IL- 4. IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-2 8. IL-29, IL-30, IL-31, IL-32, IL-33, IL-35, IL-36, CXCL2(MCP-1), CXCL3(MIP-1α), CXCL4(MIP-1β), CXCL5(RANTES), IFN-α, IFN-β, IFN-γ, granzyme-B, perforin and TNF-α. In various embodiments, the level of a protein associated with tumor growth inhibition, anti-tumor activity, or pro-inflammatory activity is reduced by 5-100% (e.g., by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%, including all values ​​and ranges between these values), 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, 50%, or 100%, as compared to one or more samples collected from one or more healthy tissues or one or more tumor samples collected from a subject suffering from cancer and responding to treatment. Several methods have been described for analyzing tumor samples for proteins, including immunohistochemistry, immunofluorescence, Western blotting, intracellular flow cytometry, and ELISA.

[0547] In various embodiments, the tumor characteristics of a subject suffering from cancer are determined from a tumor proportion score (TPS) of PD-L1 expression in one or more tumor samples from the subject. In various embodiments, the TPS of a subject having one or more tumors characterized as immune-refractory, immune-protective, or immune "cold" is between 1 and 50 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50, including all ranges between these values). In various embodiments, a subject having one or more tumors characterized as immune-refractory, immune-protective, or immune-"cold" has a TPS ≤ 1. The TPS for PD-L1 expression is defined as the percentage of viable tumor cells that exhibit partial or complete membrane staining according to immunohistochemical analysis.

[0548] In various embodiments, the combined positive score (CPS) of PD-L1 expression in one or more tumor samples from the subject is used to determine the tumor characteristics of the subject suffering from cancer. In various embodiments, the CPS of a subject with one or more tumors characterized as immune refractory, immune protective or immune "cold" is ≤10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, including all ranges between these values). In various embodiments, CPS ≤ 1. The CPS of PD-L1 expression is determined from an immunohistochemical assay of the percentage of live tumor cells, lymphocytes, and macrophages positive for PD-L1 as a percentage of all live tumor cells.

[0549] In various embodiments, the tumor characteristics of a subject suffering from cancer are determined from a microsatellite instability test of one or more tumor samples from the subject. In various embodiments, the tumor characteristics of a subject suffering from cancer are determined by comparing the microsatellite instability test of one or more tumor samples with the microsatellite stability test of one or more healthy tissues of the subject. In various embodiments, the microsatellite instability test is an analysis of microsatellite markers. In various embodiments, the microsatellite instability test is an analysis of mismatch repair markers. In various embodiments, the microsatellite marker is selected from the group consisting of: BAT25, BAT26, D2S123, D5S346 and D17S250. In various embodiments, the mismatch repair marker is selected from the group consisting of: MLH1, MSH2, MLH6 and PMS2. In various embodiments, the subject has one or more immune refractory, immune protective or immune "cold" tumors, and the tumor is determined to be low in microsatellite instability. In various embodiments, the subject has one or more immune refractory, immune protective or immune "cold" tumors, and the tumor is determined to be stable in microsatellites. In various embodiments, the subject has one or more immune-refractory, immune-protective, or immune-"cold" tumors that are mismatch repair intact.

[0550] In various embodiments, analysis of one or more tumor samples from subjects having one or more tumors characterized as immune-refractory, immune-protective, or immune-"cold" demonstrates increased levels of neutrophil extracellular traps (NETs). In various embodiments, analysis of one or more tumor samples from subjects having one or more tumors characterized as immune-refractory, immune-protective, or immune-"cold" demonstrates increased levels of neutrophil extracellular traps (NETs) compared to analysis of one or more tumor samples from one or more healthy subjects. In various embodiments, the level of NET in one or more tumor samples from a subject suffering from cancer is increased by 5-100% (e.g., by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%, including all values ​​and ranges between these values), 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, 50%, or 100% compared to one or more tissue samples from one or more healthy subjects or subjects suffering from cancer and responding to treatment. In various embodiments, the level of NET in one or more tumor samples of a subject suffering from cancer is increased by 2-100 times (e.g., about 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 times, including all values ​​and ranges between these values) compared to one or more tissue samples from one or more healthy subjects or subjects suffering from cancer and responding to treatment. Several methods for analyzing NET have been described in the literature, including Western blotting, ELISA, and flow cytometry.

[0551] In various embodiments, the tumor characteristics of a subject suffering from cancer are determined from the analysis of nucleic acids in one or more tumor samples of the subject. In various embodiments, the tumor characteristics of a subject suffering from cancer are determined by comparing the analysis of nucleic acids in one or more tumor samples from a subject suffering from cancer with the analysis of one or more tissue samples from one or more healthy subjects or subjects suffering from cancer and responding to treatment. In various embodiments, nucleic acid is selected from the group comprising: DNA, ssDNA, RNA, mRNA, dsRNA, siRNA, miRNA and lncRNA. In various embodiments, nucleic acid analysis is performed by PCR, RT-PCR, qRT-PCR, next generation sequencing (NGS), RNA-seq, ATAC-seq, exome sequencing, Southern blot, microarray analysis or single cell sequencing.

[0552] In various embodiments, tumor mutational load is determined using analysis of nucleic acids from one or more tumor samples from a subject suffering from cancer. In various embodiments, analysis of nucleic acids from one or more tumor samples from a subject having one or more tumors characterized as immune-refractory, immune-protective, or immune-"cold" demonstrates a low tumor mutational load. In various embodiments, analysis of nucleic acid from one or more tumor samples from subjects having one or more tumors characterized as immune-refractory, immune-protective, or immune "cold" demonstrates a tumor mutation load of between 5 and 0.001 (e.g., about 5, about 4, about 3, about 2, about 1, about 0.9, about 0.8, about 0.7, about 0.6, about 0.5, about 0.4, about 0.3, about 0.2, about 0.1, about 0.09, about 0.08, about 0.07, about 0.06, about 0.05, about 0.4, about 0.03, about 0.02, about 0.01, about 0.009, about 0.008, about 0.007, about 0.006, about 0.005, about 0.004, about 0.003, about 0.002, or 0.001, including all values ​​and ranges between these values) individual cell mutations per million base pairs. In various embodiments, nucleic acid analysis is performed by PCR, RT-PCR, qRT-PCR, next generation sequencing (NGS), RNA-seq, ATAC-seq, exome sequencing, Southern blotting, microarray analysis, or single cell sequencing.

[0553] In various embodiments, analysis of nucleic acids in one or more tumor samples from subjects having one or more tumors characterized as immune-refractory, immune-protective, or immune-"cold" demonstrates increased expression of genes associated with tumor-promoting, tumor-permissive, anti-inflammatory, or immunosuppressive activity compared to analysis of one or more tissue samples from one or more healthy subjects or subjects suffering from cancer and responding to treatment. In various embodiments, genes associated with tumor-promoting, tumor-permissive, anti-inflammatory, or immunosuppressive activity are selected from the group consisting of: CD39, CD47, CD79, CD140a, CD163, CD206, FOXP3, FAP, PD-1, PD-L1, PD-L2, CSF-1R, A 1 R.A. 2A R.A. 2B R.A. 3 R, TIM-1, TIM-3, TIM-4, TIGIT, CSFR, SIGLEC, MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11 , MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP18, MMP19, MMP20, MMP21, MMP23A, MMP23B, MMP2 4. MMP25, MMP26, MMP27, MMP28, CXCL12, GM-CSF, G-CSF, FAP, TGF-β1, TGF-β2, TGF-β3, arginase, asparaginase, kynurenase, indoleamine 2,3 dioxygenase (IDO1 and IDO2), tryptophan 2,3 dioxygenase (TDO), myeloperoxidase (MPO), neutrophil elastase (NE), and IL4I1. In various embodiments, the expression of genes associated with tumor promoting, tumor permissive, anti-inflammatory or immunosuppressive activity is increased by 5-100% (e.g., by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%, including all values ​​and ranges between these values), 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, 50%, or 100% compared to one or more tissue samples from one or more healthy subjects or subjects suffering from cancer and responding to treatment. In various embodiments, gene expression analysis is performed by PCR, RT-PCR, qRT-PCR, next generation sequencing (NGS), RNA-seq, ATAC-seq, exome sequencing, Southern blotting, microarray analysis, or single cell sequencing.

[0554] In various embodiments, analysis of nucleic acids in one or more tumor samples from subjects with one or more tumors characterized as immune-refractory, immune-protective, or immune-"cold" demonstrates low or reduced expression of genes associated with tumor suppressive, anti-tumor, or pro-inflammatory activity. In various embodiments, analysis of nucleic acids in one or more tumor samples from subjects with cancer demonstrates the absence of expression of genes associated with tumor suppressive, anti-tumor, or pro-inflammatory activity. In various embodiments, genes associated with tumor suppressor, anti-tumor or pro-inflammatory activity are selected from the group consisting of: CD44, CD56, CD103c, CD69, KG2A, NKG2B, NKG2C, NKG2D, NKG2E, NKG2F, NKG2H, ICOS, ICOS-L, SLAM, SLAMF2, OX-40, OX-40L, GITR, GITRL, TL-1A, HVEM, 41-BB, 41BB-L, TRAF1, TRAF2, TRAF3, TRAF5, BAFF, BAFF-R, APRIL, TRAIL, RANK, AITR, TRAMP, cell surface IL-15, IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10 -4. IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-35, IL-36, CXCL2(MCP-1), CXCL3(MIP-1α), CXCL4(MIP-1β), CXCL5(RANTES), IFN-α, IFN-β, IFN-γ, granzyme-B, perforin, TNF-α, and p53. In various embodiments, the expression of genes associated with tumor suppressor, anti-tumor or pro-inflammatory activity is reduced by 5-100% (e.g., by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%, including all values ​​and ranges between these values), 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, 50%, compared to healthy subjects or subjects suffering from cancer who are responsive to treatment.In various embodiments, gene expression analysis is performed by PCR, RT-PCR, qRT-PCR, next generation sequencing (NGS), RNA-seq, ATAC-seq, exome sequencing, Southern blotting, microarray analysis, or single cell sequencing.

[0555] In some embodiments, the methods of the present disclosure include analyzing a first and a second biological sample (e.g., a blood sample or a tumor sample) obtained from a subject, wherein "first" and "second" refer to the order in which the samples are collected. In some embodiments, a first, second, third, fourth, or fifth biological sample may be obtained and analyzed. The biological samples may be collected a few days, weeks, or months apart. Two or more biological samples may be analyzed as previously described herein. For example, a first biological sample may be obtained from a subject before administering a RAS inhibitor described herein and a second biological sample may be obtained from a subject after administering a RAS inhibitor described herein.

[0556] In various embodiments, the present disclosure provides a method for treating cancer in a subject, the method comprising administering to the subject a RAS inhibitor or a combination of compounds described herein, wherein the subject has one or more tumors resistant or unresponsive to treatment. In various embodiments, the subject has one or more tumors resistant or unresponsive to one or more treatments selected from the group consisting of: surgery, radiation, chemotherapy, biological agents, small molecules, cell-based therapy, hormone therapy, and immunotherapy. In various embodiments, treatment is standard care therapy, first-line therapy, second-line therapy, or third-line therapy. In various embodiments, the subject has one or more tumors that progress during one or more treatments, wherein treatment is standard care therapy, first-line therapy, second-line therapy, or third-line therapy.

[0557] First-line therapy is defined as the treatment administered to a subject suffering from cancer who has not yet received any existing treatment. Second-line therapy is defined as the treatment administered to a subject suffering from cancer who has received existing first-line therapy but has experienced disease progression during first-line therapy. Third-line therapy is defined as the treatment administered to a subject suffering from cancer who has received existing first-line and second-line therapy but has experienced disease progression during second-line therapy. Each specific type of cancer has first-line, second-line and third-line therapy. First-line, second-line and third-line therapy for cancer types are known in the art. In addition, the drug label approved by the FDA will indicate whether a specific drug is approved as first-line, second-line or third-line therapy.

[0558] Several criteria and definitions published in the literature can be used to determine the effect of one or more treatments on a tumor in a subject suffering from cancer. According to these criteria, a tumor is defined as "responsive," "stable," or "progressive" when it improves, remains the same, or worsens during treatment, respectively.

[0559] Examples of commonly used criteria published in the literature include Response Evaluation Criteria in Solid Tumors (RECIST), modified Response Evaluation Criteria in Solid Tumors (mRECIST), PET Response Criteria in Solid Tumors (PERCIST), Choi Criteria, Lugano Response Criteria, European Association for the Study of the Liver (EASL) Criteria, Response Evaluation Criteria in Liver Cancer (RECICL), and WHO Tumor Response Criteria.

[0560] In various embodiments, the present disclosure provides a method for treating cancer in a subject, the method comprising administering a RAS inhibitor or a combination of compounds described herein to a subject, wherein the subject cannot tolerate standard care therapy, first-line therapy, second-line therapy, or third-line therapy. In various embodiments, the present disclosure provides a method for treating cancer in a subject, the method comprising administering a RAS inhibitor or a combination of compounds described herein to a subject, wherein the subject has experienced tumor recurrence after surgical resection of a primary tumor. In various embodiments, the present disclosure provides a method for treating cancer in a subject, the method comprising administering a RAS inhibitor or a combination of compounds described herein to a subject, wherein the subject has a tumor that cannot be surgically removed. In various embodiments, the present disclosure provides a method for treating cancer in a subject, the method comprising administering a RAS inhibitor or a combination of compounds described herein to a subject, wherein the subject has no available treatment options.

[0561] Several therapies (such as chemotherapy) for the treatment of cancer are cytotoxic and are related to the significant side effects and toxicity related to adverse reactions to treatment with adverse results.Before applying such treatments, clinicians rely on several assessment tools to help determine that the subject suffering from cancer experiences the risk of treatment-related toxicity and adverse events.According to the results of these assessments, if the subject suffering from cancer is determined to experience the risk of treatment-related toxicity and adverse events that cause adverse results, then it is considered that they are intolerant to therapy.The example of the commonly used assessment tool for determining treatment intolerance includes Karnofsky Performance Status (KPS), Eastern Cooperative Oncology Group Functional Status (ECOGPS), Timed Stand and Walk (TUG), Simple Physical Energy Scale (SPPB), Comprehensive Geriatric Assessment (CGA), Cancer Aging Research Group (CARG) scoring and Chemotherapy Risk Assessment Scale for Elderly Patients (CRASH).

[0562] Treatment Outcomes and Clinical End Points

[0563] In various embodiments, the present disclosure provides a method for treating lung cancer in a subject, the method comprising administering a RAS inhibitor or a combination of compounds described herein to a subject, wherein the subject has one or more immune-refractory tumors. In various embodiments, the administration changes tumor immune infiltration. In various embodiments, the administration changes anti-tumor immune responses. In various embodiments, the administration changes the tumor microenvironment comprising tumor cells, immune cells, cancer stem cells, and matrix. In various embodiments, the administration converts immune-cold tumors into immune-hot tumors. In various embodiments, the administration reduces tumor size or inhibits tumor growth. In various embodiments, the administration induces tumor cell death, apoptosis, or necrosis by direct particle uptake by tumor cells.

[0564] In various embodiments, the present disclosure provides a method of treating cancer in a subject, the method comprising administering to the subject a RAS inhibitor or a combination of compounds described herein, wherein the subject has one or more tumors characterized as immunoprotective or immunorefractory. In various embodiments, the administration alters a tumor-associated matrix comprising fibroblasts, cancer-associated fibroblasts, adipocytes, pericytes, endothelium, vasculature, lymphatic vessels, tumor-associated vasculature, mesenchymal stromal cells, mesenchymal stem cells, and extracellular matrix.

[0565] It is contemplated that the method herein reduces the tumor size or tumor load of a subject, or reduces the metastasis in a subject. In various embodiments, the method reduces tumor size by 10%, 20%, 30% or more. In various embodiments, the method reduces tumor size by 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%, or includes all values ​​and scopes between these values.

[0566] When a tumor becomes immune refractory, the abundance of certain biomarkers may decrease. It is contemplated herein that after treatment with a RAS inhibitor or compound combination as described herein, the level of one or more of the biomarkers increases in the range of about 1.1 to about 10 times (e.g., about 1.1, about 1.5, about 2, about 2.5, about 3, about 3.5, about 4, about 4.5, about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5 or about 10 times). Similarly, when a tumor becomes immune refractory, the abundance of certain biomarkers increases. After treatment with a RAS inhibitor or compound combination described herein, the level of one or more of such biomarkers is reduced by an amount ranging from about 1.1-fold to about 10-fold (e.g., about 1.1, about 1.5, about 2, about 2.5, about 3, about 3.5, about 4, about 4.5, about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5, or about 10-fold). In various embodiments, administration of a RAS inhibitor or compound combination described herein to a subject having one or more tumors characterized as immune-refractory, immune-protective, or immune-"cold" reduces the level of immunosuppressive cells in the blood. In various embodiments, the suppressor cells are myeloid-derived suppressor cells (MDSCs), tumor-associated macrophages (TAMs), neutrophils, T cells, or T cells. 调控 Cells and B 调控Cells. In various embodiments, MDSCs are monocytic MDSCs (M-MDSCs) and polymorphonuclear MDSCs (PMN-MDSCs). In various embodiments, TAMs are M2 TAMs. In various embodiments, the immunosuppressive cells are CAFs. In various embodiments, the level of immunosuppressive cells is reduced by about 5-100% (e.g., by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%, including all values ​​and ranges between these values), 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, or 50%, as compared to one or more blood samples collected from the subject prior to treatment. In various embodiments, the level of immunosuppressive cells is reduced by about 2-100 times (e.g., about 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 times, including all values ​​and ranges between these values) compared to one or more blood samples collected from the subject before treatment. In various embodiments, immunosuppressive cells are identified by analyzing cell surface protein expression. In various embodiments, administration of a RAS inhibitor or combination of compounds described herein to a subject having one or more tumors characterized as immune-refractory, immune-protective, or immune "cold" increases the level of activated pro-inflammatory immune cells by 5-100% (e.g., by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%, including all values ​​and ranges between these values), 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, or 50%, as compared to one or more blood samples collected from the subject prior to treatment. In various embodiments, administration of a RAS inhibitor or combination of compounds described herein to a subject having one or more tumors characterized as immune-refractory, immune-protective, or immune-"cold" increases the level of activated pro-inflammatory immune cells by 2-100 fold (e.g., about 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 fold, including all values ​​and ranges in between these values), compared to one or more blood samples collected from the subject prior to treatment.In various embodiments, activated proinflammatory cells are dendritic cells (DC), macrophages, M1 macrophages, T cells, B cells, NK cells, NK-T cells and iNK cells. In various embodiments, in all white blood cells analyzed by one or more blood samples collected from a subject, the frequency of proinflammatory immune cells increases to 10-50% (e.g., about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50%, including all values ​​and ranges between these values). In various embodiments, activated proinflammatory immune cells are identified by analyzing cell surface protein expression.

[0567] In various embodiments, the analysis of cells in one or more blood samples of a subject suffering from cancer is performed by analyzing cell surface proteins. In various embodiments, the cell surface proteins are selected from the group consisting of receptor tyrosine kinase (RTK), CD1c, CD2, CD3, CD4, CD5, CD8, CD9, CD10, CD11b, CD11c, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD24, TACI, CD25, CD27, CD28, CD30, CD30L, CD31, CD32, CD32b, CD34, CD33, CD38, CD39, CD40, CD40-L, CD41b, CD42a, CD42b, CD43, CD44, CD48 , CD47, CD45RA, CD45RO, CD48, CD52, CD55, CD56, CD58, CD61, CD66b, CD70, CD72, CD79, CD68, CD84, CD86, CD93, CD94, CD95, CRACC, BLAME, BCMA, CD103 , CD107, CD112, CD120a, CD120b, CD123, CD125, CD134, CD135, CD140a, CD141, CD154, CD155, CD160, CD163, CD172a, XCR1, CD203c, CD204, CD206, CD207CD226、CD244、CD267、CD268、CD269、CD355、CD358、NKG2A、NKG2B、NKG2 C、NKG2D、NKG2E、NKG2F、NKG2H、KIR2DL1、KIR2DL2、KIR2DL3、KIR2DL5A、 KIR2DL5B、KIR3DL1、KIR3DL2、KIR3DL3、KIR3DL4、KIR2DS1、KIR2DS2、KI R2DS3、KIR2DS4、KIR2DS5、DAP12、KIR3DS、NKp44、NKp46、TCR、BCR FcβεRI、MHC-I、MHC-II、IL-1R、IL-2Rα、IL-2Rβ、IL-2Rγ、IL-3Rα、CSF2 RB、IL-4R、IL-5Rα、IL-6Rα、gp130、IL-7Rα、IL-9R、IL-12Rβ1、IL-12Rβ2 、IL-13Rα1、IL-13Rα2、IL-15Rα、IL-21R、IL-23R、IL-27Rα、IL-31Rα、OS MR、CSF-1R、Fluorescence IL-15、IL-10Rα、IL-10Rβ、IL-20Rα、IL-20Rβ、IL-22Rα 1、IL-22Rα2、IL-22Rβ、IL-28RA、PD-1、PD-1H、BTLA、CTLA-4、PD-L1、PD -L2、2B4、B7-1、B7-2、B7-H1、B7-H4、B7-DC、DR3、LIGHT、LAIR、LTα1β2、L TβR、TIM-1、TIM-3、TIM-4、TIGIT、LAG-3、ICOS、ICOS-L、SLAM、SLAMF2、O X-40、OX-40L、GITR、GITRL、TL1A、HVEM、41-BB、41BB-L、TL-1A、TRAF1、T RAF2、TRAF3、TRAF5、BAFF、BAFF-R、APRIL、TRAIL、RANK、AITR、TRAMP、C CR1、CCR2、CCR3、CCR4、CCR5、CCR6、CCR7、CCR8、CCR9、CCR10、CCR11、CXC R1、CXCR2、CXCR3、CXCR4、CXCR5、CXCR6、CXCR7、CLECL9a、DC-SIGN、IGSF 4A、SIGLEC、EGFR、PDGFR、VEGFR、FAPα-SMA is also associated with FAS-L-F c,ICAM-1,ICAM-2,ICAM-3,ICAM-4,ICAM-5,PECAM-1,MICA,MICB,UL16,ULBP1,ULBP2,ILBP3,ULBP4,ULBP5,ULBP6,MULT1,RAE1α,β,γ,δ and ε,A 1 R.A. 2A R.A. 2B R and A 3 In various embodiments, the integrin is selected from the group consisting of α1, α2, αIIb, α3, α4, α5, α6, α7, α8, α9, α10, α11, αD, αE, αL, αM, αV, αX, β1, β2, β3, β4, β5, β6, β7, β8 or a combination thereof. In various embodiments, the TCR is selected from the group consisting of α, β, γ, δ, ε and ζTCR. Several methods for analyzing cell surface protein expression have been described in the literature, including flow cytometry and massive blood cell counting (CyTOF). The presence or abundance of one or more of these cell surface proteins indicates that the patient is responsive to treatment with the methods disclosed herein.

[0568] In various embodiments, the neutrophil to lymphocyte ratio (NLR) in one or more blood samples is reduced from high to medium, or from high to low, to a subject with one or more tumors characterized as immune refractory, immune protective or immune "cold" by applying RAS inhibitors or compound combinations described herein. In various embodiments, NLR is reduced to between 1-2 (e.g., 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 and 2, including all values ​​and ranges between these values) from the analysis of cells of one or more blood samples collected from subjects with one or more tumors characterized as immune refractory, immune protective or immune "cold". In various embodiments, after applying RAS inhibitors or compound combinations described herein, NLR is reduced. In various embodiments, after applying RAS inhibitors or compound combinations described herein, NLR<2.

[0569] In various embodiments, administration of a RAS inhibitor or combination of compounds described herein to a subject having one or more tumors characterized as immune refractory, immune protective, or immune "cold" reduces the level of CTCs in one or more blood samples. In various embodiments, the level of CTCs in the blood is reduced to 5, 4, 3, 2, 1, or 0 per 7.5 ml of blood, including all values ​​and ranges between these values.

[0570] In various embodiments, administration of a RAS inhibitor or combination of compounds described herein to a subject having one or more tumors characterized as immunorefractory, immunoprotective, or immunologically "cold" reduces the level of a tumor-promoting, anti-inflammatory, or immunosuppressive protein in one or more blood samples of the subject. In various embodiments, the tumor-promoting, anti-inflammatory, or immunosuppressive protein is selected from the group consisting of: CD39, CD79, MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP18, MMP19, MMP20, MMP21, MMP23A, MMP23B , MMP24, MMP25, MMP26, MMP27 and MMP28, CXCL12, GM-CSF, G-CSF, TGF-β1, TGF-β2 and TGF-β3, arginase, asparaginase, kynurenase, indoleamine 2,3 dioxygenase (IDO1 and IDO2), tryptophan 2,3 dioxygenase (TDO), myeloperoxidase (MPO), neutrophil elastase (NE) and IL4I1. In various embodiments, the level of a tumor promoting, anti-inflammatory or immunosuppressive protein in one or more blood samples from a subject is reduced by 5-100% (e.g., by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%, including all values ​​and ranges between these values), 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, 50%, or 100%, as compared to one or more blood samples collected prior to treatment. In various embodiments, the level of a tumor promoting, anti-inflammatory, or immunosuppressive protein in one or more blood samples from the subject is reduced by 2-100 fold compared to one or more blood samples collected from the subject prior to treatment (e.g., by about 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 fold relative to one or more samples collected prior to treatment, including all values ​​and ranges in between these values).

[0571] In various embodiments, administration of a RAS inhibitor or combination of compounds described herein to a subject having one or more tumors characterized as immune-refractory, immune-protective, or immune-"cold" increases the level of a tumor suppressor, anti-tumor, or pro-inflammatory protein in one or more blood samples collected from the subject. In various embodiments, the tumor suppressor, anti-tumor, or pro-inflammatory protein is selected from the group consisting of IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-34, IL-35, IL-36, IL-37, IL-38, IL-39, IL-40, IL-41, IL-42, IL-43, IL-44, IL-45, IL-46, IL-47, IL-48, IL-49, IL-50 4. IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-35, IL-36, cell surface IL-15, CXCL2 (MCP-1), CXCL3 (MIP-1α), CXCL4 (MIP-1β), CXCL5 (RANTES), IFN-α, IFN-β, IFN-γ, granzyme-B, perforin and TNF-α. In various embodiments, the level of an anti-tumor or pro-inflammatory protein is increased by 5-100% (e.g., by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%, including all values ​​and ranges between these values), 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, 50%, or 100%, as compared to one or more blood samples collected prior to treatment. In various embodiments, the level of the anti-tumor or pro-inflammatory protein is increased by 2-100 fold (e.g., about 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 fold relative to one or more samples collected prior to treatment, including all values ​​and ranges between these values) compared to one or more blood samples collected from the subject prior to treatment. Several methods for analyzing blood samples for proteins have been described in the literature, including Western blotting and ELISA.

[0572] In various embodiments, administration of a RAS inhibitor or combination of compounds described herein to a subject having one or more tumors characterized as immune-refractory, immune-protective, or immune-"cold" reduces the levels of neutrophil extracellular traps (NETs) in one or more blood samples collected from the subject. In various embodiments, the level of NET in one or more blood samples is reduced by 5-100% (e.g., by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%, including all values ​​and ranges between these values), 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, 50%, or 100%, compared to one or more blood samples collected prior to treatment. In various embodiments, the level of NET in one or more blood samples is reduced by 2-100 times (e.g., about 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 times, including all values ​​and ranges between these values) compared to one or more blood samples collected from the subject before treatment. Several methods for analyzing NET in blood samples have been described in the literature, including Western blotting, ELISA, and flow cytometry.

[0573] In various embodiments, administration of a RAS inhibitor or combination of compounds described herein to a subject having one or more tumors characterized as immune-refractory, immune-protective, or immune-"cold" reduces the expression of tumor-promoting, tumor-permissive, or immune-suppressive genes in one or more blood samples of the subject. In one or more embodiments, the expression of a tumor promoting, tumor permissive or immunosuppressive gene is reduced by 5-100% (e.g., by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%, including all values ​​and ranges between these values), 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, 50% or 100% relative to the levels in one or more blood samples collected prior to treatment). In one or more embodiments, expression of tumor promoting, tumor permissive or immunosuppressive genes is reduced by 2-100 fold (e.g., by about 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 fold, including all values ​​and ranges between these values), compared to one or more blood samples collected from the subject prior to treatment.

[0574] In various embodiments, administration of a RAS inhibitor or combination of compounds described herein to a subject having one or more tumors characterized as immune-refractory, immune-protective, or immune-"cold" increases the expression of tumor suppressor, anti-tumor, or pro-inflammatory genes in one or more samples collected from the subject. In one or more embodiments, the expression of tumor suppressor, anti-tumor or pro-inflammatory genes is increased by 5-100% (e.g., by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95% or about 100%, including all values ​​and ranges between these values), 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, 50% or 100% as compared to one or more blood samples collected prior to treatment. In various embodiments, the expression of tumor suppressor, anti-tumor or pro-inflammatory genes is increased by 2-100 fold (e.g., about 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 fold relative to one or more samples collected prior to treatment, including all values ​​and ranges between these values) compared to one or more blood samples collected from the subject prior to treatment. In various embodiments, gene expression analysis is performed by PCR, RT-PCR, qRT-PCR, next generation sequencing (NGS), RNA-seq, ATAC-seq, exome sequencing, Southern blotting, microarray analysis or single cell sequencing.

[0575] In various embodiments, administration of a RAS inhibitor or compound combination described herein to a subject having one or more tumors characterized as immune refractory, immune protective, or immune "cold" increases the level of leukocytes in the tumor. In various embodiments, the level of leukocytes in the tumor core or tumor periphery increases. In various embodiments, the leukocytes are increased by 5-100% (e.g., an increase of about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%, including all values ​​and ranges between these values), 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, or 50%, as compared to one or more tumor samples collected from the subject prior to treatment. In various embodiments, the level of leukocytes is increased by 2-100 times (e.g., about 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 times, including all values ​​and ranges between these values) compared to one or more tumor samples collected from the subject before treatment. In various embodiments, the frequency of leukocytes in the tumor core or tumor periphery is ≥5%, ≥10%, ≥15%, ≥20%, ≥25%, ≥30%, ≥35%, ≥40%, ≥45%, or ≥50, including all values ​​and ranges between these values, among all cells analyzed.

[0576] In various embodiments, administration of a RAS inhibitor or combination of compounds described herein to a subject having one or more tumors characterized as immune-refractory, immune-protective, or immune-"cold" reduces the level of immunosuppressive cells in the tumor. In various embodiments, the level of immunosuppressive cells in the tumor core or tumor periphery is reduced. In various embodiments, the suppressor cells are myeloid-derived suppressor cells (MDSCs), tumor-associated macrophages (TAMs), neutrophils, T cells, or T cells. 调控 Cells and B 调控Cells. In various embodiments, MDSCs are monocytic MDSCs (M-MDSCs) and polymorphonuclear MDSCs (PMN-MDSCs). In various embodiments, TAMs are M2 TAMs. In various embodiments, the immunosuppressive cells are CAFs. In various embodiments, the level of immunosuppressive cells is reduced by about 5-100% (e.g., by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%, including all values ​​and ranges between these values), 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, or 50%, compared to one or more tumor samples collected from the subject prior to treatment. In various embodiments, the level of immunosuppressive cells is reduced by about 2-100 times (e.g., about 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 times, including all values ​​and ranges between these values) compared to one or more tumor samples collected from the subject before treatment. In various embodiments, immunosuppressive cells are identified by analyzing cell surface protein expression.

[0577] The level of leukocytes in tumor samples can be assessed by several methods, including flow cytometry and immunohistochemistry. In various embodiments, administration of a RAS inhibitor or compound combination described herein to a subject having one or more tumors characterized as immune refractory, immune protective, or immune "cold" increases the level of activated proinflammatory immune cells in the tumor. In various embodiments, the level of activated proinflammatory cells in the tumor core or tumor periphery is increased.

[0578] In various embodiments, administration of a RAS inhibitor or combination of compounds described herein to a subject having one or more tumors characterized as immune-refractory, immune-protective, or immune "cold" increases the level of activated pro-inflammatory immune cells in the tumor by 5-100% (e.g., by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%, including all values ​​and ranges between these values), 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, or 50%, as compared to one or more tumor samples collected from the subject prior to treatment. In various embodiments, administration of a RAS inhibitor or combination of compounds described herein to a subject having one or more tumors characterized as immune-refractory, immune-protective, or immune-"cold" increases the level of activated pro-inflammatory immune cells by 2-100 fold (e.g., about 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 fold, including all values ​​and ranges between these values), compared to one or more tumor samples collected from the subject prior to treatment. In various embodiments, the activated pro-inflammatory cells are dendritic cells (DCs), macrophages, M1 macrophages, T cells, B cells, NK cells, NK-T cells, and NK cells. In various embodiments, the frequency of pro-inflammatory immune cells is between about 10-50% (e.g., about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%, including all values ​​and ranges between these values) of all leukocytes analyzed from one or more tumor samples collected from a subject. In various embodiments, activated pro-inflammatory immune cells are identified by analyzing cell surface protein expression.

[0579] In various embodiments, the analysis of cells in one or more tumor samples of a subject suffering from cancer is performed by analyzing cell surface proteins. In various embodiments, the cell surface proteins are selected from the group consisting of receptor tyrosine kinase (RTK), CD1c, CD2, CD3, CD4, CD5, CD8, CD9, CD10, CD11b, CD11c, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD24, TACI, CD25, CD27, CD28, CD30, CD30L, CD31, CD32, CD32b, CD34, CD33, CD38, CD39, CD40, CD40-L, CD41b, CD42a, CD42b, CD43, CD44, CD48 , CD47, CD45RA, CD45RO, CD48, CD52, CD55, CD56, CD58, CD61, CD66b, CD70, CD72, CD79, CD68, CD84, CD86, CD93, CD94, CD95, CRACC, BLAME, BCMA, CD103 , CD107, CD112, CD120a, CD120b, CD123, CD125, CD134, CD135, CD140a, CD141, CD154, CD155, CD160, CD163, CD172a, XCR1, CD203c, CD204, CD206, CD207CD226、CD244、CD267、CD268、CD269、CD355、CD358、NKG2A、NKG2B、NKG2 C、NKG2D、NKG2E、NKG2F、NKG2H、KIR2DL1、KIR2DL2、KIR2DL3、KIR2DL5A、 KIR2DL5B、KIR3DL1、KIR3DL2、KIR3DL3、KIR3DL4、KIR2DS1、KIR2DS2、KI R2DS3、KIR2DS4、KIR2DS5、DAP12、KIR3DS、NKp44、NKp46、TCR、BCR FcβεRI、MHC-I、MHC-II、IL-1R、IL-2Rα、IL-2Rβ、IL-2Rγ、IL-3Rα、CSF2 RB、IL-4R、IL-5Rα、IL-6Rα、gp130、IL-7Rα、IL-9R、IL-12Rβ1、IL-12Rβ2 、IL-13Rα1、IL-13Rα2、IL-15Rα、IL-21R、IL-23R、IL-27Rα、IL-31Rα、OS MR、CSF-1R、Fluorescence IL-15、IL-10Rα、IL-10Rβ、IL-20Rα、IL-20Rβ、IL-22Rα 1、IL-22Rα2、IL-22Rβ、IL-28RA、PD-1、PD-1H、BTLA、CTLA-4、PD-L1、PD -L2、2B4、B7-1、B7-2、B7-H1、B7-H4、B7-DC、DR3、LIGHT、LAIR、LTα1β2、L TβR、TIM-1、TIM-3、TIM-4、TIGIT、LAG-3、ICOS、ICOS-L、SLAM、SLAMF2、O X-40、OX-40L、GITR、GITRL、TL1A、HVEM、41-BB、41BB-L、TL-1A、TRAF1、T RAF2、TRAF3、TRAF5、BAFF、BAFF-R、APRIL、TRAIL、RANK、AITR、TRAMP、C CR1、CCR2、CCR3、CCR4、CCR5、CCR6、CCR7、CCR8、CCR9、CCR10、CCR11、CXC R1、CXCR2、CXCR3、CXCR4、CXCR5、CXCR6、CXCR7、CLECL9a、DC-SIGN、IGSF 4A、SIGLEC、EGFR、PDGFR、VEGFR、FAPα-SMA is also associated with FAS-L-F c,ICAM-1,ICAM-2,ICAM-3,ICAM-4,ICAM-5,PECAM-1,MICA,MICB,UL16,ULBP1,ULBP2,ILBP3,ULBP4,ULBP5,ULBP6,MULT1,RAE1α,β,γ,δ and ε,A 1 R.A. 2A R.A. 2B R and A 3 In various embodiments, the integrin is selected from the group consisting of α1, α2, αIIb, α3, α4, α5, α6, α7, α8, α9, α10, α11, αD, αE, αL, αM, αV, αX, β1, β2, β3, β4, β5, β6, β7, β8 or a combination thereof. In various embodiments, the TCR is selected from the group consisting of α, β, γ, δ, ε and ζTCR. Several methods for analyzing cell surface protein expression have been described in the literature, including flow cytometry and massive blood cell counting (CyTOF). The presence or abundance of one or more of these cell surface proteins indicates that the patient is responsive to treatment with the methods disclosed herein.

[0580] In various embodiments, administration of a RAS inhibitor or combination of compounds described herein to a subject having one or more tumors characterized as immunorefractory, immunoprotective, or immunologically "cold" results in a decrease in the level of a tumor promoting, anti-inflammatory, or immunosuppressive protein in one or more tumor samples from the subject. In various embodiments, the tumor promoting, anti-inflammatory, or immunosuppressive protein is selected from the group consisting of: CD39, CD79, MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP18, MMP19, MMP20, MMP21, MMP23A, MMP23B , MMP24, MMP25, MMP26, MMP27, MMP28, CXCL12, GM-CSF, G-CSF, TGF-β1, TGF-β2, TGF-β3, arginase, asparaginase, kynurenase, indoleamine 2,3 dioxygenase (IDO1 and IDO2), tryptophan 2,3 dioxygenase (TDO), myeloperoxidase (MPO), neutrophil elastase (NE), and IL4I1. In various embodiments, the level of a tumor-promoting, anti-inflammatory, or immunosuppressive protein in one or more tumor samples from a subject is reduced by 5-100% (e.g., by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%, including all values ​​and ranges between these values), 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, 50%, or 100%, as compared to one or more tumor samples collected prior to treatment. In various embodiments, the level of a tumor promoting, anti-inflammatory, or immunosuppressive protein in one or more tumor samples from the subject is reduced by 2-100 fold compared to one or more tumor samples collected from the subject prior to treatment (e.g., reduced by about 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 fold relative to one or more samples collected prior to treatment, including all values ​​and ranges in between these values).

[0581] In various embodiments, administration of a RAS inhibitor or combination of compounds described herein to a subject having one or more tumors characterized as immune-refractory, immune-protective, or immune-"cold" increases the level of a protein associated with tumor growth inhibition, anti-tumor activity, or pro-inflammatory activity. In various embodiments, the protein associated with tumor growth inhibition, anti-tumor activity, or pro-inflammatory activity is selected from the group consisting of CD44, CD56, CD103c, CD69, KG2A, NKG2B, NKG2C, NKG2D, NKG2E, NKG2F, NKG2H, ICOS, ICOS-L, SLAM, SLAMF2, OX-40, OX-40L, GITR, GITRL, TL-1A, HVEM, 41-BB, 41BB-L, TRAF1, TRAF2, TRAF3, TRAF5, BAFF, BAFF-R, APRIL, TRAIL, RANK, AITR, TRAMP, IL-1α, IL-1β, IL-2, IL-3, IL- 4. IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-2 8. IL-29, IL-30, IL-31, IL-32, IL-33, IL-35, IL-36, CXCL2(MCP-1), CXCL3(MIP-1α), CXCL4(MIP-1β), CXCL5(RANTES), IFN-α, IFN-β, IFN-γ, granzyme-B, perforin and TNF-α. In various embodiments, the level of a protein associated with tumor growth inhibition, anti-tumor activity, or pro-inflammatory activity is increased by 5-100% (e.g., by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%, including all values ​​and ranges between these values), 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, 50%, or 100%, compared to one or more tumor samples collected prior to treatment.In various embodiments, the level of a protein associated with tumor growth inhibition, anti-tumor activity, or pro-inflammatory activity is increased 2-100 fold compared to one or more tumor samples collected from the subject prior to treatment (e.g., decreased by about 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 fold relative to one or more samples collected prior to treatment, including all values ​​and ranges between these values). Several methods for analyzing proteins in tumor samples have been described in the literature, including Western blotting and ELISA.

[0582] In various embodiments, administration of a RAS inhibitor or combination of compounds described herein to a subject having one or more tumors characterized as immune-refractory, immune-protective, or immune-"cold" reduces the levels of neutrophil extracellular traps (NETs) in one or more tumor samples collected from the subject. In various embodiments, the level of NET in one or more tumor samples is reduced by 5-100% (e.g., by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%, including all values ​​and ranges between these values), 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, 50%, or 100%, compared to one or more tumor samples collected prior to treatment. In various embodiments, the level of NET in one or more tumor samples is reduced by 2-100 times (e.g., about 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 times, including all values ​​and ranges between these values, relative to one or more samples collected from the subject prior to treatment). Several methods for analyzing NET in tumor samples have been described in the literature, including Western blotting, ELISA, and flow cytometry.

[0583] In various embodiments, administration of a RAS inhibitor or combination of compounds described herein to a subject having one or more tumors characterized as immune-refractory, immune-protective, or immune-"cold" results in reduced expression of tumor-promoting, tumor-permissive, or immune-suppressive genes in one or more tumor samples from the subject. In one or more embodiments, the expression of tumor promoting, tumor permissive or immunosuppressive genes is reduced by 5-100% (e.g., by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%, including all values ​​and ranges between these values), 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, 50% or 100% as compared to one or more tumor samples collected prior to treatment. In one or more embodiments, expression of a tumor promoting, tumor permissive, or immunosuppressive gene is reduced by 2-100 fold compared to one or more tumor samples collected from the subject prior to treatment (e.g., by about 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 fold relative to one or more samples collected prior to treatment, including all values ​​and ranges therebetween).

[0584] In various embodiments, administration of a RAS inhibitor or combination of compounds described herein to a subject having one or more tumors characterized as immune-refractory, immune-protective, or immune-"cold" increases the expression of tumor suppressor, anti-tumor, or pro-inflammatory genes in one or more samples collected from the subject. In one or more embodiments, the expression of a tumor suppressor, anti-tumor or pro-inflammatory gene is increased by 5-100% (e.g., by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95% or about 100%, including all values ​​and ranges between these values), 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, 50% or 100% as compared to one or more tumor samples collected prior to treatment. In various embodiments, the expression of tumor suppressor, anti-tumor or pro-inflammatory genes is increased by 2-100 times (e.g., by about 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 times, including all values ​​and ranges between these values) compared to one or more tumor samples collected from the subject before treatment. In various embodiments, gene expression analysis is performed by PCR, RT-PCR, qRT-PCR, next generation sequencing (NGS), RNA-seq, ATAC-seq, exome sequencing, Southern blot, microarray analysis or single cell sequencing.

[0585] In various embodiments, a subject with lung cancer is treated with a RAS inhibitor or a combination of compounds described herein to convert a cold tumor to a hot tumor. Such conversions can be detected using methods described herein and known in the art. If a subject has been diagnosed with a tumor, and the tumor has been converted from a cold tumor to a hot tumor, then treatment can be continued by administering a RAS inhibitor or a combination of compounds described herein, wherein the RAS inhibitor or a combination of compounds described herein can be used to treat a hot tumor or an immune cell-enriched or immunogenic tumor. In other embodiments, when a tumor has been converted from a cold tumor to a hot tumor, the patient stops treating with a RAS inhibitor or a combination of compounds described herein, and the patient begins treatment with a cancer therapeutic agent, which can be used to treat a hot tumor or an immune cell-enriched or immunogenic tumor. Such cancer therapeutic agents include chemotherapeutics, cytokines, angiogenesis inhibitors, enzymes, immune checkpoint regulators, and monoclonal antibodies, hormone therapy, including one or more cell-based therapies, such as adoptive cell transfer, tumor-infiltrating leukocyte therapy, chimeric antigen receptor T cell therapy (CAR-T), NK cell therapy, and stem cell therapy, or oncolytic viruses or oncolytic bacteria.

[0586] In various embodiments, immune checkpoint regulators target programmed cell death protein 1 (PD-1), programmed cell death protein ligand-1 (PD-L1), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), T cell immunoglobulin and mucin domain-3 (TIM-3), lymphocyte activation gene 3 (LAG-3) or TIGIT (T cell immune receptor with lg and ITIM domains). In various embodiments, immune checkpoint regulators are antibodies selected from the group consisting of: ipilimumab, tremelimumab, pembrolizumab, nivolumab, atezolizumab, avelumab, cemilimab and durvalumab.

[0587] In various embodiments, subjects diagnosed with cold tumors and receiving therapy using a RAS inhibitor or combination of compounds described herein are monitored regularly to determine whether the tumor has converted to a hot tumor. Monitoring can be performed as necessary as determined by the physician, for example, monthly, every two months, every three months, every six months, or annually.

[0588] In various embodiments, the subject has been previously treated with immunotherapy but has developed resistance to the immunotherapy or has converted from a hot tumor to a cold tumor. Also provided is a method of treating a subject with cancer that has developed resistance to immunotherapy or has developed a cold tumor, the method comprising administering to the subject a RAS inhibitor or compound combination described herein.

[0589] Cancers with oncogenic RAS mutations

[0590] In some embodiments, the present invention discloses a method of treating lung cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a compound of the present invention or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising such a compound or salt, wherein the cancer is immune-refractory lung cancer.

[0591] Methods for detecting Ras mutations are known in the art. Such means include, but are not limited to, direct sequencing and the use of highly sensitive diagnostic analysis (using CE-IVD marking), such as described in Domagala et al., Pol J Pathol 3: 145-164 (2012), which is incorporated herein by reference in its entirety, including TheraScreen PCR; AmoyDx; PNAClamp; RealQuality; EntroGen; LightMix; StripAssay; HybcellplexA; Devyser; Surveyor; Cobas; and TheraScreen Pyro. See also, for example, WO 2020 / 106640.

[0592] In some embodiments, the cancer is non-small cell lung cancer or any of the lung cancers described herein, and the Ras mutation comprises a K-Ras G12C mutation, an H-Ras G12C mutation, or an N-Ras G12C mutation. In some embodiments, the cancer is non-small cell lung cancer or any of the lung cancers described herein, and the Ras mutation comprises a K-Ras G12C mutation.

[0593] Also provided is a method of inhibiting Ras protein in a cell, the method comprising contacting the cell with an effective amount of a compound of the present invention or a pharmaceutically acceptable salt thereof. Also provided is a method of inhibiting RAF-Ras binding, the method comprising contacting the cell with an effective amount of a compound of the present invention or a pharmaceutically acceptable salt thereof. The cell may be a cancer cell. The cancer cell may belong to any cancer type described herein. The cell may be in vivo or in vitro.

[0594] Lung cancer

[0595] In some embodiments, the present invention discloses a method for treating lung cancer. In some embodiments, the lung cancer is immune-refractory lung cancer.

[0596] Lung cancer can be classified using different systems. In one system, lung cancer includes adenocarcinoma (mixed, acinar, papillary, solid, micropapillary, lepidic nonmucinous, and lepidic mucinous), squamous cell carcinoma, large cell carcinoma (e.g., non-small cell lung cancer (NSCLC) (e.g., advanced or non-advanced large cell carcinoma with neuroendocrine morphology (LCNEM), NSCLC-not otherwise specified (NOS) / adenosquamous carcinoma, sarcomatoid carcinoma, adenosquamous carcinoma, and large cell neuroendocrine carcinoma (LCNEC)); and small cell lung cancer / carcinoma (SCLC)).

[0597] Alternatively, in different systems, lung cancer can be classified as preinvasive lesions, minimally invasive adenocarcinomas, and invasive adenocarcinomas (invasive mucinous adenocarcinoma, mucinous bronchioloalveolar carcinoma (BAC), colloid adenocarcinoma, fetal adenocarcinoma (low-grade and high-grade), and intestinal-type adenocarcinoma). Non-small cell lung cancer includes adenocarcinoma, squamous cell carcinoma, large cell carcinoma, or large cell neuroendocrine tumors.

[0598] More often, lung cancer can be classified as small cell lung cancer ("SCLC") or non-small cell lung cancer ("NSCLC"). NSCLC can be further classified as squamous or non-squamous. An example of non-squamous NSCLC is adenocarcinoma.

[0599] In some embodiments, the lung cancer is bronchogenic carcinoma (squamous cell, undifferentiated small cell, undifferentiated large cell adenocarcinoma), alveolar (bronchiolar) carcinoma, bronchial adenoma, sarcoma, lymphoma, enchondromatous hamartoma, or mesothelioma.

[0600] Lung cancer can be newly diagnosed and untreated, or can be relapsed, refractory, relapsed and refractory, locally advanced, or metastatic. In some cases, lung cancer comprises relapsed or refractory lung cancer. In some cases, lung cancer comprises metastatic lung cancer. In some cases, the subject is diagnosed as having relapsed or refractory lung cancer. In other cases, the subject is diagnosed as having metastatic lung cancer.

[0601] Combination therapy

[0602] The methods of the present invention may include the compounds of the present invention used alone or in combination with one or more other therapies (e.g., non-drug treatments or therapeutic agents). The dosage of one or more other therapies (e.g., non-drug treatments or therapeutic agents) may be reduced relative to the standard dosage when administered alone. For example, the dosage may be determined empirically based on drug combinations and permutations, or may be inferred by isoradiometric analysis (e.g., Black et al., Neurology 65: S3-S6 (2005)).

[0603] The compounds of the present invention may be administered before, after or in parallel with one or more of such other therapies. When combined, the dose of the compound of the present invention and the dose of one or more other therapies (e.g., non-drug therapy or therapeutic agent) provide a therapeutic effect (e.g., a synergistic or additive therapeutic effect). The compounds of the present invention and another therapy (such as an anticancer agent) may be administered together, such as in the form of a single pharmaceutical composition, or administered separately, and when administered separately, this may be performed simultaneously or sequentially. Such sequential administration times may be close or distant.

[0604] In some embodiments, the additional therapy is the administration of a side effect limiting agent (e.g., an agent intended to reduce the occurrence or lessen the severity of a side effect of the treatment. For example, in some embodiments, the compounds of the invention may also be used in combination with a therapeutic agent for treating nausea. Examples of agents useful for treating nausea include: dronabinol, granisetron, metoclopramide, ondansetron, and prochlorperazine, or a pharmaceutically acceptable salt thereof.

[0605] In some embodiments, one or more other therapies include non-drug treatments (e.g., surgery or radiotherapy). In some embodiments, one or more other therapies include therapeutic agents (e.g., compounds or biological agents as anti-angiogenic agents, signal transduction inhibitors, antiproliferative agents, glycolysis inhibitors, or autophagy inhibitors). In some embodiments, one or more other therapies include non-drug treatments (e.g., surgery or radiotherapy and therapeutic agents (e.g., compounds or biological agents as anti-angiogenic agents, signal transduction inhibitors, antiproliferative agents, glycolysis inhibitors, or autophagy inhibitors). In other embodiments, one or more other therapies include two therapeutic agents. In other embodiments, one or more other therapies include three therapeutic agents. In some embodiments, one or more other therapies include four or more therapeutic agents.

[0606] In this combination therapy section, all references to the agents described are incorporated herein by reference, whether or not expressly stated as such.

[0607] Non-drug treatments

[0608] Examples of non-drug treatments include, but are not limited to, radiation therapy, cryotherapy, hyperthermia, surgery (eg, surgical removal of tumor tissue), and T-cell adoptive transfer (ACT) therapy.

[0609] In some embodiments, the compounds of the invention may be used as adjuvant therapy after surgery. In some embodiments, the compounds of the invention may be used as neoadjuvant therapy before surgery.

[0610] In a subject (e.g., a mammal (e.g., a human)), radiotherapy can be used to inhibit abnormal cell growth or treat hyperproliferative disorders, such as cancer. The techniques for administering radiotherapy are known in the art. Radiotherapy can be administered by one of several methods or a combination of methods, including but not limited to external beam therapy, internal radiotherapy, implant radiation, stereotactic radiosurgery, systemic radiotherapy, radiotherapy, and permanent or temporary interstitial brachytherapy. As used herein, the term "brachytherapy" refers to radiotherapy delivered by inserting a spatially confined radioactive material into a tumor or other proliferative tissue disease site in the body or near it. The term is intended to include, but is not limited to, exposure to radioisotopes (e.g., At-211, I-131, I-125, Y-90, Re-186, Re-188, Sm-153, Bi-212, P-32, and Lu radioisotopes). Radioactive sources suitable for use as cell regulators of the present invention include both solids and fluids. By way of non-limiting example, the radiation source may be a radionuclide such as I-125, I-131, Yb-169, Ir-192 as a solid source, I-125 as a solid source, or other radionuclides that emit photons, beta particles, gamma radiation, or other therapeutic rays. The radioactive material may also be a fluid made of any solution of one or more radionuclides (e.g., a solution of I-125 or I-131), or a slurry of a suitable fluid containing small particles of solid radionuclides (such as Au-198 or Y-90) may be used to produce a radioactive fluid. In addition, one or more radionuclides may be contained in a gel or radioactive microspheres.

[0611] In some embodiments, the compounds of the present invention can make abnormal cells more sensitive to radiation therapy to kill such cells or inhibit their growth. Therefore, the present invention also relates to a method for sensitizing abnormal cells in mammals to radiation therapy, the method comprising administering to the mammal an amount of the compound of the present invention, the amount being effective in sensitizing abnormal cells to radiation therapy. The amount of the compound in this method can be determined according to the means for determining the effective amount of such compounds described herein. In some embodiments, the compounds of the present invention can be used as an adjuvant therapy after radiation therapy or as a neoadjuvant therapy before radiation therapy.

[0612] In some embodiments, non-drug therapy is T cell adoptive transfer (ACT) therapy. In some embodiments, T cells are activated T cells. T cells may be modified to express chimeric antigen receptors (CAR). CAR-modified T (CAR-T) cells may be produced by any method known in the art. For example, CAR-T cells may be produced by introducing a suitable expression vector encoding CAR into T cells. Before the amplification and gene modification of T cells, T cell sources are obtained from subjects. T cells can be obtained from many sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue at the site of infection, ascites, pleural effusion, spleen tissue, and tumors. In certain embodiments of the present invention, any number of T cell lines available in the art may be used. In some embodiments, T cells are autologous T cells. Before or after genetically modifying T cells to express a desired protein (e.g., CAR), T cells can generally be activated and expanded using methods such as those described in, for example, U.S. Patents 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 7,572,631; 5,883,223; 6,905,874; 6,797,514; and 6,867,041.

[0613] Therapeutic agents

[0614] A therapeutic agent may be a compound used to treat cancer or symptoms associated therewith.

[0615] For example, the therapeutic agent may be a steroid. Thus, in some embodiments, the one or more other therapies include a steroid. Suitable steroids may include, but are not limited to, 21-acetoxypregnenolone, alclometasone, algestone, amcinonide, beclomethasone, betamethasone, budesonide, chloroprednisone, clobetasol, clocortolone, cloprednol, corticosterone, cortisone, cortivazole, cortivazol, deflazacort, desonide, desoximetasone, dexamethasone, diflorasone, diflucortolone, difuprednate, enoxolone, fluazacort, fiucloronide, flumethasone, flunisolide, fluocinolone acetate acetonide, fluocinonide, fluocortin butyl, fluocortolone, fluorometholone, fluperolone acetate, fluprednideneacetate, fluprednisolone, flurandrenolide, fluticasone propionate, formocortal, halcinonide, halobetasol propionate, halometasone, hydrocortisone, loteprednol etabonate, mazipredone, medroxyprogesterone, meprednisone, methylprednisolone, mometasone furoatefuroate, paramethasone, prednicarbate, prednisolone, prednisolone 25-diethylaminoacetate, prednisolone sodium phosphate, prednisone, prednival, prednylidene, rimexolone, tixocortol, triamcinolone, triamcinolone acetonide, triamcinolone benetonide, triamcinolone hexacetonide, and salts or derivatives thereof.

[0616] Other examples of therapeutic agents that can be used in combination therapy with the compounds of the present invention include compounds described in U.S. Patent Nos. 6,258,812, 6,630,500, 6,515,004, 6,713,485, 5,521,184, 5,770,599, 5,747,498, 5,990,141, 6,235,764, and 8,623,885, and International Patent Application WO01 / 37820, WO01 / 32651, WO02 / 68406, WO02 / 66470, WO02 / 55501, WO04 / 05279, WO04 / 07481, WO04 / 07458, WO04 / 09784, WO02 / 59110, WO99 / 45009, WO00 / 59509, WO99 / 61422, WO00 / 12089 and WO00 / 02871.

[0617] The therapeutic agent may be a biological agent (e.g., a cytokine (e.g., an interferon or an interleukin, such as IL-2)) used to treat cancer or symptoms associated therewith. In some embodiments, the biological agent is an immunoglobulin-based biological agent that agonizes a target to stimulate an anti-cancer response or antagonizes an antigen important to cancer, such as a monoclonal antibody (e.g., a humanized antibody, a fully human antibody, an Fc fusion protein, or a functional fragment thereof). Antibody-drug conjugates are also included.

[0618] The therapeutic agent may be a T cell checkpoint inhibitor. In one embodiment, the checkpoint inhibitor is an inhibitory antibody (e.g., a monospecific antibody, such as a monoclonal antibody). The antibody may be, for example, a humanized antibody or a fully human antibody. In some embodiments, the checkpoint inhibitor is a fusion protein, such as an Fc-receptor fusion protein. In some embodiments, the checkpoint inhibitor is an agent that interacts with the checkpoint protein, such as an antibody. In some embodiments, the checkpoint inhibitor is an agent that interacts with the ligand of the checkpoint protein, such as an antibody. In some embodiments, the checkpoint inhibitor is an inhibitor (e.g., an inhibitory antibody or a small molecule inhibitor) of CTLA-4 (e.g., an anti-CTLA-4 antibody or a fusion protein). In some embodiments, the checkpoint inhibitor is an inhibitor or antagonist (e.g., an inhibitory antibody or a small molecule inhibitor) of PD-1. In some embodiments, the checkpoint inhibitor is an inhibitor or antagonist (e.g., an inhibitory antibody or a small molecule inhibitor) of PD-L1. In some embodiments, the checkpoint inhibitor is an inhibitor or antagonist (e.g., an inhibitory antibody or an Fc fusion or a small molecule inhibitor) of PD-L2 (e.g., an inhibitory antibody or an Fc fusion or a small molecule inhibitor) (e.g., a PD-L2 / Ig fusion protein). In some embodiments, the checkpoint inhibitor is an inhibitor or antagonist (e.g., an inhibitory antibody or small molecule inhibitor) of B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK 1, CHK2, A2aR, B-7 family ligand, or a combination thereof. In some embodiments, the checkpoint inhibitor is pembrolizumab, nivolumab, PDR001 (NVS), REGN2810 (Sanofi / Regeneron), a PD-L1 antibody such as avelumab, durvalumab, atezolizumab, pidilizumab, JNJ-63723283 (JNJ), BGB-A317 (BeiGene and Celgene); or Preusser, M. et al. (2015) Checkpoint inhibitors disclosed in Nat. Rev. Neurol., including but not limited to ipilimumab, tremelimumab, nivolumab, pembrolizumab, AMP224, AMP514 / MEDI0680, BMS936559, MED14736, MPDL3280A, MSB0010718C, BMS986016, IMP321, lilizumab, IPH2101, 1-7F9 and KW-6002. Other checkpoint inhibitors are described herein.

[0619] The therapeutic agent may be an anti-TIGIT antibody, such as MBSA43, BMS-986207, MK-7684, COM902, AB154, MTIG7192A, or OMP-313M32 (etigilimab).

[0620] The therapeutic agent may be an agent that treats cancer or symptoms associated therewith (e.g., a cytotoxic agent, a non-peptide small molecule, or other compounds useful for treating cancer or symptoms associated therewith, collectively referred to as "anti-cancer agents"). The anti-cancer agent may be, for example, a chemotherapeutic agent or a targeted therapy agent.

[0621] Anticancer agents include mitotic inhibitors, intercalated antibiotics, growth factor inhibitors, cell cycle inhibitors, enzymes, topoisomerase inhibitors, biological response regulators, alkylating agents, antimetabolites, folic acid analogs, pyrimidine analogs, purine analogs and related inhibitors, vinca alkaloids, epipodophyllotoxins, antibiotics, L-asparaginase, topoisomerase inhibitors, interferons, platinum coordination complexes, anthracenedione substituted ureas, methylhydrazine derivatives, adrenocortical inhibitors, adrenal steroids, progesterone, estrogen, antiestrogens, androgens, antiandrogens and gonadotropin-releasing hormone analogs. Other anticancer agents include folinic acid (LV), irinotecan, oxaliplatin, capecitabine, paclitaxel and docetaxel. In some embodiments, one or more other therapies include two or more anticancer agents. Two or more anticancer agents can be used in the form of a mixture to be administered in combination or administered alone. Suitable dosing regimens for combined anticancer agents are known in the art and described, for example, in Saltz et al., Proc. Am. Soc. Clin. Oncol. 18:233a (1999) and Douillard et al., Lancet 355(9209):1041-1047 (2000).

[0622] Other non-limiting examples of anticancer agents include (Imatinib Mesylate); (carfilzomib); (bortezomib); Casodex (bicalutamide); (gefitinib); alkylating agents such as thiotepa and cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethyleneimines and methylmelamines, including hexamethylmelamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolmelamine; polyacetyl groups (especially bratacin) camptothecins (including the synthetic analogues topotecan); bryostatin; callystatin; CC-1065 (including its synthetic analogues adozelesin, carzelesin and bizelesin); cryptophycin (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including its synthetic analogues KW-2189 and CB-1065); 1-TM1); eleutherobin; cypermethrin; stolon A; spongestatin; nitrogen mustards such as chlorambucil, naphthyl mustard, cholephosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitroureas such as carmustine ustine), chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; antibiotics such as enediyne antibiotics (e.g., calicheamicins such as calicheamicin γll and calicheamicin ωll (see, e.g., Agnew, Chem. Intl. Ed Engl. 33:183-186 (1994)); dynemicins such as dynemicin A; bisphosphonates such as clodronate; esperamicin;New carcinostomycin chromophores and related chromoprotein enediyne antibiotic chromophores, aclacinomycin, actinomycin, authramycin, azaserine, bleomycin, actinomycin C, calicheamicin, carabicin, caminomycin, carminomycin, carmomycin, chromomycin, dactinomycin, daunorubicin, detoxibics detorubicin, 6-diazo-5-oxo-L-norleucine, adriamycin (doxorubicin), morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, deoxydoxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins (such as mitomycin C), mycophenolic acid (such as mycophenolic acid), acid, nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptozocin, tuberculin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as folinic acid, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopropionic acid, pyridoxine, oxadiazine, dapoxet ... Purines, thioimidazole, thioguanine; pyrimidine analogs such as cyclocytidine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, deoxyfluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epithioandrostrol, mepitiostane, testolactone; antiadreners such as aminoglutethimide, mitotane, trilostane; folic acid supplements such as folinic acid; acetylglucuronolactone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfomithine; elliptinium acetate acetate; epothilones such as epothilone B; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansinoids such as maytansine and ansamitocin; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; mustard; pirarubicin; losoxantrone; podophyllic acid; 2-ethylhydrazine; procarbazine; Polysaccharide complex (JHS Natural Products, Eugene, OR); razoxane; rhizoxin; sizofiran; spirogermanamine; tricholomanic acid; triazoquinone; 2,2',2"-trichlorotriethylamine; trichothecenes such as T-2 toxin, verrucosporin A, baculosporin A, and serpentin; carbamates; vindesine; dacarbazine; mannitol mustard; dibromomannitol; dibromodulanol; pipobroman; garcitocin; arabinoside ("cytarabine"); cyclophosphamide; thiotepa; taxanes, such as (Pacitaxel), (Cremophor-free albumin-engineered nanoparticle formulation of paclitaxel) and (docetaxel); chlorambucil; tamoxifen (Nolvadex TM ); raloxifene; aromatase inhibiting 4(5)-imidazoles; 4-hydroxytamoxifen, trioxifene; keoxifene; LY117018; onapristone; toremifene Flutamide, nilutamide, bicamamide, leuprolide, goserelin; mustard phenylbutyric acid; gemcitabine; 6-thioguanine; mercaptopurine; platinum coordination complexes, such as cisplatin, oxaliplatin, and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; (vinorelbine); dihydroxyanthraquinone; teniposide; edatrexate; daunomycin; aminopterin; ibandronate; irinotecan (e.g., CPT-11); topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoids, such as retinoic acid; esperamicin; capecitabine (e.g., ); and pharmaceutically acceptable salts of any one of the above.

[0623] Other non-limiting examples of anticancer agents include trastuzumab Bevacizumab Cetuximab Rituximab ABVD, avicine, abagovomab, acridinium carboxamide, adecatumumab, 17-N-allylamino-17-demethoxygeldanamycin, alpharadin, avocidib, 3-aminopyridine-2-carboxaldehyde thiosemicarbazone, amonafide, anthracenedione, anti-CD22 immunotoxin, antineoplastic agents (e.g., cell cycle non-specific antineoplastic agents and other antineoplastic agents described herein), antineoplastic herbal medicines, apaziquone, atiprimod, azathiopurines, belotecan, bendamustine, BIBW 2992, biricodar, brostallicin, diostostatin, buthionine sulfoximine, CBV (chemotherapy), calyculin, dichloroacetic acid, pelargonide, elsamitrucin, enocitabine, eribulin, exatecan, exisulind, ferrugol, forodesine, fosfestrol, ICE chemotherapy regimen, IT-101, imexon, imiquimod, indolecarbazole, irofulven, laniquidar, lalostat larotaxel, lenalidomide, lucanthone, lurtotecan, mafosfamide, mitozolomide, nafoxidine, nedaplatin, olaparib, ortataxel, PAC-1, papaya, pixantrone, proteasome inhibitors, rebeccamycin, resiquimod, rubitecan, SN-38, salinosporamide A, sapacitabine, StanfordV, swainsonine, talaporfin, tariquidar, tegafur-uracil, temodar, tesetaxel, triplatinium tetranitrate, tris(2-chloroethyl)amine, troxacitabine, uramustine, vadimezan, vinflunine, ZD6126, and zosuquidar.

[0624] Other non-limiting examples of anticancer agents include natural products such as vinca alkaloids (e.g., vinblastine, vincristine, and vinorelbine), epipodophyllotoxins (e.g., etoposide and teniposide), antibiotics (e.g., dactinomycin (actinomycin D), daunorubicin, and idarubicin), anthracyclines, mitoxantrone, bleomycin, plicamycin (mithramycin), mitomycins, enzymes (e.g., L-asparaginase, which metabolizes L-asparagine systemically and entrains cells that do not have the ability to synthesize their own asparagine), antiplatelet agents, antiproliferative / Anti-mitotic alkylating agents such as nitrogen mustards (e.g., mechlorethamine, cyclophosphamide and analogs, melphalan, and mechlorethamine phenylbutyrate), ethyleneimines and methylmelamines (e.g., hexamethylmelamine and thiotepa), CDK inhibitors (e.g., CDK4 / 6 inhibitors such as abemaciclib, ribociclib, palbociclib; seliciclib, UCN-01, P1446A-05, PD-0332991, dinacilib, clib), P27-00, AT-7519, RGB286638, and SCH727965), alkyl sulfonates (e.g., busulfan), nitrosoureas (e.g., carmustine (BCNU) and analogs and streptozocin), trazenes-dacarbazinine (DTIC), antiproliferative / antimitotic antimetabolites such as folic acid analogs, pyrimidine analogs (e.g., fluorouracil, floxuridine, and cytarabine), purine analogs and related inhibitors (e.g., mercaptopurine, thioguanine, pentostatin (pentopurine), and thioguanine). statin and 2-chlorodeoxyadenosine), aromatase inhibitors (e.g., anastrozole, exemestane, and letrozole), and platinum coordination complexes (e.g., cisplatin and carboplatin), procarbazine, hydroxyurea, mitotane, aminoglutethimide, histone deacetylase (HDAC) inhibitors (e.g., trichostatin, sodium butyrate, apicidan, suberoylanilide hydroxamic acid, vorinostat, LBH 589, romidepsin, ACY-1215, and panobinostat), mTOR inhibitors (e.g., vistusertib, temsirolimus, everolimus, ridaforolimus, and sirolimus), KSP (Eg5) inhibitors (e.g., Array 520), DNA binding agents (e.g., ), PI3K inhibitors (such as PI3Kδ inhibitors (e.g., GS-1101 and TGR-1202), PI3Kδ and γ inhibitors (e.g., CAL-130)), copanlisib, alpelisib, and idelalisib; multikinase inhibitors (e.g., TG02 and sorafenib), hormones (e.g., estrogens), and hormone agonists, such as luteinizing hormone-releasing hormone (LHRH) agonists (e.g., goserelin, leuprorelin, and triptorelin), BAFF neutralizing antibodies (e.g., LY2127399), IKK inhibitors agents, p38MAPK inhibitors, anti-IL-6 (e.g. CNT0328), telomerase inhibitors (e.g. GRN1631), Aurora kinase inhibitors (e.g. MLN8237), cell surface monoclonal antibodies (e.g. anti-CD38 (HUMAX-CD38), anti-CS1 (e.g. elotuzumab), HSP90 inhibitors (e.g. 17AAG and KOS953), P13K / Akt inhibitors (e.g. perifosine), AKT inhibitors (e.g. GSK-2141795), PKC inhibitors (e.g. enzastaurin), FTIs (e.g. Zarnestra TM ), anti-CD138 (e.g. BT062), Torcl / 2 specific kinase inhibitors (e.g. INK128), ER / UPR targeting agents (e.g. MKC-3946), cFMS inhibitors (e.g. ARRY-382), JAK1 / 2 inhibitors (e.g. CYT387), PARP inhibitors (e.g. olaparib and veliparib (ABT-888)), and BCL-2 antagonists.

[0625] In some embodiments, the anticancer agent is selected from dichloromethane, camptothecin, ifosfamide, tamoxifen, raloxifene, gemcitabine, Sorafenib or any analogs or derivatives thereof.

[0626] In some embodiments, the anticancer agent is a HER2 inhibitor. Non-limiting examples of HER2 inhibitors include monoclonal antibodies such as trastuzumab and pertuzumab Small molecule tyrosine kinase inhibitors, such as gefitinib Erlotinib pilitinib, CP-654577, CP-724714, canertinib (CI 1033), HKI-272, lapatinib (GW-572016; ), PKI-166, AEE788, BMS-599626, HKI-357, BIBW2992, ARRY-334543, and JNJ-26483327.

[0627] In some embodiments, the anticancer agent is an ALK inhibitor. Non-limiting examples of ALK inhibitors include ceritinib, TAE-684 (NVP-TAE694), PF02341066 (crizotinib or 1066), alectinib; brigatinib; entrectinib; ensartinib (X-396); lorlatinib; ASP3026; CEP-37440; 4SC-203; TL-398; PLB1003; TSR-011; CT-707; TPX-0005 and AP26113. Other examples of ALK kinase inhibitors are described in Examples 3-39 of WO05016894.

[0628] In some embodiments, the anticancer agent is an inhibitor of a downstream member of a receptor tyrosine kinase (RTK) / growth factor receptor (e.g., a SHP2 inhibitor (e.g., SHP099, TNO155, RMC-4550, RMC-4630, JAB-3068, JAB-3312, RLY-1971, ERAS-601, SH3809, PF-07284892, or BBP-398, or other SHP2 inhibitors described herein), a SOS1 inhibitor (e.g., BI-1701963, BI-3406, SDR5, RMC-5845, MRTX-0902, or BAY-293), a Raf inhibitor, a MEK inhibitor, an ERK inhibitor, a PI3K inhibitor, a PTEN inhibitor, an AKT inhibitor, or an mTOR inhibitor (e.g., an mTORC1 inhibitor or an mTORC2 inhibitor). In some embodiments, the anticancer agent is JAB-3312.

[0629] In some embodiments, the anticancer agent is another Ras inhibitor or a Ras vaccine, or is designed to directly or indirectly reduce another treatment mode of the oncogenic activity of Ras. In some embodiments, the anticancer agent is another Ras inhibitor. In some embodiments, the Ras inhibitor targets Ras with its active or GTP-bound state. In some embodiments, the Ras inhibitor targets Ras with its inactive or GDP-bound state. In some embodiments, the Ras inhibitor is an inhibitor of K-Ras G12C, such as AMG510, MRTX1257, MRTX849, JNJ-74699157, LY3499446, ARS-1620, ARS-853, BPI-421286, LY3537982, JDQ443, JAB-21822, JAB-21000, IBI351, ERAS-3490 or GDC-6036. In some embodiments, the Ras inhibitor is an inhibitor of K-Ras G12D, such as MRTX1133 or JAB-22000. In some embodiments, the Ras inhibitor is a K-Ras G12V inhibitor, such as JAB-23000. In some embodiments, the Ras inhibitor is RMC-6236. In some embodiments, the Ras inhibitor is selected from the Ras (open) inhibitors (i.e., Ras in a GTP-bound state) disclosed in the following patents incorporated herein by reference in their entirety, or a pharmaceutically acceptable salt, solvate, isomer (e.g., stereoisomer), prodrug, or tautomer thereof: WO 2022235870, WO 2022235864, WO2021091982, WO 2021091967, WO 2021091956, and WO 2020132597. Other examples of Ras inhibitors that can be combined with the Ras inhibitors of the present invention are provided in the following patents, which are incorporated herein by reference in their entirety: WO2023287896, WO 2023287730, WO 2023284881, WO 2023284730, WO 2023284537, WO2023283933, WO 2023283213, WO 2023280960, WO 2023280280, WO 2023280136, WO2023280026, WO 2023278600, WO 2023274383, WO 2023274324, WO 2023020523, WO 2023020521, WO 2023020519, WO 2023020518、WO 2023018812、WO 2023018810、WO2023018809、WO 2023018699、WO 2023015559、WO2023014979, WO 2023014006, WO2023010121, WO 2023009716, WO 2023009572, WO 2023004102, WO 2023003417, WO2023001141, WO 2023001123, WO 2022271923, WO 2022271823, WO 2022271810, WO2022271658, WO 2022269508, WO 2022266167, WO 2022266069, WO 2022266015, WO2022265974, WO 2022261154, WO 2022261154、WO 2022251576、WO 2022251296、WO2022237815、WO 2022232332、WO 2022232331、WO 2022232320、WO 2022232318、WO2022223037、WO 2022221739、WO 2022221528、WO 2022221386、WO 2022216762、WO2022192794、WO 2022192790、WO 2022188729、WO 2022187411、WO 2022184178、WO2022173870、WO 2022173678、WO 2022135346、WO 2022133731、WO 2022133038、WO2022133345、WO 2022132200、WO 2022119748、WO 2022109485、WO 2022109487, WO2022066805, WO 2022002102, WO 2022002018, WO 2021259331, WO 2021257828, WO2021252339, WO 2021248095, WO 2021248090, WO 2021248083、WO 2021248082, WO2021248079, WO 2021248055, WO 2021245051, WO 2021244603, WO 2021239058, WO2021231526, WO 2021228161, WO 2021219090, WO 2021219090, WO 2021219072, WO2021218939, WO 2021217019, WO 2021216770, WO 2021215545, WO2021215544、WO2021211864、WO 2021190467、WO 2021185233、WO 2021180181、WO 2021175199、2021173923、WO 2021169990、WO 2021169963、WO 2021168193、WO 2021158071、WO2021155716、WO 2021152149、WO 2021150613、WO 2021147967、WO 2021147965、WO2021143693、WO 2021142252、WO 2021141628、WO 2021139748、WO 2021139678、WO2021129824、WO 2021129820、WO 2021127404、WO 2021126816、WO 2021126799、WO2021124222、WO 2021121371、WO 2021121367, WO 2021121330, WO 2020050890, WO2020047192, WO 2020035031, WO 2020028706, WO 2019241157, WO 2019232419, WO2019217691, WO 2019217307、WO 2019215203, WO 2019213526, WO 2019213516, WO2019155399, WO 2019150305, WO 2019110751, WO 2019099524, WO 2019051291, WO2018218070, WO 2018217651、WO 2018218071、WO 2018218069、WO 2018206539、WO2018143315、WO 2018140600、WO 2018140599、WO 2018140598、WO 2018140514、WO2018140513、WO 2018140512、WO 2018119183、WO 2018112420、WO 2018068017、WO2018064510、WO 2017201161、WO 2017172979、WO 2017100546、WO 2017087528、WO2017058807、WO 2017058805、WO 2017058728、WO 2017058902、WO 2017058792、WO2017058768、WO2017058915、WO 2017015562、WO 2016168540、WO 2016164675、WO2016049568、WO 2016049524、WO 2015054572、WO 2014152588、WO 2014143659 and WO2013155223.

[0630] In some embodiments, the therapeutic agent that can be combined with the compounds of the invention is an inhibitor of the MAP kinase (MAPK) pathway (or "MAPK inhibitor"). MAPK inhibitors include, but are not limited to, one or more of the MAPK inhibitors described in Cancers (Basel) 2015 Sep; 7(3): 1758–1784. For example, the MAPK inhibitor can be selected from one or more of the following: trametinib, binimetinib, selumetinib, cobimetinib, LErafAON (NeoPharm), ISIS 5132; vemurafenib, pimasertib, TAK733, RO4987655 (CH4987655); CI-1040; PD-0325901; CH5126766; MAP855; AZD6244; refametinib (RDEA 119 / BAY 86-9766); GDC-0973 / XL581; AZD8330 (ARRY-424704 / ARRY-704); RO5126766 (Roche, PLoS One. 2014 Nov 25; 9(11)); and GSK1120212 (or JTP-74057, described in Clin Cancer Res. 2011 Mar 1; 17(5):989-1000). The MAPK inhibitor may be PLX8394, LXH254, GDC-5573 or LY3009120.

[0631] In some embodiments, the anticancer agent is a disruptor or inhibitor of the RAS-RAF-ERK or PI3K-AKT-TOR or PI3K-AKT signaling pathway. PI3K / AKT inhibitors may include, but are not limited to, one or more PI3K / AKT inhibitors described in Cancers (Basel) 2015 September; 7(3): 1758–1784. For example, the PI3K / AKT inhibitor may be selected from one or more of the following: NVP-BEZ235; BGT226; XL765 / SAR245409; SF1126; GDC-0980; PI-103; PF-04691502; PKI-587; GSK2126458.

[0632] In some embodiments, the anticancer agent is a PD-1 or PD-L1 antagonist.

[0633] In some embodiments, other therapeutic agents include ALK inhibitors, HER2 inhibitors, EGFR inhibitors, IGF-1R inhibitors, MEK inhibitors, PI3K inhibitors, AKT inhibitors, TOR inhibitors, MCL-1 inhibitors, BCL-2 inhibitors, SHP2 inhibitors, proteasome inhibitors and immunotherapy, such as immune checkpoint inhibitors. In some embodiments, the therapeutic agent may be a full RTK inhibitor, such as afatinib.

[0634] IGF-1R inhibitors include linsitinib or a pharmaceutically acceptable salt thereof.

[0635] EGFR inhibitors include, but are not limited to, small molecule antagonists, antibody inhibitors, or specific antisense nucleotides or siRNA. Available EGFR antibody inhibitors include cetuximab Panitumumab Zalutumumab, nimotuzumab, and matuzumab. Other antibody-based EGFR inhibitors include any anti-EGFR antibody or antibody fragment that can partially or completely block EGFR activation by natural ligands. Non-limiting examples of antibody-based EGFR inhibitors include those described in Modjtahedi et al., Br. J. Cancer 1993, 67: 247-253; Teramoto et al., Cancer 1996, 77: 639-645; Goldstein et al., Clin. Cancer Res. 1995, 1: 1311-1318; Huang et al., 1999, Cancer Res. 15: 59 (8): 1935-40; and Yang et al., Cancer Res. 1999, 59: 1236-1243. The EGFR inhibitor may be the monoclonal antibody Mab E7.6.3 (Yang, 1999 supra) or Mab C225 (ATCC Accession No. HB-8508) or an antibody or antibody fragment having the binding specificity thereof.

[0636] Small molecule EGFR antagonists include gefitinib Erlotinib Lapatinib See, for example, Yan et al., Pharmacogenetics and Pharmacogenomics in Oncology Therapeutic Antibody Development, BioTechniques 2005, 39(4):565-8; and Paez et al., EGFR Mutations in Lung Cancer Correlation With Clinical Response To Gefitinib Therapy, Science 2004, 304(5676):1497-500. In some embodiments, the EGFR inhibitor is osimertinib. Other non-limiting examples of small molecule EGFR inhibitors include any of the EGFR inhibitors described in the following patent publications, and all pharmaceutically acceptable salts of such EGFR inhibitors: EP0520722; EP 0566226; WO96 / 33980; U.S. Pat. No. 5,747,498; WO96 / 30347; EP 0787772; WO97 / 30034; WO97 / 30044; WO97 / 38994; WO97 / 49688; EP 837063; WO98 / 02434; WO97 / 38983; WO95 / 19774; WO95 / 19970; WO97 / 13771; WO98 / 02437; WO98 / 02438; WO97 / 32881; DE 19629652; WO98 / 33798; WO97 / 32880; WO97 / 32880; EP 682027; WO97 / 02266; WO97 / 27199; WO98 / 07726; WO97 / 34895; WO96 / 31510; WO98 / 14449; WO98 / 14450; WO98 / 14451; WO95 / 09847; WO97 / 19065; WO98 / 17662; U.S. Patent No. 5,789,427; U.S. Patent No. 5,650,415; U.S. Patent No. 5,656,643; WO99 / 35146; WO99 / 35132; WO99 / 07701; and WO92 / 20642. Other non-limiting examples of small molecule EGFR inhibitors include any of the EGFR inhibitors described in Traxler et al., Exp. Opin. Ther. Patents 1998, 8(12): 1599-1625. In some embodiments, the EGFR inhibitor is an ERBB inhibitor. In humans, the ERBB family contains HER1 (EGFR, ERBB1), HER2 (NEU, ERBB2), HER3 (ERBB3), and HER (ERBB4).

[0637] MEK inhibitors include but are not limited to pamosenterib, selumetinib, and cometinib trametinib and bemetinib In some embodiments, the MEK inhibitor targets a MEK mutation that is a class I MEK1 mutation selected from the group consisting of: D67N; P124L; P124S; and L177V. In some embodiments, the MEK mutation is a class II MEK1 mutation selected from the group consisting of: ΔE51-Q58; ΔF53-Q58; E203K; L177M; C121S; F53L; K57E; Q56P; and K57N.

[0638] PI3K inhibitors include, but are not limited to, wortmannin; 17-hydroxywortmannin analogs described in WO06 / 044453; 4-[2-(1H-indazol-4-yl)-6-[[4-(methylsulfonyl)piperazin-1-yl]methyl]thiopheno[3,2-d]pyrimidin-4-yl]morpholine (also known as pictilisib or GDC-0941 and described in WO09 / 036082 and WO09 / 055730); 2-methyl-2-[4-[3-methyl-2-oxo-8-(quinolin-3-yl)-2,3-dihydroimidazo[4,5-c]quinolin-1-yl]phenyl]propionitrile (also known as BEZ 235 or NVP-BEZ 235, and described in WO06 / 122806); (S)-1-(4-((2-(2-aminopyrimidin-5-yl)-7-methyl-4-morpholinothieno[3,2-d]pyrimidin-6-yl)methyl)piperazin-1-yl)-2-hydroxypropan-1-one (described in WO08 / 070740); LY294002 (2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (available from Axon Medchem); PI103 hydrochloride (3-[4-(4-morpholinylpyrido-[3',2':4,5]furo[3,2-d]pyrimidin-2-yl]phenol hydrochloride (available from Axon Medchem); Medchem); PIK75 (2-methyl-5-nitro-2-[(6-bromoimidazo[1,2-a]pyridin-3-yl)methylene]-1-methylhydrazide-benzenesulfonic acid monohydrochloride) (available from Axon Medchem); PIK 90 (N-(7,8-dimethoxy-2,3-dihydro-imidazo[1,2-c]quinazolin-5-yl)-nicotinamide (available from Axon Medchem); AS-252424 (5-[1-[5-(4-fluoro-2-hydroxy-phenyl)-furan-2-yl]-methyl(Z)-ylidene]-thiazolidine-2,4-dione (available from Axon Medchem); Medchem); TGX-221 (7-methyl-2-(4-morpholinyl)-9-[1-(phenylamino)ethyl]-4H-pyrido[1,2-a]pyrimidin-4-one (available from Axon Medchem); XL-765; and XL-147.Other PI3K inhibitors include demethoxyviridin, perifosine, CAL101, PX-866, BEZ235, SF1126, INK1117, IPI-145, BKM120, XL147, XL765, Palomid529, GSK1059615, ZSTK474, PWT33597, IC87114, TGI00-115, CAL263, PI-103, GNE-477, CUDC-907, and AEZS-136.

[0639] AKT inhibitors include, but are not limited to, Akt-1-1 (inhibits Akt1) (Barnett et al., Biochem. J. 2005, 385 (Pt. 2): 399-408); Akt-1-1,2 (inhibits Ak1 and 2) (Barnett et al., Biochem. J. 2005, 385 (Pt. 2): 399-408); API-59CJ-Ome (e.g., Jin et al., Br. J. Cancer 2004, 91: 1808-12); 1-H-imidazo[4,5-c]pyridinyl compounds (e.g., WO 05 / 011700); indole-3-methanol and its derivatives (e.g., U.S. Pat. No. 6,656,963; Sarkar and Li J Nutr. 2004, 134(12 increase): 3493S-3498S); perifosine (e.g., interferes with Akt membrane localization; Dasmahapatra et al. Clin. Cancer Res. 2004, 10(15): 5242-52); phosphatidylinositol ether lipid analogs (e.g., Gills and Dennis Expert. Opin. Investig. Drugs 2004, 13: 787-97); and triciribine (TCN or API-2 or NCI identifier: NSC 154020; Yang et al., Cancer Res. 2004, 64: 4394-9).

[0640] mTOR inhibitors include, but are not limited to, ATP-competitive mTORC1 / mTORC2 inhibitors, such as PI-103, PP242, PP30; Torin 1; FKBP12 enhancer; 4H-1-benzopyran-4-one derivatives; and rapamycin (also known as sirolimus) and its derivatives, including: temsirolimus Everolimus ( WO 94 / 09010); remdeforolimus (also known as deforolimus or AP23573); rapamycin analogs (rapalogs), for example as disclosed in WO98 / 02441 and WO01 / 14387, such as AP23464 and AP23841; 40-(2-hydroxyethyl)rapamycin; 40-[3-hydroxy(hydroxymethyl)methylpropionic acid]-rapamycin (also known as CC1779); 40-epi(tetrazolyl)-rapamycin (also known as ABT578); 32-deoxyrapamycin; 16-pentynyloxy-32(S)-dihydrorapamycin; derivatives disclosed in WO05 / 005434; Derivatives disclosed in U.S. Pat. Nos. 5,258,389, 5,118,677, 5,118,678, 5,100,883, 5,151,413, 5,120,842, and 5,256,790, and WO94 / 090101, WO92 / 05179, WO93 / 111130, WO94 / 02136, WO94 / 02485, WO95 / 14023, WO94 / 02136, WO95 / 16691, WO96 / 41807, WO96 / 41807, and WO2018204416; and phosphorus-containing rapamycin derivatives (e.g., WO05 / 016252). In some embodiments, the mTOR inhibitor is a bisteric inhibitor (see, e.g., WO2018204416, WO2019212990, and WO2019212991), such as RMC-5552, having the following structure:

[0641]

[0642] BRAF inhibitors that can be used in combination with the compounds of the present invention include, for example, vemurafenib, dabrafenib, and encorafenib. BRAF may include 3 types of BRAF mutations. In some embodiments, the 3 types of BRAF mutations are selected from one or more of the following amino acid substitutions in human BRAF: D287H; P367R; V459L; G466V; G466E; G466A; S467L; G469E; N581S; N581I; D594N; D594G; D594A; D594H; F595L; G596D; G596R and A762E.

[0643] MCL-1 inhibitors include, but are not limited to, AMG-176, MIK665, and S63845. Myeloid cell leukemia-1 (MCL-1) protein is one of the major anti-apoptotic members of the B-cell lymphoma-2 (BCL-2) protein family. Overexpression of MCL-1 is closely associated with tumor progression and resistance not only to traditional chemotherapy but also to targeted therapeutics including BCL-2 inhibitors (such as ABT-263).

[0644] In some embodiments, other therapeutic agents are selected from the group consisting of: MEK inhibitors, HER2 inhibitors, SHP2 inhibitors, CDK4 / 6 inhibitors, mTOR inhibitors, SOS1 inhibitors, and PD-L1 inhibitors. In some embodiments, other therapeutic agents are selected from the group consisting of: MEK inhibitors, SHP2 inhibitors, and PD-L1 inhibitors. See, for example, Hallin et al., Cancer Discovery, DOI: 10.1158 / 2159-8290 (October 28, 2019) and Canon et al., Nature, 575: 217 (2019). In some embodiments, the Ras inhibitors of the present invention are used in combination with MEK inhibitors and SOS1 inhibitors. In some embodiments, the Ras inhibitors of the present invention are used in combination with PD-L1 inhibitors and SOS1 inhibitors. In some embodiments, the Ras inhibitors of the present invention are used in combination with PD-L1 inhibitors and SHP2 inhibitors. In some embodiments, the Ras inhibitors of the present invention are used in combination with MEK inhibitors and SHP2 inhibitors. In some embodiments, the cancer is colorectal cancer and the treatment comprises administering a Ras inhibitor of the invention in combination with a second or third therapeutic agent.

[0645] Proteasome inhibitors include but are not limited to carfilzomib Bortezomib and oprozomib.

[0646] Immunotherapies include, but are not limited to, monoclonal antibodies, immunomodulatory imides (IMiDs), GITR agonists, genetically engineered T cells (e.g., CAR-T cells), bispecific antibodies (e.g., BiTEs), and anti-PD-1, anti-PD-L1, anti-CTLA4, anti-LAG1, and anti-OX40 agents).

[0647] Immunomodulators (IMiDs) are a class of immunomodulatory drugs (drugs that modulate immune responses) that contain an imide group. The IMiD class includes thalidomide and its analogs (lenalidomide, pomalidomide, and apremilast).

[0648] Exemplary anti-PD-1 antibodies and methods of use thereof are described by Goldberg et al., Blood 2007, 110(1):186-192; Thompson et al., Clin. Cancer Res. 2007, 13(6):1757-1761; and WO06 / 121168 A1) and elsewhere herein.

[0649] GITR agonists include, but are not limited to, GITR fusion proteins and anti-GITR antibodies (e.g., bivalent anti-GITR antibodies), such as the GITR fusion proteins described in U.S. Pat. No. 6,111,090, U.S. Pat. No. 8,586,023, WO2010 / 003118, and WO2011 / 090754; or, for example, U.S. Pat. No. 7,025,962, EP1947183, U.S. Pat. No. 7,812,135, U.S. Pat. No. 8,388,967, U.S. Pat. No. 8 ,591,886, U.S. Patent No. 7,618,632, EP1866339 and WO2011 / 028683, WO2013 / 039954, WO05 / 007190, WO07 / 133822, WO05 / 055808, WO99 / 40196, WO01 / 03720, WO99 / 20758, WO06 / 083289, WO05 / 115451 and anti-GITR antibodies described in WO2011 / 051726.

[0650] Another example of a therapeutic agent that can be used in combination with the compounds of the present invention is an anti-angiogenic agent. Anti-angiogenic agents include, but are not limited to, chemical compositions, antibodies, antigen binding regions, radionuclides, and combinations and conjugates thereof prepared by in vitro synthesis. Anti-angiogenic agents can be agonists, antagonists, allosteric modulators, toxins, or more generally can act to inhibit or stimulate their targets (e.g., receptor or enzyme activation or inhibition), and thereby promote cell death or inhibit cell growth. In some embodiments, one or more other therapies include anti-angiogenic agents.

[0651] Anti-angiogenic agents can be MMP-2 (matrix-metalloproteinase 2) inhibitors, MMP-9 (matrix-metalloproteinase 9) inhibitors and COX-II (cyclooxygenase 11) inhibitors. Non-limiting examples of anti-angiogenic agents include rapamycin, temsirolimus (CCI-779), everolimus (RAD001), sorafenib, sunitinib and bevacizumab. Examples of available COX-II inhibitors include alecoxib, valdecoxib and rofecoxib. Examples of useful matrix metalloproteinase inhibitors are described in WO96 / 33172, WO96 / 27583, WO98 / 07697, WO98 / 03516, WO98 / 34918, WO98 / 34915, WO98 / 33768, WO98 / 30566, WO90 / 05719, WO99 / 52910, WO99 / 52889, WO99 / 29667, WO99007675, EP0606046, EP0780386, EP1786785, EP1181017, EP0818442, EP1004578 and US20090012085 and U.S. Pat. Nos. 5,863,949 and 5,861,510. Preferred MMP-2 and MMP-9 inhibitors are those that have little activity inhibiting MMP-1. More preferred are those that selectively inhibit MMP-2 or AMP-9 relative to other matrix-metalloproteinases (i.e., MAP-1, MMP-3, MMP-4, MMP-5, MMP-6, MMP-7, MMP-8, MMP-10, MMP-11, MMP-12, and MMP-13). Some specific examples of MMP inhibitors are AG-3340, RO32-3555, and RS13-0830.

[0652] Other exemplary anti-angiogenic agents include KDR (kinase domain receptor) inhibitors (e.g., antibodies and antigen binding regions that specifically bind to kinase domain receptors), anti-VEGF agents (e.g., antibodies or antigen binding regions that specifically bind to VEGF (e.g., bevacizumab) or soluble VEGF receptors or their ligand binding regions (such as VEGF-TRAP TM ) and anti-VEGF receptor agents (e.g., antibodies or antigen-binding regions that specifically bind thereto), EGFR inhibitors (e.g., antibodies or antigen-binding regions that specifically bind thereto) (such as (panitumumab), erlotinib ), anti-Ang1 and anti-Ang2 agents (e.g., antibodies or antigen binding regions that specifically bind to them or their receptors (e.g., Tie2 / Tek)), and anti-Tie2 kinase inhibitors (e.g., antibodies or antigen binding regions that specifically bind to them). Other anti-angiogenic agents include Campath, IL-8, B-FGF, Tek antagonists (US2003 / 0162712; US6,413,932), anti-TWEAK agents (e.g., specific binding antibodies or antigen binding regions, or soluble TWEAK receptor antagonists; see US6,727,225), ADAM disintegrin domains that antagonize the binding of integrins to their ligands (US 2002 / 0042368), specifically binding anti-eph receptor or anti-ephrin antibodies or antigen binding regions (U.S. Patent Nos. 5,981,245; 5,728,813; 5,969,110; 6,596,852; 6,232,447; 6,057,124 and members of their patent families), and anti-PDGF-BB antagonists (e.g., specifically binding antibodies or antigen binding regions), and antibodies or antigen binding regions that specifically bind to PDGF-BB ligands, and PDGFR kinase inhibitors (e.g., antibodies or antigen binding regions that specifically bind thereto). Other antiangiogenic agents include: SD-7784 (Pfizer, USA); cilengitide (Merck KGaA, Germany, EPO 0770622); pegaptanib octasodium (Gilead Sciences, USA); Alphastatin (BioActa, UK); M-PGA (Celgene, USA, US 5712291); ilomastat (Arriva, USA, US 5892112); emaxanib (Pfizer, USA, US 5792783); vatalanib (Novartis, Switzerland); 2-methoxyestradiol (EntreMed, USA); TLC ELL-12 (Elan, Ireland); anecortave acetate (Celgene, USA, US 5712291); ilomastat (Arriva, USA, US 5892112); emaxanib (Pfizer, USA, US 5792783); vatalanib (Novartis, Switzerland); 2-methoxyestradiol (EntreMed, USA); TLC ELL-12 (Elan, Ireland); anecortave acetate (Celgene, USA, US 5712291); acetate) (Alcon, USA); α-D148 Mab (Amgen, USA); CEP-7055 (Cephalon, USA); anti-VnMab (Crucell, Netherlands), DAC anti-angiogenic agent (ConjuChem, Canada); Angiocidin (InKine Pharmaceutical, USA); KM-2550 (Kyowa Hakko, Japan);SU-0879 (Pfizer, USA); CGP-79787 (Novartis, Switzerland, EP 0970070); ARGENT technology (Ariad, USA); YIGSR-Stealth (Johnson & Johnson, USA); fibrinogen-E fragment (BioActa, UK); angiogenesis inhibitor (Trigen, UK); TBC-1635 (Encysive Pharmaceuticals, USA); SC-236 (Pfizer, USA); ABT-567 (Abbott, USA); metastasis inhibitor (Metastatin) (EntreMed, USA); maspin (Sosei, Japan); 2-methoxyestradiol (Oncology Sciences Corporation, USA); ER-68203-00 (IV AX, USA); BeneFin (Lane Labs, USA); Tz-93 (Tsumura, Japan); TAN-1120 (Takeda, Japan); FR-111142 (Fujisawa, Japan, JP 02233610); platelet factor 4 (RepliGen, USA, EP 407122); vascular endothelial growth factor antagonist (Borean, Denmark); bevacizumab (pINN) (Genentech, USA); angiogenesis inhibitor (SUGEN, USA); XL 784 (Exelixis, USA); XL 647 (Exelixis, USA); MAb α5β3 integrin second generation (Applied Molecular Evolution, USA and Medlmmune, USA); enzastaurin hydrochloride (Lilly, USA); CEP 7055 (Cephalon, USA and Sanofi-Synthelabo, France); BC 1 (Genoa Institute of Cancer Research, Italy); rBPI 21 and BPI-derived antiangiogenic agents (XOMA, USA); PI 88 (Progen, Australia); Cilengitide (Merck KGaA, Germany; Munich Technical University, Germany, Scripps Clinic and Research Foundation, USA); AVE 8062 (Ajinomoto, Japan);AS 1404 (Cancer Research Laboratory, New Zealand); SG 292 (Telios, USA); endostatin (Boston Childrens Hospital, USA); ATN161 (Attenuon, USA); 2-methoxyestradiol (Boston Childrens Hospital, USA); ZD 6474, (AstraZeneca, UK); ZD 6126 (Angiogene Pharmaceuticals, UK); PPI2458 (Praecis, USA); AZD9935 (AstraZeneca, UK); AZD2171 (AstraZeneca, UK); vatalanib (pINN) (Novartis, Switzerland and Schering AG, Germany); tissue factor pathway inhibitor (EntreMed, USA); pegaptanib (Pinn) (Gilead Sciences, USA); xanthorrhizol (Yonsei University, South Korea); gene-based VEGF-2 vaccine (Scripps Clinic and Research Foundation, USA); SPV5.2 (Supratek, Canada); SDX 103 (University of California at San Diego, USA); PX 478, (ProlX, USA); METASTATIN (EntreMed, USA); troponin I (Harvard University, USA); SU 6668 (SUGEN, USA); OXI 4503 (OXiGENE, USA); orthoguanidine (Dimensional Pharmaceuticals, USA); motopramide C (British Columbia University, Canada); CDP 791 (Celltech Group, UK); atiprimod (pINN) (GlaxoSmithKline, UK); E 7820 (Eisai, Japan); CYC 381 (Harvard University, USA); University, USA); AE941 (Aeterna, Canada); angiogenesis vaccine (EntreMed, USA); urokinase plasminogen activator inhibitor (Dendreon, USA);oglufanide (pINN) (Melmotte, USA); HIF-lα inhibitor (Xenova, UK); CEP 5214 (Cephalon, USA); BAYRES 2622 (Bayer, Germany); Angiocidin (InKine, USA); A6 (Angstrom, USA); KR 31372 (Korea Research Institute of Chemical Technology, South) Korea); GW2286 (GlaxoSmithKline, UK); EHT0101 (ExonHit, France); CP868596 (Pfizer, USA); CP564959 (OSI, USA); CP 547632 (Pfizer, USA); 786034 (GlaxoSmithKline, UK); KRN633 (Kirin Brewery, Japan); intraocular 2-methoxyestradiol drug delivery system; anginex (Maastricht University, Netherlands and Minnesota University, USA); ABT 510 (Abbott, USA); AAL 993 (Novartis, Switzerland); VEGI (ProteomTech, USA); tumor necrosis factor-α inhibitor; SU 11248 (Pfizer, USA and SUGEN USA); ABT 518 (Abbott, USA); YH16 (Yantai Rongchang, China); S-3APG (Boston Childrens Hospital, USA and EntreMed, USA); MAb KDR (ImClone Systems, USA); MAb α5β (Protein Design, USA); KDR kinase inhibitor (Celltech Group, UK and Johnson & Johnson, USA); GFB 116 (South Florida University, USA and Yale University, USA); CS 706 (Sankyo, Japan); combretastatin A4 prodrug (Arizona State University, USA); chondroitinase AC (IBEX, Canada); BAY RES 2690 (Bayer, Germany);AGM 1470 (Harvard University, USA, Takeda, Japan and TAP, USA); AG13925 (Agouron, USA); tetrathiomolybdate (University of Michigan, USA); GCS100 (Wayne State University, USA) CV 247 (Ivy Medical, UK); CKD 732 (Chong Kun Dang, South Korea); irsogladine (Nippon Shinyaku, Japan); RG 13577 (Aventis, France); WX 360 (Wilex, Germany); squalamine (Genaera, USA); RPI 4610 (Sirna, USA); heparanase inhibitor (InSight, Israel); KL 3106 (Kolon, South Korea); Honokiol (Emory University, USA); ZK CDK (Schering AG, Germany); ZK Angio (Schering AG, Germany); ZK229561 (Novartis, Switzerland and Schering AG, Germany); XMP 300 (XOMA, USA); VGA 1102 (Taisho, Japan); VE-cadherin-2 antagonist (ImClone Systems, USA); Vasostatin (National Institutes of Health, USA); Flk-1 (ImClone Systems, USA); TZ 93 (Tsumura, Japan); Tumstatin (Beth Israel Hospital, USA); Truncated soluble FLT 1 (vascular endothelial growth factor receptor 1) (Merck & Co, USA); Tie-2 ligand (Regeneron, USA); and thrombospondin 1 inhibitor (Allegheny Health, Education and Research Foundation, USA). ;

[0653] Other examples of therapeutic agents that can be used in combination with the compounds of the invention include agents (e.g., antibodies, antigen binding regions, or soluble receptors) that specifically bind to and inhibit the activity of growth factors, such as antagonists of hepatocyte growth factor (HGF, also known as scatter factor), and antibodies or antigen binding regions that specifically bind to its receptor c-Met.

[0654] Another example of a therapeutic agent that can be used in combination with the compounds of the present invention is an autophagy inhibitor. Autophagy inhibitors include, but are not limited to, chloroquine, 3-methyladenine, hydroxychloroquine (Plaquenil TM ), bafilomycin A1, 5-amino-4-imidazolecarboxamide ribonucleoside (AICAR), okadaic acid, autophagy-inhibiting algal toxins that inhibit type 2A or type 1 protein phosphatases, analogs of cAMP, and drugs that increase cAMP levels, such as adenosine, LY204002, N6-mercaptopurine riboside, and vinblastine. In addition, antisense or siRNA that inhibits protein expression including but not limited to ATG5 (which is related to autophagy) can also be used. In some embodiments, one or more other therapies include autophagy inhibitors.

[0655] Another example of a therapeutic agent that can be used in combination with the compounds of the present invention is an anti-neoplastic agent. In some embodiments, the one or more additional therapies include an anti-neoplastic agent. Non-limiting examples of anti-neoplastic agents include acemannan, aclarubicin, aldesleukin, alemtuzumab, alitretinoin, hexamethylmelamine, amifostine, aminolevulinic acid, amrubicin, amsacrine, anagrelide, anastrozole, ancer, ancestim, arglabin, arsenic trioxide, BAM-002 (Novelos), bexarotene, bicalutamide, bromuridine, capecitabine, celmoleukin, cetrorelix, cladribine, clotrimazole, cytarabine octadecyl phosphate, DA 3030 (Dong-A), daclizumab, denileukindiftitox, deslorelin, dexrazoxane, dilazep, docetaxel, docosanol, doxercalciferol, deoxyfluridine, doxorubicin,...

Claims

1. A method of treating immune-refractory lung cancer in a subject, the method comprising administering to the subject a RAS G12C (Turn on) inhibitor.

2. A method for transforming the tumor microenvironment of an immune-cold lung cancer in a subject in need thereof, the method comprising administering to the subject a RAS G12C (Turn on) inhibitor.

3. The method of claim 1 or 2, wherein the RAS G12C (Open) Inhibitors for triple-complex RAS G12C (Turn on) inhibitor.

4. A method of treating immune-refractory lung cancer in a subject, the method comprising administering to the subject a RAS inhibitor of Formula I: or a pharmaceutically acceptable salt thereof, where the dashed lines represent zero, one, two, three, or four nonadjacent double bonds; A is -N(H or CH3)C(O)-(CH2)-, wherein the amino nitrogen is bound to -CH(R 10 )-, an optionally substituted 3- to 6-membered cycloalkylene group, an optionally substituted 3- to 6-membered heterocycloalkylene group, an optionally substituted 6-membered arylene group, or an optionally substituted 5- to 10-membered heteroarylene group; B does not exist, is -CH(R 9 )-、>C=CR 9 R 9’ or>CR 9 R 9’ wherein the carbon is bonded to -N(R 11 )C(O)-, an optionally substituted 3- to 6-membered cycloalkylene group, an optionally substituted 3- to 6-membered heterocycloalkylene group, an optionally substituted 6-membered arylene group, or a 5- to 6-membered heteroarylene group; G is an optionally substituted C1-C4 alkylene group, an optionally substituted C1-C4 alkenylene group, an optionally substituted C1-C4 heteroalkylene group, -C(O)O-CH(R 6 )-, where C is bound to -C(R 7 R 8 )-、-C(O)NH-CH(R 6 )-, where C is bound to -C(R 7 R 8 )-, optionally substituted C1-C4 heteroalkylene or 3 to 8 membered heteroarylene; L is a linker, wherein the linker is acyclic or comprises a monocyclic, fused bicyclic, fused polycyclic, bridged bicyclic, or bridged polycyclic group; W is a crosslinking group comprising a vinyl ketone, a vinyl sulfone, an alkynyl ketone, a haloacetyl group, or an alkynyl sulfone; X 1 is an optionally substituted C1-C2 alkylene group, NR, O or S(O) n ; X 2 is O or NH; X 3 N or CH; n is 0, 1 or 2; R is hydrogen, cyano, optionally substituted C1-C4 alkyl, optionally substituted C2-C4 alkenyl, optionally substituted C2-C4 alkynyl, C(O)R', C(O)OR', C(O)N(R')2, S(O)R', S(O)2R' or S(O)2N(R')2; Each R ’ is independently H or optionally substituted C1-C4 alkyl; Y 1 is C, CH or N; Y 2 , Y 3 , Y 4 and Y 7 are independently C or N; Y 5 is CH, CH2 or N; Y 6 is C(O), CH, CH2 or N; R 1 is cyano, optionally substituted C1-C6 alkyl, optionally substituted C1-C6 heteroalkyl, optionally substituted 3- to 6-membered cycloalkyl, optionally substituted 3- to 6-membered cycloalkenyl, optionally substituted 3- to 6-membered heterocycloalkyl, optionally substituted 6- to 10-membered aryl, or optionally substituted 5- to 10-membered heteroaryl, or R 1 and R 2 and the atoms to which they are attached are combined to form an optionally substituted 3- to 14-membered heterocycloalkyl; R 2 R is absent, hydrogen, optionally substituted C1-C6 alkyl, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 alkynyl, optionally substituted 3- to 6-membered cycloalkyl, optionally substituted 3- to 7-membered heterocycloalkyl, optionally substituted 6-membered aryl, optionally substituted 5- or 6-membered heteroaryl; 3 does not exist, or R 2 and R 3 and the atoms to which it is attached are combined to form an optionally substituted 3- to 8-membered cycloalkyl or an optionally substituted 3- to 14-membered heterocycloalkyl; R 4 is absent, hydrogen, halogen, cyano or methyl optionally substituted with 1 to 3 halogens; R 5 is hydrogen, C1-C4 alkyl optionally substituted with halogen, cyano, hydroxy or C1-C4 alkoxy, cyclopropyl or cyclobutyl; R 6 is hydrogen or methyl; R 7 is hydrogen, halogen or optionally substituted C1-C3 alkyl, or R 6 and R 7 and the carbon atoms to which they are attached form an optionally substituted 3- to 6-membered cycloalkyl or an optionally substituted 3- to 7-membered heterocycloalkyl; R 8 is hydrogen, halogen, hydroxy, cyano, optionally substituted C1-C3 alkoxy, optionally substituted C1-C3 alkyl, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 alkynyl, optionally substituted 3- to 8-membered cycloalkyl, optionally substituted 3- to 14-membered heterocycloalkyl, optionally substituted 5- to 10-membered heteroaryl, or optionally substituted 6- to 10-membered aryl, or R 7 and R 8 Combined with the carbon atom to which it is attached, it forms C=CR 7’ R 8’ ; C=N(OH), C=N(O-C1-C3 alkyl), C=O, C=S, C=NH, optionally substituted 3- to 6-membered cycloalkyl, or optionally substituted 3- to 7-membered heterocycloalkyl; R 7a and R 8a are independently hydrogen, halo, optionally substituted C1-C3 alkyl, or combined with the carbon to which they are attached to form a carbonyl; R 7’ is hydrogen, halogen or optionally substituted C1-C3 alkyl; R 8’ is hydrogen, halogen, hydroxy, cyano, optionally substituted C1-C3 alkoxy, optionally substituted C1-C3 alkyl, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 alkynyl, optionally substituted 3- to 8-membered cycloalkyl, optionally substituted 3- to 14-membered heterocycloalkyl, optionally substituted 5- to 10-membered heteroaryl, or optionally substituted 6- to 10-membered aryl, or R 7’ and R 8’ and the carbon atoms to which they are attached form an optionally substituted 3- to 6-membered cycloalkyl or an optionally substituted 3- to 7-membered heterocycloalkyl; R 9 is H, F, optionally substituted C1-C6 alkyl, optionally substituted C1-C6 heteroalkyl, optionally substituted 3- to 6-membered cycloalkyl, or optionally substituted 3- to 7-membered heterocycloalkyl, or R 9 and L, combined with the atoms to which they are attached, form an optionally substituted 3- to 14-membered heterocycloalkyl; R 9’ is hydrogen or optionally substituted C1-C6 alkyl; or R 9 and R 9’ and the atoms to which they are attached form a 3- to 6-membered cycloalkyl group or a 3- to 6-membered heterocycloalkyl group; R 10 is hydrogen, halogen, hydroxy, C1-C3 alkoxy or C1-C3 alkyl; R 10a is hydrogen or halogen; R 11 is hydrogen or C1-C3 alkyl; and R 21 It is H or C1-C3 alkyl.

5. A method of transforming the tumor microenvironment of an immune-cold lung cancer in a subject in need thereof, the method comprising administering to the subject a RAS inhibitor of Formula I: or a pharmaceutically acceptable salt thereof, where the dashed lines represent zero, one, two, three, or four nonadjacent double bonds; A is -N(H or CH3)C(O)-(CH2)-, wherein the amino nitrogen is bound to -CH(R 10 )-, an optionally substituted 3- to 6-membered cycloalkylene group, an optionally substituted 3- to 6-membered heterocycloalkylene group, an optionally substituted 6-membered arylene group, or an optionally substituted 5- to 10-membered heteroarylene group; B does not exist, is -CH(R 9 )-、>C=CR 9 R 9’ or>CR 9 R 9’ wherein the carbon is bonded to -N(R 11 )C(O)-, an optionally substituted 3- to 6-membered cycloalkylene group, an optionally substituted 3- to 6-membered heterocycloalkylene group, an optionally substituted 6-membered arylene group, or a 5- to 6-membered heteroarylene group; G is an optionally substituted C1-C4 alkylene group, an optionally substituted C1-C4 alkenylene group, an optionally substituted C1-C4 heteroalkylene group, -C(O)O-CH(R 6 )-, where C is bound to -C(R 7 R 8 )-、-C(O)NH-CH(R 6 )-, where C is bound to -C(R 7 R 8 )-, optionally substituted C1-C4 heteroalkylene or 3 to 8 membered heteroarylene; L is a linker, wherein the linker is acyclic or comprises a monocyclic, fused bicyclic, fused polycyclic, bridged bicyclic, or bridged polycyclic group; W is a crosslinking group comprising a vinyl ketone, a vinyl sulfone, an alkynyl ketone, a haloacetyl group, or an alkynyl sulfone; X 1 is an optionally substituted C1-C2 alkylene group, NR, O or S(O) n ; X 2 is O or NH; X 3 N or CH; n is 0, 1 or 2; R is hydrogen, cyano, optionally substituted C1-C4 alkyl, optionally substituted C2-C4 alkenyl, optionally substituted C2-C4 alkynyl, C(O)R', C(O)OR', C(O)N(R')2, S(O)R', S(O)2R' or S(O)2N(R')2; Each R ’ is independently H or optionally substituted C1-C4 alkyl; Y 1 is C, CH or N; Y 2 , Y 3 , Y 4 and Y 7 are independently C or N; Y 5 is CH, CH2 or N; Y 6 is C(O), CH, CH2 or N; R 1 is cyano, optionally substituted C1-C6 alkyl, optionally substituted C1-C6 heteroalkyl, optionally substituted 3- to 6-membered cycloalkyl, optionally substituted 3- to 6-membered cycloalkenyl, optionally substituted 3- to 6-membered heterocycloalkyl, optionally substituted 6- to 10-membered aryl, or optionally substituted 5- to 10-membered heteroaryl, or R 1 and R 2 and the atoms to which they are attached are combined to form an optionally substituted 3- to 14-membered heterocycloalkyl; R 2 R is absent, hydrogen, optionally substituted C1-C6 alkyl, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 alkynyl, optionally substituted 3- to 6-membered cycloalkyl, optionally substituted 3- to 7-membered heterocycloalkyl, optionally substituted 6-membered aryl, optionally substituted 5- or 6-membered heteroaryl; 3 does not exist, or R 2 and R 3 and the atoms to which it is attached are combined to form an optionally substituted 3- to 8-membered cycloalkyl or an optionally substituted 3- to 14-membered heterocycloalkyl; R 4 is absent, hydrogen, halogen, cyano or methyl optionally substituted with 1 to 3 halogens; R 5 is hydrogen, C1-C4 alkyl optionally substituted with halogen, cyano, hydroxy or C1-C4 alkoxy, cyclopropyl or cyclobutyl; R 6 is hydrogen or methyl; R 7 is hydrogen, halogen or optionally substituted C1-C3 alkyl, or R 6 and R 7 and the carbon atoms to which they are attached form an optionally substituted 3- to 6-membered cycloalkyl or an optionally substituted 3- to 7-membered heterocycloalkyl; R 8 is hydrogen, halogen, hydroxy, cyano, optionally substituted C1-C3 alkoxy, optionally substituted C1-C3 alkyl, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 alkynyl, optionally substituted 3- to 8-membered cycloalkyl, optionally substituted 3- to 14-membered heterocycloalkyl, optionally substituted 5- to 10-membered heteroaryl, or optionally substituted 6- to 10-membered aryl, or R 7 and R 8 Combined with the carbon atom to which it is attached, it forms C=CR 7’ R 8’ ; C=N(OH), C=N(O-C1-C3 alkyl), C=O, C=S, C=NH, optionally substituted 3- to 6-membered cycloalkyl, or optionally substituted 3- to 7-membered heterocycloalkyl; R 7a and R 8a are independently hydrogen, halo, optionally substituted C1-C3 alkyl, or combined with the carbon to which they are attached to form a carbonyl; R 7’ is hydrogen, halogen or optionally substituted C1-C3 alkyl; R 8’ is hydrogen, halogen, hydroxy, cyano, optionally substituted C1-C3 alkoxy, optionally substituted C1-C3 alkyl, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 alkynyl, optionally substituted 3- to 8-membered cycloalkyl, optionally substituted 3- to 14-membered heterocycloalkyl, optionally substituted 5- to 10-membered heteroaryl, or optionally substituted 6- to 10-membered aryl, or R 7’ and R 8’ and the carbon atoms to which they are attached form an optionally substituted 3- to 6-membered cycloalkyl or an optionally substituted 3- to 7-membered heterocycloalkyl; R 9 is H, F, optionally substituted C1-C6 alkyl, optionally substituted C1-C6 heteroalkyl, optionally substituted 3- to 6-membered cycloalkyl, or optionally substituted 3- to 7-membered heterocycloalkyl, or R 9 and L, combined with the atoms to which they are attached, form an optionally substituted 3- to 14-membered heterocycloalkyl; R 9’ is hydrogen or optionally substituted C1-C6 alkyl; or R 9 and R 9’ and the atoms to which they are attached form a 3- to 6-membered cycloalkyl group or a 3- to 6-membered heterocycloalkyl group; R 10 is hydrogen, halogen, hydroxy, C1-C3 alkoxy or C1-C3 alkyl; R 10a is hydrogen or halogen; R 11 is hydrogen or C1-C3 alkyl; and R 21 It is H or C1-C3 alkyl.

6. The method of claim 5, wherein the subject is resistant to an immune checkpoint inhibitor prior to transforming the tumor microenvironment.

7. The method of claim 5 or 6, wherein administration of the RAS inhibitor transforms the tumor microenvironment thereby sensitizing the cancer to treatment with an immune checkpoint inhibitor.

8. The method of any one of claims 1 to 7, further comprising administering to the subject a SHP2 inhibitor.

9. A method of treating immune-refractory lung cancer in a subject, the method comprising administering to the subject a RAS G12C (Open) inhibitors, SHP2 inhibitors, and immune checkpoint inhibitors.

10. A method of transforming the tumor microenvironment of an immune-cold lung cancer in a subject in need thereof, the method comprising administering to the subject a RAS G12C (Open) inhibitors, SHP2 inhibitors, and immune checkpoint inhibitors.

11. The method of any one of claims 1 to 3 and 8 to 10, wherein the RAS G12C The (opening) inhibitor is a compound of formula I: or a pharmaceutically acceptable salt thereof, where the dashed lines represent zero, one, two, three, or four nonadjacent double bonds; A is -N(H or CH3)C(O)-(CH2)-, wherein the amino nitrogen is bound to -CH(R 10 )-, an optionally substituted 3- to 6-membered cycloalkylene group, an optionally substituted 3- to 6-membered heterocycloalkylene group, an optionally substituted 6-membered arylene group, or an optionally substituted 5- to 10-membered heteroarylene group; B does not exist, is -CH(R 9 )-、>C=CR 9 R 9’ or>CR 9 R 9’ wherein the carbon is bonded to -N(R 11 )C(O)-, an optionally substituted 3- to 6-membered cycloalkylene group, an optionally substituted 3- to 6-membered heterocycloalkylene group, an optionally substituted 6-membered arylene group, or a 5- to 6-membered heteroarylene group; G is an optionally substituted C1-C4 alkylene group, an optionally substituted C1-C4 alkenylene group, an optionally substituted C1-C4 heteroalkylene group, -C(O)O-CH(R 6 )-, where C is bound to -C(R 7 R 8 )-、-C(O)NH-CH(R 6 )-, where C is bound to -C(R 7 R 8 )-, optionally substituted C1-C4 heteroalkylene or 3 to 8 membered heteroarylene; L is a linker, wherein the linker is acyclic or comprises a monocyclic, fused bicyclic, fused polycyclic, bridged bicyclic, or bridged polycyclic group; W is a crosslinking group comprising a vinyl ketone, a vinyl sulfone, an alkynyl ketone, a haloacetyl group, or an alkynyl sulfone; X 1 is an optionally substituted C1-C2 alkylene group, NR, O or S(O) n ; X 2 is O or NH; X 3 N or CH; n is 0, 1 or 2; R is hydrogen, cyano, optionally substituted C1-C4 alkyl, optionally substituted C2-C4 alkenyl, optionally substituted C2-C4 alkynyl, C(O)R', C(O)OR', C(O)N(R')2, S(O)R', S(O)2R' or S(O)2N(R')2; Each R ’ is independently H or optionally substituted C1-C4 alkyl; Y 1 is C, CH or N; Y 2 , Y 3 , Y 4 and Y 7 are independently C or N; Y 5 is CH, CH2 or N; Y 6 is C(O), CH, CH2 or N; R 1 is cyano, optionally substituted C1-C6 alkyl, optionally substituted C1-C6 heteroalkyl, optionally substituted 3- to 6-membered cycloalkyl, optionally substituted 3- to 6-membered cycloalkenyl, optionally substituted 3- to 6-membered heterocycloalkyl, optionally substituted 6- to 10-membered aryl, or optionally substituted 5- to 10-membered heteroaryl, or R 1 and R 2 and the atoms to which they are attached are combined to form an optionally substituted 3- to 14-membered heterocycloalkyl; R 2 R is absent, hydrogen, optionally substituted C1-C6 alkyl, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 alkynyl, optionally substituted 3- to 6-membered cycloalkyl, optionally substituted 3- to 7-membered heterocycloalkyl, optionally substituted 6-membered aryl, optionally substituted 5- or 6-membered heteroaryl; 3 does not exist, or R 2 and R 3 and the atoms to which it is attached are combined to form an optionally substituted 3- to 8-membered cycloalkyl or an optionally substituted 3- to 14-membered heterocycloalkyl; R 4 is absent, hydrogen, halogen, cyano or methyl optionally substituted with 1 to 3 halogens; R 5 is hydrogen, C1-C4 alkyl optionally substituted with halogen, cyano, hydroxy or C1-C4 alkoxy, cyclopropyl or cyclobutyl; R 6 is hydrogen or methyl; R 7 is hydrogen, halogen or optionally substituted C1-C3 alkyl, or R 6 and R 7 and the carbon atoms to which they are attached form an optionally substituted 3- to 6-membered cycloalkyl or an optionally substituted 3- to 7-membered heterocycloalkyl; R 8 is hydrogen, halogen, hydroxy, cyano, optionally substituted C1-C3 alkoxy, optionally substituted C1-C3 alkyl, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 alkynyl, optionally substituted 3- to 8-membered cycloalkyl, optionally substituted 3- to 14-membered heterocycloalkyl, optionally substituted 5- to 10-membered heteroaryl, or optionally substituted 6- to 10-membered aryl, or R 7 and R 8 Combined with the carbon atom to which it is attached, it forms C=CR 7’ R 8’ ; C=N(OH), C=N(O-C1-C3 alkyl), C=O, C=S, C=NH, optionally substituted 3- to 6-membered cycloalkyl, or optionally substituted 3- to 7-membered heterocycloalkyl; R 7a and R 8a are independently hydrogen, halo, optionally substituted C1-C3 alkyl, or combined with the carbon to which they are attached to form a carbonyl; R 7’ is hydrogen, halogen or optionally substituted C1-C3 alkyl; R 8’ is hydrogen, halogen, hydroxy, cyano, optionally substituted C1-C3 alkoxy, optionally substituted C1-C3 alkyl, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 alkynyl, optionally substituted 3- to 8-membered cycloalkyl, optionally substituted 3- to 14-membered heterocycloalkyl, optionally substituted 5- to 10-membered heteroaryl, or optionally substituted 6- to 10-membered aryl, or R 7’ and R 8’ and the carbon atoms to which they are attached form an optionally substituted 3- to 6-membered cycloalkyl or an optionally substituted 3- to 7-membered heterocycloalkyl; R 9 is H, F, optionally substituted C1-C6 alkyl, optionally substituted C1-C6 heteroalkyl, optionally substituted 3- to 6-membered cycloalkyl, or optionally substituted 3- to 7-membered heterocycloalkyl, or R 9 and L, combined with the atoms to which they are attached, form an optionally substituted 3- to 14-membered heterocycloalkyl; R 9’ is hydrogen or optionally substituted C1-C6 alkyl; or R 9 and R 9’ and the atoms to which they are attached form a 3- to 6-membered cycloalkyl group or a 3- to 6-membered heterocycloalkyl group; R 10 is hydrogen, halogen, hydroxy, C1-C3 alkoxy or C1-C3 alkyl; R 10a is hydrogen or halogen; R 11 is hydrogen or C1-C3 alkyl; and R 21 It is H or C1-C3 alkyl.

12. The method of any one of claims 1 to 11, wherein the RAS inhibitor is a compound of Table 1 or a pharmaceutically acceptable salt thereof.

13. The method of any one of claims 1 to 12, wherein the RAS inhibitor is or a pharmaceutically acceptable salt thereof.

14. The method of any one of claims 1 to 8 and 11 to 13, further comprising administering an immune checkpoint inhibitor to the subject.

15. A method of treating immune-refractory lung cancer in a subject, the method comprising administering to the subject a RAS inhibitor, a SHP2 inhibitor, and an immune checkpoint inhibitor, wherein the RAS inhibitor is: or a pharmaceutically acceptable salt thereof, and The SHP2 inhibitor is: or a pharmaceutically acceptable salt thereof.

16. The method of any one of claims 9, 10, 14 and 15, wherein the immune checkpoint inhibitor is a PD-1 inhibitor.

17. The method of any one of claims 1 to 16, wherein the subject has previously been administered an immune checkpoint inhibitor.

18. The method of any one of claims 1 to 17, wherein the subject is resistant to treatment with an immune checkpoint inhibitor.

19. The method of claim 18, wherein the subject has acquired resistance to treatment with an immune checkpoint inhibitor.

20. The method of any one of claims 1 to 19, wherein administration of the RAS inhibitor sensitizes the cancer to treatment with an immune checkpoint inhibitor.

21. The method of any one of claims 8 to 20, wherein the inhibitors are administered simultaneously or sequentially.

22. The method of any one of claims 8 to 21, wherein the inhibitor is administered as a single formulation or in divided formulations.

23. The method of any one of claims 1 to 22, wherein the subject has one or more tumors with a low tumor mutation burden.

24. The method of any one of claims 1 to 23, wherein the subject has one or more microsatellite stable tumors.

25. The method of any one of claims 1 to 24, wherein the subject has one or more tumors with low microsatellite instability.

26. The method of any one of claims 1 to 25, wherein the subject has one or more tumors with low tumor immune infiltration.

27. The method of any one of claims 1 to 26, wherein administration of the RAS inhibitor alters the tumor immune infiltrate relative to the tumor immune infiltrate in the absence of the RAS inhibitor.

28. The method of claim 26 or 27, wherein the tumor immune infiltrate comprises antigen presenting cells, myeloid cells, or lymphoid cells.

29. The method of any one of claims 1 to 28, wherein administration of the RAS inhibitor alters an anti-tumor immune response.

30. The method of any one of claims 1 to 29, wherein administration of the RAS inhibitor alters the tumor microenvironment relative to the tumor microenvironment in the absence of the RAS inhibitor.

31. The method of any one of claims 1 to 30, wherein administration of the RAS inhibitor converts an immune-cold tumor into an immune-hot tumor.

32. The method of any one of claims 1 to 31, wherein the method reduces tumor size or inhibits tumor growth.

33. The method of any one of claims 1 to 32, wherein the immune-refractory lung cancer is non-small cell lung cancer or small cell lung cancer.

34. The method of any one of claims 1 to 33, wherein the immune-refractory lung cancer comprises a K-RasG12C, H-Ras G12C, or N-Ras G12C mutation.

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