GLP-1r use during immune checkpoint inhibitor treatment for nsclc in overweight patients

Abstract

The disclosure relates to a method of treating cancer by administering to an overweight human subject at least one glucagon-like peptide-1 receptor agonist (GLP-1RA) and at least one immune checkpoint inhibitor. The disclosure also relates to a method for treating an overweight human subject having cancer by administering the subject a therapeutically effective combination of at least one GLP-1RA and at least one immune checkpoint inhibitor. The method may also reduce resistance to immune checkpoint inhibition. The cancer being treated may be non-small cell lung cancer, including non-small cell lung cancer that is resistant to immune checkpoint inhibition. The GLP-1RA may be liraglutide or semaglutide and the immune checkpoint inhibitor may be pembrolizumab.

Description

GLP-1R USE DURING IMMUNE CHECKPOINT INHIBITOR TREATMENT FOR NSCLC IN OVERWEIGHT PATIENTSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to and the benefit of co-pending U.S. Provisional PatentApplication Serial No. 63 / 673,478, entitled Glucagon-like Peptide-1 Receptor Agonist Use DuringImmune Checkpoint Inhibitor Treatment for Non-Small Cell Lung Cancer in Overweight Patients, filed July 19, 2024, which is incorporated herein by reference in its entirety.TECHNICAL FIELD

[0002] The disclosure relates to an improved cancer treatment. In particular, the disclosure is directed to methods of treating cancer by administering glucagon-like peptide receptor agonists(GLP-1RA) to patients who are overweight to increase the effectiveness of immune checkpoint inhibitors in treating the cancer.BACKGROUND

[0003] Glucagon-like peptide-1 Receptor Agonists (GLP-lRAs), initially introduced as a class of anti-diabetic drugs, have entered widespread use after their recent approval for the treatment of obesity and obesity-related conditions (1). Several retrospective studies further associate GLP-IRA use with altered cancer incidence, yet the underlying mechanisms remain unclear. (2, 3). A correlation exists between excess visceral fat and obesity-related lung cancer risk (4, 5).

[0004] Visceral adiposity induces multiple changes in metabolic pathways, resulting in chronic meta-inflammation and suppression of anti-tumor immunity. Specifically, it is known to hamper the function of CD8+ T cells, key mediators of the immune response against cancer, while broadly upregulating the expression of inhibitory immune checkpoint molecules such as programmed cell death protein 1 (PD-1) (7) and enhancing suppressive leukocyte populations (8).

[0005] Given that GLP-lRAs are effective in promoting weight loss and reducing visceral adiposity(10), GLP-lRAs may similarly counteract the detrimental effects of obesity on anti-tumorimmunity and disease progression, leading to improved cancer outcomes, including non-small cell lung cancer (NSCLC) outcomes.SUMMARY

[0006] A first aspect of the present invention is directed to method of treating cancer, consisting of: administering to an overweight human subject at least one glucagon-like peptide-1 receptor agonist (GLP-1RA), and administering to the human subject at least one immune checkpoint inhibitor.

[0007] Another aspect of the present invention is directed to a method for treating an overweight human subject having cancer by administering to the human subject a therapeutically effective combination of at least one glucagon-like peptide-1 receptor agonist (GLP-1RA) and at least one immune checkpoint inhibitor.

[0008] Another aspect of the present invention is directed to a method of reducing resistance to immune checkpoint inhibition by administering to a human subject having cancer at least one glucagon-like peptide-1 receptor agonist (GLP-1RA) before treating the human subject with an immune checkpoint inhibitor.BRIEF DESCRIPTION OF THE DRAWINGS

[0009] FIG. 1 shows data indicating that liraglutide suppresses tumor growth in mice. 7.5x10A4 cells of the KPN1.1 cell lines were injected subcutaneously in the flanks of normal-weight (n=8 / group) or obese (n=8 / group) C57BL / 6 mice fed a control or high-fat diet for 12 weeks, respectively. Once tumors were palpable, mice received daily intraperitoneal injections of liraglutide or a phosphate-buffered saline (PBS) vehicle. The volumes and weights of tumors in normal-weight mice were not significantly different (FIGs. 1A, IB). Tumors induced in obese mice had significantly higher volume and end weight in control mice than in liraglutide-treated mice (FIGs. 1C, ID). Shown are representative findings from at least two independent experiments (n=8 mice / group / trial). SEM = Standard error of mean. *<p=0.05.

[0010] FIG. 2 shows gene expression changes in tumor cells from obese mice and altered pathways with liraglutide treatment. FIG. 2A is a Volcano plot: Differentially expressed genes in liraglutide-treated (green) vs vehicle-treated tumors (red). X-axis: Log2 fold change; Y-axis: Statistical significance. FIG. 2B shows changes in biological processes, immune, cell signaling, andmetabolic pathways, as seen by alterations in gene sets, expressed as normalized enrichment score (NES).

[0011] FIGs. 3A and 3B characterize the impact of GLP-IRa treatment on tumor-infiltrating leukocyte populations and phenotypes. Tumor tissues were recovered from the obese mice described in FIG. 2, and, after enzymatic digestion, single-cell suspensions were generated. Immunostaining followed by spectral flow cytometry analysis revealed the effects of LIRA on T cell (panels A-G) and myeloid cell (panels H-J) populations. Flow analysis was also performed to assess intracellular cytokine production by leukocytes re-stimulated ex vivo with PMA / lonomycin and brefeldin A (K). Shown are the mean + / -SEM results from one of two independent experiments and representative flow plots (n=5 per group / trial). *p < 0.05, **<0.02, ***<0.01, ****<0.001 by t test.

[0012] FIG. 4 shows the effect of semaglutide in an autochthonous mouse model. FIG. 4A is a schematic illustrating the experimental design in the diet-induced obesity (DIO) KP mouse. FIG. 4B shows a Kaplan-Meier survival analysis of DIO-KP mice receiving vehicle or semaglutide following tumor initiation (n = 7 per group). Log-rank test (Veh vs Sem): *, P = 0.0119. FIG. 4C consists of representative pCT images of lung tumor burden in DIO-KP mice at endpoint, comparing semaglutide- and vehicle-treated groups.

[0013] FIG. 5 shows clinical outcomes with adjuvant GLP-1 RA use after resection in non-small cell lung cancer (NSCLC). Recurrence-free survival (FIG. 5A) and overall survival (FIG. 5B) is shown for overweight and obese patients who underwent resection for non-small cell lung cancer (NSCLC) and received GLP-1 RA. GLP-IRA use is associated with improved recurrence-free survival (RFS) after propensity score matching for age, sex, race, smoking, histology, stage, and GLP-IRA use as covariates.

[0014] FIG. 6 shows outcomes with GLP-IRA use in advanced-stage non-small cell lung cancer (NSCLC) patients on immune checkpoint inhibitors (ICI). Progression-free survival (FIG. 6A) and overall survival (FIG. 6B) is shown for overweight and obese patients receiving ICI for advanced NSCLC after propensity score matching for age, sex, race, smoking, histology, stage, first-line therapy, and GLP-IRA use as covariates.DETAILED DESCRIPTION

[0015] A reduced risk of multiple cancers occurs with glucagon-like peptide-1 receptor agonists (GLP-lRAs), drugs widely used for glycemic control and weight reduction that modulate severalkey regulators of metabolism, particularly with non-small cell lung cancer (NSCLC). Two clinical cohorts of overweight and obese patients with NSCLC— one undergoing surgical resection (n=l,177, 71 GLP-1RA users) and another receiving immune checkpoint inhibitors (ICIs; n=300, 10 GLP-1RA users), were propensity score matched for relevant covariates and analyzed for clinical outcomes. GLP-1RA use was associated with increased recurrence-free survival in overweight and obese patients (HR=0.41 [95%CI=0.16- 1.04], p=0.026) after lobectomy. GLP-1RA treatment reduced tumor burden in obese but not normal-weight mice and altered the frequency and phenotypes of leukocyte populations and gene expression patterns in obese tumors, crucial to cancer progression and anti-tumor immunity. Concurrent GLP-1RA and immunotherapy was also associated with improved overall (0.41 [0.16-1.01], 0.027) and progression-free survival (HR=0.31, [0.10-0.94], 0.019) for patients with advanced NSCLC. GLP-lRAs may enhance lung cancer-specific clinical outcomes and augment immunotherapy efficacy. Preclinical evidence suggests this effect is obesity restricted and mediated by immune modulation of the tumor microenvironment.

[0016] GLP-lRAs counteract the detrimental effects of obesity on anti-tumor immunity and disease progression, leading to improved NSCLC outcomes. GLP-1RA has little effect on in vitro cell proliferation, suggesting that significant inhibitory effects on malignant cells are unlikely. Both implanted and autochthonous mouse lung cancer models treated with GLP-1RA show consistent and significantly reduced tumor burden. Thus, the reduction in tumor growth seen in obese mice treated with GLP1-RA may be linked to biological pathways active within the immune tumor microenvironment (TME).

[0017] Parallel transcriptomic analysis substantiated an immune-stimulating effect of GLP-1RA, revealing significant treatment-associated alterations in pathways related to oncogenesis, tumor cell proliferation, and metabolism, which may modulate complex biological processes intrinsic as well as extrinsic to malignant cells themselves. The subcutaneous tumor model of the current disclosure showed that liraglutide, a widely used GLP-1RA, inhibited tumor growth selectively in obese mice and also revealed multifaceted immune-related changes in the tumor microenvironment consistent with enhanced anti-tumor immunity. These included increased CD4+ T cell and NK cell frequencies, bolstered pools of memory-like T cells, reduced Treg populations, and apparent improvements in APC functionality. Changes in gene expression profiles closely resembled those observed in individuals with elevated total fat areas (36). That study, which examined genomic alterations in NSCLC patients with high versus low body fat, revealed similar changes in transcripts associated with cell proliferation, signaling, and apoptosis,apart from genes regulating fatty acid metabolism. These findings indicate that liraglutide may exert its effects by modulating cellular pathways in a manner that counteracts obesity-induced metabolic adaptations, potentially leading to improved clinical outcomes.

[0018] The observed immunological effects run surprisingly counter to much of the existing literature speaking to the immunomodulatory properties of GLP-1 and GLP-1RA, which generally support a role in immune suppression in various settings. For example, GLP-1 has been convincingly characterized as a mediator of negative costimulation in T cells, with agonism leading to suppression of proliferation and allograft rejection in mouse studies. In contrast, GLP-1R antagonism, via exe9, bolstered anti-tumor immunity when tested in a colorectal cancer model(37). Additional studies have characterized the activity of GLP-1RA drugs as anti-inflammatory(38), and reports have suggested both high expression of the GLP-1 receptor by Treg cells (20) and a role for GLP-1 signaling in the biology of these suppressive cells (21, 39).

[0019] The retrospective clinical studies disclosed herein provide evidence that this effect translates into human NSCLC. In early-stage NSCLC patients undergoing surgery, GLP-1RA in the postoperative period improved recurrence-free survival (RFS). Following the animal experiments and in vitro assays, analysis of GLP-1RA use in advanced-stage NSCLC patients receiving ICI demonstrated augmented benefit, with improvement in both progression free survival (PFS) and overall survival (OS).

[0020] Liraglutide does not impair murine lung cancer cell proliferation in vitro.

[0021] The in vitro effect of liraglutide on KPN 1.1 (Kraslox-stop-lox(lsl)-G12D / +; p53flox / flox- NINJA) and Lewis Lung Carcinoma (LLC) cell proliferation was investigated. Based on reported steady-state plasma concentrations of liraglutide ranging from 20-40 nM for typical clinical doses (0.6-1.8 mg), a range of concentrations: 0, 10, 30, 90, 270, and 810 nM was examined. Cell proliferation was assessed by measuring the fold change in absorption values after 48 hours of treatment. There was no significant difference in cell proliferation for KPN1.1 cells across liraglutide concentrations, while LLC cells exhibited increased proliferation at higher drug concentrations.

[0022] Liraglutide inhibits the progression of implanted tumor growth in obese, but not normal weight mice.

[0023] The effect of liraglutide on tumor growth in vivo was investigated. Here, normal weight (lean) C57BL / 6 mice and those with diet-induced obesity (DIO) were challenged with subcutaneous (s.c.) injection of KPN1.1 lung cancer cells. Upon detection of palpable s.c. tumors,mice received daily liraglutide (0.2 pig / g body weight) or a PBS vehicle intraperitoneally. While liraglutide-treated mice exhibited a slight decrease in body weight during the experiment, and control mice showed an increase (weight change: -0.78 g vs. +0.98 g, respectively), no statistically significant difference was observed in mean tumor volumes or final tumor weight (0.98 g vs. 1.04 g; p = 0.67) or in normal weight / lean mice (FIG. 1A, IB). In contrast, liraglutide administration significantly inhibited the progression of tumor growth, as evidenced by reductions in tumor volumes and weight (1.07 g vs. 1.86 g; p = 0.03) relative to the vehicle group (FIG. 1C, ID). Of note, liraglutide-treated DIO mice demonstrated greater weight reduction compared to their respective controls (13.46 g vs. 4.06 g, respectively).

[0024] RNA sequencing reveals GLP1-RA altered pro-tumor gene expression in mice with implanted lung cancers.

[0025] Tumor gene expression was compared in liraglutide- and vehicle-treated s.c. tumors harvested in DIO mouse model studies using RNA Sequencing (RNASeq). Of the 35,141 transcripts assessed, T1 were differentially expressed significantly (adjusted p-value < 0.05 and | logFC | > 0.58, FIG. 2A). The transcripts upregulated with liraglutide treatment (FIG. 2A, green) included Igfbp7, which induces G1 phase cell cycle arrest by upregulating cyclin-dependent kinase inhibitors. Additionally, treatment was associated with increased Creb3ll, a regulator of angiogenesis (11), and Nfix, which encodes a factor with pro- and anti-tumor roles that are also involved with immune cell development and differentiation (12). Conversely, liraglutide exposure was also associated with decreased AnxalO, a well-known marker of poor prognosis with a role in various cell-signaling pathways (13). Further, the drug was linked to reduced expression of the pro-tumor genes Higdla and Rpl39, associated with hypoxia cellular stress and metabolism, and cell proliferation and migration, respectively.

[0026] Gene set enrichment analysis (GSEA) revealed significant treatment effects on several pathways relevant to cancer outcomes (normalized enrichment score > 2, q-value < 0.05) spanning cell proliferation, cellular respiration, immune signaling, and cell signaling (FIG. 2B). Genes linked to signaling triggered by interferon-gamma (an anti-tumor immunity-promoting cytokine) and growth factor (Insulin-derived growth factor) signaling were significantly upregulated with liraglutide treatment. Several metabolic pathways important for tumor and leukocyte biology, including fatty acid (sphingolipids) and amino acid metabolism, were enhanced and disrupted, respectively, and genes important for translation-associated processes were also apparently suppressed with treatment, as were those associated with poor lung cancer survival.Finally, multiple cell signaling pathways well appreciated for driving tumor development and progression were also suppressed in GLP-1RA treated tumors, including those involving KRAS, EGFR, MYC, and E2F transcription factors (FIG. 2B). Overall, these findings link liraglutide with gene expression changes in the tumor relevant to immunity, metabolism, and tumor progression, explaining the effects of GLP-1RA seen in mice and patients.

[0027] GLP-1RA treatment enhances anti-tumor immune responses in mice.

[0028] Flow cytometry was used to characterize the phenotypic and functional changes among the tumor-infiltrating leukocytes (TILs) associated with treatment in harvested s.c. tumors. Increased CD4+ T cell and Natural-killer (NK) cell frequencies in the tumors of GLP-lRA-treated obese mice were seen (FIGs. 3A, 3B). Proportions of conventional (i.e., non-regulatory) CD4+ T cells (Tcon), marked by the activation marker CD69, were also elevated in liraglutide-treated mice (FIG. 3C)— potentially indicating an enhanced anti-tumor immune response. The fraction of intratumor CD8+ and Tcon compartments with an effector surface marker profile (CD44high / CD62Llow) was apparently reduced in favor of CD44+ / CD62L+ T cells (FIGs. 3D, 3E), suggesting a potential shift towards a memory-like phenotype in these cells.

[0029] Liraglutide-triggered changes among tumor Tregs were also assessed. Proportions of Foxp3+ / CD25+ cells within the TIL CD4+ pool were significantly reduced with treatment (FIG. 3F). The functionally potent, activated (CD44+ / CD62L-) or "eTreg" subpopulation made up a smaller portion of the TIL-Treg pool from liraglutide-treated mice compared to vehicle-treated control mice, in favor of a population with memory-like potential (i.e., CD44+ / CD62L+ surface staining) (FIG. 5G). In agreement with a prior study (20), GLP-1RA treatment upregulated PD-1 and PD-L1 on tumor Tregs, however increased levels of mTOR activity (indicated by phosphorylated mTOR (p-mTOR)+ / Foxp3+ T cells) and Tbet expression were found among GLP-lRA-treated Tregs as well. This combination indicates an activated but unstable Treg phenotype (22-25), less capable of restricting robust anti-tumor immune response suppression. Collectively, these results suggest the ability of GLP-1RA to undermine immune suppression in obese mice.

[0030] Additional effects of GLP-1RA treatment were evident in antigen-presenting cell (APC) populations in the tumor niche. Dendritic cells (DCs) and macrophages from treated mice displayed upregulated MHC II levels, an effect that approached and achieved statistical significance in these populations, respectively (FIG. 3H). Conversely, TILs of the GLP-1RA harbored markedly fewer APCs producing indoleamine 2,3-dioxygenase (IDO) (FIG. 31), a mediator of tumor- associated immune suppression and a mechanism of Treg expansion and differentiation (26, 27).The mice treated with liraglutide also displayed a relative scarcity of arginase-expressing myeloid- derived suppressor cells (MDSCs) (FIG. 5J). GLP-1RA triggers APC phenotypes poised to effectively stimulate T cell responses and cell-mediated anti-tumor immunity. Further supporting this notion, the pool of TIL-isolated CD4+ T cells recovered from liraglutide-treated mice capable of producing pro-inflammatory, tumoricidal cytokines ex vivo was expanded (FIG. 3K). These findings support a potent immunostimulatory effect of the GLP-1RA liraglutide impacting several leukocyte populations that may be responsible for the improved tumor growth control and clinical outcomes linked to the drug.

[0031] Semaglutide inhibits tumor progression and improves survival in a KRAS-driven mouse lung cancer model.

[0032] The effects of GLP-1RA administration on murine tumor growth were also examined in an independent autochthonous mouse model with conditional KrasG12D activation and Trp53 deletion (KP) using the GLP-1RA semaglutide. To induce obesity, tumor-free KP mice were pre-fed a high-fat Western diet (WD; 60% kcal from fat) supplemented with sugar water (23.1 g / L D- fructose and 18.9 g / L D-glucose) for 13 weeks prior to tumor initiation (FIG. 4A). Lung adenocarcinoma was initiated via intratracheal delivery of Cre recombinase, precipitating spontaneous tumor growth which more closely mimics the geographic pattern of human lung cancer metastases compared to subcutaneous implantation. The KP mice with DIO and induced lung adenocarcinoma were treated with semaglutide ( N=7) or vehicle control ( N=7).

[0033] Semaglutide treatment significantly extended overall survival (HR: 0.21, 95% Cl: 0.057- 0.79, p = 0.012) compared to vehicle-treated control (Fig. 4B). Longitudinal pCT revealed significantly reduced tumor burden in the semaglutide groups (Fig. 4C), mirroring the results of the heterotopic models, where GLP-RA reduced tumor growth and progression in obesity- associated lung cancer.

[0034] GLP-1RA use is associated with improved Recurrence Free Survival (RFS) after non- small cell lung cancer (NSCLC) resection.

[0035] Clinical outcomes in a database of patients undergoing surgery for non-small cell lung cancer (NSCLC) at Roswell Park Cancer Institute (RPCI) were examined between 2015 and 2024. A total of 1,177 patients met criteria for inclusion in the post-surgical cohort. Within this cohort, 71 patients were GLP-RA users. Demographic characteristics, including age, clinical stage, race, and smoking history were generally well balanced between the GLP-1RA users and non-users, however GLP-1RA users had significantly higher mean Body Mass Index (BMI) at the time ofresection than the non-users (35.07 vs 30.6; p = 0.001). Most patients across both groups had clinical-stage I disease 970 (82.4%), while 193 (16.4%) had stage II, and 14 (1.2%) had stage III lung cancers.

[0036] In an unmatched, univariate cox-regression model including the entire cohort, GLP-1RA use was associated with improved recurrence free survival (RFS) (HR: 0.41; 95% Cl: 0.17- 1.02; p = 0.027). No such association was noted for overall survival (OS) (HR: 0.70; 95% Cl: 0.43- 1.16; p = 0.09).

[0037] In a propensity matched analysis, where propensity scores were calculated using age, sex, race, BMI, cancer stage, smoking status, and histology as covariates and GLP-1RA use as the dependent variable, a matched cohort of 629 was identified which included 560 GLP-1RA nonusers and 69 GLP-1RA users. This cohort did not include any patients with stage III disease. Univariate Cox regression of matched cohort showed that GLP-1RA use was associated with improved recurrence free survival (RFS) (HR = 0.41, 95% Cl: 0.16-1.04, p = 0.026; Figure 5A). GLP- 1RA use was not significantly associated with OS (HR = 0.78, 95% Cl: 0.44-1.38, p = 0.20; Figure 5B). Results of univariate analyses for all the covariates in both unmatched and matched cohorts are presented in Table 1.

[0038] Table 1 shows the overall survival (OS) and recurrence-free survival (RFS) analyses for overweight and non-overweight NSCLC patients who underwent resection. Univariate analyses were performed using the cox proportional hazards model before and after propensity score matching for age, sex, race, smoking status, stage, histology, and history of GLP-1 RA use (users vs. non-users).

[0039] GLP-1RA use improved survival in overweight patients receiving immune checkpoint inhibitors (ICI) for advanced non-small cell lung cancer (NSCLC).

[0040] The benefits of GLP1-RA use extend to patients on immune checkpoint inhibitor (ICI) therapy. A cohort of 300 patients with advanced lung cancer treated with ICI at Roswell Park Cancer Institute (RPCI) between 2015 and 2023 was identified, of which 10 patients were prescribed concurrent GLP-1RA. All GLP-1RA users in the cohort had diabetes (reflecting FDA approved indications during the study period), whereas 25% of non-users were diabetic. Most patients (78.7%) had stage IV disease; the remainder of the cohort was comprised of stage III NSCLC not amenable to local treatments. Treatment included immunotherapy alone for 158 patients (52.7%) and a combination of immuno- and chemotherapy for 142 (47.3%). Patientsreceiving GLP-1RA were significantly younger; other characteristics such as gender, BMI, race, smoking status, tumor histology, and treatment modality were similar between groups.

[0041] In an unmatched analysis, univariate cox-regression modeling demonstrated that GLP- 1RA use was associated with improved progression free survival (PFS) (HR: 0.39; 95% Cl: 0.13- 1.14; p = 0.044) and overall survival (OS) (HR: 0.40; 95% Cl: 0.15- 1.04; p = 0.031).

[0042] In a propensity matched analysis, propensity scores were calculated using age, sex, race, smoking status, BMI, tumor histology, first-line therapy, stage at the start of ICI therapy, as covariates with GLP-1RA use as the dependent variable. The matched cohort (n=89) had 79 non- users and 10 GLP-1RA users. Univariate Cox regression of the matched cohort showed that GLP- 1RA use was associated with improved progression free survival (PFS) (HR = 0.31, 95% Cl: 0.1- 0.94, p = 0.019; FIG. 6A) and overall survival (OS) (HR = 0.41, 95% Cl: 0.16-1.01, p = 0.027; FIG. 6B). Results of univariate analyses for all covariates in both unmatched and matched cohorts are presented in Table 2.

[0043] Table 2 shows overall survival (OS) and progression-free survival (PFS) analyses for overweight and non-overweight NSCLC patients on immune checkpoint inhibitors. Univariate analyses were performed using the Cox proportional hazards model before and after propensity score matching for age, sex, race, smoking status, stage, histology, first-line therapy, and history of GLP-1 RA use (users vs. non-users).REFERENCES

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Lu et aL, Liraglutide activates nature killer cell-mediated antitumor responses by inhibiting IL-6 / STAT3 signaling in hepatocellular carcinoma. Transl Oncol 14, 100872 (2021).41. D. Chen, H. Liang, L. Huang, H. Zhou, Z. Wang, Liraglutide enhances the effect of checkpoint blockade through the inhibition of neutrophil extracellular traps in murine lung and liver cancers. FEBS Open Bio n / a.42. D. Chen et aL, Exenatide enhanced the antitumor efficacy on PD-1 blockade by the attenuation of neutrophil extracellular traps. Biochem Biophys Res Commun 619, 97-103 (2022).43. B. Fitzgerald et aL, A mouse model for the study of anti-tumor T cell responses in Kras- driven lung adenocarcinoma. Cell Rep Methods 1, (2021).44. B. Langmead, Aligning short sequencing reads with Bowtie. Curr Protoc Bioinformatics Chapter 11, Unit 11.17 (2010).45. S. Ghosh, C. K. Chan, Analysis of RNA-Seq Data Using TopHat and Cufflinks. Methods Mol Biol 1374, 339-361 (2016).46. Y. Liao, G. K. Smyth, W. Shi, The Subread aligner: fast, accurate and scalable read mapping by seed-and-vote. Nucleic Acids Res 41, el08 (2013).47. M. I. Love, W. Huber, S. Anders, Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biology 15, 550 (2014).48. J. Reimand et al., Pathway enrichment analysis and visualization of omics data using g:Profiler, GSEA, Cytoscape and EnrichmentMap. Nat Protoc 14, 482-517 (2019).49. A. Liberzon et al., Molecular signatures database (MSigDB) 3.0. Bioinformatics 27, 1739- 1740 (2011).50. E. L. Jackson et al., The differential effects of mutant p53 alleles on advanced murine lung cancer. Cancer Res 65, 10280-10288 (2005).51. M. DuPage, A. L. Dooley, T. Jacks, Conditional mouse lung cancer models using adenoviral or lentiviral delivery of Cre recombinase. Nat Protoc 4, 1064-1072 (2009).52. M. M. Winslow et aL, Suppression of lung adenocarcinoma progression by Nkx2-1. Nature 473, 101-104 (2011).53. E. L. Jackson et al., Analysis of lung tumor initiation and progression using conditional expression of oncogenic K-ras. Genes Dev 15, 3243-3248 (2001).54. T. Tammela et al., A Wnt-producing niche drives proliferative potential and progression in lung adenocarcinoma. Nature 545, 355-359 (2017).55. Y. Zhang et al., Activity-balanced GLP-1 / GDF15 dual agonist reduces body weight and metabolic disorder in mice and non-human primates. Cell Metab 35, 287-298. e284 (2023).56. E. Meylan et al., Requirement for NF-kappaB signalling in a mouse model of lung adenocarcinoma. Nature 462, 104-107 (2009).57. S. Stapleton, G. Welch, L. DiBerardo, L. R. Freeman, Sex differences in a mouse model of diet-induced obesity: the role of the gut microbiome. Biology of Sex Differences 15, 5 (2024).58. J. Oraha, R. F. Enriquez, H. Herzog, N. J. Lee, Sex-specific changes in metabolism during the transition from chow to high-fat diet feeding are abolished in response to dieting in C57BL / 6J mice. 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[0045] Disclosed is a method of treating cancer, consisting of: administering to an overweight human subject at least one glucagon-like peptide-1 receptor agonist (GLP-1RA), and administering to the human subject at least one immune checkpoint inhibitor. In embodiments of the disclosed method of treatment, the GLP-1RA is any of Dulaglutide (Trulicity®); Exenatide (Byetta®); Exenatide extended-release (Bydureon®); Liraglutide (Victoza®); Lixisenatide (Adlyxin®); Semaglutide injection (Ozempic®); Semaglutide tablets (Rybelsus®), or a combination thereof. Other embodiments may use tirzepatide (Zepbound®) alone or in combination with other GLP- 1RA drugs. Any suitable formulation that mimics naturally-occurring GLP-1 and boosts signaling at GLP-1 receptors may also be used. GPL-1RA drugs are generally described in Zheng, Z., Zong, Y., Ma, Y. et al. Glucagon-like peptide-1 receptor: mechanisms and advances in therapy. Sig Transduct Target Ther 9, 234 (2024). https: / / doi.arfi / 10.1038 / s41392-024-01931-z.

[0046] In some embodiments, the dosage of the GLP1-RA may range from 0.25 mg to 14 mg, or 10 mcg to 20 mcg. In some embodiments, the dosage of the GLP1-RA is 0.25 mg, 0.5 mg, 0.6 mg, 1.0 mg 1.2 mg, 1.7 mg, 1.8 mg, 2.4 mg, 3.0 mg, 7 mg, or 14 mg. In some embodiments, the dosage of the GLP1-RA may range from 0.6 mg to 1.8 mg. In some embodiments, the dosage of the GLP1- RA is 0.6 mg, 1.2 mg, or 1.8 mg. In some embodiments, the dosage of the GLP1-RA ranges from2.5 mg-15 mg. In some embodiments, the dosage of the GLP1-RA is 2.5 mg, 5 mg, 7.5 mg, 10 mg,12.5 mg, or 15 mg

[0047] Proven immune checkpoint inhibitors (ICIs) in clinical application include cytotoxic T- lymphocyte-associated protein 4 (CTLA-4) inhibitors and programmed cell death protein-1 (PD-1) receptor inhibitors / programmed cell death ligand 1 (PD-L1) inhibitors. CTLA-4 is a transmembrane receptor on T cells, which can compete with CD28 to prevent co-stimulation and induce T cell cycle arrest. CTLA-4 inhibitors block the above process and restore the function of T cells to eradicate tumor cells. CTLA-4 inhibitors approved by the U.S. Food and Drug Administration (FDA) include: ipilimumab and tremelimumab. PD-1 is expressed on tumor-infiltrating lymphocytes (mainly CD4+ T cells), B cells, natural killer cells, monocytes, and dendritic cells, while PD-L1 is highly expressed on tumor cells. The binding of PD-1 to PD-L1 mediates a co-inhibitory signal of T cell activation, thus leading to tumor immune escape. PD-1 / PD-L1 inhibitors block the PD-1 signaling pathway, partially restoring T-cells recognition of tumors, and inducing immune normalization. FDA-approved PD-1 inhibitors include: nivolumab, pembrolizumab, cemiplimab, and dostarlimab. Approved PD-L1 inhibitors include: atezolizumab, avelumab, durvalumab. See Jiang M, Hu Y, Lin G, Chen C. Dosing Regimens of Immune Checkpoint Inhibitors: Attempts atLower Dose, Less Frequency, Shorter Course. Front Oncol. 2022 Jun 20;12:906251. doi: 10.3389 / fonc.2022.906251. PMID: 35795044; PMCID: PMC9251517.

[0048] ICIs comprise monoclonal antibodies and their pharmacokinetic / pharmacodynamic properties are distinctly different from those of traditional cytotoxic and small molecule drugs. ICIs were initially administered based on body weight, and as population pharmacokinetic data accumulated, fixed-dose regimens were found to improve convenience and reduce waste while preserving efficacy. The FDA has approved nivolumab 240 mg Q2W equivalent to 3 mg / kg Q2W and pembrolizumab 200 mg Q3W equivalent to 2 mg / kg Q3W. High-dose, extended-interval dosing regimens (e.g., nivolumab 480 mg Q4W and pembrolizumab 400 mg Q6W) were subsequently added to all approved adult indications. The choice of average body weight is not consistent across ICIs: 240mg fixed dose of nivolumab is numerically equivalent to 3mg / kg dose for 80kg patients, while 200mg pembrolizumab corresponds to 2mg / kg for 100kg patients and 750mg durvalumab corresponds to 10mg / kg for 75kg patients. The average weight of patients using ICI is about 75 kg. Clinicians may reduce the dose or delay the administration of ICIs concerns about the patient's physical condition or adverse effects of drugs, as well as for economic reasons or patient requests, but significant survival benefits can still be seen, which may provide information in optimizing the dosing regimens. Dosing regimens for ICIs are discussed in Jiang M, Hu Y, Lin G, Chen C. Dosing Regimens of Immune Checkpoint Inhibitors: Attempts at Lower Dose, Less Frequency, Shorter Course. Front Oncol. 2022 Jun 20;12:906251. doi: 10.3389 / fonc.2022.906251. PMID: 35795044; PMCID: PMC9251517.

[0049] In embodiments of the disclosed method of treatment, the immune checkpoint inhibitor inhibits at least one of PD1, PD-L1 or PD-L2, or CTLA4. In an embodiment, the immune checkpoint inhibitor comprises pembrolizumab. The immune checkpoint inhibitor may be any inhibitor effective against the cancer being treated. The GLP-1RA and the immune checkpoint inhibitor may be the only agents administered to the individual to elicit an anti-cancer response, or the described approach can be combined with other anti-cancer and other agents, such as antidiabetic agents other than the described GLP-lRAs.

[0050] Examples of administration of the GLP-1RA drugs used in the disclosed methods may include (with SC=subcutaneously) those listed in Table 3.

[0051] TABLE 3

[0052] In the disclosed methods, a therapeutically effective amount of a combination of an immune checkpoint inhibitor and a GLP-1RA may be used. In the disclosed methods, a therapeutically effective amount of an immune checkpoint inhibitor may be used with a therapeutically effect amount of a GLP-1RA. A precise dosage of the described agents can be selected by the individual physician in view of the patient to be treated. A therapeutically effective amount is an amount that reduces one or more signs or symptoms of a disease, and / or reduces the severity of the disease. In some embodiments, a therapeutically effective amount is an amount that reduces or eliminates cancer cells from an individual. In some embodiments, a therapeutically effective dose inhibits growth of cancer cells. In some embodiments, a therapeutically effective dose inhibits metastasis.

[0053] The immune checkpoint inhibitor and the GLP-1RA may be administered concurrently or sequentially. In one embodiment, the at least one GLP-1RA is administered prior to the administering of the at least one immune checkpoint inhibitor, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12. 13. 14. 15. 16. 17. 18. 19, 20, 21, 22, 23, 24 hours or >24 hours prior the administration of the immune checkpoint inhibitor(s). In another embodiment, the immune checkpoint inhibitor is administered prior to the at least one GLP-1RA, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16. 17. 18. 19, 20, 21, 22, 23, 24 hours or >24 hours prior the administration of the GLP-1RA.

[0054] The treated subject may be an individual who has not previously received a GLP-1RA, and / or an immune checkpoint inhibitor, and / or a combination thereof. The treatment may beadministered to a subject that is resistant to the immune checkpoint inhibitor. In one embodiment, combined administration of a GLP-1RA reduces or eliminates resistance to immune checkpoint inhibition. In one embodiment of the method, the GLP1-RA is liraglutide. In one embodiment of the method, the GLP1-RA is semaglutide. In one embodiment of the method, the GLP1-RA is liraglutide and the immune checkpoint inhibitor is pembrolizumab. In one embodiment of the method, the GLP1-RA is semaglutide and the immune checkpoint inhibitor is pembrolizumab.

[0055] The described methods of treatment may be applied to an individual who is overweight and has been diagnosed with cancer, including in a preferred embodiment, non-small cell lung cancer (NSCLC). In embodiments of the disclosed method, the overweight human subject has a Body Mass Index (BMI) greater than or equal to 25 to 29.9 kg / mA2. In other embodiments, the overweight human subject is obese and has a BMI greater than or equal to 30 kg / mA2. In other embodiments, the overweight human subject has Obesity class I (BMI 30 to 34.9 kg / mA2); or Obesity class II (BMI 35 to 39.9 kg / mA2) or Obesity class III (BMI greater than or equal to 40 kg / mA2), which may also be referred to as severe, extreme, or massive obesity.

[0056] In some embodiments, the overweight individual has non-small cell lung cancer (NSLC) that is resistant to immune checkpoint inhibition. In one embodiment, the individual's cancer is resistant to anti-PD-1 therapy.

[0057] The disclosed methods may be efficacious to treat different types of cancer. The cancers may include bladder cancer, bone cancer, brain cancer, breast cancer, cervical cancer, colon cancer, esophageal cancer, gastric cancer, gastrointestinal cancer, gallbladder cancer, head and neck cancers, Hodgkin's Lymphoma, glioblastoma, acute myeloid leukemia, leukemia, liver cancer, lung cancer, melanoma, mesothelioma, multiple myeloma, myelodysplastic syndrome, Non-Hodgkin's Lymphoma, ovarian cancer, pancreatic cancer, prostate cancer, rectal cancer, renal cancer, sarcoma, skin cancer, testicular cancer, throat cancer, thyroid cancer and uterine cancer. The human subject may have solid tumors.

[0058] When administering the at least one GLP-1RA in combination with at least one immune checkpoint inhibitor, the administering can be carried out in vitro, in vivo, or ex vivo to a subject. When administering the at least one GLP-1RA in combination with at least one immune checkpoint inhibitor, the normal, monotherapeutic dose of the immune checkpoint inhibitor may be significantly reduced. In one embodiment, the dose of the immune checkpoint inhibitor for thesubject being treated is reduced by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or >50% of the monotherapeutic dose of that subject having cancer.

[0059] In accordance all aspects of the present invention, the subject being treated is a mammal, preferably a human, but can also be an animal in need of veterinary treatment, e.g., companion animals (e.g., dogs, cats, and the like), farm animals (e.g., cows, sheep, pigs, horses, and the like) and laboratory animals (e.g., rats, mice, guinea pigs and the like).

[0060] For the at least one GLP-1RA or the at least one checkpoint inhibitor, the administering step is to be carried out systemically or via direct or local administration. By way of example, suitable modes of systemic administration include, without limitation orally, topically, transdermally, parenterally, intradermally, intramuscularly, intraperitoneally, intravenously, subcutaneously, or by intranasal instillation, by intracavitary or intravesical instillation, intraocularly, intraarterialy, intralesionally, or by application to mucous membranes. Suitable modes of local administration include, without limitation, catheterization, implantation, direct injection, dermal / transdermal application, or portal vein administration to relevant tissues, or by any other local administration technique, method or procedure generally known in the art. The mode of affecting delivery of the GLP-1RA or the one checkpoint inhibitor will vary depending on the type of therapeutic agent and the cancer to be treated.MATERIALS AND METHODS

[0061] In vitro effects of liraglutide on murine lung cancer cell lines.

[0062] The murine lung cancer cell lines KPN 1.1 (43) (provided by Dr. N. Joshi) and Lewis Lung Carcinoma (LLC; ATCC # CRL-1642-LUC2) were cultured in vitro and harvested in the logarithmic phase by trypsinization, followed by centrifugation. Cell concentration was adjusted and added to strip-well plates at 3000 cells per well, which were then incubated at 37°C. After 24 hours, the cells were treated with liraglutide at 10, 30, 90, 270, and 810 nM concentrations. To measure the cell proliferation rate, the wells were washed, and 95 μL of cell culture medium and 5 μL of CCK- 8 reagent (Dojindo Laboratories) were added to each well. The strips were allowed to incubate at 37 °C for 2 h, and the absorbance value (OD value) was measured and recorded using a microplate reader at a wavelength of 450 nm at 0, 24, 48, 72, and 96 hr. The cell proliferation rate was calculated as follows: cell proliferation rate = (OD value at a given concentration at 48-hour timepoint-blank) / (OD value on day 0-blank).

[0063] Effects of GLP1-RA on murine survival, subcutaneous tumor dynamics, gene expression and tumor immune microenvironment in an implanted lung cancer model.

[0064] Age-matched obese and non-obese male C57BL / 6 mice were purchased (The Jackson Laboratory, ME, US; strain numbers 380050, 380056) or generated in-house by feeding normal weight mice with high fat (60% calories from fat, 5.4 kCal / g) or control chow ("'7.2% calories from fat, 3.9 kCal / g) (Bio-Serv, NJ, USA, products #53282 and #F4031, respectively). For s.c. tumor challenge experiments, KPN 1.1 cells or LLC cells were injected into the flanks of 7-17-week-old obese and non-obese mice (75,000 cells per mouse). Mice with palpable tumors (typically detected 6-7 days post-implantation) received intraperitoneal injections of liraglutide (0.2 ug per gram of mouse body weight) or Phosphate-buffered saline (PBS) vehicle daily. The mice were euthanized after 19-22 days of cell inoculation, and tissues were harvested for further analysis. All animal experiments conducted were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Roswell Park Comprehensive Cancer (Protocol 1487).

[0065] RNA sequencing was performed on excised, subcutaneous KPN1.1 tumors from obese mice treated with GLP-1RA. At the time of mouse tumor harvest, a fragment was flash frozen and was later used for RNA isolation using the miRNeasy mini kit (Qiagen). The sequencing libraries were prepared with the RNA HyperPrep Kit with RiboErase (HMR) kit (Roche Sequencing Solutions), from 500ng total RNA. Following the manufacturer's instructions, the first step depletes rRNA from total RNA. After ribosomal depletion, the remaining RNA is DNase-digested to remove any gDN A contamination. Samples are then purified, fragmented, and primed for cDNA synthesis. Fragmented RNA is then reverse-transcribed into first-strand cDNA using random primers.

[0066] The next step removes the RNA template and synthesizes a replacement strand, incorporating dUTP in place of dTTP to generate ds cDNA. Pure Beads (KAPA BIOSYSTEMS) are used to separate the ds cDNA from the second-strand reaction mix, resulting in blunt-ended cDNA. A single 'A' nucleotide is then added to the 3' ends of the blunt fragments. Multiple indexing adapters, containing a single 'T' nucleotide on the 3' end of the adapter, are ligated to the ends of the ds cDNA, preparing them for hybridization onto a flow cell. Adapter-ligated libraries are amplified by PCR, purified using Pure Beads, and validated for appropriate size on a 4200 Tapestation D1000 Screentape (Agilent Technologies, Inc.). The DNA libraries are quantified using KAPA Biosystems qPCR kit, and are pooled together in an equimolar fashion, following experimental design criteria. Each pool is denatured and diluted to 350pM with 1% PhiX controllibrary added. The resulting pool is then loaded into the appropriate NovaSeq Reagent cartridge for 100 paired-end sequencing and sequenced on a NovaSeq6000 following the manufacturer's recommended protocol (Illumina Inc.). Raw reads that passed the Illumina RTA quality filter were demultiplexed and pre-processed using FastQC for sequencing base quality control. Reads were then mapped to the latest version of a mouse reference (GRCm39 / mm39) using Bowtie (v1.0.1) (44) and TopHat (v2.0.13) aligner (45). Mapped reads were quantified at the gene level as a raw counts matrix using featurecounts from Subread (46). Samples were filtered after quality assessment, and a total of 10 samples were used in subsequent analyses (n=5 / group). Raw feature counts were normalized, and differential expression analysis was carried out using DESeq2 (47), and visualized using volcano plots. Differential expression rank order was used for subsequent gene set enrichment analysis (GSEA) (48), performed using the cluster profile package in R, and visualized via lollipop plots. Gene sets queried included the Hallmark, Canonical pathways, and GO Biological Processes Ontology collections available through the Molecular Signatures Database (MSigDB) (49).

[0067] Flow cytometry analysis was performed on the s.c. KPN1.1 tumors excised from obese mice treated with GLP-1RA. Tumors were cleaned of skin and fat before mechanical (GentleMACS dissociation) and enzymatic digestion in a collagenase / hyaluronidase mix (Stem Cell Technologies) following the manufacturer's protocol. The resulting cell suspensions were filtered (100 μm cell strainer), washed, and pelleted before RBC lysis in 1 mL ACK buffer (Thermo Fisher Scientific). Cells were then washed with PBS containing 1% Fetal Bovine Serum (FBS) and 2mM EDTA before incubation with fluorochrome-conjugated antibodies diluted in like buffer. After surface immunostaining, intracellular markers (i.e., F0XP3) were stained after fixation and permeabilized using eBioscience's F0XP3 staining kit. For intracellular cytokine staining, cell suspensions were re-stimulated in media containing PMA and lonomycin in the presence of Golgi- Plug (BD) for 5 hours at 37 °C before surface staining, fixation / permeabilization, and internal staining with anti-IFNγ and TNFα antibodies. Samples were run on an Aurora spectral flow cytometer (Cytek) before data analysis via OMIQ.

[0068] Effect of semaglutide on survival in an autochthonous KRAS-driven murine lung cancer model.

[0069] KrasLSL-G12D; Trp53fl / fl (KP) mice (50) (Jackson Laboratory #032435) were fed a high-fat Western diet (WD; 60% kcal fat, Research Diets #012492) with sugar water (23.1 g / L D-fructose + 18.9 g / L D-glucose) ad libitum for 13 weeks to induce diet-induced obesity (DIO). Lung tumorswere initiated via intratracheal adenoviral Cre recombinase (Ad-Cre; 2.5x107 PFU; University of Iowa Viral Core) (51-54).Tumor-bearing DIO-KP mice were randomized to receive intraperitoneal semaglutide (60 nmol / kg / 0.25 pig / gm body weight, 3x / week; Cat #S9697; Selleck Chemicals, Houston, TX, USA) or saline (pH 7.4; Cytiva, Marlborough, MA, USA) (55). Imaging was conducted through the Oncology Precision Therapeutics and Imaging Core (OPTIC Core) at the Columbia University Herbert Irving Comprehensive Cancer Center. High-resolution microcomputed tomography (pCT) was performed to assess lung tumor burden, as previously described (56). All procedures complied with Columbia University IACUC guidelines (Protocol #AABV8661).

[0070] Sex as a biological variable.

[0071] Male mice were used in these lung cancer preclinical models to facilitate creation of DIO models as male C57BL / 6 mice more uniformly display significant weight gain on high fat diets (relative to normal diet controls) than females (57-59). This approach is consistent with established protocols in the field and facilitates direct comparison with previous studies.

[0072] Effects of GLP1-RA on survival outcomes in two retrospective clinical cohorts.

[0073] Two distinct single-center clinical cohorts were manually curated and analyzed.

[0074] In a post-surgical cohort, medical charts for patients undergoing surgical resection atRoswell Park Cancer Institute (RPCI) between 2015-2024 were manually curated. Patients were included for analysis if they had (1) histologically confirmed non-small cell lung cancer (NSCLC) (2) BMI >25. Patients were defined as GLP-1RA users if they had GLP-1RA prescribed for >6 months during the period between resection and any event (defined as disease recurrence, death, or loss to follow up).

[0075] In an advanced disease cohort, medical charts for patients undergoing treatment for advanced or metastatic lung cancer at Roswell Park Cancer Institute (RPCI) between 2015-2023 were manually curated. Patients were included for analysis if they had: (1) histologically confirmed advanced or metastatic NSCLC, (2) received at least 3 doses of an anti-PD-(L)l and / or anti-CTLA-4 immune checkpoint inhibitor (3), received no definitive local therapy (surgery or radiotherapy) and (4) had a BMI >25. Patients were again defined as GLP-1RA users if they had GLP-1RA prescribed for >6 months during the period between the start of ICI treatment and any event (defined as disease progression, death, or loss to follow up).

[0076] In both cohorts, patient demographics, including age, sex (male or female), race (white and non-white), smoking status (current, former, never smokers), and BMI were collected.

[0077] Visceral Fat Indexes and abdominal circumference were not routinely available in this retrospective database. For the surgical cohort, recurrence free survival (RFS) was calculated from the date of resection until disease recurrence, death, or censoring at loss to follow up. For the advanced disease cohort, progression free survival (PFS) was used, calculated from the date of treatment initiation until disease progression, death, or censoring at loss to follow up. Overall survival (OS) was defined from the point of resection in the post-surgical cohort and start of treatment in the advanced disease cohort until the date of death or censoring at loss to follow up.

[0078] Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Unless specified to the contrary, it is intended that every maximum numerical limitation given throughout this description includes every lower numerical limitation, as if such lower numerical limitations were expressly written herein. Every minimum numerical limitation given throughout this specification will include every higher numerical limitation, as if such higher numerical limitations were expressly written herein. Every numerical range given throughout this specification will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.

[0079] The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention.

[0080] While embodiments of the present disclosure have been particularly shown and described with reference to certain examples and features, it will be understood by one skilled in the art that various changes in detail may be effected therein without departing from the spirit and scope of the present disclosure as defined by claims that can be supported by the written description and drawings. Further, where exemplary embodiments are described with reference to a certain number of elements it will be understood that the exemplary embodiments can be practiced utilizing either less than or more than the certain number of elements.

Claims

Claims1. A method of treating cancer, consisting of: administering to an overweight human subject at least one glucagon-like peptide-1 receptor agonist (GLP-1RA), and administering to the human subject at least one immune checkpoint inhibitor.

2. A method for treating an overweight human subject having cancer by administering to the human subject a therapeutically effective combination of at least one glucagon-like peptide-1 receptor agonist (GLP-1RA) and at least one immune checkpoint inhibitor.

3. A method of reducing resistance to immune checkpoint inhibition by administering to a human subject having cancer at least one glucagon-like peptide-1 receptor agonist (GLP-1RA) before treating the patient with at least one immune checkpoint inhibitor.

4. The method of any of claims 1-3, wherein the GLP1-RA is any of dulaglutide; exenatide; exenatide extended-release; liraglutide; lixisenatide; semaglutide injection; semaglutide tablets; or a combination thereof.

5. The method of any of claims 1-3, wherein the GLP1-RA is tirzepatide.

6. The method of any of claims 1-3, wherein the dosage of the GLP1-RA ranges from 0.25 mg to 14 mg, or 10 mcg to 20 mcg.

7. The method of claim 6, wherein the dosage of the GLP1-RA is 0.25 mg, 0.5 mg, 0.6 mg, 1.0 mg 1.2 mg, 1.7 mg, 1.8 mg, 2.4 mg, 3.0 mg, 7 mg, or 14 mg.

8. The method of claims 1-3, wherein the dosage of the GLP1-RA ranges from 0.6 mg to 1.8 mg.

9. The method of claim 8, wherein the dosage of the GLP1-RA is 0.6 mg, 1.2 mg, or 1.8 mg,10. The method of claim 5, wherein the dosage of the GLP1-RA ranges from 2.5 mg-15 mg.

11. The method of claim 10, wherein the dosage of the GLP1-RA is 2.5 mg, 5 mg, 7.5 mg, 10 mg, 12.5 mg, or 15 mg.

12. The method of any of claims 1-11, wherein the checkpoint inhibitor inhibits at least one of PD1, PD-L1 or PD-L2, or CTLA4.

13. The method of any of claims 1-11, wherein the checkpoint inhibitor is ipilimumab, tremelimumab, nivolumab, pembrolizumab, cemiplimab, dostarlimab, atezolizumab, avelumab, or durvalumab, or combinations thereof.

14. The method of claim 13, wherein the checkpoint inhibitor comprises pembrolizumab.

15. The method of any of claims 1-14, wherein the human subject is resistant to the immune checkpoint inhibitor prior to administration of the GLP1-RA.

16. The method of any of claims 1-14, wherein the human subject has not previously been administered a GLP1-RA, and / or an immune checkpoint inhibitor, and / or a combination thereof.

17. The method of any of claims 1-16, wherein the immune checkpoint inhibitor and / or the GLP1- RA are administered concurrently.

18. The method of any of claims 1-16, wherein the immune checkpoint inhibitor and / or the GLP1- RA are administered sequentially.

19. The method of any of claims 1-18 wherein the human subject has a body mass index of 25 to29.9 kg / mA2.

20. The method of any of claims 1-18, wherein the human subject has a body mass index greater than or equal to 30 kg / mA2.

21. The method of claim 20, wherein the human subject has a body mass index of 30 to 34.9 kg / mA2.

22. The method of claim 20, wherein the human subject has a body mass index of 35 to 39.9 kg / mA2.

23. The method of claim 20, wherein the human subject has a body mass index of greater than or equal to 40 kg / mA2.

24. The method of any of claims 1-23, wherein the cancer is non-small cell lung cancer.

25. The method of any of claims 1-23, wherein the cancer is non-small cell lung cancer that is resistant to immune checkpoint inhibition.

26. The method of any of claims 1-25, wherein the cancer is resistant to anti-PD-1 therapy.

27. The method of any of claims 1-26 wherein the GLP-1RA is liraglutide or semaglutide and the immune checkpoint inhibitor is pembrolizumab.TABLE 1TABLE 2

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