Methods for treating cancer using incretin receptor agonists

Abstract

Compositions and methods for the treatment of cancer are described. The compositions include one or more incretin agonists, preferably multi-incretin receptor agonists. Therapeutic methods using multi-incretin receptor agonists such as incretin triple agonists for the treatment and prevention of cancers and related metabolic-oncologic disorders are provided. In one embodiment, a dosage formulation and method thereof for treating or preventing a malignancy in a subject comprises administering to the subject a therapeutically effective amount of a incretin triple hormone receptor agonist targeting a glucagon-like peptide-1 receptor (GLP1R), a glucose-dependent insulinotropic polypeptide receptor (GIPR), and a glucagon receptor (GCGR), wherein the administration is effective to reduce tumor engraftment and attenuate tumor progression.

Description

[0001] ATTORNEY DOCKET NO. UTRF 25094-05 PCT

[0002] METHODS FOR TREATING CANCER USING INCRETIN RECEPTOR AGONISTS CROSS REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 63 / 761,094, filed February 20, 2025, the entire contents of which are incorporated by reference herein.

[0004] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0005] This invention was made with government support under federal grant numbers NCI R01CA253329 and U01CA272541 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0006] FIELD OF THE INVENTION

[0007] The invention relates to therapeutic methods using incretin agonists for the treatment and prevention of cancers and related metabolic-oncologic disorders.

[0008] BACKGROUND OF THE INVENTION

[0009] Obesity-driven metabolic abnormalities — specifically insulin resistance, chronic inflammation, and disrupted energy homeostasis — significantly increase cancer risk, progression, and treatment resistance. While weight reduction with lifestyle interventions can mitigate these risks, they often fail to achieve the durable, long-term results necessary for clinical impact. Consequently, there remains a critical need for pharmacological therapies that provide sustained weight loss while simultaneously targeting the underlying oncogenic mechanisms of obesity.

[0010] Incretin-based therapies targeting the glucagon-like peptide- 1 (GLP-1) receptor and the glucose-dependent insulinotropic polypeptide (GIP) receptor have improved the treatment of obesity and type 2 diabetes. Dual GLP-l / GIP receptor agonists have been shown to induce clinically meaningful weight loss and improve metabolic parameters. However, these therapies were developed primarily to treat metabolic disease and are not specifically designed to address the biological pathways linking obesity to cancer development and progression. As a result, residual cancer risk may persist in individuals with severe obesity and associated inflammatory and metabolic dysfunction.

[0011] More recently, incretin triple agonists that engage GLP-1, GIP, and glucagon receptors have been investigated for their enhanced metabolic effects. In preclinical models, triple agonists such as retatrutide (LY3437943) have been reported to produce greater weight loss than single-or dual-receptor agonists and to reduce obesity-associated tumor burden. These findings suggest that broader incretin receptor engagement may influence pathways relevant to obesity-associated malignancies.

[0012] 45828227.1 1ATTORNEY DOCKET NO. UTRF 25094-05 PCT

[0013] Current incretin-based therapies also present limitations related to weight-loss magnitude, treatment tolerability, and durability of effect following treatment discontinuation. In some patients, cessation of therapy is associated with rapid weight regain, potentially diminishing both metabolic and oncologic benefits.

[0014] Thus, there remains a need for pharmacologic methods that provide sustained weight reduction and more effectively target the metabolic and inflammatory mechanisms through which obesity contributes to cancer initiation and progression.

[0015] Accordingly, compositions and methods of providing weight reduction and / or effectively reducing cancer initiation and progression are described.

[0016] Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present disclosure as it existed before the priority date of each claim of this application.

[0017] BRIEF SUMMARY OF THE INVENTION

[0018] Compositions and pharmaceutical formulations of one or more incretin agonists, preferably multi -incretin agonists, and methods of use thereof are described. A dosage formulation includes one or more incretin agonists in an amount effective to prevent cancer onset, reduce cancer risk, delay tumor engraftment or onset, slow tumor progression, reduce tumor burden or volume, improve response to cancer therapy, and / or improve survival in a subject in need thereof, and one or more pharmaceutically acceptable excipients. The one or more incretin agonists activate one or more receptors of Glucagon-like peptide- 1 (GLP-1), Glucose-dependent insulinotropic polypeptide (GIP), and Glucagon (GCG); and preferably, the one or more incretin agonists activate at least two receptors of Glucagon-like peptide-1 (GLP-1), Glucose-dependent insulinotropic polypeptide (GIP), and Glucagon (GCG). In some embodiments, the one or more incretin agonists activate all three receptors of Glucagon-like peptide- 1 (GLP-1), Glucose-dependent insulinotropic polypeptide (GIP), and Glucagon (GCG). In one embodiment, the one or more incretin agonists are retatrutide shown in Structure I, or an analog, a variant, and a derivative thereof.

[0019] 45828227.1 2ATTORNEY DOCKET NO. UTRF 25094-05 PCT

[0020]

[0021] Structure I

[0022] Generally, the dosage is formulated for administration by parenteral, enteral, or topical routes of administration. Exemplary parenteral administrations include intramuscular, intraperitoneal, intravenous or subcutaneous injection. In some cases, the one or more incretin agonists are present in an amount between 0.0001 mg and 20 mg per kg of body weight of the human, preferably between 0.001 mg and 10 mg per kg of body weight of the human. In other embodiments, the dosage is formulated for administration once daily, weekly, or monthly. Typically, the subject in need thereof is diagnosed with cancer or as being at enhanced risk of cancer, such as an obesity-associated cancer or a non-obesity-associated cancer. Exemplary cancer includes pancreatic ductal adenocarcinoma (PDAC), breast cancer (BC), or lung adenocarcinoma (LUAD).

[0023] Methods for preventing cancer onset, reducing cancer risk, delaying tumor engraftment or onset, slowing tumor progression, reducing tumor burden or volume, improving response to cancer therapy, and / or improving survival are also provided. The methods include administering 45828227.1 3ATTORNEY DOCKET NO. UTRF 25094-05 PCT

[0024] to a subject in need thereof an effective amount of one or more incretin agonists. The one or more incretin agonists activate one or more receptors of Glucagon-like peptide- 1 (GLP-1), Glucose-dependent insulinotropic polypeptide (GIP), and Glucagon (GCG); and preferably, the one or more incretin agonists activate at least two receptors of Glucagon- like peptide- 1 (GLP-1), Glucose-dependent insulinotropic polypeptide (GIP), and Glucagon (GCG). In some embodiments, the one or more incretin agonists activate all three receptors of Glucagon-like peptide-1 (GLP-1), Glucose-dependent insulinotropic polypeptide (GIP), and Glucagon (GCG). In one embodiment, the one or more incretin agonists are retatrutide shown in Structure I, or an analog, a variant, and a derivative thereof.

[0025] In preferred embodiments, the method administers an effective amount of the one or more incretin agonists to enrich pro-inflammatory gene signatures, reduce immunosuppressive suppressor cells, increase MHC Il-high antigen-presenting macrophages, elevate pro-inflammatory cytokines such as interleukin-6, and / or enhance cytotoxic CD8+ T-cell activation. Exemplary immunosuppressive suppressor cells include one or more of immunosuppressive monocytic myeloid-derived suppressor cells (M-MDSCs) and polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs). In some embodiments, the administration of the one or more incretin agonists improves glycemic control by lowering fasting blood glucose and insulin levels, reducing indices of insulin resistance, including the homeostatic model assessment of insulin resistance (HOMA-IR), and increasing insulin sensitivity indices, including the quantitative insulin sensitivity check index (QUICKI). Preferably, the method administers the one or more incretin agonists in an amount effective to achieve a body-weight reduction of about 1% to about 40%, or about 5% to about 30%, or about 10% to about 30%, thereby establishing a metabolic environment associated with reduced cancer risk and improved therapeutic response. In one embodiment, the method is effective to reduce tumor size in the subject.

[0026] The methods can be used in combination with at least one additional cancer therapy to the subject. Additional therapy may be selected from chemotherapy, targeted therapy, anti-infective agents, adoptive T cell therapy, a cancer vaccine, surgery, radiation therapy or immune checkpoint inhibition. The incretin agonist is administered before, during, or after the additional therapy to improve metabolic status, enhance anti-tumor immunity, and reduce tumor burden or recurrence. Exemplary immune checkpoint modulators include PD-1 antagonists, PD-1 ligand antagonists, and CTLA4 antagonists. In one embodiment, the immune checkpoint modulator is PD-1 antagonists.

[0027] Additional advantages of the disclosed method and compositions will be set forth in part in the description which follows, and in part will be understood from the description, or can be 45828227.1 4ATTORNEY DOCKET NO. UTRF 25094-05 PCT

[0028] learned by practice of the disclosed method and compositions. The advantages of the disclosed method and compositions will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention as claimed.

[0029] BRIEF DESCRIPTION OF THE DRAWINGS

[0030] The accompanying drawings illustrate several embodiments of the disclosed method and compositions and together with the description, serve to explain the principles of the disclosed method and compositions.

[0031] FIGs. 1A-1I show that retatrutide (RETA) or semaglutide (SEMA) treatment reduces body weight, improves fasted blood glucose, and attenuates tumor outcomes in the KPCY model of obesity-associated PDAC. FIG. 1A shows percent body weight changes from baseline. FIG. IB, Epididymal white adipose tissue weight was quantified at endpoint. FIG. 1C. Fasted blood glucose was quantified at baseline and throughout the study at indicated timepoints until endpoint. FIG. ID. Tumor engraftment or “tumor take” is reported. FIG. IE. Survival curve is plotted as % of mice tumor-free for each group. FIG. IF. Tumor progression over 15 days after injecting KPCY cells is measured by volume recorded using digital caliper. FIG. 1G. Fold change in tumor volume compared to Veh at endpoint are reported. FIG.1H. Endpoint tumor volumes, and FIG.1I. Tumor weights (normalized to body weights) were quantified. Data are represented as mean ± SEM (N = 9 mice Veh, N = 10 mice RETA, N = 9 mice SEMA, N = 10 mice WM-CR). FIG.1B, FIG.1G, FIG.1H, FIG.1I Statistical significance was determined by one-way ANOVA with Tukey’s multiple comparison test (MCT) and is denoted as *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. FIG.1C, FIG.1F. Statistical significance was determined by two-way ANOVA with Sidak’s MCT. FIG.1C Statistical significance is denoted as *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, Veh versus RETA;AP < 0.05,AAP < 0.01,AAAAP < 0.0001 Veh versus SEMA, OP < 0.05 Veh versus WM-CR; $$$$P < 0.0001 RETA versus SEMA; - P < 0.001, - P < 0.0001 RETA versus WM-CR; ####P < 0.0001 SEMA versus WM-CR. FIG.1F. Statistical significance is denoted as *P < 0.05 Veh versus RETA; $ P < 0.05 RETA versus SEMA; # P < 0.05 RETA versus WM-CR.

[0032] FIGs. 2A-2H show that RETA withdrawal causes a reversal in body weight, adiposity, and fasted blood glucose, with only partial loss of anti-tumor effects in KPCY model of obesity-associated PDAC. FIG. 2A. Percent body weight changes from baseline are reported. FIG. 2B.

[0033] Epididymal white adipose tissue weight was quantified at endpoint. FIG.2C. Fasted blood glucose was quantified at baseline and throughout the study at indicated time points until 45828227.1 5ATTORNEY DOCKET NO. UTRF 25094-05 PCT

[0034] endpoint. FIG. 2D Insulin resistance was calculated by Homeostatic Model Assessment for Insulin Resistance (HOMA-IR) score. FIG.2E. The quantitative insulin sensitivity check index (QUICKI) was calculated. FIG. 2F. Tumor engraftment or “tumor take” is reported. FIG. 2G.

[0035] Survival curve is plotted as % of mice tumor-free for each group. FIG.2H. Tumor progression over 18 days after injecting KPCY cells was measured by volume recorded using digital caliper.

[0036] FIG. 21 Endpoint tumor volumes, and FIG.2J. Tumor weights (normalized to body weights) were quantified. Data are represented as mean ± SEM (N = 10 mice Veh, N = 12 mice RETA, N = 14 mice RETA-w / d). FIG. 2B, FIG. 2D, FIG. 2E, FIG. 21, FIG. 2J. Statistical significance was determined by one-way ANOVA with Tukey’s test and is denoted as **P < 0.01, ***P < 0.001, ****P < 0.0001. FIG. 2G. Log-rank (Mantel-Cox) test for survival curves (P < 0.0001).

[0037] FIG. 2C, FIG. 2H. Statistical significance was determined by two-way ANOVA with repeated measures using a mixed-effects model and Tukey’s multiple comparison test. FIG.2C ***P < 0.001, ****P < 0.0001, Veh versus RETA;AP < 0.05,AAAAP < 0.0001, Veh versus RETA-w / d; $P < 0.05, P < 0.0001, RETA versus RETA-w / d. FIG. 2H **P < 0.01, ***P < 0.001, ****P < 0.0001, Veh versus RETA;AP < 0.05,AAP < 0.01,AAP < 0.001, Veh versus RETA-w / d; $ P < 0.05, $$P < 0.01, RETA versus RETA-w / d.

[0038] FIG. 3 shows that RETA treatment significantly alters gene expression in KPCY tumors which is restored with RETA-w / d. Principal component analysis (PCA) plots of gene expression analysis using RNAseq data are shown.

[0039] FIGs. 4A-4B show that RETA treatment significantly modulates Hallmark pathways involved in improved tumor outcomes, which is reversed with RETA-w / d in the KPCY model. Modulation of various immunometabolic Hallmark pathways influencing tumor outcomes are reported for (FIG. 4A), RETA versus Veh and (FIG. 4B), RETA-w / d versus RETA.

[0040] FIGs. 5A-5J show that RETA treatment reduces body weights and adiposity, improves fasted blood glucose, and attenuates tumor outcomes in LLC model. FIG. 5A. Percent body weight changes from baseline are reported. FIG. 5B. Epididymal white adipose tissue were weighed at endpoint and normalized to total body weight. FIG.5C. Fasted blood glucose was quantified at baseline and throughout the study at indicated timepoints until end-point. FIG. 5D.

[0041] Tumor engraftment or “tumor take” is reported. FIG.5E. Survival curve plotted as % of mice tumor- free for each group. FIG. 5F. Tumor progression over 19 days after injecting LLC cells measured by volume recorded using digital caliper. FIG. 5G. Endpoint tumor volumes, and (FIG. 5H), tumor weights (normalized to body weights) were quantified. Data are represented as mean ± SEM (N = 7 mice Veh, N = 8 mice RETA). FIG.51, IL-6 level (pg / mL) in Veh and RETA-treated groups, and FIG.5J leptin level (pg / mL) in Veh and RETA-treated groups. FIG.

[0042] 45828227.1 6ATTORNEY DOCKET NO. UTRF 25094-05 PCT

[0043] 5A Statistical significance was determined by Two-way ANOVA and is denoted as ****P < 00001 FIGs. 5B, 5G, 5H, 51 and 5J Statistical significance was determined by Student’s t-test with Mann- Whitney test and is denoted as **P < 0.01, ***P < 0.001. FIGs. 5C and 5F Statistical significance was determined by two-way ANOVA with repeated measures using a mixed-effects model and Sidak’s multiple comparison test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. FIG. 5E Ixig-rank (Mantel-Cox) test for survival curves (P < 0.0001).

[0044] FIGs. 6A-6I show that RETA significantly improves antigen presentation and reduces immunosuppression in the LLC model. Frequencies of (FIG. A), CD45+ cells / live cells (FIG.

[0045] 6B), CDllb+ cells / CD45+ cells, (FIG. 6C), M-MDSCs / CD45+ cells, (FIG. 6D), PMN-MDSCs / CD45+ cells, (FIG.6E), macrophages / CD45+ cells, FIG. 6F, MHC II high macrophages / macrophages, FIG. 6G CD3+ cells / CD45+ cells, and (FIG. 6H), CD8+ cclls / CD3+ cells arc shown. FIG. 61 The mean fluorescence intensity for PD-1 is shown for CD8+ cells. Data is represented as mean ± SEM (N = 7 mice Veh, N = 8 mice RETA). Statistical significance was determined by Student’s t-test with Mann-Whitney test and is denoted as *P <0.05; ***P<0.001; ****P <0.0001.

[0046] FIG. 7 is a survival curve plotted as % of mice tumor-free in Veh or RETA-treated groups in the - C3(l)-T antigen genetic model of breast cancer. Statistical significance was determined by Logrank (Mantel-Cox) test and is denoted as *P < 0.05.

[0047] FIGs. 8A shows body weights in groups treated with three doses at 1 nmol / Kg, 1.5 nmol / Kg, and 2.0 nmol / Kg. FIG. 8B. Fasted blood glucose was quantified at baseline and throughout the study at indicated timepoints.

[0048] FIGs. 9A-9C show 1 nmol / Kg RETA - / + immunotherapy (anti-PD-1) in the KPCY (PDAC) cancer model. FIG. 9A. Body weights in groups treated with Veh + IgG, RETA + IgG, Veh + anti-PD-1, and RETA + anti-PD-1, using dose at 1 nmol / Kg RETA and IgG / anti-PD-1 immunotherapy. FIG. 9B. Tumor progression over 16 days after injecting KPCY cells was measured by volume recorded using digital caliper. FIG. 9C. Endpoint tumor volumes in groups treated with Veh + IgG, RETA + IgG, Veh + anti-PD-1, and RETA + anti-PD-1. Statistical significance was determined by two-way ANOVA with repeated measures using a mixed-effects model and Tukey’s multiple comparison test. *P < 0.05; **P < 0.01.

[0049] DETAILED DESCRIPTION OF THE INVENTION

[0050] The disclosed method and compositions can be understood more readily by reference to the following detailed description of particular embodiments and the Examples included therein and to the Figures and their previous and following description.

[0051] 45828227.1 7ATTORNEY DOCKET NO. UTRF 25094-05 PCT

[0052] It is to be understood that the disclosed method and compositions are not limited to specific synthetic methods, specific analytical techniques, or to particular reagents unless otherwise specified, and, as such, can vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0053] I. DEFINITIONS

[0054] Throughout this specification, the word “comprise” or variations such as “comprises” or “comprising” will be understood to imply the inclusion of a stated integer (or components) or group of integers (or components), but not the exclusion of any other integer (or components) or group of integers (or components).

[0055] The singular forms “a,” “an,” and “the” include the plurals unless the context clearly dictates otherwise.

[0056] The term “including" is used to mean “including but not limited to.” “Including” and “including but not limited to” are used interchangeably.

[0057] The terms “patient,” “subject,” and “individual” may be used interchangeably and refer to either a human or a non-human animal. These terms include mammals such as humans, primates, livestock animals (e.g., bovines, porcines), companion animals (e.g., canines, felines) and rodents (e.g., mice and rats). The terms do not denote a particular age or sex. Thus, adult and newborn subjects, as well as fetuses, whether male or female, are intended to be covered. A patient refers to a subject afflicted with a disease or disorder.

[0058] The term “agonist” as employed in the context of the invention refers to a substance (ligand) that activates the receptor type in question.

[0059] The term “multi-incretin receptor agonist” means a compound that has agonist activity at two or more incretin-related receptors, preferably at GIP, GLP-1, and glucagon receptors, sufficient to produce metabolic and anti-tumor effects when administered to a subject as described herein.

[0060] The term “incretin analog” includes peptide analogs, variants, and derivatives structurally related to endogenous GIP, GLP-1, and / or glucagon, including chimeric or hybrid peptides, that activate at least two, preferably three, of the above receptors.

[0061] The term “obesity associated cancer” includes cancers for which increased incidence or mortality is associated with elevated body fatness, such as PDAC and other malignancies identified epidemiologically.

[0062] The terms “immunomodulatory agent” or “immunotherapeutic agent” refer to an active agent that can be administered to regulate, enhance, reduce, prolong, decrease or otherwise alter 45828227.1 8ATTORNEY DOCKET NO. UTRF 25094-05 PCT

[0063] one or more factors of the innate or adaptive immune response in the recipient. Generally, immunomodulatory agents can modulate immune microenvironment for a desired immunological response by targeting one or more immune cells or cell types at a target site, and thus, are not necessarily specific to any cancer type. For example, the blockade of a single molecule, programmed cell-death protein 1 (PD-1) on immune cells, has resulted in anti -tumor activity. In some embodiments, the immunomodulatory agents are specifically delivered to inhibit or reduce suppressive immune cells such as tumor associated macrophages for an enhanced anti-tumor response at a tumor site.

[0064] The terms “high,” “higher,” “increases,” “elevates,” or “elevation” refer to increases above basal levels, e.g., as compared to a control. The terms “low,” “lower,” “reduces,” or “reduction” refer to decreases below basal levels, e.g., as compared to a control.

[0065] The term “inhibit” means to reduce or decrease in activity or expression. This can be a complete inhibition of activity or expression, or a partial inhibition. Inhibition can be compared to a control or to a standard level. Inhibition can be 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, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64,65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%.

[0066] The term “in need of treatment” as used herein refers to a judgment made by a caregiver (e.g. physician, nurse, nurse practitioner, or individual in the case of humans; veterinarian in the case of animals, including non-human mammals) that a subject requires or will benefit from treatment. This judgment is made based on a variety of factors that are in the realm of a care giver's expertise, but that include the knowledge that the subject is ill, or will be ill, as the result of a condition that is treatable by the disclosed compounds.

[0067] By “treatment” and “treating” is meant the medical management of a subject with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder. This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder. In addition, this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed 45828227.1 9ATTORNEY DOCKET NO. UTRF 25094-05 PCT

[0068] toward the improvement of the associated disease, pathological condition, or disorder. It is understood that treatment, while intended to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder, need not actually result in the cure, amelioration, stabilization or prevention. The effects of treatment can be measured or assessed as described herein and as known in the art as is suitable for the disease, pathological condition, or disorder involved. Such measurements and assessments can be made in qualitative and / or quantitative terms. Thus, for example, characteristics or features of a disease, pathological condition, or disorder and / or symptoms of a disease, pathological condition, or disorder can be reduced to any effect or to any amount. The term “treating cancer’’ includes preventing cancer onset, reducing cancer risk, delaying tumor engraftment or onset, slowing tumor progression, reducing tumor burden or volume, improving response to cancer therapy, and / or improving survival.

[0069] By the term “effective amount’’ of a compound as provided herein is meant a nontoxic but sufficient amount of the compound to provide the desired result. As will be pointed out below, the exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the disease that is being treated, the particular compound used, its mode of administration, and the like. Thus, it is not possible to specify an exact “effective amount.’’ However, an appropriate effective amount can be determined by one of ordinary skill in the art using only routine experimentation.

[0070] The dosages or amounts of the compounds described herein are large enough to produce the desired effect in the method by which delivery occurs. The dosage should not be so large as to cause adverse side effects, such as unwanted cross-reactions, anaphylactic reactions, and the like. Generally, the dosage will vary with the age, condition, sex and extent of the disease in the subject and can be determined by one of skill in the art. The dosage can be adjusted by the individual physician based on the clinical condition of the subject involved. The dose, schedule of doses and route of administration can be varied.

[0071] The efficacy of administration of a particular dose of the compounds or compositions according to the methods described herein can be determined by evaluating the particular aspects of the medical history, signs, symptoms, and objective laboratory tests that are known to be useful in evaluating the status of a subject in need for the treatment of cancer or other diseases and / or conditions. These signs, symptoms, and objective laboratory tests will vary, depending upon the particular disease or condition being treated or prevented, as will be known to any clinician who treats such patients or a researcher conducting experimentation in this field. For example, if, based on a comparison with an appropriate control group and / or knowledge of the normal progression of the disease in the general population or the particular individual: (1) a 45828227.1 10ATTORNEY DOCKET NO. UTRF 25094-05 PCT

[0072] subject’s physical condition is shown to be improved (e.g., a tumor has partially or fully regressed), (2) the progression of the disease or condition is shown to be stabilized, or slowed, or reversed, or (3) the need for other medications for treating the disease or condition is lessened or obviated, then a particular treatment regimen will be considered efficacious.

[0073] By “pharmaceutically acceptable” is meant a material that is not biologically or otherwise undesirable, i.e., the material can be administered to a subject along with the selected compound without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained.

[0074] Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein.

[0075] Use of the term “about” is intended to describe values either above or below the stated value in a range of approx. + / - 10%; in other embodiments the values may range in value either above or below the stated value in a range of approx. + / - 5%; in other embodiments the values may range in value either above or below the stated value in a range of approx. + / - 2%; in other embodiments the values may range in value either above or below the stated value in a range of approx. + / - 1%. The preceding ranges are intended to be made clear by context, and no further limitation is implied. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any nonclaimed element as essential to the practice of the invention.

[0076] Disclosed are materials, compositions, and components that can be used for, can be used in conjunction with, can be used in preparation for, or are products of the disclosed method and compositions. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutation of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein. For example, if a ligand is disclosed and discussed and a number of modifications that can be made to a number of molecules including the ligand are discussed, each and every combination and permutation of ligand and the modifications that are possible are specifically contemplated unless specifically indicated to the contrary. Thus, if a class of 45828227.1 11ATTORNEY DOCKET NO. UTRF 25094-05 PCT

[0077] molecules A, B, and C are disclosed as well as a class of molecules D, E, and F and an example of a combination molecule, A-D is disclosed, then even if each is not individually recited, each is individually and collectively contemplated. Thus, in this example, each of the combinations A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are specifically contemplated and should be considered disclosed from disclosure of A, B, and C; D, E, and F; and the example combination A-D.

[0078] Eikewise, any subset or combination of these is also specifically contemplated and disclosed. Thus, for example, the sub-group of A-E, B-F, and C-E are specifically contemplated and should be considered disclosed from disclosure of A, B, and C; D, E, and F; and the example combination A-D. Further, each of the materials, compositions, components, etc. contemplated and disclosed as above can also be specifically and independently included or excluded from any group, subgroup, list, set, etc. of such materials.

[0079] These concepts apply to all aspects of this application including, but not limited to, steps in methods of making and using the disclosed compositions. Thus, if there are a variety of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific embodiment or combination of embodiments of the disclosed methods, and that each such combination is specifically contemplated and should be considered disclosed.

[0080] Unless otherwise indicated, the disclosure encompasses conventional techniques of molecular biology, microbiology, cell biology and recombinant DNA, which are within the skill of the art. Unless otherwise noted, technical terms are used according to conventional usage, and in the art, such as in the references cited herein, each of which is specifically incorporated by reference herein in its entirety.

[0081] IL COMPOSITIONS

[0082] The compositions include one or more incretin agonists activating one or more of three different receptors of Glucagon- like peptide- 1 (GLP-1), Glucose-dependent insulinotropic polypeptide (GIF), and Glucagon (GCG).

[0083] A. Incretin Agonist

[0084] Incretin agonist includes peptide analogs, variants, and derivatives structurally related to endogenous GIP, GLP-1, and / or glucagon, including chimeric or hybrid peptides, that activate at least one, preferably two to three, of the above receptors. In preferred embodiments, the incretin analog is a triple agonist that activates three different receptors simultaneously: Glucagon-like peptide- 1 (GLP-1), Glucose-dependent insulinotropic polypeptide (GIP), and Glucagon (GCG). In one embodiment, the multi-receptor incretin agonist is a triple agonist peptide, including

[0085] 45828227.1 12ATTORNEY DOCKET NO. UTRF 25094-05 PCT

[0086] retatrutide (also referred to as LY3437943), or a pharmaceutically acceptable salt, solvate, prodrug, metabolite, conjugate, derivative, fragment, or functional variant thereof.

[0087] 1. Retatrutide (also referred to as LY3437943)

[0088] Retatrutide (LY3437943) is an agonist of the glucose-dependent insulinotropic polypeptide, glucagon-like peptide 1, and glucagon receptors.

[0089] As shown in the Examples, in pre-clinical models with significant retatrutide (RETA, LY3437943)-induced weight loss, pancreatic cancer engraftment was reduced, tumor onset was delayed, and progression was attenuated resulting in a 14-fold reduction in tumor volume compared to only 4-fold reduction in single agonist semaglutide-treated mice. Despite weight re-gain after RETA withdrawal, the anti-tumor benefits of RETA persisted. Remarkably, RETA-induced protection extends to a lung cancer model with 50% reduced tumor engraftment, significantly delayed tumor onset, and mitigated tumor progression, with a 17-fold reduction in tumor volume compared to controls. RETA induced immune reprogramming occurs systemically and in the tumor microenvironment with durable anti-tumor immunity evidenced by elevated circulating IL-6, increased antigen presenting cells, reduced immunosuppressive cells, and activation of pro-inflammatory pathways.

[0090] 45828227.1 13ATTORNEY DOCKET NO. UTRF 25094-05 PCT

[0091]

[0092] Structure I: Retatrutide

[0093] Structures of incretin agonists, such as incretin triple agonists retatrutide and analogs thereof are described in U.S. Patent No. 7,968,686, W02009120530A1, WO 2022 / 178366 Al, the entirety of which is incorporated by reference.

[0094] B. Pharmaceutical Formulations

[0095] Pharmaceutical compositions including the one or more incretin agonists may be formulated in a conventional manner using one or more physiologically acceptable carriers including excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen. In preferred embodiments, the compositions are formulated for parenteral delivery. In some embodiments, the compositions are formulated for intratumoral injection. Typically, the compositions will be formulated in sterile saline or buffered solution for injection into the tissues or cells to be treated. The compositions can be stored lyophilized in

[0096] 45828227.1 14ATTORNEY DOCKET NO. UTRF 25094-05 PCT

[0097] single use vials for rehydration immediately before use. Other means for rehydration and administration are known to those skilled in the art.

[0098] Pharmaceutical formulations contain the disclosed compositions in combination with one or more pharmaceutically acceptable excipients. Representative excipients include solvents, diluents, pH modifying agents, preservatives, antioxidants, suspending agents, wetting agents, viscosity modifiers, tonicity agents, stabilizing agents, and combinations thereof. Suitable pharmaceutically acceptable excipients are preferably selected from materials which are generally recognized as safe (GRAS), and may be administered to an individual without causing undesirable biological side effects or unwanted interactions.

[0099] Generally, pharmaceutically acceptable salts can be prepared by reaction of the free acid or base forms of an active agent with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; generally, non-aqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred. Pharmaceutically acceptable salts include salts of an active agent derived from inorganic acids, organic acids, alkali metal salts, and alkaline earth metal salts as well as salts formed by reaction of the drug with a suitable organic ligand (e.g., quaternary ammonium salts). Lists of suitable salts are found, for example, in Remington’s Pharmaceutical Sciences, 20th ed., Lippincott Williams & Wilkins, Baltimore, MD, 2000, p. 704. Examples of drugs sometimes administered in the form of a pharmaceutically acceptable salt include timolol maleate, brimonidine tartrate, and sodium diclofenac.

[0100] The compositions are preferably formulated in dosage unit form for ease of administration and uniformity of dosage. The phrase "dosage unit form" refers to a physically discrete unit of conjugate appropriate for the patient to be treated. It will be understood, however, that the total single administration of the compositions will be decided by the attending physician within the scope of sound medical judgment. The therapeutically effective dose can be estimated initially either in cell culture assays or in animal models, usually mice, rabbits, dogs, or pigs. The animal model is also used to achieve a desirable concentration range and route of administration. Such information should then be useful to determine useful doses and routes for administration in humans. Therapeutic efficacy and toxicity of conjugates can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED50 (the dose is therapeutically effective in 50% of the population) and LD50 (the dose is lethal to 50% of the population). The dose ratio of toxic to therapeutic effects is the therapeutic index and it can be expressed as the ratio, LD50 / ED50. Pharmaceutical compositions which exhibit large

[0101] 45828227.1 15ATTORNEY DOCKET NO. UTRF 25094-05 PCT

[0102] therapeutic indices are preferred. The data obtained from cell culture assays and animal studies can be used in formulating a range of dosages for human use.

[0103] In certain embodiments, the compositions are administered locally, for example, by injection directly into a site to be treated. In some embodiments, the compositions are injected, topically applied, or otherwise administered directly into the vasculature onto vascular tissue at or adjacent to a site of injury, surgery, or implantation. For example, in embodiments, the compositions are topically applied to vascular tissue that is exposed, during a surgical or implantation, or transplantation procedure. Typically, local administration causes an increased localized concentration of the compositions which is greater than that which can be achieved by systemic administration.

[0104] Pharmaceutical compositions formulated for administration by parenteral (intramuscular, intraperitoneal, intravenous (IV) or subcutaneous injection), enteral, and topical routes of administration are described.

[0105] 1. Parenteral Administration

[0106] In some embodiments, the compositions are formulated to be administered parenterally. The phrases “parenteral administration” and “administered parenterally” are art-recognized terms, and include modes of administration other than enteral and topical administration, such as injections, and include without limitation intravenous, intramuscular, intrapleural, intravascular, intrapericardial, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradennal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrastemal injection and infusion. In some embodiments, the compositions are administered parenterally, for example, by subdural, intravenous, intrathecal, intraventricular, intraarterial, intra-articular, intra-synovial, intra-amniotic, intraperitoneal, or subcutaneous routes.

[0107] For liquid formulations, pharmaceutically acceptable carriers may be, for example, aqueous or non-aqueous solutions, suspensions, emulsions or oils. Parenteral vehicles (for subcutaneous, intravenous, intraarterial, or intramuscular injection) include, for example, sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's and fixed oils. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, and injectable organic esters such as ethyl oleate. Aqueous carriers include, for example, water, alcoholic / aqueous solutions, cyclodextrins, emulsions or suspensions, including saline and buffered media. The compositions can also be administered in an emulsion, for example, water in oil. Examples of oils are those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, mineral oil, olive oil, sunflower oil, fish- liver oil, sesame oil, 45828227.1 16ATTORNEY DOCKET NO. UTRF 25094-05 PCT

[0108] cottonseed oil, com oil, olive, petrolatum, and mineral. Suitable fatty acids for use in parenteral formulations include, for example, oleic acid, stearic acid, and isostearic acid. Ethyl oleate and isopropyl myristate are examples of suitable fatty acid esters.

[0109] Formulations suitable for parenteral administration can include antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. Intravenous vehicles can include fluid and nutrient replenishers, electrolyte replenishers such as those based on Ringer’s dextrose. In general, water, saline, aqueous dextrose and related sugar solutions, and glycols such as propylene glycols or polyethylene glycol are preferred liquid carriers, particularly for injectable solutions.

[0110] Injectable pharmaceutical carriers for injectable compositions arc well-known to those of ordinary skill in the art (see, e.g., Pharmaceutics and Pharmacy Practice, J.B. Lippincott Company, Philadelphia, PA, Banker and Chalmers, eds., pages 238-250 (1982), and ASHP Handbook on Injectable Drugs, Trissei, 15th ed., pages 622-630 (2009)).

[0111] 2. Enteral Administration

[0112] In some embodiments, the compositions are formulated to be administered enterally. The carriers or diluents may be solid earners or diluents for solid formulations, liquid carriers or diluents for liquid formulations, or mixtures thereof.

[0113] For liquid formulations, pharmaceutically acceptable carriers may be, for example, aqueous or non-aqueous solutions, suspensions, emulsions or oils. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, and injectable organic esters such as ethyl oleate. Aqueous carriers include, for example, water, alcoholic / aqueous solutions, cyclodextrins, emulsions or suspensions, including saline and buffered media.

[0114] Examples of oils are those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, mineral oil, olive oil, sunflower oil, fish-liver oil, sesame oil, cottonseed oil, corn oil, olive, petrolatum, and mineral. Suitable fatty acids for use in parenteral formulations include, for example, oleic acid, stearic acid, and isostearic acid. Ethyl oleate and isopropyl myristate are examples of suitable fatty acid esters.

[0115] Vehicles include, for example, sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's and fixed oils. Formulations include, for example, aqueous and non-aqueous, isotonic sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, 45828227.1 17ATTORNEY DOCKET NO. UTRF 25094-05 PCT

[0116] solubilizers, thickening agents, stabilizers, and preservatives. Vehicles can include, for example, fluid and nutrient replenishers, electrolyte replenishers such as those based on Ringer's dextrose. In general, water, saline, aqueous dextrose and related sugar solutions are preferred liquid carriers. These can also be formulated with proteins, fats, saccharides and other components of infant formulas.

[0117] In preferred embodiments, the compositions are formulated for oral administration. Oral formulations may be in the form of chewing gum, gel strips, tablets, capsules or lozenges.

[0118] Encapsulating substances for the preparation of enteric-coated oral formulations include cellulose acetate phthalate, polyvinyl acetate phthalate, hydroxypropyl methylcellulose phthalate and methacrylic acid ester copolymers. Solid oral formulations such as capsules or tablets are preferred. Elixirs and syrups also are well known oral formulations.

[0119] 3. Topical Administration

[0120] In some forms, the compositions are formulated to be administered topically. Topical administration can include application directly to exposed tissue, vasculature, mucosa or to tissues or prostheses, for example, during surgery. The preferred tissue for topical administration is tumor.

[0121] C. Additional Active Agents to be Delivered

[0122] In some embodiments, the compositions are used to deliver one or more additional active agents, particularly one or more active agents to prevent or treat one or more symptoms of cancer.

[0123] 1. Therapeutic agents

[0124] Representative therapeutic agents include, but are not limited to, chemotherapeutic agents, anti-infectious agents, and combinations thereof.

[0125] Additional therapeutic agents include conventional cancer therapeutics such as chemotherapeutic agents, cytokines, chemokines, and radiation therapy. The majority of chemotherapeutic drugs can be divided into alkylating agents, antimetabolites, anthracyclines, plant alkaloids, topoisomerase inhibitors, and other antitumor agents. These drugs affect cell division or DNA synthesis and function in some way. Additional therapeutics include monoclonal antibodies and the tyrosine kinase inhibitors e.g., imatinib mesylate (GLEEVEC® or GLIVEC®), which directly targets a molecular abnormality in certain types of cancer (chronic myelogenous leukemia, gastrointestinal stromal tumors).

[0126] Representative chemotherapeutic agents include, but are not limited to, amsacrine, bleomycin, busulfan, camptothecin, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, clofarabine, crisantaspase, cyclophosphamide, cytarabine, dacarbazine, 45828227.1 18ATTORNEY DOCKET NO. UTRF 25094-05 PCT

[0127] dactinomycin, daunorubicin, docetaxel, doxorubicin, epipodophyllotoxins, epirubicin, etoposide, etoposide phosphate, fludarabine, fluorouracil, gemcitabine, hydroxycarb amide, idarubicin, ifosfamide, innotecan, leucovorin, liposomal doxorubicin, liposomal daunorubici , lomustine, mechlorethamine, melphalan, mercaptopurine, mesna, methotrexate, mitomycin, mitoxantrone, oxaliplatin, paclitaxel, pemetrexed, pentostatin, procarbazine, raltitrexed, satraplatin, streptozocin, teniposide, tegafur-uracil, temozolomide, teniposide, thiotepa, tioguanine, topotecan, treosulfan, vinblastine, vincristine, vindesine, vinorelbine, vorinostat, taxol, trichostatin A and derivatives thereof, trastuzumab (HERCEPTIN®), cetuximab, and rituximab (RITUXAN® or MABTHERA®), bevacizumab (AVASTIN®), and combinations thereof. Representative pro-apoptotic agents include, but are not limited to, fludarabinetaurosporine, cycloheximide, actinomycin D, lactosylceramide, 15d-PG.T(2)5 and combinations thereof.

[0128] In some embodiments, the active agents arc histone deacetylase (HD AC) inhibitors. In one embodiment, the active agent is vorinostat. In other embodiments, the active agents are topoisomerase I and / or II inhibitors. In a particular embodiment, the active agent is etoposide or camptothecin.

[0129] Additional anti-cancer agents include, but are not limited to, irinotecan, exemestane, octreotide, carmofur, clarithromycin, zinostatin, tamoxifen, tegafur, toremifene, doxifluridine, nimustine, vindensine, nedaplatin, pirarubicin, flutamide, fadrozole, prednisone, medroxyprogesterone, mitotane, mycophenolate mofetil, and mizoribine.

[0130] Representative anti-angiogenesis agents include, but are not limited to, antibodies to vascular endothelial growth factor (VEGF) such as bevacizumab (AVASTIN®) and rhuFAb V2 (ranibizumab, LUCENTIS®), and other anti-VEGF compounds including aflibercept (EYLEA®); MACUGEN® (pegaptanim sodium, anti-VEGF aptamer or EYE001) (Eyetech Pharmaceuticals); pigment epithelium derived factor(s) (PEDF); COX-2 inhibitors such as celecoxib (CELEBREX®) and rofecoxib (VIOXX®); interferon alpha; interleukin- 12 (IL-12); thalidomide (THALOMID®) and derivatives thereof such as lenalidomide (REVLIMID®); squalamine; endostatin; angiostatin; ribozyme inhibitors such as ANGIOZYME® (Sima Therapeutics); multifunctional antiangiogenic agents such as NEOVASTAT® (AE-941) (Aetema Laboratories, Quebec City, Canada); receptor tyrosine kinase (RTK) inhibitors such as sunitinib (SUTENT®); tyrosine kinase inhibitors such as sorafenib (Nexavar®) and erlotinib (Tarceva®); antibodies to the epidermal grown factor receptor such as panitumumab (VECTIBIX®) and cetuximab (ERBITUX®), as well as other anti-angiogenesis agents known in the art.

[0131] 45828227.1 19ATTORNEY DOCKET NO. UTRF 25094-05 PCT

[0132] In some cases, the active agent is an anti-infectious agent. Exemplary anti-infectious agents include antiviral agents, antibacterial agents, antiparasitic agents, and anti-fungal agents. Exemplary antibiotics include moxifloxacin, ciprofloxacin, erythromycin, levofloxacin, cefazolin, vancomycin, tigecycline, gentamycin, tobramycin, ceftazidime, ofloxacin, gatifloxacin; antifungals: amphotericin, voriconazole, natamycin.

[0133] D. Kits

[0134] The materials described above as well as other materials can be packaged together in any suitable combination as a kit useful for performing, or aiding in the performance of, the disclosed method. It is useful if the kit components in a given kit are designed and adapted for use together in the disclosed method. For example, disclosed are kits for treating cancer, the kit comprising one or more incretin agonists, or a pharmaceutically acceptable salt thereof. The disclosed kits can also include instructions for use.

[0135] III. Methods of Using

[0136] The present disclosure relates to the methods of using one or more incretin agonists, preferably incretin triple agonists having activity activating the glucagon-like peptide- 1 receptor (GLP-1R), glucose-dependent insulinotropic polypeptide receptor (GIPR), and glucagon receptor (GCGR) for the treatment, interception, and prevention of cancers and related metabolic-oncologic disorders. More particularly, the disclosure provides methods for preventing, intercepting, and treating cancer by administering a therapeutically effective amount of a compound having agonist activity at the glucagon-like peptide- 1 receptor (GLP-1R), the glucose-dependent insulinotropic polypeptide receptor (GIPR), and the glucagon receptor (GCGR). In a preferred embodiment, cancer includes obesity-associated cancer and non-obesity-associated cancer.

[0137] In some embodiments, the composition includes a multi-receptor incretin agonist. In one embodiment, the multi-receptor incretin agonist is a triple agonist peptide, including retatrutide (also referred to as LY3437943), or a pharmaceutically acceptable salt, solvate, prodrug, metabolite, conjugate, derivative, fragment, or functional variant thereof.

[0138] The one or more incretin agonists are particularly suitable for preventing, intercepting, and treating cancer. In some embodiments, the compositions are suited to interrupt the process of carcinogenesis by targeting precancerous cells, early lesions, or molecular pathways before they develop into invasive cancer. In some embodiments, the compositions are administered in an amount effective to arrest, reverse, and / or eliminate early-stage, often high-risk lesions.

[0139] The disclosed methods apply to subjects with diet-induced obesity or other forms of obesity, as well as to subjects with normal body weight who have cancers responsive to the 45828227.1 20ATTORNEY DOCKET NO. UTRF 25094-05 PCT

[0140] described treatment and are effective for treating both obesity-associated cancers and non-obesity-associated cancers. The term "obesity-associated cancer" refers to malignancies whose risk or progression is exacerbated by an obese state, including but not limited to pancreatic ductal adenocarcinoma (PDAC). "Non-obesity associated cancer" refers to malignancies that may occur independent of metabolic state but remain responsive to the therapeutic methods described herein, such as lung adenocarcinoma (LUAD).

[0141] The disclosed methods are effective to induce systemic metabolic and immune reprogramming, uniquely reducing white fat, increasing cecal content mass, modulating the tumor microenvironment, and / or improving glycemic control. In preferred embodiments, the disclosed methods enrich pro-inflammatory gene signatures, reduce immunosuppressive myeloid-derived suppressor cells, increase MHC Il-high antigen-presenting macrophages, elevate cytokines including intcrlcukin-6, and / or enhance cytotoxic CD8+ T-cell activation. The methods administer an effective amount of the incretin agonist to reduce tumor engraftment, delay tumor onset, attenuate progression of tumor growth / metastasis, and / or reduce tumor volume. Preferably, the effects are durable, persisting after treatment discontinuation despite partial metabolic rebound, enabling use in prevention, interception, adjuvant therapy, and treatment of established diseases in subjects with or without obesity.

[0142] Methods for modulating tumor microenvironment, enhancing tumor immunogenicity and / or inducing an anti-tumor immune response are described. In some embodiments, treatment using the compositions reduces or inhibits the number or activity of immunosuppressive immune cells, for example, MDSCs, relative to the number or activity of the immunosuppressive immune cells prior to administration of the disclosed composition.

[0143] The method induces a profound shift in the tumor transcriptomic profile. As demonstrated in the Examples, the method was effective in significantly enriching pro-inflammatory and immune-activation pathways, including TNFa signaling via NFKB (normalized enrichment score [NES] = 1.95), Interferon Gamma Response (NES = 1.84), Interferon Alpha Response (NES = 1.76), Inflammatory Response (NES = 1.62), and IL-2 / STAT5 signaling (NES = 1.54). Concurrently, the method significantly down-regulated hallmarks of cancer progression and metabolic demand, including E2F Targets (NES = -2.31), MYC Targets VI (NES = -2.14), G2M Checkpoint (NES = -2.10), Oxidative Phosphorylation (NES = -1.82), Fatty Acid Metabolism (NES = -1.68), and Glycolysis (NES = -1.54). Thus, in accordance with one embodiment of the present disclosure, the method includes administering a multi -incretin receptor agonist in an amount effective to modulate immune pathways and cell populations both systemically and within a tumor microenvironment (TME), wherein the method 45828227.1 21ATTORNEY DOCKET NO. UTRF 25094-05 PCT

[0144] induces a comprehensive reprogramming of tumor biology and host immunity characterized by enriching pro-inflammatory gene signatures including, but not limited to, tumor gene expression associated with TNFA signaling via NFKB, interferon alpha and gamma responses, inflammatory response, IL-2 / STAT5 signaling, and allograft rejection, while simultaneously down-regulating proliferative and metabolic gene signatures associated with E2F targets, MYC targets, glycolysis, fatty acid metabolism, and oxidative phosphorylation.

[0145] In accordance with the present disclosure, the method establishes a unique systemic cytokine and adipokine signature characterized by the reciprocal modulation of Interleukin-6 (IL-6) and leptin. Specifically, the administration of the triple agonist induces a significant elevation in circulating anti-tumor IL-6 concentrations while simultaneously affecting a profound reduction in pro-tumor leptin concentrations. This reciprocal shift from a pro-tumorigcnic adipokine profile (high leptin) to an anti-tumorigcnic cytokine profile (high IL-6) serves as a systemic biomarker for the successful induction of a host environment hostile to tumor engraftment and progression. Thus, in preferred embodiments, the methods administer an effective of the disclosed composition to increase IL-6 levels, and / or reduce leptin levels in the subject under treatment.

[0146] As shown in the Examples, the method systemically altered the composition of the myeloid and lymphoid compartments. In the LLC model, the method significantly reduced the frequency of immunosuppressive polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) within the spleen (2.22% of CD45+cells in treated vs. 6.04% of CD45+cells in vehicle; ***P < 0.001). Within the tumor microenvironment (TME), the method increased the frequency of MHC Il-high macrophages (50.29% of macrophages in treated vs. 37.59% of macrophages in vehicle; p < 0.05), indicating enhanced antigen-presenting capacity.

[0147] Furthermore, the method increased the activation of cytotoxic CD8+ T cells, as evidenced by a significant elevation in the mean fluorescent intensity (MFI) of PD-1 expression on CD8+ T cells (p < 0.05), and a corresponding increase in circulating IL-6 levels (p < 0.0001), which remained elevated even in the withdrawal subjects.

[0148] Accordingly, methods of depleting, inhibiting, or reducing one or more of immunosuppressive monocytic and polymorphonuclear myeloid-derived suppressor cells (M-MDSCs and PMN-MDSCs) at tumor tissues in a subject are described. Myeloid-derived suppressor cells (MDSCs) have emerged as major regulators of immune responses in cancer and other pathological conditions. Two major subsets based on their phenotypic and morphological features: polymorphonuclear (PMN) and monocytic (M)-MDSC. PMN-MDSC is also known as granulocytic MDSC (gMDSC). Immune suppression is a main feature of MDSC. The methods 45828227.1 22ATTORNEY DOCKET NO. UTRF 25094-05 PCT

[0149] include administering to the subject the disclosed composition including one or more incretin agonists in an effective amount to deplete, inhibit, or reduce activity, quantity, and / or function of MDSCs at tumor tissues. In preferred embodiments, the methods effectively reduce both PMN-MDSCs and M-MDSC within the tumor microenvironment.

[0150] Methods of increasing the presence of MHC class II high macrophages systemically and / or within the tumor microenvironment in a subject are also provided. The methods include administering to the subject the disclosed composition including one or more incretin agonists in an effective amount to increase the presence of MHC class II high macrophages.

[0151] Methods of increasing cytotoxic CD8+ T-cell activation systemically and / or within the tumor microenvironment in a subject are also provided. The methods include administering to the subject the disclosed composition including one or more incretin agonists in an effective amount to increase the cytotoxic CD8+ T-ccll activation.

[0152] In some embodiments, the compositions are administered in an amount effective to decrease monocytic myeloid derived suppressor cells (M-MDSCs) and granulocytic or polymorphonuclear MDSCs (PMN-MDSCs), and / or enrich MHC II high macrophages. In further embodiments, the compositions are administered in an amount effective to provide systemic immune modification by reducing the frequency of immunosuppressive M-MDSCs and PMN-MDSCs and increasing the presence of MHC class II high macrophages, while concurrently enhancing PD-1 expression on CD8+ T cells to facilitate increased cytotoxic T-cell activation, thereby creating a host environment hostile to tumor engraftment and progression.

[0153] In accordance with another embodiment of the present disclosure, there is provided a method for establishing durable anti-tumor protection through a finite course of treatment. The method can include an induction phase and a withdrawal phase, where the induction phase includes administering a triple incretin receptor agonist in a dosing regimen sufficient to achieve a target threshold of weight loss or a predefined metabolic improvement plateau; and where the withdrawal phase includes discontinuing administration of the triple agonist and allowing for a partial or complete rebound of body weight or metabolic parameters; and further, notwithstanding said rebound of metabolic parameters, the method provides a sustained attenuation of tumor progression and delay of tumor onset, thereby indicating a durable reprogramming of systemic immunity and tumor biology that persists in the absence of active pharmacological intervention.

[0154] 1. Subject to be Treated

[0155] In general, the compositions and methods of use thereof are useful in the context of cancer. In some embodiments, the subject to be treated is a human. All the methods described 45828227.1 23ATTORNEY DOCKET NO. UTRF 25094-05 PCT

[0156] can include the step of identifying and selecting a subject in need of treatment, or a subject who would benefit from administration with the compositions.

[0157] In some embodiments, the cancer is selected from an obesity-associated cancer or a nonobesity-associated cancer.

[0158] i. Cancer

[0159] In some embodiments, the compositions are administered to a subject having a proliferative disease, such as a benign or malignant tumor. In some embodiments, the subjects to be treated have been diagnosed with stage I, stage II, stage III, or stage IV cancer.

[0160] The term cancer refers specifically to a malignant tumor. In addition to uncontrolled growth, malignant tumors exhibit metastasis. In this process, small clusters of cancerous cells dislodge from a tumor, invade blood or lymphatic vessels, and are carried to other tissues, where they continue to proliferate. In this way a primary tumor at one site can give rise to a secondary tumor at another site.

[0161] The compositions and methods are useful for treating subjects that have benign or malignant tumors by delaying or inhibiting the growth of a tumor in a subject, reducing the growth or size of the tumor, inhibiting or reducing metastasis of the tumor, and / or inhibiting or reducing symptoms associated with tumor development or growth.

[0162] The compositions and methods are particularly suited to interrupt the process of carcinogenesis by targeting precancerous cells, early lesions, or molecular pathways before they develop into invasive cancer. In some embodiments, the compositions are administered in an amount effective to arrest, reverse, and / or eliminate early-stage, often high-risk lesions. In one embodiment, the compositions and methods are effective in cancer prevention or interception for those at elevated risk of PDAC and LU AD.

[0163] Malignant tumors which may be treated are classified according to the embryonic origin of the tissue from which the tumor is derived. Carcinomas are tumors arising from endodermal or ectodermal tissues such as skin or the epithelial lining of internal organs and glands. The compositions are particularly effective in treating carcinomas. Sarcomas, which arise less frequently, are derived from mesodermal connective tissues such as bone, fat, and cartilage. The leukemias and lymphomas are malignant tumors of hematopoietic cells of the bone marrow. Leukemias proliferate as single cells, whereas lymphomas tend to grow as tumor masses.

[0164] Malignant tumors may show up at numerous organs or tissues of the body to establish cancer.

[0165] The types of cancer that can be treated with the compositions and methods include, but are not limited to, cancers such as vascular cancer such as multiple myeloma, adenocarcinomas and sarcomas, of bone, bladder, brain, breast, cervical, colorectal, kidney, liver, lung, 45828227.1 24ATTORNEY DOCKET NO. UTRF 25094-05 PCT

[0166] nasopharangeal, pancreatic, prostate, skin, stomach, and uterine. In some embodiments, the compositions are used to treat multiple cancer types concurrently. The compositions can also be used to treat metastases or tumors at multiple locations.

[0167] Exemplary tumor cells include tumor cells of cancers, including leukemias including, but not limited to, acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemias such as myeloblastic, promyelocytic, myelomonocytic, monocytic, erythroleukemia leukemias and myelodysplastic syndrome, chronic leukemias such as, but not limited to, chronic myelocytic (granulocytic) leukemia, chronic lymphocytic leukemia, hairy cell leukemia; polycythemia vera; lymphomas such as, but not limited to, Hodgkin’s disease, non- Hodgkin’s disease; multiple myelomas such as, but not limited to, smoldering multiple myeloma, nonsecretory myeloma, osteosclerotic myeloma, plasma cell leukemia, solitary plasmacytoma and extramedullary plasmacytoma; Waldenstrom’s macroglobulincmia; monoclonal gammopathy of undetermined significance; benign monoclonal gammopathy; heavy chain disease; bone and connective tissue sarcomas such as, but not limited to, bone sarcoma, osteosarcoma, chondrosarcoma, Ewing’s sarcoma, malignant giant cell tumor, fibrosarcoma of bone, chordoma, periosteal sarcoma, soft-tissue sarcomas, angiosarcoma (hemangiosarcoma), fibrosarcoma, Kaposi’s sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, neurilemmoma, rhabdomyosarcoma, synovial sarcoma; brain tumors including, but not limited to, glioma, astrocytoma, brain stem glioma, ependymoma, oligodendroglioma, nonglial tumor, acoustic neurinoma, craniopharyngioma, medulloblastoma, meningioma, pineocytoma, pineoblastoma, primary brain lymphoma; breast cancer including, but not limited to, adenocarcinoma, lobular (small cell) carcinoma, intraductal carcinoma, medullary breast cancer, mucinous breast cancer, tubular breast cancer, papillary breast cancer, Paget’s disease, and inflammatory breast cancer; adrenal cancer, including, but not limited to, pheochromocytom and adrenocortical carcinoma; thyroid cancer such as but not limited to papillary or follicular thyroid cancer, medullary thyroid cancer and anaplastic thyroid cancer; pancreatic cancer, including, but not limited to, insulinoma, gastrinoma, glucagonoma, vipoma, somatostatin-secreting tumor, and carcinoid or islet cell tumor; pituitary cancers including, but not limited to, Cushing’s disease, prolactin-secreting tumor, acromegaly, and diabetes insipius; eye cancers including, but not limited to, ocular melanoma such as iris melanoma, choroidal melanoma, and ciliary body melanoma, and retinoblastoma: vaginal cancers, including, but not limited to, squamous cell carcinoma, adenocarcinoma, and melanoma; vulvar cancer, including, but not limited to, squamous cell carcinoma, melanoma, adenocarcinoma, basal cell carcinoma, sarcoma, and Paget’s disease; cervical cancers including, but not limited to, squamous cell carcinoma, and adenocarcinoma; 45828227.1 25ATTORNEY DOCKET NO. UTRF 25094-05 PCT

[0168] uterine cancers including, but not limited to, endometrial carcinoma and uterine sarcoma; ovarian cancers including, but not limited to, ovarian epithelial carcinoma, borderline tumor, germ cell tumor, and stromal tumor; esophageal cancers including, but not limited to, squamous cancer, adenocarcinoma, adenoid cystic carcinoma, mucoepidermoid carcinoma, adenosquamous carcinoma, sarcoma, melanoma, plasmacytoma, verrucous carcinoma, and oat cell (small cell) carcinoma; stomach cancers including, but not limited to, adenocarcinoma, fungating (polypoid), ulcerating, superficial spreading, diffusely spreading, malignant lymphoma, liposarcoma, fibrosarcoma, and carcinosarcoma; colon cancers; rectal cancers; liver cancers including, but not limited to, hepatocellular carcinoma and hepatoblastoma, gallbladder cancers including, but not limited to, adenocarcinoma; cholangiocarcinomas including, but not limited to, papillary, nodular, and diffuse; lung cancers including, but not limited to, non-small cell lung cancer, squamous cell carcinoma (epidermoid carcinoma), adenocarcinoma, large-cell carcinoma and small-cell lung cancer; testicular cancers including, but not limited to, germinal tumor, seminoma, anaplastic, classic (typical), spermatocytic, nonseminoma, embryonal carcinoma, teratoma carcinoma, choriocarcinoma (yolk-sac tumor), prostate cancers including, but not limited to, adenocarcinoma, leiomyosarcoma, and rhabdomyosarcoma; penal cancers; oral cancers including, but not limited to, squamous cell carcinoma; basal cancers; salivary gland cancers including, but not limited to, adenocarcinoma, mucoepidermoid carcinoma, and adenoidcystic carcinoma; pharynx cancers including, but not limited to, squamous cell cancer, and verrucous; skin cancers including, but not limited to, basal cell carcinoma, squamous cell carcinoma and melanoma, superficial spreading melanoma, nodular melanoma, lentigo malignant melanoma, acral lentiginous melanoma; kidney cancers including, but not limited to, renal cell cancer, adenocarcinoma, hypernephroma, fibrosarcoma, transitional cell cancer (renal pelvis and / or ureter); Wilms’ tumor; bladder cancers including, but not limited to, transitional cell carcinoma, squamous cell cancer, adenocarcinoma, carcinosarcoma. In one embodiment, cancer is brain metastasis in patients with leukemia.

[0169] Cancers that can be prevented, treated or otherwise diminished by the compositions include myxosarcoma, osteogenic sarcoma, endotheliosarcoma, lymphangioendotheliosarcoma, mesothelioma, synovioma, hemangioblastoma, epithelial carcinoma, cystadenocarcinoma, bronchogenic carcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, and gastric cancer (for a review of such disorders, see Fishman et al., 1985, Medicine, 2d Ed., J.B. Lippincott Co., Philadelphia and Murphy et al., 1997, Informed Decisions: The Complete Book of Cancer Diagnosis, Treatment, and Recovery, Viking Penguin, Penguin Books U.S.A., Inc., United States of America).

[0170] 45828227.1 26ATTORNEY DOCKET NO. UTRF 25094-05 PCT

[0171] In some embodiments, the cancers are characterized as being triple negative breast cancer, or having one or more KRAS-mutations, EGFR mutations, ALK mutations, RBI mutations, HIF mutations, KEAP mutations, NRF mutations, or other metabolic-related mutations, or combinations thereof.

[0172] The methods and compositions described are useful for both prophylactic and therapeutic treatment.

[0173] Therapeutic treatment involves administering to a subject a therapeutically effective amount of the compositions or pharmaceutically acceptable salts thereof as described after cancer is diagnosed.

[0174] In further embodiments, the compositions are used for prophylactic use i.e. prevention, delay in onset, diminution, eradication, or delay in exacerbation of signs or symptoms after onset, and prevention of relapse. For prophylactic use, a therapeutically effective amount of the disclosed compounds and compositions or pharmaceutically acceptable salts thereof as described are administered to a subject prior to onset (e.g., before obvious signs of cancer), during early onset (e.g., upon initial signs and symptoms of cancer), or after an established development of cancer. Prophylactic administration can occur for several days to years prior to the manifestation of symptoms. Prophylactic administration can be used, for example, those diagnosed with early-stage malignancies, and for subgroups with susceptibilities (e.g., family, racial, and / or occupational) to particular cancers.

[0175] In some embodiments, the subject to be treated is one with one or more solid tumors. A solid tumor is an abnormal mass of tissue that usually does not contain cysts or liquid areas. Solid tumors may be benign (not cancer), or malignant (cancer). Examples of solid tumors are sarcomas, carcinomas, and lymphomas. In preferred embodiments, the compositions and methods are effective in treating one or more symptoms of cancers of the skin, lung, liver, pancreas, brain, kidney, breast, prostate, colon and rectum, bladder, etc. In further embodiment, the tumor is a focal lymphoma or a follicular lymphoma.

[0176] In some embodiments, the subject to be treated has pancreatic ductal adenocarcinoma (PDAC). Therefore, in some embodiments, the compositions and methods are effective for treating PDAC in a subject in need thereof. The subject can be diagnosed as having PDAC, or be identified as being at enhanced risk of PDAC. Compositions and methods are useful for treating subjects having PDAC by delaying or inhibiting the growth of a tumor in a subject, reducing the growth or size of the tumor, inhibiting or reducing metastasis of the tumor, and / or inhibiting or reducing symptoms associated with tumor development or growth.

[0177] 45828227.1 27ATTORNEY DOCKET NO. UTRF 25094-05 PCT

[0178] In some embodiments, the subject to be treated has lung adenocarcinoma (LUAD). Therefore, in some embodiments, the compositions and methods are effective for treating LUAD in a subject in need thereof. The subject can be diagnosed as having LUAD or be identified as being at enhanced risk of LUAD. The compositions and methods are useful for treating subjects having LUAD by delaying or inhibiting the growth of a tumor in a subject, reducing the growth or size of the tumor, inhibiting or reducing metastasis of the tumor, and / or inhibiting or reducing symptoms associated with tumor development or growth.

[0179] In some embodiments, the subject to be treated has breast cancer (BC). Therefore, in some embodiments, the compositions and methods are effective for treating BC in a subject in need thereof. The subject can be diagnosed as having BC or be identified as being at enhanced risk of BC. Compositions and methods are useful for treating subjects having BC by delaying or inhibiting the growth of a tumor in a subject, reducing the growth or size of the tumor, inhibiting or reducing metastasis of the tumor, and / or inhibiting or reducing symptoms associated with tumor development or growth.

[0180] 2. Combination Therapies and Procedures

[0181] In some embodiments, the method further includes administering at least one additional cancer therapy to the subject. Additional therapy may be selected from chemotherapy, radiotherapy, targeted therapy, or immune checkpoint inhibition. The multi-incretin agonist is administered before, during, or after the additional therapy to improve metabolic status, enhance anti-tumor immunity, and reduce tumor burden or recurrence.

[0182] In some embodiments, conventional therapy includes administration of one or more of the compositions in combination with one or more additional active agents. The combination therapies can include administration of the active agents together in the same admixture, or in separate admixtures. Therefore, in some embodiments, the pharmaceutical composition includes two, three, or more active agents. The additional active agent(s) can have the same, or different mechanisms of action. In some embodiments, the combination results in an additive effect on the treatment of cancer. In some embodiments, the combinations result in a more than additive effect on the treatment of the disease or disorder.

[0183] In some embodiments, the formulation is formulated for intravenous, subcutaneous, or intramuscular administration to the subject, or for enteral administration. In some embodiments, the formulation is administered prior to, in conjunction with, subsequent to, or in alternation with treatment with one or more additional therapies or procedures. In some embodiments the additional therapy is performed between drug cycles or during a drug holiday that is part of the

[0184] 45828227.1 28ATTORNEY DOCKET NO. UTRF 25094-05 PCT

[0185] disclosed composition dosage regime. For example, in some embodiments, the additional therapy or procedure is surgery, radiation therapy, or chemotherapy.

[0186] Additional therapeutic agents include conventional cancer therapeutics such as chemotherapeutic agents, cytokines, chemokines, and radiation therapy. The majority of chemotherapeutic drugs can be divided into alkylating agents, antimetabolites, anthracyclines, plant alkaloids, topoisomerase inhibitors, and other antitumor agents. These drugs affect cell division or DNA synthesis and function in some way. Additional therapeutics include monoclonal antibodies and the tyrosine kinase inhibitors e.g., imatinib mesylate (GLEEVEC® or GLIVEC®), which directly targets a molecular abnormality in certain types of cancer (chronic myelogenous leukemia, gastrointestinal stromal tumors).

[0187] Representative chemotherapeutic agents include, but are not limited to, amsacrine, bleomycin, busulfan, camptothccin, capccitabinc, carboplatin, carmustinc, chlorambucil, cisplatin, cladribine, clofarabine, crisantaspase, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunorubicin, docetaxel, doxorubicin, epipodophyllotoxins, epirubicin, etoposide, etoposide phosphate, fludarabine, fluorouracil, gemcitabine, hydroxycarb amide, idarubicin, ifosfamide, innotecan, leucovorin, liposomal doxorubicin, liposomal daunorubici , lomustine, mechlorethamine, melphalan, mercaptopurine, mesna, methotrexate, mitomycin, mitoxantrone, oxaliplatin, paclitaxel, pemetrexed, pentostatin, procarbazine, raltitrexed, satraplatin, streptozocin, teniposide, tegafur-uracil, temozolomide, teniposide, thiotepa, tioguanine, topotecan, treosulfan, vinblastine, vincristine, vindesine, vinorelbine, vorinostat, taxol, trichostatin A and derivatives thereof, trastuzumab (IIERCEPTIN®), cetuximab, and rituximab (RITUXAN® or MABTHERA®), bevacizumab (AVASTIN®), and combinations thereof. Representative pro-apoptotic agents include, but are not limited to, fludarabinetaurosporine, cycloheximide, actinomycin D, lactosylceramide, 15d-PGJ(2)5 and combinations thereof.

[0188] In some embodiments, the compositions and methods are used prior to or in conjunction with an immunotherapy such inhibition of checkpoint proteins such as components of the PD-1 / PD-L1 axis or CD28-CTLA-4 axis using one or more immune checkpoint modulators (e.g., PD-1 antagonists, PD-1 ligand antagonists, and CTLA4 antagonists), adoptive T cell therapy, and / or a cancer vaccine. Exemplary immune checkpoint modulators used in immunotherapy include Pembrolizumab (anti-PDl mAb), Durvalumab (anti-PDLl mAb), PDR001 (anti-PDl mAb), Atezolizumab (anti-PDLl mAb), Nivolumab (anti-PDl mAb), Tremelimumab (anti-CTLA4 mAb), Avelumab (anti-PDLl mAb), and RG7876 (CD40 agonist mAb).

[0189] Methods of adoptive T cell therapy are known in the art and used in clinical practice. Generally adoptive T cell therapy involves the isolation and ex vivo expansion of tumor specific 45828227.1 29ATTORNEY DOCKET NO. UTRF 25094-05 PCT

[0190] T cells to achieve greater number of T cells than what could be obtained by vaccination alone. The tumor specific T cells are then infused into patients with cancer in an attempt to give their immune system the ability to overwhelm remaining tumor via T cells, which can attack and kill the cancer. Several forms of adoptive T cell therapy can be used for cancer treatment including, but not limited to, culturing tumor infiltrating lymphocytes or TIL; isolating and expanding one particular T cell or clone; and using T cells that have been engineered to recognize and attack tumors. In some embodiments, the T cells are taken directly from the patient's blood. Methods of priming and activating T cells in vitro for adaptive T cell cancer therapy are known in the art. See, for example, Wang, et al, Blood, 109(11):4865-4872 (2007) and Hervas-Stubbs, et al, J. Immunol., 189(7):3299-310 (2012).

[0191] Historically, adoptive T cell therapy strategies have largely focused on the infusion of tumor antigen specific cytotoxic T cells (CTL) which can directly kill tumor cells. However, CD4+ T helper (Th) cells such as Thl, Th2, Tfh, Treg, and Thl7 can also be used. Th can activate antigen-specific effector cells and recruit cells of the innate immune system such as macrophages and dendritic cells to assist in antigen presentation (APC), and antigen primed Th cells can directly activate tumor antigen-specific CTL. As a result of activating APC, antigen specific Thl have been implicated as the initiators of epitope or determinant spreading which is a broadening of immunity to other antigens in the tumor. The ability to elicit epitope spreading broadens the immune response to many potential antigens in the tumor and can lead to more efficient tumor cell kill due to the ability to mount a heterogeneic response. In this way, adoptive T cell therapy can be used to stimulate endogenous immunity.

[0192] In some embodiments, the T cells express a chimeric antigen receptor (CARs, CAR T cells, or CARTs). Artificial T cell receptors are engineered receptors, which graft a particular specificity onto an immune effector cell. Typically, these receptors are used to graft the specificity of a monoclonal antibody onto a T cell and can be engineered to target virtually any tumor associated antigen. First generation CARs typically had the intracellular domain from the CD3 chain, which is the primary transmitter of signals from endogenous TCRs. Second generation CARs add intracellular signaling domains from various costimulatory protein receptors (e.g., CD28, 41BB, ICOS) to the cytoplasmic tail of the CAR to provide additional signals to the T cell, and third generation CARs combine multiple signaling domains, such as CD3z-CD28-41BB or CD3z-CD28-OX40, to further enhance effectiveness.

[0193] In some embodiments, the compositions and methods are used prior to or in conjunction with a cancer vaccine, for example, a dendritic cell cancer vaccine. Vaccination typically includes administering a subject an antigen e.g., a cancer antigen) together with an adjuvant to 45828227.1 30ATTORNEY DOCKET NO. UTRF 25094-05 PCT

[0194] elicit therapeutic T cells in vivo. In some embodiments, the cancer vaccine is a dendritic cell cancer vaccine in which the antigen delivered by dendritic cells primed ex vivo to present the cancer antigen. Examples include PROVENGE® (sipuleucel-T), which is a dendritic cell-based vaccine for the treatment of prostate cancer (Ledford, et al., Nature, 519, 17-18 (05 March 2015). Such vaccines and other compositions and methods for immunotherapy are reviewed in Palucka, et al., Nature Reviews Cancer, 12, 265-277 (April 2012).

[0195] In some embodiments, the compositions and methods are used prior to or in conjunction with surgical removal of tumors, for example, in preventing primary tumor metastasis. In some embodiments, the compositions and methods are used to enhance body’s own anti -tumor immune functions.

[0196] 3. Treatment Regimen

[0197] In certain embodiments, administration of the multi-rcccptor incrctin agonist is performed according to a regimen effective to induce weight loss and improve glycemic control in the subject. In some embodiments, the dosing regimen is sufficient to induce a systemic metabolic state characterized by a reduction in adiposity and adipokine signaling, including decreased epididymal or visceral adipose depot mass, reduced liver weight, and lowered circulating concentrations of adiposity-associated factors such as leptin and resistin.

[0198] In further embodiments, administration improves glycemic control by lowering fasting blood glucose and insulin levels, reducing indices of insulin resistance, including the homeostatic model assessment of insulin resistance (HOMA-IR), and increasing insulin sensitivity indices, including the quantitative insulin sensitivity check index (QUICKI). In certain embodiments involving human subjects, the method includes administering the multireceptor agonist in an amount effective to achieve a body-weight reduction of approximately 10% to about 30%, thereby establishing a metabolic environment associated with reduced cancer risk and improved therapeutic response.

[0199] In some embodiments, administration of the triple hormone receptor agonist is effective to modulate gastrointestinal physiology, specifically by slowing gastric transit and delaying gastric emptying. The modulation of gastric motility is characterized by a significant increase in cecal content weight, which serves as a physiological proxy for delayed gastric emptying. In certain embodiments, administration of retatrutide induces a significantly greater increase in cecal mass compared to single-receptor agonists such as semaglutide, indicating a more potent effect on gastric transit associated with triple hormone receptor agonism.

[0200] In one embodiment, the incretin triple hormone receptor agonist is retatrutide (RETA) (LY3437943) or a pharmaceutically acceptable salt thereof.

[0201] 45828227.1 31ATTORNEY DOCKET NO. UTRF 25094-05 PCT

[0202] In some embodiments, the cancer is selected from an obesity-associated cancer or a non-obesity-associated cancer.

[0203] In some embodiments, cancer is selected from a group consisting of pancreatic cancer, lung cancer, and breast cancer, including spontaneous malignancies driven by genetic factors.

[0204] In certain embodiments, RETA is administered before detectable tumor formation, thereby reducing tumor engraftment. In other embodiments, RETA is administered after tumor cell exposure or early tumor establishment, thereby reducing tumor progression.

[0205] In some embodiments, RETA is administered as a cancer interceptive agent to a subject at risk of cancer, wherein the administration delays tumor onset or reduces the probability of tumor engraftment.

[0206] In one embodiment, the triple hormone receptor agonist uniquely reduces white fat mass compared to single-receptor agonists or caloric restriction, and delays tumor latency significantly as measured by time to first palpable tumor onset.

[0207] In certain embodiments, the triple hormone receptor agonist is administered to a subject at a weight-neutral or low-dose concentration, such as a dose between 1 nmol / kg and 2 nmol / kg, which is effective to reduce fasted blood glucose (e.g., from about 200 mg / dL to about 100 mg / dL) and attenuate tumor progression without requiring significant systemic weight loss.

[0208] In other embodiments, the triple hormone receptor agonist is administered in combination with an additional therapeutic agent, such as an immune checkpoint inhibitor. In a preferred embodiment, the triple hormone receptor agonist is administered with an anti-PD-1 antibody, wherein the triple agonist provides anti-tumor efficacy matching that of immunotherapy even at weight-neutral doses.

[0209] In further embodiments, the reduction in tumor engraftment and / or tumor progression persists after discontinuation of RETA administration. The persistence of tumor suppression occurs despite partial or complete reversal of RETA-induced weight loss. In another embodiment, the administration is effective in providing a durable anti-tumor effect that persists following discontinuation of the agonist and subsequent weight rebound.

[0210] In certain embodiments, administration of RETA induces systemic immune modulation associated with the reduction in tumor engraftment or tumor progression. The immune modulation may comprise one or more of: increased antigen-presenting immune cells, reduced immunosuppressive myeloid cell populations, or increased activation of pro-inflammatory signaling pathways.

[0211] In an aspect described herein, the systemic immune modulation comprises a reciprocal shift in the subject’s cytokine-adipokine profile, wherein circulating IL-6 is increased (e.g., a 4.6- 45828227.1 32ATTORNEY DOCKET NO. UTRF 25094-05 PCT

[0212] fold increase) and circulating leptin is significantly decreased. This reciprocal relationship is effective in inducing anti-tumor immunity that persists despite the partial reversal of leptin levels following treatment discontinuation.

[0213] In some embodiments, the immune modulation persists following discontinuation of RETA administration.

[0214] In one embodiment, the efficacy of the incretin triple agonist in reducing tumor volume is greater than the efficacy of a single GLP-1 receptor agonist, such as semaglutide, under comparable conditions.

[0215] i. Dosage and Effective Amounts

[0216] Dosage and dosing regimens are dependent on the severity and location of the cancer to be treated and / or methods of administration, and are known to those skilled in the art. A therapeutically effective amount of the composition used in the treatment of cancer is typically sufficient to reduce or alleviate one or more symptoms of cancer.

[0217] Symptoms of cancer may be physical, such as tumor burden, or biological such as proliferation of cancer cells. Accordingly, the amount of the composition can be effective to, for example, kill tumor cells or inhibit proliferation or metastasis of the tumor cells. Preferably the active agents do not target or otherwise modulate the activity or quantity of healthy cells not within or associated with tumor tissues, or do so at a reduced level compared to cancer or cancer-associated cells. In this way, by-products and other side effects associated with the compositions are reduced, preferably leading directly or indirectly to cancer cell death. In some embodiments, the composition directly or indirectly reduces cancer cell migration, angiogenesis, immune escape, radioresistance, or a combination thereof.

[0218] In some in vivo approaches, the compositions are administered to a subject in a therapeutically effective amount to reduce tumor size. In some embodiments, an effective amount of the composition is used to put cancer in remission and / or keep the cancer in remission. Also provided are effective amounts of the compositions to reduce or stop cancer stem cell proliferation. In preferred cases, it is an amount of a multi-incretin receptor agonist sufficient to produce a clinically meaningful improvement in a cancer-related endpoint and / or a metabolic endpoint in the treated subject.

[0219] The actual effective amounts of the composition can vary according to factors including the specific active agent administered, the particular composition formulated, the mode of administration, and the age, weight, condition of the subject being treated, as well as the route of administration and the disease or disorder. The subjects are typically mammals, most preferably, humans. Generally, for intravenous injection or infusion, the dosage may be lower.

[0220] 45828227.1 33ATTORNEY DOCKET NO. UTRF 25094-05 PCT

[0221] In general, the timing and frequency of administration will be adjusted to balance the efficacy of a given treatment or diagnostic schedule with the side-effects of the given delivery system. Exemplary dosing frequencies include continuous infusion, single and multiple administrations such as hourly, daily, weekly, monthly or yearly dosing.

[0222] In one embodiment, the dosing frequency is once weekly via subcutaneous injection, for example about 0.0001 mg / kg to about 100 mg / kg, preferably about 0.001 mg / kg to about 20 mg / kg, more preferably about 0.001 mg / kg to about 10 mg / kg.

[0223] ii. Controls

[0224] The therapeutic result of the compositions including can be compared to a control.

[0225] Suitable controls are known in the art and include, for example, untreated cells or an untreated subject. A typical control is a comparison of a condition or symptom of a subject prior to and after administration of the targeted agent. The condition or symptom can be a biochemical, molecular, physiological, or pathological readout. For example, the effect of the composition on a particular symptom, pharmacologic, or physiologic indicator can be compared to an untreated subject, or the condition of the subject prior to treatment. In some embodiments, the symptom, pharmacologic, or physiologic indicator is measured in a subject prior to treatment, and again one or more times after treatment is initiated. In some embodiments, the control is a reference level, or average determined based on measuring the symptom, pharmacologic, or physiologic indicator in one or more subjects that do not have the disease or condition to be treated (e.g., healthy subjects). In some embodiments, the effect of the treatment is compared to a conventional treatment that is known the art.

[0226] Any of the disclosed incretin agonists can be used therapeutically in combination with a pharmaceutically acceptable carrier. The compounds described herein can be conveniently formulated into pharmaceutical compositions composed of one or more of the compounds in association with a pharmaceutically acceptable carrier. See, e.g., Remington's Pharmaceutical Sciences, latest edition, by E.W. Martin Mack Pub. Co., Easton, PA, which discloses typical earners and conventional methods of preparing pharmaceutical compositions that can be used in conjunction with the preparation of formulations of the compounds described herein. These most typically would be standard carriers for administration of compositions to humans. In one aspect, humans and non-humans, including solutions such as sterile water, saline, and buffered solutions at physiological pH. Other compounds will be administered according to standard procedures used by those skilled in the art.

[0227] The pharmaceutical compositions described herein can include, but are not limited to, carriers, thickeners, diluents, buffers, preservatives, surface active agents and the like in addition 45828227.1 34ATTORNEY DOCKET NO. UTRF 25094-05 PCT

[0228] to the molecule of choice. Pharmaceutical compositions can also include one or more active ingredients such as antimicrobial agents, anti-inflammatory agents, anesthetics, and the like.

[0229] The compounds and pharmaceutical compositions described herein can be administered to the subject in a number of ways depending on whether local or systemic treatment is desired, and on the area to be treated. Thus, for example, a pharmaceutical composition described herein can be administered as intratumoral injection. Moreover, a pharmaceutical composition can be administered to a subject vaginally, rectally, intranasally, orally, by inhalation, or parenterally, for example, by intradermal, subcutaneous, intramuscular, intraperitoneal, intrarectal, intraarterial, intralymphatic, intravenous, intrathecal and intratracheal routes. Parenteral administration, if used, is generally characterized by injection and includes intravenous (IV), subcutaneous (SC), intramuscular (IM), epidural and intra-articular injection, as well as surgical insertion of depots in the organ or tissue of interest (Bittner, ct al., BioDrugs., 32:425-440 (2018); Lee et al., J. Phami. Investig., 49: 459-476 (2019); Chaudhary et al., Crit. Rev. Ther. Drug Carrier Syst., 36:137-181 (2019)). Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions (Park et al., J Control Release, 342:53-65 (2022); Nkanga, et al., Advanced Drug Delivery Reviews, 167:19-46, (2020); Sheikh, et al., Asian Journal of Pharmaceutics, 10(4):S465-S471 (2016); Rhee et al., Pharmaceutical Technology Drug Delivery, p.S6 (2010)). An exemplary approach for parenteral administration involves use of a slow release or sustained release system such that a constant dosage is maintained. See for example, U.S. Patent No. 9,700,630, WO 2006 / 125620, and KR 101898816.

[0230] Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions which can also contain buffers, diluents and other suitable additives. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives can also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.

[0231] Formulations for topical administration can include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like can be necessary or desirable.

[0232] 45828227.1 35ATTORNEY DOCKET NO. UTRF 25094-05 PCT

[0233] Compositions for oral administration can include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers, dispersing aids or binders can be desirable.

[0234] The disclosed compositions and methods can be further understood through the following numbered paragraphs and Examples.

[0235] 1. A dosage formulation comprising one or more incretin agonists in an amount effective to prevent cancer onset, reduce cancer risk, delay tumor engraftment or onset, slow tumor progression, reduce tumor burden or volume, improve response to cancer therapy, and / or improve survival in a subject in need thereof, and one or more pharmaceutically acceptable excipients.

[0236] 2. The dosage formulation of paragraph 1 , wherein the one or more incretin agonists activate one or more receptors of Glucagon- like peptide- 1 (GLP-1), Glucosc-dcpcndcnt insulinotropic polypeptide (GIP), and Glucagon (GCG).

[0237] 3. The dosage formulation of paragraph 1 or 2, wherein the one or more incretin agonists activate at least two receptors of Glucagon- like peptide- 1 (GLP-1), Glucose-dependent insulinotropic polypeptide (GIP), and Glucagon (GCG).

[0238] 4. The dosage formulation of any one of paragraphs 1-3, wherein the one or more incretin agonists activate all three receptors of Glucagon-like peptide- 1 (GLP-1), Glucose-dependent insulinotropic polypeptide (GIP), and Glucagon (GCG).

[0239] 5. The dosage formulation of any one of paragraphs 1-4, wherein the one or more incretin agonists comprise retatrutide shown in Structure I, or an analog, a variant, and a derivative thereof.

[0240] 45828227.1 36ATTORNEY DOCKET NO. UTRF 25094-05 PCT

[0241]

[0242] Structure I:

[0243] 6. The dosage formulation of any one of paragraphs 1-5, wherein the dosage is formulated for administration by parenteral, enteral, or topical routes of administration.

[0244] 7. The dosage formulation of any one of paragraphs 1-6, wherein the dosage is formulated for administration by intramuscular, intraperitoneal, intravenous or subcutaneous injection. 8. The dosage formulation of any one of paragraphs 1-7, wherein the one or more incretin agonists are present in an amount between 0.005 mg and 20 mg per kg of body weight of the human.

[0245] 9. The dosage formulation of any one of paragraphs 1-8, wherein the one or more incretin agonists are present in an amount between 0.0001 mg and 20 mg per kg of body weight of the human.

[0246] 10. The dosage formulation of any one of paragraphs 1-9, formulated for administration once daily, weekly, or monthly.

[0247] 45828227.1 37ATTORNEY DOCKET NO. UTRF 25094-05 PCT

[0248] 11. The dosage formulation of any one of paragraphs 1-10, wherein the subject in need thereof is diagnosed with cancer or as being at enhanced risk of cancer.

[0249] 12. The dosage formulation of paragraph 11, wherein the cancer is an obesity-associated cancer or a non-obesity-associated cancer.

[0250] 13. The dosage formulation of paragraph 11 or 12, wherein the cancer is pancreatic ductal adenocarcinoma (PDAC) or lung adenocarcinoma (LU AD).

[0251] 14. A method for preventing cancer onset, reducing cancer risk, delaying tumor engraftment or onset, slowing tumor progression, reducing tumor burden or volume, improving response to cancer therapy, and / or improving survival, comprising administering to a subject in need thereof an effective amount of one or more incretin agonists.

[0252] 15. The method of paragraph 14, wherein the one or more incretin agonists activate one or more receptors of Glucagon-like peptide- 1 (GLP-1), Glucose-dependent insulinotropic polypeptide (GIF), and Glucagon (GCG).

[0253] 16. The method of paragraph 14 or 15, wherein the one or more incretin agonists activate at least two receptors of Glucagon-like peptide- 1 (GLP-1), Glucose-dependent insulinotropic polypeptide (GIF), and Glucagon (GCG).

[0254] 17. The method of any one of paragraphs 14-16, wherein the one or more incretin agonists activate all three receptors of Glucagon-like peptide- 1 (GLP-1), Glucose-dependent insulinotropic polypeptide (GIP), and Glucagon (GCG).

[0255] 18. The method of any one of paragraphs 14-17, wherein the one or more incretin agonists comprise retatrutide shown in Structure I, or an analog, a variant, and a derivative thereof. 19. The method of any one of paragraphs 14-18, wherein the one or more incretin agonists are formulated with one or more pharmaceutically acceptable excipients.

[0256] 20. The method of any one of paragraphs 14-19, wherein the one or more incretin agonists are administered by parenteral, enteral, or topical routes of administration.

[0257] 21. The method of any one of paragraphs 14-20, wherein the one or more incretin agonists are administered by intramuscular, intraperitoneal, intravenous or subcutaneous injection.

[0258] 22. The method of any one of paragraphs 14-21, wherein the one or more incretin agonists are administered in an amount between 0.0001 mg and 20 mg per kg of body weight of the human.

[0259] 23. The method of any one of paragraphs 14-22, wherein the one or more incretin agonists are administered in an amount between 0.0001 mg and 20 mg per kg of body weight of the human.

[0260] 45828227.1 38ATTORNEY DOCKET NO. UTRF 25094-05 PCT

[0261] 24. The method of any one of paragraphs 14-23, wherein the administration is once daily, weekly, or monthly.

[0262] 25. The method of any one of paragraphs 14-24, wherein the subject in need thereof is diagnosed with cancer or as being at enhanced risk of cancer.

[0263] 26. The method of any one of paragraphs 14-25, wherein the cancer is an obesity-associated cancer or a non-obesity-associated cancer.

[0264] 27. The method of any one of paragraphs 14-26, wherein: (a) the cancer is pancreatic ductal adenocarcinoma (PDAC), breast cancer (BC) or lung adenocarcinoma (LU AD) and / or (b) the subject in need thereof is obese or non-obese.

[0265] 28. The method of any one of paragraphs 14-27, wherein the method administers an effective amount of the one or more incretin agonists to enrich pro-inflammatory gene signatures, reduce immunosuppressive suppressor cells, increase MHC Il-high antigen-presenting macrophages, elevate pro-inflammatory cytokines, and / or enhance cytotoxic CD8+ T-cell activation.

[0266] 29. The method of paragraph 28, wherein the immunosuppressive suppressor cells comprise one or more of immunosuppressive monocytic myeloid-derived suppressor cells (M-MDSCs) and polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs).

[0267] 30. The method of paragraph 28, wherein the pro-inflammatory cytokines comprise interleukin-6.

[0268] 31. The method of any one of paragraphs 14-30, wherein the administration of the one or more incretin agonists improves glycemic control by lowering fasting blood glucose and insulin levels, reducing indices of insulin resistance, including the homeostatic model assessment of insulin resistance (HOMA-IR), and increasing insulin sensitivity indices, including the quantitative insulin sensitivity check index (QUICKI).

[0269] 32. The method of any one of paragraphs 14-31, wherein the method administers the one or more incretin agonists in an amount effective, optionally, to achieve a body-weight reduction of about 1% to about 40%, or about 5% to about 30%, or about 10% to about 30%, thereby establishing a metabolic environment associated with reduced cancer risk and improved therapeutic response.

[0270] 33. The method of any one of paragraphs 14-32, wherein the method is effective to reduce tumor size in the subject.

[0271] 34. The method of any one of paragraphs 14-33, further comprising administering to the subject one or more selected from the group consisting of an immune checkpoint modulator, a chemotherapeutic agent, an anti-infective agent, adoptive T cell therapy, a cancer vaccine, surgery, radiation therapy.

[0272] 45828227.1 39ATTORNEY DOCKET NO. UTRF 25094-05 PCT

[0273] 35. The method of paragraph 34, wherein the immune checkpoint modulator is selected from the group consisting of PD-1 antagonists, PD-1 ligand antagonists, and CTLA4 antagonists. 36. The method of paragraph 35, wherein the immune checkpoint modulator is PD-1 antagonists.

[0274] 37. The method of any one of paragraphs 14-36, further comprising:

[0275] (a) an administration phase, and

[0276] (b) a withdrawal phase,

[0277] wherein the administrative phase comprises administering an effective amount of one or more incretin agonists for an effective amount of time, and

[0278] the withdrawal phase comprises a period of time after no administration of one or more incretin agonists wherein one or more incretin agonists are not administered, allowing increase in body weight compared to the administration phase, wherein the subject has one or more of elevated circulating IL-6, increased antigen presenting cells, and reduced immunosuppressive cells, compared to levels prior to the administration phase, during said withdrawal phase.

[0279] Examples

[0280] Example 1: RE TA reduced body weight, improved metabolic parameters,

[0281] and attenuated tumor outcomes in obesity-associated pancreatic ductal adenocarcinoma (PDAC)

[0282] Impacts on PDAC demonstrated that both RETA and SEMA led to significant weight loss beginning one day after administration compared to vehicle (Veh, water) controls which remained weight stable (FIG. 1A). All mice remained on high fat diet throughout the duration of the study. Weight loss in the RETA group was substantial and plateaued after 2 weeks of RETA intervention at 38% of initial weight at baseline, which was maintained until endpoint. In contrast, SEMA showed a gradual but “oscillatory” weight loss which fluctuated between 16 and 20% of initial weight which remained stable until endpoint (FIG.1A). Although diet interventions in humans are difficult to maintain weight loss, mice respond to caloric restriction (CR) well; CR reduces cancer onset and progression in several models. Therefore, weight loss through CR was induced to establish a weight matched control (WM-CR). Body weights were matched to the SEMA-treated mice to delineate the role of weight loss by CR as opposed to the role of GLP-1 agonism. Importantly, a WM-CR group for RETA could not be established because of the extreme weight loss observed with RETA which could not be implemented through CR alone. RETA and SEMA treatment was continued for the duration of the study. Results demonstrated that RETA and SEMA induced an immediate but transient reduction in food intake that rebounded after 2 weeks of treatment to control levels of intake quantified in the 45828227.1 40ATTORNEY DOCKET NO. UTRF 25094-05 PCT

[0283] Veh group. The food intake in WM-CR group was - by design - controlled to match weight loss in SEMA group. Incretin agonism leads to delayed gastric emptying, which is one mechanism proposed to contribute to weight loss. Cecal content weight at endpoint was quantified as an approximation for gastric emptying. Despite identical food intake and fasting at endpoint, while WM-CR did not alter cecal content mass compared to Veh controls, incretin agonism significantly elevated cecal content weight at endpoint. RETA demonstrated the most cecal content with 6.3-fold greater mass than Veh, and SEMA had half the mass found in RETA. Importantly, none of the mice in any of the groups displayed poor body condition score.

[0284] Next, body composition and metabolic parameters were examined to determine improvement of metabolic dysfunction with incretin agonism. RETA led to remarkable improvements in body composition with a significant reduction in epididymal adipose mass quantified at endpoint as compared to Veh (FIG. IB). Interestingly, despite significant weight loss observed in SEMA and WM-CR groups compared to Veh (FIG. 1A), no differences in epididymal fat mass were observed. The lack of loss in fat mass could be due to the “oscillatory” weight loss in SEMA group attributed to the alternate day dosing, as opposed to previous studies where SEMA was administered daily. Similarly, CR in this study was designed to match body weights with SEMA group, while other CR approaches may differ. Other factors may also impact differences such as age, source of mice, animal facility temperature, diet, and the microbiome which have demonstrated impacts on both obesity and cancer. RETA treatment also resulted in striking improvements in systemic metabolism with a significant reduction in fasted blood glucose concentrations. RETA reduced blood glucose from - 190 mg / dL to approximately 80 mg / dL after 1 week, which steadily rose after 2 weeks of treatment over the remainder of the study to -130 mg / dL while Veh controls maintained blood glucose - 180 mg / dL from baseline to endpoint (FIG. 1C). Despite a significant reduction in fasted blood glucose with SEMA treatment identical to RETA after 1 week, a striking rebound in blood glucose to almost baseline levels was observed by week 2 after treatment, which gradually reduced by endpoint. In contrast to incretin treatment, WM-CR showed a gradual decrease in fasted blood glucose throughout the study. Interestingly, all intervention groups showed similarly reduced fasted blood glucose concentrations at study endpoint that were significantly lower than Veh (FIG. 1C). Endpoint brown adipose tissue and liver weights were significantly reduced to similar levels by RETA, SEMA, and WM-CR compared to Veh; however, no differences in spleen weights were observed.

[0285] Following robust weight loss with SEMA and even more dramatic weight loss with RETA, the impacts on PDAC were determined. KrasLSL G12D / +;Trp53LSL R172H / +;Pdxl- 45828227.1 41ATTORNEY DOCKET NO. UTRF 25094-05 PCT

[0286] Cre;Rosa26YFP / YFP(KPCY) cancer cells were injected when RE TA / SEMA weight loss plateaued after 2 weeks (FIG. 1A). Quantification of tumor engraftment or “tumor take” demonstrated that RETA significantly reduced the number of resultant tumors after injection (FIG. ID). While every mouse injected with cancer cells in the Veh and WM-CR groups had tumors engraft (N = 9 / 9, 100%Veh; N = 10 / 10, 100% WM-CR), in the RETA group, only 70% of the mice (N = 7 / 10) had tumors take while 30% mice rejected tumor cells at injection or failed to take. SEMA treatment showed higher tumor engraftment (N = 8 / 9, 88%) compared to RETA (FIG. ID). Furthermore, thrice weekly palpation of KPCY injection sites revealed that RETA treatment significantly delayed tumor onset compared to Veh, SEMA and WM-CR (FIG.1E), Tumors were allowed to progress for two weeks. While tumors in Veh group showed an exponential increase in volumes post day 7 after tumor cell injection, tumor volumes remained significantly blunted in RETA treated mice (FIG. IF). SEMA treated mice showed tumor onset and progression at intermediate levels compared to RETA and Veh (FIGS. IE- IF). WM-CR paralleled SEMA tumor onset and growth (FIGS. 1E-1F). Tumor volume at endpoint was most reduced by RETA, with a 14-fold reduction compared to Veh, while SEMA and WM-CR volumes did not differ with both at 3-fold reduction relative to Veh. (FIG. 1G-1H). Endpoint tumor weights further demonstrated that compared to Veh, RETA exhibited the most pronounced effect among the treatment groups (FIG. II).

[0287] Male C57BL / 6J mice were maintained on a 60% high-fat diet (HFD) for 16 weeks to achieve an obese phenotype. Subjects were administered a triple agonist (retatrutide) subcutaneously at a dose of 30 nmol / kg thrice weekly. Control groups received either a vehicle or a single-receptor GLP-1 agonist (semaglutide, 30 nmol / kg). Once a weight loss plateau of approximately 38-40% was reached in the triple agonist group (at day 14), subjects were subcutaneously injected with 2 x 105KPCY pancreatic cancer cells into the right flank. Body weight and glucose levels were monitored throughout the study.

[0288] The method significantly reduced tumor "take," with only 70% of treated subjects showing successful engraftment compared to 100% in the vehicle and weight-matched (WM) controls. At the study endpoint (day 35 post-injection), the method achieved a 14-fold reduction in endpoint tumor volume (49.5 mm3) compared to vehicle controls (772.89 mm3). In contrast, the single-agonist method (semaglutide) resulted in only a 4-fold reduction (254.77 mm3).

[0289] Furthermore, at the study endpoint, the method successfully lowered fasted blood glucose from an average of 184.56 mg / dL in vehicle controls to approximately 32.57 mg / dL, and significantly reduced the mass of epididymal white adipose tissue (eWAT) and liver weights compared to vehicle controls.

[0290] 45828227.1 42ATTORNEY DOCKET NO. UTRF 25094-05 PCT

[0291] Furthermore, the triple agonist significantly impacted gastric motility as measured by endpoint biomarkers. Despite identical food intake among groups, the method resulted in an approximately 6.3-fold increase in cecal content mass compared to vehicle controls. This elevation in cecal weight, used as an approximation for delayed gastric emptying, was significantly more pronounced in the triple-agonist group than in the single-agonist (semaglutide) group, which exhibited only half the cecal mass found in the retatrutide-treated subjects.

[0292] Example 2: RETA withdrawal reversed metabolic benefits, but tumor suppression remained persistent

[0293] Patients often do not tolerate the side effects of medical weight loss, including nausea or gut discomfort, or may not have access to the prescriptions due to financial burdens or lack of availability. To determine if weight rebound would reverse the profound protective effects of RETA to reduce PDAC onset and burden, the effect of RETA withdrawal (RETA-w / d) on metabolic parameters and PDAC was examined. A second group of mice was examined for RETA-induced weight loss and RETA-w / d compared to Veh controls. Similar to the first study, RETA intervention led to a transient reduction in food intake and a 40% reduction in body weight that stabilized after two weeks for the duration of the study (FIG.2A). Mice subjected to RETA-w / d displayed an immediate weight regain almost to baseline body weights (FIG.2A).

[0294] Food consumption after RETA withdrawal significantly increased and, in fact, surpassed that of Veh for 2 weeks before returning to baseline levels of intake at study endpoint, not significantly different from Veh or RETA intake. Similarly to the first study, RETA delayed gastric emptying, whereas RETA-w / d completely reversed cecal retention and returned to reduced cecal content weights comparable to Veh. The fact that the cecal contents of RETA-w / d mice are not different than Veh controls demonstrates that RETA withdrawal was effective as the half-life in mice is 21 h.

[0295] RETA again led to significant improvements in body composition that were partially retained after RETA withdrawal despite the dramatic weight rebound in the RETA-w / d group. A significant reduction in epididymal fat mass was quantified at endpoint after RETA and reflected by an almost ablated concentration of circulating leptin, whereas after RETA withdrawal, white adipose mass and leptin concentrations were partially reversed compared to Veh (FIG.2B). Similarly, endpoint brown adipose and liver weights were reduced by RETA and regained after RETA-w / d to Veh weights; however, spleen weights were increased with RETA treatment and restored with RETA withdrawal.

[0296] 45828227.1 43ATTORNEY DOCKET NO. UTRF 25094-05 PCT

[0297] Next, metabolic parameters after incretin agonist withdrawal were examined to determine the extent of improvement of metabolic dysfunction. Veh treated mice displayed glucose concentrations of >200 mg / dL throughout the study (FIG. 2C). The striking improvements in systemic metabolism with RETA treatment were partially reversed after RETA-w / d. Although RETA induced a significant reduction in fasted blood glucose concentrations after 1 week, which steadily rose over time but remained significantly reduced compared to Veh, RETA-w / d caused a sharp increase in fasting blood glucose (FIG. 2C). Compared to Veh, blood glucose levels were 40% lower in RETA and 16% lower after RETA-w / d at endpoint (FIG. 2C). Next, to examine functional responses to glucose after RETA or RETA-w / d, oral glucose tolerance tests (OGTT) were performed. RETA improved glucose tolerance compared to baseline tolerance tests at the start of the study, as well as compared to Veh at study midpoint. Interestingly, by endpoint, all three groups displayed elevated glucose concentrations to the same extent 15 mins after glucose administration despite significantly different fasted concentrations at time 0.

[0298] Despite glucose concentrations peaking at approximately 400 mg / dL during the OGTT, both RETA and RETA-w / d groups disposed of glucose effectively by the end of the study compared to DIO mice treated with Veh, which displayed glucose intolerance. Importantly, RETA also significantly reduced fasting plasma insulin concentrations compared to Veh at endpoint, which were only partially reversed after RETA-w / d. C-peptide, a peptide released from pro-insulin to generate mature insulin, is a proxy measure for insulin release. Likewise, C-peptide concentrations paralleled those of insulin with a significant reduction in the RETA group, which was only partially reversed to Veh concentrations after RETA-w / d. Resistin, an adipokine which may play a role in insulin resistance, was also significantly downregulated with RETA-induced weight loss but was restored to Veh concentrations after RETA-w / d. A calculated estimate of insulin resistance using the homeostatic model assessment for insulin resistance (HOMA-IR) demonstrated a significant reduction in the HOMA-IR score to almost undetectable levels with RETA treatment, an approximation suggesting a loss of insulin resistance (FIG. 2D). With RETA withdrawal, endpoint analysis revealed that HOMA-IR was restored to just 40% of the Veh group. Furthermore, a significantly higher quantitative insulin-sensitivity check index (QUICKI) was observed for RETA-treated mice compared to vehicle and RETA-w / d groups, suggesting improved insulin sensitivity with RETA (FIG. 2E).

[0299] Following the remarkable findings in PDAC with RETA treatment, the effect of RETA withdrawal on PDAC tumor onset and progression was determined. Tumor take after KPCY injection was significantly reduced by RETA with only 66% tumor engraftment (N = 8 / 12), as compared to 100% in Veh (N = 10 / 10) (FIG. 2F), similar to FIG. ID. Of note, the protection 45828227.1 44ATTORNEY DOCKET NO. UTRF 25094-05 PCT

[0300] from RETA to reject tumors was completely lost after RETA withdrawal with all tumors engrafting (N = 14 / 14, 100%, FIG. 2F). RETA again led to significantly delayed tumor onset and blunted tumor growth over the course of the study (FIGs. 2G-2H), as in the first Example (FIGs. 1E-1F) Likewise, tumor volume and weights were significantly reduced at endpoint by RETA compared to Veh controls (FIGs. 2I-2K). Tumor onset and growth dynamics after RETA withdrawal showed partial protection in tumor onset and persistent protection in tumor progression with significantly reduced endpoint tumor volume and weight compared to Veh. Moreover, tumor progression after RETA withdrawal was also significantly different relative to RETA-treated mice (FIGs. 2G-2J). Lastly, classic circulating markers of obesity-associated inflammation that are associated with cancer were examined. Despite the striking differences observed in metabolic measures at endpoint, no changes were seen in circulating inflammatory markers TNF-a and MCP-1 / CCL2 in RETA or RETA-w / d groups compared to Veh. However, RETA moderately increased IL-6 concentrations, whereas withdrawal caused significantly elevated plasma IL-6 concentrations compared to Veh. These findings demonstrate that RETA leads to persistent changes that impair tumor progression even after discontinuing treatment.

[0301] The effects of RETA and RETA-w / d on the tumor microenvironment were evaluated to determine if there is a persistent effect of RETA. Principal Component Analysis (PCA) of KPCY tumor RNA-seq transcriptomic data revealed significantly different and highly distinct clustering of RETA treated mice compared to Veh controls (FIG. 3). Interestingly, tumors from the RETA-w / d group completely resembled the Veh group (FIG. 3). Gene Set Enrichment Analysis (GSEA) uncovered unique impacts of RETA on key biological processes. Compared to Veh, RETA treatment significantly enriched the expression of genes associated with several Hallmark pathways such as TNFA signaling via NFKB, interferon gamma and alpha responses, and inflammatory response which are associated with enhanced anti-tumor immunity (FIG. 4A). Additionally, Hallmark pathways such as IL2 STAT5 signaling and allograft rejection were upregulated, reflecting the heightened immune activation in tumors which could foster a hostile environment for tumor growth. In contrast, downregulation of hallmark E2F targets and MYC targets VI with RETA denotes impaired cancer cell proliferation and tumor suppression (FIG.

[0302] 4A). Lastly, significant downregulation of metabolic pathways such as bile acid metabolism, glycolysis, fatty acid metabolism, and oxidative phosphorylation likely contribute to tumor suppression by regulating anti-tumor immunity and tumor growth (FIG.4A).

[0303] Interestingly, RETA withdrawal reversed the regulation of almost all of the Hallmark pathways regulated by RETA. Hallmark pathways such as oxidative phosphorylation, glycolysis, fatty acid metabolism, MYC targets VI, adipogenesis, and bile acid metabolism were 45828227.1 45ATTORNEY DOCKET NO. UTRF 25094-05 PCT

[0304] significantly upregulated in tumors from mice subjected to RETA-w / d (FIG.4B). In contrast, Hallmark pathways such as TNFA signaling via NFKB, interferon gamma and alpha responses, inflammatory response, and allograft rejection were down-regulated (FIG.4B).

[0305] The method was evaluated to determine if the anti-tumor therapeutic benefits persist following the cessation of administration, even after metabolic parameters rebound.

[0306] A RETA-withdrawal (RETA-w / d) protocol was implemented wherein DIO mice were treated with the triple agonist (30 nmol / kg, thrice weekly) for 14 days to achieve an approximately 40% reduction in body weight. Upon reaching this plateau, administration was completely discontinued, and subjects were immediately challenged with an orthotopic injection of KPCY cancer cells. Subjects were monitored for weight regain and tumor progression for 21 days post-withdrawal.

[0307] Upon withdrawal of the triple agonist, subjects experienced a rapid "rebound" effect, with body weight returning to baseline (obese) levels within 21 days. Fasted blood glucose concentrations and adiposity (eWAT mass) similarly returned to levels observed in vehicle -treated obese controls. Despite this complete reversal of metabolic improvements, the anti-tumor effects remained significantly persistent. Endpoint tumor weights in the withdrawal group were significantly lower (tumors weighing 2.45 mg / g body weight in RETA-withdrawal group compared to vehicle controls with tumors weighing 5.10 mg / g body weight; **P < 0.01). These data indicate that the method induces a durable "metabolic memory" or systemic immune reprogramming that continues to suppress tumor progression in the absence of active pharmacological intervention.

[0308] Notably, the Example demonstrates that while anti-tumor benefits persisted after drug withdrawal, the gastric effects did not. In the withdrawal group (RETA-w / d), the cessation of treatment led to a complete reversal of cecal retention, with cecal content weights returning to reduced levels comparable to vehicle controls. This reversal confirms that the triple agonist was effectively cleared from the system (consistent with a 21-hour half-life) and suggests that the persistent anti-tumor phenotype is not dependent on continued delay of gastric emptying or active modulation of gastrointestinal transit.

[0309] Importantly, the reciprocal cytokine / adipokine signature demonstrated unique durability during the withdrawal phase. While leptin concentrations — which are closely tied to fat mass — partially reversed toward vehicle levels upon weight rebound, the elevation of circulating IL-6 persisted. In fact, subjects in the withdrawal group (RETA-w / d) exhibited significantly higher plasma IL-6 concentrations than those in the vehicle group. This sustained or further elevated IL-6 signaling, occurring even as metabolic benefits such as leptin suppression were partially lost, suggests that 45828227.1 46ATTORNEY DOCKET NO. UTRF 25094-05 PCT

[0310] IE-6-mediated immune reprogramming is a critical component of the persistent anti-tumor phenotype that remains active after pharmacological intervention has ceased.

[0311] Example 3: RETA treatment improved metabolic factors, boosted anti-tumor immunity, and potently blunted tumor progression in non-obesity associated lung adenocarcinoma Based on the dramatic findings in PDAC, an obesity-associated cancer, whether RETA could also be effective in a non-obesity associated cancer was evaluated. RETA treatment was studied in a Lewis lung carcinoma (LLC) mouse model of LU AD using an identical study design to KPCY above in Example 2 in the same strain of mice, C57BL / 6 J with DIO. In line with the KPCY model, RETA effectively reduced body weights within the first two weeks, plateauing at 41% loss of baseline body weight (FIG. 5A). Significant reductions in epididymal white adipose mass and circulating leptin were found in RETA compared to Veh (FIGs. 5B and 5J). Like above, RETA significantly reduced gastric emptying in LLC model with RETA cecal content greatly elevated compared to Veh. Similarly, endpoint brown adipose tissue and liver weights were significantly reduced with RETA, however there were no differences in spleen weight. RETA also improved systemic metabolism in LLC model with a significant reduction in fasted blood glucose concentrations 1 week after starting the treatment continuing until endpoint where glucose concentrations were 41% lower compared to Veh (FIG. 5C), which was similar to findings in the KPCY studies above. Compared to Veh, RETA also showed significantly lower concentrations of plasma insulin at endpoint and reduced HOMA-IR score, whereas the QUICKI score was significantly increased with RETA. Taken together, in this LLC model, RETA induced identical physiological improvements compared to the KPCY cohorts, demonstrating highly reproducible impacts on metabolic outcomes.

[0312] Once improved metabolic parameters were established after RETA treatment the impact of RETA on LUAD was assessed. After two weeks of RETA treatment, LLC cells were injected when weight loss plateaued as in above studies. Importantly, tumor take was remarkably reduced with RETA (N = 4 / 8, 50%) compared to Veh (N = 7 / 7, 100%) (FIG. 5D), more than observed with RETA treatment in the KPCY model. RETA treatment dramatically delayed LLC tumor onset, with unpalpable tumors in the RETA treated group until day 16, compared to onset observed in Veh treated mice where tumors were palpated starting on day 10 (Fig.5E). RETA also blunted tumor progression with significantly reduced tumor volumes and weights at endpoint compared to Veh (FIGs. 5F-5H). Lastly, circulating cytokines and chemokines were measured with minor non-significant elevations in plasma concentrations of TNF-a and MCP-1 after RETA treatment. However, IL-6 was significantly 4.6-fold elevated with RETA treatment in the LLC model (FIG.51).

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[0314] Last, to assess the role of the anti-tumor immunity to RETA-mediated improved LLC tumor outcomes, systemic quantities of key innate and adaptive immune cells were examined. A significant reduction in CD1 lb+ cells and macrophages was observed with RETA when quantified out of CD45, which did not differ between intervention and Veh (FIG. 6A-6B).

[0315] Intriguingly, RETA significantly decreased monocytic myeloid derived suppressor cells (M-MDSCs) and granulocytic or polymorphonuclear MDSCs (PMN-MDSCs) and enriched MHC II high macrophages (FIGs. 6C-F), suggesting reduced immunosuppression and increased antigen presentation with RETA, which aligns with reduced tumor burden. Lastly, while no significant changes were observed in CD3+ or CD8+ cells, RETA significantly increased PD-1 mean fluorescent intensity (MFI) in CD8+ T cells compared to Veh, suggesting elevated activation of cytotoxic T cells (FIGs. 6G-6I).

[0316] The method was evaluated in a Lewis lung carcinoma (LLC) model to assess efficacy in malignancies not traditionally categorized as obesity driven.

[0317] DIO mice were treated with the triple agonist (30 nmol / kg, every other day, throughout the study). Following a two- week priming period with the triple agonist, subjects were subcutaneously injected into the flank with 106LLC cells.

[0318] The method achieved a 50% reduction in tumor engraftment (4 / 8 mice) compared to the vehicle group (7 / 7 mice). Tumor onset was significantly delayed, with no palpable tumors observed in the triple agonist group until day 16, whereas vehicle tumors were palpable by day 8. At the study endpoint (day 21), the method resulted in a 17-fold reduction in tumor volume (52.67 mm3) relative to vehicle controls (925.93 mm3). Tumor weights were similarly reduced (0.70 mm3in treated vs. 11.65 mm3in vehicle).

[0319] Analysis of systemic markers in the non-obesity-associated LLC model confirmed this immune-metabolic reprogramming. Treatment with the triple agonist resulted in a 4.6-fold increase in plasma IL-6 concentrations compared to vehicle-treated controls. Simultaneously, the treatment significantly suppressed circulating leptin levels, corresponding with the reduction in epididymal white adipose tissue mass. This 4.6-fold elevation of IL-6, a key mediator of antitumor immunity, highlights the technology’s ability to promote a robust systemic anti-cancer response even in malignancies where obesity is not the primary driver.

[0320] Example 4: Spontaneous Tumor Suppression in a Genetic Model of Breast Cancer

[0321] To demonstrate the effectiveness of triple incretin agonism in a spontaneous, non-injection-based malignancy model, the method was evaluated using the C3(l)-T antigen genetic model of breast cancer. Unlike the syngeneic models described in Examples 1 and 3, this

[0322] 45828227.1 48ATTORNEY DOCKET NO. UTRF 25094-05 PCT

[0323] transgenic model involves the spontaneous development of mammary adenocarcinomas, which closely mimics the multi-stage progression and genetic drivers of human breast cancer.

[0324] Transgenic mice were administered the triple agonist according to the dosing protocols established in Example 1. Preliminary results demonstrated that triple agonism significantly delayed the onset of palpable mammary tumors and reduced the total tumor burden compared to vehicle-treated transgenic controls. FIG.7 shows survival curve plotted as % of mice tumor-free for each group in the genetic model of breast cancer-C3(l)-T antigen. This finding confirms that the anti-tumor effects of the triple agonist extend to genetic drivers of malignancy and are not limited to models requiring orthotopic or syngeneic cell-line transplantation.

[0325] Example 5: Weight-Neutral Efficacy and Combination with Immune Checkpoint Inhibition

[0326] The method was evaluated to determine if anti-tumor efficacy could be achieved at sub-weight-loss doses and to assess synergy with standard-of-care immunotherapy. Doseoptimization studies were conducted in male DIO mice with PDAC (KPCY model) at 1.0, 1.5, and 2.0 nmol / kg of RETA. For the therapy study, a low dose of 1 nmol / kg RETA was administered alone or in combination with anti-PD-1 immunotherapy.

[0327] Results demonstrated that at these optimized lower doses, body weights were maintained with minimal weight loss. Despite the lack of significant weight reduction, RETA administration effectively improved metabolic signaling, reducing fasting blood glucose concentrations from 200 mg / dL to 100 mg / dL. Notably, low-dose RETA reduced tumor progression to volumes that matched the efficacy of anti-PD-1 immunotherapy. While the combination of RETA and anti-PD-1 did not further boost the effectiveness of the immunotherapy at this specific dosing and timing, the ability of RETA to independently match the performance of a primary checkpoint inhibitor — while remaining weight-neutral — confirms that the anti-cancer benefits are driven by unique immune-metabolic signaling rather than caloric restriction or extreme weight loss.

[0328] Example 6: Systemic Biomarkers of Response (IL-6 and Leptin Ratios)

[0329] To identify systemic signatures of the protective phenotype, circulating cytokine and adipokine levels were quantified in the LLC model. The method induced a reciprocal shift in the systemic environment characterized by a 4.6-fold increase in anti-tumor IL-6 (FIG.51) and a simultaneous significant decrease in pro-tumor leptin (FIG.5J). This systemic signature (High IL-6 / Low Leptin) was consistently associated with the 50% reduction in tumor engraftment and the 17-fold reduction in tumor volume observed in treated subjects, regardless of the primary metabolic driver of the malignancy.

[0330] Example 7: Dose optimization to minimize weight loss to test for therapeutic response 45828227.1 49ATTORNEY DOCKET NO. UTRF 25094-05 PCT

[0331] RETA therapy trial dose pilot study was earned out. Body weights were maintained with very minimal weight loss across all 3 doses from 1, 1.5, 2.0 nmol / Kg (FIG.8A). Glucoses were reduced in all 3 doses from 200 to 100 mg / dL (FIG.8B).

[0332] Material and Methods

[0333] Reagents

[0334] Unless otherwise specified, all reagents were purchased from Sigma-Aldrich (St. Eouis, MO). Retatrutide (RETA) (LY3437943, Cat. No. HY-P3506, MedChemExpress, Monmouth, NJ), Semaglutide (SEMA, Cat. No. HY-114118, MedChemExpress, Monmouth, NJ) and dextrose (CAS No. 50-99-7, Fisher Scientific, Waltham, MA) were purchased from Fisher Scientific Company, Waltham, MA. PCR grade water was purchased from Alpha Teknova (Product number W333O, CAS No. 7732-18-5, Alpha Teknova, Inc., Hollister, CA). Flow cytometry antibodies (Table 1), compensation beads, and reagents were from Tonbo Biosciences, Inc. (San Diego, CA) and Biolegend (San Diego, CA).

[0335] Table 1. Antibodies for flow cytometric analysis of spleens with myeloid and T cell panels.

[0336]

[0337] Animal experiments

[0338] The protocol (IACUC #24-0504.0) used for animal studies was approved by the University of Tennessee Health Science Center’s (UTHSC) Institutional Animal Care and Use Committee (IACUC) under Animal Welfare Assurance Number A3325-01 and in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Diet-induced obese (DIO) C57BL / 6 J male mice were purchased from Jackson Laboratory (#380050, Bar Harbor, ME) at 16 weeks of age. Upon receipt, mice were housed in groups of five and acclimated for 3 weeks. The mice were maintained on the same diet used to induce DIO by Jackson Lab, 60 kcal% from high-fat diet (D12492i, Research Diets Inc., New Brunswick, NJ, 45828227.1 5QATTORNEY DOCKET NO. UTRF 25094-05 PCT

[0339] USA) with ad libitum access and sterile water was provided from the Lixit System. A 12 h light:dark cycle and temperature at 24 ± 2 °C was maintained. The health of the mice was monitored using body condition scoring (BCS) to observe any signs of weakness or inactivity.

[0340] RETA and SEMA administration

[0341] Mice were randomized to intervention at the start of the study at the cage level. Mice were assigned to respective study groups to comprehensively evaluate multiple medical weight loss approaches with various controls: 1) vehicle (Veh, sterile water) as control, 2) SEMA to induce weight loss using semaglutide, 2) WM-CR to induce weight loss by caloric restriction to match weight loss in SEMA, 3) continuous RETA to induce weight loss, or 4) short-term RETA to induce weight loss then regain, termed “RETA withdrawal” (RETA-w / d). Mice were subcutaneously (SQ) administered either Veh, 30 nmol / kg RETA, or 30 nmol / kg SEMA every other day before the initiation of the dark cycle at 5 pm. In the RETA-w / d group, 14 days after RETA initiation, RETA administration was stopped, and this subset of mice was switched to SQ Veh injections. The body weight of mice and food intake were recorded daily.

[0342] Cell culture

[0343] KPCY 2838 mouse pancreatic cancer cell line was acquired from Kerafast® (Shirley, MA). KPCY cells were cultured in DMEM (Cat. No. 11965092, Gibco, Waltham, MA) supplemented with 1% penicillin-streptomycin (Cat. No. SV30010, HyClone™, Cytiva, Marlborough, MA), 1% GlutaMAX (Cat. No. 35050061, Gibco, Waltham, MA), and 10% FBS (Cat. No. 26140-087 Gibco, Waltham, MA) per instructions from Kerafast®. The cells were cultured in 150 mm2culture dishes containing 25 mL media under aseptic conditions in a CO2 incubator at 37 °C. The cells were passaged after reaching a sub-confluent stage. LLC cell line was a gift from J. A. Carson (UTHSC, Memphis, TN) and was cultured in DMEM as described previously. Cell lines were confirmed negative for mycoplasma.

[0344] Tumor models and endpoint

[0345] DIO mice were injected SQ with either 2 x IO3KPCY cells or 106LLC cells in 100 pL sterile PBS in the right flank. Mice were maintained on Veh, SEMA, WM-CR, or RETA. Tumor implantation (tumor take) was recorded. The progression of the tumors was monitored by palpating every 3 days after injecting the cells until tumors were palpable, after which the tumors were measured by digital caliper every other day. Tumor size was monitored by measuring the length and width, and endpoint was 2.5 weeks after cell injection. Tumor volume was calculated using the formula: volume = (length) x (width)2 / 2. At the endpoint, mice were euthanized using isoflurane. Blood was collected via cardiac puncture into EDTA-coated syringes. Plasma was separated from other blood components by centrifugation at 1200 x g for 45 min at 10 °C.

[0346] 45828227.1 51ATTORNEY DOCKET NO. UTRF 25094-05 PCT

[0347] Tumors were excised and tumor mass was recorded. Cecal content was collected and weighed. Epididymal white adipose tissue (WAT), brown adipose tissue (BAT), liver, and spleen were collected and weighed. Samples were snap-frozen in liquid nitrogen and stored at -80 °C until further analysis.

[0348] Oral glucose tolerance test (OGTT)

[0349] For the KPCY model, oral glucose tolerance tests (OGTT) were performed to evaluate glucose tolerance. OGTTs were performed two days before beginning the RETA treatment (baseline OGTT), 12 days after beginning RETA / Veh treatment (midpoint OGTT) before KPCY cell injections, and 2 days before endpoint (endpoint OGTT). Briefly, mice were fasted for five hours in fresh cages with ad libitum access to drinking water. Blood glucose levels were measured using tail vein prick using a lancet and a Glucometer (Contour™ Next EZ, Ascensia Diabetes Care Holdings AG, Basel, CH). Blood glucose readings for 0 min time point were recorded, then 2 g / kg dextrose was administered using a 20% glucose solution prepared in sterile distilled water by oral gavage. Blood glucose was measured at 15, 30, 60, 90, and 120 min. Bleeding was stopped by applying Kwik Stop® Styptic powder (Miracle Care Products, Dayton, OH) using sterile cotton swabs.

[0350] Cytokine profiling using immunology multiplex assay

[0351] MCP-1 / CCL2, IL-6, and TNF-alpha (TNFa) were measured in fasted plasma collected at endpoint using the Milliplex MAP Mouse Metabolic Hormone Magnetic Bead Panel in the Luminex MAGPIX system (EMD Millipore, Billerica, MA).

[0352] Bulk RNAseq analysis

[0353] Reads from RNAseq data were processed using the nf-core / RNAseq v3.17.0 pipeline and the GRCm39 genome assembly. Aligned transcript counts were imported into R v4.2.2 using Tximport vl.32.0. Low capture genes with less than ten total counts summed across all samples were removed. Differential expression was performed using DESeq2. Gene set enrichment analysis was performed using fgsea v 1.30.0 using mouse-ortholog Hallmark genesets from MsigDB loaded with msigdbr v7.5.1.

[0354] Flow cytometry

[0355] Excised spleens were crushed in RPMI following established protocols. Digested tissue was filtered through 70 pm strainer to obtain a single cell suspension. Antibodies used (Table 1) were titrated and the separation index was calculated using FlowJo version 10 software for every study. Following red blood cell lysis (BioLegend), viability was determined by staining with Ghost dye (Tonbo Biosciences, Inc.) followed by FcR-blocking (Tonbo Biosciences, Inc.). Cells were stained with fluorescently labeled antibodies and fixed in Perm / fix buffer (Tonbo 45828227.1 52ATTORNEY DOCKET NO. UTRF 25094-05 PCT

[0356] Biosciences Inc.). Stained cells were analyzed using a Bio-Rad ZE5 flow cytometer at the UTHSC Flow Cytometry and Cell Sorting Core. A minimum number of 1000 events were considered for analysis. Fluorescence minus one (FMO) stained cells and single color Ultracomp Beads (Invitrogen, Carlsbad, CA) were used as negative and positive controls, respectively. Data were analyzed using FlowJo version 10 software with FlowAI plug in clean up. Gating schemata were drawn in FlowJo version 10 software.

[0357] Statistical methods

[0358] Statistical analysis was conducted using One-way or Two-way ANOVA followed by Tukey’s or Sidak’s multiple comparison test or using Student’s t-test as specified in figure legends. Data are presented as mean ± SEM. P value less than 0.05 was considered statistically significant. Data and statistical analysis were performed using GraphPad Prism 10.4.0 (Graphpad Software, Inc., La Jolla CA). Animals were assigned to study groups based on their starting body weights to ensure equal unbiased distribution at the cage level. For analysis of circulating peptide hormones, data below the detection limit was considered as an outlier and omitted from data analysis.

[0359] Data Availability

[0360] It is understood that the disclosed method and compositions are not limited to the particular methodology, protocols, and reagents described as these can vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims.

[0361] Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, many equivalents to the specific embodiments of the method and compositions described herein. Such equivalents are intended to be encompassed by the following claims.

[0362] 45828227.1 53

Claims

ATTORNEY DOCKET NO. UTRF 25094-05 PCTCLAIMSWe claim:

1. A dosage formulation comprising one or more incretin agonists in an amount effective to prevent cancer onset, reduce cancer risk, delay tumor engraftment or onset, slow tumor progression, reduce tumor burden or volume, improve response to cancer therapy, and / or improve survival in a subject in need thereof, and one or more pharmaceutically acceptable excipients.

2. The dosage formulation of claim 1, wherein the one or more incretin agonists activate one or more receptors of Glucagon- like peptide- 1 (GLP-1), Glucose-dependent insulinotropic polypeptide (GIP), and Glucagon (GCG).

3. The dosage formulation of claim 1 or 2, wherein the one or more incretin agonists activate at least two receptors of Glucagon-like pcptidc-1 (GLP-1), Glucosc-dcpcndcnt insulinotropic polypeptide (GIP), and Glucagon (GCG).

4. The dosage formulation of any one of claims 1-3, wherein the one or more incretin agonists activate all three receptors of Glucagon-like peptide- 1 (GLP-1), Glucose-dependent insulinotropic polypeptide (GIP), and Glucagon (GCG).

5. The dosage formulation of any one of claims 1-4, wherein the one or more incretin agonists comprise retatrutide shown in Structure I, or an analog, a variant, and a derivative thereof.45828227.1 54ATTORNEY DOCKET NO. UTRF 25094-05 PCTStructure I:

6. The dosage formulation of any one of claims 1-5, wherein the dosage is formulated for administration by parenteral, enteral, or topical routes of administration.

7. The dosage formulation of any one of claims 1-6, wherein the dosage is formulated for administration by intramuscular, intraperitoneal, intravenous or subcutaneous injection.

8. The dosage formulation of any one of claims 1-7, wherein the one or more incretin agonists are present in an amount between 0.005 mg and 20 mg per kg of body weight of the human.

9. The dosage formulation of any one of claims 1-8, wherein the one or more incretin agonists are present in an amount between 0.0001 mg and 20 mg per kg of body weight of the human.

10. The dosage formulation of any one of claims 1-9, formulated for administration once daily, weekly, or monthly.45828227.1 55ATTORNEY DOCKET NO. UTRF 25094-05 PCT11. The dosage formulation of any one of claims 1-10, wherein the subject in need thereof is diagnosed with cancer or as being at enhanced risk of cancer.

12. The dosage formulation of claim 11, wherein the cancer is an obesity-associated cancer or a non-obesity-associated cancer.

13. The dosage formulation of claim 11 or 12, wherein the cancer is pancreatic ductal adenocarcinoma (PDAC) or lung adenocarcinoma (LU AD).

14. A method for preventing cancer onset, reducing cancer risk, delaying tumor engraftment or onset, slowing tumor progression, reducing tumor burden or volume, improving response to cancer therapy, and / or improving survival, comprising administering to a subject in need thereof an effective amount of one or more incretin agonists.

15. The method of claim 14, wherein the one or more incretin agonists activate one or more receptors of Glucagon-like peptide- 1 (GLP-1), Glucose-dependent insulinotropic polypeptide (GIP), and Glucagon (GCG).

16. The method of claim 14 or 15, wherein the one or more incretin agonists activate at least two receptors of Glucagon-like peptide- 1 (GLP-1), Glucose-dependent insulinotropic polypeptide (GIP), and Glucagon (GCG).

17. The method of any one of claims 14-16, wherein the one or more incretin agonists activate all three receptors of Glucagon-like peptide- 1 (GLP-1), Glucose-dependent insulinotropic polypeptide (GIP), and Glucagon (GCG).

18. The method of any one of claims 14-17, wherein the one or more incretin agonists comprise retatrutide shown in Structure I, or an analog, a variant, and a derivative thereof.

19. The method of any one of claims 14-18, wherein the one or more incretin agonists are formulated with one or more pharmaceutically acceptable excipients.

20. The method of any one of claims 14-1 , wherein the one or more incretin agonists are administered by parenteral, enteral, or topical routes of administration.

21. The method of any one of claims 14-20, wherein the one or more incretin agonists are administered by intramuscular, intraperitoneal, intravenous or subcutaneous injection.

22. The method of any one of claims 14-21, wherein the one or more incretin agonists are administered in an amount between 0.0001 mg and 20 mg per kg of body weight of the human.

23. The method of any one of claims 14-22, wherein the one or more incretin agonists are administered in an amount between 0.0001 mg and 20 mg per kg of body weight of the human.

24. The method of any one of claims 14-23, wherein the administration is once daily, weekly, or monthly.45828227.1 56ATTORNEY DOCKET NO. UTRF 25094-05 PCT25. The method of any one of claims 14-24, wherein the subject in need thereof is diagnosed with cancer or as being at enhanced risk of cancer.

26. The method of any one of claims 14-25, wherein the cancer is an obesity-associated cancer or a non-obesity-associated cancer.

27. The method of any one of claims 14-26, wherein the cancer is pancreatic ductal adenocarcinoma (PDAC), breast cancer (BC) or lung adenocarcinoma (LUAD).

28. The method of any one of claims 14-27, wherein the subject in need thereof is obese or non-obese.

29. The method of any one of claims 14-28, wherein the method administers an effective amount of the one or more incretin agonists to enrich pro-inflammatory gene signatures, reduce immunosuppressive suppressor cells, increase MHC Il-high antigen-presenting macrophages, elevate pro-inflammatory cytokines, and / or enhance cytotoxic CD8+ T-ccll activation.

30. The method of claim 29, wherein the immunosuppressive suppressor cells comprise one or more of immunosuppressive monocytic myeloid-derived suppressor cells (M-MDSCs) and polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs).

31. The method of claim 30, wherein the pro-inflammatory cytokines comprise interleukin-6.

32. The method of any one of claims 14-31, wherein the administration of the one or more incretin agonists improves glycemic control by lowering fasting blood glucose and insulin levels, reducing indices of insulin resistance, including the homeostatic model assessment of insulin resistance (HOMA-IR), and increasing insulin sensitivity indices, including the quantitative insulin sensitivity check index (QUICKI).

33. The method of any one of claims 14-32, wherein the method administers the one or more incretin agonists in an amount effective, optionally, to achieve a body-weight reduction of about 1% to about 40%, or about 5% to about 30%, or about 10% to about 30%, thereby establishing a metabolic environment associated with reduced cancer risk and improved therapeutic response.

34. The method of any one of claims 14-33, wherein the method is effective to reduce tumor size in the subject.

35. The method of any one of claims 14-34, further comprising administering to the subject one or more selected from the group consisting of an immune checkpoint modulator, a chemotherapeutic agent, an anti-infective agent, adoptive T cell therapy, a cancer vaccine, surgery, radiation therapy.

36. The method of claim 35, wherein the immune checkpoint modulator is selected from the group consisting of PD-1 antagonists, PD-1 ligand antagonists, and CTLA4 antagonists.

37. The method of claim 36, wherein the immune checkpoint modulator is PD-1 antagonists.45828227.1 57ATTORNEY DOCKET NO. UTRF 25094-05 PCT38. The method of any one of claims 14-37, further comprising:(a) an administration phase, and(b) a withdrawal phase,wherein the administrative phase comprises administering an effective amount of one or more incretin agonists for an effective amount of time, andthe withdrawal phase comprises a period of time after no administration of one or more incretin agonists wherein one or more incretin agonists are not administered, allowing increase in body weight compared to the administration phase, wherein the subject has one or more of elevated circulating IL-6, increased antigen presenting cells, and reduced immunosuppressive cells, compared to levels prior to the administration phase, during said withdrawal phase.45828227.1 58

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