Compositions and methods for treating lung cancer

Abstract

The disclosure features methods for treating lung cancer using RAS(ON) inhibitors. The disclosure also relates to uses of RAS(ON) multi-selective inhibitors in combination with RAS(ON) G12C- selective inhibitors and optionally one or more additional therapeutic agents, such as immune checkpoint inhibitors.

Description

[0001] PATENT

[0002] ATTORNEY DOCKET NO.: 51432-077WO2

[0003] COMPOSITIONS AND METHODS FOR TREATING LUNG CANCER

[0004] Field

[0005] The present disclosure relates to the treatment of RAS G12C mutant lung cancer, e.g., locally 5 advanced or metastatic non-small cell lung cancer (NSCLC).

[0006] Background

[0007] Lung cancer remains one of the most prevalent and deadly forms of cancer worldwide. According to the World Health Organization (WHO), lung cancer accounts for approximately 1.8 million deaths 10 annually, making it the leading cause of cancer-related mortality. Despite advances in diagnostic and therapeutic strategies, the overall survival rate for lung cancer patients remains low, primarily due to late- stage diagnosis, high rates of metastasis, and the aggressive nature of the disease.

[0008] The immune system plays a critical role in the body's ability to detect and destroy cancer cells. However, many lung cancers, particularly those classified as "cold tumors," lack sufficient infiltration by 15 immune cells, leading to poor patient outcomes. The lack of immunogenicity, low IFN-γ gene signature, and absence of effective T-cell infiltration in cold tumors renders many immune-based therapies, such as immune checkpoint inhibitors, less effective. Immune checkpoint inhibitors, including those targeting PD- 1 / PD-L1 and CTLA-4, have shown promise in treating several cancers by reinvigorating exhausted T- cells. Nonetheless, their efficacy in lung cancer has been limited to a subset of patients, often those 20 whose tumors display a higher level of immune cell infiltration or express certain biomarkers.

[0009] RAS mutations, particularly in the KRAS gene, are among the most common oncogenic drivers in lung cancer, found in approximately 28% of lung adenocarcinomas. The presence of RAS mutations is associated with aggressive disease progression and poor prognosis. Furthermore, RAS-mutant lung cancers tend to exhibit resistance to various forms of therapy, including immune checkpoint inhibitors. 25 This resistance may be due in part to the immunosuppressive microenvironment often associated with RAS-mutant tumors, which further contributes to the "cold" tumor phenotype by promoting the exclusion of cytotoxic T-cells and the accumulation of immunosuppressive cell types such as regulatory T-cells (Tregs) and myeloid-derived suppressor cells (MDSCs).

[0010] The discovery and FDA approval of the KRASG12Cmutant-selective inhibitors sotorasib and 30 adagrasib has reshaped the therapeutic landscape of KRASG12C-mutant advanced NSCLC. These inhibitors selectively target the KRASG12Cprotein in the GDP-bound inactive state (KRASG12C(OFF)) via covalent modification of the mutated cysteine residue. However, the clinical outcomes from KRASG12C(OFF) inhibitor treatment are limited by primary and acquired resistance mechanisms, indicating the need for improved therapeutic agents and effective combinations.

[0011] 35 There remains a need for compositions and methods for treating RAS G12C mutant lung cancer and in particular immune refractory tumors.

[0012] Summary

[0013] The present disclosure provides, in part, compositions and uses thereof for treating immune 40 refractory lung cancer. The disclosure is based, at least in part, on the observation that treating immune refractory lung cancer with a combination therapy including a RAS inhibitor therapy (e.g., a RAS(ON)

[0014] 1 PATENT

[0015] ATTORNEY DOCKET NO.: 51432-077WO2

[0016] multi-selective inhibitor and a RAS(ON) G12C-selective inhibitor) and an immune checkpoint inhibitor is unexpectedly more effective than using a single agent RAS(ON) inhibitor combined with an immune checkpoint inhibitor. In some embodiments, the RAS inhibitor therapy comprises a RAS(ON) G12C- selective inhibitor which inhibits RAS having an oncogenic G12C mutation. In some embodiments, the 5 RAS(ON) inhibitor therapy further comprises a RAS(ON) multi-selective inhibitor. In some embodiments, treatment with a RAS inhibitor therapy and immune checkpoint inhibitor is synergistic. In some embodiments, the combination therapy described herein is administered to a subject in need thereof who has previously failed immunotherapy treatment, such as immunotherapy treatment with an immune checkpoint inhibitor.

[0017] 10 In an aspect, the disclosure provides a method of treating an immune refractory lung cancer in a subject by administering to the subject in need thereof a RAS inhibitor therapy (e.g., a RAS(ON) multi- selective inhibitor and a RAS(ON) G12C-selective inhibitor) and an immune checkpoint inhibitor.

[0018] In an aspect, the disclosure provides a method of modulating tumor cell MHC class I expression by administering to a subject in need thereof or contacting a tumor cell with a RAS inhibitor therapy (e.g., 15 a RAS(ON) multi-selective inhibitor and a RAS(ON) G12C-selective inhibitor) and an immune checkpoint inhibitor.

[0019] In some embodiments of the methods described herein, the subject in need thereof is administered an immune checkpoint inhibitor that is a PD-1 inhibitor.

[0020] In some embodiments, the subject in need thereof has previously been administered an immune 20 checkpoint inhibitor. In some embodiments, the subject in need thereof is resistant to treatment with an immune checkpoint inhibitor. In some embodiments, the subject in need thereof has acquired resistance to treatment with an immune checkpoint inhibitor.

[0021] In some embodiments, the immune refractory lung cancer is a non-small cell lung cancer. In some embodiments, the immune refractory non-small cell lung cancer includes a G12C RAS mutation.

[0022] 25 In some embodiments, the disclosure provides methods of treating a subject afflicted with a RAS G12C mutant cancer, the methods generally comprise administering to the subject a therapeutically effective amount of Compound B (elironrasib), and avoiding co-administration of a proton pump inhibitor, wherein the subject is also in need of the proton pump inhibitor.

[0023] In some embodiments, the disclosure provides methods of treating a subject afflicted with a RAS 30 G12C mutant cancer, the methods generally comprise discontinuing administration of a proton pump inhibitor to avoid an adverse drug interaction with Compound B (elironrasib), and administering to the subject a therapeutically effective amount of Compound B (elironrasib).

[0024] In some embodiments, the disclosure provides methods of treating a subject afflicted with a RAS G12C mutant cancer, wherein said subject is also in need of a proton pump inhibitor, the methods 35 generally comprise administering to the subject a therapeutically effective amount of Compound B (elironrasib) while avoiding proton pump inhibitor co-administration, and any one or more of the following: (a) advising the subject that proton pump inhibitors should be avoided or discontinued, (b) advising the subject that co-administration of Compound B (elironrasib) with drugs that are proton pump inhibitors can alter the therapeutic effect or adverse reaction profile of Compound B (elironrasib), (c) advising the 40 subject that co-administration of Compound B (elironrasib) with proton pump inhibitors can alter the therapeutic effect or adverse reaction profile of Compound B (elironrasib), (d) advising the subject that

[0025] 2 PATENT

[0026] ATTORNEY DOCKET NO.: 51432-077WO2

[0027] use of Compound B (elironrasib) in subjects being treated with proton pump inhibitors is contraindicated, or (e) advising the subject that proton pump inhibitors should be used with caution in subjects receiving Compound B (elironrasib) due to the potential for an adverse reaction profile.

[0028] It is specifically contemplated that any limitation discussed with respect to one embodiment of the 5 disclosure may apply to any other embodiment of the disclosure. Furthermore, any compound or composition of the disclosure may be used in any method of the disclosure, and any method of the disclosure may be used to produce or to utilize any compound or composition of the disclosure.

[0029] Brief Description of the Figures

[0030] 10 FIG.1 is a graph showing the anti-tumor activity of Compound A (RMC-6236, daraxonrasib), Compound B (RMC-6291, elironrasib), the RAS(ON) doublet combination (i.e., Compound A + Compound B), and the corresponding combinations with anti-PD-1 following repeated oral administration in C57BL / 6J mice bearing the NSCLC syngeneic model e3LL (KRASG12C / G12C, NRAS- / -) shown as mean tumor volumes with standard error means.

[0031] 15 FIG.2 graphically depicts anti-tumor activity of Compound A, Compound B, the RAS(ON) doublet combination, and the corresponding combination with anti-PD-1 following repeated oral administration in C57BL / 6J mice bearing the NSCLC syngeneic model e3LL (KRASG12C / G12C, NRAS- / -) shown as individual tumor growth curves. Numbers indicate number of complete regressions per number of injected mice.

[0032] FIG.3 shows a Kaplan-Meier graph of treatment response durability of Compound A, Compound 20 B, the RAS(ON) doublet combination, and the corresponding combination with anti-PD-1 following repeated oral administration in C57BL / 6J mice bearing the NSCLC syngeneic model e3LL (KRASG12C / G12C, NRAS- / -).

[0033] FIG.4 graphically depicts mouse body weight change following repeated oral administration of Compound A, Compound B, the RAS(ON) doublet combination, and the corresponding combination with 25 anti-PD-1 following repeated oral administration in C57BL / 6J mice bearing the NSCLC syngeneic model e3LL (KRASG12C / G12C, NRAS- / -).

[0034] FIG.5 shows immune cell composition in the NSCLC syngeneic e3LL (KRASG12C / G12C, NRAS- / -) model represented as percentage of CD45+ cells and expression of cell surface markers on viable, CD45- large cells (assessed as tumor cells), 24h post 8 days of treatment.

[0035] 30 FIG.6A-6C show the RAS(ON) inhibitor doublet induces deep and durable responses in KRASG12C-mutant NSCLC xenograft models. FIG.6A shows a tumor response waterfall plot of 8 KRASG12C-mutant NSCLC subcutaneous xenograft models upon daily treatment of Compound B at 30, 100 or 200 mg / kg and Compound A at 25 mg / kg as single agents or in combination (n = 3-15 per group in each model). Average % mean tumor volume change ± SEM from baseline at response calling date are 35 shown. mRECIST criteria were used to call tumor response as indicated on the right-hand side of the waterfall plot. Oncoplots illustrating gene alterations and expression levels in critical genes linked to the clinicopathologic characteristics of the indicated models are shown below the waterfall. Color coding represents dark gray for mutations and light gray for the absence of mutations. The symbol denotes that mRNA of corresponding genes is not expressed, defined as having a gene-expression value of ≤0.5 40 CPM. The bottom row indicates the KRASG12Cgene copy number (CN) in each model as assessed by digital PCR. ^ and # symbols indicate that Compound B was dosed at 30 or 200 mg / kg in specific group,

[0036] 3 PATENT

[0037] ATTORNEY DOCKET NO.: 51432-077WO2

[0038] respectively. FIG.6B shows a Kaplan-Meier analysis of time to tumor doubling on treatment in individual tumor-bearing animals from 8 KRASG12C-mutant NSCLC subcutaneous xenograft models upon daily treatment of vehicle, Compound B at 30, 100 or 200 mg / kg and Compound A at 25 mg / kg as single agents or in combination for up to 90 days (n = 46 animals in control, n = 55 in Compound B and

[0039] 5 Compound A single agent groups, n = 54 in Compound B and Compound A doublet combination group).

[0040] Time to event was determined by the time on treatment until tumor volume doubling from baseline on the survival plot by Kaplan-Meier analysis. Log-rank test was used to compare specific treatment groups (****, P < 0.0001). FIG.6C shows the antitumor activity of sotorasib, Compound D (RMC-4998), Compound C (RMC-7977) and the combination of Compound D (RMC-4998) with Compound C (RMC-7977) in 3 10 KRASG12C-mutant NSCLC subcutaneous PDX models including CRUK-NBC-M17 (n = 5-7 per group), CRUK-NBC-KRAS3 (n = 5-6 per group) and CRUK-NBC-KRAS1 (n = 4-7 per group). Tumor-bearing mice were treated with vehicle or inhibitors (sotorasib at 100 mg / kg po qd, Compound D (RMC-4998) at 100 mg / kg po qd, Compound C (RMC-7977) at 10 mg / kg po qd and the combination of Compound D (RMC- 4998) at 100 mg / kg po qd plus Compound C (RMC-7977) at 10 mg / kg po qd) for up to 28 days followed 15 by off-treatment measurements. Mean tumor volumes of each group were plotted over the course of treatment. The horizontal dotted line indicates the initial average tumor volume. The vertical dotted line indicates treatment stop. Error bars, SEM.

[0041] FIG.7A-7G show the RAS(ON) inhibitor doublet forestalls resistance driven by elevated RAS pathway flux. FIG.7A shows the antitumor activity of Compound A and Compound B as single agents or 20 in combination in NCI-H2122 (KRASG12C / G12CSTK11mutKEAP1mut, NSCLC) subcutaneous xenograft model (n = 8-15 per group). Tumor-bearing mice were treated with vehicle or RAS(ON) inhibitors (Compound B at 100 mg / kg po qd and Compound A at 25 mg / kg po qd as single agents or in combination) for 17 to 56 days. Mean tumor volumes of each group were plotted over the course of treatment. Vehicle control and specific treatment group were compared by two-way repeated-measures 25 ANOVA on the last measurement day of the vehicle group (***, P < 0.001; ****, P < 0.0001). The dotted line indicates the initial average tumor volume. Error bars, SEM. FIG.7B show the antitumor activity of Compound A and Compound B as single agents or in combination in NCI-H2030 (KRASG12C / G12CSTK11mutKEAP1mut, NSCLC) subcutaneous xenograft model (n = 8 per group). Tumor-bearing mice were treated with vehicle or RAS(ON) inhibitors (Compound B at 100 mg / kg po qd and Compound A at 25 30 mg / kg po qd as single agents or in combination) for 21 to 89 days. Mean tumor volumes of each group were plotted over the course of treatment. Vehicle control and specific treatment group were compared by two-way repeated-measures ANOVA on the last measurement day of the vehicle group (****, P < 0.0001). The dotted line indicates the initial average tumor volume. Error bars, SEM. FIG.7C shows a histopathology analysis of HER2 in 8 KRASG12C-mutant NSCLC subcutaneous xenograft tumors at 35 baseline included in the tumor response waterfall plot and Kaplan-Meier analysis in FIG.6.

[0042] Representative images are shown at 60× magnification. Scale bars, 50 µm. FIG.7D shows the antitumor activity of Compound A, Compound B, and Compound A plus Compound B combination in CTG-2536 (KRASG12C / WT, HER2High, NSCLC) subcutaneous PDX model (n = 4-6 per group). Tumor-bearing mice were treated with vehicle or RAS(ON) inhibitors (Compound B at 200 mg / kg po qd, Compound A at 25 40 mg / kg po qd and combination of Compound B at 200 mg / kg po qd plus Compound A at 25 mg / kg po qd) for 16 to 90 days. Mean tumor volumes of each group were plotted over the course of treatment. Vehicle

[0043] 4 PATENT

[0044] ATTORNEY DOCKET NO.: 51432-077WO2

[0045] control and specific treatment group were compared by two-way repeated-measures ANOVA on the last measurement day of the vehicle group, and none of the comparisons were statistically significant. The dotted line indicates the initial average tumor volume. Error bars, SEM. FIG.7E shows the antitumor activity of Compound A, Compound B, and Compound A plus Compound B combination in LUN055 5 (KRASG12C Amp, NSCLC) subcutaneous PDX model (n = 3 per group). Tumor-bearing mice were treated with vehicle or RAS(ON) inhibitors (Compound B at 100 mg / kg po qd, Compound A at 25 mg / kg po qd and combination of Compound B at 30 mg / kg po qd plus Compound A at 25 mg / kg po qd) for 21 to 91 days. Mean tumor volumes of each group were plotted over the course of treatment. Vehicle control and specific treatment group were compared by two-way repeated-measures ANOVA on the last

[0046] 10 measurement day of the vehicle group (*, P < 0.05). The dotted line indicates the initial average tumor volume. Error bars, SEM. Two tumor volume plots were from two different studies. FIG.7F shows the relative copy numbers of KRASWTor KRASG12Cin LUN055 (KRASG12C Amp, NSCLC) subcutaneous PDX tumors (n = 2-3 per group) collected at the end of tumor growth studies as shown in (FIG.7E). Tumors used for ddPCR analysis were collected at 3 hours post the last dose of vehicle or inhibitors. The relative 15 gene copy numbers were determined by ddPCR and normalized to RPP30. FIG.7G shows DNA fluorescence in situ hybridization (FISH), RNA in situ hybridization (ISH) and histopathology analysis of LUN055 (KRASG12C Amp, NSCLC) subcutaneous PDX tumors collected at the end of tumor growth studies as shown in (FIG.7E). Representative images are shown at 63×, 40× and 40× magnification from ROIs closest to the mean of the group for FISH, RNA ISH and IHC, respectively. Tumors used for

[0047] 20 representative images were collected at 3 hours post the last dose of vehicle or Compound B. Cen12: centromere 12. Scale bars, 50 µm.

[0048] FIG.8A-8F show the RAS(ON) inhibitor doublet drives sustained RAS signaling suppression, inhibits cell proliferation and induces apoptosis in vivo. FIG.8A shows target engagement of KRASG12Cprotein by Compound B in NCI-H2122 (KRASG12C / G12CSTK11mutKEAP1mut, NSCLC) subcutaneous 25 xenograft tumors, shown as the percentage of crosslinked KRASG12Cby Compound B relative to controls.

[0049] Tumor-bearing mice were treated with 17 consecutive daily doses of Compound B at 100 mg / kg as single agent (black line) or in combination with Compound A at 25 mg / kg (gray line). Tumors were harvested at indicated time points (n = 2-3 per time point). Values are plotted as mean ± SEM. FIG.8B shows PD in NCI-H2122 (KRASG12C / G12CSTK11mutKEAP1mut, NSCLC) subcutaneous xenograft tumors, shown as the 30 relative change in human DUSP6 mRNA expression. Tumor-bearing mice were treated with a single dose (qd x1, solid lines) or 7 consecutive daily doses (qd x7, dashed lines) of Compound B at 100 mg / kg and Compound A at 25 mg / kg as single agents or in combination. Tumors were harvested at indicated time points (n = 2-3 per time point). Data from Compound B single agent, Compound A single agent and RAS(ON) inhibitor doublet groups are plotted. Values are plotted as mean ± SEM. FIG.8C and FIG.8D 35 show histopathology analysis of NCI-H2122 subcutaneous xenograft tumors treated with a single dose (qd x1, circles in the quantification plot) or 7 consecutive daily doses (qd x7, dots in the quantification plot) of Compound B at 100 mg / kg and Compound A at 25 mg / kg as single agents or in combination and collected at indicated time points (n= 3 per time point). (FIG.8C) Representative images are shown at 20× magnification from samples closest to the mean of the group. Tumors used for representative images 40 of pERK, pS6 and Ki67 were collected at 48 hours post 7 consecutive daily doses of RAS(ON) inhibitors.

[0050] Tumors used for representative images of CC3 were collected at 24 hours post a single dose of RAS(ON)

[0051] 5 PATENT

[0052] ATTORNEY DOCKET NO.: 51432-077WO2

[0053] inhibitors. Scale bars, 100 µm. (FIG.8D) pERK, pS6 and CC3 staining in tumor areas and Ki67 staining in tumor cell nucleuses were quantified and compared with vehicle using one-way ANOVA followed by Dunnett multiple comparison test (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001). FIG.8E shows PD in CTG-2536 (KRASG12C / WT, HER2High, NSCLC) subcutaneous PDX tumors, shown as the 5 relative change in human DUSP6 mRNA expression. Tumor-bearing mice were treated with a single dose of Compound B at 200 mg / kg and Compound A at 25 mg / kg as single agents or in combination. Tumors were harvested at indicated time points (n = 2-3 per time point). Data from Compound B single agent, Compound A single agent and RAS(ON) inhibitor doublet groups are plotted. Values are plotted as mean ± SEM. FIG.8F shows histopathology analysis of CTG-2536 subcutaneous PDX tumors treated with a 10 single dose of Compound B at 200 mg / kg and Compound A at 25 mg / kg as single agents or in combination and collected at indicated time points (n= 3 per time point). Representative images are shown at 20× magnification from samples closest to the mean of the group. Tumors used for representative images of pERK and CC3 were collected at 48 hours and 24 hours post a single dose of RAS(ON) inhibitors, respectively. Scale bars, 100 µm.

[0054] 15 FIG.9A-9D show PK / TE / PD modeling of complementary mechanisms predicts the superiority of the RAS(ON) inhibitor doublet over monotherapies in the context of KRASG12Coverexpression. FIG.9A shows the model captures the blood PK of Compound A at 25 mg / kg and plasma PK of Compound B at 100 mg / kg upon single and repeated daily administration. The simulated PK is shown by the solid lines. The observed data are shown by dots. FIG.9B shows the model captures the target engagement (TE) 20 and pharmacodynamics (PD) upon single and repeated daily administration of Compound B at 100 mg / kg and Compound A at 25 mg / kg as single agents or in combination, as measured by the levels of non- crosslinked KRASG12Cprotein and DUSP6 mRNA expression, respectively. Simulated TE and PD are shown by the dash and solid lines, respectively. The observed TE and PD data are shown by circles and diamonds, respectively. FIG.9C shows the steady-state simulation of RAS pathway suppression as 25 measured by DUSP6 mRNA expression relative to the baseline following daily administration of Compound B at 30 or 100 mg / kg and Compound A at 25 mg / kg as single agents or in combination. FIG.

[0055] 9D shows the steady-state simulation of RAS pathway suppression with different levels of KRASG12Cprotein as measured by DUSP6 mRNA expression relative to the baseline without KRASG12Coverexpression, following daily administration of Compound B at 30 or 100 mg / kg and Compound A at 25 30 mg / kg as single agents or in combination.

[0056] FIG.10A-10D show Compound B and Compound A drive immune-dependent complete regressions, favorably modulate the TME and synergize with anti-PD-1 in the immunogenic KPAR1.3 model. FIG.10A shows tumor growth of subcutaneous tumors treated for 32 days (dashed line indicated treatment stop) with vehicle, Compound B at 100 mg / kg, Compound A at 25 mg / kg po qd in combination 35 with mouse IgG2a (isotype control) or anti-PD-1 at 10 mg / kg ip biw in C57BL / 6 mice shown as individual tumor volume (n=10). Number of complete regressions indicated. FIG.10B shows representative lung IHC images (large image - scale bar 500 µm, zoomed in image – scale bar 100 µm, # denotes enlarged area, dashed lines highlight large tumor nodules), percentage tumor area per lung area at 4 hours post 4 days of treatment with Compound B at 30 mg / kg or Compound A at 25 mg / kg po qd (four lung sections 40 were evaluated for each mouse) and quantification of CD8+ or CD4+ T cells as percentage of all cells within the tumor region in orthotopic lung tumors. Each point is the analysis of the tumor mass / section of

[0057] 6 PATENT

[0058] ATTORNEY DOCKET NO.: 51432-077WO2

[0059] lung. Four lung sections were evaluated for each mouse. Asterix indicates Analysis was performed using one-way ANOVA. For all statistical analysis *p < 0.05, **p < 0.01, ***p<0.001. FIG.10C shows tumor growth of subcutaneous tumors treated for 32 days (dashed line indicated treatment stop) with Compound B at 100 mg / kg in combination with Compound A at 25 mg / kg po qd and mouse IgG2a 5 (isotype control) or anti-PD-1 at 10 mg / kg ip biw in C57BL / 6 mice shown as individual tumor volume (n=10). FIG.10D shows tumor growth of subcutaneous tumors treated for 32 days with Compound B at 100 mg / kg or Compound A at 25 mg / kg po qd in the presence of CD4, CD8 T cell and NK cell depleting antibodies administered at 10 mg / kg at day -1, 0 and every 6 days post dosing start. CR – complete regressions defined as tumor volume at 0 mm3.

[0060] 10 FIG.11A-11F shows the RAS(ON) doublet maximizes pathway suppression and favorably modulates the TME of the ICI-refractory 3LL-ΔNRAS model. FIG.11A shows PD of Compound B, Compound A and the RAS(ON) doublet in subcutaneous tumors shown as the relative change in pERK expression in tumor cells. Tumor-bearing mice were treated with a single dose Compound B at 30 mg / kg, Compound A at 25 mg / kg and the RAS(ON) doublet and tumors were collected at indicated time points.

[0061] 15 FIG.11B shows PROGENy MAPK and G2M score calculated based on bulkRNA sequencing of whole tumors collected at 24 hours post 8 days of treatment. FIG.11C shows quantification of as percentage of total cells in tumors collected at 24 hours post 8 days of treatment. FIG.11D shows frequency of CD8 T cells, CD4 T cells, NK cells, gMDSCs (Ly6G+), M2 macrophages (CD11b+, F4 / 80+, CD206+) based on live, CD45+ cells and H2Kbcell surface expression on tumor cells (large CD45-, Periostin-) identified by 20 multiparameter spectral flow cytometry in tumors collected at 24 hours post 8 days of treatment. FIG.11E shows representative IHC images (scale bar 100 µm). FIG.11F shows quantification of CD4, CD8 and Granzyme B positive cells as percentage of total in tumors collected at 24 hours post 8 days of treatment. Values are plotted as mean ± SD. Each point is an individual tumor. Analysis was performed using one- way ANOVA. For all statistical analysis *p < 0.05, **p < 0.01, ***p<0.001, ****p<0.0001.

[0062] 25 FIG.12A-12F show the RAS(ON) doublet maximizes antigen presentation and sensitizes the ICI- refractory 3LL-ΔNRAS tumor model to anti-PD-1. FIG.12A shows a heat map of individual antigen presentation machinery signature genes. Differential expression in tumors (n=5 per treatment) collected at 24 hours post 8 days of treatment with vehicle, Compound B at 30 mg / kg, Compound A at 25 mg / kg or the RAS(ON) doublet. FIG.12B, FIG.12C and FIG.12D show immuno-sequencing (TCRB assay) of 30 gDNA extracted from tumors collected at 24 hours post 8 days of treatment FIG.12B shows the correlation of percent change in the tumor volume from pre-dose to day 8 with the increase in T cell abundance in the treated tumors. FIG.12C shows the sum frequency of convergent clones in the tumor.

[0063] FIG.12D shows the number of shared clones (amino acid + V-gene) within each treatment group that were found in each of the ten tumors / group. FIG.12E shows tumor growth of subcutaneous tumors 35 treated for 72 days (dashed line represents treatment stop) with vehicle, Compound B at 30 mg / kg or Compound A at 25 mg / kg po qd in combination with mouse IgG2a (isotype control) or anti-PD-1 at 10 mg / kg ip biw in C57BL / 6 mice shown as individual tumor volume (n=10). Number of complete regressions indicated. FIG.12F shows tumor growth for the mice from FIG.12E with durable CR for 76 days that were rechallenged on the opposite flank. Table indicates treatment that the primary tumor received and number 40 of mice with immune rejections. Naïve mice were used as controls. CR – complete regressions defined as tumor volume at 0 mm3.

[0064] 7 PATENT

[0065] ATTORNEY DOCKET NO.: 51432-077WO2

[0066] Detailed Description

[0067] Oncogenic mutations in the canonical RAS genes, KRAS, NRAS and HRAS, drive malignant growth in various human cancers with the highest prevalence in pancreatic ductal adenocarcinoma 5 (PDAC, >90%), colorectal cancer (CRC, ~50%), and non-small cell lung cancer (NSCLC, ~30%), accounting for approximately 200,000 new cancers diagnosed in the United States each year. As one of the common hotspot mutations in KRAS, the glycine-to-cysteine amino acid substitution at residue 12 (G12C) is predominant in patients with NSCLC harboring KRAS mutations, accounting for about 40% of these cases.

[0068] 10 Frontline therapy for patients with advanced NSCLC, including those harboring KRAS mutations, includes administration of immune checkpoint inhibitors (ICI), e.g., anti-PD-1 / anti-PD-L1 immunotherapy, often in combination with chemotherapy and sometimes with anti-CTLA-4 agents. Patients with KRASG12C-mutant NSCLC, which is commonly linked to smoking, tend to exhibit higher tumor mutational burden and elevated PD-L1 expression compared to non-G12C KRAS mutants, possibly contributing to 15 increased sensitivity to immunotherapies. However, a significant subset of these patients fail to benefit from ICI, with MHC class I downregulation being a common mechanism of immune evasion in NSCLC. In addition, mutant KRAS have been shown to sustain an immunosuppressive tumor microenvironment (TME) and facilitate cancer cell immune escape by several mechanisms including: secretion of anti- inflammatory chemokines and cytokines, differentiation and infiltration of immunosuppressive regulatory 20 cells, inhibition of T cell activation, and suppression of cytotoxic CD8+ T cell-mediated tumor killing via MHC class I downregulation and PD-L1 upregulation on the tumor cell membrane.

[0069] The present disclosure relates generally to compositions and methods for the treatment of RAS G12C mutant lung cancer including immune refractory lung cancer. In part, the inventors have discovered the combination of a RAS(ON) G12C-selective and RAS(ON) multi-selective inhibitor mitigates clinical 25 resistance mechanisms to KRASG12C(OFF) inhibitors and enhances tumor immune recognition, overcoming immune checkpoint inhibitor resistance. The disclosure is based, at least in part, on the observation of synergy in the treatment of immune refractory lung cancer with a RAS inhibitor therapy comprising a RAS(ON) G12C-selective inhibitor and a RAS(ON) multi-selective inhibitor and an immune checkpoint inhibitor. In some embodiments, the immune refractory lung cancer comprises a G12C RAS 30 mutation. In some embodiments a combination therapy described herein is administered to a subject in need thereof who has previously failed immunotherapy treatment, such as treatment with an immune checkpoint inhibitor. These findings support the evaluation of a combination strategy with a RAS(ON) multi-selective inhibitor, a RAS(ON) G12C-selective inhibitor and one or more immune checkpoint inhibitors as a targeted therapy regimen for patients with RAS G12C mutant lung cancer (e.g., NSCLC).

[0070] 35 Additional aspects of the disclosure are described below.

[0071] Definitions

[0072] In this application, unless otherwise clear from context, (i) the term “a” means “one or more”; (ii) the term "or" is used to mean "and / or" unless explicitly indicated to refer to alternatives only or the 40 alternative are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and "and / or”; (iii) the terms “comprising” and “including” are understood to encompass

[0073] 8 PATENT

[0074] ATTORNEY DOCKET NO.: 51432-077WO2

[0075] itemized components or steps whether presented by themselves or together with one or more additional components or steps; and (iv) where ranges are provided, endpoints are included.

[0076] As used herein, the term “about” is used to indicate that a value includes the standard deviation of error for the device or method being employed to determine the value. In certain embodiments, the 5 term “about” refers to a range of values that fall within 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) of a stated value, unless otherwise stated or otherwise evident from the context (e.g., where such number would exceed 100% of a possible value).

[0077] Note that when a range or amount is provided in the disclosure herein, ± 5% of each range 10 endpoint or specific amount is included, unless otherwise indicated. For example, a range of 100 mg to 400 mg of Compound A is understood to encompass 100 ± 5% mg to 400 ± 5% mg, e.g., 95 mg to 420 mg of Compound A.

[0078] As used herein, the term “administration” refers to the administration of a composition comprising a compound described herein to a subject or system. Administration also includes administering a 15 prodrug derivative or analog or pharmaceutically acceptable salt to the subject, which can form an equivalent amount of active compound within the subject’s body. Administration to an animal subject (e.g., to a human) may be by any appropriate route. For example, in some embodiments, administration may be bronchial (including by bronchial instillation), buccal, enteral, intradermal, intra-arterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, 20 intraventricular, mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (including by intratracheal instillation), transdermal, vaginal or vitreal. In some embodiments, a composition comprising a RAS(ON) multi-selective inhibitor or a RAS(ON) G12C-selective inhibitor disclosed herein is administered orally.

[0079] The term “combination therapy” refers to a method of treatment including administering to a 25 subject at least two active therapeutic agents (e.g., a RAS(ON) multi-selective inhibitor, a RAS (ON) G12C-selective inhibitor, and one or more immune checkpoint inhibitors), as one or more pharmaceutical compositions, as part of a therapeutic regimen. For example, a combination therapy may include administration of a single pharmaceutical composition including at least two therapeutic agents and one or more pharmaceutically acceptable carrier, excipient, diluent, or surfactant. A combination therapy may 30 include administration of two or more pharmaceutical compositions, each composition including one or more therapeutic agent and one or more pharmaceutically acceptable carrier, excipient, diluent, or surfactant. A combination therapy may include administration of a single pharmaceutical composition including three therapeutic agents and one or more pharmaceutically acceptable carrier, excipient, diluent, or surfactant. A combination therapy may include administration of three pharmaceutical

[0080] 35 compositions, each composition including a therapeutic agent and one or more pharmaceutically acceptable carrier, excipient, diluent, or surfactant. The two or more therapeutic agents may optionally be administered simultaneously (as a single or as separate compositions) or sequentially (as separate compositions). The therapeutic agents may be administered in an effective amount. The therapeutic agent may be administered in a therapeutically effective amount. In some embodiments, the effective 40 amount of one or more of the therapeutic agents may be lower when used in a combination therapy than

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[0083] the therapeutic amount of the same therapeutic agent when it is used as a monotherapy, e.g., due to an additive or synergistic effect of combining the two or more therapeutics.

[0084] As used herein, the term “dosage form” refers to a physically discrete unit of a compound disclosed herein for administration to a subject. Each unit contains a predetermined quantity of

[0085] 5 compound. In some embodiments, such quantity is a unit dosage amount (or a whole fraction thereof) appropriate for administration in accordance with a dosing regimen that has been determined to correlate with a desired or beneficial outcome when administered to a relevant population (i.e., with a therapeutic dosing regimen). Those of ordinary skill in the art appreciate that the total amount of a therapeutic composition or the compound administered to a particular subject is determined by one or more attending 10 physicians and may involve administration of multiple dosage forms.

[0086] The terms "inhibit," "block," and "suppress" are used interchangeably and refer to any statistically significant decrease in a biological activity, including full blocking of the activity. As used herein, the term “inhibitor” refers to a compound that prevents a biomolecule, (e.g., a protein, nucleic acid) from completing or initiating a reaction. An inhibitor can inhibit a reaction by competitive, uncompetitive, or non-15 competitive means, for example. With respect to 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 mimetics, peptides, peptidomimetics, antibodies, small molecules, chemicals, analogs that mimic the binding site of an enzyme, receptor, or other protein. In some embodiments, the inhibitor is a small molecule, e.g., a low molecular weight organic compound, 20 e.g., an organic compound having a molecular weight (MW) of less than 1200 Daltons (Da). In some embodiments, the MW is less than 1100 Da. In some embodiments, the MW is less than 1000 Da. In some embodiments, the MW is less than 900 Da. In some embodiments, the range of the MW of the small molecule is between 800 Da and 1200 Da. Small molecule inhibitors include cyclic and acyclic compounds. Small molecules inhibitors include natural products, derivatives, and analogs thereof.

[0087] 25 As used herein “patient” and “subject” are used interchangeably and refer to a mammal, for whom diagnosis, prognosis, or therapy is desired. Mammalian subjects include, but are not limited to, humans, domestic animals, farm animals, sports animals, and zoo animals including, for example, humans, non- human primates, dogs, cats, guinea pigs, rabbits, rats, mice, horses, and cattle. In certain embodiments, the subject has been diagnosed with cancer. In certain embodiments, the subject is a human afflicted with 30 a tumor (e.g., cancer) who has been diagnosed with a need for treatment for a tumor (e.g., cancer).

[0088] As used herein, the term “pharmaceutical composition” refers to a compound, such as Compound A or Compound B disclosed herein, or a pharmaceutically acceptable salt, polymorph, hydrate, or solvate thereof, formulated together with a pharmaceutically acceptable excipient.

[0089] A “pharmaceutically acceptable excipient,” as used herein, refers to any inactive ingredient (for 35 example, a vehicle capable of suspending or dissolving the active compound) having the properties of being nontoxic and noninflammatory in a subject. Typical excipients include, for example: anti-adherents, antioxidants, binders, coatings, compression aids, disintegrants, dyes (colors), emollients, emulsifiers, fillers (diluents), film formers or coatings, flavors, fragrances, glidants (flow enhancers), lubricants, preservatives, printing inks, sorbents, suspensing or dispersing agents, sweeteners, or waters of 40 hydration. Excipients include, but are not limited to: butylated optionally substituted hydroxyltoluene (BHT), calcium carbonate, calcium phosphate (dibasic), calcium stearate, croscarmellose and salts

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[0092] thereof, crosslinked polyvinyl pyrrolidone and salts thereof, citric acid, crospovidone, cysteine, ethylcellulose, gelatin, optionally substituted hydroxylpropyl cellulose and salts thereof, optionally substituted hydroxylpropyl methylcellulose and salts thereof, lactose, magnesium stearate, maltitol, mannitol, methionine, methylcellulose, methyl paraben, microcrystalline cellulose, polyethylene glycol, 5 polyvinyl pyrrolidone, povidone, pregelatinized starch, propyl paraben, retinyl palmitate, shellac, silicon dioxide, carboxymethyl cellulose and salts thereof, 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 of ordinary skill in the art are familiar with a variety of agents and materials useful as excipients. See, e.g., Ansel, et al., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems.

[0093] 10 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, a composition includes at least two different pharmaceutically acceptable excipients.

[0094] The term “pharmaceutically acceptable salt,” as use herein, refers to those salts of the

[0095] 15 compounds described herein that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and other animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, pharmaceutically acceptable salts are described in: Berge et al., J. Pharmaceutical Sciences 66:119, 1977 and in Pharmaceutical Salts: Properties, Selection, and 20 Use, (Eds. P.H. Stahl and C.G. Wermuth), WileyVCH, 2008. The salts can be prepared in situ during the final isolation and purification of the compounds described herein or separately by reacting the free base group with a suitable organic acid.

[0096] The terms “RAS pathway” and “RAS / MAPK pathway” are used interchangeably herein to refer to a signal transduction cascade downstream of various cell surface growth factor receptors in which 25 activation of RAS (and its various isoforms and allotypes) is a central event that drives a variety of cellular effector events that determine the proliferation, activation, differentiation, mobilization, and other functional properties of the cell. For example, SHP2 conveys positive signals from growth factor receptors to the RAS activation / deactivation cycle, which is modulated by guanine nucleotide exchange factors (GEFs, such as SOS1) that load GTP onto RAS to produce functionally active GTP-bound RAS as well as 30 GTP-accelerating proteins (GAPs, such as NF1) that facilitate termination of the signals by conversion of GTP to GDP. GTP-bound RAS produced by this cycle conveys essential positive signals to a series of serine / threonine kinases including RAF and MAP kinases, from which emanate additional signals to various cellular effector functions.

[0097] The terms “RAS inhibitor” and “inhibitor of [a] RAS” are used interchangeably to refer to any 35 inhibitor that targets, that is, selectively binds to or inhibits a RAS protein. A RAS inhibitor may be RO7673396, for example.

[0098] As used herein, the term “RAS(ON) inhibitor” refers to an inhibitor that targets, that is, selectively binds to or inhibits, the GTP-bound, active state of RAS (e.g., selective over the GDP-bound, inactive state of RAS). Inhibition of the GTP-bound, active state of RAS includes, for example, the inhibition of 40 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

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[0101] embodiments, RAS(ON) inhibitors may also bind to or inhibit the GDP-bound, inactive state of RAS (e.g., with a lower affinity or inhibition constant than for the GTP-bound, active state of RAS). In certain embodiments, a RAS(ON) inhibitor useful in the present disclosure may form a high affinity three- component complex, or conjugate, between a synthetic ligand and two intracellular proteins which do not 5 interact under normal physiological conditions: the target protein of interest (e.g., RAS), and a widely expressed cytosolic chaperone (presenter protein) in the cell (e.g., cyclophilin A). More specifically, in some embodiments, the inhibitors of RAS described herein induce a new binding pocket in RAS by driving formation of a high affinity tri-complex, or conjugate, between the RAS protein and the widely expressed cytosolic chaperone, cyclophilin A (CypA). A RAS(ON) inhibitor may be an antibody-drug 10 conjugate. See also doi.org / 10.1021 / acs.jmedchem.4c02929.

[0102] As used herein, the term “RAS(OFF) inhibitor” refers to an inhibitor that targets, that is, selectively binds to or inhibits, the GDP-bound, inactive state of RAS (e.g., selective over the GTP-bound, active state of RAS). RAS(OFF) inhibitors are known in the art and described. Non-limiting examples of RAS(OFF) inhibitors include A2A-03, ABREV01, ADT-007, ABT-200, ADT-030, ADT-1004, BBP-454, 15 BGB-53038, BI-2865, BI-2493, BI 3706674, BRSD-143, ERAS-4, ERAS-254, ERAS-4001, HB-700 (G12X+G13D), HZ-V068, ID12241161, JAB-23400, LY4066434, OC211, PF-07985045, PF-07934040, PF-4040, QTX2024, QTX3034, RSC-1255, SIL204, SYNB021225, YL-17231, and ZG2001. Non-limiting examples of RASG12C(OFF) inhibitors include adagrasib (MRTX849), divarasib (RG6330 / GDC-6036), fulzerasib (IBI351 / GFH925), garsorasib (D-1553), glecirasib (JAB-21822), olomorasib (LY3537982), 20 opnurasib (JDQ443), sotorasib (AMG 510), ARS-853, ARS-1620, BI-0474, BI 1823911, BPI-421286, D3S-001, ERAS-3490, GEC255, GH35, HBI-2438, HS-10370, JAB-21000, JAB-21822, JMKX001899, JNJ-74699157 (ARS-3248), MK-1084, SK-17, and YL-15293. Non-limiting examples of RASG12D(OFF) inhibitors include AST2169, BPI-501836, DN022150, ERAS-4693, ERAS-5024, GDC-7035 (RG6620), HBW-012-D, HBW-012-E, HBW-012336, HRS-4642, HS-10529, INCB186748, JAB-22000, KD-8, KRB-25 456, LY3962673, MRTX282, MRTX1133, Q2a, QLC1101, RNK08954, SHR1127, TH-Z827, TH-Z835, TSN1611, and VRTX153, HJ-119, JR-6000, NKT-G12D, FWD-K02, JAB-BX600, EB-TM1, ABSK141, and BPI-2491. Non-limiting examples of RASG12V(OFF) inhibitors include JAB-23000 and QTX3544.

[0103] As used herein, the terms “RAS(ON) multi-selective inhibitor,” “RASMULTI inhibitor,” “RASMULTI(ON) inhibitor,” and “RAS(MULTI) inhibitor” refer to a RAS inhibitor of at least three RAS 30 isoforms, including wild-type and / or variants with missense mutations at one of the following positions: 12, 13, 59, 61, or 146. In some embodiments, a RAS(ON) multi-selective inhibitor (e.g., daraxonrasib or RMC-6236) refers to a RAS inhibitor of at least three RAS variants with missense mutations at one of the following positions: 12, 13, and 61. Exemplary RAS(ON) multi-selective inhibitors include but are not limited to compounds described in the following patent applications, and as otherwise described herein: 35 WO 2025162395, WO 2025119392, WO2025087431, WO 2025051241, WO 2025045233, WO 2024249299, WO 2024222864, WO 2024206858, WO 2024169914, WO 2024153208, WO 2024149214, WO 2024104364, WO 2024067857, WO 2024060966, WO 2024017859, WO 2024008834, WO 2023240263, WO 2023025832, WO 2022060836, WO 2021091956, CN 120441594, CN 119350371, CN 117903169, CN 117720556, CN 117720555, CN 117720554, CN 117534687, CN 117534685, and CN 40 117534684, each of which is incorporated herein by reference in its entirety, including the compound structures disclosed therein. Non-limiting examples of RAS(ON) multi-selective inhibitors also include

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[0106] daraxonrasib (RMC-6236), AN9025, BPI-572270, compound 6A of WO 2024067857, ERAS-0015, GFH276, GFH547, HJ-099, Compound C (RMC-7977), RCZY-680, or RCZY-690.

[0107] As used herein, the terms “RAS(ON) mutant-selective inhibitor” refers to a RAS inhibitor selective for a RAS(ON) variant with missense mutation at one of the following positions: 12, 13, or 61. Non-limiting 5 examples of RAS(ON) mutant-selective inhibitors include RAS(ON) G12C-selective inhibitors (e.g., elironrasib or RMC-6291), RAS(ON) G12D-selective inhibitors (e.g., zoldonrasib or RMC-9805), RAS(ON) Q61H-selective inhibitors (e.g., RMC-0708), RAS(ON) G12V-selective inhibitors (e.g. RMC-5127), and RAS(ON) G13D-selective inhibitors. RAS(ON) mutant-selective inhibitors can be found in any one of the following patent applications, and as otherwise described herein: WO 2025104149, WO 2025093625, 10 WO 2025080946, WO 2024249299, WO 2024211663, WO 2024211712, WO 2024208934, WO 2024149819, WO 2024008610, WO 2024102421, WO 2023240263, WO 2023133543, WO 2023015559, WO 2023086341, WO 2023208005, WO 2023232776, WO 2023086341, WO 2023060253, WO 2023015559, WO 2022235870, WO 2022235864, WO 2021091967, WO 2021091982, WO 2021108683, WO 2020132597, each of which is incorporated herein by reference in its entirety, including the

[0108] 15 compound structures disclosed therein.

[0109] A “therapeutic agent” is any substance, e.g., a compound or composition, capable of treating a disease or disorder. In some embodiments, therapeutic agents that are useful in connection with the present disclosure including RAS inhibitors and cancer chemotherapeutics. Many such therapeutic agents are known in the art and are disclosed herein.

[0110] 20 The term “therapeutically effective amount” means an amount that is sufficient, when administered to a population suffering from or susceptible to a disease, disorder, or condition in accordance with a therapeutic dosing regimen, to treat the disease, disorder, or condition. In some embodiments, a therapeutically effective amount is one that reduces the incidence or severity of, or delays onset of, one or more symptoms of the disease, disorder, or condition. Those of ordinary skill in 25 the art will appreciate that the term “therapeutically effective amount” does not in fact require successful treatment be achieved in a particular individual. Rather, a therapeutically effective amount may be that amount that provides a particular desired pharmacological response in a significant number of subjects when administered to patients in need of such treatment. It is specifically understood that particular subjects may, in fact, be “refractory” to a “therapeutically effective amount.” In some embodiments, 30 reference to a therapeutically effective amount may be a reference to an amount as measured in one or more specific tissues (e.g., a tissue affected by the disease, disorder, or condition) or fluids (e.g., blood, saliva, serum, sweat, tears, urine). Those of ordinary skill in the art will appreciate that, in some embodiments, a therapeutically effective amount may be formulated or administered in a single dose. In some embodiments, a therapeutically effective amount may be formulated or administered in a plurality of 35 doses, for example, as part of a dosing regimen.

[0111] The term “treatment” (also “treat” or “treating”), in its broadest sense, refers to any administration of a substance (e.g., a RAS(ON) multi-selective inhibitor compound) that partially or completely alleviates, ameliorates, relieves, inhibits, delays onset of, reduces severity of, or reduces incidence of one or more symptoms, features, or causes of a particular disease, disorder, or condition. In some embodiments, such 40 treatment may be administered to a subject who is diagnosed with the disease, disorder or condition but does not exhibit signs of the relevant disease, disorder, or condition or of a subject who exhibits only early

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[0114] signs of the disease, disorder, or condition. Alternatively, or additionally, in some embodiments, treatment may be administered to a subject who exhibits one or more established signs of the relevant disease, disorder or condition. In some embodiments, treatment may be of a subject who has been diagnosed as suffering from the relevant disease, disorder, or condition. In any treatment method herein, a patient or 5 subject may be in need of such treatment.

[0115] The term “wild type” refers to an entity having a structure or activity as found in nature in a “normal” (as contrasted with mutant, diseased, altered, etc.) state or context. Those of ordinary skill in the art will appreciate that wild type genes and polypeptides often exist in multiple different forms (e.g., alleles).

[0116] 10

[0117] I. Compositions

[0118] The present disclosure provides, in part, synergistic combination therapy composition and uses thereof as described herein. Combination therapy compositions of the disclosure comprises a RAS(ON) inhibitor therapy and an immune checkpoint inhibitor. The RAS(ON) inhibitor therapy comprises two or 15 more RAS(ON) inhibitor compounds. In some embodiments, the RAS(ON) inhibitors useful in the compositions and uses according to the disclosure include a RAS(ON) G12C-selective inhibitor and a RAS(ON) multi-selective inhibitor.

[0119] (a) RAS(ON) inhibitor therapy

[0120] 20 Provided herein are compositions comprising compounds that inhibit RAS and uses thereof. Also provided are pharmaceutical compositions including a RAS(ON) inhibitor therapy (e.g., a RAS(ON) G12C- selective inhibitor and a RAS(ON) multi-selective inhibitor), or a pharmaceutically acceptable salts thereof, and a pharmaceutically acceptable excipient. RAS(ON) inhibitor compounds may be used in methods of inhibiting RAS (e.g., in a subject or in a cell) and methods of treating cancer, as described 25 herein. In some embodiments, a compound of the present disclosure is or acts as a prodrug, such as with respect to administration to a cell or to a subject in need thereof.

[0121] RAS proteins (KRAS, HRAS, and NRAS) play an essential role in various human cancers and are therefore appropriate targets for anticancer therapy. Indeed, mutations in RAS proteins account for approximately 30% of all human cancers in the United States, many of which are fatal. Dysregulation of 30 RAS proteins by activating mutations, overexpression, or upstream activation is common in human tumors, and activating mutations in RAS are frequently found in human cancer. RAS converts between a GDP-bound “off” and a GTP-bound “on” state. The conversion between states is facilitated by interplay between a guanine nucleotide exchange factor (GEF) protein (e.g., SOS1), which loads RAS with GTP, and a GTPase-activating protein (GAP) protein (e.g., NF1), which hydrolyzes GTP, thereby inactivating 35 RAS. Additionally, the SH2 domain-containing protein tyrosine phosphatase-2 (SHP2) associates with the receptor signaling apparatus and becomes active upon RTK activation, and then promote RAS activation. Mutations in RAS proteins can lock the protein in the “on” state resulting in a constitutively active signaling pathway that leads to uncontrolled cell growth. For example, activating mutations at codon 12 in RAS proteins function by inhibiting both GAP-dependent and intrinsic hydrolysis rates of GTP,

[0122] 40 significantly skewing the population of RAS mutant proteins to the “on” (GTP-bound) state (RAS(ON)), leading to oncogenic MAPK signaling. Notably, RAS exhibits a picomolar affinity for GTP, enabling RAS

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[0125] to be activated even in the presence of low concentrations of this nucleotide. Mutations at codons 13 (e.g., G13D) and 61 (e.g., Q61K) of RAS are also responsible for oncogenic activity in some cancers.

[0126] Oncogenic pathways, like KRAS, and the presence of immunosuppressive cell populations, like tumor associated macrophages (TAMs) and myeloid-derived suppressor cells (MDSCs), have emerged 5 as biological mechanisms of T cell exclusion (Liu et al, Theranostics 2021).

[0127] KRAS mutations predominate in lung, pancreatic cancers (e.g., pancreatic adenocarcinoma or pancreatic ductal adenocarcinoma) and colon cancers and dictate the immunosuppressive tumor microenvironments (TME) in these tumors (Gu et al., Cancers 2021). Oncogenic KRAS mutations mediate autocrine effects and crosstalk with the TME by inducing several inflammatory cytokines, 10 chemokines and signaling pathways that promote carcinogenesis and resistance to immunotherapies (Hamarsheh et al, Nat. Commun.2020).

[0128] The combination therapy described in this disclosure works synergistically to treat lung cancer, including challenging cases of immune refractory lung cancer. While single-agent RAS(ON) inhibitor therapy favorably modulates the tumor microenvironment as does a single-agent RAS(ON) inhibitor plus 15 immune checkpoint inhibitor doublet combination, the combination of a RAS(ON) G12C-selective inhibitor with a RAS(ON) multi-selective inhibitor has been found to synergize effectively with an immune checkpoint inhibitor in treating immune refractory lung cancer. Without wishing to be bound by theory, it is believed that this combination increases tumor cell MHC class I expression, enhancing antigen presentation and recognition by T cells. In some embodiments, the RAS(ON) inhibitor therapy described 20 herein may sensitize a subject to subsequent immunotherapy, such as checkpoint inhibitor therapy.

[0129] Accordingly, provided herein are compositions comprising a RAS(ON) multi-selective inhibitor. (e.g., Compound A, AB-23400, BBP-454, BI-2852, Compound C (RMC-7977), RM-034, GFH547, ERAS- 0015 and compound 6A of WO 2024 / 067857). Exemplary RAS(ON) multi-selective inhibitors useful in combinations according to the present disclosure include but are not limited to daraxonrasib (Compound 25 A or RMC-6236), AN9025, BPI-572270, compound 6A of WO 2024 / 067857, ERAS-0015, GFH276, GFH547, HJ-099, Compound C (RMC-7977), RCZY-680, or RCZY-690. One or more RAS(ON) multi- selective inhibitors useful according to the present disclosure can be found in any of the following patent applications: WO 2025162395, WO 2025119392, WO2025087431, WO 2025051241, WO 2025045233, WO 2024249299, WO 2024222864, WO 2024206858, WO 2024169914, WO 2024153208, WO

[0130] 30 2024149214, WO 2024104364, WO 2024067857, WO 2024060966, WO 2024017859, WO 2024008834, WO 2023240263, WO 2023025832, WO 2022060836, WO 2021091956, CN 120441594, CN 119350371, CN 117903169, CN 117720556, CN 117720555, CN 117720554, CN 117534687, CN 117534685, CN 117534684, each of which is incorporated herein by reference in its entirety, including the compound structures disclosed therein.

[0131] 35 In some embodiments, the RAS(ON) multi-selective inhibitor is Compound A (daraxonrasib or RMC-6236)

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[0134]

[0135] .

[0136] In some embodiments, the RAS(ON) multi-selective inhibitor is Compound C (RMC-7977)

[0137] 5

[0138]

[0139] .

[0140] In some embodiments, the RAS(ON) multi-selective inhibitor is RM-034.

[0141] The RAS(ON) multi-selective compounds useful according to the present disclosure exhibit inhibitory activities across a variety of RAS mutants. In some embodiments, a RAS(ON) multi-selective compound inhibits wild type RAS. In some embodiments, a RAS(ON) multi-selective compound inhibits 10 wild type KRAS. In some embodiments, a RAS(ON) multi-selective compound inhibits a RAS mutant with one or more mutations at G12X, G13X, and / or Q61X, wherein X represents any naturally occurring amino acid residue. In certain instances, X is A, C, D, V, S, R, H, K, or L amino acid residue.

[0142] In certain embodiments, a RAS(ON) multi-selective compound inhibits a RAS mutant with one or more mutations at G12X, wherein X represents any naturally occurring amino acid residue. In certain 15 instances, X is A, C, D, V, S or R amino acid residue.

[0143] In other embodiments, a RAS(ON) multi-selective compound inhibits a RAS mutant with one or more mutations at G13X, wherein X is any naturally occurring amino acid residue. In certain instances, X is A, C, D, V, S or R amino acid residue.

[0144] In other embodiments, a RAS(ON) multi-selective compound inhibits a RAS mutant with one or 20 more mutations at Q61X, wherein X is any naturally occurring amino acid residue. In certain instances, X is A, C, D, V, S, R, H, K, or L amino acid residue. In other instances, X is H, K, R, or L amino acid residue.

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[0147] A variety of RAS proteins may be inhibited by a RAS(ON) multi-selective compound (e.g., KRAS, NRAS, HRAS, and mutants thereof at positions 12, 13 and 61, such as G12A, G12C, G12D, G12V, G12S, G12R, G13C, G13D, Q61H, Q61K, Q61R and Q61L, and others described herein, or a combination thereof). In some embodiments, a RAS(ON) multi-selective compound inhibits a G12A, 5 G12C, G12D, G12R, G12S, G12V, or Q61H mutant of RAS, or a combination thereof.

[0148] A RAS(ON) multi-selective inhibitor, such as Compound A, may exist as a conformational stereoisomer, such as an atropisomer. Pharmaceutically acceptable salts of Compound A are also contemplated, as are solvates, hydrates and polymorphs. See, e.g., WO 2022060836, incorporated herein by reference in its entirety. Compound A can be prepared as generally described in WO

[0149] 10 2021091956 or as specifically described in WO 2022060836, each incorporated herein by reference in its entirety.

[0150] A RAS(ON) multi-selective inhibitor, such as Compound A can be present as a pharmaceutically acceptable isotopically labeled version, wherein one or more atoms is replaced by atoms having the same atomic number, but an atomic mass or mass number different from the atomic mass or mass 15 number usually found in nature. Examples of isotopes that can be incorporated into Compound A include isotopes of hydrogen, carbon, nitrogen, oxygen, and fluorine, such as2H,3H,11C,13C,14C,13N,15N,15O,17O, and18O, respectively. These radio-labeled compounds could be useful to help determine or measure the effectiveness of Compound A, by characterizing, for example, the site or mode of action. Certain isotopically labeled versions of Compound A, for example, those incorporating a radioactive isotope, are 20 useful in drug and / or substrate tissue distribution studies. The radioactive isotopes tritium, i.e.,3H, and carbon-14, i.e.,14C, are particularly useful for this purpose in view of their ease of incorporation and ready means of detection. Non-limiting examples of such incorporation can be seen in, e.g., WO 2022060836.

[0151] Substitution with heavier isotopes such as deuterium, i.e.,2H, may afford certain therapeutic advantages resulting from greater metabolic stability, for example, increased in vivo half-life or reduced 25 dosage requirements. Substitution with positron emitting isotopes, such as11C,15O and13N, can be useful in Positron Emission Topography (PET) studies.

[0152] Further, provided herein are compositions comprising a RAS(ON) G12C-selective inhibitor (e.g., Compound B). Exemplary RAS(ON) G12C-selective inhibitors useful in combinations according to the present disclosure can be found in any one of the following patent applications: WO 2024249299, WO 30 2024211663, WO 2024008610, WO 2024102421, WO 2023240263, WO 2023133543, WO 2023015559, WO 2023086341, WO 2023208005, WO 2023232776, WO 2023060253, WO 2022235870, WO 2022235864, WO 2021091967, WO 2021091982, WO 2021108683, WO 2020132597, International Patent Application Numbers PCT / US2024 / 023208, and PCT / US2024 / 30993, each of which is incorporated herein by reference in its entirety, including the compound structures disclosed therein. 35 In some embodiments, the RAS(ON) G12C-selective inhibitor is Compound D (RMC-4998)

[0153]

[0154] .

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[0157] In some embodiments, the RAS(ON) G12C-selective inhibitor is a Compound B

[0158]

[0159] (also known as RMC-6291 or RM-046, Schulze et. al., Science. 2023 Aug 18; 381(6659): 794–799).

[0160] A RAS(ON) G12C-selective inhibitor, such as a Compound B, may exist as a conformational 5 stereoisomer, such as an atropisomer. Pharmaceutically acceptable salts of Compound B are also contemplated, as are solvates, hydrates and polymorphs. See, e.g., WO 2024216016 and WO 2021091982, incorporated herein by reference in their entirety. Compound B can be prepared as described in WO 2021091982 and WO 2022235864, each incorporated herein by reference in its entirety.

[0161] A RAS(ON) G12C-selective inhibitor, such as Compound B, can be present as a

[0162] 10 pharmaceutically acceptable isotopically labeled version, wherein one or more atoms is replaced by atoms having the same atomic number, but an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes that can be incorporated into Compound B include isotopes of hydrogen, carbon, nitrogen, oxygen, and fluorine, such as2H,3H,11C,13C,14C,13N,15N,15O,17O, and18O, respectively. These radio-labeled compounds could be useful to help 15 determine or measure the effectiveness of Compound B, by characterizing, for example, the site or mode of action. Certain isotopically labeled versions of Compound B, for example, those incorporating a radioactive isotope, are useful in drug and / or substrate tissue distribution studies. The radioactive isotopes tritium, i.e.,3H, and carbon-14, i.e.,14C, are particularly useful for this purpose in view of their ease of incorporation and ready means of detection.

[0163] 20 Substitution with heavier isotopes such as deuterium, i.e.,2H, may afford certain therapeutic advantages resulting from greater metabolic stability, for example, increased in vivo half-life or reduced dosage requirements. Substitution with positron emitting isotopes, such as11C,15O and13N, can be useful in Positron Emission Topography (PET) studies.

[0164] The RAS(ON) inhibitor compounds described herein may be made from commercially available 25 starting materials or synthesized using known organic, inorganic, or enzymatic processes. By way of example, the RAS(ON) compounds can be synthesized using the methods described in WO 2022060836, WO 2021091956, or WO 2021091982, or any of the other RAS(ON) references cited herein, together with synthetic methods known in the art of synthetic organic chemistry, or variations thereon as appreciated by those skilled in the art.

[0165] 30 In some embodiments, the RAS(ON) inhibitor therapy for use in the combination therapy according to the present disclosure comprises, consists essentially of, or consists of Compound A and Compound B. In some embodiments, the composition and uses thereof comprise a total daily dose of 100 mg to 300 mg of Compound A and a total daily dose of 200 mg to 400 mg of Compound B. In some embodiments, Compound A is administered once daily (QD), and Compound B is administered twice 35 daily (BID). In some embodiments, Compound A is administered in a total daily dose of 100 mg and compound B is administered in a total daily dose of 200 mg. In some embodiments, Compound A is administered in a total daily dose of 100 mg and Compound B is administered in a total daily dose of 300

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[0168] mg. In some embodiments, Compound A is administered in a total daily dose of 100 mg and Compound B is administered in a total daily dose of 400 mg. In some embodiments, Compound A is administered in a total daily dose of 200 mg and Compound B is administered in a total daily dose of 200 mg. In some embodiments, Compound A is administered in a total daily dose of 200 mg and Compound B is

[0169] 5 administered in a total daily dose of 300 mg. In some embodiments, Compound A is administered in a total daily dose of 200 mg and Compound B is administered in a total daily dose of 400 mg. In some embodiments, Compound A is administered in a total daily dose of 300 mg and Compound B is administered in a total daily dose of 200 mg. In some embodiments, Compound A is administered in a total daily dose of 300 mg and compound B in a total daily dose of 300 mg. In some embodiments, 10 Compound A is administered in a total daily dose of 300 mg and Compound B is administered in a total daily dose of 400 mg.

[0170] (b) Immune Checkpoint Inhibitors

[0171] Compositions and uses thereof described herein may include an immune checkpoint inhibitor 15 (ICI). An immune checkpoint inhibitor may be administered or formulated in combination with a RAS(ON) inhibitor therapy as described herein.

[0172] Immune checkpoints refer to a plethora of inhibitory pathways hardwired into the immune system, which, under normal physiological conditions are crucial for maintaining self-tolerance and modulating the duration and amplitude of physiological immune responses in peripheral tissues to minimize collateral 20 tissue damage in response to pathogenic infection. However, the expression of immune checkpoint proteins is often dysregulated by tumors as an important immune resistance and escape mechanism.

[0173] Because many of the immune checkpoints are initiated by ligand-receptor interactions, they can be readily blocked by antibodies or modulated by recombinant forms of ligands or receptors. Thus, inhibition of these pathways has been used to activate therapeutic anti-tumor immunity. For example, 25 cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) antibodies were the first of this class of immunotherapeutics to achieve US Food and Drug Administration (FDA) approval. Preliminary clinical findings with inhibitors of additional immune-checkpoint proteins, such as programmed cell death protein 1 (PD-1), indicate broad and diverse opportunities to enhance anti-tumor immunity with the potential to produce durable clinical responses.

[0174] 30 T cell activation through blockade of immune checkpoints has been a major focus of efforts to therapeutically manipulate endogenous anti-tumor immunity, owing to the capacity of T cells for the selective recognition of peptides derived from proteins in all cellular compartments; their capacity to directly recognize and kill antigen-expressing cells (by CD8+ effector T cells; also known as cytotoxic T lymphocytes (CTLs)); and their ability to orchestrate diverse immune responses (by CD4+ helper T cells), 35 which integrate adaptive and innate effector mechanisms. Thus, agonists of co-stimulatory receptors or antagonists of inhibitory signals, both of which result in the amplification of antigen-specific T cell responses, are currently agents of interest in clinical testing.

[0175] Table 1. Non-limiting list of immune checkpoint targets.

[0176]

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[0179]

[0180] CTLA4, cytotoxic T-lymphocyte-associated antigen 4; LAG3, lymphocyte activation gene 3; PD-1, programmed cell death protein 1; PD-L1, PD-1 ligand; TIM3, T cell membrane protein 3; VISTA, V- domain immunoglobulin (Ig)-containing suppressor of T-cell activation; KIR, killer lgG-like receptor.

[0181] 5 ICIs approved or in development include, but are not limited to, YERVOY® (ipilimumab), OPDIVO® (nivolumab), KEYTRUDA® (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 numerous other antibodies or fusion proteins directed to the 10 immune checkpoint proteins noted in Table 3. Common immune checkpoint proteins that may be targeted by ICIs include, but are not limited to B7.1, B7-H3, LAG3, CD137, KIR, CCR4, CD27, OX40, GITR, CD40, CTLA4, PD-1, and PD-L1. In some embodiments, the immune checkpoint inhibitor is an inhibitor of a target selected from the group comprising or consisting of programmed cell death protein- 1, ligand of PD-1, cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), T cell immunoglobulin and mucin-domain 15 containing-3 (TIM-3), V-domain Ig suppressor of T cell activation (VISTA), lymphocyte-activation gene 3 (LAG-3), T cell immunoglobulin and ITIM domain (TIGIT), B7 homolog 3 protein (B7-H3), B- and T- lymphocyte attenuator (BTLA), Sialic acid binding Ig-like lectin 15 (Siglec-15), cytokine-inducible SH2- containing protein (CISH), and combination thereof.

[0182] In some embodiments, the ICI therapy is selected from one or more of anti-PD-1, anti-PD-L1, 20 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, the ICI therapy is an antibody (e.g., a monoclonal antibody selective for any of the targets in Table 1). In some embodiments the ICI is an anti-PD-1 antibody. The antibody may be, e.g., humanized or fully human. In some embodiments, the checkpoint inhibitor is a fusion protein, e.g., an Fc-receptor fusion protein. In 25 some embodiments, the checkpoint inhibitor is an agent, such as an antibody, which interacts with a checkpoint protein. In some embodiments, the checkpoint inhibitor is an agent, such as an antibody,

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[0185] which interacts with the ligand of a checkpoint protein. In some embodiments, the checkpoint inhibitor is an inhibitor (e.g., an inhibitory antibody or small molecule inhibitor) of CTLA-4 (e.g., an anti-CTLA-4 antibody or fusion a protein). In some embodiments, the checkpoint inhibitor is an inhibitor or antagonist (e.g., an inhibitory antibody or small molecule inhibitor) of PD-1. In some embodiments, the checkpoint 5 inhibitor is an inhibitor or antagonist (e.g., an inhibitory antibody or small molecule inhibitor) of PD-L1. In some embodiments, the checkpoint inhibitor is an inhibitor or antagonist (e.g., an inhibitory antibody or Fc fusion or small molecule inhibitor) of PD-L2 (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, 10 CHK2, A2aR, B-7 family ligands, or a combination thereof. In some embodiments, the checkpoint inhibitor is pembrolizumab, nivolumab, PDR001 (NVS), REGN2810 (Sanofi / Regeneron), a PD-L1 antibody such as, e.g., avelumab, durvalumab, atezolizumab, pidilizumab, JNJ-63723283 (JNJ), BGB- A317 (also known as tislelizumab; BeiGene & Celgene) or a checkpoint inhibitor disclosed in Preusser, M. et al. (2015) Nat. Rev. Neurol., including, without limitation, ipilimumab, tremelimumab, nivolumab, 15 pembrolizumab, AMP224, AMP514 / MEDI0680, BMS936559, MEDl4736, MPDL3280A, MSB0010718C, BMS986016, IMP321, lirilumab, IPH2101, 1-7F9, and KW-6002.

[0186] In some embodiments, the immune checkpoint inhibitor is an inhibitor of programmed cell death protein- 1 (PD-1) or an inhibitor of the ligand of PD-1 (PDL-1).

[0187] Programmed cell death protein-1 is herein interchangeably referred to as PD-1, PD1, PDCD1, 20 PDCD-1, SLEB2, SLE1 and CD279.

[0188] In humans, PD-1 typically has the sequence as disclosed in UniProtKB Ref. Q15116, incorporated herein by reference.

[0189] Programmed death-ligand 1 is herein interchangeably referred to as PDL-1, PD-Ll, PDL1, PDCD1L1, PDCD1LG1, CD274, B7-H1, B7-H, B7H1.

[0190] 25 In humans, PD-L1 typically has the sequence as disclosed in UniProtKB Ref. Q9NZQ7, incorporated herein by reference.

[0191] In some embodiments, the anti-PD1 antibody is cemiplimab, nivolumab, pembrolizumab, pidilizumab, spartalizumab, camrelizumab, sintilimab, tislelizumab, toripalimab, dostarlimab, sasanlimab, retifanlimab, tebotelimab, ABBV-181, AK104, AK105, BCD-100, BI-754091, CBT-501, CC-90006, GLS-30 010, HLX10, IBI-308, JNJ-3283, JS001, LZM009, MEDI0680 (AMP-514), REGN-2810, SHR-1210, Sym021, TSR-042, or XmAb20717.

[0192] In some embodiments, the PD-1 inhibitor is a bispecific antibody specific for PD-1 and VEGF. In some embodiment, the bispecific antibody is ivonescimab (SMT112).

[0193] In some embodiments, the anti-PDL1 antibody is atezolizumab, avelumab, durvalumab,

[0194] 35 envafolimab, FS118, BCD-135, BGB-A333, BGBA-317, CBT-502, CK-301, CS1001, FAZ053, MDX-1105, MSB2311, SHR-1316, M7824, LY3415244, CA-170, or CX-07Z.

[0195] In some embodiments, the methods include administering RAS(ON) inhibitor combinations described herein (including the amounts described herein) and a PD-1 / PD-L 1 inhibitor, wherein the methods include simultaneous, separate or sequential administration. In one embodiment, the PD-1 / PD-40 L1 inhibitor is administered prior to administration of the RAS(ON) inhibitor combinations. In another embodiment, the PD-1 / PDL1 inhibitor or a pharmaceutical composition thereof is administered after

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[0198] administration of the RAS(ON) inhibitor combination. In another embodiment, the PD-1 / PD-L1 inhibitor, or a pharmaceutical composition thereof, is administered at about the same time as administration of the RAS(ON) inhibitor combination. In one embodiment, the PD-1 / PD-L1 inhibitor or a pharmaceutical composition thereof and the RAS(ON) inhibitor combination, can be formulated into separate or individual 5 dosage forms which can be co-administered simultaneously or one after the other.

[0199] Separate administration of each inhibitor, at different times and by different routes, in some cases would be advantageous. Thus, the components of the combination, i.e. RAS(ON) multi-selective inhibitor, RAS(ON) G12C-selective inhibitor and the PD-1 / PD-L1 inhibitor or a pharmaceutical composition thereof, need not be necessarily administered at essentially the same time or in any order.

[0200] 10 In one embodiment, a single dose of the PD-1 / PD-L1 inhibitor is administered. In one embodiment, the PD-1 / PD-L1 inhibitor is administered once every two weeks. In one embodiment, the PD-1 / PD-L1 inhibitor is administered once every three weeks. In one embodiment, the PD-1 / PD-L1 inhibitor is administered once every four weeks. In one embodiment, the PD-1 / PD-L1 inhibitor is nivolumab and is administered once every two weeks. In one embodiment, the PD-1 / PD-L1 inhibitor is 15 nivolumab and is administered once every four weeks. In one embodiment, the PD-1 / PD-L1 inhibitor is pembrolizumab and is administered once every three weeks. In one embodiment, the PD-1 / PD-L1 inhibitor is atezolizumab and is administered once every three weeks. In one embodiment, the PD-1 / PD- L1 inhibitor is cemiplimab and is administered once every three weeks. In one embodiment, the PD-1 / PD- L1 inhibitor is tislelizumab and is administered once every three weeks. In one embodiment, the PD-20 1 / PD-L1 inhibitor is avelumab and is administered once every two weeks. In one embodiment, the PD- 1 / PD-L1 inhibitor is durvalumab and is administered once every two weeks. In one of any of said embodiments, the PD-1 / PD-L1 inhibitor is administered intravenously.

[0201] (c) Pharmaceutical Compositions

[0202] 25 The disclosure provides pharmaceutical compositions including one or more RAS(ON) inhibitor compounds described herein, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient. In various embodiments, pharmaceutical compositions according to the disclosure may include immune check point inhibitors described herein.

[0203] In some embodiments, an active agent (e.g., a RAS(ON) inhibitor compound and / or immune 30 checkpoint inhibitor) is present in a pharmaceutical composition in unit dose amount appropriate for administration in a therapeutic regimen that shows a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population. In some embodiments, pharmaceutical compositions may be specially formulated for administration in solid or liquid form, including those adapted for the following: oral administration, for example, drenches (aqueous or 35 non-aqueous solutions or suspensions), tablets, e.g., those targeted for buccal, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue; parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation; topical application, for example, as a cream, ointment, or a controlled-release patch or spray applied to the skin, lungs, or oral cavity; intravaginally or 40 intrarectally, for example, as a pessary, cream, or foam; sublingually; ocularly; transdermally; or nasally, pulmonary, and to other mucosal surfaces.

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[0206] Active agents described herein, whether expressly stated or not, may be provided or utilized in salt form, e.g., a pharmaceutically acceptable salt form, unless expressly stated to the contrary.

[0207] An active agent of the disclosure may have ionizable groups so as to be capable of preparation as pharmaceutically acceptable salts. These salts may be acid addition salts involving inorganic or 5 organic acids or the salts may, in the case of acidic forms of the compounds of the disclosure, be prepared from inorganic or organic bases. In some embodiments, the compounds are prepared or used as pharmaceutically acceptable salts prepared as addition products of pharmaceutically acceptable acids or bases. Suitable pharmaceutically acceptable acids and bases are well-known in the art, such as hydrochloric, sulfuric, hydrobromic, acetic, lactic, citric, or tartaric acids for forming acid addition salts, and 10 potassium hydroxide, sodium hydroxide, ammonium hydroxide, caffeine, various amines, and the like for forming basic salts. Methods for preparation of the appropriate salts are well-established in the art.

[0208] Representative acid addition salts include acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate, 15 glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide, hydrochloride, hydroiodide,

[0209] 2-optionally substituted hydroxyl-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate, undecanoate, valerate salts and the 20 like. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like, as well as nontoxic ammonium, quaternary ammonium, and amine cations, including, but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine and the like.

[0210] For use as treatment of subjects, an active agent of the disclosure, or a pharmaceutically 25 acceptable salt thereof, can be formulated as pharmaceutical or veterinary compositions. Depending on the subject to be treated, the mode of administration, and the type of treatment desired, e.g., prevention, prophylaxis, or therapy, the compounds, or a pharmaceutically acceptable salt thereof, are formulated in ways consonant with these parameters. A summary of such techniques may be found in Remington: The Science and Practice of Pharmacy, 21stEdition, Lippincott Williams & Wilkins, (2005); and Encyclopedia 30 of Pharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan, 1988-1999, Marcel Dekker, New York, each of which is incorporated herein by reference.

[0211] Compositions can be prepared according to conventional mixing, granulating, or coating methods, respectively, and the present pharmaceutical compositions can contain from about 0.1% to about 99%, from about 5% to about 90%, or from about 1% to about 20% of a compound of the present 35 disclosure, or pharmaceutically acceptable salt thereof, by weight or volume. In some embodiments, compounds, or a pharmaceutically acceptable salt thereof, described herein may be present in amounts totaling 1-95% by weight of the total weight of a composition, such as a pharmaceutical composition.

[0212] The composition may be provided in a dosage form that is suitable for intraarticular, oral, parenteral (e.g., intravenous, intramuscular), rectal, cutaneous, subcutaneous, topical, transdermal, 40 sublingual, nasal, vaginal, intravesicular, intraurethral, intrathecal, epidural, aural, or ocular administration, or by injection, inhalation, or direct contact with the nasal, genitourinary, reproductive, or

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[0215] oral mucosa. Thus, the pharmaceutical composition may be in the form of, e.g., tablets, capsules, pills, powders, granulates, suspensions, emulsions, solutions, gels including hydrogels, pastes, ointments, creams, plasters, drenches, osmotic delivery devices, suppositories, enemas, injectables, implants, sprays, preparations suitable for iontophoretic delivery, or aerosols. The compositions may be formulated 5 according to conventional pharmaceutical practice.

[0216] Formulations may be prepared in a manner suitable for systemic administration or topical or local administration. Systemic formulations include those designed for injection (e.g., intramuscular, intravenous, or subcutaneous injection) or may be prepared for transdermal, transmucosal, or oral administration. A formulation will generally include a diluent as well as, in some cases, adjuvants, buffers, 10 preservatives and the like. Compounds, or a pharmaceutically acceptable salt thereof, can be administered also in liposomal compositions or as microemulsions.

[0217] For injection, formulations can be prepared in conventional forms as liquid solutions or suspensions or as solid forms suitable for solution or suspension in liquid prior to injection or as emulsions. Suitable excipients include, for example, water, saline, dextrose, glycerol, and the like. Such 15 compositions may also contain amounts of nontoxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like, such as, for example, sodium acetate, sorbitan monolaurate, and so forth.

[0218] Various sustained release systems for drugs have also been devised. See, for example, U.S. Patent No.5,624,677.

[0219] 20 Systemic administration may also include relatively noninvasive methods such as the use of suppositories, transdermal patches, transmucosal delivery and intranasal administration. Oral administration is also suitable for compounds of the disclosure, or a pharmaceutically acceptable salt thereof. Suitable forms include syrups, capsules, and tablets, as is understood in the art.

[0220] Each compound, or a pharmaceutically acceptable salt thereof, as described herein, may be 25 formulated in a variety of ways that are known in the art. For example, the first and second agents of the combination therapy may be formulated together or separately. Other modalities of combination therapy are described herein.

[0221] The individually or separately formulated agents can be packaged together as a kit. Non-limiting examples include, but are not limited to, kits that contain, e.g., two pills, a pill and a powder, a

[0222] 30 suppository, and a liquid in a vial, two topical creams, etc. The kit can include optional components that aid in the administration of the unit dose to subjects, such as vials for reconstituting powder forms, syringes for injection, customized IV delivery systems, inhalers, etc. Additionally, the unit dose kit can contain instructions for preparation and administration of the compositions. The kit may be manufactured as a single use unit dose for one subject, multiple uses for a particular subject (at a constant dose or in 35 which the individual compounds, or a pharmaceutically acceptable salt thereof, may vary in potency as therapy progresses); or the kit may contain multiple doses suitable for administration to multiple subjects (“bulk packaging”). The kit components may be assembled in cartons, blister packs, bottles, tubes, and the like.

[0223] Formulations for oral use include tablets containing the active ingredient(s) in a mixture with 40 non-toxic pharmaceutically acceptable excipients. These excipients may be, for example, inert diluents or fillers (e.g., sucrose, sorbitol, sugar, mannitol, microcrystalline cellulose, starches including potato starch,

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[0226] calcium carbonate, sodium chloride, lactose, calcium phosphate, calcium sulfate, or sodium phosphate); granulating and disintegrating agents (e.g., cellulose derivatives including microcrystalline cellulose, starches including potato starch, croscarmellose sodium, alginates, or alginic acid); binding agents (e.g., sucrose, glucose, sorbitol, acacia, alginic acid, sodium alginate, gelatin, starch, pregelatinized starch, 5 microcrystalline cellulose, magnesium aluminum silicate, carboxymethylcellulose sodium, methylcellulose, optionally substituted hydroxylpropyl methylcellulose, ethylcellulose, polyvinylpyrrolidone, or polyethylene glycol); and lubricating agents, glidants, and antiadhesives (e.g., magnesium stearate, zinc stearate, stearic acid, silicas, hydrogenated vegetable oils, or talc). Other pharmaceutically acceptable excipients can be colorants, flavoring agents, plasticizers, humectants, buffering agents, and the like.

[0227] 10 Two or more compounds may be mixed together in a tablet, capsule, or other vehicle, or may be partitioned. In one example, the first compound is contained on the inside of the tablet, and the second compound is on the outside, such that a substantial portion of the second compound is released prior to the release of the first compound.

[0228] Formulations for oral use may also be provided as chewable tablets, or as hard gelatin capsules 15 wherein the active ingredient is mixed with an inert solid diluent (e.g., potato starch, lactose, microcrystalline cellulose, calcium carbonate, calcium phosphate or kaolin), or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example, peanut oil, liquid paraffin, or olive oil. Powders, granulates, and pellets may be prepared using the ingredients mentioned above under tablets and capsules in a conventional manner using, e.g., a mixer, a fluid bed apparatus or a spray 20 drying equipment.

[0229] Dissolution or diffusion-controlled release can be achieved by appropriate coating of a tablet, capsule, pellet, or granulate formulation of compounds, or by incorporating the compound, or a pharmaceutically acceptable salt thereof, into an appropriate matrix. A controlled release coating may include one or more of the coating substances mentioned above or, e.g., shellac, beeswax, glycowax, 25 castor wax, carnauba wax, stearyl alcohol, glyceryl monostearate, glyceryl distearate, glycerol palmitostearate, ethylcellulose, acrylic resins, dl-polylactic acid, cellulose acetate butyrate, polyvinyl chloride, polyvinyl acetate, vinyl pyrrolidone, polyethylene, polymethacrylate, methylmethacrylate, 2-optionally substituted hydroxylmethacrylate, methacrylate hydrogels, 1,3 butylene glycol, ethylene glycol methacrylate, or polyethylene glycols. In a controlled release matrix formulation, the matrix material 30 may also include, e.g., hydrated methylcellulose, carnauba wax and stearyl alcohol, carbopol 934, silicone, glyceryl tristearate, methyl acrylate-methyl methacrylate, polyvinyl chloride, polyethylene, or halogenated fluorocarbon.

[0230] The liquid forms in which the compounds, or a pharmaceutically acceptable salt thereof, and compositions of the present disclosure can be incorporated for administration orally include aqueous 35 solutions, suitably flavored syrups, aqueous or oil suspensions, and flavored emulsions with edible oils such as cottonseed oil, sesame oil, coconut oil, or peanut oil, as well as elixirs and similar pharmaceutical vehicles.

[0231] Generally, when administered to a human, the oral dosage of any of the compounds of the disclosure, or a pharmaceutically acceptable salt thereof, will depend on the nature of the compound, and 40 can readily be determined by one skilled in the art. A dosage may be, for example, about 0.001 mg to about 2000 mg per day, about 1 mg to about 1000 mg per day, about 5 mg to about 500 mg per day,

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[0234] about 100 mg to about 1500 mg per day, about 500 mg to about 1500 mg per day, about 500 mg to about 2000 mg per day, or any range derivable therein.

[0235] In some embodiments, the pharmaceutical composition may further include an additional compound having antiproliferative (e.g., anti-cancer) activity. Depending on the mode of administration, 5 compounds, or a pharmaceutically acceptable salt thereof, will be formulated into suitable compositions to permit facile delivery. Each compound, or a pharmaceutically acceptable salt thereof, of a combination therapy may be formulated in a variety of ways that are known in the art. For example, the first and second agents of the combination therapy may be formulated together or separately. Desirably, the first and second agents are formulated together for the simultaneous or near simultaneous administration of 10 the agents.

[0236] It will be appreciated that the compounds and pharmaceutical compositions of the present disclosure can be formulated and employed in combination therapies, that is, the compounds and pharmaceutical compositions can be formulated with or administered concurrently with, prior to, or subsequent to, one or more other desired therapeutics or medical procedures. The particular combination 15 of therapies (therapeutics or procedures) to employ in a combination regimen will take into account compatibility of the desired therapeutics or procedures and the desired therapeutic effect to be achieved. It will also be appreciated that the therapies employed may achieve a desired effect for the same disorder, or they may achieve different effects (e.g., control of any adverse effects).

[0237] Administration of each drug in a combination therapy, as described herein, can, independently, 20 be one to four times daily for one day to one year, and may even be for the life of the subject. Chronic, long-term administration may be indicated.

[0238] II. Methods of Use

[0239] In some embodiments, the disclosure provides a method of treating a subject having a lung 25 cancer (e.g., an immune refractory lung cancer), the method generally comprises, administering to the subject in need thereof a combination therapy comprising a RAS(ON) inhibitor therapy and an immune check point inhibitor. Compositions suitable for the combination therapy of the disclosure are described in section I and incorporated into the section by reference. In some embodiments, the subject is administered a RAS(ON) multi-selective inhibitor, a RAS(ON) G12C-selective inhibitor and an immune 30 check point inhibitor (e.g., PD-1 / PD-L1 inhibitor).

[0240] In some embodiments, the disclosure provides a method of sensitizing an immune refractory cancer to immunotherapy in a subject in need thereof, the method generally comprises, administering to the subject a combination therapy comprising a RAS(ON) inhibitor therapy and an immune check point inhibitor. In some embodiments, the subject is administered a RAS(ON) multi-selective inhibitor, a 35 RAS(ON) G12C-selective inhibitor and an immune check point inhibitor (e.g., PD-1 / PD-L1 inhibitor).

[0241] In some embodiments, the disclosure provides a method of boosting the efficacy of other cancer therapies when administered in combination to the subject in need thereof. In some embodiments, the subject is administered a combination therapy comprising a RAS(ON) inhibitor therapy and an immune check point inhibitor. In some embodiments, the subject is administered a RAS(ON) multi-selective 40 inhibitor, a RAS(ON) G12C-selective inhibitor and an immune check point inhibitor (e.g., PD-1 / PD-L1 inhibitor).

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[0244] In each of the above embodiments, the methods of the present disclosure can alter MHC class I expression resulting in an enhanced anti-tumor effect of the combination therapy of the disclosure. In each of the above embodiments, the methods of the present disclosure can alter the tumor immune infiltrate comprising of T-cells, B-cells, APCs, monocytes, MDSCs, TAMs, neutrophils, other monocyte- 5 derived cells, tumor-associated stroma, cancer stem cells, and mesenchymal stem cells and result in an enhanced anti-tumor therapeutic effect.

[0245] Identification of Tumor Types

[0246] In various embodiments, the disclosure provides a method of treating lung cancer in a subject in 10 need thereof comprising administering to the subject a combination therapy described herein, wherein the subject has one or more tumors that are characterized as immune refractory, immune evasive, immunologically protected, immunologically “cold,” microsatellite stable, microsatellite instability low, comprising a low immune infiltrate, comprising a low tumor mutational burden, or exhibiting heterogeneity.

[0247] In various embodiments, the disclosure provides a method for treating tumors (e.g., lung cancer) 15 that are characterized as immune evasive, immunologically protected, immunologically “cold,” microsatellite stable, microsatellite instability low, comprising a low immune infiltrate, comprising a low tumor mutational burden or exhibiting heterogeneity in a subject, comprising (i) diagnosing the subject as having an immune refractory, immune evasive tumor, immunologically protected tumor, immunologically “cold” tumor, microsatellite stable tumor, microsatellite instability low tumor, a tumor comprising a low 20 immune infiltrate, a tumor comprising a low tumor mutational burden or a tumor exhibiting heterogeneity, and (ii) administering a combination therapy described herein to the subject. In various embodiments, the diagnosing comprises assaying biomarkers / characteristics associated with tumors that are characterized as immune refractory, immune evasive, immunologically protected, immunologically “cold,” microsatellite stable, microsatellite instability low, comprising a low immune infiltrate, comprising a low tumor mutational 25 burden, or exhibiting heterogeneity. In various embodiments, the method further comprises (iii) determining if the subject's tumor becomes immune responsive, and then (iv) administering an immune checkpoint inhibitor, optionally in combination with the RAS(ON) inhibitor therapy described herein. In some embodiments, the subject is previously diagnosed as having a tumor characterized as immune evasive, immunologically protected, immunologically “cold,” microsatellite stable, microsatellite instability 30 low, comprising a low immune infiltrate, comprising a low tumor mutational burden, or exhibiting heterogeneity.

[0248] Also provided herein is a method for determining if a subject is likely to be responsive to treatment with a combination therapy described herein and treating the subject accordingly. In various embodiments, a patient diagnosed with cancer undergoes testing to identify the tumor as a cold tumor, 35 e.g., using methods described herein and others described in the art. The disclosure provides a method for treating a subject having a cancer, e.g., an immune refractory cancer, with a combination therapy described herein, the method comprising obtaining a tumor sample from a subject, conducting assays to determine if the tumor is a cold tumor, and treating the subject with a combination therapy described herein if the tumor is identified as a cold tumor. Assays to determine whether the tumor is a cold tumor 40 include, but are not limited to determining the expression level of MHC class I, tumor mutational burden analysis, microsatellite instability (MSI) testing, the degree of immune cell (e.g., CD4+T-cells, CD8+T- 27 PATENT

[0249] ATTORNEY DOCKET NO.: 51432-077WO2

[0250] cells, NK1.1+NK cells, APCs, monocytes, and neutrophils) infiltration into the tumor, immune cell phenotype (e.g., PD-1+, PD-L1+, and PD-L2+), immune cell function (e.g., expression of IFN-γ, IL-12, and IL-15), and ratio of pro-inflammatory and anti-inflammatory mediators in the tumor microenvironment (TME).

[0251] 5 Multiple diagnostic tools designed to characterize tumors at the cellular and molecular level are FDA-approved and commercially available. Examples of approved diagnostics include FOUNDATIONONE® CDX, FOUNDATIONONE® LIQUID, FOUNDATIONONE® HEME, BRACAnalysis CDx, therascreen EGFR RGQ PCR kit, cobase EGFR Mutation Test v2, PD-L1 IHC 22C3 pharmDx, Abbott RealTime IDH1, MRDx BCR-ABL test, VENTANA ALK (D5F3) CDx Assay, Abbott RealTime IDH2, 10 Praxis Extended RAS Panel, Oncomine Dx Target Test, LeukoStrat CDx FLT3 Mutation Assay, FoundationFocus CDxBRCA Assay, Vysis CLL FISH Probe Kit, KIT D816V Mutation Detection, PDGFRB FISH, cobas KRAS Mutation Test, therascreen KRAS RGQ PCR 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 CISH pharmDx Kit, INFORM HER2 DUAL ISH DNA Probe Cocktail,

[0252] 15 HercepTest, HER2 FISH pharmDx Kit, THXID BRAF Kit, Vysis ALK Break Apart 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.

[0253] In various embodiments, the subject is screened for eligibility for treatment with one or more immunotherapies described herein. In various embodiments, subjects that are not eligible for treatment 20 with such immunotherapies (e.g., are non-responsive to one or more immunotherapies or have a cancer characterized as non-responsive to one or more immunotherapies) may be treated with a combination therapy described herein. Non-limiting examples of immunotherapies include Pembrolizumab (KEYTRUDA®, Merck Sharp & Dohme Corp), Nivolumab (OPDIVO®, Bristol-Myers Squibb), Atezolizumab (TECENTRIQ®), Avelumab (BAVENCIO®), and Durvalumab (IMFINZI®). Eligibility criteria 25 for these immunotherapies are known in the art. For example, without limitation, pembrolizumab (KEYTRUDA®), nivolumab (OPDIVO®), and atezolizumab (TECENTRIQ®) have eligibility criteria based on PD-L1 expression levels. PD-L1 expression criteria and methods of measuring the same may be found at keytrudahcp.com / biomarker-testing / pd-11-expression-testing / (pembrolizumab; KEYTRUDA®), or the FDA-approved prescribing information for pembrolizumab (KEYTRUDA®, as revised 1 / 2020), 30 atezolizumab (e.g., TECENTRIQ®, as revised 5 / 2020), and nivolumab (e.g., OPDIVO®, as revised on 6 / 2020). Each of these publications are herein incorporated by reference in its entirety for all purposes. As described herein, treating such patients with a combination therapy described herein may work synergistically to treat the tumor that is not eligible for treatment with an immunotherapy to an immunogenic tumor. In various embodiments, the tumors of subjects that are not eligible for an

[0254] 35 immunotherapy can be monitored throughout the course of treatment with a combination therapy described herein.

[0255] In various embodiments, the disclosure provides a method of treating lung cancer in a subject in need thereof comprising administering to the subject a combination therapy described herein, wherein the subject has one or more tumors with a low immune infiltration. In various embodiments, the administering 40 to a subject with one or more lung tumors with a low immune infiltrate alters the tumor immune infiltrate.

[0256] In various embodiments, the tumor immune infiltrate comprises antigen-presenting cells, myeloid cells,

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[0259] and lymphoid cells. In various embodiments, antigen-presenting cells in the tumor immune infiltrate comprise macrophages or dendritic cells. In various embodiments, myeloid cells in the tumor immune infiltrate comprise monocytes, neutrophils, myeloid-derived suppressor cells (MDSCs), and tumor- associated macrophages (TAMs). In various embodiments, the TAMs in the tumor immune infiltrate 5 comprise M1 macrophages, M2 macrophages, and MARCO+macrophages. In various embodiments, lymphoid cells in the tumor immune infiltrate comprise T-cells, B-cells, NK T-cells, and NK cells.

[0260] Qualitative and quantitative methods have been described for the characterization of the tumor immune infiltrate, including but not limited, microscopic analyses, histological assays, cytological assays, flow cytometry, polymerase chain reaction (PCR), quantitative polymerase chain reaction (qPCR), RNA 10 sequencing (RNA-seq), single-cell RNA sequencing (scRNA-seq), next-generation sequencing, whole- exome sequencing, epigenetic sequencing, ATAC-seq, microarray analysis, and mass cytometry or CyTOF. Biomarkers can be used, alone or in combination, for the evaluation of immune cells and include cell surface markers and secreted proteins. Exemplary biomarkers for the characterization of the tumor immune infiltrate include, but are not limited to, CD45, CD3, CD4, CD8, CD25, CD44, CD134, CD252, 15 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, 20 and RORγt.

[0261] Cancer stem cells (CSCs) have been described as a subset of cells found within solid and hematologic tumors that are tumorigenic, and capable of self-renewal, differentiation. Several reports have described the importance of CSCs in the pathogenesis of a variety of tumors, tumor relapse after therapy, and development of therapeutic resistance. A number of cell surface markers can be used to 25 distinguish CSCs within solid and hematologic tumors. CSC markers include, but are not limited to, CD19, CD20, CD24, CD34, CD38, CD44, CD90, CD133, Aldehyde dehydrogenase 1, CEACAM-6 / CD66c, BMl- 1, Connexin 43 / GJA1, DLL4, EpCAM / TROP1, GLl-1, GLl-2, Integrins, PON1, PTEN, ALCAM / CD166, DPPIV / CD26, Lgr5, Musashi-1, A20, ABCG2, CD15, Fractalkine, HIF-2α, L1CAM, c-MAF, Nestin, Podoplanin, SOX2, CD96, CD117, FLT3, AFP, CD13, CD90, NF2 / Merlin, ABCB5, NGFR, Syndecan-1, 30 Endoglin, STRO-1, and PON1.

[0262] In various embodiments, the disclosure provides a method of treating cancer in a subject in need thereof comprising administering to the subject a combination therapy described herein, wherein the subject has one or more immune refractory tumors. In various embodiments, the subject has one or more immunologically protected tumors. In various embodiments, the subject has one or more microsatellite 35 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 moderate microsatellite instability. In various embodiments, the subject has one or more tumors with a low tumor mutational burden. In various embodiments, the subject has one or more tumors with a moderate tumor mutational burden. In various embodiments, the subject has one or more tumors resistant to therapy. In various embodiments, the 40 subject has one or more immunologically heterogeneous tumors. In various embodiments, the subject has genetically heterogeneous tumors. In various embodiments, the subject has one or more refractory

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[0265] tumors. In one or more embodiments, the subject has a tumor that develops resistance during the course of treatment.

[0266] In various embodiments, the tumor characteristic is determined from one or more biological samples from a subject suffering from cancer. In various embodiments, the tumor characteristic is 5 determined by comparing one or more biological samples from a subject suffering from cancer to one or more biological samples from one or more healthy subjects. In various embodiments, the tumor characteristic is determined from one or more biological samples selected from the group consisting of blood, cerebrospinal fluid, urine, stool, buccal swab, nasal swab, lavage, tissue biopsy, bone marrow biopsy, and tumor biopsy. In various embodiments, the tumor characteristic is determined from the 10 analysis of cells, proteins, or nucleic acids in one or more biological samples from a subject suffering from cancer. In various embodiments, the tumor characteristic is determined by comparing the analysis of cells, proteins, or nucleic acids in one or more biological samples from a subject suffering from cancer to the analysis of one or more biological samples from one or more healthy subjects. In various embodiments, the tumor characteristic is determined by comparing the analysis of cells, proteins, or 15 nucleic acids in one or more biological samples from a subject suffering cancer to the analysis of one or more biological samples from one or more subjects suffering from cancer and responsive to treatment. In various embodiments, the cells are 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), 20 hematopoietic stem cells, and hematopoietic progenitor cells. In various embodiments, the proteins are selected from the group consisting of cytokines, chemokines, growth factors, signal transduction proteins, enzymes, proteases, and nucleases. In various embodiments, the 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, the nucleic acid analysis is performed by PCR, RT-PCR, qRT-25 PCR, next-generation sequencing (NGS), RNA-seq, ATAC-seq, exome sequencing, Southern Blot, microarray analysis, or single-cell sequencing.

[0267] In various embodiments, the tumor characteristic of a subject suffering from cancer is determined from the analysis of one or more blood samples collected from the subject. In various embodiments, the tumor characteristic of a subject suffering from cancer is determined from the analysis of cells, proteins, 30 or nucleic acids in one or more blood samples collected from the subject. In various embodiments, the tumor characteristic of a subject suffering from cancer is determined by comparing the analysis of cells, proteins, or nucleic acids in one or more blood samples from the subject suffering from cancer to 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, 35 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 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

[0268] 40 lymphoid cells are T cells, B cells, NK cells, NK-T cells, or iNK cells.

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[0271] In various embodiments, the analysis of cells from one or more blood samples collected from a subject suffering from cancer demonstrates increased levels of immune suppressive cells compared to the analysis of cells from one or more healthy subjects or one or more subjects suffering from cancer and responsive to treatment (e.g., responsive to an immunotherapy such as an immune checkpoint inhibitor).

[0272] 5 In various embodiments, the immune suppressive cells are myeloid derived suppressor cells (MDSCs), tumor-associated macrophages (TAMs), neutrophils, Treg cells, and Breg cells. In various embodiments, MDSCs are monocytic MDSCs (M-MDSCs) and polymorphonuclear MDSCs (PMN-MDSCs). In various embodiments, the TAMs are M2 TAMs. In various embodiments, the immune suppressive cells are CAFs. In various embodiments, the levels of immune suppressive cells in one or more blood samples of a 10 subject having one or more tumors characterized as immune refractory, immunologically protected, or immunologically “cold” are 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%, inclusive of all values and ranges between these values), 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 15 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 responsive to treatment. In various embodiments, the levels of immune suppressive cells in one or more blood samples of a subject having one or more tumors characterized as immune refractory, immunologically protected, or immunologically “cold” are increased by about 2-100 fold (e.g., about 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 20 70, 75, 80, 85, 90, 95, or 100 fold, inclusive of 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 responsive to treatment. In various embodiments, immune suppressive cells are identified by the assay of cell-surface proteins expression. In various embodiments, the analysis of cells from one or more blood samples collected from a subject having one or more tumors characterized as 25 immune refractory, immunologically protected, or immunologically “cold” demonstrates reduced levels or absence of activated pro-inflammatory immune cells (e.g., reduced relative to a healthy subject or a subject suffering from cancer and responsive to treatment 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%, 30 inclusive of all values and ranges between these values).

[0273] In various embodiments, the analysis of cells from one or more blood samples collected from a subject having one or more tumors characterized as immune refractory, immunologically protected, or immunologically “cold” demonstrates reduced levels or absence of activated pro-inflammatory immune cells (e.g., reduced relative to a healthy subject or a subject suffering from cancer and responsive to 35 treatment 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 inclusive of all values and ranges between these values). 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 iNK cells. In various embodiments, the frequency of pro-inflammatory immune cells is ≤10% (e.g., about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, or 40 about 1%) of all leukocytes analyzed from one or more blood samples collected from the subject. In

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[0276] various embodiments, activated pro-inflammatory immune cells are identified by the assay of cell-surface protein expression.

[0277] In various embodiments, the analysis of cells in one or more blood samples of a subject suffering from cancer is performed by the assay of cell-surface proteins. In various embodiments, the cell-surface 5 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, 10 CRACC, BLAME, BCMA, CD103, CD107, CD112, CD120a, CD120b, CD123, CD125, CD134, CD135, CD140a, CD141, CD154, CD155, CD160, CD163, CD172a, XCR1, CD203c, CD204, CD206, CD207 CD226, CD244, CD267, CD268, CD269, CD355, CD358, NKG2A, NKG2B, NKG2C, NKG2D, NKG2E, NKG2F, NKG2H, KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL5A, KIR2DL5B, KIR3DL1, KIR3DL2, KIR3DL3, KIR3DL4, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5, DAP12, KIR3DS, NKp44, NKp46, TCR, 15 BCR, Integrins, FcβεRI, MHC-I, MHC-II, IL-1R, IL-2Rα, IL-2Rβ, IL-2Rγ, IL-3Rα, CSF2RB, 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, OSMR, CSF-1R, cell-surface IL-15, IL-10Rα, IL-10Rβ, IL-20Rα, IL-20Rβ, IL-22Rα1, 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, LTβR, TIM-1, TIM-3, TIM-4, TIGIT, LAG-3, ICOS, ICOS-L, SLAM, 20 SLAMF2, OX-40, OX-40L, GITR, GITRL, TL1A, HVEM, 41-BB, 41BB-L, TL-1A, TRAF1, TRAF2, TRAF3, TRAF5, BAFF, BAFF-R, APRIL, TRAIL, RANK, AITR, TRAMP, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CCR11, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CXCR7, CLECL9a, DC-SIGN, IGSF4A, SIGLEC, EGFR, PDGFR, VEGFR, FAP, α-SMA, Vimentin, Laminin, FAS, FAS-L, Fc, ICAM-1, ICAM-2, ICAM-3, ICAM-4, ICAM-5, PECAM-1, MICA, MICB, UL16, 25 ULBP1, ULBP2, ILBP3, ULBP4, ULBP5, ULBP6, MULT1, RAE1 α, β, γ, δ, and ε, A1R, A2AR, A2BR, and A3R, H60a, H60b, and H60c. In various embodiments, Integrins are 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 combinations thereof. In various embodiments, TCR is selected from the group consisting of a, β, γ, δ, ε, and ζ TCR. Several methods have been described in the literature for assaying of cell-surface protein 30 expression, including Flow Cytometry and Mass Cytometry (CyTOF). The presence or abundance of one or more of these cell-surface proteins indicates that the patient is amendable to treatment with the methods disclosed herein.

[0278] In various embodiments, the analysis of cells from one or more blood samples collected from a subject having one or more tumors characterized as immune refractory, immunologically protected, or 35 immunologically “cold” demonstrates a high neutrophil to lymphocyte ratio (NLR). In various embodiments, the analysis of cells from one or more blood samples collected from a subject having one or more tumors characterized as immune refractory, immunologically protected, or immunologically “cold” demonstrates an NLR ≥ 2. In various embodiments, the analysis of cells from one or more blood samples collected from a subject having one or more tumors characterized as immune refractory, immunologically 40 protected, or immunologically “cold” demonstrates NLR of between 2 and 10 (e.g., NLR of 2, 3, 4, 5, 6, 7, 8, 9, and 10, inclusive of all values and ranges between these values). In various embodiments, NLR is

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[0281] used to determine the prognosis for a subject suffering from cancer and having one or more tumors characterized as immune refractory, immunologically protected, or immunologically “cold.” In various embodiments, NLR ≥ 2 determines a poor prognosis.

[0282] In various embodiments, the cells analyzed from one or more blood samples collected from a 5 subject suffering from cancer are circulating tumor cells (CTCs). In various embodiments, the assay of one or more blood samples collected from a subject suffering from cancer demonstrates increased frequency of CTCs compared to the analysis of one or more blood samples collected from one or more healthy subjects or one or more subjects suffering from cancer and responsive to treatment. In various embodiments, the frequency of circulating tumor cells in one or more blood samples of a subject having 10 one or more tumors characterized as immune refractory, immunologically protected, or immunologically “cold” is ≥ 3 or ≤ 5 CTCs per 7.5 ml blood.

[0283] In various embodiments, the tumor characteristic of a subject suffering from cancer is determined from the analysis of proteins in one or more blood samples of the subject. In various embodiments, the tumor characteristic of a subject suffering from cancer is determined by comparing the analysis of 15 proteins in one or more blood samples from the subject suffering from cancer to the analysis of one or more blood samples from one or more healthy subjects or one or more subjects suffering from cancer and responsive 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 cytokines, chemokines, growth factors, enzymes, proteases, and nucleases. In various embodiments, cytokines and 20 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, CCL12, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CXCL1, CXCL2 (MCP-1), CXCL3 25 (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 factors are 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 30 protein is a protease is selected from the group consisting of aspartic protease, a cysteine protease, a metalloprotease, a serine protease, or a threonine protease. In some embodiments, the protein is a protease is 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, 35 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, kynurinase, 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, proteins associated with apoptosis are 40 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,

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[0286] 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 have been described in the literature for assaying proteins from blood samples, including western blot, and ELISA.

[0287] In various embodiments, the analysis of proteins from one or more blood samples of a subject 5 having one or more tumors that are characterized as immune refractory, immunologically protected, or immunologically “cold” demonstrates increased levels of tumor promoting, anti-inflammatory, or immune suppressive proteins. In various embodiments, the tumor promoting, anti-inflammatory, or immune suppressive proteins are cell-surface proteins, intracellular proteins, or secreted proteins. In various embodiments, the tumor promoting, anti-inflammatory, or immune suppressive proteins are selected from 10 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, kyneurinase, indoleamine 2,3 dioxygenase (IDO1 and IDO2), tryptophan 2,3 dioxygenase (TDO), myeloperoxidase (MPO), neutrophil elastase (NE), and IL4I1. In 15 various embodiments, the levels of tumor promoting, anti-inflammatory, or immune suppressive proteins in one or more blood samples of a subject having one or more tumors that are characterized as immune refractory, immunologically protected, or immunologically “cold” are increased by 5-100% (e.g., increased 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%, 20 about 90%, about 95%, or about 100%, inclusive of 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 suffering from cancer and responsive to treatment. In various embodiments, the levels of tumor promoting, anti- inflammatory, or immune suppressive proteins in one or more blood samples of a subject having one or 25 more tumors that are characterized as immune refractory, immunologically protected, or immunologically “cold” are 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, inclusive of 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 responsive to treatment. In various embodiments, the analysis of proteins from one or more 30 blood samples of a subject having one or more tumors that are characterized as immune refractory, immunologically protected, or immunologically “cold” demonstrate reduced levels, low levels, or absence of tumor inhibiting, anti-tumor, or pro-inflammatory proteins. In various embodiments, tumor inhibiting, anti-tumor, or pro-inflammatory proteins 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-35 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, 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 levels of tumor inhibiting, anti-tumor, or pro- inflammatory proteins in one or more blood samples of a subject having one or more tumors that are characterized as immune refractory, immunologically protected, or immunologically “cold” are decreased 40 by 5-100% (e.g., reduced relative to a healthy subject or a subject suffering from cancer and responsive to treatment by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about

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[0290] 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%, inclusive of 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 5 subjects suffering from cancer and responsive to treatment. In various embodiments, the levels of tumor inhibiting, anti-tumor, or pro-inflammatory proteins in one or more blood samples of a subject having one or more tumors that are characterized as immune refractory, immunologically protected, or immunologically “cold” are decreased 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, inclusive of all values and ranges between these values) 10 compared to one or more blood samples collected from one or more healthy subjects or subjects suffering from cancer and responsive to treatment. Several methods have been described in the literature for assaying proteins from blood samples, including western blot, and ELISA.

[0291] In various embodiments, the analysis of one or more blood samples of a subject having one or more tumors that are characterized as immune refractory, immunologically protected, or immunologically 15 “cold” demonstrates increased levels of neutrophil extracellular traps (NETs). In various embodiments, the analysis of one or more blood samples of a subject having one or more tumors that are characterized as immune refractory, immunologically protected, or immunologically “cold” demonstrates increased levels of neutrophil extracellular traps (NETs) compared to the analysis of one or more blood samples from one or more healthy subjects or one or more subjects suffering from cancer and responsive to treatment. In 20 various embodiments, the levels of NETs in one or more blood samples of a subject having one or more tumors that are characterized as immune refractory, immunologically protected, or immunologically “cold” are increased by 5-100% (e.g., increased 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%, inclusive of all values 25 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 suffering from cancer and responsive to treatment. In various embodiments, the levels of NETs in one or more blood samples of a subject having one or more tumors that are characterized as immune refractory, immunologically protected, or immunologically “cold” are increased 30 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, inclusive of 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 responsive to treatment. Several methods have been described in the literature for assaying NETs from blood samples, including western blot, ELISA, and flow cytometry.

[0292] 35 In various embodiments, the tumor characteristic of a subject suffering from cancer is determined from the analysis of nucleic acids in one or more blood samples of the subject. In various embodiments, the tumor characteristic of a subject suffering from cancer is determined by comparing the analysis of nucleic acids in one or more blood samples from the subject suffering from cancer to the analysis of one or more blood samples from one or more healthy subjects or one or more subjects suffering from cancer 40 and responsive to treatment. In various embodiments, the nucleic acid is selected from the group comprising DNA, ssDNA, circulating tumor DNA (ctDNA), RNA, mRNA, dsRNA, siRNA, miRNA, and

[0293] 35 PATENT

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[0295] lncRNA. In various embodiments, the analysis of ctDNA from one or more blood samples of a subject having one or more tumors that are characterized as immune refractory, immunologically protected, or immunologically “cold” demonstrates low levels or absence of one or more tumor mutations, tumor antigens, or neoantigens. In various embodiments, the analysis of ctDNA from one or more blood 5 samples of a subject suffering from cancer demonstrates a low or no tumor mutation burden. In various embodiments, the analysis of ctDNA from one or more blood samples of a subject having one or more tumors that are characterized as immune refractory, immunologically protected, or immunologically “cold” demonstrates a tumor mutation burden of between 5 and 0.001 somatic mutations per mega base pairs (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, 10 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 0.04, 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, inclusive of all values and ranges between these values). In various embodiments, the nucleic acid analysis is performed by PCR, RT-PCR, qRT- PCR, next- generation sequencing (NGS), RNA-seq, ATAC-seq, exome sequencing, Southern Blot, 15 microarray analysis, or single-cell sequencing.

[0296] In various embodiments, the tumor characteristic of a subject is determined from the gene expression analysis from nucleic acids in one or more blood samples of a subject suffering from cancer. In various embodiments, gene expression analysis from nucleic acids in one or more blood samples of a subject having one or more tumors that are characterized as immune refractory, immunologically 20 protected, or immunologically “cold” demonstrates increased expression of tumor promoting, tumor permissive, or immune suppressive genes compared to the analysis of one or more blood samples from one or more healthy subjects or one or more subjects suffering from cancer and responsive to treatment. In various embodiments, the expression of tumor promoting, tumor permissive, or immune suppressive genes in one or more blood samples of a subject having one or more tumors that are characterized as 25 immune refractory, immunologically protected, or immunologically “cold” is increased by 5-100% (e.g., increased 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%, inclusive of 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 30 or more blood samples collected from one or more healthy subjects or one or more subjects suffering from cancer and responsive to treatment. In various embodiments, the expression of tumor promoting, tumor permissive, or immune suppressive genes in one or more blood samples of a subject having one or more tumors that are characterized as immune refractory, immunologically protected, or immunologically “cold” 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, 35 85, 90, 95, or 100 fold, inclusive of 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 responsive to treatment. In various embodiments, the analysis of nucleic acids in one or more blood samples of a subject having one or more tumors that are characterized as immune refractory, immunologically protected, or immunologically “cold” demonstrates decreased expression of tumor 40 inhibiting, anti-tumor, or pro-inflammatory genes compared to the analysis of one or more blood samples from one or more healthy subjects or one or more subjects suffering from cancer and responsive to

[0297] 36 PATENT

[0298] ATTORNEY DOCKET NO.: 51432-077WO2

[0299] treatment. In various embodiments, the analysis of nucleic acids in one or more blood samples of a subject having one or more tumors that are characterized as immune refractory, immunologically protected, or immunologically “cold” demonstrates low or no expression of tumor inhibiting, anti-tumor, or anti-inflammatory genes compared to the analysis of one or more blood samples from one or more 5 healthy subjects or one or more subjects suffering from cancer and responsive to treatment. In various embodiments, the expression of tumor inhibiting, anti- tumor, or pro-inflammatory genes in one or more blood samples of a subject having one or more tumors that are characterized as immune refractory, immunologically protected, or immunologically “cold” is decreased by 5-100% (e.g., decreased by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, 10 about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%, inclusive of 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 a healthy subject or a subject suffering from cancer who is responsive to therapy. In various embodiments, the expression of tumor inhibiting, anti-tumor, or pro-inflammatory genes in one or more blood samples of a subject having 15 one or more tumors that are characterized as immune refractory, immunologically protected, or immunologically “cold” is reduced by 2-100 fold (e.g., reduced relative to a healthy subject or a subject suffering from cancer and responsive to therapy 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, inclusive of all values and ranges between these values). In various embodiments, the gene expression analysis is performed by PCR, RT- PCR, qRT-PCR, next-20 generation sequencing (NGS), RNA-seq, ATAC-seq, exome sequencing, Southern Blot, microarray analysis, or single-cell sequencing.

[0300] In various embodiments, the tumor characteristic of a subject suffering from cancer is determined from 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 characteristic of a subject suffering from 25 cancer is determined from the analysis of cells, proteins, or nucleic acids in one or more tumor samples collected from the subject. In various embodiments, the tumor characteristic of a subject suffering from cancer is determined by comparing the analysis of cells, proteins, or nucleic acids in one or more tumor samples from the subject suffering from cancer to the analysis of tissue samples from one or more healthy subjects or one or more subjects suffering from cancer and responsive to treatment. In various 30 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, the 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 35 lymphoid cells are T cells, B cells, NK cells, NK-T cells, or iNK cells.

[0301] In various embodiments, the analysis of cells from one or more tumor samples collected from a subject having one or more tumors that are characterized as immune refractory, immunologically protected, or immunologically “cold” demonstrates the presence of immune suppressive cells. In various embodiments, the analysis of one or more tumor samples collected from a subject having one or more 40 tumors that are characterized as immune refractory, immunologically protected, or immunologically “cold” demonstrates the presence of immune suppressive cells in the tumor core. In various embodiments, the

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[0303] ATTORNEY DOCKET NO.: 51432-077WO2

[0304] analysis of cells from one or more tumor samples collected from a subject having one or more tumors that are characterized as immune refractory, immunologically protected, or immunologically “cold” demonstrates increased levels of immune suppressive cells. In various embodiments, the analysis of one or more tumor samples demonstrates increased levels of immune suppressive cells in the tumor core. In 5 various embodiments, the immune suppressive cells are myeloid derived suppressor cells (MDSCs), tumor-associated macrophages (TAMs), neutrophils, Treg cells, and Breg cells. In various embodiments, MDSCs are monocytic MDSCs (M-MDSCs) and polymorphonuclear MDSCs (PMN-MDSCs). In various embodiments, the TAMs are M2 TAMs. In various embodiments, the immune suppressive cells are CAFs. In various embodiments, the levels of immune suppressive cells in one or more tumor samples of a 10 subject having one or more tumors that are characterized as immune refractory, immunologically protected, or immunologically “cold” are increased by 5-100% (e.g., increased 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%, inclusive of all values and ranges between these values), 10-95%, 15-90%, 20-85%, 25-15 75%, 30-70%, 35-65%, 40-60%, 45-55%, or 50% compared to tissue samples of one or more healthy subjects or one or more subjects suffering from cancer and responsive to treatment. In various embodiments, the levels of immune suppressive cells in one or more tumor samples of a subject having one or more tumors that are characterized as immune refractory, immunologically protected, or immunologically “cold” are increased by 2-100-fold (e.g., increased relative to a healthy subject or a 20 subject suffering from cancer and responsive to treatment 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, inclusive of all values and ranges between these values) compared to one or more tissue samples or one or more healthy subjects or one or more subjects suffering from cancer and responsive to treatment.

[0305] In various embodiments, the analysis of cells from one or more tumor samples collected from a 25 subject having one or more tumors that are characterized as immune refractory, immunologically protected, or immunologically “cold” demonstrates the absence of leukocytes. In various embodiments, the analysis of cells from one or more tumor samples collected from a subject having one or more tumors that are characterized as immune refractory, immunologically protected, or immunologically “cold” demonstrates the reduced or low levels of leukocytes. In various embodiments, the frequency of

[0306] 30 leukocytes is ≤50%, ≤ 40%, ≤ 30%, ≤ 20%, ≤ 10%, or ≤ 5%, inclusive of all values and ranges between these values, of all cells analyzed.

[0307] In various embodiments, the analysis of cells from one or more tumor samples collected from a subject having one or more tumors that are characterized as immune refractory, immunologically protected, or immunologically “cold” demonstrates the absence of activated pro-inflammatory immune 35 cells. In various embodiments, the analysis of cells from one or more tumor samples collected from a subject having one or more tumors that are characterized as immune refractory, immunologically protected, or immunologically “cold” demonstrates the absence of activated pro-inflammatory immune cells from the tumor core. In various embodiments, the analysis of cells from one or more tumor samples collected from a subject having one or more tumors that are characterized as immune refractory, 40 immunologically protected, or immunologically “cold” demonstrates low or reduced levels of activated pro- inflammatory immune cells. In various embodiments, the analysis of cells from one or more tumor

[0308] 38 PATENT

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[0310] samples collected from a subject having one or more tumors that are characterized as immune refractory, immunologically protected, or immunologically “cold” demonstrates low or reduced levels of activated pro- inflammatory immune cells in the tumor core. 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 5 iNK cells. In various embodiments, the frequency of pro-inflammatory immune cells is ≤ 50%, ≤ 40%, ≤ 30%, ≤ 20%, ≤ 10%, or ≤ 5%, inclusive of all values and ranges between these values, of all cells analyzed.

[0311] In various embodiments, the tumor characteristic of a subject suffering from cancer is determined from the analysis of the location of immune cells in one or more tumor samples of the subject. In various 10 embodiments, immune cells in one or more tumor samples of a subject having one or more immune refractory, immunologically protected, or immunologically “cold” are located in the tumor periphery. In various embodiments, immune cells in one or more tumor samples of a subject having one or more immune refractory, immunologically protected, or immunologically “cold” are absent from the tumor core. In various embodiments, immune cells in one or more tumor samples of a subject having one or more 15 immune refractory, immunologically protected, or immunologically “cold” are reduced in the tumor core. In various embodiments, immune cells in the tumor core are reduced by 5-100% (e.g., relative to a healthy subject or a subject suffering from cancer and responsive to treatment 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 20 100%, inclusive of 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 samples from one or more healthy subjects or one or more subjects suffering from cancer and responsive to treatment.

[0312] In various embodiments, the tumor characteristic of a subject suffering from cancer is determined from the analysis of the location of stromal cells in one or more tumor samples of the subject. In various 25 embodiments, the stromal cells are CAFs, pericytes, adipocytes, and endothelial cells. In various embodiments, CAFs in one or more tumor samples of a subject having one or more immune refractory, immunologically protected, or immunologically “cold” tumors are increased in the tumor periphery. In various embodiments, CAFs in one or more tumor samples of a subject having one or more immune refractory, immunologically protected, or immunologically “cold” tumors are increased in the tumor core. In 30 various embodiments, the frequency of CAFs in the tumor periphery is increased by 5-100% (e.g., increased 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%, inclusive of 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%

[0313] 35 compared to one or more tissue sample from one or more healthy subjects or subjects suffering from cancer and responsive to treatment. In various embodiments, the frequency of CAFs in the tumor periphery is increased by 2-100 fold (e.g., increased by about 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 fold, inclusive of all values and ranges between these values) compared to one or more tissue samples from one or more healthy subjects or one or more subjects suffering from 40 cancer and responsive to treatment. In various embodiments, the frequency of CAFs in the tumor core is increased by 5-100% (e.g., increased by about 5%, about 10%, about 15%, about 20%, about 25%,

[0314] 39 PATENT

[0315] ATTORNEY DOCKET NO.: 51432-077WO2

[0316] 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%, inclusive of 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 of one or more healthy subjects or one or 5 more subjects suffering from cancer and responsive to treatment. In various embodiments, the frequency of CAFs in the tumor core is increased by 2-100 fold (e.g., increased relative to a healthy subject or a subject suffering from cancer and responsive to treatment by about 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 fold, inclusive of all values and ranges between these values) compared to one or more healthy tissue samples.

[0317] 10 In various embodiments, the analysis of cells in one or more tumor samples of a subject suffering from cancer is performed by the assay of 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, 15 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, CD207 CD226, CD244, CD267, CD268, CD269, CD355, CD358, NKG2A, NKG2B, NKG2C, NKG2D, NKG2E, 20 NKG2F, NKG2H, KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL5A, KIR2DL5B, KIR3DL1, KIR3DL2, KIR3DL3, KIR3DL4, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5, DAP12, KIR3DS, NKp44, NKp46, TCR, BCR, Integrins, FcβcRI, MHC-I, MHC-II, IL-1R, IL-2Rα, IL-2Rβ, IL-2Rγ, IL-3Rα, CSF2RB, 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, OSMR, CSF-1R, cell-surface IL-15, IL-10Rα, IL-10Rβ, IL-20Rα, IL-20Rβ, IL-22Rα1, IL-25 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, LTβR, TIM-1, TIM-3, TIM-4, TIGIT, LAG-3, ICOS, ICOS-L, SLAM, SLAMF2, OX-40, OX-40L, GITR, GITRL, TL1A, HVEM, 41-BB, 41BB-L, TL-1A, TRAF1, TRAF2, TRAF3, TRAF5, BAFF, BAFF-R, APRIL, TRAIL, RANK, AITR, TRAMP, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CCR11, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, 30 CXCR7, CLECL9a, DC-SIGN, IGSF4A, SIGLEC, EGFR, PDGFR, VEGFR, FAP, α-SMA, Vimentin, Laminin, FAS, FAS-L, Fc, 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, A1R, A2AR, A2BR, and A3R, H60a, H60b, and H60c. In various embodiments, Integrins are 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, 35 or combinations thereof. In various embodiments, TCR is selected from the group consisting of a, β, γ, δ, ε, and ζ TCR. Several methods have been described in the literature for assaying of cell-surface protein expression from tumor samples, including immunohistochemistry, immunofluorescence, western blot, flow cytometry, and Mass Cytometry (CyTOF).

[0318] Tumor core is generally described as the densely packed, central, bulk-forming and differentiated 40 region of the tumor. In contrast, the tumor periphery is generally described as the invasive edge of the tumor that interacts with the surrounding stroma and parenchyma.

[0319] 40 PATENT

[0320] ATTORNEY DOCKET NO.: 51432-077WO2

[0321] In various embodiments, the tumor characteristic of a subject suffering from cancer is determined from the analysis of proteins in one or more tumor samples of the subject. In various embodiments, the tumor characteristic of a subject suffering from cancer is determined by comparing the analysis of proteins in one or more tumor samples from the subject suffering from cancer to the analysis of one or 5 more tissues from one or more healthy subjects or one or more subjects suffering from cancer and responsive 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, 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-10 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, CCL12, 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, 15 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 factors are 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 protease, a cysteine protease, a metalloprotease, a serine protease, or a 20 threonine protease. In some embodiments, the protein is a protease is 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, 25 MMP27, and MMP28. In various embodiments, the protein is an enzyme selected from the group consisting of arginase, asparaginase, kynurinase, 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, proteins associated with apoptosis are selected from the group consisting of P53, Caspase 1, Caspase 2, Caspase 3, Caspase 4, Caspase 5, Caspase 6, Caspase 7, 30 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 have been described in the literature for assaying proteins from tumor samples, including immunohistochemistry, immunofluorescence, western blot, and ELISA.

[0322] In various embodiments, the analysis of proteins from one or more tumor samples of a subject 35 having one or more tumors characterized as immune refractory, immunologically protected, or immunologically “cold” demonstrates increased levels of proteins associated with tumor progression, anti- inflammatory activity, or immune suppression. In various embodiments, proteins associated with tumor progression, anti-inflammatory activity, or immune suppression are cell-surface proteins, intracellular proteins, or secreted proteins. In various embodiments, proteins associated with tumor progression, anti-40 inflammatory activity, or immune suppression are selected from the group consisting of CD39, CD47, CD79, CD140a, CD163, CD206, FOXP3, FAP, PD-1, PD-L1, PD-L2, CSF-1R, A1R, A2AR, A2BR, A3R,

[0323] 41 PATENT

[0324] ATTORNEY DOCKET NO.: 51432-077WO2

[0325] 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, MMP24, MMP25, MMP26, MMP27, MMP28, CXCL12, GM-CSF, G-CSF, FAP, TGF- β1, TGF-β2, TGF-β3, arginase, asparaginase, kyneurinase, indoleamine 2,3 dioxygenase (IDO1 and 5 IDO2), tryptophan 2,3 dioxygenase (TDO), myeloperoxidase (MPO), neutrophil elastase (NE), and IL4I1.

[0326] In various embodiments, the levels of proteins associated with tumor progression, anti-inflammatory activity, or immune suppression in one or more tumor samples of a subject having one or more tumors characterized as immune refractory, immunologically protected, or immunologically “cold” are increased by 5-100% (e.g., increased by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, 10 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%, inclusive of 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 responsive to treatment. In various embodiments, the levels of 15 proteins associated with tumor progression, anti-inflammatory activity, or immune suppression in one or more tumor samples of a subject having one or more tumors characterized as immune refractory, immunologically protected, or immunologically “cold” are increased by 2-100 fold (e.g., increased relative to a healthy subject or a subject suffering from cancer and responsive to treatment by about 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 fold, inclusive of all values and ranges 20 between these values) compared to one or more tissue samples from one or more healthy subjects or subjects suffering from cancer and responsive to treatment.

[0327] In various embodiments, the analysis of proteins from one or more tumor samples of a subject having one or more tumors characterized as immune refractory, immunologically protected, or immunologically “cold” demonstrate reduced levels, low levels, or absence of proteins associated with 25 tumor growth inhibition, anti-tumor activity, or pro- inflammatory activity. In various embodiments, proteins associated with tumor growth inhibition, anti-tumor activity, 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, IL-1α, IL-1β, IL-30 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, 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 levels of proteins associated with tumor growth inhibition, anti-tumor activity, or pro-inflammatory activity are reduced by 5-100% (e.g., reduced by about 35 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%, inclusive of 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 samples collected from one or more healthy tissues or one or more tumor samples collected from a 40 subject suffering from cancer and responsive to treatment. Several methods have been described for

[0328] 42 PATENT

[0329] ATTORNEY DOCKET NO.: 51432-077WO2

[0330] assaying proteins from tumor samples, including immunohistochemistry, immunofluorescence, western blot, intracellular flow cytometry, and ELISA.

[0331] In various embodiments, the tumor characteristic of a subject suffering from cancer is determined from the Tumor Proportion Score (TPS) for PD-L1 expression in one or more tumor samples from the 5 subject. In various embodiments, the TPS of a subject having one or more tumors that are characterized as immune refractory, immunologically protected, or immunologically “cold” of 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, inclusive of all ranges between these values). In various embodiments, the TPS of a subject having one or more tumors that are

[0332] 10 characterized as immune refractory, immunologically protected, or immunologically “cold” is ≤ 1. TPS for PD-L1 expression is defined as the percentage of viable tumor cells demonstrating partial or complete membrane staining by immunohistochemical analysis.

[0333] In various embodiments, the tumor characteristic of a subject suffering from cancer is determined from the Combined Positivity Score (CPS) for PD-L1 expression in one or more tumor samples from the 15 subject. In various embodiments, the CPS of a subject having one or more tumors that are characterized as immune refractory, immunologically protected, or immunologically “cold” is ≤ 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, inclusive of all ranges between these values). In various embodiments, the CPS is ≤ 1. CPS for PD-L1 expression is determined from the immunohistochemical determination of the number of viable tumor cells, lymphocytes, and macrophages positive for PD-L1 as a percentage of all viable tumor cells.

[0334] 20 In various embodiments, the tumor characteristic of a subject suffering from cancer is determined from microsatellite instability testing of one or more tumor samples from the subject. In various embodiments, the tumor characteristic of a subject suffering from cancer is determined by comparing microsatellite instability testing of one or more tumor samples to microsatellite stability testing from one or more healthy tissues of the subject. In various embodiments, the microsatellite instability testing is the 25 assay of microsatellite markers. In various embodiments, the microsatellite instability testing is the assay of mismatch repair markers. In various embodiments, the microsatellite markers are selected from the group consisting of BAT25, BAT26, D2S123, D5S346, and D17S250. In various embodiments, the mismatch repair markers are selected from the group consisting of MLH1, MSH2, MLH6, and PMS2. In various embodiments, the subject has one or more immune refractory, immunologically protected, or 30 immunologically “cold” tumors that are determined to be microsatellite instability low. In various embodiments, the subject has one or more immune refractory, immunologically protected, or immunologically “cold” tumors that are determined to be microsatellite stable. In various embodiments, the subject has one or more immune refractory, immunologically protected, or immunologically “cold” tumors that are mismatch repair proficient.

[0335] 35 In various embodiments, the analysis of one or more tumor samples of a subject having one or more tumors characterized as immune refractory, immunologically protected, or immunologically “cold” demonstrates increased levels of neutrophil extracellular traps (NETs). In various embodiments, the analysis of one or more tumor samples of a subject having one or more tumors characterized as immune refractory, immunologically protected, or immunologically “cold” demonstrates increased levels of 40 neutrophil extracellular traps (NETs) compared to the analysis of one or more tumor samples from one or more healthy subjects. In various embodiments, the levels of NETs in one or more tumor samples of a

[0336] 43 PATENT

[0337] ATTORNEY DOCKET NO.: 51432-077WO2

[0338] subject suffering from cancer are increased by 5-100% (e.g., increased 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%, inclusive of all values and ranges between these values), 10-95%, 15-90%, 20-85%, 25-75%, 30- 5 70%, 35-65%, 40-60%, 45-55%, 50%, or 100% compared to one or more tissue samples from one or more healthy subjects or subject suffering from cancer and responsive to treatment. In various embodiments, the levels of NETs in one or more tumor samples of a subject suffering from cancer are increased by 2-100 fold (e.g., increased relative to a healthy subject or a subject suffering from cancer and responsive to treatment by about 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 10 90, 95 fold, inclusive of 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 responsive to treatment. Several methods have been described in the literature for assaying NETs, including western blot, ELISA, and flow cytometry.

[0339] In various embodiments, the tumor characteristic of a subject suffering from cancer is determined 15 from the analysis of nucleic acids in one or more tumor samples of the subject. In various embodiments, the tumor characteristic of a subject suffering from cancer is determined by comparing the analysis of nucleic acids in one or more tumor samples from the subject suffering from cancer to the analysis of one or more tissue samples from one or more healthy subjects or subjects suffering from cancer and responsive to treatment. In various embodiments, the nucleic acid is selected from the group comprising 20 DNA, ssDNA, RNA, mRNA, dsRNA, siRNA, miRNA, and lncRNA. In various embodiments, the 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.

[0340] In various embodiments, the analysis of nucleic acids from one or more tumor samples of a subject suffering from cancer is used to determine the tumor mutation burden. In various embodiments, 25 the analysis of nucleic acids from one or more tumor samples of a subject having one or more tumors characterized as immune refractory, immunologically protected, or immunologically “cold” demonstrates a low tumor mutation burden. In various embodiments, the analysis of nucleic acids from one or more tumor samples a subject having one or more tumors characterized as immune refractory, immunologically protected, or immunologically “cold” demonstrates a tumor mutation burden of between 5 and 0.001 30 somatic mutations per mega base pairs (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 0.04, 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, inclusive of all values and ranges between these values). In various embodiments, the nucleic acid 35 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.

[0341] In various embodiments, the analysis of nucleic acids in one or more tumor samples of a subject having one or more tumors characterized as immune refractory, immunologically protected, or immunologically “cold” demonstrates increased expression of genes associated with tumor promoting, 40 tumor permissive, anti-inflammatory, or immune suppressive activity compared to the analysis of one or more tissue samples from one or more healthy subjects or subjects suffering from cancer and responsive

[0342] 44 PATENT

[0343] ATTORNEY DOCKET NO.: 51432-077WO2

[0344] to treatment. In various embodiments, genes associated with tumor promoting, tumor permissive, anti- inflammatory, or immune suppressive activity are selected from the group consisting of CD39, CD47, CD79, CD140a, CD163, CD206, FOXP3, FAP, PD-1, PD-L1, PD-L2, CSF-1R, A1R, A2AR, A2BR, A3R, TIM-1, TIM-3, TIM-4, TIGIT, CSFR, SIGLEC, MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, 5 MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP18, MMP19, MMP20, MMP21, MMP23A, MMP23B, MMP24, MMP25, MMP26, MMP27, MMP28, CXCL12, GM-CSF, G-CSF, FAP, TGF- β1, TGF-β2, TGF-β3, arginase, asparaginase, kyneurinase, 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, 10 anti-inflammatory, or immune suppressive activity is increased by 5-100% (e.g., increased 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%, inclusive of 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 15 samples of one or more healthy subjects or subjects suffering from cancer and responsive to treatment.

[0345] In various embodiments, the 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.

[0346] In various embodiments, the analysis of nucleic acids in one or more tumor samples of a subject 20 having one or more tumors characterized as immune refractory, immunologically protected, or immunologically “cold” demonstrates low or decreased expression of genes associated with tumor inhibiting, anti-tumor, or pro-inflammatory activity. In various embodiments, the analysis of nucleic acids in one or more tumor samples of a subject suffering from cancer demonstrates no expression of genes associated with tumor inhibiting, anti-tumor, or pro-inflammatory activity. In various embodiments, genes 25 associated with tumor inhibiting, 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, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, 30 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 genes associated with tumor inhibiting, anti-tumor, or pro- inflammatory activity is decreased by 5-100% (e.g., decreased by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 35 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%, inclusive of 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 a healthy subject or a subject suffering from cancer responsive to treatment. In various embodiments, the gene expression analysis is performed by PCR, RT-PCR, qRT- PCR, next-generation sequencing (NGS), RNA-seq, ATAC-seq, 40 exome sequencing, Southern Blot, microarray analysis, or single-cell sequencing.

[0347] 45 PATENT

[0348] ATTORNEY DOCKET NO.: 51432-077WO2

[0349] In some embodiments, a method of the present disclosure comprises 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 were collected. In some embodiments, a first, second, third, fourth or fifth biological sample can be obtained and analyzed. The biological samples may 5 be collected days, weeks, or months apart. The two or more biological samples can be analyzed as previously described herein. For example, a first biological sample can be obtained from the subject before administration of a RAS(ON) inhibitor therapy described herein and the second biological sample obtained from the subject after administration of a RAS(ON) inhibitor therapy described herein.

[0350] In various embodiments, the disclosure provides a method of treating cancer in a subject 10 comprising administering to the subject a combination therapy described herein, wherein the subject has one or more tumors that are resistant or unresponsive to treatment. In various embodiments, the subject has one or more tumors that are resistant or unresponsive to one or more treatments selected from the group consisting of surgery, radiation, chemotherapy, biologic agents, small molecules, cell-based therapy, hormone therapy, and immunotherapy. In various embodiments, treatment is a standard of care 15 therapy, first-line therapy, second-line therapy, or third- line therapy. In various embodiments, the subject has one or more tumors that have progressed during one or more treatments, wherein the treatments are standard of care therapy, first-line therapy, second-line therapy, or third-line therapy.

[0351] First-line therapy is defined as a treatment that is administered to a subject suffering from cancer who has not received any prior treatment. Second-line therapy is defined as treatment that is

[0352] 20 administered to a subject suffering from cancer who has received prior first-line therapy but experienced disease progression during first-line treatment. Third-line therapy is defined as treatment that is administered to a subject suffering from cancer who has received prior first and second-line treatment but has experienced disease progression during second-line treatment. Each particular type of cancer has a first-line, second-line, and third-line therapy. The first-, second-, and third-line therapies for types of 25 cancer are known in the art. In addition, FDA approved drug labels will indicate if a particular drug is approved as a first-, second-, or third- line therapy.

[0353] Several criteria and definitions published in the literature can be used to determine the effect of one or more treatments on tumors in a subject suffering from cancer. Based on these criteria, tumors are defined as “responsive,” “stable,” or “progressive” when they improve, remain the same, or worsen during 30 treatment, respectively.

[0354] Examples of the 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 the Cancer of the 35 Liver (RECICL), and WHO Criteria in Tumor Response.

[0355] In various embodiments, the disclosure provides a method of treating cancer in a subject in need thereof comprising administering to the subject a combination therapy described herein, wherein the subject cannot tolerate standard of care therapy, first-line therapy, second-line therapy, or third-line therapy. In various embodiments, the disclosure provides a method of treating cancer in a subject 40 comprising administering to the subject a combination therapy described herein, wherein the subject has experienced tumor recurrence after surgical resection of the primary tumor. In various embodiments, the

[0356] 46 PATENT

[0357] ATTORNEY DOCKET NO.: 51432-077WO2

[0358] disclosure provides a method of treating cancer in a subject comprising administering to the subject a combination therapy described herein, wherein the subject has a tumor that cannot be surgically removed. In various embodiments, the disclosure provides a method of treating cancer in a subject comprising administering to the subject a combination therapy described herein, wherein the subject has 5 no treatment options available.

[0359] Several therapies used in the treatment of cancer (e.g., chemotherapies) are cytotoxic and are associated with significant side-effects and toxicities that are associated with poor outcomes and poor response to treatment. Prior to administering such treatments, clinicians rely on several assessment tools to help determine the risk of a subject suffering from cancer experiencing treatment related toxicities and 10 adverse events. Based on the results of these assessments, a subject suffering from cancer is considered intolerant to therapy if they are determined to be at increased risk of experiencing therapy- related toxicities and adverse events resulting in poor outcomes. Examples of commonly used assessment tools used in the determination of therapy intolerance include Karnofsky Performance Status (KPS), Eastern Cooperative Oncology Group Performance Status (ECOG PS), Timed Get Up and Go 15 (TUG), Short Physical Performance Battery (SPPB), Comprehensive Geriatric Assessment (CGA), Cancer Aging Research Group (CARG) Score, and Chemotherapy Risk Assessment Scale for High-Age Patients (CRASH).

[0360] Treatment Outcomes and Clinical Endpoints

[0361] 20 In various embodiments, the disclosure provides a method of treating lung cancer in a subject in need thereof comprising administering to the subject a combination therapy described herein, wherein the subject has one or more immune refractory tumors. In various embodiments, the administering alters the tumor immune infiltrate. In various embodiments, the administering alters the anti-tumor immune response. In various embodiments, the administering alters the tumor microenvironment comprising 25 tumor cells, immune cells, cancer stem cells, and stroma. In various embodiments, the administering transforms an immunologically cold tumor into an immunologically hot tumor. In various embodiments, the administering reduces tumor size or inhibits tumor growth. In various embodiments, the administering induces tumor cell death, apoptosis, or necrosis via direct particle uptake by tumor cells.

[0362] In various embodiments, the disclosure provides a method of treating cancer in a subject in need 30 thereof comprising administering to the subject a combination therapy described herein, wherein the subject has one or more tumors that are characterized as immunologically protected or immune refractory. In various embodiments, the administering alters the tumor-associated stroma comprising fibroblasts, cancer-associated fibroblasts, adipocytes, pericytes, endothelium, vasculature, lymphatic vessels, tumor-associated vasculature, mesenchymal stromal cells, mesenchymal stem cells, and 35 extracellular matrix.

[0363] It is contemplated that the methods herein reduce tumor size or tumor burden in the subject or reduce metastasis in the subject. In various embodiments, the methods reduce the tumor size by 10%, 20%, 30% or more. In various embodiments, the methods reduce tumor size by 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%, or including all 40 values and ranges that lie in between these values.

[0364] 47 PATENT

[0365] ATTORNEY DOCKET NO.: 51432-077WO2

[0366] Certain biomarkers may decrease in abundance when a tumor becomes immune refractory. It is contemplated herein that after treatment with a combination therapy described herein, the level of one or more of biomarkers increases by an amount in the range of 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 5 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5, or about 10 fold. Similarly, certain biomarkers increase in abundance when a tumor becomes immune refractory. After treatment with a combination therapy described herein, the level of one or more of such biomarkers decrease by an amount in the range of 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, 10 about 8.5, about 9, about 9.5, or about 10 fold. In various embodiments, administering a combination therapy described herein to a subject having one or more tumors characterized as immune refractory, immunologically protected, or immunologically “cold” reduces the levels of immune suppressive cells in blood. In various embodiments, the suppressive cells are myeloid derived suppressor cells (MDSCs), tumor-associated macrophages (TAMs), neutrophils, Treg cells, and Breg cells. In various embodiments, 15 MDSCs are monocytic MDSCs (M-MDSCs) and polymorphonuclear MDSCs (PMN-MDSCs). In various embodiments, the TAMs are M2 TAMs. In various embodiments, the immune suppressive cells are CAFs. In various embodiments, the levels of immune suppressive cells are reduced by about 5-100% (e.g., reduced 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%, 20 about 85%, about 90%, about 95%, or about 100%, inclusive of 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 the subject prior to treatment. In various embodiments, the levels of immune suppressive cells are reduced by about 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, inclusive of all values and ranges between these 25 values) compared to one or more blood samples collected from the subject prior to treatment. In various embodiments, immune suppressive cells are identified by the assay of cell-surface proteins expression. In various embodiments, administering a combination therapy described herein to a subject having one or more tumors characterized as immune refractory, immunologically protected, or immunologically “cold” increases the levels of activated pro-inflammatory immune cells by 5-100% (e.g., increased by about 5%, 30 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%, inclusive of 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 the subject prior to treatment. In various embodiments, administering a combination 35 therapy described herein to a subject having one or more tumors characterized as immune refractory, immunologically protected, or immunologically “cold” increases the levels 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, inclusive of all values and ranges between these values) compared to one or more blood samples collected from the subject prior to treatment. In various embodiments, the activated pro-40 inflammatory 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 pro-inflammatory immune cells is

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[0369] increased to 10-50% (e.g., about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%, inclusive of all values and ranges between these values) of all leukocytes analyzed from one or more blood samples collected from the subject. In various embodiments, activated pro-inflammatory immune cells are identified by the assay of cell-surface protein expression.

[0370] 5 In various embodiments, the analysis of cells in one or more blood samples of a subject suffering from cancer is performed by the assay of 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, 10 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, CD207 CD226, CD244, CD267, CD268, CD269, CD355, CD358, NKG2A, NKG2B, NKG2C, NKG2D, NKG2E, 15 NKG2F, NKG2H, KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL5A, KIR2DL5B, KIR3DL1, KIR3DL2, KIR3DL3, KIR3DL4, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5, DAP12, KIR3DS, NKp44, NKp46, TCR, BCR, Integrins, FcβεRI, MHC-I, MHC-II, IL-1R, IL-2Rα, IL-2Rβ, IL-2Rγ, IL-3Rα, CSF2RB, 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, OSMR, CSF-1R, cell-surface IL-15, IL-10Rα, IL-10Rβ, IL-20Rα, IL-20Rβ, IL-22Rα1, IL-20 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, LTβR, TIM-1, TIM-3, TIM-4, TIGIT, LAG-3, ICOS, ICOS-L, SLAM, SLAMF2, OX-40, OX-40L, GITR, GITRL, TL1A, HVEM, 41-BB, 41BB-L, TL-1A, TRAF1, TRAF2, TRAF3, TRAF5, BAFF, BAFF-R, APRIL, TRAIL, RANK, AITR, TRAMP, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CCR11, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, 25 CXCR7, CLECL9a, DC-SIGN, IGSF4A, SIGLEC, EGFR, PDGFR, VEGFR, FAP, α-SMA, Vimentin, Laminin, FAS, FAS-L, Fc, ICAM-1, ICAM-2, ICAM-3, ICAM-4, ICAM-5, PECAM-1, MICA, MICB, UL16, ULBP1, ULBP2, ILBP3, ULBP4, ULBP5, ULBP6, MULT1, RAE1 α, β, γ, δ, and ε, A1R, A2AR, A2BR, and A3R, H60a, H60b, and H60c. In various embodiments, Integrins are 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, 30 or combinations thereof. In various embodiments, TCR is selected from the group consisting of a, β, γ, δ, ε, and ζ TCR. Several methods have been described in the literature for assaying of cell-surface protein expression, including Flow Cytometry and Mass Cytometry (CyTOF). The presence or abundance of one or more of these cell-surface proteins indicates that the patient is responsive to treatment with the method disclosed herein.

[0371] 35 In various embodiments, administering a combination therapy described herein to a subject having one or more tumors characterized as immune refractory, immunologically protected, or immunologically “cold” reduces the neutrophil to lymphocyte (NLR) in one or more blood samples from high to moderate, or high to low. In various embodiments, the analysis of cells from one or more blood samples collected from a subject having one or more tumors characterized as immune refractory, 40 immunologically protected, or immunologically “cold” reduces NLR to between 1-2 (e.g., between 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, and 2 inclusive of all values and ranges between these values). In

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[0374] various embodiments, NLR, after administration of the combination therapy described herein, is reduced. In various embodiments, NLR, after administration of the combination therapy described herein is < 2.

[0375] In various embodiments, administering a combination therapy described herein to a subject having one or more tumors characterized as immune refractory, immunologically protected, or

[0376] 5 immunologically “cold” reduces the levels of CTCs in one or blood samples. In various embodiments, the levels of CTCs in blood are reduced to 5, 4, 3, 2, 1, or 0 per 7.5 ml blood inclusive of inclusive of all values and ranges between these values.

[0377] In various embodiments, administering a combination therapy described herein to a subject having one or more tumors characterized as immune refractory, immunologically protected, or

[0378] 10 immunologically “cold” decreases the levels of tumor promoting, anti-inflammatory, or immune suppressive proteins in one or more blood samples of the subject. In various embodiments, the tumor promoting, anti- inflammatory, or immune suppressive 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, 15 MMP26, MMP27 and MMP28, CXCL12, GM-CSF, G-CSF, TGF-β1, TGF-β2, and TGF-β3, arginase, asparaginase, kyneurinase, indoleamine 2,3 dioxygenase (IDO1 and IDO2), tryptophan 2,3 dioxygenase (TDO), myeloperoxidase (MPO), neutrophil elastase (NE), and IL4I1. In various embodiments, the levels of tumor promoting, anti-inflammatory, or immune suppressive proteins in one or more blood samples of the subject are decreased by 5-100% (e.g., decreased by about 5%, about 10%, about 15%, about 20%, 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%, inclusive of 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 levels of tumor promoting, anti-inflammatory, or immune suppressive proteins 25 in one or more blood samples of the subject are decreased by 2-100 fold (e.g., decreased relative to one or more samples collected prior to treatment 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, inclusive of all values and ranges between these values) compared to one or more blood samples collected from the subject prior to treatment.

[0379] In various embodiments, administering a combination therapy described herein to a subject 30 having one or more tumors characterized as immune refractory, immunologically protected, or immunologically “cold” increases the levels of tumor inhibiting, anti-tumor, or pro-inflammatory proteins in one or more blood samples collected from the subject. In various embodiments, tumor inhibiting, anti- tumor, or pro-inflammatory proteins 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-35 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, 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 levels of anti-tumor, or pro-inflammatory proteins are increased by 5-100% (e.g., increased 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 40 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%, inclusive of all values and ranges between these values), 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45- 50 PATENT

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[0381] 55%, 50%, or 100% compared to one or more blood samples collected prior to treatment. In various embodiments, the levels of anti-tumor, or pro-inflammatory proteins are increased by 2-100 fold (e.g., increased relative to one or more samples collected prior to treatment 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, inclusive of all values and ranges between 5 these values) compared to one or more blood samples collected from the subject prior to treatment.

[0382] Several methods have been described in the literature for assaying proteins from blood samples, including western blot, and ELISA.

[0383] In various embodiments, administering a combination therapy described herein to a subject having one or more tumors characterized as immune refractory, immunologically protected, or

[0384] 10 immunologically “cold” decreases the levels of neutrophil extracellular traps (NETs) in one or more blood samples collected from the subject. In various embodiments, the levels of NETs in one or more blood samples is decreased by 5-100% (e.g., decreased 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%, inclusive of 15 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 levels of NETs in one or more blood samples is decreased 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, inclusive of all values and ranges between these values) compared to one or more blood samples collected from the 20 subject prior to treatment. Several methods have been described in the literature for assaying NETs from blood samples, including western blot, ELISA, and flow cytometry.

[0385] In various embodiments, administering a combination therapy described herein to a subject having one or more tumors characterized as immune refractory, immunologically protected, or immunologically “cold” decreases the expression of tumor promoting, tumor permissive, or immune 25 suppressive genes in one or more blood samples of the subject. In one or more embodiments, the expression of tumor promoting, tumor permissive, or immune suppressive genes is decreased by 5-100% (e.g., decreased relative to levels in one or more blood samples collected prior to treatment 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%, 30 about 95%, or about 100%, inclusive of 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 one or more embodiments, the expression of tumor promoting, tumor permissive, or immune suppressive genes is decreased by 2-100-fold (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, inclusive of all values 35 and ranges between these values) compared to one or more blood samples collected from the subject prior to treatment.

[0386] In various embodiments, administering a combination therapy described herein to a subject having one or more tumors characterized as immune refractory, immunologically protected, or immunologically “cold” increases the expression of tumor inhibiting, anti-tumor, or pro-inflammatory genes 40 in one or more samples collected from the subject. In one or more embodiments, the expression of tumor inhibiting, anti-tumor, or pro-inflammatory genes is increased by 5-100% (e.g., increased by about 5%,

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[0389] 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%, inclusive of 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 5 samples collected prior to treatment. In various embodiments, the expression of tumor inhibiting, anti- tumor, or pro-inflammatory genes is increased by 2-100-fold (e.g., increased relative to one or more samples collected prior to treatment 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, inclusive of all values and ranges between these values) compared to one or more blood samples collected from the subject prior to treatment. In various embodiments, the gene 10 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.

[0390] In various embodiments, administering a combination therapy described herein to a subject having one or more tumors characterized as immune refractory, immunologically protected, or immunologically “cold” increases the levels of leukocytes in the tumor. In various embodiments, the levels 15 of leukocytes are increased in the tumor core or tumor periphery. In various embodiments, the leukocytes are increased by 5-100% (e.g., increased 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%, inclusive of all values and ranges between these values), 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-20 55%, or 50% compared to one or more tumor samples collected from the subject prior to treatment. In various embodiments, the levels of leukocytes are 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, inclusive of 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 frequency of leukocytes in the tumor core or tumor periphery is ≥ 25 5%, ≥ 10%, ≥ 15%, ≥ 20%, ≥ 25%, ≥ 30%, ≥ 35%, ≥ 40%, ≥ 45%, or ≥ 50, inclusive of all values and ranges between these values, of all cells analyzed.

[0391] In various embodiments, administering a combination therapy described herein to a subject having one or more tumors characterized as immune refractory, immunologically protected, or immunologically “cold” reduces the levels of immune suppressive cells in the tumor. In various

[0392] 30 embodiments, the levels of immune suppressive cells are reduced in the tumor core or tumor periphery.

[0393] In various embodiments, the suppressive cells are myeloid derived suppressor cells (MDSCs), tumor associated macrophages (TAMs), neutrophils, Treg cells, and Breg cells. In various embodiments, MDSCs are monocytic MDSCs (M-MDSCs) and polymorphonuclear MDSCs (PMN-MDSCs). In various embodiments, the TAMs are M2 TAMs. In various embodiments, the immune suppressive cells are CAFs.

[0394] 35 In various embodiments, the levels of immune suppressive cells are reduced by about 5-100% (e.g., reduced 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%, inclusive of 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 40 or more tumor samples collected from the subject prior to treatment. In various embodiments, the levels of immune suppressive cells are reduced by about 2-100 fold (e.g., about 2, 5, 10, 15, 20, 25, 30, 35, 40,

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[0397] 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 fold, inclusive of all values and ranges between these values) compared to one or more tumor samples collected from the subject prior to treatment. In various embodiments, immune suppressive cells are identified by the assay of cell-surface proteins expression.

[0398] Levels of leukocytes in a tumor sample can be evaluated by several methods including flow 5 cytometry and immunohistochemistry. In various embodiments, administering a combination therapy described herein to a subject having one or more tumors characterized as immune refractory, immunologically protected, or immunologically “cold” increases the levels of activated pro-inflammatory immune cells in the tumor. In various embodiments, the levels of activated pro-inflammatory cells are increased in the tumor core or tumor periphery.

[0399] 10 In various embodiments, administering a combination therapy described herein to a subject having one or more tumors characterized as immune refractory, immunologically protected, or immunologically “cold” increases the levels of activated pro-inflammatory immune cells in the tumor by 5- 100% (e.g., increased 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%, 15 about 80%, about 85%, about 90%, about 95%, or about 100%, inclusive of 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, administering a combination therapy described herein to a subject having one or more tumors characterized as immune refractory, immunologically protected, or immunologically “cold” 20 increases the levels 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, inclusive of 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

[0400] 25 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%, inclusive of all values and ranges between these values) of all leukocytes analyzed from one or more tumor samples collected from the subject. In various embodiments, activated pro-inflammatory immune cells are identified by the assay of cell-surface protein expression.

[0401] 30 In various embodiments, the analysis of cells in one or more tumor samples of a subject suffering from cancer is performed by the assay of 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, 35 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, CD207 CD226, CD244, CD267, CD268, CD269, CD355, CD358, NKG2A, NKG2B, NKG2C, NKG2D, NKG2E, 40 NKG2F, NKG2H, KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL5A, KIR2DL5B, KIR3DL1, KIR3DL2, KIR3DL3, KIR3DL4, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5, DAP12, KIR3DS, NKp44, NKp46, TCR,

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[0404] BCR, Integrins, FcβεRI, IL-6Rα, gp130, IL-7Rα, I

[0405]

[0406] 27Rα, IL-31Rα, OSMR, CSF-1R, cell-surface IL-15, IL-10Rα, IL-10Rβ, IL-20Rα, IL-20Rβ, IL-22Rα1, 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, 5 B7-DC, DR3, LIGHT, LAIR, LTα1β2, LTβR, TIM-1, TIM-3, TIM-4, TIGIT, LAG-3, ICOS, ICOS-L, SLAM, SLAMF2, OX-40, OX-40L, GITR, GITRL, TL1A, HVEM, 41-BB, 41BB-L, TL-1A, TRAF1, TRAF2, TRAF3, TRAF5, BAFF, BAFF-R, APRIL, TRAIL, RANK, AITR, TRAMP, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CCR11, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CXCR7, CLECL9a, DC-SIGN, IGSF4A, SIGLEC, EGFR, PDGFR, VEGFR, FAP, α-SMA, Vimentin, 10 Laminin, FAS, FAS-L, Fc, ICAM-1, ICAM-2, ICAM-3, ICAM-4, ICAM-5, PECAM-1, MICA, MICB, UL16, ULBP1, ULBP2, ILBP3, ULBP4, ULBP5, ULBP6, MULT1, RAE1 α, β, γ, δ, and ε, A1R, A2AR, A2BR, and A3R, H60a, H60b, and H60c. In some embodiments, the cell-surface protein is MHC-I. In various embodiments, Integrins are 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 combinations thereof. In various 15 embodiments, TCR is selected from the group consisting of a, β, γ, δ, ε, and ζ TCR. Several methods have been described in the literature for assaying of cell-surface protein expression, including Flow Cytometry and Mass Cytometry (CyTOF). The presence or abundance of one or more of these cell- surface proteins indicates that the patient is responsive to treatment with the method disclosed herein.

[0407] In various embodiments, administering a combination therapy described herein to a subject 20 having one or more tumors characterized as immune refractory, immunologically protected, or immunologically “cold” decreases the levels of tumor promoting, anti-inflammatory, or immune suppressive proteins in one or more tumor samples of the subject. In various embodiments, the tumor promoting, anti- inflammatory, or immune suppressive proteins are selected from the group consisting of CD39, CD79, MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, 25 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, kyneurinase, indoleamine 2,3 dioxygenase (IDO1 and IDO2), tryptophan 2,3 dioxygenase (TDO), myeloperoxidase (MPO), neutrophil elastase (NE), and IL4I1. In various embodiments, the levels of tumor promoting, anti-inflammatory, or immune suppressive proteins in one or more tumor samples of 30 the subject are decreased by 5-100% (e.g., decreased 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%, inclusive of 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 35 various embodiments, the levels of tumor promoting, anti-inflammatory, or immune suppressive proteins in one or more tumor samples of the subject are decreased by 2-100 fold (e.g., decreased relative to one or more samples collected prior to treatment 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, inclusive of all values and ranges between these values) compared to one or more tumor samples collected from the subject prior to treatment.

[0408] 40 In various embodiments, administering a combination therapy described herein to a subject having one or more tumors characterized as immune refractory, immunologically protected, or

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[0411] immunologically “cold” increases the levels of proteins associated with tumor growth inhibition, anti-tumor activity, or pro-inflammatory activity. In various embodiments, proteins associated with tumor growth inhibition, anti-tumor activity, 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, 5 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-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, and TNF-α. In various 10 embodiments, the levels of proteins associated with tumor growth inhibition, anti-tumor activity, or pro- inflammatory activity are increased by 5-100% (e.g., increased 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%, inclusive of all values and ranges between these values), 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 15 35-65%, 40-60%, 45-55%, 50%, or 100% compared to one or more tumor samples collected prior to treatment. In various embodiments, the levels of proteins associated with tumor growth inhibition, anti- tumor activity, or pro-inflammatory activity are increased by 2-100 fold (e.g., decreased relative to one or more samples collected prior to treatment 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, inclusive of all values and ranges between these values) compared to one 20 or more tumor samples collected from the subject prior to treatment. Several methods have been described in the literature for assaying proteins from tumor samples, including western blot, and ELISA.

[0412] In various embodiments, administering a combination therapy described herein to a subject having one or more tumors characterized as immune refractory, immunologically protected, or immunologically “cold” decreases the levels of neutrophil extracellular traps (NETs) in one or more tumor 25 samples collected from the subject. In various embodiments, the levels of NETs in one or more tumor samples is decreased by 5-100% (e.g., decreased 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%, inclusive of all values and ranges between these values), 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-30 60%, 45-55%, 50%, or 100% compared to one or more tumor samples collected prior to treatment. In various embodiments, the levels of NETs in one or more tumor samples is decreased by 2-100-fold (e.g., decreased relative to one or more samples collected prior to treatment 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, inclusive of all values and ranges between these values) compared to one or more tumor samples collected from the subject prior to treatment. 35 Several methods have been described in the literature for assaying NETs from tumor samples, including western blot, ELISA, and flow cytometry.

[0413] In various embodiments, administering a combination therapy described herein to a subject having one or more tumors characterized as immune refractory, immunologically protected, or immunologically “cold” decreases the expression of tumor promoting, tumor permissive, or immune 40 suppressive genes in one or more tumor samples of the subject. In one or more embodiments, the expression of tumor promoting, tumor permissive, or immune suppressive genes is decreased by 5-100%

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[0416] (e.g., decreased 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%, inclusive of 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% 5 compared to one or more tumor samples collected prior to treatment. In one or more embodiments, the expression of tumor promoting, tumor permissive, or immune suppressive genes is decreased by 2-100- fold (e.g., decreased relative to one or more samples collected prior to treatment 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, inclusive of all values and ranges between these values) compared to one or more tumor samples collected from the subject prior to 10 treatment.

[0417] In various embodiments, administering a combination therapy described herein to a subject having one or more tumors characterized as immune refractory, immunologically protected, or immunologically “cold” increases the expression of tumor inhibiting, anti-tumor, or pro-inflammatory genes in one or more samples collected from the subject. In one or more embodiments, the expression of tumor 15 inhibiting, anti-tumor, or pro-inflammatory genes is increased by 5-100% (e.g. increased 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%, inclusive of 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 20 samples collected prior to treatment. In various embodiments, the expression of tumor inhibiting, anti- tumor, or pro-inflammatory genes is increased by 2-100-fold (e.g., increased 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, inclusive of 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 gene expression analysis is performed by PCR, RT-PCR, qRT-25 PCR, next-generation sequencing (NGS), RNA-seq, ATAC-seq, exome sequencing, Southern Blot, microarray analysis, or single-cell sequencing.

[0418] In various embodiments, treatment of a subject having lung cancer with a combination therapy described herein switches the cold tumor to a hot tumor. Such switch can be detected using the methods described herein and known in the art. If the subject has been diagnosed with a tumor that has switched 30 from cold to hot tumor, treatment may continue by administering a combination therapy described herein, wherein the combination described herein is useful in treating hot tumors, or tumors that are immune cell rich or immunogenic.

[0419] In various embodiments, the subject in need thereof is administered the combination therapy of the disclosure for at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 35 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 15 months, at least 18 months, at least 21 months, or at least 23 months, e.g., for 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 15 months, 18 months, 21 months, 24 months or longer. In various embodiments, the subject is administered the combination therapy of the disclosure for 40 at least 1 month. In various embodiments, the subject is administered the combination therapy of the disclosure for at least 3 months. In various embodiments, the subject is administered the combination

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[0422] therapy of the disclosure for at least 6 months. In various embodiments, the subject is administered the combination therapy of the disclosure for at least 8 months. In various embodiments, the subject is administered the combination therapy of the disclosure for at least 10 months. In various embodiments, the subject is administered the combination therapy of the disclosure for at least 12 months.

[0423] 5 In various embodiments, administering to a subject in need thereof the combination therapy disclosed herein, synergistically increases depth or duration of response relative to a control subject or subject population. In some embodiments, duration is measured as progression-free survival. In some embodiments, depth of response is measured as a partial or a complete response.

[0424] A control subject or subject population refers to a subject or subject population having the same 10 cancer type as the treated subject (e.g., an immune refractory lung cancer or immunologically cold lung cancer) and is treated or has been treated with a RAS(ON) multi-selective monotherapy, a RAS(ON) G12C-selective monotherapy, a RAS(ON) multi-selective inhibitor plus an immune checkpoint inhibitor, or a RAS(ON) G12C-selective inhibitor plus an immune checkpoint inhibitor.

[0425] Response rates or results for subjects administered the combination therapy of the disclosure in 15 the methods disclosed herein can be measured in various ways, after the subject has been taking the combination therapy of the disclosure for a suitable length of time, as is known to those of skill in the art.

[0426] The subject can respond to the therapy as measured by at least a stable disease (SD), as determined by Response Evaluation Criteria in Solid Tumors (RECIST) 1.1 protocol (Eisenhauer, et al., 2009). RECIST v1.1 is discussed in detail in the examples below. An at least stable disease is one that is 20 a stable disease, has shown a partial response (PR) or has shown a complete response (CR) (i.e., “at least SD” = SD+PR+CR, often referred to as disease control). In various embodiments, the stable disease has neither sufficient shrinkage to qualify for partial response (PR) nor sufficient increase to qualify for progressive disease (PD). In various embodiments, the subject exhibits at least a partial response (i.e., “at least PR” = PR+CR, often referred to as objective response).

[0427] 25 Response can be measured by one or more of decrease in tumor size, suppression or decrease of tumor growth, decrease in target or tumor lesions, delayed time to progression, no new tumor or lesion, a decrease in new tumor formation, an increase in survival or progression-free survival (PFS), and no metastases. In various embodiments, the progression of a subject’s disease can be assessed by measuring tumor size, tumor lesions, or formation of new tumors or lesions, by assessing the subject 30 using a computerized tomography (CT) scan, a positron emission tomography (PET) scan, a magnetic resonance imaging (MRI) scan, an X-ray, ultrasound, or some combination thereof.

[0428] Several criteria and definitions published in the literature can be used to determine the effect of one or more treatments on tumors in a subject suffering from cancer. Based on these criteria, tumors are defined as “responsive,” “stable,” or “progressive” when they improve, remain the same, or worsen during 35 treatment, respectively. The amount of a tumor in an individual is the "tumor burden" which can be measured as the number, volume, and / or weight of the tumor.

[0429] Examples of the 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 40 Association for the Study of the Liver (EASL) Criteria, Response Evaluation Criteria in the Cancer of the Liver (RECICL), and WHO Criteria in Tumor Response.

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[0432] As used herein, "progression free survival" or “PFS” is the time from treatment to the date of the first confirmed disease progression per RECIST 1.1 criteria. In various embodiments, the subject exhibits a PFS of at least 1 month. In various embodiments, the subject exhibits a PFS of at least 3 months. In some embodiments, the subject exhibits a PFS of at least 6 months. In some embodiments, the subject 5 exhibits a PFS of at least 8 months. In some embodiments, the subject exhibits a PFS of at least 10 months. In some embodiments, the subject exhibits a PFS of at least 12 months.

[0433] “RECIST” shall mean an acronym that stands for “Response Evaluation Criteria in Solid Tumors” and is a set of published rules that define when cancer subjects improve (“respond”), stay the same (“stable”) or worsen (“progression”) during treatments. Response as defined by RECIST criteria have 10 been published, for example, a Journal of the National Cancer Institute, Vol.92, No.3, Feb.2, 2000 and RECIST criteria can include other similar published definitions and rule sets. One skilled in the art would understand definitions that go with RECIST criteria, as used herein, such as “Partial Response (PR),” “Complete Response (CR),” “Stable Disease (SD)” and “Progressive Disease (PD).”

[0434] As used herein, "survival" refers to the subject remaining alive, and includes overall survival as 15 well as progression free survival.

[0435] In various embodiments, the immune checkpoint modulators 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 lmmunoglobulin and mucin-domain containing-3 (TIM-3), Lymphocyte- activation Gene 3 (LAG-3), or TIGIT (T cell immunoreceptor with lg and ITIM domains). In various 20 embodiments, the immune checkpoint modulator is an antibody selected from the group consisting of ipilimumab, tremelimumab, pembrolizumab, nivolumab, atezolizumab, avelumab, cemiplimab, and durvalumab.

[0436] In various embodiments, the subject has previously been treated with immunotherapy but has developed resistance to immunotherapy or had a shift from a hot tumor to a cold tumor. Also provided is a 25 method of treating a subject having cancer that has developed resistance to immunotherapy or developed a cold tumor comprising administering to the subject a combination therapy described herein.

[0437] Cancers having an Oncogenic RAS Mutation

[0438] In some embodiments, the invention discloses a method of treating lung cancer in a subject in 30 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 an immune refractory lung cancer.

[0439] Methods of detecting RAS mutations are known in the art. Such means include, but are not 35 limited to direct sequencing, and utilization of a high-sensitivity diagnostic assay (with CE-IVD mark), e.g., as described in Domagala, et al., Pol J Pathol 3: 145-164 (2012), incorporated herein by reference in its entirety, including TheraScreen PCR; AmoyDx; PNAClamp; RealQuality; EntroGen; LightMix; StripAssay; Hybcell plexA; Devyser; Surveyor; Cobas; and TheraScreen Pyro. See, also, e.g., WO 2020 / 106640.

[0440] In some embodiments, the cancer is non-small cell lung cancer, or any of the lung cancers 40 described herein, and the RAS mutation comprises a KRAS G12C mutation, an HRAS G12C mutation, or

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[0443] an NRAS 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 KRAS G12C mutation.

[0444] Also provided is a method of inhibiting a RAS protein in a cell, the method comprising contacting the cell with an effective amount of a combination therapy of the disclosure. A method of inhibiting 5 RAF-RAS binding, the method comprising contacting the cell with an effective amount of a combination therapy of the disclosure, is also provided. The cell may be a cancer cell. The cancer cell may be of any type of cancer described herein. The cell may be in vivo or in vitro.

[0445] Lung Cancers

[0446] 10 In some embodiments, the invention discloses a method of treating a lung cancer. In some embodiments, the lung cancer is an immune refractory lung cancer.

[0447] 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 cancers (NSCLC) 15 (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)).

[0448] Alternatively, in a different system, lung cancer can be classified into preinvasive lesions, 20 minimally invasive adenocarcinoma, and invasive adenocarcinoma (invasive mucinous adenocarcinoma, mucinous bronchioloalveolar carcinoma (BAC), colloid, fetal (low and high grade), and enteric). Non-small cell lung cancer comprises adenocarcinoma, squamous cell carcinoma, large cell carcinoma, or large cell neuroendocrine tumors.

[0449] More frequently, lung cancer may be categorized as either small cell lung cancer (“SCLC”) or 25 non-small cell lung cancer (“NSCLC”). NSCLCs may be further categorized as squamous or non- squamous. An example of a non-squamous NSCLC is adenocarcinoma.

[0450] In some embodiments, lung cancer is bronchogenic carcinoma (squamous cell, undifferentiated small cell, undifferentiated large cell, adenocarcinoma), alveolar (bronchiolar) carcinoma, bronchial adenoma, sarcoma, lymphoma, chondromatous hamartoma, or mesothelioma.

[0451] 30 The lung cancer may be newly diagnosed and naïve to treatment, or may be relapsed, refractory, relapsed and refractory, locally advanced, or metastatic. In some instances, the lung cancer comprises a relapsed or refractory lung cancer. In some instances, the lung cancer comprises a metastatic lung cancer. In some cases, the subject is diagnosed with a relapsed or refractory lung cancer. In additional cases, the subject is diagnosed with a metastatic lung cancer.

[0452] 35 The compounds described herein can be used in combination with the agents disclosed herein or other suitable agents, depending on the condition being treated. Hence, in some embodiments the one or more compounds of the disclosure will be co-administered with other therapies as described herein. When used in combination therapy, the compounds described herein may be administered with the second agent simultaneously or separately. This administration in combination can include simultaneous 40 administration of the two agents in the same dosage form, simultaneous administration in separate dosage forms, and separate administration. That is, a compound described herein and any of the agents

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[0455] described herein can be formulated together in the same dosage form and administered simultaneously. Alternatively, a compound of the invention and any of the therapies described herein can be simultaneously administered, wherein both the agents are present in separate formulations. In another alternative, a compound of the present disclosure can be administered and followed by any of the 5 therapies described herein, or vice versa. In some embodiments of the separate administration protocol, a compound of the invention and any of the therapies described herein are administered a few minutes apart, or a few hours apart, or a few days apart.

[0456] In some embodiments of any of the methods described herein, the first therapy (e.g., a compound of the invention) and one or more additional therapies are administered simultaneously or sequentially, in 10 either order. The first therapeutic agent may be administered immediately, up to 1 hour, up to 2 hours, up to 3 hours, up to 4 hours, up to 5 hours, up to 6 hours, up to 7 hours, up to, 8 hours, up to 9 hours, up to 10 hours, up to 11 hours, up to 12 hours, up to 13 hours, 14 hours, up to hours 16, up to 17 hours, up 18 hours, up to 19 hours up to 20 hours, up to 21 hours, up to 22 hours, up to 23 hours, up to 24 hours, or up to 1-7, 1-14, 1-21 or 1-30 days before or after the one or more additional therapies.

[0457] 15 In some embodiments, the present disclosure provides methods of administering Compound B to a subject in need thereof (e.g., a subject afflicted with a RAS G12C mutant cancer, e.g., lung cancer (NSCLC), pancreatic cancer (e.g., pancreatic adenocarcinoma or pancreatic ductal adenocarcinoma), and colorectal cancer), comprising administering to the subject a therapeutically effective amount of Compound B, and avoiding co-administration of a proton pump inhibitor, wherein said subject is also in 20 need of the proton pump inhibitor. Exemplary proton pump inhibitors include but are not limited to omeprazole, lansoprazole, dexlansoprazole, rabeprazole, pantoprazole, and esomeprazole. In some embodiments, the proton pump inhibitor is esomeprazole.

[0458] In some embodiments, the present disclosure provides methods of administering Compound B to a subject in need thereof (e.g. a subject afflicted with a RAS G12C mutant cancer, e.g., lung cancer 25 (NSCLC), pancreatic cancer (e.g., pancreatic adenocarcinoma or pancreatic ductal adenocarcinoma), and colorectal cancer), comprising discontinuing administration of a proton pump inhibitor to avoid an adverse drug interaction with Compound B, and administering to the subject a therapeutically effective amount of Compound B. In some embodiments, the proton pump inhibitor is discontinued within 1 month prior to being administered Compound B. In some embodiments, the proton pump inhibitor is

[0459] 30 discontinued within 2 weeks prior to being administered Compound B. In some embodiments, the subject is advised that co-administration of Compound B and the proton pump inhibitor can alter the therapeutic effect or adverse reaction profile of Compound B.

[0460] In some embodiments, the present disclosure provides methods of administering Compound B to a subject in need thereof (e.g. a subject afflicted with a RAS G12C mutant cancer, e.g., lung cancer 35 (NSCLC), pancreatic cancer (e.g., pancreatic adenocarcinoma or pancreatic ductal adenocarcinoma), and colorectal cancer), wherein said subject is also in need of a proton pump inhibitor, comprising administering to the subject a therapeutically effective amount of Compound B while avoiding proton pump inhibitor co-administration, and any one or more of the following:

[0461] (a) advising the subject that proton pump inhibitors should be avoided or discontinued,

[0462] 40 (b) advising the subject that co-administration of Compound B with drugs that are proton pump inhibitors can alter the therapeutic effect or adverse reaction profile of Compound B,

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[0465] (c) advising the subject that co-administration of Compound B with proton pump inhibitors can alter the therapeutic effect or adverse reaction profile of Compound B,

[0466] (d) advising the subject that use of Compound B in subjects being treated with proton pump inhibitors is contraindicated, or

[0467] 5 (e) advising the subject that proton pump inhibitors should be used with caution in subjects receiving Compound B due to the potential for an adverse reaction profile. In some embodiments, the methos further comprising avoiding administering a proton pump inhibitor. In some embodiments, the methods further comprising discontinuing administration of a proton pump inhibitor.

[0468] The invention also features kits including (a) a pharmaceutical composition including an agent 10 (e.g., a compound of the invention) described herein, and (b) a package insert with instructions to perform any of the methods described herein. In some embodiments, the kit includes (a) a pharmaceutical composition including an agent (e.g., a compound of the invention) described herein, (b) one or more additional therapies (e.g., non-drug treatment or therapeutic agent), and (c) a package insert with instructions to perform any of the methods described herein.

[0469] 15 As one aspect of the present invention contemplates the treatment of the disease or symptoms associated therewith with a combination of pharmaceutically active compounds that may be administered separately, the invention further relates to combining separate pharmaceutical compositions in kit form. The kit may comprise two separate pharmaceutical compositions: a compound of the present invention, and one or more additional therapies. The kit may comprise a container for containing the separate 20 compositions such as a divided bottle or a divided foil packet. Additional examples of containers include syringes, boxes, and bags. In some embodiments, the kit may comprise directions for the use of the separate components. The kit form is particularly advantageous when the separate components are preferably administered in different dosage forms (e.g., oral, and parenteral), are administered at different dosage intervals, or when titration of the individual components of the combination is desired by the 25 prescribing health care professional.

[0470] III. Combination Therapy

[0471] The methods of the disclosure may include a combination therapy of the present disclosure (e.g., RAS(ON) multi-selective, RAS(ON) G12C-selective, and ICI) used alone or in combination with one or 30 more additional therapies (e.g., non-drug treatments or therapeutic agents). The dosages of one or more of the additional therapies (e.g., non-drug treatments or therapeutic agents) may be reduced from standard dosages when administered alone. For example, doses may be determined empirically from drug combinations and permutations or may be deduced by isobolographic analysis (e.g., Black et al., Neurology 65:S3-S6 (2005)).

[0472] 35 A combination therapy of the present disclosure may be administered before, after, or concurrently with one or more of such additional therapies. When combined, dosages of a combination therapy of the present disclosure and dosages of the one or more additional therapies (e.g., non-drug treatment or therapeutic agent) provide a therapeutic effect (e.g., synergistic or additive therapeutic effect). A combination therapy of the present disclosure and an additional therapy, such as an anti-cancer 40 agent, may be administered together, such as in a unitary pharmaceutical composition, or separately and,

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[0475] when administered separately, this may occur simultaneously or sequentially. Such sequential administration may be close or remote in time.

[0476] In certain embodiments, compositions of the disclosure comprise a combination therapy of the present disclosure and one additional therapeutic agent. In certain embodiments, compositions of the 5 disclosure comprise a combination therapy of the present disclosure and two additional therapeutic agents. In certain embodiments, compositions of the disclosure comprise a combination therapy of the present disclosure and three additional therapeutic agents. In certain embodiments, compositions of the disclosure comprise a combination therapy of the present disclosure and four or more additional therapeutic agents.

[0477] 10 Also provided are pharmaceutical compositions including the combinations, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient. Compositions comprising a combination of therapeutic agents may be used in methods of modulating RAS (e.g., in a subject or in a cell) and in methods of treating RAS related diseases and disorders (e.g., cancer), as described herein. The present disclosure provides, inter alia, compositions, methods, and kits for treating or preventing a 15 RAS related disease or disorder.

[0478] Exemplary agents that may be used in combination with a compound of the present disclosure are described below. All references herein are incorporated by reference for the agents described, including compound or molecular structures disclosed therein, whether explicitly stated as such or not.

[0479] RAS / MAPK Inhibitors

[0480] 20 Compositions and methods described herein may include a compound of the present disclosure in combination with one or more RAS / MAPK pathway inhibitors. The RAS / MAPK pathway is a 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 the proliferation, activation, differentiation, mobilization, and other functional 25 properties of the cell. SHP2 conveys positive signals from growth factor receptors to the RAS activation / deactivation cycle, which is modulated by guanine nucleotide exchange factors (GEFs, such as SOS1) that load GTP onto RAS to produce functionally active GTP-bound RAS as well as GTP- accelerating proteins (GAPs, such as NF1) that facilitate termination of the signals by conversion of GTP to GDP. GTP-bound RAS produced by this cycle conveys essential positive signals to a series of 30 serine / threonine kinases including RAF and MAP kinases, from which emanate additional signals to various cellular effector functions. In some embodiments, a therapeutic agent that may be combined with a RAS(ON) inhibitor is an inhibitor of the MAP kinase (MAPK) pathway (or “MAPK pathway inhibitor”). MAPK pathway inhibitors include, but are not limited to, one or more MAPK pathway inhibitors described in Cancers (Basel) 2015 Sep; 7(3): 1758–1784. For example, the MAPK inhibitor may be selected from 35 one or more of trametinib, binimetinib, selumetinib, cobimetinib, LErafAON (NeoPharm), ISIS 5132; vemurafenib, pimasertib, TAK733, RO4987655 (CH4987655); CI-1040; PD-0325901; CH5126766;

[0481] MAP855; AZD6244; refametinib (RDEA 119 / BAY 86-9766); GDC-0973 / XL581; AZD8330 (ARRY- 424704 / ARRY-704); RO5126766 (Roche, described in 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 pathway inhibitor 40 may be PLX8394, LXH254, GDC-5573, or LY3009120. A MAPK pathway inhibitor may be a PI3Kα:RAS breaker, such as BBO-10203.

[0482] 62 PATENT

[0483] ATTORNEY DOCKET NO.: 51432-077WO2

[0484] i) RAS(OFF) inhibitors, RAS(OFF) degraders and other RAS inhibitor types Compositions and methods described herein may include a compound of the present disclosure in combination with one or more RAS(OFF) inhibitors. Numerous mutant-selective and pan-KRAS inhibitors have been disclosed and are known in the art. A RAS(OFF) inhibitor may be administered or 5 formulated in combination with a RAS(ON) inhibitor described herein. RAS(OFF) inhibitors are designed to inhibit RAS activity by targeting different regions of the RAS protein in its inactive state (GDP bound state), preventing its activation and downstream signaling.

[0485] In some embodiments, a RAS(OFF) inhibitor is a KRAS(OFF) inhibitor that has a molecular weight of under 700 Da. In some embodiments, the KRAS(OFF) inhibitor is specific for a KRASG12C10 mutation. KRASG12C(OFF) inhibitors use a covalent binding group that allows them to selectively target the KRASG12Cmutant protein, and many such inhibitors comprise a pyrimidine core. KRASG12C(OFF) inhibitors all target the same cysteine residue in the KRASG12Cmutant protein, leading to a conformational change that locks the protein in an inactive state. KRASG12C(OFF) inhibitors include, but are not limited to, adagrasib (MRTX849), divarasib (RG6330 / GDC-6036), fulzerasib (IBI351 / GFH925), garsorasib (D-1553), 15 glecirasib (JAB-21822), olomorasib (LY3537982), opnurasib (JDQ443), sotorasib (AMG 510), ARS-853, ARS-1620, BI-0474, BI 1823911, BPI-421286, D3S-001, ERAS-3490, GEC255, GH35, HBI-2438, HS- 10370, JAB-21000, JAB-21822, JMKX001899, JNJ-74699157 (ARS-3248), MK-1084, SK-17, and YL- 15293, HRS-7058. In some embodiments, the KRAS(OFF) inhibitor is selected from AMG510 and MRTX849. In some embodiments, the KRAS(OFF) inhibitor is AMG510. In some embodiments, the 20 KRAS(OFF) inhibitor is MRTX849. In some embodiments, the KRAS(OFF) inhibitor is GDC-6036. A RAS(OFF) inhibitor may be an antibody-drug conjugate. See also doi.org / 10.1021 / acs.jmedchem.4c02929.

[0486] In some embodiments, a KRAS(OFF) inhibitor is specific for a KRASG12Dmutation. Non-limiting examples of KRASG12D(OFF) inhibitors include AST2169, BPI-501836, DN022150, ERAS-4693, ERAS-25 5024, GDC-7035 (RG6620), HBW-012-D, HBW-012-E, HBW-012336, HRS-4642, HS-10529, INCB186748, JAB-22000, KD-8, KRB-456, LY3962673, MRTX282, MRTX1133, Q2a, QLC1101, RNK08954, SHR1127, TH-Z827, TH-Z835, TSN1611, and VRTX153, HJ-119, JR-6000, NKT-G12D, FWD-K02, JAB-BX600, EB-TM1, ABSK141, BPI-2491, HRS-6093, HRS-7172, .

[0487] In some embodiments, the small molecule RAS(OFF) inhibitor is specific for a KRASG12V30 mutation (e.g., JAB-23000, QTX3544). In some embodiments, the small molecule RAS(OFF) inhibitor is specific for a KRASG13Dmutation.

[0488] In some embodiments, reference to the term RAS(OFF) inhibitor includes any such RAS(OFF) inhibitor disclosed in any one of the following patent applications: WO 2025179058, WO 2025170938, WO 2025168072, WO 2025167948, WO 2025165972, WO 2025162091, WO 2025157289, WO

[0489] 35 2025157260, WO 2025157246, WO 2025157162, WO 2025153038, WO 2025151738, WO 2025151594, WO 2025148979, WO 2025146194, WO 2025136346, WO 2025132549, WO 2025130912, WO 2025129002, WO 2025124415, WO 2025123318, WO 2025123007, WO 2025122619, WO 2025117828, WO 2025111586, WO 2025111582, WO 2025108443, WO 2025106905, WO 2025106901, WO 2025101776, WO 2025096984, WO 2025096957, WO 2025096738, WO 2025092986, WO 2025092798, 40 WO 2025085748, WO 2025085580, WO 2025080653, WO 2025077770, WO 2025077663, WO 2025076523, WO 2025072649, WO 2025072457, WO 2025072451, WO 2025067459, WO 2025067453,

[0490] 63 PATENT

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[0492] WO 2025064848, WO 2025064542, WO 2025061125, WO 2025059366, WO 2025059040, WO 2025054530, WO 2025054347, WO 2025054270, WO 2025053850, WO 2025051242, WO 2025045141, WO 2025049641, WO 2025049619, WO 2025049402, WO 2025049274, WO 2025040767, WO 2025038936, WO 2025036475, WO 2025036470, WO 2025034883, WO 2025034849, WO 2025026903, 5 WO 2025019688, WO 2025018418, WO 2025016899, WO 2025016432, WO 2025011443, WO 2025010415, WO 2025007000, WO 2025006967, WO 2025006962, WO 2025006720, WO 2025006704, WO 2025002430, WO 2025002302, WO 2024259169, WO 2024254404, WO 2024254334, WO 2024255795, WO 2024246099, WO 2024243025, WO 2024238633, WO 2024238343, WO 2024236452, WO 2024235286, WO 2024235225, WO 2024233776, WO 2024230734, WO 2024230707, WO

[0493] 10 2024229447, WO 2024229444, WO 2024229442, WO 2024229317, WO 2024227091, WO 2024220645, WO 2024220532, WO 2024218686, WO 2024215862, WO 2024215754, WO 2024213979, WO 2024213122, WO 2024208305, WO 2024209339, WO 2024206766, WO 2024206747, WO 2024197503, WO 2024193698, WO 2024192424, WO 2024179546, WO 2024178313, WO 2024178304, WO 2024173842, WO 2024167922, WO 2024160225, WO 2024159471, WO 2024159470, WO 2024158778, 15 WO 2024158242, WO 2024153119, WO 2024153116, WO 2024138486, WO 2024138206, WO 2024138052, WO 2024131829, WO 2024125642, WO 2024125600, WO 2024123913, WO 2024123102, WO 2024120433, WO 2024120419, WO 2024119277, WO 2024118926, WO 2024109233, WO 2024112654, WO 2024104453, WO 2024104425, WO 2024107686, WO 2024104453, WO 2024103010, WO 2024097559, WO 2024091409, WO 2024088273, WO 2024085661, WO 2024083258, WO

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[0496] 40 2023173016, WO 2023173014, WO 2023172737, WO 2023171781, WO 2023159087, WO 2023159086, WO 2023154766, WO 2023152255, WO 2023151674, WO 2023151621, WO 2023150394, WO

[0497] 64 PATENT

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[0499] 2023150284, WO 2023143623, WO 2023143605, WO 2023143352, WO 2023143352, WO 2023143312, WO 2023141570, WO 2023141300, WO 2023138662, WO 2023138601, WO 2023138589, WO 2023138524, WO 2023133183, WO 2023133181, WO 2023130012, WO 2023125989, WO 2023125627, WO 2023122662, WO 2023122154, WO 2023120742, WO 2023119677, WO 2023117681, WO

[0500] 5 2023116934, WO 2023116895, WO 2023114733, WO 2023105491, WO 2023104018, WO 2023103906, WO 2023103523, WO 2023101928, WO 2023099624, WO 2023099624, WO 2023099620, WO 2023099612, WO 2023099608, WO 2023099592, WO 2023098832, WO 2023098425, WO 2023097227, WO 2023081840, WO 2023081476, WO 2023078424, WO 2023077441, WO 2023072297, WO 2023072188, WO 2023066371, WO 2023064857, WO 2023061463, WO 2023061294, WO 2023057985, 10 WO 2023056951, WO 2023056421, WO 2023051586, WO 2023049697, WO 2023046135, WO 2023045960, WO 2023041059, WO 2023041059, WO 2023040989, WO 2023040513, WO 2023039240, WO 2023039020, WO 2023036282, WO 2023034290, WO 2023030517, WO 2023030495, WO 2023030385, WO 2023030495, WO 2023030517, WO 2023030685, WO 2023030687, WO 2023034290, WO 2023036282, WO 2023039240, WO 203020347, WO 2023025116, WO 2023287896, WO

[0501] 15 2023287730, WO 2023284881, WO 2023284730, WO 2023284537, WO 2023283933, WO 2023283213, WO 2023280280, WO 2023280136, WO 2023280026, WO 2023278600, WO 2023274383, WO 2023327324, WO 2023040989, WO 2023039240, WO 2023039020, WO 2023036282, WO 2023034290, WO 2023030517, WO 2023030495, WO 2023030385, WO 2023025116, WO 2023020523, WO 2023020521, WO 2023020519, WO 2023020518, WO 2023020347, WO 2023018812, WO 2023018810, 20 WO 2023018809, WO 2023018699, WO 2023014979, WO 2023014006, WO 2023004102, WO 2023003417, WO 2023001141, WO 2023001123, WO 2022271658, WO 2022269508, WO 2022266167, WO 2022266069, WO 2022266015, WO 2022265974, WO 2022261154, WO 2022261154, WO 2022251576, WO 2022251296, WO 2022237815, WO 2022232332, WO 2022232331, WO 2022232320, WO 2022232318, WO 2022223037, WO 2022221739, WO 2022221528, WO 2022221386, WO

[0502] 25 2022216762 (e.g., Compound 44 or Compound 66a), WO 2022212894, WO 2022192794, WO 2022192790, WO 2022188729, WO 2022187411, WO 2022184178, WO 2022173870, WO 2022173678, WO 2022135346, WO 2022133731, WO 2022133038, WO 2022133345, WO 2022132200, WO 2022119748, WO 2022109485, WO 2022109487, WO 2022066805, WO 2022002102, WO 2022002018, WO 2021259331, WO 2021257828, WO 2021252339, WO 2021248095, WO 2021248090, WO

[0503] 30 2021248083, WO 2021248082, WO 2021248079, WO 2021248055, WO 2021245051, WO 2021244603, WO 2021239058, WO 2021231526, WO 2021228161, WO 2021219090, WO 2021219090, WO 2021219072, WO 2021218939, WO 2021217019, WO 2021216770, WO 2021215545, WO 2021215544, WO 2021211864, WO 2021190467, WO 2021185233, WO 2021180181, WO 2021175199, 2021173923, WO 2021169990, WO 2021169963, WO 2021168193, WO 2021158071, WO 2021155716, WO

[0504] 35 2021152149, WO 2021150613, WO 2021147967, WO 2021147965, WO 2021143693, WO 2021142252, WO 2021141628, WO 2021139748, WO 2021139678, WO 2021129824, WO 2021129820, WO 2021127404, WO 2021126816, WO 2021126799, WO 2021124222, WO 2021121371, WO 2021121367, WO 2021121330, WO 2021113595, WO 2021107160, WO 2021106231, WO 2021088458, WO 2021086833, WO 2021085653, WO 2021081212, WO 2021058018, WO 2021057832, WO 2021055728, 40 WO 2021031952, WO 2021027911, WO 2021023247, WO 2020259513, WO 2020259432, WO 2020234103, WO 2020233592, WO 2020216190, WO 2020178282, WO 2020146613, WO 2020118066,

[0505] 65 PATENT

[0506] ATTORNEY DOCKET NO.: 51432-077WO2

[0507] WO 2020113071, WO 2020106647, WO 2020102730, WO 2020101736, WO 2020097537, WO 2020086739, WO 2020081282, WO 2020050890, WO 2020047192, WO 2020035031, WO 2020028706, WO 2019241157, WO 2019232419, WO 2019217691, WO 2019217307, WO 2019215203, WO 2019213526, WO 2019213516, WO 2019155399, WO 2019150305, WO 2019110751, WO 2019099524, 5 WO 2019051291, WO 2018218070, WO 2018218071, WO 2018218069, WO 2018217651, WO 2018206539, WO 2018143315, WO 2018140600, WO 2018140599, WO 2018140598, WO 2018140514, WO 2018140513, WO 2018140512, WO 2018119183, WO 2018112420, WO 2018068017, WO 2018064510, WO 2017201161, WO 2017172979, WO 2017100546, WO 2017087528, WO 2017058807, WO 2017058805, WO 2017058728, WO 2017058902, WO 2017058792, WO 2017058768, WO

[0508] 10 2017058915, WO 2017015562, WO 2016168540, WO 2016164675, WO 2016049568, WO 2016049524, WO 2015054572, WO 2014152588, WO 2014143659, WO 2013155223, KR 20250100539, KR102819454, KR 20240159370, KR 20240101190, KR 20240101189, KR 20240041720, KR 20240041719, JP 2025100453, CN 120535536, CN 120535527, CN 120441596, CN 120230123, CN 119607214, CN 119930639, CN 119909188, CN 119751476, CN 119733053, CN 119684316, CN 15 119684315, CN 119684314, CN 119661556, CN 119661555, CN 119661539, CN 119606974, CN 119528902, CN 119528810, CN 119504612, CN 119490514, CN 119490512, CN 119462648, CN 119371353, CN 119350242, CN 119264124, CN 119241566, CN 119060049, CN 119060066, CN 119019382, CN 118994158, CN 118994031, CN 118806919, CN 118791505, CN 118772176, CN 118754899, CN 118745175, CN 118666870, CN118666869, CN 118580238, CN 118307563, CN 20 118221700, CN 118221699, CN 118221698, CN 118221685, CN 118126064, CN 118078802, CN 118078801, CN 118005656, CN 117986263, CN 117986263, CN 117946135, CN 117924327, CN 117903117, CN 117800990, CN 117800989, CN 117800976, CN 117736226, CN 117683051, CN 117645627, CN 117624194, CN 117624190, CN 117586280, CN 117486901, CN 117466917, CN 117462688, CN 117362315, CN 117327102, CN 117327094, CN 117327074, CN 117285590, CN 25 117263959, CN 117247382, CN 117186095, CN 117164605, CN 116969977, CN 116925075, CN 116891489, CN 116731045, CN 116731044, CN 116554208, CN 116514846, CN 116478184, CN 116478141, CN 116410145, CN 116375742, CN 116354988, CN 116332948, CN 116332938, CN 116327956, CN 116262759, CN 116217592, CN 116199703, CN 116162099, CN 116143806, CN 116143805, CN 116120315, CN 116102559, CN 115960105, CN 115894520, CN 115872979, CN 30 115850267, CN 115785199, CN 115785124, CN 115724842, CN 115724842, CN 115721720, CN 115716840, CN 115703775, CN 115611923, CN 115611898, CN 115583937, CN 115572278, CN 115557949, CN 115521312, CN 115504976, CN 115490709, CN 115466272, CN 115433183, CN 115433179, CN 115403575, CN 115385938, CN 115385937, CN 115385912, CN 115381786, CN 115368383, CN 115368382, CN 115368381, CN 115353506, CN 115322158, CN 115304623, CN 35 115304602, CN 115197245, CN 115181106, CN 114989195, CN 114989166, CN 114989147, CN 114920741, CN 114920739, CN 114907387, CN 114874234, CN 114874201, CN 114716436, CN 114716435, CN 114685532, CN 114685460, CN 114591319, CN 114539293, CN 114539286, CN 114539246, CN 114437107, CN 114437084, CN 114409653, CN 114380827, CN 114195804, CN 114195788, CN 114437107, CN 114409653, CN 114380827, CN 114195804, CN 114057776, CN 40 114057744, CN 114057743, CN 113999226, CN 113980032, CN 113980014, CN 113960193, CN 113929676, CN 113754653, CN 113683616, CN 113563323, CN 113527299, CN 113527294, CN

[0509] 66 PATENT

[0510] ATTORNEY DOCKET NO.: 51432-077WO2

[0511] 113527293, CN 113493440, CN 113429405, CN 113321654, CN 113248521, CN 113087700, CN 113024544, CN 113004269, CN 112920183, CN 112778284, CN 112390818, CN 112390788, CN 112300196, CN 112300194, CN 112300173, CN 112225734, CN 112142735, CN 112110918, CN 112094269, CN 112047937, CN 109574871, US 12331063, US 2025115603, US 2025114346, US 5 2025114339, US 20240358702, US 2024270736, EP 4574151, or EP 4389751, each of which is incorporated herein by reference in its entirety, including the RAS compound structures disclosed therein which are specifically incorporated herein by reference.

[0512] In some embodiments, reference to the term RAS(OFF) inhibitor refers to a pan-KRAS inhibitor, such as selected from one disclosed in any of the following: WO 2025165972, WO 2025153038, WO 10 2025151594, WO 2025136346, WO 2025130912, WO 2025129002, WO 2025123007, WO 2025117828, WO 2025106905, WO 2025106901, WO 2025101776, WO 2025096738, WO 2025092798, WO 2025085748, WO 2025077770, WO 2025077663, WO 2025076523, WO 2025064848, WO 2025059366, WO 2025059040, WO 2025049641, WO 2025049619, WO 2025049402, WO 2025045141, WO 2025038936, WO 2025026903, WO 2025016899, WO 2025007000, WO 2025006967, WO 2025006962, 15 WO 2025006720, WO 2025006704, WO 2024255795, WO 2024254404, WO 2024246099, WO 2024238633, WO 2024238343, WO 2024236452, WO 2024235286, WO 2024235225, WO 2024230734, WO 2024220645, WO 2024220532, WO 2024218686, WO 2024215754, WO 2024213979, WO 2024213122, WO 2024209339, WO 2024206766, WO 2024206747, WO 2024192424, WO 2024178313, WO 2024178304, WO 2024173842, WO2024153180, WO 2024119277, WO 2024120433, WO

[0513] 20 2024115890, WO 2024112654, WO 2024104453, WO 2024104425, WO 2024107686, WO 2024104453, WO 2024103010, WO 2024085661, WO 2024083246, WO 2024083168, WO 2024067575, WO 2024064335, WO 2024063578, WO 2024063576, WO 2024051852, WO 2024051763, WO 2024046370, WO 2024044667, WO 2024041621, WO 2024041606, WO 2024041589, WO 2024040131, WO 2024040109, WO 2024032747, WO 2024032704, WO 2024032703, WO 2024032702, WO 2024031088, 25 WO 2024030647, WO 2024030633, WO 2024015262, WO 2024009191, WO 2024008068, WO 2024002373, WO 2023287896, WO 2023274324, WO 2023246914 (e.g., compound 14), WO 2023246777, WO 2023230190, WO 2023215802, WO 2023215801, WO 2023197984, WO 2023190748, WO 2023183585, WO 2023179703, WO 2023173017, WO 2023173016, WO 2023173014, WO 2023172737, WO 2023154766, WO 2023143352, WO 2023143312, WO 2023138589, WO 2023133183, 30 WO 2023122662, WO 2023114733, WO 2023099624, WO 2023099623, WO 2023099612, WO 2023099608, WO 2023099592, WO 2023097227, WO 2023064857, WO 2023056421, WO 2023049697, WO 2023046135, WO 2023039240, WO 2023034290, WO 2023020523, WO 2023020521, WO 2023020519, WO 2023020518, WO 2023001123, WO 2022271823, WO 2022261210, WO 2022258974, WO 2022256459, WO 2022250170, WO 2022248885, WO 2022228543, WO 2022216762, WO

[0514] 35 2022072783, WO 2016161361, KR 20250100539, KR 20240101190, KR 20240101189, KR 20240041720, KR 20240041719, JP 2025100453, CN 120535536, CN 120441596, CN 120365289, CN 119751476, CN 119661539, CN 119371353, CN 119019382, CN 118791505, CN 118221700, CN 118126064, CN 117924327, CN 117946135, CN 117800990, CN 117800989, CN 117683051, CN 117486901, CN 117263959, CN 116969977, CN 116332948, or US 12331063, each of which is 40 incorporated herein by reference in its entirety, including the compound structures disclosed therein. In some embodiments, combination comprising a pan-KRAS inhibitor therapy comprises ERAS-4001. In

[0515] 67 PATENT

[0516] ATTORNEY DOCKET NO.: 51432-077WO2

[0517] some embodiments, the pan-KRAS inhibitor is a pan-KRAS inhibitor in a patent application filed in the name of Medshine Discovery, Inc. In some embodiments, a combination comprising a pan-KRAS inhibitor therapy includes A2A-03, ABREV01, ADT-007, ABT-200, ADT-030, ADT-1004, BBP-454, BGB-53038, BI-2865, BI-2493, BI 3706674, BRSD-143, ERAS-4, ERAS-254, ERAS-4001, HB-700 (G12X+G13D), HZ- 5 V068, ID12241161, JAB-23400, LY4066434, OC211, PF-07985045, PF-07934040, PF-4040, QTX2024, QTX3034, RSC-1255, SIL204, SYNB021225, YL-17231, ZG2001, WEF-001.

[0518] In some embodiments, a RAS inhibitor binds to the OFF form as well as the ON form. Non- limiting examples of such inhibitors include, e.g., pan-KRAS: ALTA3263, AMG 410, BBO-11818, HBW- 012462, HBW-016-K, HEC211909, JAB-23E73, JAB-23425, JAB-23E73; or a compound disclosed in 10 Zheng, Q.; Shen, T.; Pampel, J.; Shokat, K.M.; Distal Covalent Targeting Suppresses Signaling of Oncogenic KRas-(G13C) in Cancer Cells. ACS Chem. Biol.2025, 20, 7, 1696-1706; G12C: BBO-8520, FMC-376; G12D: AZD0022, GFH375 (VS-7375), INCB161734, QTX3046, TSN1611, TH-Z835, TLN-372.

[0519] In some embodiments, a RAS inhibitor binds to the ON form of RAS but is not a tri-complex inhibitor, such as pan-KRAS inhibitors JTX-102 and JTX-105.

[0520] 15 In any embodiment employing a RAS(OFF) inhibitor herein, a RAS(OFF) degrader targeting the OFF state of RAS may be employed. These degraders are known in the art, such as ASP3082 (G12D) and ASP5834 (pan-KRAS). RAS degraders may be found, for example, in one or more of the following applications: WO 2025169901, WO 2025168124, WO 2025168051, WO 2025162250, WO 2025159142, WO 2025151765, WO 2025125630, WO 2025108479, WO 2025107579, WO 2025103476, WO

[0521] 20 2025096855, WO 2025085815, WO 2025083472, WO 2025078984, WO 2025076044, WO 2025058008, WO 2025053850, WO 2025024732, WO 2025019823, WO 2025006783, WO 2025006753, WO 2024263586, WO 2024261257, WO 2024261256, WO 2024241248, WO 2024233838, WO 2024199266, WO 2024188281, WO 2024 / 59164, WO 2024152247, WO 2024149214, WO 2024131777, WO 2024120424, WO 2024119278, WO 2024118966, WO 2024118960, WO 2024083258, WO 2024083256, 25 WO 2024055112, WO 2024054625, WO 2024050742, WO 2024044334, WO 2024040080, WO 2024034657, WO 2024034593, WO 2024034591, WO 2024034123, WO 2024029613, WO 2024020159, WO 2024019103, WO 2024017392, WO 2023215906, WO 2023185864, WO 2023171781, WO 2023141570, WO 2023138524, WO 2023130012, WO 2023116934, WO 2023099620, WO 2023081476, WO 2023077441, WO 2022260482, CN 120535501, CN 120463820, CN 120441554, CN 120365263, 30 CN 119219669, CN 119161349, CN 118955610, CN 118772249, CN 118725012, CN 118496502, CN 118496300, CN 118126040, CN 115785199, or US 2025213706, each of which is incorporated herein by reference in its entirety. Non-limiting examples of RAS degraders include: ASP3082 (KRAS G12D); ASP4396 (KRAS G12D); BPI-585725 (G12X and WT), LT-010366 (G12D); PT0253 (G12D), RD0255359 (KRAS G12C / D / V); RP03707 (G12D), JR-9000, 356A, SH1718, IPS-06061, HDB-82.

[0522] 35 In some embodiments, the RAS(OFF) inhibitor is a peptide-based inhibitor. Peptide-based RAS(OFF) inhibitors have been developed that target specific regions of the RAS protein, such as the Switch II region or the RAS-effector interface. Non-limiting examples include the K-Ras-binding peptide (Krpep-2d), the Ras inhibitory peptide (RasIn) and LUNA18 (NCT05012618). Peptide-based RAS(OFF) inhibitors are a class of compounds that target the RAS protein by disrupting its interaction with its 40 downstream effectors or other signaling proteins. These inhibitors are typically designed to mimic the binding motifs of RAS-interacting proteins or other RAS effectors, such as RAF or PI3K. By binding to

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[0525] RAS at the same site as these effectors, peptide-based inhibitors can effectively compete with these proteins and prevent the activation of downstream signaling pathways. See, e.g., WO 2025162428, WO 2025127968, WO 2025018418, WO 2024219480, WO 2024219446, WO 2024176153, WO 2024101402, WO 2024101386, WO 2023214576, WO 2023140329, WO 2022234853, WO 2022234852, WO

[0526] 5 2022234851, WO 2022234639 and CN 120040551, each of which is incorporated herein by reference in its entirety.

[0527] Peptide-based RAS(OFF) inhibitors can be further classified into two main categories: those that target the RAS-effector interface, and those that target other regions of the RAS protein. Peptide-based inhibitors that target the RAS-effector interface are designed to bind to the switch regions of RAS that are 10 critical for its interaction with downstream effectors, such as RAF or PI3K. These inhibitors typically contain amino acid residues that are similar to those found in the binding motifs of RAS-interacting proteins or effectors and are often designed to form hydrogen bonds or other interactions with key residues on the surface of RAS.

[0528] Peptide-based RAS(OFF) inhibitors that target other regions of the RAS protein are typically 15 designed to disrupt other interactions that are critical for the activation or signaling of RAS. For example, some peptide-based inhibitors are designed to bind to the hypervariable region of RAS, which is thought to play a role in membrane localization and anchoring of the protein. By binding to this region, peptide- based inhibitors can prevent the proper localization of RAS to the plasma membrane, which is necessary for its activation and signaling.

[0529] 20 Several common motifs have been identified as important for the binding of RAS-interacting proteins and effectors and are often used in the design of peptide-based inhibitors. One example is the RAF-binding domain (RBD), which is found in many RAS-interacting proteins and is important for the interaction of RAS with downstream effectors such as RAF. The RBD contains a conserved amino acid sequence (Arg-Xaa-Arg) that is critical for binding to RAS, and this motif has been incorporated into 25 several peptide-based inhibitors designed to disrupt the RAS-RAF interaction. Another example is the RAS-binding domain (RBD) of PI3K, which is important for the interaction of RAS with this downstream effector. The RBD of PI3K contains several conserved amino acid residues (such as Arg-Arg-Trp) that are critical for binding to RAS, and these motifs have been used in the design of peptide-based inhibitors that target the RAS-PI3K interaction. Other common motifs used in peptide-based RAS(OFF) inhibitors 30 include the Ras-binding domain (RBD) of other RAS-interacting proteins such as RalGDS and SOS, as well as sequences that mimic the structure of the switch regions of RAS itself. These motifs are typically used to optimize the binding affinity and selectivity of the inhibitor for the desired target protein or interaction.

[0530] In some embodiments, the RAS(OFF) inhibitor is an antibody or antigenic binding peptide specific 35 for RAS(OFF). Antibodies have been developed that bind to specific regions of the RAS protein, such as the Switch II region or the RAS-effector interface. For example, some antibodies have been developed that target the switch regions of RAS proteins, which are critical for the activation of these proteins and their interaction with downstream effectors. Binding of these antibodies to the switch regions can prevent the conformational changes required for RAS activation and downstream signaling. Another approach 40 involves the use of antibodies that target RAS-interacting proteins or downstream effectors, such as RAF or PI3K. Binding of these antibodies to their target proteins can disrupt the RAS-dependent signaling

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[0533] pathways and inhibit the growth and survival of cancer cells. Additionally, some antibodies have been developed that can induce the internalization and degradation of RAS proteins, leading to their depletion and inhibition of downstream signaling. For example, some antibodies have been developed that recognize the unique structure of mutant RAS proteins and target them for degradation via the ubiquitin- 5 proteasome pathway. Non-limiting examples of KRAS(OFF)-specific inhibitory antibodies include anti- p21ser, and K27 (DARPin) (see, e.g., Khan et al, Biochim Biophys Acta Mol Cell Res.2020 Feb;1867(2):118570). See also WO 2024136608 and WO 2024111590, each of which is incorporated herein by reference in its entirety.

[0534] Antibody-drug conjugates may also be constructed using RAS inhibitors (e.g., RAS(OFF) 10 inhibitors), such as WO 2024189481, which is incorporated herein by reference in its entirety, including the compound structures disclosed therein.

[0535] Vaccines may also be used in combination with compounds of the present invention. Non-limiting examples include: AFNT-111 (KRAS G12V), AFNT-211 (KRAS G12V), AFNT-212 (KRAS G12D), ELI- 002 (KRAS G12 / 13X), HB-700, NT-112 (KRAS G12D), and TG01 (pan-KRAS).

[0536] 15 Other RAS modalities useful in combination with compounds of the present invention include:

[0537] ADGN-123, ADGN-121 (gene editing peptide-RNA nanoparticles G12D); ADT-030 (Ras / B-catenin inhibitor); BBO-10203 (PI3Kα:RAS breaker); BI 1701963 (Pan-KRAS:SOS1); mRNA-5671 (nucleic acid) and RO7673396 (RAS inhibitor), AZD0240 (TCR-T cell product targeting G12D), MDG2021 (TCR-T cell product targeting G12D), ADGN-121 (peptide-sgRNA nanoparticles), BION-302 (antibody based) LIB111 20 (antibody based), SIL-204 (ASO / siRNA-based).

[0538] ii) SOS1 inhibitors

[0539] In some embodiments, compositions and methods described herein may include a combination therapy of the present disclosure in combination with one or more SOS1 inhibitors. A SOS1 inhibitor may be administered or formulated in combination with a combination therapy of the present disclosure and / or 25 any additional therapeutic agent described herein. In some embodiments, a SOS1 inhibitor is one or more of RMC-5845, RMC-4948, RMC-0331, BI-1701963, BI-1918455, BI-3406, SDR5, MRTX-0902, ZG2001, and BAY-293. In some embodiments, reference to the term SOS1 inhibitor includes any such SOS1 inhibitor disclosed in any one of the following patent applications: WO 2025070947, WO 2025067316, WO 2025062157, WO 2025059046, WO 2025038785, WO 2025003694, WO 2025000265, WO

[0540] 30 2024255827, WO 2024172632, WO 2024172631, WO 2024119028, WO 2024102952, WO 2024083257, WO 2024083255, WO 2024079252, WO 2024075070, WO 2024067744, WO 2024035921, WO 2024027762, WO 2024008185, WO 2023250165, WO 2023215257, WO 2023215256, WO 2023180345, WO 2023109929, WO 2023059597, WO 2023041049, WO 2023029833, WO 2023022497, WO 2022184116, WO 2022171184, WO 2022170952, WO 2022170917, WO 2022170802, WO 2022161461, 35 WO 2022157629, WO 2022139304, WO 2022121813, WO 2022028506, WO 2021228028, WO 2019122129, KR 20240128541, CN 119431234, CN 119039237, CN 119039234, CN 118812510, CN 117800922, CN117143175, CN 117143176, CN 116462669, CN 116444447, CN 115806560, CN 115677702, CN 115215847, CN 115028644, CN 114685488, and CN 111393519 each of which is incorporated herein by reference in its entirety, including the compound structures disclosed therein which 40 are specifically incorporated herein by reference.

[0541] iii) SHP inhibitors

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[0544] In some embodiments, compositions and methods described herein may include a combination therapy of the present disclosure in combination with one or more SHP inhibitors. A SHP inhibitor may be administered or formulated in combination with a combination therapy of the present disclosure and / or any additional therapeutic agent described herein. In some embodiments, the SHP inhibitor is an inhibitor 5 of SHP1. In some embodiments, the SHP inhibitor is an inhibitor of SHP2. In some embodiments, the SHP1 inhibitor is SB8091 or SB6299 aka DA-4511. In some embodiments, a SHP2 inhibitor is one or more of SHP099, TNO155, RMC-4550, RMC-4630, JAB-3068, JAB-3312, RLY-1971, ERAS-601, SH3809, PF-07284892, ARRY-558, or BBP-398. In some embodiments, reference to the term SHP2 inhibitor includes any such SHP2 inhibitor disclosed in any one of the following patent applications: WO 10 2025075693, WO 2025019666, WO 2025011568, WO 2025011480, WO 2024258652, WO 2024193439, WO 2024175081, WO 2024147703, WO 2024125603, WO 2023282702, WO 2023280283, WO 2023280237, WO 2023018155, WO 2023011513, WO 2022271966, WO 2022271964, WO 2022271911, WO 2022259157, WO 2022242767, WO 2022241975, WO 2022237676, WO 2022237367, WO 2022237178, WO 2022235822, WO 20222084008, WO 2022135568, WO 2022063190, WO

[0545] 15 2022043865, WO 2022042331, WO 2022033430, WO 2022017444, WO 2022007869, WO 2021259077, WO 2021249449, WO 2021249057, WO 2021244659, WO 2021218755, WO 2021176072, WO 2021171261, WO 2021149817, WO 2021148010, WO 2021147879, WO 2021143823, WO 2021143701, WO 2021143680, WO 2021281752, WO 2021121397, WO 2021119525, WO 2021115286, WO 2021110796, WO 2021088945, WO 2021073439, WO 2021061706, WO 2021061515, WO 2021043077, 20 WO 2021033153, WO 2021028362, WO 2021033153, WO 2021028362, WO 2021018287, WO 2020259679, WO 2020249079, WO 2020210384, WO 2020201991, WO 2020181283, WO 2020177653, WO 2020165734, WO 2020165733, WO 2020165732, WO 2020156243, WO 2020156242, WO 2020108590, WO 2020104635, WO 2020094104, WO 2020094018, WO 2020081848, WO 2020073949, WO 2020073945, WO 2020072656, WO 2020065453, WO 2020065452, WO 2020063760, WO

[0546] 25 2020061103, WO 2020061101, WO 2020033828, WO 2020033286, WO 2020022323, WO 2019233810, WO 2019213318, WO 2019183367, WO 2019183364, WO 2019182960, WO 2019167000, WO 2019165073, WO 2019158019, WO 2019152454, WO 2019051469, WO 2019051084, WO 2018218133, WO 2018172984, WO 2018160731, WO 2018136265, WO 2018136264, WO 2018130928, WO 2018129402, WO 2018081091, WO 2018057884, WO 2018013597, WO 2017216706, WO 2017211303, 30 WO 2017210134, WO 2017156397, WO 2017100279, WO 2017079723, WO 2017078499, WO 2016203406, WO 2016203405, WO 2016203404, WO 2016196591, WO 2016191328, WO 2015107495, WO 2015107494, WO 2015107493, WO 2014176488, WO 2014113584, CN 116332908, CN 119264153, CN 117069698, CN 117143107, CN 115677661, CN 115677660, CN 115611869, CN 115521305, CN 115490697, CN 115466273, CN 115394612, CN 115304613, CN 115304612, CN 115300513, CN 35 115197225, CN 114957162, CN 114920759, CN 114716448, CN 114671879, CN 114539223, CN 114524772, CN 114213417, CN 114195799, CN 114163457, CN 113896710, CN 113248521, CN 113248449, CN 113135924, CN 113024508, CN 112920131, CN 112823796, CN 112409334, CN 112402385, CN 112174935, 111848599, CN 111704611, CN 111393459, CN 111265529, CN 110143949, CN 108113848, US 11179397, US 11044675, US 11034705, US 11033547, US 11001561, 40 US 10988466, US 10954243, US 10934302, or US 10858359, each of which is incorporated herein by

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[0549] reference in its entirety, including the compound structures disclosed therein which are specifically incorporated herein by reference.

[0550] iv) MEK inhibitors

[0551] In some embodiments, compositions and methods described herein may include a combination 5 therapy of the present disclosure in combination with one or more MEK inhibitors. A MEK inhibitor may be administered or formulated in combination with a combination therapy of the present disclosure and / or any additional therapeutic agent described herein. In some embodiments, a MEK inhibitor is one or more of pimasertib, IMM-1-104, selumetinib, cobimetinib (Cotellic®), trametinib (Mekinist®), and binimetinib (Mektovi®). In some embodiments, a MEK inhibitor targets a MEK mutation that is a Class I MEK1 10 mutation selected from D67N; P124L; P124S; and L177V. In some embodiments, the MEK mutation is a Class II MEK1 mutation selected from ΔE51-Q58; ΔF53-Q58; E203K; L177M; C121S; F53L; K57E; Q56P; and K57N. In some embodiments, reference to the term MEK inhibitor includes any such MEK inhibitor disclosed in any one of the following patent applications: WO 2022221866, WO 2022125941, WO 2022208391, WO 2022015736, WO 2022177557, WO 2021018866, WO 2021069486, WO

[0552] 15 2021142144, WO 2021168283, WO 2021234097, WO 2019076947, WO 2018233696, WO 2016188472, WO 2014063024, WO 2013019906, WO 2011047238, WO 2007044515, US 2023032403, and CN 115813930, each of which is incorporated herein by reference in its entirety, including the compound structures disclosed therein which are specifically incorporated herein by reference.

[0553] v) RAF inhibitors

[0554] 20 In some embodiments, compositions and methods described herein may include a combination therapy of the present disclosure in combination with one or more RAF inhibitors. A RAF inhibitor may be administered or formulated in combination with a combination therapy of the present disclosure and / or any additional therapeutic agent described herein. In some embodiments, a RAF inhibitor is VS-6766 or BTDX-4933. In some embodiments, a RAF inhibitor is a BRAF inhibitor. BRAF inhibitors that may be 25 used in combination with a compound of the present disclosure include, for example, VS-6766, IK-595, vemurafenib, dabrafenib, and encorafenib. BRAF may comprise a Class 3 BRAF mutation. In some embodiments, the Class 3 BRAF mutation is 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. In some embodiments, 30 reference to the term RAF inhibitor includes any such RAF inhibitor disclosed in any one of the following patent applications: WO 2023076991, WO 2022226626, WO 2022226261, WO 2019084459, WO 2018203219, WO 201851306, WO 2017212442, WO 2015075483, WO 2013134243, WO 2013134298, WO 2011047238, WO 2011025965, WO 2011025947, WO 2011025951, WO 2011025940, WO 2011025938, WO 2010065893, WO 2009016460, WO 2009130015, WO 2009111278, WO 2009111279, 35 WO 2008028141, and WO 2006024834, each of which is incorporated herein by reference in its entirety, including the compound structures disclosed therein which are specifically incorporated herein by reference.

[0555] vi) ERK inhibitors

[0556] In some embodiments, compositions and methods described herein may include a combination 40 therapy of the present disclosure in combination with one or more ERK inhibitors. An ERK inhibitor may be administered or formulated in combination with a combination therapy of the present disclosure and / or

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[0559] any additional therapeutic agent described herein. In some embodiments, an ERK inhibitor is an ERK1 / 2 inhibitor, such as ERAS-007. In some embodiments, an ERK inhibitor is an ERK 5 inhibitor. In some embodiments, an ERK inhibitor is one or more of ASTX-029 or I-75. In some embodiments, reference to the term ERK inhibitor includes any such ERK inhibitor disclosed in any one of the following patent 5 applications: WO 2023076305, WO 2022259222, WO 2022221547, WO 2021110169, WO 2021110168, WO 2021252316, WO 2020102686, WO 2020228817, WO 2020107987, WO 2019233456, WO 2019233457, WO 2016025561, WO 2016192063, WO 2016106029, WO 2016106009, WO 2015051341, WO 2014124230, WO 2014052563, WO 2011041152, WO 200910550, WO 2008153858, CN114315837, CN 115057860, CN 107973783, each of which is incorporated herein by reference in its entirety, 10 including the compound structures disclosed therein which are specifically incorporated herein by reference.

[0560] vii) MAPK inhibitors

[0561] In some embodiments, compositions and methods described herein may include a combination therapy of the present disclosure in combination with one or more Mitogen-Activated Protein Kinase 15 (MAPK) inhibitors. A MAPK inhibitor may be administered or formulated in combination with a compound of the present disclosure and / or any additional therapeutic agent described herein. In some embodiments, a MAPK inhibitor is a p38MAPK inhibitor or a MAP3K8 inhibitor. In some embodiments, the MAPK inhibitor is one or more of Tilpisertib (GS-4875) and neflamapidmod (VX-745). In some embodiments, reference to the term MAPK inhibitor includes any such MAPK inhibitor disclosed in any 20 one of the following patent applications: WO 2016029263, CN 114767674, CN 115850179, and CN 1743006, each of which is incorporated herein by reference in its entirety, including the compound structures disclosed therein which are specifically incorporated herein by reference.

[0562] In some embodiments, a therapeutic agent that may be combined with a compound of the present disclosure is an inhibitor of MAP2K4. A non-limiting example of a MAP2K4 inhibitor useful 25 according to the disclosure is HRX-0233.

[0563] Kinase Inhibitors

[0564] Compositions and methods described herein may include a compound of the present disclosure in combination with one or more kinase inhibitors. Tyrosine kinases and serine / threonine kinases play a crucial role in various cellular processes such as cell signaling, growth, and differentiation. Kinase 30 inhibitors known in the art have been developed as a treatment for various types of cancer in addition to therapies for conditions such as neurodegenerative diseases, autoimmune disorders, and inflammation.

[0565] i) PKA inhibitors

[0566] In some embodiments, compositions and methods described herein may include one or more Protein Kinase A (PKA) inhibitors. A PKA inhibitor may be administered or formulated in combination with 35 a combination therapy of the present disclosure and / or any additional therapeutic agent described herein.

[0567] In some embodiments, a PKA inhibitor is H89. In some embodiments, reference to the term PKA inhibitor includes any such PKA inhibitor disclosed in any one of the following patent applications: CN 106620678 and CN 114632155, each of which is incorporated herein by reference in its entirety, including the compound structures disclosed therein which are specifically incorporated herein by reference.

[0568] 40 ii) FAK inhibitors

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[0571] In some embodiments, compositions and methods described herein may include a compound of the present disclosure in combination with one or more Focal Adhesion Kinase (FAK) inhibitors. A FAK inhibitor may be administered or formulated in combination with a combination therapy of the present disclosure and / or any additional therapeutic agent described herein. In some embodiments, a FAK 5 inhibitor is one or more of BI853520, defactinib, GSK2256098, PF-00562271, and VS-4718. In some embodiments, reference to the term FAK inhibitor includes any such FAK inhibitor disclosed in any one of the following patent applications: WO 2022152315, WO 2021098679, WO 2020135442, WO 2020191448, WO 2012022408, WO 2013134353, WO 2012110774, WO 2010062578, CN 111072571, and KR 101691536, each of which is incorporated herein by reference in its entirety, including the 10 compound structures disclosed therein which are specifically incorporated herein by reference.

[0572] iii) ROCK inhibitors

[0573] In some embodiments, compositions and methods described herein may include a combination therapy of the present disclosure in combination with one or more Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitors. A ROCK inhibitor may be administered or formulated in combination 15 with a compound of the present disclosure and / or any additional therapeutic agent described herein. In some embodiments, a ROCK inhibitor is GSK269962A. In some embodiments, reference to the term ROCK inhibitor includes any such ROCK inhibitor disclosed in any one of the following patent applications: WO 2023051753, WO 2022237892, WO 2022012409, WO 2021093795, WO 2021214200, WO 2020177292, WO 202011751, WO 2019014304, WO 2019179525, WO 2019089868, WO

[0574] 20 2019014300, WO 2018108156, WO 2018009627, WO 2018009625, WO 2018009622, WO 2017123860, WO 2017205709, WO 2016112236, WO 2014068035, WO 2013030367, WO 2012146724, WO 2012067965, WO 2011107608, CN 108129453, CN 108191821, CN 110917352, CN 108558823, CN108047193, CN107973777, CN108047197, CN108129448, CN 115869304, and GB202214708, each of which is incorporated herein by reference in its entirety, including the compound structures disclosed 25 therein which are specifically incorporated herein by reference.

[0575] iv) MSK1 inhibitors

[0576] In some embodiments, compositions and methods described herein may include a combination therapy of the present disclosure in combination with one or more Mitogen- and stress-activated kinase (MSK1) inhibitors. A MSK1 inhibitor may be administered or formulated in combination with a compound 30 of the present disclosure and / or any additional therapeutic agent described herein. In some embodiments, a MSK1 inhibitor is one or more of SB-747651A, SB 747651A, Ro 320432, CGP 57380, GSK2830371, SR1664, LY-3214996, PFI-4, MSC-2363318A, and AS601245.

[0577] v) RSK inhibitors

[0578] In some embodiments, compositions and methods described herein may include a combination 35 therapy of the present disclosure in combination with one or more ribosomal S6 kinase (RSK) inhibitors. A RSK1 inhibitor may be administered or formulated in combination with a compound of the present disclosure and / or any additional therapeutic agent described herein. In some embodiments, a RSK inhibitor is one or more of BI-D1870, LJH685, SL0101-1, FMK, BRD7389, BIX 02565, LJI308, LJI308-S, LJI308-1, and LJH685-S. In some embodiments, a RSK inhibitor is PMD-026. In some embodiments, 40 reference to the term RSK inhibitor includes any such RSK inhibitor disclosed in any one of the following patent applications: WO 2021249558, WO 2020165646, WO 2017141116, and CN 113801139, each of

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[0581] which is incorporated herein by reference in its entirety, including the compound structures disclosed therein which are specifically incorporated herein by reference.

[0582] vi) ALK inhibitors

[0583] In some embodiments, compositions and methods described herein may include a combination 5 therapy of the present disclosure in combination with one or more Anaplastic Lymphoma Kinase (ALK) inhibitors. An ALK inhibitor may be administered or formulated in combination with a compound of the present disclosure and / or any additional therapeutic agent described herein. In some embodiments, an ALK inhibitor is one or more of Crizotinib (Xalkori), Ceritinib (Zykadia), Alectinib (Alecensa), Brigatinib (Alunbrig), Lorlatinib (Lorbrena), Ensartinib (X-396), TAE684, ASP3026, TPX-0131, LDK378 (Ceritinib 10 analog), CEP-37440; 4SC-203, TL-398, PLB1003, TSR-011, CT-707, TPX-0005, and AP26113.

[0584] Additional examples of ALK kinase inhibitors are described in examples 3-39 of WO05016894. In some embodiments, reference to the term ALK inhibitor includes any such ALK inhibitor disclosed in any one of the following patent applications: WO 2019142095, WO 2019179482, WO 2018130928, WO 2018127184, WO 2017101803, WO 2016192132, WO 2014100431, WO 2012082972, CN 111138492, 15 CN 110526914, CN 109836415, CN 105801603, CN107987056, and CN 105878248, each of which is incorporated herein by reference in its entirety, including the compound structures disclosed therein which are specifically incorporated herein by reference.

[0585] d) Receptor tyrosine kinase inhibitors

[0586] Compositions and methods described herein may include a combination therapy of the present 20 disclosure in combination with one or more receptor tyrosine kinase inhibitors. A receptor tyrosine kinase (RTK) inhibitor is a type of molecule (e.g., small molecule, antibody, and nucleic acid) that binds to and blocks the activity of receptor tyrosine kinases or their ligands. RTKs are proteins found on the surface of cells that play a critical role in cell signaling and growth and have been developed as therapeutics for a range of diseases, including cancer, diabetes, and autoimmune disorders. In some embodiments, a 25 therapeutic agent may be a pan-RTK inhibitor, such as afatinib.

[0587] i) EGFR inhibitors

[0588] In some embodiments, compositions and methods described herein may include a combination therapy of the present disclosure in combination with one or more EGFR inhibitors. An EGFR inhibitor may be administered or formulated in combination with a combination therapy of the present disclosure 30 and / or any additional therapeutic agent described herein. EGFR inhibitors include, but are not limited to, small molecule antagonists, antibody inhibitors, or specific antisense nucleotide or siRNA. Useful antibody inhibitors of EGFR include cetuximab (Erbitux®), panitumumab (Vectibix®), zalutumumab, nimotuzumab, and matuzumab. Further antibody-based EGFR inhibitors include any anti-EGFR antibody or antibody fragment that can partially or completely block EGFR activation by its natural ligand. In some 35 embodiments, the antibody-based EGFR inhibitor is a bispecific antibody. In some embodiments, the bispecific is selective for EGFR and MET. In some embodiments, the bispecific is amivantamab. 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.

[0589] 1995, 1:1311-1318; Huang et al., 1999, Cancer Res.15:59(8):1935-40; and Yang et al., Cancer 40 Res.1999, 59:1236-1243. The EGFR inhibitor can be monoclonal antibody Mab E7.6.3 (Yang, 1999

[0590] 75 PATENT

[0591] ATTORNEY DOCKET NO.: 51432-077WO2

[0592] supra), or Mab C225 (ATCC Accession No. HB-8508), or an antibody or antibody fragment having the binding specificity thereof.

[0593] Small molecule antagonists of EGFR include gefitinib (Iressa®), Lazertinib, erlotinib (Tarceva®), and lapatinib (TykerB®). See, e.g., Yan et al., Pharmacogenetics and Pharmacogenomics In Oncology 5 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 (Tagrisso®). In some embodiments, an EGFR inhibitor is one or more of cetuximab, gefitinib (Iressa), erlotinib (Tarceva), and afatinib (Gilotrif). Additional non-limiting examples of small molecule EGFR inhibitors include any of the EGFR inhibitors 10 described in Traxler et al., Exp. Opin. Ther. Patents 1998, 8(12):1599-1625. An EGFR inhibitor may be ERAS-801. In some embodiments, an EGFR inhibitor is an ERBB inhibitor. In humans, the ERBB family contains HER1 (EGFR, ERBB1), HER2 (NEU, ERBB2), HER3 (ERBB3), and HER (ERBB4). In some embodiments, the EGFR inhibitor may be bosutinib, crizotinib, dasatinib, erlotinib, gefitinib, lapatinib, pazopanib, ruxolitinib, sunitinib, vemurafenib, abrocitinib, asciminib, futibatinib, ibrutinib, imatinib, 15 pacritinib, or sorafenib. In some embodiments, reference to the term EGFR inhibitor includes any such EGFR inhibitor disclosed in any one of the following patent applications: WO 2023041071, WO 2023049312, WO 2023020600, WO 2023284747, WO 2022206797, WO 2022258977, WO 2022033416, WO 2022033410, WO 2022105908, WO 2022100641, WO 2022014639, WO 2022007841, WO 2021018009, WO 2021057882, WO 2021252661, WO 2021018003, WO 2021073498, WO 2021238827, 20 WO 2020254547, WO 2020216371, WO 2020147838, WO 2020207483, WO 2020254572, WO 2020001350, WO 2021001351, WO 2019164948, WO 2019218958, WO 2019046775, WO 2019015655, WO 2018121758, WO 2018218963, WO 2017220007, WO 2017205459, WO 2017161937, WO 2016192609, WO 199633980, WO 199630347, WO 199730034, WO 199730044, WO 199738994, WO 199749688, WO 199802434, WO 199738983, WO 199519774, WO 199519970, WO 199713771, WO 25 199802437, WO 199802438, WO 199732881, WO 199833798, WO 199732880, WO 199732880, WO 199702266, WO 199727199, WO 199807726, WO 1997 / 34895, WO 199631510, WO 199814449, WO 199814450, WO 199814451, WO 199509847, WO 199719065, WO 199817662, WO 199935146, WO 199935132, WO 199907701, WO 199220642, DE 19629652, EP 682027, EP 837063, EP 0787772, EP 0520722, EP 0566226, CN 115960018, CN 110283162, CN 114044774, CN111973601, CN 111973602, 30 and CN113896744, each of which is incorporated herein by reference in its entirety, including the compound structures disclosed therein which are specifically incorporated herein by reference.

[0594] ii) HER2 inhibitors

[0595] In some embodiments, compositions and methods described herein may include a combination therapy of the present disclosure in combination with one or more HER2 inhibitors. A HER2 inhibitor may 35 be administered or formulated in combination with a combination therapy of the present disclosure and / or any additional therapeutic agent described herein. In some embodiments, an HER2 inhibitor is one or more of tucatinib, rastuzumab (Herceptin™), pertuzumab (Perjeta™), lapatinib (Tykerb™), ado-trastuzumab emtansine (Kadcyla™), and neratinib (Nerlynx™). Non-limiting examples of HER2 inhibitors include monoclonal antibodies such as trastuzumab (Herceptin®) and pertuzumab (Perjeta®); small molecule 40 tyrosine kinase inhibitors such as gefitinib (Iressa®), erlotinib (Tarceva®), pilitinib, CP-654577, CP- 724714, canertinib (CI 1033), HKI-272, lapatinib (GW-572016; Tykerb®), PKI-166, AEE788, BMS- 76 PATENT

[0596] ATTORNEY DOCKET NO.: 51432-077WO2

[0597] 599626, HKI-357, BIBW 2992, ARRY-334543, and JNJ-26483327. In some embodiments, reference to the term HER2 inhibitor includes any such HER2 inhibitor disclosed in any one of the following patent applications: WO 2021156178, WO 2021156180, WO 2021213800, WO 2021088987, WO 2013561183, and WO 2013056108, each of which is incorporated herein by reference in its entirety, including the 5 compound structures disclosed therein which are specifically incorporated herein by reference.

[0598] iii) MET inhibitors

[0599] In some embodiments, compositions and methods described herein may include a combination therapy of the present disclosure in combination with one or more MET inhibitors. A MET inhibitor may be administered or formulated in combination with a combination therapy of the present disclosure and / or 10 any additional therapeutic agent described herein. In some embodiments, the MET inhibitor is an anti- MET antibody-drug-conjugate such as MYTX-011, TR1801-ADC, ABBV-399, and SHR-A1403. In some embodiments, a MET inhibitor is one or more of Crizotinib (Xalkori™), Cabozantinib (Cometriq,Cabometyx™), Capmatinib (Tabrecta™), Tepotinib (Tepmetko™), Savolitinib (Volitinib™), Onartuzumab(MetMab™), Foretinib (GSK1363089), MGCD-265 (Amuvatinib), SU11274, and SU5416. In some 15 embodiments, reference to the term MET inhibitor includes any such MET inhibitor disclosed in any one of the following patent applications: WO 2022226168, WO 2021222045, WO 2020047184, WO 2020015744, WO 2020244654, WO 2020156453, WO 2019206268, WO 2018077227, WO 2017012539, WO 2016015653, WO 2016012963, WO 2012015677, WO 2011162835, WO 2010089507, WO 2009091374, WO 2009056692, WO 2008051547, WO 2007130468, US 2012237524, CN 103497177, 20 CN 107311983, CN 107382968, CN 110218191, and TW201331206, each of which is incorporated herein by reference in its entirety, including the compound structures disclosed therein which are specifically incorporated herein by reference.

[0600] iv) AXL inhibitors

[0601] In some embodiments, compositions and methods described herein may include a combination 25 therapy of the present disclosure in combination with one or more AXL inhibitors. An AXL inhibitor may be administered or formulated in combination with a combination therapy of the present disclosure and / or any additional therapeutic agent described herein. AXL is a receptor tyrosine kinase that belongs to the TAM family of receptors, which also includes TYRO3 and MERTK. In some embodiments, an AXL inhibitor is one or more of bemcentib, BGB324, R428, SGI-7079, TP-0903, BMS-777607, UNC2025, and 30 TP-0903. In some embodiments, reference to the term AXL inhibitor includes any such AXL inhibitor disclosed in any one of the following patent applications: WO 2023045816, WO 2022237843, WO 2022246179, WO 2021012717, WO 2021088787, WO 2021067772, WO 2021239133, WO 2021204713, WO 2020238802, WO 2019039525, WO 2019101178, WO 2019074116, WO 2017146236, WO 2016097918, WO 2015012298, WO 2010005876, WO 2010083465, CN 115073367, and JP

[0602] 35 2022171109, each of which is incorporated herein by reference in its entirety, including the compound structures disclosed therein which are specifically incorporated herein by reference.

[0603] v) IGFR inhibitors

[0604] In some embodiments, compositions and methods described herein may include a combination therapy of the present disclosure in combination with one or more insulin-like growth factor receptor 1 40 (IGF-1R) inhibitors. An IGFR inhibitor may be administered or formulated in combination with a combination therapy of the present disclosure and / or any additional therapeutic agent described herein.

[0605] 77 PATENT

[0606] ATTORNEY DOCKET NO.: 51432-077WO2

[0607] IGFR inhibitors have been developed to target the IGFR receptor, which plays a critical role in cancer progression and metastasis. In some embodiments, an IGFR inhibitor is one or more of linsitinib, AXL1717, OSI-906 (Linsitinib), BMS-754807, BI 836845, AZ12253801, PQIP (Pyrrolo[1,2-a]quinoxaline), and NVP-AEW541. In some embodiments, reference to the term IGFR inhibitor includes any such IGFR 5 inhibitor disclosed in any one of the following patent applications: WO 2022115946, WO 2022217923, WO 2021203861, WO 2021246413, WO 2020116398, WO 2019046600, WO 2018195250, WO 2018221521, WO 2018204872, WO 2017072196, WO 2016173682, WO 2015162291, WO 2015162292, WO 2010066868, WO 2006069202, and CN 112125916, each of which is incorporated herein by reference in its entirety, including the compound structures disclosed therein which are specifically 10 incorporated herein by reference.

[0608] vi) RET inhibitors

[0609] In some embodiments, compositions and methods described herein may include a combination therapy of the present disclosure in combination with one or more Rearranged during transfection (RET) inhibitors. An RET inhibitor may be administered or formulated in combination with a combination therapy 15 of the present disclosure and / or any additional therapeutic agent described herein. RET plays a critical role in various cellular processes, including cell growth, differentiation, survival, and migration. RET is activated by binding of its ligands, such as glial cell line-derived neurotrophic factor (GDNF) family ligands, which leads to the activation of downstream signaling pathways that promote these cellular processes. In some embodiments, a RET inhibitor is one or more of pralsetinib, selpercatinib (LOXO-20 292), BLU-667, RXDX-105, TPX-0046, GSK3179106, molidustat (BAY 85-3934), and RPI-1 (Retrophin).

[0610] In some embodiments, reference to the term RET inhibitor includes any such RET inhibitor disclosed in any one of the following patent applications: WO 2021211380, WO 2021057963, WO 2021043209, WO 2021222017, WO 2020035065, WO 2020114487, WO 2020200314, WO 2020200316, WO 2020114494, WO 2018071447, WO 2018213329, WO 2017079140, WO 2014050781, CN 113943285, CN 113683610, 25 CN 113683611, CN 113620944, CN 113620945, CN 113527291, CN 113527292, CN 113527290, CN 113135896, CN 111057075, CN111233899, and CN111362923, each of which is incorporated herein by reference in its entirety, including the compound structures disclosed therein which are specifically incorporated herein by reference.

[0611] vii) ROS1 inhibitors

[0612] 30 In some embodiments, compositions and methods described herein may include a combination therapy of the present disclosure in combination with one or more c-ros oncogene 1 (ROS1) inhibitors. A ROS1 inhibitor may be administered or formulated in combination with a combination therapy of the present disclosure and / or any additional therapeutic agent described herein. ROS1 is a receptor tyrosine kinase that belongs to the insulin receptor family and plays a role in various cellular processes, including 35 cell growth, differentiation, survival, and migration. In some embodiments, a ROS1 inhibitor is one or more of taletrectinib, DS-6051b, TPX-0131, GZD824, and PF-06463922. In some embodiments, reference to the term ROS1 inhibitor includes any such ROS1 inhibitor disclosed in any one of the following patent applications: WO 2021098703, WO 2020024825, and US 2017079972, each of which is incorporated herein by reference in its entirety, including the compound structures disclosed therein which 40 are specifically incorporated herein by reference.

[0613] viii) PDGFR inhibitors

[0614] 78 PATENT

[0615] ATTORNEY DOCKET NO.: 51432-077WO2

[0616] In some embodiments, compositions and methods described herein may include a combination therapy of the present disclosure in combination with one or more platelet-derived growth factor receptor (PDGFR) inhibitors. A PDGFR inhibitor may be administered or formulated in combination with a compound of the present disclosure and / or any additional therapeutic agent described herein. PDGFR is 5 a family of receptor tyrosine kinases that consists of two members, PDGFRα and PDGFRβ. They are activated by binding to their ligands, such as platelet-derived growth factor (PDGF), which leads to the activation of downstream signaling pathways that promote cell growth, proliferation, and survival. In some embodiments, a PDGFR inhibitor is one or more of CP-673451, imatinib, nintedanib (Ofev™), sunitinib(Sutent™), pazopanib (Votrient™), regorafenib (Stivarga™), and dasatinib (Sprycel™).

[0617] 10 ix) FGF inhibitors

[0618] In some embodiments, compositions and methods described herein may include a combination therapy of the present disclosure in combination with fibroblast growth factor (FGF) inhibitors. An FGF inhibitor may be administered or formulated in combination with a compound of the present disclosure and / or any additional therapeutic agent described herein. FGFRs are a family of receptor tyrosine kinases 15 that consists of four members, FGFR1-4. FGFRs are activated by binding to their ligands, fibroblast growth factors (FGFs), which leads to the activation of downstream signaling pathways that promote cell growth, differentiation, and survival. In some embodiments, the FGFR inhibitor is an inhibitor of FGFR2. In some embodiments, the FGFR inhibitor is an inhibitor of FGFR4. In some embodiments, an FGFR inhibitor is one or more of futibatinib (TAK-659), erdafitinib (Balversa™), infigratinib (Truseltiq™), Debio 20 1347, and rogaratinib (BAY 1163877). In some embodiments, reference to the term FGFR inhibitor includes any such FGFR inhibitor disclosed in any one of the following patent applications: WO 2022033472, WO 2022152274, WO 2022166469, WO 2022206939, WO 2021037219, WO 2021089005, WO 2021113462, WO 2020185532, WO 2019213544, WO 2020164603, WO 2019154364, WO 2019034076, WO 2019213506, WO 2019223766, WO 2018028438, WO 2018153373, WO 2018121650, 25 WO 2018010514, WO 2017028816, WO 2017118438, WO 2016134320, WO 2015008844, WO 2014172644, WO 2014007951, WO 2013179033, WO 2013087578, WO 2012047699, CN 105906630, CN 115869315, CN 115141176, CN 115043832, and CN 115028634, each of which is incorporated herein by reference in its entirety. In some embodiments, the FGF pathway inhibitor targets an FGF ligand. Such FGF pathway inhibitors include FGF ligand traps and antibodies. Non-limiting examples 30 include, FP-1039, an FGF ligand trap consisting of the extracellular domain of FGFR1 fused to the Fc portion of human IgG1, designed to sequester FGF ligands and inhibit FGF signaling, and MFGR1877S, a monoclonal antibody targeting FGF ligands, designed to block FGF-mediated signaling, including the compound structures disclosed therein which are specifically incorporated herein by reference.

[0619] x) VEGF inhibitors

[0620] 35 In some embodiments, compositions and methods described herein may include a combination therapy of the present disclosure in combination with one or more vascular endothelial growth factor (VEGF) signaling inhibitors. VEGF (vascular endothelial growth factor) signaling inhibitors are a class of drugs that target the signaling pathway mediated by VEGF and its receptors. VEGF plays a critical role in angiogenesis, the process of forming new blood vessels from existing ones, and it is overexpressed in 40 many types of cancer, making it an attractive target for cancer therapy. A VEGF inhibitor may be administered or formulated in combination with a compound of th...

Claims

1. PATENT2.ATTORNEY DOCKET NO.: 51432-077WO23.Claims1. A method of treating a RAS G12C mutant lung cancer in a subject in need thereof, the method comprising administering to the subject a RAS(ON) multi-selective inhibitor and a RAS(ON) G12C-selective inhibitor.

2. The method of claim 1, wherein the method further comprises administering to the subject an immune checkpoint inhibitor.

3. The method of claim 1 or 2, wherein the RAS(ON) multi-selective inhibitor is one or more of Compound A (daraxonrasib), AB-23400, BBP-454, BI-2852, Compound C (RMC-7977), RM-034, GFH547, ERAS-0015 and compound 6A of WO 2024 / 067857.

4. The method of any one of claims 1 to 3, wherein RAS(ON) G12C-selective inhibitor is one or more of Compound B (elironrasib) and Compound D (RMC-4998) ,5. The method of any one of claims 2 to 4, wherein the immune checkpoint inhibitor is one or more of anti-PD1, anti-PDL1, anti-CTLA4, 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.

6. The method of claim 5, wherein the anti-PD1 is one or more of cemiplimab, nivolumab, pembrolizumab, pidilizumab, spartalizumab, camrelizumab, sintilimab, tislelizumab, toripalimab, dostarlimab, sasanlimab, retifanlimab, tebotelimab, ivonescimab, ABBV-181, AK104, AK105, BCD-100, BI-754091, CBT-501, CC-90006, GLS-010, HLX10, IBI-308, JNJ-3283, JS001, LZM009, MEDI0680 (AMP-514), REGN-2810, SHR-1210, Sym021, TSR-042, or XmAb20717.

7. The method of claim 6, wherein the anti-PD1 is pembrolizumab or ivonescimab.

8. The method of any one of claims 1 to 7, wherein the subject is resistant to an immune checkpoint inhibitor prior to treatment with the method of claim 1.

9. The method of any one of claims 1 to 8, wherein the subject has previously been administered an immune checkpoint inhibitor.

10. The method of any one of claims 1 to 9, wherein the subject is resistant to treatment with an immune checkpoint inhibitor.

11. The method of claim 10, wherein the subject has acquired resistance to treatment with an immune checkpoint inhibitor.

12. The method of any one of claims 1 to 11, wherein the RAS G12C mutation is KRASG12C, HRASG12C, or NRASG12C.16.125 PATENT17.ATTORNEY DOCKET NO.: 51432-077WO213. The method of any one of claims 1 to 12, wherein the lung cancer is non-small cell lung cancer (NSCLC).

14. The method of claim 13, wherein the NSCLC is lung adenocarcinoma.

15. The method of any one of claims 1 to 14, wherein the inhibitors are administered simultaneously or sequentially.

16. The method of any one of claims 1 to 14, wherein the inhibitors are administered as a single formulation or in separate formulations.

17. The method of any one of claims 2 to 16, wherein administration of the RAS(ON) multi-selective inhibitor, RAS(ON) G12C-selective inhibitor and immune checkpoint inhibitor transforms a tumor microenvironment of an immunologically cold lung cancer.

18. The method of any one of claims 1 to 17, wherein administration of the RAS(ON) multi-selective inhibitor, RAS(ON) G12C-selective inhibitor and immune checkpoint inhibitor increases MHC class I expression of an immune refractory lung cancer in a subject.

19. A method of treating a subject afflicted with a RAS G12C mutant cancer comprising administering to the subject a therapeutically effective amount of Compound B (elironrasib) and avoiding co-administration of a proton pump inhibitor, wherein the subject is also in need of the proton pump inhibitor.

20. The method of claim 19, wherein the cancer is a non-small cell lung cancer (NSCLC), a pancreatic adenocarcinoma, a pancreatic ductal adenocarcinoma, or a colorectal cancer.

21. The method of claim 20, wherein the proton pump inhibitor is one or more of omeprazole, lansoprazole, dexlansoprazole, rabeprazole, pantoprazole, and esomeprazole.

22. The method of claim 21, wherein the proton pump inhibitor is esomeprazole.

23. A method of treating a subject afflicted with a RAS G12C mutant cancer, comprising discontinuing administration of a proton pump inhibitor to avoid an adverse drug interaction with Compound B (elironrasib), and administering to the subject a therapeutically effective amount of Compound B (elironrasib).

24. The method of claim 23, wherein the proton pump inhibitor is discontinued within 1 month prior to administering Compound B (elironrasib).30.126 PATENT31.ATTORNEY DOCKET NO.: 51432-077WO225. The method of claim 23, wherein the proton pump inhibitor is discontinued within 2 weeks prior to administering Compound B (elironrasib).

26. The method of any one of claims 23 to 25, further comprising advising the subject that co-administration of Compound B (elironrasib) and the proton pump inhibitor can alter the therapeutic effect or adverse reaction profile of Compound B (elironrasib).

27. A method of treating a subject afflicted with a RAS G12C mutant cancer, wherein said subject is also in need of a proton pump inhibitor, comprising administering to the subject a therapeutically effective amount of Compound B (elironrasib) while avoiding proton pump inhibitor co-administration, and any one or more of the following:35.(a) advising the subject that proton pump inhibitors should be avoided or discontinued,36.(b) advising the subject that co-administration of Compound B (elironrasib) with drugs that are proton pump inhibitors can alter the therapeutic effect or adverse reaction profile of Compound B (elironrasib), (c) advising the subject that co-administration of Compound B (elironrasib) with proton pump inhibitors can alter the therapeutic effect or adverse reaction profile of Compound B (elironrasib), (d) advising the subject that use of Compound B (elironrasib) in subjects being treated with proton pump inhibitors is contraindicated, or37.(e) advising the subject that proton pump inhibitors should be used with caution in subjects receiving Compound B (elironrasib) due to the potential for an adverse reaction profile.

28. The method of claim 27, wherein the method further comprises avoiding administering a proton pump inhibitor.

29. The method of claim 27, wherein the method further comprises discontinuing administration of a proton pump inhibitor.40.127